Method for producing polymer compound

Abstract

In one aspect of the present disclosure, provided is a method for producing a polymer compound, comprising a step of reacting dimethyl sulfoxide in the presence of a base and a reducing agent, the method is characterized in that a polypeptide containing at least one mercapto group and at least one compound selected from the group consisting of a polyether, a polyester and a polycarbonate having two structures represented by general formula (1) (in general formula (1), M represents any one of H, Na, K, NHEt3 or NHEtiPr2) are heated and reacted. [Chemical Formula 1] # imgabs0 #

Description

[Technical field]

[0001] The present disclosure relates to a method for producing a polymer compound, a method for producing a polymer compound solution, a method for producing a film, and a method for producing a fiber, as well as the film and the fiber. [Background Technology]

[0002] Because structural proteins such as silk fibroin and spider silk fibroin exhibit excellent strength, they have attracted attention as alternatives to structural materials composed of synthetic resins, etc. In recent years, with the development of recombinant technology, mass production technologies for recombinant structural proteins that mimic these structural proteins are being developed (e.g., Patent Document 1).

[0003] Because proteins contain numerous amide groups on their main chains and numerous hydrogen-bonding functional groups such as carboxyl, amino, hydroxyl, and thiol groups on their side chains, they can form numerous hydrogen bonds between molecules. Therefore, while molded articles obtained by heat-pressing structural proteins are believed to have excellent mechanical strength, they also exhibit a relatively fragile nature, susceptible to cracks and crazing due to impacts. This situation hinders the use of proteins as alternatives to conventional general-purpose plastics.

[0004] As a means of protein modification, methods are currently under investigation for conjugating compounds with properties different from those of proteins. For example, Patent Document 2 discloses a method for producing a protein material that involves conjugating a substance with excellent water resistance to a specially modified silk fibroin.

[0005] For example, Patent Document 3 discloses a method for producing a polymer, wherein a thioether bond is generated by utilizing a 1,4-addition reaction (Michael addition reaction) between the thiol group of cysteine ​​constituting the polypeptide backbone and the carbon-carbon double bond of maleimide or a maleic acid derivative, thereby introducing at least one functional group selected from the group consisting of a free polyether group, a polyester group, a polycarbonate group, a polyamide group, a polyol group, and a modified polysaccharide group into the polypeptide backbone. [Prior art literature] [Patent Document]

[0006] Patent Document 1: International Publication No. 2015 / 178466 Patent Document 2: International Publication No. 2019 / 194146 Patent Document 3: International Publication No. 2021 / 187502 Patent Document 4: Japanese Patent Application Laid-Open No. 05-336963 Patent Document 5: International Publication No. 1994 / 023021 Patent Document 6: International Publication No. 2019 / 194263 [Summary of the invention] [Problems to be solved by the invention]

[0007] Although the manufacturing method described in Patent Document 3 can introduce various functional groups into the polypeptide backbone and has practical value, due to the different structures of the Michael addition acceptor that can undergo Michael addition reaction with the thiol group possessed by the polypeptide backbone, more stringent control of the reaction and reaction efficiency are required. Therefore, as a synthetic technology for industrial raw materials, there is still room for improvement.

[0008] For example, when using a maleimide group as a Michael addition acceptor, the high reactivity of the compound constituting the Michael addition acceptor can result in the formation of not only the intended molecule but also polymers with unintended long-chain growth or branching. This can increase the viscosity of the reaction solution and even make the reaction system difficult to control. In this case, the conversion rate of the starting peptide to the target polymer is low, resulting in a downward trend in the conversion rate of the starting material. Furthermore, even by adjusting reaction conditions such as the acidity of the reaction solution, reaction temperature, and stirring speed, it is still difficult to fully control the reaction system.

[0009] Therefore, it is possible to consider using a less reactive Michael addition acceptor as the reaction substrate. However, due to the reduced reactivity of the substrate itself, the conversion rate of the raw material may be reduced.

[0010] The purpose of the present disclosure is to provide a production method that can improve the raw material conversion rate compared with the traditional method in the production process of the above-mentioned polymer compound. Methods used to solve problems

[0011] The inventors have discovered that when using conventional manufacturing methods on an industrial scale, if the non-protonic polar solvent dimethyl sulfoxide is used as a solvent for the reaction of introducing specific functional groups into a polypeptide, heating during synthesis will promote the formation of disulfide bonds between the sulfhydryl groups possessed by the polypeptide, making it difficult to bind the functional group to the polypeptide through a Michael addition reaction utilizing the sulfhydryl group. It is speculated that when the rate of the Michael addition reaction is significantly faster than the rate of the disulfide bond formation reaction, no obvious problems will arise. However, in practice, it has been confirmed that, for example, in the reaction of a substrate containing a (meth)acryloyloxy group and a polypeptide, the rate of the Michael addition reaction is slower, while the disulfide bond formation reaction between the polypeptides is promoted, resulting in the inability to fully synthesize the target polymer compound. The present disclosure is based on the above insights.

[0012] The present disclosure provides the following [1] to

[34] . [1] A method for producing a polymer compound, comprising the following steps: In dimethyl sulfoxide, in the presence of a base and a reducing agent, a polypeptide containing at least one thiol group is heated to react. and at least one compound selected from the group consisting of polyether, polyester, and polycarbonate having two structures represented by the following general formula (1). [Chemical Formula 1] [In the general formula (1), M represents any one of H, Na, K, NHEt3, or NHEtiPr2.] [2] A method for producing a polymer compound, comprising the following steps: In dimethyl sulfoxide, in the presence of alkali, reducing agent and polymerization inhibitor, a polypeptide containing at least one thiol group, and at least one compound selected from the group consisting of polyether, polyester, and polycarbonate having two structures represented by the following general formula (2). [Chemical Formula 2] [In general formula (2), R 1 represents a hydrogen atom or a methyl group.] [3] A method for producing a polymer compound, comprising the following steps: In dimethyl sulfoxide, in the presence of a reducing agent, a polypeptide containing at least one sulfhydryl group, and at least one compound selected from the group consisting of polyether, polyester, and polycarbonate having two structures represented by the following general formula (1). [Chemical Formula 3] [In the general formula (1), M represents any one of H, Na, K, NHEt3, or NHEtiPr2.] [4] A method for producing a polymer compound, comprising the following steps: In dimethyl sulfoxide, in the presence of a reducing agent and an inhibitor, a polypeptide containing at least one thiol group is heated to react and at least one compound selected from the group consisting of polyether, polyester, and polycarbonate having two structures represented by the following general formula (2). [Chemical Formula 4] [In general formula (2), R1 represents a hydrogen atom or a methyl group.] [5] The method for producing a polymer compound according to any one of [1] to [4], wherein the reducing agent is at least one selected from the group consisting of dithiol, sodium sulfate, sodium sulfite, and sodium dithionite. [6] The production method according to any one of [1] to [5], wherein the amount of the reducing agent prepared is 0.5 equivalents or more relative to the thiol groups of the polypeptide. [7] The production method according to any one of [1] to [6], wherein the reaction between the polypeptide and the compound is carried out at 50°C or above. [8] A method for producing a polymer compound solution, comprising the steps of isolating the polymer compound obtained by the production method according to any one of [1] to [7] and then dissolving the resulting polymer compound in a solvent. [9] A method for producing a thin film, comprising the step of forming a thin film from a polymer compound solution obtained by the production method described in [8].

[10] A method for producing fibers, comprising the step of spinning a polymer compound solution obtained by the production method described in [8].

[11] A method for manufacturing a thin film, comprising the following steps: A polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates having two structures represented by the following general formula (1) are subjected to a heating reaction in dimethyl sulfoxide in the presence of a base and a reducing agent to obtain a polymer compound solution; The polymer compound solution is formed into a film. [Chemical Formula 5] [In the general formula (1), M represents any one of H, Na, K, NHEt3, or NHEtiPr2.]

[12] A method for manufacturing a thin film, comprising the following steps: A polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates having two structures represented by the following general formula (2) are reacted by heating in dimethyl sulfoxide in the presence of a base, a reducing agent, and a polymerization inhibitor to obtain a polymer compound solution; The polymer compound solution is formed into a film. [Chemical Formula 6] [In general formula (2), R 1 represents a hydrogen atom or a methyl group.]

[13] A method for producing a fiber, comprising the following steps: A polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates having two structures represented by the following general formula (1) are subjected to a heating reaction in dimethyl sulfoxide in the presence of a base and a reducing agent to obtain a polymer compound solution; The polymer compound solution is spun. [Chemical Formula 7] [In the general formula (1), M represents any one of H, Na, K, NHEt3, or NHEtiPr2]

[14] A method for producing a fiber, comprising the following steps: A polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates having two structures represented by the following general formula (2) are reacted by heating in dimethyl sulfoxide in the presence of a base, a reducing agent, and a polymerization inhibitor to obtain a polymer compound solution; The polymer compound solution is spun. [Chemical Formula 8] [In general formula (2), R 1 represents a hydrogen atom or a methyl group.]

[15] A film containing a polymer compound, wherein: The polymer compound has the following structure: Peptide part, And at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure is directly bonded via the structure represented by the following general formula (3), the following general formula (4), or the following general formula (5), so that the polypeptide portion is bonded to S of the following general formula (3), the following general formula (4), or the following general formula (5). [Chemical Formula 9] [Chemical Formula 10] [Chemical Formula 11] [In general formula (3) and general formula (4), M represents any one of H, Na, K, NHEt3, or NHEtiPr2, and in general formula (5), R 1 Indicates H or Me.] Its elongation is above 400%, and its breaking strength is greater than its yield point strength.

[16] A fiber containing a polymer compound, wherein: The polymer compound has the following structure: Peptide part, And at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure is directly bonded via the structure represented by the following general formula (3), the following general formula (4), or the following general formula (5), so that the polypeptide portion is bonded to S of the following general formula (3), the following general formula (4), or the following general formula (5). [Chemical Formula 12] [Chemical Formula 13] [Chemical Formula 14] [In general formula (3) and general formula (4), M represents any one of H, Na, K, NHEt3, or NHEtiPr2, and in general formula (5), R 1 Indicates H or Me.] Its elongation is above 400%, and its breaking strength is greater than its yield point strength.

[17] A fiber comprising a synthetic polymer obtained by the production method described in any one of [1] to [8], having an elongation of 400% or more and a ratio of breaking strength to yield strength greater than 1.

[18] A film comprising a synthetic polymer obtained by the production method described in any one of [1] to [8], wherein the film has an elongation of 400% or more and a ratio of breaking strength to yield strength greater than 1.

[19] A method for producing a solution-state adhesive, comprising dissolving a polymer compound obtained by the method described in any one of [1] to [8] in a solvent.

[20] A method for producing a water-dispersible adhesive, comprising dispersing a polymer compound obtained by the method described in any one of [1] to [8] in an aqueous medium. [twenty one] A method for producing a film-like adhesive, comprising the step of forming a film from a solution obtained by dissolving a polymer compound obtained by the method described in any one of [1] to [8]. [twenty two] A method for producing a powdered adhesive, comprising the step of obtaining a powder composition containing a polymer compound obtained by the method described in any one of [1] to [8]. [twenty three] A method for producing an adherend, characterized in that: a polymer compound obtained by the method described in any one of [1] to [8] is dissolved in a solvent to form a solution, and the solution is placed between a plurality of adherends, and then the solvent is removed from the solution to solidify the polymer compound, thereby bonding the adherends to each other. [twenty four] A method for producing an adherend, characterized in that: a polymer compound obtained by the method described in any one of [1] to [8] is dispersed in an aqueous medium to form an aqueous dispersion, the aqueous dispersion is placed between a plurality of adherends, and then the aqueous medium is removed from the aqueous dispersion to solidify the polymer compound, thereby bonding the adherends to each other.

[25] A method for manufacturing an adherend, characterized in that a film containing a polymer compound obtained by any one of the methods described in [1] to [8] is softened by swelling or heating, and the film is placed between a plurality of adherends, and then the film is hardened while being pressed against the adherends, thereby bonding the adherends to each other.

[26] A method for producing an adherend, characterized in that a powder composition containing a polymer compound obtained by the method described in any one of [1] to [8] is placed between a plurality of adherends, and while heating the powder composition, the powder composition is pressurized through the adherends to solidify the powder composition, thereby bonding the adherends.

[27] A method for producing a solution coating, comprising the step of dissolving a polymer compound obtained by the method described in any one of [1] to [8] in a solvent.

[28] A method for producing a water-dispersible coating, comprising dispersing a polymer compound obtained by the method described in any one of [1] to [8] in an aqueous medium.

[29] A method for producing a thin film coating, comprising the step of forming a thin film from a solution obtained by dissolving a polymer compound obtained by the method described in any one of [1] to [8].

[30] A method for producing a powdered coating, comprising the step of obtaining a powder composition containing a polymer compound obtained by the method described in any one of [1] to [8].

[31] A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on a surface of the substrate, characterized in that: A solution in which a polymer compound obtained by any one of the methods described in any one of [1] to [8] is dissolved in a solvent is supplied to at least a portion of the surface of the substrate, at least a portion of the surface of the substrate is coated with the solution, the solvent is removed from the solution, and the polymer compound is solidified, thereby forming a coating layer on at least a portion of the surface of the substrate.

[32] A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on a surface of the substrate, characterized in that: An aqueous dispersion formed by dispersing a polymer compound obtained by any one of the methods described in any one of [1] to [8] in an aqueous medium is supplied to at least a portion of the surface of the substrate. After coating at least a portion of the surface of the substrate with the aqueous dispersion, the aqueous medium is removed from the aqueous dispersion, and the polymer compound is solidified, thereby forming a coating layer on at least a portion of the surface of the substrate.

[33] A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on a surface of the substrate, characterized in that: A film containing a polymer compound obtained by any one of the methods described in [1] to [8] is softened by swelling or heating, and after being placed on at least a portion of the surface of the substrate, the film is hardened while being pressed against the substrate, thereby forming the coating layer on at least a portion of the surface of the substrate.

[34] A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on a surface of the substrate, characterized in that: In a state where a powder composition containing a polymer compound obtained by the method described in any one of [1] to [8] is placed on at least a portion of the surface of the substrate, the powder composition is heated and simultaneously pressurized between a pressurizing body and the substrate to solidify the powder composition, thereby forming the coating layer on at least a portion of the surface of the substrate.

[0014] One aspect of the present disclosure provides a method for producing a polymer compound, comprising the following steps: heating a polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyether, polyester and polycarbonate having two structures represented by the following general formula (1) in dimethyl sulfoxide in the presence of an alkali and a reducing agent. [Chemical Formula 15] [In the general formula (1), M represents any one of H, Na, K, NHEt3, or NHEtiPr2.]

[0015] In one example of the above-mentioned manufacturing method, when the polypeptide reacts with the specific compound of the present invention, a method in which a base and a reducing agent are present in the reaction system is adopted. The reducing agent can exist stably in dimethyl sulfoxide itself, is not easy to undergo Michael addition reaction with the functional group shown in formula (1), and can inhibit the formation of disulfide bonds between the sulfhydryl groups possessed by the polypeptide. Moreover, the base can activate the sulfhydryl groups possessed by the polypeptide, thereby promoting the Michael addition reaction with the functional group represented by formula (1). Through this effect, even by the method of heating in dimethyl sulfoxide, a skeleton such as polyether can be introduced into the polypeptide, thereby obtaining a polymer compound with higher efficiency. Under other manufacturing conditions that are the same, the effect expected by the present invention can be enhanced to a higher level by adding a base, but depending on the standard of the desired effect, the use of the base can also be omitted.

[0016] One aspect of the present disclosure provides a method for producing a polymer compound, comprising the following steps: heating a polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyether, polyester and polycarbonate having two structures represented by the following general formula (2) in dimethyl sulfoxide in the presence of an alkali, a reducing agent and a polymerization inhibitor. [Chemical Formula 16] [In general formula (2), R 1 represents a hydrogen atom or a methyl group.]

[0017] In one example of the above-mentioned manufacturing method, when the polypeptide reacts with the specific compound of the present invention, a method in which a base and a reducing agent are present in the reaction system is adopted. Among them, the functional group represented by the general formula (2) may undergo free radical polymerization due to heating in dimethyl sulfoxide, thereby causing at least one compound selected from the group consisting of polyether, polyester and polycarbonate having two structures represented by the above-mentioned general formula (2) to self-polymerize. However, according to the manufacturing method of the present invention, by making the above-mentioned reaction system simultaneously contain an inhibitor, the free radical polymerization reaction of the functional group represented by the general formula (2) can be suppressed. Through this effect, even by the method of heating in dimethyl sulfoxide, a skeleton such as polyether can be introduced into the polypeptide, thereby obtaining a polymer compound with higher efficiency. Under the same manufacturing conditions as other conditions, the effect expected by the present invention can be enhanced to a higher level by adding a base, but depending on the different standards of the desired effect, the use of the base can also be omitted.

[0018] In the above production method, the reducing agent may be at least one selected from the group consisting of dithiols, sodium sulfate, sodium sulfite, and sodium dithionite. This specific reducing agent has high stability in dimethyl sulfoxide and can more effectively inhibit the formation of disulfide bonds between sulfhydryl groups in the polypeptide.

[0019] In the above production method, the reducing agent may be prepared in an amount of 0.5 equivalents or greater relative to the thiol groups of the polypeptide. By controlling the reducing agent amount within this range, the formation of disulfide bonds between thiol groups of the polypeptide can be more effectively suppressed. In this manner, the production efficiency of the target synthetic polymer can be further improved.

[0020] In the above production method, the reaction between the polypeptide and the compound can be carried out at 50° C. or higher.

[0021] One aspect of the present disclosure provides a method for producing a polymer compound solution, comprising the step of dissolving the polymer compound obtained by the above-mentioned production method in a solvent.

[0022] A method for producing a polymer film comprises forming a polymer solution obtained by the above-mentioned method into a film. The film can be formed by, for example, casting.

[0023] One aspect of the present disclosure provides a method for producing polymer fibers, comprising a step of spinning a polymer compound solution obtained by the above-mentioned production method.

[0024] One aspect of the present disclosure provides a film containing a polymer compound, wherein the polymer compound has the following structure: a polypeptide portion and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure, directly bonded via a structure represented by the following formula (3), the following formula (4), or the following general formula (5), so that the polypeptide portion is bonded to S of the following general formula (3), the following general formula (4), or the following general formula (5), and the film has an elongation of greater than 400% and a breaking strength greater than the yield strength. In general formulas (3) and (4), M represents any one of H, Na, K, NHEt3, or NHEtiPr2, and in general formula (5), R1 represents H or Me.

[0025] [Chemical Formula 17]

[0026] [Chemical Formula 18]

[0027] [Chemical Formula 19]

[0028] One aspect of the present disclosure provides a fiber containing a polymer compound, wherein the polymer compound has the following structure: a polypeptide portion and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure, directly bonded via a structure represented by the following general formula (3), the following general formula (4), or the following general formula (5), so that the polypeptide portion is bonded to S of the following general formula (3), the following general formula (4), or the following general formula (5), and the film elongation is greater than 400%, and the breaking strength is greater than the yield point strength. In the general formulas (3) and (4), M represents any one of H, Na, K, NHEt3, or NHEtiPr2, and in the general formula (5), R1 represents H or Me.

[0029] [Chemical Formula 20]

[0030] [Chemical Formula 21]

[0031] [Chemical Formula 22] Effects of the Invention

[0032] According to the present disclosure, a production method can be provided that can improve the raw material conversion rate in the production of the polymer compound as described above compared to conventional methods. [Specific implementation method]

[0033] Unless otherwise specified, the materials exemplified in this specification may be used alone or in combination of two or more. When there are multiple substances corresponding to each component in the composition, the content of each component in the composition refers to the total amount of the multiple substances present in the composition, unless otherwise specified.

[0034] In this specification, a molding material refers to a material used to prepare a molded body. The molding material involved in the present disclosure can also be referred to as a material for chemically modifying a polypeptide. The shape of the molded body in this specification is not particularly limited, and can be, for example, a film, plate, block, sponge, or fiber. The form of the molding material in this specification is also not limited in any way, and can be, for example, a powder, granules, liquid, or gel. In addition, the molding material involved in the present disclosure can be a molded body of various shapes made by, for example, heat and pressure molding, casting molding, and spinning. During molding, a mold can also be used as needed.

[0035] The method for producing a polymer compound according to the present disclosure utilizes the reaction between a thiol group (nucleophilic functional group) possessed by a polypeptide and a polyether or other backbone having a specific unsaturated bond (electrophilic functional group).

[0036] A first embodiment of a method for producing a polymer compound includes the following steps: a polypeptide containing at least one thiol group (hereinafter referred to as component (A)) and at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates having two structures represented by the following formula (1) (hereinafter referred to as component (B1). Together with component (B2) described later, they are collectively referred to as component (B)). These compounds are subjected to a heating reaction in dimethyl sulfoxide in the presence of a base (hereinafter referred to as component (C)) and a reducing agent (hereinafter referred to as component (D)). The heating reaction is a reaction between component (A) and component (B) and can be carried out, for example, by heating to 50°C or above. In addition, the timing of heating is not limited to after mixing components (A) to (D). For example, components (A) to (D) can be added to pre-heated dimethyl sulfoxide; heating can also be started after adding components (C) and (D) to dimethyl sulfoxide, and after reaching a predetermined temperature, components (A) and (B) are added to react; or one of components (A) and (B) and components (C) and (D) are added first, and then heating is started. After reaching a predetermined temperature, the remaining component (A) and (B) are added to react. As for component (C), under other manufacturing conditions that are the same, the effect expected by the present disclosure can be enhanced to a higher level by adding component (C), but depending on the standard of the desired effect, the use of component (C) can also be omitted.

[0037] [Chemical Formula 23]

[0038] The number of thiol groups possessed by the polypeptide (component (A)) is one or more per molecule, but may also be, for example, two or more, four or more, eight or more, or 16 or more per molecule. When the number of thiol groups is within the above range, the probability of contact at the reaction sites increases, thereby enabling a more efficient reaction. The number of thiol groups possessed by component (A) may be, for example, 64 or fewer, or 32 or fewer per molecule. When the number of thiol groups is within the above range, the formation of by-products by reactions between the thiol groups of the polypeptide can be suppressed, while the target polymer compound can be obtained more efficiently.

[0039] The number of amino acid residues constituting component (A) can be, for example, more than 50, more than 100, more than 150, more than 200, more than 250, more than 300, more than 350, more than 400, more than 450 or more than 500. The number of amino acid residues can, for example, be less than 5000, less than 4500, less than 4000, less than 3500, less than 3000, less than 2500, less than 2000, less than 1500, less than 1000. The fewer the number of amino acid residues, the higher the solubility in dimethyl sulfoxide. When the number of amino acid residues constituting component (A) is, for example, less than 5000 or less than 2500, when the polypeptide is dissolved in a solvent and the polypeptide is reacted with the above-mentioned compound (component (B1)), the generation efficiency of the target polymer compound can be further improved. In addition, the molded body consisting of the obtained polymer compound can exert a higher level of mechanical strength and flexibility.

[0040] Component (A) can be, for example, a hydrophobic polypeptide. If component (A) is a hydrophobic polypeptide, since component (A) is easier to prepare, the raw material costs of the above-mentioned production method can be further reduced. In this case, the molded body composed of the resulting polymer compound can exhibit even greater flexibility. In addition, the molded body can also exhibit even greater water resistance. In this case, the service life of the resulting molded body can be further extended. In addition, by adjusting the hydrophobicity of component (A), the hydrophobicity of the polymer compound can be arbitrarily adjusted.

[0041] The hydrophobicity of component (A) can be estimated using the average hydrophilic index (hereinafter also referred to as "average HI") as an indicator. The average HI value of component (A) can be greater than 0, for example, 0.10 or greater, 0.20 or greater, 0.22 or greater, 0.25 or greater, 0.30 or greater, 0.35 or greater, 0.40 or greater, 0.45 or greater, 0.50 or greater, 0.55 or greater, 0.60 or greater, 0.65 or greater, or 0.70 or greater. The upper limit of the average HI value of component (A) is not particularly limited and can be, for example, 1.00 or less, or 0.7 or less.

[0042] Component (A) is preferably a component having low solubility in a 60°C lithium bromide aqueous solution (concentration: 9 M). The solubility can be evaluated by using a polypeptide equivalent to component (A). The maximum concentration when the above-mentioned polypeptide is dissolved in a 60°C lithium bromide aqueous solution (concentration: 9 M) can be, for example, less than 30% by mass, less than 25% by mass, less than 20% by mass, less than 15% by mass, less than 10% by mass, less than 5% by mass, or less than 1% by mass. In addition, as component (A), a substance that is completely insoluble in a 60°C lithium bromide aqueous solution (concentration: 9 M) can be used. The polymer compound obtained by the production method involved in the present disclosure can use a polypeptide obtained by decomposing the polymer compound and separating only the hydrophobic polypeptide, thereby confirming the above-mentioned solubility of the polypeptide portion possessed by the polymer compound.

[0043] Component (A) is preferably a component having a larger contact angle of water. The contact angle of water can be evaluated by using a polypeptide equivalent to component (A) on a substrate. More specifically, a film composed of the above-mentioned polypeptide can be formed and evaluated using the film. As component (A), it is preferably a polypeptide that is configured to form a thin film having a contact angle of 55° or more after 5 seconds when water is dripped onto the surface of the film. The above-mentioned contact angle can be, for example, 60° or more, 65° or more, or 70° or more. The polymer compound obtained by the manufacturing method involved in the present disclosure can use a polypeptide obtained by decomposing the polymer compound and separating only the hydrophobic polypeptide, thereby confirming the above-mentioned contact angle of the polypeptide portion possessed by the polymer compound.

[0044] Component (A) is preferably a component having excellent hot water resistance. Hot water resistance can be evaluated by using a polypeptide equivalent to component (A). As component (A), it is preferred that a polypeptide is prepared by preparing a dispersion consisting of the above-mentioned polypeptide and water, wherein the content of the above-mentioned polypeptide is 5% by mass, and the dispersion is heated at 100°C for 5 hours without decomposition. The polymer compound obtained by the production method involved in the present disclosure can use a polypeptide obtained by decomposing the polymer compound and isolating only the hydrophobic polypeptide, thereby confirming the above-mentioned hot water resistance of the polypeptide portion of the polymer compound.

[0045] The weight-average molecular weight of component (A) is, for example, preferably 200 to 1,000,000, more preferably 300 to 900,000, further preferably 400 to 800,000, even more preferably 500 to 700,000, further preferably 600 to 600,000, further preferably 1,000 to 600,000, further preferably 3,000 to 600,000, further preferably 5,000 to 600,000, further preferably 10,000 to 600,000, and further preferably 5,000 to 100,000. Furthermore, if the weight-average molecular weight of component (A) is less than 200, the polypeptide serving as the hard segment may be too small relative to the polyether structure (soft segment) of component (B1). In this case, the rigidity of a molded article molded from the resulting polymer compound (molding material) may be reduced, making it difficult to use, for example, as a structural article.

[0046] The weight-average molecular weight in this specification is the value measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis was performed according to the following procedure. First, 200 μL of 2 M lithium chloride DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to 2 mg of a powdered sample and heated at 80°C for 60 minutes, and then at 95°C for 10 minutes while stirring, to dissolve the sample. The sample was then diluted 50-fold with a 10 M urea solution, diluted 2-fold with a sample buffer (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and heated at 95°C for 5 minutes to denature the protein. Next, an SDS-PAGE gel (manufactured by Bio-lad) was mounted on an electrophoresis apparatus (manufactured by Bio-lad), filled with SDS buffer, and connected to a power supply (manufactured by Biocraft). The denatured sample was added to each well of the SDS-PAGE gel with 10 μL, and the current was circulated under the conditions of 30 mA / 1 sheet and 30 minutes. After electrophoresis, the SDS-PAGE gel was taken out from the device, immersed in Oriole fluorescent gel dye (manufactured by Bio-lad), and shaken for 1 hour. The gel was then placed on a UV sample tray (manufactured by Bio-lad) and a staining image was obtained using a Gel Doc EZ gel imaging device (manufactured by Bio-lad). In addition, XL ladder Broad (manufactured by APRO Science) was used as a standard sample.

[0047] The molecular weight of component (A) can be, for example, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more. The molecular weight of component (A) can be, for example, 500 kDa or less, 400 kDa or less, less than 360 kDa, 300 kDa or less, or 200 kDa or less. The molecular weight of component (A) can be adjusted within the above range, for example, 2 to 500 kDa. In addition, the molecular weight can be measured by liquid chromatography-mass spectrometry.

[0048] Component (A) may be a natural protein or an artificial protein. Furthermore, component (A) may be a protein (modified protein) obtained by chemically modifying a natural or artificial protein (e.g., alkylation, etherification, esterification, amidation, thioetherification, and thioesterification). As long as the natural, artificial, or modified protein has at least one thiol group, its amino acid sequence is not particularly limited.

[0049] Component (A) can be selected, for example, from proteins having similar physical properties, depending on the desired properties of the polymer compound for its intended use. Examples of polypeptides used in this embodiment include those for medical applications and those for industrial applications. "Industrial applications" refers to, for example, various general-purpose materials that can be used indoors or outdoors.

[0050] Specific examples of polypeptides that can be used for medical purposes include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens for vaccination, vaccines, antigen-binding proteins, immunostimulatory proteins, allergens, full-length antibodies or antibody fragments or their derivatives.

[0051] As specific examples of polypeptides that can be used for industrial purposes, structural proteins can be cited. So-called structural proteins refer to proteins related to the structure of an organism, or proteins that constitute a structure produced by an organism, or proteins derived from them. Structural proteins refer to proteins that self-condense under certain conditions to form structures such as fibers, films, resins, gels, micelles, and nanoparticles. In addition, structural proteins can also refer to proteins whose motifs composed of characteristic amino acid sequences or amino acid residues are repeated to form the skeleton of organisms and materials. As specific examples of structural proteins, spider silk (spider silk), silkworm silk, keratin, collagen, elastin, arthropod elastic protein, and proteins derived from them can be cited. As structural proteins, it can be artificial silk fibroin or artificial spider silk fibroin (artificially modified spider silk fibroin).

[0052] Structural proteins can also be artificial structural proteins. These include synthetic proteins and recombinant structural proteins produced by microorganisms using genetic recombination techniques. In other words, as used herein, "artificial structural proteins" refer to artificially produced structural proteins. Artificial structural proteins can have the same amino acid sequence as naturally occurring structural proteins, or they can be modified structural proteins by partially modifying the amino acid sequence of naturally occurring structural proteins for productivity or formability.

[0053] The artificial structural protein may have a glycine residue content of 10% to 55%. For example, the glycine residue content may be 13% to 55%, 15% to 55%, 18% to 55%, 20% to 55%, 22% to 55%, or 25% to 55%. In this specification, "glycine residue content" refers to the value represented by the following formula.

[0054] Glycine residue content = (number of glycine residues in the artificial structural protein / total number of amino acid residues in the polypeptide) × 100 (%)

[0055] The number of amino acid residues in the artificial structural protein may be 150 or more. The number of amino acid residues may be, for example, 200 or more or 250 or more, preferably 300 or more, 350 or more, 400 or more, 450 or more, or 500 or more.

[0056] The artificial structural protein may contain at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine (i.e., any one of the serine residue content, the threonine residue content, the tyrosine residue content, the sum of the serine residue content and the threonine residue content, the sum of the serine residue content and the tyrosine residue content, the sum of the threonine residue content and the tyrosine residue content, and the sum of the serine residue content, the threonine residue content, and the tyrosine residue content), the alanine residue content, and the glycine residue content (total content) of 40% or more. This total content may be, for example, 45% or more, 50% or more, 55% or more, or 60% or more. The upper limit of this total content is not particularly limited, and for example, it may be 90% or less, 85% or less, or 80% or less.

[0057] The sum of the serine residue content, threonine residue content, and tyrosine residue content of the artificial structural protein may be 4% or greater, 4.5% or greater, 5% or greater, 5.5% or greater, 6% or greater, 6.5% or greater, or 7% or greater. The sum of the serine residue content, threonine residue content, and tyrosine residue content may be, for example, 35% or less, 33% or less, 30% or less, 25% or less, or 20% or less.

[0058] The distribution of serine residues, threonine residues or tyrosine residues in the artificial structural protein is even. In any consecutive 20 amino acid residues, the total content of serine residues, threonine residues and tyrosine residues can be greater than 4%, greater than 5%, greater than 10% or greater than 15%, or less than 50%, less than 40%, less than 30% or less than 20%.

[0059] Furthermore, the alanine residue content, the serine residue content, the threonine residue content, and the tyrosine residue content have the same meanings as in the above formula except that the alanine residue is replaced by a glycine residue, a serine residue, a threonine residue, and a tyrosine residue, respectively.

[0060] The artificial structural protein may be a protein having a repeating sequence. That is, the artificial structural protein may contain multiple amino acid sequences (repeating sequence units) with high sequence identity within the artificial structural protein. The number of amino acid residues in the repeating sequence unit is preferably 6 to 200.

[0061] In the above-mentioned repeating sequence units, the ratio of the total number of glycine residues, serine residues, glutamine residues and alanine residues to the total number of amino acid residues may be 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more or 70% or more. In addition, the sequence identity between repeating sequence units may be, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0062] In addition, the hydrophobicity degree (hydrophilic index) of the above-mentioned repeating sequence unit can be, for example, greater than -0.80, greater than -0.70, greater than -0.60, greater than -0.50, greater than -0.40, greater than -0.30, greater than -0.20, greater than -0.10, greater than 0.00, greater than 0.22, greater than 0.25, greater than 0.30, greater than 0.35, greater than 0.40, greater than 0.45, greater than 0.50, greater than 0.55, greater than 0.60, greater than 0.65 or greater than 0.70. In addition, the upper limit of the hydrophobicity degree of the repeating sequence unit is not particularly limited, and for example, it can be less than 1.0 or less than 0.7.

[0063] The artificial structural protein may be a protein containing an (A)n motif. As used herein, the (A)n motif refers to an amino acid sequence primarily composed of alanine residues. The number of amino acid residues in the (A)n motif may be 2 to 27, or may be an integer from 2 to 20, 2 to 16, or 2 to 12.

[0064] In addition, as long as the ratio of the number of alanine residues in the (A)n motif to the total number of amino acid residues is 40% or more, it can be 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues).

[0065] In the (A)n motif, the ratio of the total number of alanine residues, serine residues, threonine residues, and valine residues to the total number of amino acid residues in the (A)n motif can be 80% or greater, preferably 85% or greater, more preferably 90% or greater, even more preferably 95% or greater, and even more preferably 100% (meaning that it is composed solely of one or more amino acid residues selected from alanine residues, serine residues, threonine residues, and valine residues). The multiple (A)n motifs present in the recombinant structural protein of this embodiment may have the same or different amino acid sequences. Furthermore, since the (A)n motif primarily contains alanine residues, it is likely to form an α-helical structure or a β-sheet structure. By including the (A)n motif in the repeating sequence unit, the artificial structural protein of this embodiment repeatedly possesses these secondary structures. Therefore, as described below, when the artificial structural protein is formed into a molded article such as a fiber, film, or resin, it is expected to exhibit high strength due to these secondary structures.

[0066] Furthermore, when molding structural proteins, amino acids with relatively small side chains are more likely to form hydrogen bonds, making it easier to obtain stronger molded products. Furthermore, since the side chains of alanine and glycine residues are non-polar amino acids, they tend to be positioned inward during the folding process of the resulting polypeptide, facilitating the formation of α-helical or β-sheet structures. Therefore, it is desirable to increase the proportion of amino acids such as glycine and alanine residues. To achieve a molded product with even greater strength, the alanine residue content can be, for example, 10-40%, and can be 12-40%, 15-40%, 18-40%, 20-40%, or 22-40%. To achieve a molded product with even greater strength, the glycine residue content can be, for example, 10-55%, and can be 11-55%, 13-55%, 15-55%, 18-55%, 20-55%, 22-55%, or 25-55%.

[0067] In this specification, the "alanine residue content ratio" refers to the ratio of the number of alanine residues to the total number of amino acid residues constituting a protein, and is a value represented by the following formula.

[0068] Alanine residue content = (number of alanine residues in the protein / total number of amino acid residues in the protein) x 100 (%)

[0069] In addition, the glycine residue content rate, serine residue content rate, threonine residue content rate, proline residue content rate and tyrosine residue content rate have the same meanings as when alanine residues in the above formula are replaced with glycine residues, serine residues, threonine residues, proline residues and tyrosine residues, respectively.

[0070] Structural proteins preferably contain a certain amount of amino acids with larger side chains or amino acids with flexibility uniformly throughout their sequence. Specifically, structural proteins may also contain a motif containing tyrosine, threonine, and proline residues that is repeatedly and periodically incorporated. Such structural proteins can easily hinder the formation of strong intermolecular hydrogen bonds during processing of the resulting molded product, thereby facilitating processing. For example, the total content of proline, threonine, and tyrosine residues in any consecutive sequence of 20 amino acid residues may be 5% or greater, greater than 5.5%, 6.0% or greater, greater than 6.5%, 7.0% or greater, greater than 7.5%, 8.0% or greater, greater than 8.5%, 9.0% or greater, 10.0% or greater, or 15.0% or greater. Furthermore, for example, the total content of proline, threonine, and tyrosine residues in any consecutive sequence of 20 amino acid residues may be less than 50%, less than 40%, less than 30%, or less than 20%.

[0071] The artificial structural protein, similar to the proteins described above, may have a total content of serine residues, threonine residues, and tyrosine residues of, for example, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 6.5% or more, or 7% or more, in order to improve the productivity of the reaction-modified protein. The total content of serine residues, threonine residues, and tyrosine residues may be, for example, 35% or less, 33% or less, 30% or less, 25% or less, or 20% or less.

[0072] In the above-mentioned production method, the amount of the above-mentioned polypeptide (component (A)) can be, for example, greater than 1 part by mass, 2 parts by mass or more, 5 parts by mass or more, greater than 5 parts by mass, 6 parts by mass or more, 7 parts by mass or more, or 8 parts by mass or more per 100 parts by mass of dimethyl sulfoxide. Because the production method involved in the present disclosure can inhibit the formation of disulfide bonds between polypeptides, the production of high-molecular-weight compounds can be carried out at an increased polypeptide concentration, thereby achieving more efficient production. For example, the amount of component (A) can be 50 parts by mass or less, 30 parts by mass or less, 20 parts by mass or less, or 15 parts by mass or less per 100 parts by mass of dimethyl sulfoxide. If the upper limit of the amount of component (A) is within the above-mentioned range, the reaction can be promoted while more effectively suppressing the formation of by-products and gelation. In the above-mentioned production method, the proportion of the above-mentioned polypeptide (component (A)) in the dimethyl sulfoxide solution can be, for example, 15% by mass or less, less than 15% by mass, 14% by mass or less, 12.5% ​​by mass or less, or 11.5% by mass or less.

[0073] At least one compound (component (B1)) having two structures represented by the general formula (1) selected from the group consisting of polyether, polyester and polycarbonate can react with the thiol group of the above-mentioned polypeptide due to its structure represented by the general formula (1). It is a compound that can introduce a polyether structure, a polyester structure or a polycarbonate structure into the polypeptide.

[0074] Examples of polyethers having two structures represented by the general formula (1) include polyethylene glycol, polytetramethylene glycol, polypropylene glycol, and ethylene glycol-propylene glycol copolymers. Examples of polyesters having two structures represented by the general formula (1) include polylactic acid, polyglycolic acid, polybutylene succinate, polycaprolactone, and polyhydroxy fatty acid esters containing copolymers of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid. Examples of polycarbonates having two structures represented by the general formula (1) include aliphatic polycarbonates such as polyethylene carbonate, polypropylene carbonate, and polytrimethyl carbonate. Since these compounds are more prone to molecular motion than the above-mentioned polypeptides, when the above-mentioned compounds are used, the flexibility of the molded body composed of the obtained polymer compound can be further improved.

[0075] In the above-mentioned production method, the weight average molecular weight of the above-mentioned compound (component (B1)) may be 100 or more, 1000 or more, or 2000 or more. By using the component (B1) having a weight average molecular weight within the above-mentioned range, the influence of the bulkiness between molecules can be more effectively suppressed, thereby promoting the reaction more efficiently. In the above-mentioned production method, the weight average molecular weight of the component (B1) may be, for example, 200,000 or less, 100,000 or less, 50,000 or less, or 10,000 or less. By using the component (B1) having a weight average molecular weight within the above-mentioned range, the decrease in the solubility of the above-mentioned compound in the solvent and the decrease in the reactivity with the polypeptide can be more fully suppressed.

[0076] In the above-mentioned production method, the weight average molecular weight of the above-mentioned compound ((B1) component) is based on the weight average molecular weight of the above-mentioned polypeptide, for example, it can be more than 0.005 times, more than 0.01 times, more than 0.05 times or more than 0.1 times. By controlling the relationship between the weight average molecular weight of the above-mentioned polypeptide and the (B1) component within the above-mentioned range, a synthetic polymer having a polyether structure of sufficient length on the polypeptide portion can be produced. A molded body composed of such a synthetic polymer can be a molded body with excellent flexibility. In the above-mentioned production method, the weight average molecular weight of the (B1) component is based on the weight average molecular weight of the above-mentioned polypeptide, for example, it can be less than 20 times, less than 10 times, less than 5 times or less than 2 times. By controlling the relationship between the weight average molecular weight of the above-mentioned polypeptide and the (B) component within the above-mentioned range, the reaction can be promoted while more effectively suppressing the reduction in reactivity. In the above production method, the weight average molecular weight of component (B1) can be adjusted within the above range, for example, based on the weight average molecular weight of the above polypeptide, it can be 0.005 to 20 times, 0.01 to 10 times, 0.05 to 5 times, or 0.1 to 2 times.

[0077] In the above-mentioned production method, the ratio of the number of moles of the structure represented by the general formula (1) possessed by the above-mentioned compound (component (B1)) to the number of moles of the thiol groups possessed by the above-mentioned polypeptide (component (A)) can be, for example, 0.1 times or more, 0.2 times or more, 0.5 times or more, or 0.7 times or more. By controlling the above-mentioned ratio within the above-mentioned range, the reaction can be promoted while the reactivity is more effectively suppressed from decreasing. In the above-mentioned production method, the ratio of the number of moles of the structure represented by the general formula (1) possessed by component (B1) to the number of moles of the thiol groups possessed by component (A) can be, for example, 5.0 times or less, 3.0 times or less, 1.5 times or less, or 0.8 times or less. By controlling the above-mentioned ratio within the above-mentioned range, the reaction can be promoted while the generation of by-products and gelation are more effectively suppressed. By adjusting the above-mentioned molar ratio, the number of polyether structures, etc. in the obtained polymer compound can be controlled. In the obtained polymer compound, the total number of polyether structures and the like per polypeptide can be set to, for example, 1 or more, or 2 or more, or 2-10, 2-8, 2-6, or 2-4.

[0078] In this application, the molar number of thiol groups refers to the value obtained by enzymatically cleaving the polypeptide in the assay sample and converting it into amino acids, followed by separation and detection using liquid chromatography. Furthermore, if you prepare your own polypeptide, you can determine the molar number of these functional groups based on the designed molecular sequence.

[0079] In the present specification, the molar numbers of the structure represented by the general formula (1) and the structure represented by the general formula (2) refer to values ​​measured by MALDI-TOFMS (matrix-assisted laser desorption time-of-flight mass spectrometry).

[0080] In the above production method, the molar amount of the compound (component (B1)) present in dimethyl sulfoxide relative to the total molar amount of sulfhydryl groups of the polypeptide can be, for example, 0.1 times or more, 0.2 times or more, 0.5 times or more, or 0.8 times or more. By adjusting the raw material ratio so that the molar amount of component (B1) exceeds the total molar amount of sulfhydryl groups, the reaction probability between the polypeptide and the compound can be increased and the frequency of disulfide bond formation between the polypeptides can be further reduced. In the above production method, the molar amount of component (B1) present in dimethyl sulfoxide relative to the total molar amount of sulfhydryl groups of the polypeptide can be, for example, 5.0 times or less, 3.0 times or less, 1.5 times or less, or 1.0 times or less. By controlling the molar amount of component (B1) within the above range, the reaction can be promoted while more effectively suppressing the formation of by-products and gelation.

[0081] In the above-mentioned production method, the base (component (C)) is a compound that can activate the thiol group of the polypeptide and promote the Michael addition reaction between the thiol group and the functional group represented by the general formula (1). Examples of the base include primary amines, secondary amines, tertiary amines, nitrogen-containing cyclic compounds, nitrogen-containing aromatic compounds, and inorganic bases. Examples of primary amines include ethanolamine and hexamethylenediamine, and examples of secondary amines include diethylamine, methylethylamine, and N-methylbutylamine. Examples of tertiary amines include triethylamine, methyldiethylamine, and DIPEA (N,N-diisopropylethylamine). Examples of nitrogen-containing cyclic compounds include quinuclidine, DABCO (1,4-diazabicyclo[2.2.2]octane), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), and DBN (1,5-diazabicyclo[4.3.0]non-5-ene). Examples of the nitrogen-containing aromatic compound include pyridine and imidazole, and examples of the inorganic base include sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate, potassium carbonate, cesium carbonate, tripotassium phosphate, sodium acetate, and potassium acetate.

[0082] The lower limit of the amount of the above-mentioned base is based on the mass of the polypeptide, for example, it can be 0.01 mass parts or more, 0.05 mass parts or more, 0.1 mass parts or more, or 0.5 mass parts or more. By controlling the lower limit of the amount of the base within the above range, the reaction between the thiol group possessed by the polypeptide and the unsaturated bond represented by the general formula (1) or (2) possessed by the above-mentioned compound can be further promoted. The upper limit of the amount of the above-mentioned base is based on the mass of the polypeptide, for example, it can be 50 mass parts or less, 20 mass parts or less, 10 mass parts or less, or 5 mass parts or less. By controlling the upper limit of the amount of the base within the above range, the molecular decomposition caused by the residual base can be more effectively suppressed, thereby synthesizing the target polymer compound. The amount of the above-mentioned base can be adjusted within the above range, and based on the mass of the polypeptide, for example, it can be 0.05 to 50 mass parts, or 0.1 to 10 mass parts. Furthermore, the amount of the base added may be, for example, 0 to 50 parts by mass, 0 to 10 parts by mass, 0 to 1 part by mass, or 0 to 0.5 parts by mass based on the mass of the polypeptide.

[0083] In the production method, the reducing agent (component (D)) is a compound capable of stabilizing thiol groups of the polypeptide in dimethyl sulfoxide and inhibiting disulfide bond formation between these thiol groups. The reducing agent may comprise, for example, at least one selected from the group consisting of thiols, dithiols, sodium sulfite, sodium hyposulfite, sodium sulfate, and sodium dithionite. The reducing agent may comprise at least one selected from dithiols and sodium sulfite. Alternatively, the reducing agent may be a dithiol or sodium sulfite. A compound having a thiol group is preferably used as the reducing agent. A dithiol is preferably used as the reducing agent.

[0084] The compound represented by the following general formula (Y) is preferably used as the compound having a thiol group. Such compounds can form a chemically stable six-membered ring structure in the oxidized state after the reduction reaction, thereby inhibiting the reaction between the reducing agent and the Michael addition acceptor in the reaction system; in addition, by forming a ring structure between the reducing agent and the thiol group possessed by the polypeptide, the formation of disulfide bonds between the polypeptides can be inhibited, making the reaction between the thiol group possessed by the polypeptide and the Michael addition acceptor more effective, and ultimately being able to more efficiently produce a high molecular weight compound that meets the purpose of the reaction design. As dithiols, for example, dithiothreitol and 1,4-butanedithiol can be listed. Specifically, the compound represented by the general formula (Y) (dithiol) can be dithiothreitol or 1,4-butanedithiol.

[0085] [Chemical Formula 24]

[0086] Examples of other compounds having a mercapto group include 3-mercaptopropionic acid, 3-mercapto-1,2-propanediol, and pentaerythritol tetrakis-3-mercaptopropionate. In addition to the above compounds, the reducing agent may be tris(2-carbonylethyl)phosphine hydrochloride (TCEP).

[0087] The lower limit of the amount of the reducing agent to be prepared relative to the thiol groups of the polypeptide may be, for example, 0.1 equivalents or more, 0.3 equivalents or more, 0.5 equivalents or more, 0.8 equivalents or more, or 1.0 equivalents or more. By controlling the lower limit of the amount of the reducing agent to be prepared within the above range, the formation of disulfide bonds due to the reaction between the thiol groups of the polypeptide can be more effectively suppressed. In this way, the production efficiency of the target synthetic polymer can be further improved. The upper limit of the amount of the reducing agent to be prepared relative to the thiol groups of the polypeptide may be, for example, 2.4 equivalents or less, 2.0 equivalents or less, 1.6 equivalents or less, 1.2 equivalents or less, or 1.1 equivalents or less. By controlling the upper limit of the amount of the reducing agent to be prepared within the above range, the reaction between the component (B1) and the reducing agent and the formation of side reaction products can be more effectively suppressed while promoting the reaction.

[0088] In the above-described production method, the reaction between the polypeptide (component (A)) and the compound (component (B1)) can be carried out, for example, at a temperature above 55°C. At reaction temperatures exceeding 55°C, disulfide bonds may form between the sulfhydryl groups of the polypeptide, typically in dimethyl sulfoxide. In contrast, in the production method of the present disclosure, the formation of disulfide bonds can be avoided by conducting the reaction in the presence of a reducing agent. Therefore, while suppressing disulfide bond formation, the activity of the reaction between the polypeptide and the compound can be enhanced, thereby improving the efficiency of polymer compound synthesis.

[0089] In the above-mentioned production method, the reaction temperature for the reaction between component (A) and component (B1) may be, for example, 20°C or higher, 30°C or higher, 40°C or higher, 50°C or higher, 53°C or higher, 55°C or higher, 57°C or higher, or 60°C or higher. By controlling the lower limit of the above-mentioned reaction temperature within the above-mentioned range, the reaction between component (A) and component (B1) can be more effectively promoted. In the above-mentioned production method, the upper limit of the reaction temperature for the reaction between component (A) and component (B1) may be, for example, 110°C or lower, 100°C or lower, 90°C or lower, 85°C or lower, 80°C or lower, 75°C or lower, or 70°C or lower. By controlling the upper limit of the above-mentioned reaction temperature within the above-mentioned range, the formation of bonds between thiol groups possessed by the polypeptide as a side reaction can be more effectively suppressed, thereby efficiently synthesizing the target polymer compound. The reaction temperature between component (A) and component (B1) can be adjusted within the above range, for example, 50-90°C, 53-80°C, 55-75°C, or 55-70°C. The reaction can be carried out at any temperature, but preferably at a temperature at which the DMSO solvent does not freeze and does not cause protein decomposition.

[0090] The method for producing a polymer compound may further include other steps, such as separately preparing the component (A) and separately preparing the component (B1).

[0091] The preparation method of component (A) may include a method of expressing a nucleic acid using a host transformed with an expression vector described below. In this case, component (A) may also be artificial silk fibroin. As an expression method, in addition to direct expression, secretory production, fusion protein expression, etc. may also be performed according to the methods described in the second edition of molecular cloning. When expression is performed using yeast, animal cells, or insect cells, a polypeptide with attached sugars or sugar chains can be obtained.

[0092] The artificial silk fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, allowing the artificial silk fibroin to be produced and accumulated in the culture medium, and collecting it from the culture medium. The method for culturing the host in the culture medium can be carried out according to a method commonly used for host culture.

[0093] When the host is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, as a culture medium for the host, any natural culture medium or synthetic culture medium can be used as long as it contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the host and can efficiently culture the host.

[0094] The carbon source can be any substance assimilated by the host, for example, carbohydrates such as glucose, fructose, sucrose, and molasses containing these sugars, starch, and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.

[0095] Nitrogen sources include, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other inorganic or organic acid ammonium salts, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermentation bacteria and their digestion products.

[0096] As the inorganic salt, for example, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

[0097] Cultivation of prokaryotes such as Escherichia coli or eukaryotic organisms such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40°C. The culture time is generally 16 hours to 7 days. The pH of the culture medium during cultivation is preferably maintained between 3.0 and 9.0. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like.

[0098] Additionally, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed during culturing. When culturing microorganisms transformed with an expression vector utilizing an inducible promoter, an inducer may be added to the culture medium as needed. For example, when culturing microorganisms transformed with an expression vector utilizing the lac promoter, isopropyl-β-D-thiogalactopyranoside may be added to the culture medium; when culturing microorganisms transformed with an expression vector utilizing the trp promoter, indoleacrylic acid may be added to the culture medium.

[0099] Examples of commonly used culture media for insect cells include TNM-FH medium (manufactured by Pharmingen), Sf-900 II SFM medium (manufactured by Life Technologies), ExCell400 and ExCell405 (both manufactured by JRH Biosciences), and Grace's insect medium (Nature, 195, 788 (1962)).

[0100] Insect cells can be cultured for 1 to 5 days under conditions such as a culture medium pH of 6 to 7 and a culture temperature of 25 to 30° C. During the culture, antibiotics such as gentamicin may be added to the culture medium as needed.

[0101] When the host is a plant cell, the transformed plant cell can be cultured directly or differentiated into plant organs for culture. Examples of culture media for the plant cells include commonly used Murashige & Skoog (MS) medium, White's medium, or culture media supplemented with plant hormones such as auxins and cytokinins.

[0102] Animal cells can be cultured for 3 to 60 days under conditions such as a culture medium pH of 5 to 9 and a culture temperature of 20 to 40° C. During the culture, antibiotics such as kanamycin and hygromycin can be added to the culture medium as needed.

[0103] Methods for producing fibroin using a host transformed with the above-mentioned expression vector include methods of producing the fibroin within host cells, methods of secreting the fibroin outside the host cells, and methods of producing the fibroin on the outer membrane of the host cells. These methods can be selected by varying the host cells used and the structure of the fibroin produced.

[0104] For example, when rayon is produced within host cells or on the outer membrane of host cells, the method of Paulson et al. (J. Biol. Chem., 264, 17619 (1989)), the method of Row et al. (Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)), or the methods described in Japanese Patent Application Laid-Open No. 05-336963, International Publication No. 1994 / 023021, etc. can be applied to modify the expression of rayon so that it is actively secreted outside the host cells. In other words, by using genetic recombination methods, a signal peptide is attached to a polypeptide containing the active site of rayon, and the polypeptide is expressed, thereby enabling active secretion of rayon outside the host cells.

[0105] Artificial silk fibroin produced by a host transformed with the above-described expression vector can be isolated and purified using methods commonly used for protein isolation and purification. For example, when artificial silk fibroin is expressed in a dissolved state within cells, after completion of culture, the host cells are recovered by centrifugation, suspended in an aqueous buffer, and then disrupted using an ultrasonic disruptor, a French press, a Manton-Gaulin high-pressure homogenizer, a DYNO-MILL, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a purified product can be obtained by using methods commonly used for the separation and purification of proteins, namely, solvent extraction, salting-out using ammonium sulfate or the like, desalting, precipitation using an organic solvent, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-agarose and DIAION HPA-75 (Mitsubishi Chemical Corporation), cation exchange chromatography using resins such as S-Sepharose FF (Pharmacia), hydrophobic chromatography using resins such as butyl agarose and phenyl agarose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric point electrophoresis, alone or in combination.

[0106] As the above-mentioned chromatography, column chromatography using Phenyl-Toyopearl (Tosoh), DEAE-Toyopearl (Tosoh), or Sephadex G-150 (Pharmacia Biotech) is preferably used.

[0107] Furthermore, when rayon protein is expressed as an insoluble form within cells, the host cells can be recovered and then disrupted and centrifuged to recover the insoluble form of rayon protein as a precipitate fraction. The recovered insoluble form of rayon protein can be solubilized using a protein denaturant. Following this operation, a purified product of rayon protein can be obtained using the same separation and purification method as described above.

[0108] When fibroin or a derivative of fibroin having sugar chains added thereto is secreted outside the cells, fibroin or its derivative can be recovered from the culture supernatant. Specifically, the culture is treated by centrifugation or other methods to obtain a culture supernatant, and a purified product can be obtained from the culture supernatant using the same separation and purification method as described above.

[0109] (A) component can also be artificial silk fibroin. If artificial silk fibroin is prepared separately, the method thereof can be, for example, artificial synthesis of silk fibroin of natural origin. Among them, as silk fibroin of natural origin, for example, silk fibroin produced by insects or spiders can be cited. Natural silk fibroin is a fibrous protein with a molecular weight of about 370,000, composed of two subunits, with a high content of glycine residues, alanine residues, serine residues and tyrosine residues, and these amino acid residues account for nearly 90% of the total number of amino acid residues. Natural silk fibroin has two different areas, namely a crystalline region rich in amino acid residues with smaller side chains such as glycine, alanine and serine, and an amorphous region having amino acid residues with larger side chains such as tyrosine.

[0110] More specific examples of naturally derived silk fibroin include those for which sequence information is registered in NCBI GenBank. For example, identification can be achieved by extracting sequences containing the classification code "INV" (division) in the sequence information registered in NCBI GenBank, and extracting sequences with keywords "spidroin," "ampullar gland," "fibroin," "silk and polypeptide," or "silk and protein" listed in the definition (DEFINITION), extracting character strings specifying products from the CDS, and extracting sequences with a specified character string listed in the tissue type (TISSUE TYPE) from the source (SOURCE).

[0111] In this specification, "artificial silk fibroin" refers to artificially produced silk fibroin (artificial silk fibroin). Artificial silk fibroin may be a silk fibroin having an amino acid sequence different from that of silk fibroin derived from natural sources, or a silk fibroin having an amino acid sequence identical to that of silk fibroin derived from natural sources. Artificial silk fibroin can be produced by a known method, for example, by the method described in International Publication No. 2019 / 194263.

[0112] Artificial silk fibroin may be a fibrous protein having a structure suitable for naturally derived silk fibroin, or may be a silk fibroin having a sequence identical to a repeating sequence found in naturally derived silk fibroin. The "sequence identical to a repeating sequence found in silk fibroin" may actually be a sequence found in naturally derived silk fibroin or a sequence similar thereto.

[0113] "Artificial silk fibroin" may be obtained by modifying the amino acid sequence of naturally derived silk fibroin as long as it has the amino acid sequence specified in the present disclosure (for example, silk fibroin obtained by modifying the amino acid sequence of cloned naturally derived silk fibroin gene sequence). Alternatively, it may be silk fibroin with an artificially designed amino acid sequence independent of naturally derived silk fibroin (for example, silk fibroin having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid encoding the designed amino acid sequence). In addition, silk fibroin with a modified amino acid sequence of artificial silk fibroin may be included in artificial silk fibroin as long as its amino acid sequence is different from that of naturally derived silk fibroin. Examples of artificial silk fibroin include artificial silk (silk) fibroin (fibroin with a modified amino acid sequence of silk fibroin produced by silkworms) and artificial spider silk fibroin (fibroin with a modified amino acid sequence of spider silk protein produced by spiders). Artificial silk fibroin is relatively easy to fibrillate and has high fiber-forming ability. Therefore, as a molding material, it preferably contains artificial spider silk fibroin, and more preferably consists of artificial spider silk fibroin.

[0114] The artificial silk fibroin involved in this embodiment can be a protein comprising a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif. The artificial silk fibroin may further comprise an amino acid sequence (N-terminal sequence and C-terminal sequence) at either or both of the N-terminal and C-terminal ends of the domain sequence. The N-terminal and C-terminal sequences are not limited thereto and typically do not contain a repeating region of the characteristic amino acid motif of silk fibroin and are composed of approximately 100 amino acid residues.

[0115] In this specification, a "domain sequence" refers to an amino acid sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif. The (A)n motif represents an amino acid sequence primarily composed of alanine residues, with 2 to 27 amino acid residues. The number of amino acid residues in the (A)n motif can be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. Furthermore, as long as the ratio of the number of alanine residues in the (A)n motif to the total number of amino acid residues is 40% or greater, it may be 60% or greater, 70% or greater, 80% or greater, 83% or greater, 85% or greater, 86% or greater, 90% or greater, 95% or greater, or 100% (meaning it is composed solely of alanine residues). At least 7 of the multiple (A)n motifs present in the domain sequence can be composed solely of alanine residues. REP represents an amino acid sequence consisting of 2 to 200 amino acid residues. REP may be an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer from 2 to 300, or may be an integer from 10 to 300. Multiple (A)n motifs present may be the same amino acid sequence or different amino acid sequences. Multiple REPs present may be the same amino acid sequence or different amino acid sequences.

[0116] Specific examples of artificial silk fibroin include artificial silk fibroin derived from the large ampullate gland of spiders (first artificial silk fibroin), artificial silk fibroin having a domain sequence with reduced glycine residue content (second artificial silk fibroin), artificial silk fibroin having a domain sequence with reduced (A)n motif content (third artificial silk fibroin), artificial silk fibroin having reduced glycine residue content and (A)n motif content (fourth artificial silk fibroin), artificial silk fibroin having a domain sequence with a localized region having a high hydrophobicity index (fifth artificial silk fibroin), and artificial silk fibroin having a domain sequence with reduced glutamine residue content (sixth artificial silk fibroin). The definitions of each of the first to sixth artificial silk fibroins are incorporated herein by reference to the contents of International Publication No. 2019 / 194263.

[0117] Artificial silk fibroin can contain a tag sequence at either or both of the N-terminus and the C-terminus, thereby enabling separation, immobilization, detection, and visualization of artificial silk fibroin.

[0118] As a tag sequence, for example, an affinity tag that utilizes specific affinity (binding property, affinity) with other molecules can be listed. As a specific example of an affinity tag, a histidine tag (His tag) can be listed. The His tag is a short peptide composed of about 4 to 10 histidine residues arranged in an array, and has the property of specifically binding to metal ions such as nickel, and can therefore be used for the separation of artificial silk core protein by chelating metal chromatography. As a specific example of a tag sequence, for example, the amino acid sequence shown in sequence number 8 (an amino acid sequence comprising a His tag sequence and a hinge sequence) can be listed.

[0119] Alternatively, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose binding protein (MBP) that specifically binds to maltose can be used.

[0120] Furthermore, an "epitope tag" that utilizes an antigen-antibody reaction can also be used. By adding a peptide (epitope) that represents antigenicity as a tag sequence, antibodies against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tags, myc tags, and FLAG tags. By utilizing epitope tags, fibroin can be easily purified with high specificity.

[0121] Furthermore, a tag obtained by removing the tag sequence using a specific protease may be used. Alternatively, the protein adsorbed via the tag sequence may be treated with a protease to recover the fibroin after removal of the tag sequence.

[0122] Specific examples of rayon include those shown in Table 1.

[0123]

[0124] The rayon may be a rayon having at least two or more characteristics among the characteristics of the first rayon, the second rayon, the third rayon, the fourth rayon, the fifth rayon, and the sixth rayon.

[0125] The molecular weight of artificial silk fibroin is not particularly limited, and for example, it can be 2 kDa or more and 700 kDa or less. The molecular weight of artificial silk fibroin involved in this embodiment can be, for example, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more, and can also be 700 kDa or less, 600 kDa or less, 500 kDa or less, 400 kDa or less, less than 360 kDa, 300 kDa or less, or 200 kDa or less.

[0126] As described above, when the synthetic polymer obtained by the above-mentioned manufacturing method is used to obtain a molded body with excellent flexibility, if the (B1) component is a low molecular weight compound, it is preferred to use a polypeptide with a relatively low molecular weight as the (A) component. However, when the molecular weight of the polypeptide is low (for example, below 30 kDa), the yield may be reduced during the purification process during its preparation. In particular, when a purification method for hydrophobic proteins is used, problems such as reduced purity may even occur. Therefore, in the above-mentioned manufacturing method, for example, a protein (PRT2882) having an amino acid sequence shown in sequence number 9 with a hydrophobic tag introduced at the N-terminus or C-terminus (for example, GFILGFIL in sequence number 9) can be used. This low molecular weight recombinant protein having an amino acid sequence containing a hydrophobic tag sequence can obtain a high yield and high purity product during purification, so a polymer compound with a low impurity content can be obtained by the above-mentioned manufacturing method. Moreover, in this way, the molded body manufactured using this polymer compound as a molding material can have advantages such as better mechanical properties.

[0127] In the artificial silk fibroin involved in the present embodiment, one or more amino acid residues constituting REP may be hydrophobic amino acid residues. That is, REP preferably contains hydrophobic amino acid residues. The so-called hydrophobic amino acid residue refers to an amino acid residue whose hydrophobicity index is positive. Regarding the hydrophobicity index (hydrophilic index, hereinafter also referred to as "HI") of the amino acid residue, a well-known index is used (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J.Mol.Biol., 157, pp.105-132). As hydrophobic amino acid residues, for example, isoleucine (HI: 4.5), valine (HI: 4.2), leucine (HI: 3.8), phenylalanine (HI: 2.8), methionine (HI: 1.9), and alanine (HI: 1.8) can be listed.

[0128] In the artificial silk fibroin according to the present embodiment, the domain sequence preferably has an amino acid sequence corresponding to a cysteine ​​residue inserted into REP, compared to naturally derived silk fibroin.

[0129] The domain sequence preferably has an amino acid sequence equivalent to an inserted cysteine ​​residue at a position adjacent to a glycine residue, a serine residue, or an alanine residue in REP, and more preferably has an amino acid sequence equivalent to an inserted cysteine ​​residue at a position adjacent to a glycine residue in REP. The cysteine ​​residue in REP may be located between a glycine residue, a serine residue, or an alanine residue and a glycine residue, a serine residue, or an alanine residue, or may be located between a serine residue and a glycine residue.

[0130] The domain sequence preferably has a domain sequence equivalent to the amino acid sequence of a cysteine ​​residue inserted at a position adjacent to the hydrophobic amino acid residue in REP. In this case, the hydrophobic amino acid residue is fixed between molecules by hydrophobic interactions. The cysteine ​​residue in REP can be located next to the hydrophobic amino acid residue, or between the hydrophobic amino acid residue and the amino acid residue other than the hydrophobic amino acid residue, or between the hydrophobic amino acid residue and a glycine residue, a serine residue or an alanine residue, or between the hydrophobic amino acid residue and the glycine residue. The hydrophobic amino acid residue can be one selected from the group consisting of isoleucine residues, valine residues, leucine residues, phenylalanine residues, methionine residues and alanine residues.

[0131] Compared to naturally derived fibroin, the domain sequence may also have an amino acid sequence equivalent to an insertion of a cysteine ​​residue in a REP located near the N-terminus and / or C-terminus of the domain sequence. In this case, the molecular chain can be extended. In this specification, the so-called REP located near the N-terminus of the domain sequence refers to the REP located at the 1st to 3rd position from the N-terminus of the domain sequence. For example, the cysteine ​​residue may also be located in the REP located at the 1st to 2nd position from the N-terminus of the domain sequence. In this specification, the so-called REP located near the C-terminus of the domain sequence refers to the REP located at the 1st to 3rd position from the C-terminus of the domain sequence. For example, the cysteine ​​residue may also be located in the REP located at the 1st to 2nd position from the C-terminus of the domain sequence. The cysteine ​​residue is preferably located in the REP closest to the N-terminus and / or the C-terminus of the domain sequence.

[0132] Compared to naturally derived silk fibroin, the domain sequence may also have an amino acid sequence corresponding to a cysteine ​​residue inserted at or near the center of REP. In this specification, "near the center" of the amino acid sequence in REP refers to positions 1 to 5 from the amino acid residue at the center of the REP (the amino acid residue at the N-terminus when there are two amino acid residues at the center), or positions 1 to 5 from the amino acid residue at the center of the REP (the amino acid residue at the C-terminus when there are two amino acid residues at the center). For example, the cysteine ​​residue may be located at the center of the REP, or at positions 1 to 3 or 1 to 2 from the amino acid residue at the center of the REP toward the N-terminus or C-terminus. Here, when using a first segment comprising a polypeptide backbone, a synthetic polymer can be obtained in which the first and second segments are linked in an alternating manner, wherein the polypeptide backbone has cysteine ​​residues inserted, for example, on each of the N-terminus and C-terminus of the domain sequence. The elongation of a molded article (e.g., a fiber, a film, a gel, etc.) obtained using the synthetic polymer is expected to be improved. Furthermore, when a first segment comprising a polypeptide backbone having a cysteine ​​residue inserted closer to the center than the N-terminal or C-terminal side of the domain sequence is used, the solubility of the synthetic polymer in a solvent, which is formed by linking the second segment, is expected to be improved relative to the first segment.

[0133] The hydrophobicity (hydrophilic index: hydrophobicity index) of rayon REP can be, for example, -0.80, -0.70, -0.06 or higher, -0.50 or higher, -0.40 or higher, -0.30 or higher, -0.20 or higher, -0.10 or higher, 0.00 or higher, 0.10 or higher, 0.20 or higher, 0.22 or higher, 0.25 or higher, 0.30 or higher, 0.35 or higher, 0.40 or higher, 0.45 or higher, 0.50 or higher, 0.55 or higher, 0.60 or higher, 0.65 or higher, or 0.70 or higher. The upper limit of the hydrophobicity of REP is not particularly limited and can be 1.0 or lower or 0.7 or lower.

[0134] In this specification, the "hydrophobicity of REP" is a value calculated by the following method. In the formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A) n In the fibroin having the domain sequence shown in the motif, the domain sequence is excluding the C-terminal end (A) n Among all REPs contained in the sequence from the motif to the C-terminus of the domain sequence, the sum of the hydrophobicity indexes of the amino acid residues in the region is set to e, and the residue (A) located closest to the C-terminus is removed from the domain sequence. n The sequence from the motif to the C-terminus of the domain sequence is removed (A) n When the total number of amino acid residues of all REPs obtained after the motif is set to f, the hydrophobicity of REP is calculated as e / f. In the calculation of the hydrophobicity of REP, the hydrophobicity of REP is calculated by "removing the residues (A) located at the most C-terminal side from the domain sequence". n The reason for targeting the "sequence from the motif to the C-terminus of the domain sequence" is the same as the above reason.

[0135] The domain sequence may also have an amino acid sequence equivalent to 1 or more and less than 16 cysteine ​​residues inserted into REP compared to naturally derived fibroin. That is, the total number of cysteine ​​residues equivalent to those inserted into REP may be 1 or more and less than 16. The total number of cysteine ​​residues equivalent to those inserted into REP may also be 1 or more and 12 or less, 1 or more and 10 or less, 1 or more and 8 or less, 1 or more and 6 or less, or 2 or more and 4 or less. For example, the number of cysteine ​​residues in one REP in the domain sequence may be 1 to 3, 1 to 2, or 1.

[0136] The total number of cysteine ​​residues in the rayon according to this embodiment may be 1 or more and less than 16, 1 or more and 12 or less, 1 or more and 10 or less, 1 or more and 8 or less, 1 or more and 6 or less, or 2 or more and 4 or less.

[0137] In addition to the modifications related to the cysteine ​​residues in the above-mentioned REP, the artificial silk fibroin involved in this embodiment can also be further modified in terms of amino acid sequence by substitution, deletion, insertion and / or addition of one or more amino acid residues compared to the silk fibroin from natural sources.

[0138] The rayon according to the present embodiment preferably has an amino acid sequence corresponding to an inserted cysteine ​​residue.

[0139] The preparation method of component (B1) may be a method comprising the following steps: subjecting maleic anhydride to an esterification reaction with at least one compound selected from the group consisting of polyether, polyester, and polycarbonate. During this process, an acid or base may be used, or the reaction may be carried out in the absence of a solvent. When preparing component (B1), there is no need to specifically introduce a protective group for the polyether, and a simple method may be employed. In the purification step, impurities may be removed by washing with a solvent such as cyclopentyl methyl ether, tetrahydrofuran, ethyl acetate, or ethanol.

[0140] A second embodiment of the method for producing a polymer compound comprises: heating a polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates (hereinafter referred to as component (B2)) having two structures represented by the following general formula (2) in dimethyl sulfoxide in the presence of a base, a reducing agent, and a polymerization inhibitor (hereinafter referred to as component (E)). In the following general formula (2), R 1 Represents a hydrogen atom or a methyl group. The above-mentioned heating reaction is a reaction between component (A) and component (B2), for example, carried out by heating to 50°C or above. In addition, the timing of heating is not limited to after mixing components (A) to (E). For example, components (A) to (E) can be added to pre-heated dimethyl sulfoxide; heating can also be started after adding components (C), (D) and (E) to dimethyl sulfoxide, and after reaching a predetermined temperature, components (A) and (B) are added to react; or one of components (A) and (B) and components (C), (D) and (E) are first added, and then heating is started. After reaching a predetermined temperature, the remaining component (A) and (B) are added to react.

[0141] [Chemical Formula 25]

[0142] In the following description, the description of the same contents as the first embodiment will be omitted, and only the differences will be described. In the second embodiment, the polypeptide containing at least one thiol group, the base, and the reducing agent can be applied to the description of components (A), (C), and (D) in the first embodiment.

[0143] At least one compound (component (B2)) selected from the group consisting of polyether, polyester and polycarbonate having two structures represented by general formula (2) is a compound that can introduce a polyether structure, a polyester structure or a polycarbonate structure into a polypeptide because it has a structure represented by formula (1) and can react with the thiol group possessed by the above-mentioned polypeptide.

[0144] Examples of polyethers having two structures represented by formula (2) include polyethylene glycol, polytetramethylene glycol, polypropylene glycol, and ethylene glycol-propylene glycol copolymers. Examples of polyesters having two structures represented by formula (2) include polylactic acid, polyglycolic acid, polybutylene succinate, polycaprolactone, and polyhydroxyalkanoates containing copolymers of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid. Examples of polycarbonates having two structures represented by formula (2) include aliphatic polycarbonates such as polyethylene carbonate, polypropylene carbonate, and polytrimethyl carbonate. Since these compounds are more prone to molecular motion than the above-mentioned polypeptides, when the above-mentioned compounds are used, the flexibility of the molded body composed of the obtained polymer compound can be further improved.

[0145] The amount of the component (B2) to be added can refer to the description of the component (B1) in the first embodiment, and the description of the component (B2) can be applied instead.

[0146] In the above production method, the polymerization inhibitor (component (E)) is a compound that inhibits the self-polymerization of component (B2). Examples of component (E) include hydroquinone, p-hydroxyanisole, p-tert-butylcatechol, tert-butylhydroquinone, 1,4-benzoquinone, dibutylhydroxytoluene, p-methoxyphenol, phenothiazine, and 1,1-diphenyl-2-picrylhydrazyl.

[0147] The content of component (E) can be, for example, 0.01 parts by mass or more, 0.05 parts by mass or more, 0.1 parts by mass or more, or 0.2 parts by mass or more relative to 100 parts by mass of the above-mentioned compound (component (B2)). By controlling the content of the polymerization inhibitor within the above-mentioned range, the start of free radical polymerization of the structure represented by the general formula (2) can be suppressed, thereby making the reaction between the structure represented by the general formula (2) and the thiol group possessed by the polypeptide more advantageous. Relative to 100 parts by mass of component (B2), the content of component (E) can be, for example, 25 parts by mass or less, 10 parts by mass or less, 5 parts by mass or less, or 1 part by mass or less. By controlling the content of component (E) within the above-mentioned range, the inhibitory effect of component (E) on the reaction in the system can be further suppressed.

[0148] Component (B2) can be prepared by a method comprising the following steps: esterifying (meth)acrylic acid, (meth)acrylic anhydride, or (meth)acryloyl chloride with at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates. When preparing component (B2), there is no need to specifically introduce a protective group into the polyether; a simple method can be employed. The purification step involves hot extraction with tetrahydrofuran to remove organic salts, followed by reprecipitation with n-hexane.

[0149] The polymer compound synthesized by the above-mentioned production method has the following structure: a polypeptide portion and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure are directly bonded via a structure represented by the following general formula (3), the following general formula (4), or the following general formula (5), so that the polypeptide portion is bonded to S of the following general formula (3), the following general formula (4), or the following general formula (5). In the general formulas (3) and (4), M represents any one of H (hydrogen atom), Na (sodium atom), K (potassium atom), NHEt3, or NHEtiPr2, and in the general formula (5), R 1 represents H or Me. The above-mentioned Me represents a methyl group, the above-mentioned Et represents an ethyl group, and the above-mentioned iPr represents an isopropyl group.

[0150] [Chemical Formula 26]

[0151] [Chemical Formula 27]

[0152] [Chemical Formula 28]

[0153] Because the polymer compound comprises a polypeptide and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure, it can also be referred to as a block copolymer. If the structural unit comprising a polypeptide is represented by A and the structural unit comprising at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure is represented by B, the polymer compound need only comprise at least one A and at least one B, respectively. For example, the polymer compound may be an AB diblock copolymer, an ABA triblock copolymer, an ABAB tetrablock copolymer, an ABABA pentablock copolymer, or a copolymer of AABBBABBAA, etc.

[0154] The polymer compound may be a linear polymer, a comb-shaped polymer, or may have a three-dimensional network structure. From the perspective of improving the flexibility of a molded article composed of the polymer compound, a linear polymer or a comb-shaped polymer is preferred, while from the perspective of improving the elastic modulus of the molded article, a three-dimensional network structure is preferred.

[0155] There is no particular limitation on the position of at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure introduced into the polypeptide portion. It may be at the end of the polypeptide portion or at a position other than the end of the polypeptide portion. However, from the perspective of the convenience of manufacturing the polymer compound, it is preferably at the end of the polypeptide portion.

[0156] The polymer compound synthesized by the above-mentioned production method can be used as a molding material. The shape of a molded body using the above-mentioned polymer compound as a molding material is not particularly limited, and may be, for example, a film, a fiber, or the like.

[0157] One aspect of the present disclosure provides a method for producing a polymer compound solution, comprising the step of dissolving the polymer compound obtained by the above-mentioned production method in a solvent. The polymer compound solution obtained by this method can be used as a spinning solution used in the preparation of a molded body. Examples of solvents used to prepare the spinning solution include formic acid, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, hexafluoroisopropanol, and N-methyl-2-pyrrolidone. In addition, in the above-mentioned production method, the target polymer compound can be obtained in a state of being dissolved in dimethyl sulfoxide (i.e., as a dimethyl sulfoxide solution) by the heating reaction. Therefore, in the above-mentioned method for producing the polymer compound solution, for example, a step of dissolving the polymer compound obtained from dimethyl sulfoxide by a known extraction method or separation method into a specified solvent is included.

[0158] A polymer film can also be produced by a method comprising forming the polymer compound solution obtained by the above-mentioned method for producing a polymer compound solution into a film. The film can be formed by, for example, casting.

[0159] The polymer fibers may be produced by a method including the step of spinning the polymer compound solution obtained by the above-mentioned method for producing the polymer compound solution.

[0160] Furthermore, the dimethyl sulfoxide solution containing the polymer compound obtained by the heat reaction in the above-mentioned polymer compound production method can also be used directly as a spinning solution for producing films or fibers. This makes the production of polymer films simpler and more cost-effective. The film can also be formed by, for example, casting.

[0161] In one aspect of the present disclosure, a method for producing a thin film is provided, comprising the following steps: heating a polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates having two structures represented by the following general formula (1) in dimethyl sulfoxide in the presence of an alkali and a reducing agent to obtain a polymer compound solution (a dimethyl sulfoxide solution of the polymer compound); and forming the polymer compound solution into a thin film. In the general formula (1), M represents any one of H, Na, K, NHEt3, or NHEtiPr2.

[0162] [Chemical Formula 29]

[0163] In one aspect of the present disclosure, a method for manufacturing a thin film is provided, comprising the following steps: heating a polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyether, polyester, and polycarbonate having two structures represented by the following general formula (2) in dimethyl sulfoxide in the presence of an alkali, a reducing agent, and a polymerization inhibitor to obtain a polymer compound solution (a dimethyl sulfoxide solution of a polymer compound); and molding the polymer compound solution into a thin film. In the general formula (2), R 1 represents a hydrogen atom or a methyl group.

[0164] [Chemical formula 30]

[0165] In one aspect of the present disclosure, a method for producing a fiber is provided, comprising the steps of: heating a polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates having two structures represented by the following general formula (1) in dimethyl sulfoxide in the presence of an alkali and a reducing agent to obtain a polymer compound solution (a dimethyl sulfoxide solution of the polymer compound); and spinning the polymer compound solution. In the general formula (1), M represents any one of H, Na, K, NHEt3, or NHEtiPr2.

[0166] [Chemical Formula 31]

[0167] One aspect of the present disclosure provides a method for manufacturing a fiber, comprising the following steps: heating a polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyether, polyester, and polycarbonate having two structures represented by the following general formula (2) in dimethyl sulfoxide in the presence of an alkali, a reducing agent, and a polymerization inhibitor to obtain a polymer compound solution (a dimethyl sulfoxide solution of a polymer compound); and spinning the polymer compound solution. In the general formula (2), R 1 represents a hydrogen atom or a methyl group.

[0168] [Chemical Formula 32]

[0169] One aspect of the present disclosure provides a film comprising a polymer compound, wherein the polymer compound has the following structure: a polypeptide portion and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure, directly bonded via a structure represented by the following formula (3), the following formula (4), or the following general formula (5), so that the polypeptide portion is bonded to S of the following formula (3), the following formula (4), or the following general formula (5), and the film has an elongation of more than 400%, and a ratio of breaking strength to yield strength greater than 1. In the general formulas (3) and (4), M represents any one of H, Na, K, NHEt3, or NHEtiPr2, and in the general formula (5), R 1 Indicates H or Me.

[0170] [Chemical Formula 33]

[0171] [Chemical Formula 34]

[0172] [Chemical Formula 35]

[0173] One aspect of the present disclosure provides a fiber containing a polymer compound, wherein the polymer compound has the following structure: a polypeptide portion and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure, directly bonded via a structure represented by the following formula (3), the following formula (4), or the following general formula (5), so that the polypeptide portion is bonded to S of the following formula (3), the following formula (4), or the following general formula (5), wherein the film elongation is greater than 400% and the ratio of breaking strength to yield strength is greater than 1. In the general formula (5), R1 represents a hydrogen atom or a methyl group.

[0174] [Chemical Formula 36]

[0175] [Chemical Formula 37]

[0176] [Chemical Formula 38]

[0177] In the above-mentioned film and the above-mentioned fiber, the ratio of the breaking strength to the yield point strength (breaking strength [MPa] / yield point strength [MPa]) is greater than 1, and can be, for example, 1.1 or greater, 1.2 or greater, 1.3 or greater, 1.4 or greater, or 1.5 or greater. In the above-mentioned film and the above-mentioned fiber, the ratio of the breaking strength to the yield point strength (breaking strength [MPa] / yield point strength [MPa]) can be, for example, 3 or less, or 2 or less.

[0178] The polymer compound of this embodiment can be used as an adhesive for adhering adherends or as a coating material for forming a coating layer on a substrate in the form of a solution, aqueous dispersion, film, or powder containing the polymer compound as a main component. These solutions, aqueous adhesives, aqueous coatings, film-like adhesives, film-like coatings, and powdered adhesives or powdered coatings can be obtained, for example, by the following production methods.

[0179] A solution-like adhesive or solution-like coating containing a polymer compound can be manufactured by a method comprising dissolving the polymer compound obtained by the method of this embodiment in a solvent. Using this type of method, unlike conventional solution-like adhesives or solution-like coatings containing components derived from petroleum, a solution-like adhesive or solution-like coating with biodegradability can be easily obtained. In addition, in the method for manufacturing the polymer compound involved in the present embodiment, the target polymer compound in a state dissolved in dimethyl sulfoxide (i.e., as a dimethyl sulfoxide solution) can be obtained by the heating reaction. Therefore, in the method for manufacturing the solution-like adhesive or coating involved in the present embodiment, for example, a process is included in which the polymer compound extracted from dimethyl sulfoxide by a known extraction method or separation method is dissolved in a specified solvent. In addition, the solution-like adhesive or solution-like coating involved in the present embodiment can also be composed of a dimethyl sulfoxide solution of the polymer compound obtained by the heating reaction in the method for manufacturing the polymer compound.

[0180] Examples of solvents used in the production of solution adhesives or solution coatings include water, aqueous media such as alkaline aqueous solutions, acidic aqueous solutions, and neutral aqueous solutions containing inorganic salts, which can dissolve polymer compounds, and organic solvents. Examples of organic solvents include formic acid, dimethyl sulfoxide (DMSO), hexafluoroisopropanol (HFIP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), and dihydrolevulinone.

[0181] The polymer compound contained in the solution adhesive or solution coating is obtained by the manufacturing method involved in the present embodiment, as long as it can be dissolved in the aqueous medium or organic solvent, there is no particular limitation. The adhesive or coating formed by the polymer compound being dissolved in the aqueous medium has the advantages of being more excellent in usability, etc., compared with the adhesive or coating formed by the polymer compound being dissolved in the organic solvent. In addition, the polymer compound involved in the present embodiment has a higher water solubility than protein because it is combined with a molecule that can plasticize protein on protein. Therefore, the aqueous solution adhesive or aqueous solution coating formed by the polymer compound being dissolved in the aqueous medium can improve the content of protein therein, compared with the aqueous solution adhesive or aqueous solution coating formed by only dissolving protein in the aqueous medium. Therefore, compared with using the aqueous solution adhesive or aqueous solution coating formed by dissolving protein in the aqueous medium, using the aqueous solution adhesive or aqueous solution coating formed by dissolving the polymer compound in the aqueous medium can improve the bonding strength of the adhesive or coating and the adhered surface or laminate surface of the adherend described later.

[0182] The concentration of the polymer compound in the solution-like adhesive or solution-like coating can be appropriately adjusted according to the solubility of the polymer compound in the solvent. This concentration can be, for example, 5 to 40% by mass, 10 to 35% by mass, 15 to 20% by mass, or 20 to 25% by mass. Furthermore, if necessary, the solution-like adhesive or solution-like coating can contain ingredients other than the polymer compound, such as various additives commonly found in solution-like adhesives or solution-like coatings. The upper limit of the above concentration can be, for example, less than 30% by mass, 28% or less by mass, 25% or less by mass, or 23% or less by mass.

[0183] The solution-like adhesive containing macromolecular compound is used to make the adherend of a plurality of adherends that adhere to each other. For example, the solution-like adhesive is placed between a plurality of adherends that need to adhere, and the solvent in the solution-like adhesive is removed afterwards, and the macromolecular compound is solidified, thereby adherends can be bonded to each other to obtain adherends. An advantage of using this type of method is that, an adherend can be obtained by using an adhesive with biodegradability, so that the adherend of the environmental burden when discarding can be easily manufactured. In addition, as long as the adherend can be bonded with the solution-like adhesive containing macromolecular compound, its material is not particularly limited, and can be an organic substance (cellulose products such as paper, wood or synthetic resins, protein products), or an inorganic substance (non-metals such as metals or glass).

[0184] The specific method when using solution adhesive to make adherend does not have any restriction.For example, can be coated with or drip solution adhesive on the adhered surface of at least one of the parties in the adherend that adheres to each other, or make the adhered surface of adherend contact, impregnate the liquid level of solution adhesive, make there is solution adhesive on the adhered surface, then overlap or be close to between the adhered surface of adherend, thereby solution adhesive is placed between multiple adherends.And, from the solution adhesive that is placed between multiple adherends, remove solvent and make polymer compound solidify when, for example, in order to ensure close fit between the adhered surface of overlapping or docking, can heat, air-dry or naturally dry under the state that multiple adherends are applied pressure from at least any one side of the overlapping or docking direction of the adhered surface.Certainly, when not applying such pressure and also can bond, then do not need to apply pressure.

[0185] A solution-like coating containing a polymer compound is used to manufacture a laminate formed by stacking a coating on all or part of a substrate surface. For example, the solution-like coating is supplied to at least a portion of a substrate surface that requires a stacked coating, and after coating the surface portion of the substrate with the solution-like coating, the solvent in the solution-like adhesive is removed to solidify the polymer compound. Thus, a laminate formed by stacking a coating on at least a portion of a substrate surface can be obtained. Using this type of method has the advantage that a biodegradable coating can also be used to obtain a laminate, so that a laminate that can reduce the environmental burden when discarded can be easily manufactured. In addition, as long as the substrate can be bonded with the solution-like coating containing a polymer compound, its material is not particularly limited and can be the same as the adherend bondable with the solution-like adhesive.

[0186] There is no restriction on the specific method of using solution coating to manufacture laminate. For example, the solution coating can be applied or dripped onto at least a portion of the substrate surface, or at least a portion of the substrate surface can be contacted or immersed in the liquid surface of the solution coating, and the coating can be supplied and applied to ensure that the solution coating forms a layer of a specified thickness on at least a portion of the substrate. In addition, when the polymer compound in the coating layer (solution coating) stacked on the substrate surface is solidified, the coating layer on the substrate surface can be heated, air-dried or naturally dried to remove the solvent in the coating layer. In this case, if necessary, a specified extrusion body (pressurizing body) can be placed on the coating layer in a manner that covers the entire coating layer, and the coating layer can be squeezed (pressurized) to the substrate surface side to ensure that the coating layer is closely attached to the substrate surface. At this time, it is preferred that the extrusion body does not adhere to the solution coating. For example, by applying a release agent or pasting release paper on the contact surface of the extrusion body with the coating layer, or by surface treating the contact surface to ensure that the coating does not adhere, or by using a material that does not adhere to the solution coating as the extrusion body, the solution coating can be prevented from adhering to the extrusion body.

[0187] A water-dispersible adhesive or water-dispersible coating containing a polymer compound can be manufactured by a method comprising dispersing the polymer compound obtained by the method of the present embodiment in an aqueous medium. Using such a method, unlike conventional water-dispersible adhesives or water-dispersible coatings containing components derived from petroleum, a water-dispersible adhesive or water-dispersible coating having biodegradability can be easily obtained. As previously mentioned, in the method for manufacturing the polymer compound involved in the present embodiment, the target polymer compound can be obtained in a state dissolved in dimethyl sulfoxide (i.e., as a dimethyl sulfoxide solution). Therefore, in the method for manufacturing the water-dispersible adhesive or water-dispersible coating involved in the present embodiment, for example, a process is included in which the polymer compound extracted from dimethyl sulfoxide by a known extraction method or separation method is dispersed in a specified aqueous medium.

[0188] Examples of aqueous media used for producing water-dispersible adhesives or water-dispersible coatings include water, alkaline aqueous solutions, acidic aqueous solutions, and aqueous solutions containing inorganic salts, which can disperse polymer compounds.

[0189] The polymer compound contained in the water-dispersible adhesive or water-dispersible coating is not particularly limited as long as it can be produced by the process involved in this embodiment and is dispersible in the aqueous medium. The content of the polymer compound in the water-dispersible adhesive or water-dispersible coating is appropriately adjusted based on factors such as the adhesion of the water-dispersible adhesive to the adherend, the adhesion of the water-dispersible coating to the substrate, and even the fixation. In addition, the water-dispersible adhesive or water-dispersible coating may contain ingredients other than the polymer compound, such as various additives contained in known water-dispersible adhesives or water-dispersible coatings, as necessary.

[0190] In addition, the polymer compound involved in the present embodiment has a higher affinity for aqueous media containing water than proteins due to the presence of molecules capable of plasticizing proteins in proteins. Therefore, the water-dispersible adhesive or water-dispersible coating containing such polymer compound can not only increase the content (dispersion amount) of protein, but also make the polymer compound uniformly dispersed in the aqueous medium, compared to water-dispersible adhesives or water-dispersible coatings formed by only dispersing proteins in aqueous media. Therefore, compared to water-dispersible adhesives or water-dispersible coatings formed by using proteins to be dispersed in aqueous media, water-dispersible adhesives or water-dispersible coatings formed by using polymer compounds to be dispersed in aqueous media can enhance the bonding strength of adhesive or coating to the adhered surface or laminate surface of the adherend described later.

[0191] The water-dispersible adhesive that contains macromolecular compound is used to make the adherend that a plurality of adherends adhere to each other.For example, the water-dispersible adhesive is placed between the a plurality of adherends that need to bond, and then the aqueous medium in the water-dispersible adhesive is removed, and the macromolecular compound is solidified, thereby can be bonded between the adherends and obtain the adherend. An advantage of using this type of method is that, can use the adhesive with biodegradability to obtain the adherend, therefore can easily produce the adherend that can reduce the environmental burden when discarding. In addition, as long as the adherend can be bonded with the water-dispersible adhesive that contains macromolecular compound, its material is not particularly limited, and can be the same as the adherend that is bonded with the solution-shaped adhesive that contains macromolecular compound.

[0192] The specific method for making an adherend using a water-dispersible adhesive is also not limited. For example, when placing a water-dispersible adhesive between a plurality of adherends to be bonded, the same method as when placing the solution-like adhesive between the plurality of adherends can be used. Furthermore, when removing the aqueous medium from the water-dispersible adhesive between the adherends and solidifying the polymer compound, the same method as when removing the solvent from the solution-like adhesive between the adherends and solidifying the polymer compound can be used.

[0193] A water-dispersible coating containing a polymer compound is used to manufacture a laminated body that is laminated to form a coating on all or part of a substrate surface. For example, the water-dispersible coating is supplied to at least a portion of the substrate surface to which the coating is to be laminated, and after the surface portion of the substrate is coated with a solution-like coating, the aqueous medium in the water-dispersible adhesive is removed to solidify the polymer compound. Thus, a laminated body that is laminated to form a coating on at least a portion of the substrate surface can be obtained. One advantage of using such a method is that a laminated body can also be obtained using a biodegradable coating, so that a laminated body that can reduce the environmental burden when discarded can be easily manufactured. In addition, the substrate can be made of any material that can be bonded with the water-dispersible coating containing a polymer compound, and the material is not particularly limited, and can be the same as the substrate on which the coating is formed with the solution-like coating.

[0194] The specific method for producing a laminate using a water-dispersible coating is also not limited. For example, the water-dispersible coating can be applied to at least a portion of a substrate surface using the same method as that used for applying the solution-based coating to the substrate surface. Furthermore, the aqueous medium in the water-dispersible coating applied to the substrate surface and the polymer compound cured can be removed using the same method as that used for removing the solvent from the solution-based coating applied to the substrate surface and the polymer compound cured.

[0195] A film-like adhesive or film-like coating containing a polymer compound can be produced by a method comprising forming a film from a polymer compound solution obtained by the method of this embodiment. This method, unlike conventional film-like adhesives or film-like coatings containing components derived from petroleum, allows for the convenient production of a biodegradable film-like adhesive or film-like coating. Furthermore, the film can be formed by conventional casting.

[0196] The film-forming solution used to produce a film-forming adhesive or film-forming coating preferably uses, for example, a polymer compound solution that is a solution-form adhesive or coating. Therefore, the film-forming solution can be a dimethyl sulfoxide solution of a polymer compound obtained by the polymer compound production method described in this embodiment, or, for example, a solution obtained by dissolving a polymer compound extracted from dimethyl sulfoxide using a known extraction or separation method in a designated solvent. Furthermore, casting of the polymer compound solution can be performed using the same known methods and conditions as those used for casting protein solutions.

[0197] The film-like adhesive that contains macromolecular compound is used to make the adherend that a plurality of adherends adhere to each other.For example, when the film-like adhesive is placed under the state between the a plurality of adherends that need to bond, the film-like adhesive is swollen or heated and softened, then hardened under the state that the film-like adhesive is pressed onto the adherend, thereby can be bonded between the adherend to obtain the adherend.In addition, also can in advance the film-like adhesive is swollen or heated and softened, then placed between the a plurality of adherends, then hardened under the state that the film-like adhesive is pressed onto the adherend.Use this type of method, an advantage having is, can use the adhesive with biodegradability to obtain the adherend, therefore can easily manufacture the adherend that can reduce the environmental burden when discarding.In addition, as long as the adherend can be bonded with the film-like adhesive that contains macromolecular compound, it can also be the same as the adherend that is bonded with the solution-like adhesive that contains macromolecular compound.

[0198] Film-shaped adhesives soften when they absorb water or are heated, and then harden by drying or cooling. Furthermore, if the polymer compound contained in the film-shaped adhesive, particularly one that exhibits shrinkage behavior when the molded body is in contact with water or heated, the film-shaped adhesive will also shrink when in contact with water or heated. Therefore, film-shaped adhesives containing polymer compounds, when softened by swelling or heating between adherends, can partially become trapped in the gaps on the adherend's bonded surface. Especially when modified proteins have the aforementioned shrinkage properties, they shrink while trapped in the gaps on the adherend's bonded surface. If the film-shaped adhesive hardens in this state, due to the anchoring effect, the film-shaped adhesive adheres to the adherend, thereby bonding the adherends. Furthermore, since film-shaped adhesives contain polymer compounds that combine proteins with molecules that plasticize proteins as their main component, they can exhibit sufficient flexibility. Therefore, such film-shaped adhesives can exhibit higher flexibility in a softened state, allowing them to more fully embed into the gaps on the adherend's bonded surface, thereby potentially achieving higher bond strength. Film-shaped adhesives also offer the following advantages: due to their excellent flexibility, they can be peeled from an adherend after being bonded, allowing for repeated use as a film-shaped adhesive. Furthermore, when reusing a film-shaped adhesive, it can be washed after being peeled from the adherend. Furthermore, the film-shaped adhesive's bonding effect on the adherend is likely due to the formation of hydrogen bonds between the adherend and the polymer compound.

[0199] When swelling and softening the film adhesive, it is preferable to use an aqueous liquid such as water, a water-containing solution, or a dispersion that can be absorbed by the film adhesive or that can cause the film adhesive to shrink. Alternatively, the film adhesive can be swollen and softened by, for example, dripping the aqueous liquid onto the film adhesive or immersing multiple adherends in the aqueous liquid to cause the film adhesive to absorb water. Furthermore, when hardening the film adhesive that has softened due to swelling, for example, the film adhesive can be heated, air-dried, or naturally dried while placed between adherends.

[0200] When heating the film adhesive to soften it, the film adhesive may be heated before being placed between the adherends, or the entire adherend and film adhesive may be heated after the film adhesive is placed between the adherends. The heating temperature is not particularly limited, as long as it softens the film adhesive and does not negatively affect the protein contained in the film adhesive (e.g., does not decompose it). To harden the film adhesive softened by heating, cooling using a cooling device or allowing it to cool can be used.

[0201] When making the film-like adhesive that softens because of swelling or heating fully sink into the gap of the adherend's bonded surface, preferably make the interface of softening film-like adhesive and adherend closely fit.Then, preferably for example from the overlapping direction of the adherend's surface or any one side in the docking direction, a plurality of adherends are exerted with pressure, the film-like adhesive that softens is pressed onto the adherend, and it is hardened.Here said pressurization method to adherend, can adopt common arbitrary method.

[0202] The film-like coating that contains macromolecular compound is used to manufacture the laminated body that forms coating on all or part of substrate surface.For example, under the state that the film-like coating is placed and covers at least a portion of substrate surface, the film-like coating is swollen or heated, softened, then hardened under the state that the film-like coating is pressed onto substrate surface, thereby can form coating on at least a portion of substrate surface, obtain laminated body.In addition, also can swell or heat to soften the film-like coating in advance, then place and cover at least a portion of substrate surface, then harden under the state that the film-like coating is pressed onto adherend.Use this type of method, an advantage that has is, can use coating with biodegradability to form laminated body, therefore can easily manufacture the laminated body that can reduce environmental burden when discarding.In addition, as long as substrate can be bonded with the film-like coating that contains macromolecular compound, also can use the same material as the adherend bonded with the film-like adhesive that contains macromolecular compound.

[0203] It can be thought that the mechanism that film-like coating adheres to substrate surface is identical with the mechanism that film-like adhesive adheres to the adhered surface of adherend.Therefore, the concrete method when using film-like coating to make laminate can also adopt the same method when obtaining adherend with film-like adhesive.In addition, the film-like coating after softening is pressed onto the substrate surface and when hardening, for example, the extrusion body identical with the extrusion body used when the solution-like coating applied to the substrate is extruded onto the substrate surface side can be used to extrude the film-like coating onto the substrate surface side.In addition, the film-like coating is also identical with the film-like adhesive, not only has higher bond strength to substrate, and can be peeled off after bonding, even can be reused.

[0204] In a powdered adhesive or powdered coating containing a polymer compound, a powder composition containing a polymer compound powder obtained by the method of this embodiment is used. As long as the powder composition contains the polymer compound as a main component, it can also contain various secondary components such as added residues. As mentioned above, in the method for producing the polymer compound involved in this embodiment, the target polymer compound can be obtained in a state dissolved in dimethyl sulfoxide (i.e., as a dimethyl sulfoxide solution). Therefore, in the method for producing the powdered adhesive or powdered coating involved in this embodiment, for example, a process of powdering the polymer compound extracted from dimethyl sulfoxide by a known extraction method or separation method is included.

[0205] The polymer compound contained in powdered adhesive or powdered coating, as long as it is the polymer compound manufactured by the process involved in the present embodiment, is not particularly limited.For example, the polymer compound contained in solution adhesive or solution coating, water-dispersible adhesive or water-dispersible coating, film adhesive or film coating all can be applicable.In addition, as mentioned above, the polymer compound involved in the present embodiment shows an affinity higher than protein for water-containing aqueous medium (aqueous liquid).Therefore, the powdered adhesive or powdered coating containing this polymer compound, compared to the water-dispersible adhesive or water-dispersible coating formed by only protein dispersion in aqueous medium, not only can improve the content (dispersion amount) of protein, can also make the polymer compound uniformly dispersed in aqueous medium.Therefore, compared with the water-dispersible adhesive or water-dispersible coating formed by protein dispersion in aqueous medium, the water-dispersible adhesive or water-dispersible coating formed by using polymer compound dispersion in aqueous medium can promote the bonding strength of adhesive or coating to the adhered surface of adherend described later or the laminate surface.

[0206] The powdered adhesive that contains modified protein is also used to make the adherend that a plurality of adherends adhere to each other.For example, when powdered adhesive is placed under the state between a plurality of adherends that need to bond, powdered adhesive is heated simultaneously via adherend pressurization, is solidified, thereby can be bonded between adherend and obtain adherend.An advantage of using this type of method is, can use the adhesive with biodegradability to obtain adherend, therefore can easily produce the adherend that can reduce the environmental burden when discarding.In addition, as long as adherend can be bonded with the powdered adhesive that contains macromolecular compound, for example, also can be the same as the adherend that is bonded with the solution-like adhesive that contains macromolecular compound.

[0207] The concrete method when using powdered adhesive to make adherend does not have any restriction yet.For example, when the powdered adhesive between the multiple adherends that need to be bonded is heated / pressurized, the powdered adhesive that places between the adherends can be heated together with the adherend by using metal plates etc. from the two sides opposite to the bonded surface side to clamp the adherend and heat the metal plates. Meanwhile, use manual presses etc. to pressurize the metal plates, via adherend, the powdered adhesive is carried out the pressurization of the prescribed time. Thus, the powdered adhesive is resinized and solidified, bonding adherend. In addition, the heating temperature, pressurization amount or heating / pressurization time when the powdered adhesive is solidified can be suitably selected from the scope that can make the modified protein resinization according to the kind of the modified protein that contains in the powdered adhesive.

[0208] A powdered coating containing a polymer compound is used to produce a laminated body that forms a coating layer on all or part of a substrate surface. For example, the powdered coating is placed on at least a portion of a substrate surface and then heated while being pressurized between a pressurizing body and the substrate to cure the powdered coating, thereby forming a coating layer on at least a portion of the substrate surface.

[0209] The specific method when using powdered coating to make laminated body also has no restriction.For example, configuration metal plate etc. makes it cover all powdered adhesives placed on substrate surface, and metal plate is heated, thereby heats powdered adhesive.At the same time, use manual press etc. between metal plate and substrate to carry out pressurization of powdered adhesive for prescribed time, thereby powdered coating resinization and solidification.In addition, the heating temperature, pressurization amount or heating / pressurization time of powdered coating are the same as when using powdered adhesive to obtain adherend.

[0210] Although several preferred embodiments have been described above, the present disclosure is not limited to the above embodiments. Furthermore, the descriptions of the above embodiments can be applied to each other. [Example]

[0211] Hereinafter, the present disclosure will be described in more detail based on Examples, etc. However, the present disclosure is not limited to the following Examples.

[0212] [Preparation of polypeptide] (Preparation of Expression Vector) Artificial silk fibroin having the amino acid sequence shown in SEQ ID NO: 9 (PRT2882) was designed.

[0213] Furthermore, the average hydrophilic index value of rayon having the amino acid sequence shown in SEQ ID NO: 9 (PRT2882) is 0.47.

[0214] Nucleic acids encoding fibroin having the amino acid sequence set forth in SEQ ID NO: 9 were synthesized. These nucleic acids had an NdeI site at the 5' end and an EcoRI site downstream of the stop codon. These nucleic acids were cloned into a cloning vector (pUC118). The nucleic acids were then treated with restriction enzymes NdeI and EcoRI, excised, and recombined into the polypeptide expression vector pET-22b(+) to produce an expression vector.

[0215] (Preparation of polypeptide) The obtained expression vector was used to transform Escherichia coli BLR (DE3). The transformed Escherichia coli was cultured in 2 mL LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL seed culture medium (Table 2) containing ampicillin to make the OD 600 The culture temperature was maintained at 30°C and the flask culture was continued until the OD 600 When the temperature reaches 5 (about 15 hours), a seed culture solution is obtained.

[0216] [Table 2] Seed culture medium

[0217] The seed culture was added to a fermenter containing 500 ml of production medium (Table 3) to obtain an OD 600 The culture medium temperature was maintained at 37°C and the pH was controlled to be constant at 6.9. In addition, the dissolved oxygen concentration in the culture medium was maintained at 20% of the saturated dissolved oxygen concentration.

[0218] [Table 3] Production medium

[0219] After the glucose in the production medium is completely consumed, the feed solution (glucose 455g / 1L, yeast extract 120g / 1L) is immediately added at a rate of 1mL / min. The culture temperature is maintained at 37°C and cultured at a constant pH of 6.9. In addition, the dissolved oxygen concentration in the culture solution is maintained at 20% of the saturated dissolved oxygen concentration and cultured for 20 hours. Then, 1M isopropyl-β-thiogalactopyranoside (IPTG) is added to the culture solution to make its final concentration 1mM, and expression of induced fibroin is induced. At a time point of 20 hours after the addition of IPTG, the culture solution is centrifuged and the bacteria are recovered. SDS-PAGE is performed using bacteria prepared from the culture solution before and after the addition of IPTG, and the expression of the target fibroin is confirmed based on the appearance of bands of the target fibroin size that depends on the addition of IPTG.

[0220] (Purification of polypeptides) Twenty hours after adding IPTG, the cells were recovered and washed with 20mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20mM Tris-HCl buffer (pH 7.4) containing approximately 1mM PMSF and disrupted using a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20mM Tris-HCl buffer (pH 7.4) until it reached high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10mM sodium dihydrogen phosphate, 20mM NaCl, 1mM Tris-HCl, pH 7.0) to a concentration of 100mg / mL and stirred at 60°C for 30 minutes to dissolve the cell. After dissolution, the cell was dialyzed against water using dialysis tubing (Sanko Junyaku Co., Ltd., Cellulose Tubing 36 / 32).

[0221] The white coagulated polypeptide obtained after dialysis was recovered by centrifugation, and the water was removed by a freeze dryer. The powdered artificial silk fibroin (PRT2882) was obtained by recovering the lyophilized powder. It was confirmed that the artificial silk fibroin with the inserted cysteine ​​residue formed disulfide bonds between the molecules. It was confirmed that in the artificial silk fibroin, with respect to 100 parts by mass of the monomer, it contained 13.8 parts by mass of dimer, 2.1 parts by mass of trimer, and 0.8 parts by mass of tetramer. The formation of disulfide bonds was determined by SDS-PAGE.

[0222] [Preparation of the compound having the structure represented by general formula (1)] (Synthesis of Polyethylene Glycol Bismaleate) 500g of polyethylene glycol (weight average molecular weight: 10000) and 50g of maleic anhydride are placed in a container and subjected to vacuum nitrogen replacement. After the contents are melted, 7ml of triethylamine is added, the temperature is raised to 80°C, and heated with stirring for 2.5 hours to react. The solution after the reaction is added to 500mL of cyclopentyl methyl ether (CPME) for reprecipitation to generate a precipitate and recycle it. 1500ml of CPME is added again to the obtained precipitate, and the precipitate is cleaned by stirring. The cleaned precipitate is then recovered by filtration, and 2000ml of tetrahydrofuran is added, and the above-mentioned precipitate is further cleaned by stirring. Then, the above-mentioned precipitate is recovered by filtration and vacuum dried to obtain 450g of polyethylene glycol bismaleate (polyethylene glycol with two maleic acid skeletons) (yield: 90% by mass).

[0223] [Preparation of the compound having the structure represented by general formula (2)] (Synthesis of Polyethylene Glycol Diacrylate) Prepare a 300 mL solution of 60 g of polyethylene glycol (weight-average molecular weight: 10,000) dissolved in dichloromethane, add 60 g of anhydrous sodium sulfate, and stir for 30 minutes. Collect the anhydrous sodium sulfate by filtration, then wash the filtered solution with 100 mL of dichloromethane to remove solids, thereby obtaining a washing liquid. The resulting filtrate and washing liquid are added to a 1 L four-necked flask, and the solvent is removed by evaporation under reduced pressure to obtain purified polyethylene glycol.

[0224] Next, under a nitrogen atmosphere, 300 mL of toluene, previously dehydrated with molecular sieves 4A, was added to the purified polyethylene glycol to prepare a suspension. The resulting suspension was heated to approximately 50°C under a nitrogen atmosphere to form a homogeneous solution. The toluene was evaporated from the resulting solution under reduced pressure. 300 mg of hydroquinone was added to the resulting residue as a polymerization inhibitor, and the mixture was dried under reduced pressure. The four-necked flask was then filled with nitrogen.

[0225] Next, at room temperature and under nitrogen flow, 500mL of dichloromethane that had been dehydrated with molecular sieve 4A in advance was added to a four-necked flask using a conduit. Further, at room temperature and under a nitrogen atmosphere, 8.4mmL (60mmol) of triethylamine that had been dehydrated with molecular sieve 4A in advance was added using a syringe to prepare a mixed solution. The mixed solution was maintained in a nitrogen atmosphere and cooled using an ice bath adjusted to -15°C. Under a nitrogen atmosphere, the reaction temperature was adjusted to -5°C to -3°C, and 5mL (60mmol) of acryloyl chloride was added dropwise in approximately 5 minutes. After the acryloyl chloride was added dropwise, an ice bath was maintained to allow the system to heat up naturally, and stirring was continued overnight under a nitrogen atmosphere to complete the reaction.

[0226] Under reduced pressure, the solvent is evaporated from the solution after the reaction, and the residue obtained is washed with n-hexane. 600 mg of hydroquinone is added to the washed residue, and hot extraction is performed with 700 mL of tetrahydrofuran to obtain an extract. The extract is filtered using diatomaceous earth (Celite) No. 503, and 700 mL of n-hexane is added to the filtered collection for reprecipitation. The generated precipitate is filtered and collected, dissolved in 500 mL of dichloromethane, 300 mg of hydroquinone is added, and the solvent is removed by evaporation under reduced pressure. By drying the obtained residue under reduced pressure, 58.8 g (yield: 99% by mass) of polyethylene glycol diacrylate (a polyether having two acrylate skeletons) of a light yellow powder is obtained.

[0227] Preparation of other compounds (Synthesis of Polyethylene Glycol Bismaleimide) Polyethylene glycol bismaleimide (Mal-PEG-Mal (Maleimide-PEG-Maleimide) manufactured by Funakoshi Corporation, molecular weight: 10,000, was used.

[0228] (Example I-1) To a two-necked eggplant flask, 1 g (0.1 mmol) of fibroin (polypeptide, 10 kDa) having the amino acid sequence represented by SEQ ID NO: 9 (PRT2882), prepared according to the above method, and 12.5 mg (0.08 mmol) of dithiothreitol (DTT) as a reducing agent were added. 12.5 g of dimethyl sulfoxide (DMSO) was then added, and the mixture was heated and stirred at 70°C for 30 minutes to prepare a polypeptide-containing solution A. The amount of the reducing agent was adjusted to 0.8 equivalents relative to the amount of thiol groups contained in the polypeptide.

[0229] 1 g (0.1 mmol) of polyethylene glycol bismaleate prepared according to the above method was weighed and added to a flask. 7.5 g of dimethyl sulfoxide (DMSO, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was then added and heated and stirred at 70°C for 10 minutes to dissolve the mixture. This prepared solution B containing polyethylene glycol bismaleate.

[0230] Solution A and solution B were mixed, and 110 μL (0.08 mmol) of triethylamine (TEA, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution. In addition, in the mixed solution, the amount of the polypeptide was adjusted to 7 parts by mass relative to 100 parts by mass of dimethyl sulfoxide. Based on the total mass of the polypeptide, the amount of the base was adjusted to 8 parts by mass. Next, the reaction solution was heated and stirred at 70°C for 90 minutes to promote the reaction. After the reaction, the solution was centrifuged to remove the precipitate and obtain a solution of a polymer compound containing a polypeptide portion and a polyethylene glycol portion. In addition, centrifugation was completed using a micro high-speed refrigerated centrifuge (manufactured by TOMY Industries, Ltd., product name: MX-307) at a speed of 15,000 rpm and a treatment condition of 10 minutes.

[0231] <Evaluation of Polymer Compound Production Method: State of Precipitate in Solution after Reaction> The post-reaction solution was visually inspected before centrifugation to confirm the presence of precipitate. Furthermore, the supernatant, after removing the precipitate, was centrifuged at 15,000 rpm for 10 minutes to confirm the formation of a gel-like substance. The results were evaluated according to the following criteria. The results are shown in Table 4. Furthermore, the precipitate was primarily yellowish-white and contained byproducts resulting from disulfide bond formation between the peptides. Furthermore, the gel-like substance was a brown, transparent solid. A: No precipitate was observed, and no gel-like substance was generated. B: A precipitate was observed, but no gel-like substance was produced. C: No precipitate was observed, but a gel-like substance was produced. D: A precipitate was observed, and a gel-like substance was generated.

[0232] <Evaluation of Polymer Compound Production Processes: Degree of Conversion from Raw Materials to Target Products> After degassing the reaction solution prepared in Example I-1 by centrifugal separation, 3 g of the degassed reaction solution was dropped into a disposable tray (manufactured by As One Co., Ltd., product name: Balance Dish SCC, outer dimensions 80 mm × 80 mm × 24 mm) and spread out. Subsequently, it was pre-dried at 60°C for 8 hours, and then dried under reduced pressure at 80°C for 3 hours, and a film with a thickness of 0.09 mm was prepared by evaporating the solvent. 2 mg of the obtained film and 3.1 mg of tris(2-carbonylethyl)phosphine hydrochloride (TCEP) were dissolved in a TFANa-HFIP solution (1.3 mL) containing 1000 g of HFIP containing 85.3 mg of sodium trifluoroacetate (TFANa, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a measurement sample. The measurement sample obtained by the above method was subjected to gel permeation chromatography (GPC) measurement according to the conditions described below. In the resulting chromatogram, the peak intensity (peak height) of the starting peptide was recorded as P2, and the peak intensity (peak height) of the product polymer compound was recorded as P1. The ratio (P1 / P2) was calculated. A higher ratio indicates a more complete reaction. The results are shown in Table 4.

[0233] The measurement conditions of the GPC measurement are as follows. Apparatus: Agilent 1260 Infinity II liquid chromatography system (Agilent Technologies, Inc.) ·Detector: Agilent 1260Infinity II refractive index detector (RID) (Agilent Technologies, Inc.) Chromatographic columns: 0.5 μm guard column filter (Shodex GPC HK-G, Showa Denko K.K.), styrene-divinylbenzene copolymer column (Showdex GPC HK404L × 2, inner diameter: 4.6 mm × length: 150 mm, manufactured by Showa Denko K.K.) Eluent: TFANa-HFIP solution (85.3 mg of sodium trifluoroacetate (TFANa) dissolved in 1000 g of hexafluoroisopropanol (HFIP, Central Glass Co., Ltd.) Flow rate: 0.15 mL / min ·Measurement temperature: 40℃

[0234] (Example 1-2) Except for the preparation according to the ingredients, mixing amounts and reaction temperature shown in Table 4, the rest of the operations were the same as those in Example I-1, thereby obtaining a polymer compound in which a polypeptide portion and a polyethylene glycol portion were bonded.

[0235] (Example 1-3) To a two-necked eggplant flask, 1 g (0.1 mmol) of fibroin (polypeptide, 10 kDa) having the amino acid sequence represented by SEQ ID NO: 9 (PRT2882), prepared according to the above method, and 7.2 mg (0.08 mmol) of butanediol as a reducing agent were added. 9.0 g of dimethyl sulfoxide (DMSO) was then added, and the mixture was heated at 70°C with stirring for 30 minutes to prepare a polypeptide-containing solution C. The amount of the reducing agent was adjusted to 0.8 equivalents relative to the amount of thiol groups contained in the polypeptide.

[0236] 1 g (0.1 mmol) of polyethylene glycol bismaleate prepared according to the above method was weighed and placed in a flask. 3.0 g of dimethyl sulfoxide (DMSO, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was then added and heated and stirred at 70°C for 10 minutes to dissolve the mixture. This prepared a polyethylene glycol bismaleate-containing solution D.

[0237] After mixing solution C and solution D, 110 μL (0.08 mmol) of triethylamine (TEA, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution. In addition, in the mixed solution, the amount of the polypeptide was adjusted to 7 parts by mass relative to 100 parts by mass of dimethyl sulfoxide. Based on the total mass of the polypeptide, the amount of the base was adjusted to 8 parts by mass. Next, the reaction solution was heated and stirred at 70°C for 90 minutes to promote the reaction. After the reaction, the solution was centrifuged to remove the precipitate and obtain a solution of a polymer compound containing a polypeptide portion and a polyethylene glycol portion. In addition, centrifugation was completed using a micro high-speed refrigerated centrifuge (manufactured by TOMY Industries, Ltd., product name: MX-307) at a speed of 15,000 rpm and a treatment condition of 10 minutes.

[0238] (Example 1-4) Except for the preparation according to the ingredients, mixing amounts and reaction temperature shown in Table 4, the rest of the operations were the same as those in Example I-2, and finally a solution of a polymer compound containing a polypeptide portion and a polyethylene glycol portion was obtained.

[0239] (Example 1-5) 1 g (0.1 mmol) of polyethylene glycol diacrylate prepared according to the above method and 25 mg (0.23 mmol) of hydroquinone (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as a polymerization inhibitor were weighed and added to a flask. 9.0 g of dimethyl sulfoxide (DMSO, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was then added and heated with stirring at 70°C for 10 minutes to dissolve the mixture. Solution E containing polyethylene glycol diacrylate was prepared by the above method.

[0240] After mixing solution A prepared in the same manner as in Example I-1 and the above-mentioned solution E, 110 μL (0.08 mmol) of triethylamine (TEA, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution. In addition, in the mixed solution, the amount of the polypeptide was adjusted to 7 parts by mass relative to 100 parts by mass of dimethyl sulfoxide. Next, the reaction solution was heated and stirred at 70°C for 90 minutes to promote the reaction. After the reaction, the solution was centrifuged to remove the precipitate and obtain a solution of a polymer compound containing a polypeptide portion and a polyethylene glycol portion bonded thereto. In addition, the centrifugal separation was completed using a micro high-speed refrigerated centrifuge (manufactured by TOMY Industries, Ltd., product name: MX-307) at a rotation speed of 15,000 rpm and a treatment condition of 10 minutes.

[0241] (Examples I-6 to 7) Except for the preparation according to the ingredients, mixing amounts and reaction temperature shown in Table 4, the remaining operations were the same as Example I-5, thereby obtaining a solution of a polymer compound containing a polypeptide portion and a polyethylene glycol portion.

[0242] In Examples I-2 to 7, the obtained post-reaction solutions were subjected to the same evaluation as in Example I-1. The results are shown in Table 4.

[0243] In Table 4, the amount of dimethyl sulfoxide refers to the total amount in the mixed solution (for example, the sum of the amount of DMSO in solution A and the amount of DMSO in solution B).

[0244] (Comparative Example I-1) 1 g (0.1 mmol) of polyethylene glycol bismaleimide (Mal-PEG-Mal (Maleimide-PEG-Maleimide), molecular weight 10,000, manufactured by Funakoshi Corporation) was weighed and added to a flask. 12.5 g of dimethyl sulfoxide (DMSO, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was then added and heated and stirred at 70°C for 10 minutes to dissolve the mixture. This method prepared a polyethylene glycol bismaleimide-containing solution F.

[0245] After mixing solution A prepared in the same manner as in Example I-1 and the above-mentioned solution F, 110 μL (0.08 mmol) of triethylamine (TEA, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution. In addition, in the mixed solution, the amount of the polypeptide was adjusted to 7 parts by mass relative to 100 parts by mass of dimethyl sulfoxide. Next, the reaction solution was heated and stirred at 70°C for 90 minutes to promote the reaction. The post-reaction solution was centrifuged to obtain a precipitate, which was a polymer compound in which the polypeptide portion and the polyethylene glycol portion were combined. In addition, centrifugation was performed using a micro high-speed refrigerated centrifuge (manufactured by TOMY Industries, Ltd., product name: MX-307) at a rotation speed of 15,000 rpm and a processing condition of 10 minutes.

[0246] (Comparative Example I-2) To a two-necked eggplant flask, 1 g (0.1 mmol) of fibroin (polypeptide, 10 kDa) having the amino acid sequence set forth in SEQ ID NO: 9 (PRT2882), prepared according to the above method, and 12.5 mg (0.08 mmol) of dithiothreitol (DTT) as a reducing agent were added. 12.5 g of dimethyl sulfoxide (DMSO) was then added, and the mixture was heated at 70°C with stirring for 30 minutes to prepare a polypeptide-containing solution G. The amount of the reducing agent was adjusted to 0.8 equivalents relative to the amount of thiol groups contained in the polypeptide.

[0247] 1 g (0.1 mmol) of polyethylene glycol bismaleimide (Mal-PEG-Mal (Maleimide-PEG-Maleimide), molecular weight 10,000, manufactured by Funakoshi Corporation) was weighed and added to a flask. 25.0 g of dimethyl sulfoxide (DMSO, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was then added and heated and stirred at 70°C for 10 minutes to dissolve the mixture. Solution H containing polyethylene glycol bismaleimide was prepared by the above method.

[0248] After mixing the above-mentioned solution G and the above-mentioned solution H, 110 μL (0.08 mmol) of triethylamine (TEA, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution. In addition, in the mixed solution, the amount of the polypeptide was adjusted to 7 parts by mass relative to 100 parts by mass of dimethyl sulfoxide. Next, the reaction solution was heated and stirred at 70°C for 90 minutes to promote the reaction. The solution after the reaction was centrifuged to obtain a precipitate, which was a polymer compound in which the polypeptide portion and the polyethylene glycol portion were combined. In addition, the centrifugal separation was completed using a micro high-speed refrigerated centrifuge (manufactured by TOMY Industries, Ltd., product name: MX-307) at a speed of 15,000 rpm and a processing condition of 10 minutes.

[0249] In Comparative Examples I-1 and I-2, the resulting post-reaction solutions were evaluated in the same manner as in Example I-1. The results are shown in Table 5. In Table 5, the peak intensity ratio in the GPC analysis for Comparative Example 1 is shown as "-," indicating that the presence of precipitates and gel-like substances in the reaction system precluded measurement.

[0250] [Table 5]

[0251] (Example 1-8) The reaction solution prepared in Example I-3 was degassed by centrifugation, and 3 g of the degassed solution was dropped into a disposable tray (As One Co., Ltd., product name: Balance Dish SCC, outer dimensions 80 mm × 80 mm × 24 mm) and spread. The solution was then dried at 60°C for 8 hours, and then dried under reduced pressure at 80°C for 3 hours. The solvent was evaporated to form a film with a thickness of 0.09 mm.

[0252] <Thin Film Evaluation> The elongation at break, elastic modulus and toughness of the film obtained by the above method were measured using a tensile testing machine (manufactured by Shimadzu Corporation, product name: AG-X plus 50kN). The film was stored at 20°C and a relative humidity of 65% for more than 24 hours before use. After storage, the film was die-cut into dumbbell-shaped specimens using a die-cutting machine (manufactured by Tester Industry Co., Ltd., product name: SA-1008) so that the film length at the test site was 25 mm and the width was 10 mm. The specimen was installed in a tensile testing machine and a tensile test was performed under the conditions of a load cell of 50 kN, a clamp spacing of 25 mm and a tensile speed of 10 mm / min. The results are shown in Table 6.

[0253] (Example 1-9) A film was prepared using the same procedures as in Example 1-8, except that the polymer material was changed to the material prepared in Example 1-7. The resulting film was evaluated in the same manner as in Example 1-8. The results are shown in Table 6.

[0254] (Comparative Example 1-3) A film was prepared using the same procedures as in Example 1-8, except that the polymer material was changed to the material prepared in Comparative Example 1-2. The resulting film was evaluated in the same manner as in Example 1-8. The results are shown in Table 6.

[0255] [Table 6]

[0256] As shown in Table 6 above, it was confirmed that no precipitate was generated due to the suppression of side reactions such as the formation of disulfide bonds between polypeptides. Although the polymer-containing solutions prepared in Examples I-8 and 9 were used directly for film formation without purification, films with good physical properties were still obtained.

[0257] (Example I-10) 100 g of the solution prepared by the same method as in Example I-1 was slowly added dropwise to a 300 mL beaker containing 100 g of ethyl acetate while stirring. The precipitate generated in the beaker was crushed into fine particles using a homogenizer (manufactured by IKA, product name: T-18 Digital). Subsequently, the precipitate was collected by suction filtration. The filtered collection was put into 100 g of ethyl acetate and stirred for washing. After repeating the washing with ethyl acetate twice, the precipitate was collected by filtration and vacuum dried at 40°C for 2 hours to obtain a powder of a purified polymer compound.

[0258] 6 g of the obtained powder was added to a 20 mL glass vial (As One, product name: screw-mouth sample bottle) containing a stirrer, followed by the addition of 11.46 mL of formic acid (manufactured by Nichigoku Sei Co., Ltd., concentration: 98% by mass). Then, the mixture was stirred at 45°C for 2 hours to prepare a formic acid solution (spinning solution) with a concentration of 30% by mass. The viscosity of the obtained spinning solution at 45°C was 17300 mPa·s. In this process, the viscosity of the spinning solution was measured by filling the spinning solution into a glass tube equipped with a spherical probe (φ4.7 mm). The value was measured using an EMS viscometer (manufactured by Kyoto Electronics Co., Ltd., product name: EMS-1000S) at a measurement temperature of 45°C.

[0259] 10.0 mL of the above spinning solution was filled into a 50 mL disposable syringe (manufactured by Musashi Engineering Co., Ltd., product name: PSY-50E), and then a plunger (manufactured by Musashi Engineering Co., Ltd., product name: FLP-50E) was inserted. Then, a plastic needle (manufactured by Musashi Engineering Co., Ltd., standard size: 22, inner diameter: 0.47 mm) was assembled, and an adapter tube (manufactured by Musashi Engineering Co., Ltd., product name: AT-50E-H-1.0) was installed on the upper part of the syringe. In addition, a syringe heater was installed to maintain the spinning solution temperature at 45°C.

[0260] Subsequently, while flowing 0.2 MPa nitrogen, the syringe pump, kept at 45°C, was activated to extrude the spinning solution at a linear speed of 1.00 m / min. The extruded spinning solution passed through a drying line at 200°C and then passed over a take-up roll adjusted to a rotation speed of 10 m / min.

[0261] The fibers were then passed through two rollers, dried on a 275°C drying line, and finally wound on a winder at a speed of 15 m / min to produce fibers (filaments). The mass per meter of the wound fibers was measured, and the mass per 10,000 meters (fineness, unit: decitex, dtex) was calculated. Five of these filaments were bundled together as a sample and cross-sectionally analyzed using a polarizing microscope (Nikon Corporation, product name: ECLIPSE LV100ND). The equivalent circular diameters of the five filaments were determined, and the arithmetic average was used as the equivalent circular diameter of the filaments.

[0262] <Fiber Evaluation: Measurement of Elongation at Break and Breaking Strength> The elongation at break and the breaking strength of the sample bundled with the above five filaments were evaluated using a tensile testing machine (manufactured by Shimadzu Corporation, AG-X plus 50kN). The sample was stored in an environment of 20°C and a relative humidity of 65% for more than 24 hours before use. The above sample was installed in the testing machine and stretched until it broke under the conditions of a load cell of 50kN, a clamp spacing of 50mm, and a tensile speed of 50cm / min. The stress and length at break were measured three times, and the arithmetic mean was calculated. The results are shown in Table 7.

[0263] <Fiber Evaluation: Stress Relaxation Measurement> The stress relaxation of the sample formed by the bundle of the above 5 filaments was evaluated using a tensile testing machine (manufactured by Shimadzu Corporation, product name: AG-X plus50kN). The sample was stored in an environment of 20°C and a relative humidity of 65% for more than 24 hours before use. The above sample was installed in the testing machine, and a tensile test was carried out under the conditions of a load cell of 50kN, a clamp spacing of 50mm and a tensile speed of 50cm / min. At this time, the stress X of the 50mm sample was measured when it was stretched to 300%. Subsequently, it was maintained in this state for 30 seconds, and the stress Y after maintenance was measured. This measurement was repeated three times and calculated according to the formula (XY) / X×100, and then the arithmetic mean was taken as the stress relaxation. The results are shown in Table 7.

[0264] (Reference example) The same evaluation as in Example I-10 was performed on commercially available spandex fiber (manufactured by TORAY OPELONTEX CO., LTD., product name: LYCRA T-127C). The results are shown in Table 7.

[0265] [Table 7]

[0266] (Example II-1) To 3.13 g of a solution of a polymer compound containing a polypeptide moiety and a polyethylene glycol moiety bonded together, prepared by the same method as in Example I-1, was added 50 mg of a water-soluble thermoplastic resin (ALKOX E-75, manufactured by Meisei Chemical Industry Co., Ltd.), and the mixture was stirred at 70°C for 1 hour to prepare a DMSO solution (spinning dope). The resulting spinning dope had a viscosity of 4130 mPa·s at 25°C. The viscosity of the spinning dope was measured at 25°C using a digital viscometer (Brookfield DV2TLVTJ0, manufactured by AMETEK).

[0267] The above spinning solution was transferred to a 100 mL glass beaker. Subsequently, the spinning solution was slowly stirred with a stainless steel scraper to avoid bubbles, and slowly lifted up to use its viscosity to stretch the spinning solution into a fiber shape. The stretched fibrous spinning solution was placed on a release film and placed in a vacuum oven at 80°C for 15 hours to obtain a filament (a polymer compound formed into a fibrous shape).

[0268] Five of the above filaments were bundled into one sample and cross-sectionally analyzed using a polarizing microscope (manufactured by Nikon Corporation, product name: ECLIPSE LV100ND). The equivalent circular diameters of the five filaments were determined and the arithmetic average was taken as the equivalent circular diameter of the filament.

[0269] The obtained sample, which was formed by bundling five filaments, was subjected to the same fiber evaluation as in Example I-10. The results are shown in Table 8. In Table 8, "-" indicates that the measurement was not performed.

[0270] [Table 8]

[0271] (Comparative Example 1-4) An attempt was made to prepare fibers by changing the polymer material to the material prepared in Comparative Example I-2. However, the fibers could not be successfully prepared due to the low concentration of the solution.

[0272] (Examples III-1 to III-4) Except for the preparation according to the ingredients, mixing amounts and reaction temperature shown in Table 9, the rest of the operations were the same as those in Example I-1, thereby obtaining a solution of a polymer compound containing a polypeptide portion and a polyethylene glycol portion bonded together.

[0273] In Examples III-1 to III-4, the obtained post-reaction solutions were subjected to the same evaluation as in Example I-1. The results are shown in Table 9.

[0274] [Table 9]

[0275] (Examples III-5 to III-6) Except for the preparation according to the ingredients, mixing amounts and reaction temperature shown in Table 10, the remaining operations were the same as Example I-1, thereby obtaining a solution of a polymer compound containing a polypeptide portion and a polyethylene glycol portion.

[0276] In Examples III-5 and III-6, the obtained post-reaction solutions were evaluated in the same manner as in Example I-1. The results are shown in Table 10.

[0277] (Comparative Example III-1, Examples III-7 to III-8) Except for the preparation according to the ingredients, mixing amounts and reaction temperature shown in Table 10, the remaining operations were the same as Example I-1, thereby obtaining a solution of a polymer compound containing a polypeptide portion and a polyethylene glycol portion.

[0278] In Comparative Example III-1 and Examples III-7 to III-8, the obtained post-reaction solutions were evaluated in the same manner as in Example I-1. The results are shown in Table 10.

[0279] [Table 10]

[0280] (Examples III-9 to III-11) Except for the preparation according to the ingredients, mixing amounts and reaction temperature shown in Table 11, the remaining operations were the same as Example I-1, thereby obtaining a solution of a polymer compound containing a polypeptide portion and a polyethylene glycol portion.

[0281] In Examples III-9 to III-11, the obtained reaction solutions were evaluated in the same manner as in Example I-1. The results are shown in Table 11. For reference, Table 11 also lists the results of Examples I-1 and III-2.

[0282] [Table 11]

[0283] (Example I-11) <Manufacturing of Film-Form Adhesive> To a two-necked eggplant flask were added 2.5 g (0.25 mmol) of fibroin (polypeptide, 10 kDa) having the amino acid sequence represented by SEQ ID NO: 9 (PRT2882), prepared as described above, and 31.2 mg (0.2 mmol) of dithiothreitol (DTT) as a reducing agent. 25 g of dimethyl sulfoxide (DMSO) was then added, and the mixture was heated at 70°C with stirring for 30 minutes to prepare a polypeptide-containing solution I. The amount of the reducing agent was adjusted to 0.8 equivalents relative to the amount of thiol groups contained in the polypeptide.

[0284] 2.5 g (0.025 mmol) of polyethylene glycol bismaleate prepared according to the above method was weighed and added to a flask. 20 g of dimethyl sulfoxide (DMSO, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was then added and heated and stirred at 70°C for 10 minutes to dissolve the mixture. Solution J containing polyethylene glycol bismaleate was prepared by the above method.

[0285] After mixing solution I and solution J, 275 μL (0.2 mmol) of triethylamine (TEA, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution. In addition, in the mixed solution, the amount of the polypeptide was adjusted to 6 parts by mass relative to 100 parts by mass of dimethyl sulfoxide. Based on the total mass of the polypeptide, the amount of the base was adjusted to 8 parts by mass. Next, the reaction solution was heated and stirred at 70°C for 90 minutes to promote the reaction. The post-reaction solution was degassed by centrifugation to obtain a DMSO solution containing a polymer compound containing a polypeptide portion and a polyethylene glycol portion. In addition, centrifugation was completed using a micro high-speed refrigerated centrifuge (manufactured by TOMY Industries, Ltd., product name: MX-307) at a speed of 15,000 rpm and a treatment condition of 10 minutes.

[0286] The resulting DMSO solution of the polymer compound was then dripped onto a stainless steel plate coated with a PET film (Teijin Film Solutions Co., Ltd., PUREX, Model A54, 38 μm thick). The film was then spread using a frame coater (Imoto Seisakusho, Ltd., coating width 250 mm, gap 1 mm) equipped with an analog micrometer and a coater (Imoto Seisakusho, Ltd., IMC-7370, coating speed 10 mm / sec). The film was then dried at 60°C for 8 hours and further dried under reduced pressure at 80°C for 3 hours. The DMSO evaporated to form a 0.08 mm thick adhesive film.

[0287] <Manufacturing of Adhesive Body> After the film-like adhesive that aforesaid method is obtained is cut into the size of 15mm × 15mm, the film-like adhesive cut is soaked in water for 5 minutes, it is swollen and softened. Subsequently, the film-like adhesive after the softening is placed on the zone of one end 15mm × 15mm of plank (cypress is made, 1.5mm × 40mm × 2mm), another plank of the same size is stacked on the film-like adhesive, the film-like adhesive is placed between two planks. Subsequently, with clamps, two planks are fixed, the film-like adhesive is crimped with each plank, then put into a blast constant temperature drying oven, dried 1 hour at 60 ℃, the film-like adhesive is hardened. Like this, obtain the adherend A that two planks are bonded together by the film-like adhesive. In addition, as a comparison, except replacing the film-like adhesive with commercially available double-sided tape (michael state (Nichiban), width 15mm, NiceTack NW-15), all the other operations are identical with above-mentioned, obtain the adherend B that two pieces of wood are bonded together by the double-sided tape.

[0288] <Tensile Test of Adhesive Body> Adhesives A and B obtained by the above method were each mounted on a tensile testing machine (AG-X plus 50kN, manufactured by Shimadzu Corporation) at room temperature of 20°C and a humidity of 65%, and subjected to a tensile test. The tensile test was conducted under the conditions of a 50kN load cell, a 57.5mm clamp spacing, and a tensile speed of 10mm / min. The results showed that the tensile strength of adhesive A was more than 10 times that of adhesive B. This demonstrates that film-like adhesives possess sufficiently greater adhesive force (bonding strength) than commercially available double-sided tapes and the like.

[0289] <Peel test of adhesive body> Another adhesive body A was prepared according to the above method. The excess film adhesive was pulled out from the gap in the adhesive portion of this body A. In this way, the two wooden boards bonded together could be peeled apart. Furthermore, visual inspection of the surfaces of the bonded portions of the two wooden boards revealed no changes compared to the surfaces before bonding. Furthermore, after washing the pulled-out film adhesive with water, this film adhesive was used to bond two more wooden boards using the same procedures as for making body A. The results showed that the other two wooden boards were also successfully bonded together. This demonstrates that the film adhesive can be reused.

[0290] (Example I-12) <Manufacturing of Powdered Adhesive> 319.3 g of dimethyl sulfoxide (DMSO) was weighed and added to a three-necked round-bottom flask. 419 mg (2.72 mmol) of dithiothreitol (DTT) as a reducing agent was then added and stirred to dissolve. Subsequently, 27.5 g (2.75 mmol) of artificial silk fibroin (polypeptide, 10 kDa) having the amino acid sequence represented by SEQ ID NO: 9 (PRT2882), prepared as described above, was added. The mixture was heated and stirred at 70°C for 30 minutes to prepare a polypeptide-containing solution K. The amount of reducing agent was adjusted to approximately 1 equivalent relative to the amount of thiol groups in the polypeptide.

[0291] 27.5 g (2.75 mmol) of polyethylene glycol bismaleate prepared according to the above method was weighed and added to a flask. 125.5 g of dimethyl sulfoxide (DMSO, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was then added and heated and stirred at 70°C for 10 minutes to dissolve the mixture. Solution L containing polyethylene glycol bismaleate was prepared by the above method.

[0292] After mixing solution K and solution L, 4400 mg (43.5 mmol) of triethylamine (TEA, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution. In addition, in the mixed solution, the amount of the polypeptide was adjusted to 5.5 parts by mass relative to 100 parts by mass of dimethyl sulfoxide. Based on the total mass of the polypeptide, the amount of the base was adjusted to 16 parts by mass. Next, the reaction solution was heated and stirred at 70°C for 90 minutes to promote the reaction. The post-reaction solution was centrifuged to remove the precipitate to obtain a DMSO solution of a polymer compound composed of a polypeptide portion and a polyethylene glycol portion. In addition, the centrifugal separation treatment condition was 10 minutes at a speed of 15,000 rpm.

[0293] The resulting DMSO solution of the polymer compound was washed five times in acetone and centrifuged. The supernatant was removed by decantation to separate the precipitate. The resulting precipitate was spread on a tray and naturally dried in a fume hood for several hours. It was then dried overnight in a vacuum oven at 40°C to obtain a polymer powder. The residual DMSO content in this powder was less than 3% by mass. The resulting polymer powder was used as a powdered adhesive.

[0294] <Manufacturing of Laminate> 0.5g of the powdered adhesive was weighed and applied to a portion of one side of a cotton fabric substrate. The substrate with the powdered adhesive was then sandwiched between two stainless steel plates coated with a release agent. The substrate was heated to 100°C and pressed at 10 MPa for 3 minutes using a manual press. This allowed the powdered adhesive to resinify and solidify. This produced a laminate with a coating layered over a portion of the substrate surface. [Industrial Applicability]

[0295] According to the present disclosure, a production method can be provided that can improve the raw material conversion rate in the production of the polymer compound as described above compared to conventional methods.

Claims

1. A method for producing a polymer compound, wherein: The process includes the following steps: A polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyether, polyester and polycarbonate having two structures represented by the following general formula (1) are reacted by heating in dimethyl sulfoxide in the presence of a base and a reducing agent. [Chemical Formula 1] In the general formula (1), M represents any one of H, Na, K, NHEt3, or NHEtiPr2.

2. A method for producing a polymer compound, wherein: The process includes the following steps: A polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates having two structures represented by the following general formula (2) are reacted by heating in dimethyl sulfoxide in the presence of a base, a reducing agent, and a polymerization inhibitor. [Chemical Formula 2] In the general formula (2), R 1 represents a hydrogen atom or a methyl group.

3. A method for producing a polymer compound, wherein: The process includes the following steps: A polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyether, polyester and polycarbonate having two structures represented by the following general formula (1) are reacted by heating in dimethyl sulfoxide in the presence of a reducing agent. [Chemical Formula 3] In the general formula (1), M represents any one of H, Na, K, NHEt3, or NHEtiPr2.

4. A method for producing a polymer compound, wherein: The process includes the following steps: A polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyether, polyester and polycarbonate having two structures represented by the following general formula (2) are reacted by heating in dimethyl sulfoxide in the presence of a reducing agent and a polymerization inhibitor. [Chemical Formula 4] In the general formula (2), R 1 represents a hydrogen atom or a methyl group.

5. The method for producing a polymer compound according to any one of claims 1 to 4, wherein The reducing agent is at least one selected from the group consisting of dithiol, sodium sulfate, sodium sulfite, and sodium dithionite.

6. The manufacturing method according to any one of claims 1 to 4, wherein: The reducing agent is added in an amount of 0.5 equivalents or more relative to the thiol groups of the polypeptide.

7. The manufacturing method according to any one of claims 1 to 4, wherein: The reaction between the polypeptide and the compound is carried out at a temperature above 50°C.

8. A method for producing a polymer compound solution, wherein: The method comprises the step of dissolving the polymer compound obtained by the production method according to any one of claims 1 to 4 in a solvent.

9. A method for producing a thin film, wherein: The method comprises the step of forming the polymer compound solution obtained by the production method according to claim 8 into a film.

10. A method for producing a fiber, wherein: The method comprises the step of spinning the polymer compound solution obtained by the production method according to claim 8.

11. A method for producing a thin film, wherein: The process includes the following steps: A polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates having two structures represented by the following general formula (1) are reacted by heating in dimethyl sulfoxide in the presence of a base and a reducing agent to obtain a polymer compound solution; forming the polymer compound solution into a film, [Chemical Formula 5] In the general formula (1), M represents any one of H, Na, K, NHEt3, or NHEtiPr2.

12. A method for producing a thin film, wherein: The process includes the following steps: A polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyether, polyester and polycarbonate having two structures represented by the following general formula (2) are reacted by heating in dimethyl sulfoxide in the presence of a base, a reducing agent and a polymerization inhibitor to obtain a polymer compound solution; forming the polymer compound solution into a film, [Chemical Formula 6] In the general formula (2), R 1 represents a hydrogen atom or a methyl group.

13. A method for producing a fiber, wherein: The process includes the following steps: A polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates having two structures represented by the following general formula (1) are reacted by heating in dimethyl sulfoxide in the presence of a base and a reducing agent to obtain a polymer compound solution; Spinning the polymer compound solution, [Chemical Formula 7] In the general formula (1), M represents any one of H, Na, K, NHEt3, or NHEtiPr2.

14. A method for producing a fiber, wherein: The process includes the following steps: A polypeptide containing at least one thiol group and at least one compound selected from the group consisting of polyether, polyester, and polycarbonate having two structures represented by the following general formula (2) are reacted by heating in dimethyl sulfoxide in the presence of a base, a reducing agent, and a polymerization inhibitor to obtain a polymer compound solution; Spinning the polymer compound solution, [Chemical Formula 8] In the general formula (2), R 1 represents a hydrogen atom or a methyl group.

15. A film containing a polymer compound, wherein: The polymer compound has the following structure: Peptide part, and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure, directly bonded via the structure represented by the following general formula (3), the following general formula (4), or the following general formula (5), so that the polypeptide portion is bonded to S of the following general formula (3), the following general formula (4), or the following general formula (5); [Chemical Formula 9] [Chemical Formula 10] [Chemical Formula 11] In the general formula (3) and the general formula (4), M represents any one of H, Na, K, NHEt3, or NHEtiPr2, and in the general formula (5), R 1 Indicates H, or Me; Its elongation is over 400%; The ratio of fracture strength to yield strength is greater than 1.

16. A fiber containing a polymer compound, wherein: The polymer compound has the following structure: Peptide part, and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure, directly bonded via the structure represented by the following formula (3), the following general formula (4), or the following general formula (5), so that the polypeptide portion is bonded to S of the following general formula (3), the following general formula (4), or the following general formula (5); [Chemical Formula 12] [Chemical Formula 13] [Chemical Formula 14] In the general formula (3) and the general formula (4), M represents any one of H, Na, K, NHEt3, or NHEtiPr2, and in the general formula (5), R 1 Indicates H, or Me; Its elongation is over 400%; The ratio of fracture strength to yield strength is greater than 1. 17 . A method for producing a solution-state adhesive, comprising dissolving the polymer compound obtained by the method according to claim 1 in a solvent.

18. A method for producing a water-dispersible adhesive, comprising dispersing the polymer compound obtained by the method according to any one of claims 1 to 4 in an aqueous medium.

19. A method for producing a film-like adhesive, comprising the step of forming a film from a solution in which a polymer compound obtained by the method according to any one of claims 1 to 4 is dissolved.

20. A method for producing a powdered adhesive, comprising the step of obtaining a powder composition containing a polymer compound obtained by the method according to any one of claims 1 to 4.

21. A method for producing an adhered body, characterized in that: The polymer compound obtained by the method according to any one of claims 1 to 4 is dissolved in a solvent to form a solution, and the solution is placed between a plurality of adherends. The solvent is then removed from the solution to solidify the polymer compound, thereby adhering the adherends.

22. A method for producing an adhered body, characterized in that: The polymer compound obtained by the method according to any one of claims 1 to 4 is dispersed in an aqueous medium to form an aqueous dispersion, and the aqueous dispersion is placed between a plurality of adherends. The aqueous medium is then removed from the aqueous dispersion to solidify the polymer compound, thereby adhering the adherends.

23. A method for producing an adhesive body, characterized in that: A film containing a polymer compound obtained by the method according to any one of claims 1 to 4 is softened by swelling or heating, and after the film is placed between a plurality of adherends, the film is hardened while being pressed against the adherends, thereby bonding the adherends.

24. A method for producing an adhered body, characterized in that: A powder composition containing a polymer compound obtained by the method according to any one of claims 1 to 4 is placed between a plurality of adherends, and while heating the powder composition, the powder composition is pressurized through the adherends to solidify the powder composition, thereby bonding the adherends.

25. A method for producing a solution coating, comprising the step of dissolving the polymer compound obtained by the method according to any one of claims 1 to 4 in a solvent.

26. A method for producing a water-dispersible coating, comprising the step of dispersing the polymer compound obtained by the method according to any one of claims 1 to 4 in an aqueous medium.

27. A method for producing a film-like coating material, comprising the step of forming a film from a solution obtained by dissolving a polymer compound obtained by the method according to any one of claims 1 to 4.

28. A method for producing a powdered coating, comprising the step of obtaining a powder composition containing a polymer compound obtained by the method according to any one of claims 1 to 4.

29. A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on a surface of the substrate, wherein: A solution obtained by dissolving a polymer compound obtained by the method according to any one of claims 1 to 4 in a solvent is supplied to at least a portion of the surface of the substrate, and after coating at least a portion of the surface of the substrate with the solution, the solvent is removed from the solution and the polymer compound is solidified, thereby forming a coating layer on at least a portion of the surface of the substrate.

30. A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on a surface of the substrate, wherein: An aqueous dispersion obtained by dispersing a polymer compound obtained by the method according to any one of claims 1 to 4 in an aqueous medium is supplied to at least a portion of the surface of the substrate; after coating at least a portion of the surface of the substrate with the aqueous dispersion, the aqueous medium is removed from the aqueous dispersion, and the polymer compound is cured, thereby forming a coating layer on at least a portion of the surface of the substrate.

31. A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on a surface of the substrate, wherein: A film containing a polymer compound obtained by the method described in any one of claims 1 to 4 is softened by swelling or heating, and after being placed on at least a portion of the surface of the substrate, the film is hardened while being pressed against the substrate, thereby forming the coating layer on at least a portion of the surface of the substrate.

32. A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on a surface of the substrate, wherein: In a state where a powder composition containing a polymer compound obtained by the method according to any one of claims 1 to 4 is placed on at least a portion of the surface of the substrate, the powder composition is heated and simultaneously pressurized between a pressurizing body and the substrate to solidify the powder composition, thereby forming the coating layer on at least a portion of the surface of the substrate.

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