Method for producing polyamino acids, method for producing polyamino acid derivatives, and method for producing fibers

Abstract

To provide a method for producing a polyamino acid having a dehydroalanine-like skeleton that can maintain the structure of the polyamino acid and has excellent atomic efficiency in the reaction; a method for producing a polyamino acid derivative using a polyamino acid having a dehydroalanine-like skeleton obtained by the above production method; and a method for producing fibers, including forming the polyamino acid derivative into fibers. [Solution] A method for producing a polyamino acid having a dehydroalanine-like skeleton, comprising reacting compound A, which is a compound having a peptide bond and at least one substituent selected from the group consisting of a hydroxyl group and a thiol group, with an inorganic carbonate, wherein at least one of the substituents is directly removed by the inorganic carbonate in the reaction; a method for producing a polyamino acid derivative using the polyamino acid having a dehydroalanine-like skeleton obtained by the above production method; and a method for producing a fiber comprising forming the polyamino acid derivative into a fiber.

Description

Technical Field

[0001] The present disclosure relates to methods for producing polyamino acids, methods for producing polyamino acid derivatives, and methods for producing fibers.

Background Art

[0002] Conventionally, introducing a dehydroalanine-like structure into polyamino acids (polypeptides, proteins, etc.) has been studied.

[0003] For example, Patent Document 1 describes crosslinking a protein using a polyfunctional reactant having two or more first reactive groups capable of reacting with the protein to form a bond, and the first reactive group being an electrophilic group.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Polyamino acids such as proteins and polypeptides have hydroxy groups, thiol groups, amino groups, etc. as reactive functional groups. Also, in the structural conversion of polyamino acids, many examples of reacting these groups with electrophiles have been reported (for example, Patent Document 1 mentioned above). However, there is a problem that candidates for reactants that can be used as such electrophiles are limited, and the degree of freedom in designing the structure of polyamino acids is low. Therefore, the inventors considered introducing a dehydroalanine-like structure, described later, into a polyamino acid and using it as a reactive functional group. The C=C double bond to which the carbonyl group is attached in the dehydroalanine-like structure readily reacts with nucleophiles. As a result, it is possible to use nucleophiles of various structures, and it is expected that polyamino acids with structures that were difficult to synthesize through reactions with conventional electrophiles can be obtained. Ultimately, we believe that it will be possible to investigate and put into practical use changes to the physical properties or functionality of polyamino acids in a variety of structures. Traditionally, strong bases have been used to introduce dehydroalanine-like structures into polyamino acids. However, it has been found that using strong bases can lead to problems such as cleavage of the polyamino acid's main chain and the mixing of racemic mixtures, thus affecting the three-dimensional structure. Furthermore, there have been reports of reaction systems that activate hydroxyl and thiol groups once to promote elimination in order to generate a dehydroalanine-like structure, but there was room for improvement in terms of atomic efficiency. Therefore, there was a need for a reaction system that could maintain the structure of polyamino acids in the reaction system under mild reaction conditions. Specifically, a reaction system in which the main chain of the polyamino acid is less likely to be cleaved, the three-dimensional structure of the polyamino acid is less affected, and the reaction has excellent atomic efficiency.

[0006] The present invention aims to provide a method for producing a polyamino acid having a dehydroalanine-like skeleton that can maintain the structure of the polyamino acid and has excellent atomic efficiency in the reaction, a method for producing a polyamino acid derivative using the polyamino acid having a dehydroalanine-like skeleton obtained by the above method, and a method for producing fibers, which includes forming the polyamino acid derivative into fibers. [Means for solving the problem]

[0007] Examples of typical embodiments of the present invention are shown below. <1> The process involves reacting compound A, which is a compound having a peptide bond and at least one substituent selected from the group consisting of a hydroxyl group and a thiol group, with an inorganic carbonate. In the above reaction, at least one of the substituents is directly removed by the inorganic carbonate. A method for producing polyamino acids having a dehydroalanine-like skeleton. <2> The process involves reacting compound A, which is a compound having a peptide bond and at least one substituent selected from the group consisting of a hydroxyl group and a thiol group, with an inorganic carbonate. In the above reaction, the mass of the inorganic carbonate added to the reaction system is 60 to 100% by mass of the total mass of compound A and other compounds added to the reaction system. A method for producing polyamino acids having a dehydroalanine-like skeleton. <3> The process involves reacting compound A, which is a compound having a peptide bond and at least one substituent selected from the group consisting of a hydroxyl group and a thiol group, with an inorganic carbonate. In the above reaction, the mass of the derivative-forming compound added to the reaction system is 0 to 40% by mass relative to the mass of compound A added to the reaction system. A method for producing polyamino acids having a dehydroalanine-like skeleton. <4> The above reaction is carried out by a mechanochemical method. <1> ~ <3> A method for producing a polyamino acid having a dehydroalanine-like skeleton as described in any one of the following. <5> The inorganic carbonate comprises at least one compound selected from the group consisting of cesium carbonate and potassium carbonate. <1> ~ <4> A method for producing a polyamino acid having a dehydroalanine-like skeleton as described in any one of the following. <6> The aforementioned compound A is a structural protein. <1> ~ <5> A method for producing a polyamino acid having a dehydroalanine-like skeleton as described in any one of the following. <7> The aforementioned compound A is a hydrophobic structural protein. <6> A method for producing polyamino acids having a dehydroalanine-like skeleton as described above. <8> The average hydroxyl index of the hydrophobic structural protein is greater than 0. <7> A method for producing polyamino acids having a dehydroalanine-like skeleton as described above. <9> The above reaction is carried out in an atmosphere containing air. <1> ~ <8> A method for producing a polyamino acid having a dehydroalanine-like skeleton as described in any one of the following. <10> <1> ~ <9> The method for producing a polyamino acid having a dehydroalanine-like skeleton as described in any one of the following: This includes reacting the compound A after the above reaction with a nucleophile. A method for producing polyamino acid derivatives. <11> <10> The method for producing polyamino acid derivatives described above includes, This includes forming the compound A, after reaction with the nucleophile, into fibers. A method for manufacturing fibers. [Effects of the Invention]

[0008] The present invention provides a method for producing a polyamino acid having a dehydroalanine-like skeleton that can maintain the structure of the polyamino acid and has excellent atomic efficiency in the reaction, a method for producing a polyamino acid derivative using a polyamino acid having a dehydroalanine-like skeleton obtained by the above production method, and a method for producing fibers, which includes forming the polyamino acid derivative into fibers. [Brief explanation of the drawing]

[0009] [Figure 1] This is a schematic diagram showing an example of a domain sequence of an artificial protein. [Figure 2] The 19F NMR spectra of 4-F-BnNH2, the synthesized compound, and a mixture thereof in Example 6 are shown. [Figure 3] This is the 19F NMR spectrum of the synthesized compound in Example 6 in the presence of fluorobenzene as an internal standard. [Modes for carrying out the invention]

[0010] The main embodiments of the present invention will be described below. However, the present invention is not limited to the embodiments specified. In this specification, a numerical range represented by the symbol "~" means a range including the numerals described before and after "~" as the lower limit value and the upper limit value, respectively. In this specification, the term "step" means not only an independent step but also a step that cannot be clearly distinguished from other steps as long as the intended action of the step can be achieved. In this specification, unless otherwise specified, the temperature is 23°C, the atmospheric pressure is 101,325 Pa (1 atm), and the humidity (relative humidity) is 50% RH. In this specification, Me represents a methyl group, Et represents an ethyl group, Ph represents a phenyl group, and Boc represents a t-butoxycarbonyl group, respectively. In this specification, a combination of preferred embodiments is a more preferred embodiment.

[0011] (Method for producing a polyamino acid having a dehydroalanine-like skeleton) The method for producing a polyamino acid having a dehydroalanine-like skeleton according to the first aspect of the present embodiment (hereinafter, also referred to as "the first method for producing a polyamino acid") includes reacting compound A, which is a compound having at least one substituent selected from the group consisting of a peptide bond and a hydroxy group and a thiol group, with an inorganic carbonate, and in the reaction, at least one of the substituents is directly eliminated by the inorganic carbonate. The method for producing a polyamino acid having a dehydroalanine-like skeleton according to the second aspect of the present embodiment (hereinafter, also referred to as "the second method for producing a polyamino acid") includes reacting compound A, which is a compound having at least one substituent selected from the group consisting of a peptide bond and a hydroxy group and a thiol group, with an inorganic carbonate, and in the reaction, the mass of the inorganic carbonate added to the reaction system is 60 to 100% by mass with respect to the total mass of compound A and compounds other than the solvent added to the reaction system. A third embodiment of this invention provides a method for producing a polyamino acid having a dehydroalanine-like skeleton (hereinafter also referred to as the "third method for producing a polyamino acid"), which comprises reacting compound A, a compound having a peptide bond and at least one substituent selected from the group consisting of a hydroxyl group and a thiol group, with an inorganic carbonate, wherein the mass of the derivative-forming compound added to the reaction system is 0 to 40% by mass relative to the mass of compound A added to the reaction system. Hereinafter, the first method for producing polyamino acids, the second method for producing polyamino acids, and the third method for producing polyamino acids will also be referred to collectively as "the method for producing polyamino acids of this embodiment."

[0012] The first method for producing polyamino acids involves using an inorganic carbonate as a base, and wherein a hydroxyl group or a thiol group is directly removed by the inorganic carbonate. In this configuration, the reaction proceeds under mild conditions using inorganic carbonates, thereby suppressing the stereochemical changes of the polyamino acids. Furthermore, the direct elimination of the substituents results in a highly atom-efficient production method. The second method for producing polyamino acids uses an inorganic carbonate as a base, and the amount of the inorganic carbonate added to the reaction system is 60 to 100% by mass relative to the total amount of compound A and other compounds added to the reaction system. In this configuration, the reaction with inorganic carbonate proceeds under mild conditions, thereby suppressing the stereochemical changes of the polyamino acids. Furthermore, there is little or no possibility of the presence of derivative-forming compounds to form intermediates, making it a highly atomically efficient manufacturing method. The third method for producing polyamino acids uses an inorganic carbonate as the base, and the content of the derivative-forming compound added to the reaction system is 0 to 40% by mass relative to the mass of compound A added to the reaction system. In this configuration, the reaction with inorganic carbonate proceeds under mild conditions, thereby suppressing the stereochemical changes of the polyamino acids. Furthermore, because the content of the derivative-forming compound required to form the intermediate is sufficiently small or not used at all, this method can be said to be highly atomically efficient in its reaction. Furthermore, in these embodiments, since the reaction proceeds under mild conditions, it is thought that the degradation of the polyamino acids themselves is also suppressed.

[0013] The following describes in detail the forms for implementing this disclosure. However, the following embodiments are illustrative examples for illustrating this disclosure and are not intended to limit this disclosure to the following.

[0014] <Dehydroalanine-like structure> In this embodiment, the dehydroalanine-like structure refers to the structure represented by the following formula (dha-1). [ka] In formula (dha-1), each wavy line independently represents a binding site with another structure, for example, a binding site with another amino acid residue. Alternatively, the wavy lines may represent binding sites with the ends of a polyamino acid or with residues of non-amino acid compounds introduced into the polyamino acid.

[0015] <Compound A> In the method for producing polyamino acids of this embodiment, compound A is used. Compound A is a compound having a peptide bond and at least one substituent selected from the group consisting of a hydroxyl group and a thiol group.

[0016] Compound A is a compound that belongs to the polyamino acid group. In this disclosure, polyamino acids refer to compounds in which two or more amino acids are linked by peptide bonds, and polyamino acids include polypeptides, proteins, and the like. In particular, from the viewpoint of the usefulness of the resulting polyamino acid having a dehydroalanine-like skeleton, it is preferable that the polyamino acid is a protein. Polypeptides are, for example, molecules in which 2 to 50 amino acids are linked together by peptide bonds, while proteins are, for example, molecules in which 50 or more amino acids are linked together by peptide bonds. Furthermore, compound A does not have to be formed solely from amino acids; it may also contain other resin blocks, residues of compounds, etc.

[0017] Compound A preferably has a hydroxyl group or a thiol group at the β-position of the carbonyl group in the peptide bond. Furthermore, compound A preferably contains a serine residue or a cysteine ​​residue as the amino acid residue having a hydroxyl group or a thiol group. The content of serine and cysteine ​​residues in compound A (the ratio of serine and cysteine ​​residues to the total number of amino acid residues) may be, for example, 1% or more, 1.5% or more, 2% or more, 2.5% or more, 3% or more, 3.5% or more, or 4% or more. The upper limit of the total content is not particularly limited, but considering the amino acid composition of various proteins that can be suitably used as compound A, it may be, for example, 50% or less, 35% or less, 30% or less, 25% or less, or 20% or less.

[0018] The amino acid residues contained in compound A may be natural amino acids or non-natural amino acids (derivatives of natural amino acids or artificial amino acids).

[0019] The following describes preferred embodiments, particularly when compound A is a protein.

[0020] 〔protein〕 The protein may be a natural protein or an artificial protein. It may also be a modified protein obtained by chemically modifying such natural or artificial proteins. The amino acid sequences of these natural, artificial, and modified proteins are not particularly limited. In this specification, the term "protein" encompasses natural proteins, artificial proteins, and modified proteins. Examples of proteins according to this embodiment include proteins usable for industrial purposes and proteins usable for medical purposes. "Usable for industrial purposes" means, for example, that it can be used in various general-purpose materials used indoors and outdoors. A specific example of an industrially usable protein is a structural protein. As a structural protein, for example, a protein having physical properties close to those required for a desired application can be used. Specific examples of structural proteins include spider silk, silkworm silk, keratin, collagen, elastin, and resilience, as well as proteins derived therefrom. The protein used may be artificial fibroin or artificial spider silk fibroin (artificially modified spider silk fibroin). Specific examples of proteins that can be used for medical purposes include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens used in vaccination, vaccines, antigen-binding proteins, immunostimulatory proteins, allergens, full-length antibodies or antibody fragments or derivatives.

[0021] [Artificial protein] In this specification, "artificial protein" means a protein that is artificially produced, and includes recombinant proteins and synthetic proteins. An artificial protein may have a domain sequence that differs from the amino acid sequence of a naturally occurring protein. Furthermore, an "artificial protein" may be one whose amino acid sequence is modified based on the amino acid sequence of a naturally occurring protein (for example, by modifying the gene sequence of a cloned naturally occurring protein), or it may be one that is artificially designed and synthesized without relying on a naturally occurring protein (for example, one that has a desired amino acid sequence by chemically synthesizing nucleic acid encoding a designed amino acid sequence). Unlike natural proteins, artificial proteins allow for the free design of their amino acid sequences. Therefore, when such artificial proteins are used in molding materials or molded products, the function, characteristics, and physical properties of the molding material or molded product can be arbitrarily controlled by appropriately designing the amino acid sequence of the artificial protein. In addition, because uniform molecular design is always possible, it is possible to stably obtain proteins with high homology to the target protein that are suitable for the purpose. Thus, the quality of molding materials or molded products obtained using artificial proteins can be advantageously stabilized. From this perspective, artificial structural proteins are advantageously used as artificial proteins.

[0022] "Structural proteins" are a type of protein that can be used industrially, and refer to proteins involved in the structure of living organisms, proteins that constitute structures produced by living organisms, or proteins derived from them. Structural proteins are also proteins that self-aggregate under certain conditions to form structures such as fibers, films, resins, gels, micelles, and nanoparticles. Furthermore, structural proteins can be said to be proteins that form the skeleton of living organisms or materials, as they have repeating motifs consisting of characteristic amino acid sequences or a number of amino acid residues. Specific examples of structural proteins include fibroins such as spider silk, silkworm silk, and hornet silk, as well as keratin, collagen, resilience, and proteins derived from these.

[0023] The number of amino acid residues in the artificial protein according to this embodiment is not particularly limited, but may be, for example, 5 or more. Alternatively, the number of amino acid residues may be, for example, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 400 or more, or 500 or more. The number of amino acid residues may also be, for example, 5000 or less, 2500 or less, 1000 or less, 750 or less, 500 or less, 250 or less, 1000 or less, 500 or less, 100 or less, or 50 or less. The fewer the number of amino acid residues, the higher the solubility in the solvent tends to be. Preferred numbers of amino acid residues for a protein are, for example, 5-5000, 10-4500, 15-4000, 20-3500, 20-3000, 20-2500, 30-2000, 50-1500, or 100-1000.

[0024] The molecular weight of the artificial protein according to this embodiment is not particularly limited, but may be, for example, 1 kDa to 500 kDa. Furthermore, the molecular weight may be, for example, 1 kDa or more, 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 may also be 500 kDa or less, 400 kDa or less, less than 360 kDa, 300 kDa or less, or 200 kDa or less. The molecular weight may be, for example, 1kDa to 500kDa, 2kDa to 500kDa, 3kDa to 500kDa, 4kDa to 500kDa, 5kDa to 500kDa, 6kDa to 500kDa, 7kDa to 500kDa, 8kDa to 500kDa, 9kDa to 500kDa, 2kDa to 400kDa, 2kDa to 200kDa, 2kDa to 100kDa, 2kDa to 50kDa, 2kDa to 30kDa, 3kDa to 300kDa, 3kDa to 200kDa, 3kDa to 100kDa, or 10kDa to 300kDa, 10kDa to 200kDa. The lower the molecular weight of an artificial protein, the higher its solubility in the solvent tends to be. Preferred amino acid residue ranges for artificial proteins are, for example, 1kDa to 50kDa and 2kDa to 10kDa.

[0025] The artificial protein according to this embodiment may have a glycine residue content of 10-55% based on the number of amino acid residues. The glycine residue content may be, for example, 13-55%, 15-55%, 18-55%, 20-55%, 22-55%, or 25-55%. In this specification, "glycine residue content" is the value expressed by the following formula. Glycine residue content = (Number of glycine residues in the artificial protein / Total number of amino acid residues in the polypeptide) × 100 (%)

[0026] The artificial protein may have a total content of 40% or more, which is the sum of the content of at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine (i.e., any of the content of serine residues, threonine residues, tyrosine residues, the sum of serine and threonine residues, the sum of serine and tyrosine residues, the sum of threonine and tyrosine residues, or the sum of serine, threonine and tyrosine residues), alanine residues, and glycine residues. This total content may be, for example, 45% or more, 50% or more, 55% or more, or 60% or more. There is no particular upper limit to this total content, but for example, it may be 90% or less, 85% or less, or 80% or less.

[0027] In this embodiment, the artificial protein may have a total content of serine residues, threonine residues, and tyrosine residues of 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 15% or more, or 16% or more, based on the number of amino acid residues. The total content of serine residues, threonine residues, and tyrosine residues may also be, for example, 35% or less, 33% or less, 30% or less, 25% or less, or 20% or less.

[0028] The artificial protein according to this embodiment has an average distribution of serine residues, threonine residues, or tyrosine residues, and the total content of serine residues, threonine residues, and tyrosine residues in any 20 consecutive amino acid residues may be 4% or more, 5% or more, 10% or more, or 15% or more, and may be 50% or less, 40% or less, 30% or less, or 20% or less.

[0029] Furthermore, the alanine residue content, serine residue content, threonine residue content, and tyrosine residue content are equivalent to those obtained by replacing the glycine residue in the above formula with alanine residue, serine residue, threonine residue, and tyrosine residue, respectively.

[0030] Furthermore, when forming artificial proteins, amino acids with relatively small side chains are more likely to form hydrogen bonds, making it easier to obtain molded products with higher strength. In addition, since alanine and glycine residues are nonpolar amino acids with side chains, they are positioned to face inward during the folding process in polypeptide production, making it easier to adopt an α-helix structure or a β-sheet structure. Therefore, it is desirable to have a high proportion of amino acids such as glycine and alanine residues. From the viewpoint of obtaining molded products with superior strength, the alanine residue content may be, for example, 10-40%, 12-40%, 15-40%, 18-40%, 20-40%, or 22-40%. From the viewpoint of obtaining molded products with superior strength, the glycine residue content may be, as mentioned above, 10-55%, 11-55%, 13-55%, 15-55%, 18-55%, 20-55%, 22-55%, or 25-55%.

[0031] It is preferable that the artificial protein contains a certain amount of amino acids with relatively large side chains or amino acids with flexibility, uniformly distributed throughout the sequence. Specifically, the structural protein may contain repeating motifs containing tyrosine residues, threonine residues, and proline residues in a periodic manner. Such a structural protein makes it easier to inhibit the formation of strong intermolecular hydrogen bonds during processing of the molded product obtained by molding, thereby improving processability. For example, the total content of proline residues, threonine residues, and tyrosine residues in any 20 consecutive amino acid residues may be 5% or more, more than 5.5%, 6.0% or more, more than 6.5%, 7.0% or more, more than 7.5%, 8.0% or more, more than 8.5%, 9.0% or more, 10.0% or more, or 15.0% or more. Alternatively, for example, the total content of proline residues, threonine residues, and tyrosine residues in any 20 consecutive amino acid residues may be 50% or less, 40% or less, 30% or less, or 20% or less.

[0032] [Hydrophobic artificial protein] The artificial protein according to this embodiment may be a so-called hydrophobic artificial protein, which is hydrophobic. When the artificial protein is a hydrophobic artificial protein, when a molded article such as a fiber, film, gel, or resin (heat-pressed molded article) is manufactured using a known method with the artificial protein as a raw material, the water resistance of the manufactured molded article is improved, and for example, when the molded article is used as a general-purpose industrial material, the service life can be advantageously extended.

[0033] The hydrophobicity of an artificial protein can be estimated using the average HI (Hydrophobic Index) value of each amino acid constituting the artificial protein as an indicator. In this specification, the average HI value of a hydrophobic artificial protein may be greater than 0 when considering the entire length of the amino acid sequence of the hydrophobic artificial protein, and the average HI may be, for example, 0.00 or more, 0.10 or more, 0.20 or more, 0.22 or more, 0.25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 or more, 0.50 or more, 0.55 or more, 0.60 or more, 0.65 or more, or 0.70 or more. Furthermore, although there is no particular upper limit, it may be, for example, 1.00 or less, or 0.7 or less.

[0034] The average HI value of hydrophobic artificial proteins, and the hydrophobicity of the repeating sequence units described later, are determined using known hydrophobicity indices of amino acid residues according to known methods. Known hydrophobicity indices of amino acid residues are shown in the table below. For example, the degree of hydrophobicity may be calculated according to the method described in Kyte J, Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J.Mol.Biol., 157, pp.105-132.

[0035] [Table 1]

[0036] Furthermore, the hydrophobic artificial protein may have low solubility in a lithium bromide aqueous solution (concentration: 9M) at 60°C. That is, the hydrophobic artificial protein may have a maximum concentration of, 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 when dissolved in a lithium bromide aqueous solution (concentration: 9M) at 60°C. The hydrophobic artificial protein may also be completely insoluble in a lithium bromide aqueous solution (concentration: 9M) at 60°C. Low solubility of the hydrophobic protein in a lithium bromide aqueous solution at 60°C makes it easier and more advantageous to obtain the aforementioned effects when using the hydrophobic artificial protein as a forming material for various molded products, including artificial protein fibers.

[0037] The hydrophobic artificial protein may have a water contact angle of 55° or higher. Furthermore, such a water contact angle may be 60° or higher, 65° or higher, or 70° or higher. The water contact angle can be evaluated by forming a film made of the hydrophobic artificial protein on a substrate, dropping water onto the film, and measuring the contact angle after 5 seconds. When the water contact angle of the hydrophobic artificial protein is 55° or higher, the aforementioned effects obtained when using the hydrophobic artificial protein as a forming material for various molded products, including artificial protein fibers, become more advantageous and reliable.

[0038] Furthermore, it is desirable that the protein structure includes a main sequence and hydrophobic tags bound to the N-terminus and C-terminus of the main sequence.

[0039] Specifically, the protein structure contains hydrophobic tags at both ends, and the N-terminal reaction site is located within the main sequence. This suppresses the binding of protein structures at the reaction site, thus resulting in excellent reactivity with the compound. Furthermore, the C-terminal reaction site is located within the C-terminal hydrophobic tag or within 9 residues from the C-terminus of the main sequence, which makes the reaction site more easily exposed to the surface and thus results in excellent reactivity with the compound. In addition, if the C-terminal reaction site is located within 9 residues from the C-terminus of the main sequence, in order to enhance reactivity with the compound, such a reaction site may be located within 8 residues, 7 residues, 6 residues, 5 residues, 4 residues, 3 residues, 2 residues, or at the C-terminus of the main sequence.

[0040] [Hydrophobic tag] A hydrophobic tag is a tag sequence formed from hydrophobic amino acid residues. However, the C-terminus of the hydrophobic tag may be an amino acid residue containing the above reaction site (for example, a cysteine ​​residue). Here, hydrophobic amino acids refer to alanine (A), valine (V), glycine (G), isoleucine (I), leucine (L), phenylalanine (F), proline (P), tryptophan (W), and tyrosine (Y). In other words, in this disclosure, a hydrophobic tag is a tag sequence formed solely from hydrophobic amino acid residues, which is a region where only hydrophobic amino acid residues are continuous from the terminal amino acid residue (however, if the terminal has a reaction site such as a cysteine ​​residue, the next amino acid residue after the terminal) until the first non-hydrophobic amino acid residue (a residue of an amino acid other than the hydrophobic amino acid) is detected.

[0041] Among these, the hydrophobic tag is preferably GFIL (glycine, phenylalanine, isoleucine, leucine) or two or more repetitions of GFIL, more preferably two or more repetitions of GFIL, and even more preferably GFILGFIL. In the above embodiment, the C-terminus of the hydrophobic tag may be an amino acid residue containing the above reaction site (for example, a cysteine ​​residue).

[0042] Among these, it is preferable to use hydrophobic structural proteins. Hydrophobic structural proteins are proteins that are both hydrophobic and structural proteins, as described above. The average hydroxyl index (HI) of the hydrophobic structural protein is preferably greater than 0, for example, it may be 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. Furthermore, there is no particular upper limit, but for example, it may be 1.00 or lower, or 0.7 or lower. The method for calculating the Hydropathy Index is as described above.

[0043] The hydrophobic artificial protein may also have excellent heat resistance. For example, if a 5 w / v% aqueous dispersion of hydrophobic artificial protein is prepared and heated to 100°C, the protein may not decompose for at least 5 hours. If the hydrophobic artificial protein has excellent heat resistance, the aforementioned effects obtained when using hydrophobic artificial protein as a forming material for various molded products, including artificial protein fibers, can be obtained more advantageously and reliably.

[0044] [Artificial proteins with repetitive sequences] The artificial protein according to this embodiment may have a repeat sequence. That is, the artificial protein according to this embodiment may have multiple amino acid sequences (repeat sequence units) with high sequence identity within the artificial protein. The number of amino acid residues in a repeat sequence unit may be 6 to 200. The total number of glycine residues, serine residues, glutamine residues, and alanine residues relative to the total number of amino acid residues in the repeat sequence unit may be 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more. Furthermore, the sequence identity between repeat 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. Furthermore, the hydrophobicity of the repeat sequence unit (i.e., the sum of the HI values ​​of each amino acid contained in the repeat sequence) may be, for example, -0.80 or higher, -0.70 or higher, -0.60 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, 0.22 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. While there is no particular upper limit to the hydrophobicity of the repeat sequence unit, it may be, for example, 1.0 or lower, or 0.7 or lower.

[0045] An artificial protein having a repeat sequence may have an amino acid sequence containing a domain sequence (repeat sequence unit) represented by formula 1:REP1-REP2. Such an artificial protein may contain, for example, 2 or more, 10 or more, 100 or more, or 200 or more of these domain sequences. The upper limit may be 300 or less. Furthermore, an additional amino acid sequence (N-terminal sequence and C-terminal sequence) may be added to either the N-terminal or C-terminal side, or both, of multiple consecutive domain sequences. The N-terminal and C-terminal sequences are regions that do not contain repeat sequences and consist of approximately 100 amino acid residues.

[0046] In formula 1 above, REP1 represents an amino acid sequence consisting of, for example, 2 to 27 amino acid residues. The number of amino acid residues in REP1 may be an integer between 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. REP2 represents an amino acid sequence consisting of, for example, 10 to 200 amino acid residues. Multiple REP1s may have the same amino acid sequence or different amino acid sequences. Multiple REP2s may have the same amino acid sequence or different amino acid sequences.

[0047] The domain sequence of an artificial protein having the repeating sequence units described above may be, for example, an amino acid sequence that produces crystalline and amorphous regions.

[0048] The domain sequence, which consists of amino acid sequences that give rise to crystalline and amorphous regions, may be, for example, an amino acid sequence represented by formula 2: [(A)n motif-REP3]m, or formula 3: [(A)n motif-REP3]m-(A)n motif. In an artificial protein having such an amino acid sequence, the crystalline region corresponds to, for example, the (A)n motif of the amino acid sequence, and the amorphous region corresponds to, for example, REP3 of the amino acid sequence. In addition, even in an artificial protein having an amino acid sequence containing a domain sequence represented by formula 2 or formula 3, the above-mentioned N-terminal sequence and C-terminal sequence may be further added to either the N-terminal side or the C-terminal side, or both, of the domain sequence.

[0049] Here, the (A)n motif represents an amino acid sequence having at least one alanine residue, and the number of amino acid residues may be 2 to 27. The number of amino acid residues in the (A)n motif may 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, the ratio of the number of alanine residues to the total number of amino acid residues in the (A)n motif may be 40% or more, and may be 60% 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 consists only of alanine residues). The (A)n motif may consist of at least 80% of the total number of alanine, serine, threonine, and valine residues relative to the total number of amino acid residues in the (A)n motif, but may also be at least 85%, more preferably at least 90%, may also be at least 95%, and may even be 100% (meaning it consists only of one or more amino acid residues selected from alanine, serine, threonine, and valine residues). REP3 represents an amino acid sequence consisting of 2 to 200 amino acid residues. REP3 may also consist of an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer from 2 to 300, and may also be an integer from 10 to 300. The (A)n motifs may consist of identical amino acid sequences or different amino acid sequences. Similarly, REP3 may consist of identical amino acid sequences or different amino acid sequences.

[0050] Furthermore, since the (A)n motif mainly contains alanine residues, it readily adopts an α-helix structure or a β-sheet structure. Because the (A)n motif is included in the repeating sequence unit, the artificial protein according to this embodiment repeatedly possesses these secondary structures. As will be described later, when the artificial protein is formed into a fiber, it is expected to exhibit high strength due to these secondary structures.

[0051] Artificial proteins having repeating sequence units as described above include, for example, artificial fibroin. Artificial fibroin may be a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added at one or several positions relative to the gene sequence of cloned naturally derived fibroin, as long as the original function is maintained. The term "one or several" above varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3. In this specification, "maintaining the original function" means that the gene or protein variant has a function (activity and properties) corresponding to the function (activity and properties) of the original gene or protein.

[0052] Furthermore, the artificial fibroin may be a protein having an amino acid sequence that has 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more homology to the entire amino acid sequence of the cloned naturally derived fibroin, as long as the original function is maintained. In this specification, "homology" may refer to "identity".

[0053] Furthermore, the artificial fibroin may also be a protein encoded in DNA that hybridizes under stringent conditions with a probe prepared from the nucleotide sequence of the cloned naturally derived fibroin gene described above, for example, a complementary sequence to all or part of the said nucleotide sequence, as long as the original function is maintained. Such a probe can be prepared, for example, by PCR using an oligonucleotide prepared based on the said nucleotide sequence as a primer and a DNA fragment containing the said nucleotide sequence as a template. "Stringent conditions" refers to conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. For example, conditions in which DNAs with high homology, such as 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more homology, hybridize, and DNAs with lower homology do not hybridize, or conditions in which washing is performed once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for normal Southern hybridization, which are 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS. Artificial fibroin may be a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added at one or several positions relative to the gene sequence of cloned naturally derived fibroin, as long as the original function is maintained. The term "one or several" above varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3. In this specification, "maintaining the original function" means that the gene or protein variant has a function (activity and properties) corresponding to the function (activity and properties) of the original gene or protein.

[0054] Furthermore, the artificial fibroin may be a protein having an amino acid sequence that has 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more homology to the entire amino acid sequence of the cloned naturally derived fibroin, as long as the original function is maintained. In this specification, "homology" may refer to "identity".

[0055] Furthermore, the artificial fibroin may also be a protein encoded in DNA that hybridizes under stringent conditions with a probe prepared from the nucleotide sequence of the cloned naturally derived fibroin gene described above, for example, a complementary sequence to all or part of the said nucleotide sequence, as long as the original function is maintained. Such a probe can be prepared, for example, by PCR using an oligonucleotide prepared based on the said nucleotide sequence as a primer and a DNA fragment containing the said nucleotide sequence as a template. "Stringent conditions" refers to conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. For example, conditions in which DNAs with high homology, such as 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more homology, hybridize, and DNAs with lower homology do not hybridize, or conditions in which washing is performed once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for normal Southern hybridization, which are 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0056] Specific examples of artificial fibroins include, for example, artificial fibroin derived from the large spindle bookmark filament protein produced in the large bottle gland of spiders, as described in International Publication No. 2019 / 194263 (first artificial fibroin), artificial fibroin having a domain sequence with reduced glycine residue content (second artificial fibroin), artificial fibroin having a domain sequence with reduced (A)n motif content (third artificial fibroin), artificial fibroin with reduced glycine residue content and (A)n motif content (fourth artificial fibroin), artificial fibroin having a domain sequence containing a region with locally high hydrophobicity (fifth artificial fibroin), and artificial fibroin having a domain sequence with reduced glutamine residue content (sixth artificial fibroin). The definitions of each of the first to sixth artificial fibroins are incorporated herein by reference to the content described in International Publication No. 2019 / 194263. Artificial fibroin may contain a tag sequence at either the N-terminus, C-terminus, or both. This enables the isolation, immobilization, detection, and visualization of the artificial fibroin.

[0057] Examples of tag sequences include affinity tags that utilize specific affinity (binding affinity) with other molecules. A specific example of an affinity tag is the histidine tag (His tag). The His tag is a short peptide consisting of about 4 to 10 histidine residues, and because it has the property of specifically binding to metal ions such as nickel, it can be used for the isolation of artificial fibroin by chelating metal chromatography. A specific example of a tag sequence is the amino acid sequence shown in Sequence ID No. 8 (an amino acid sequence including the His tag sequence and a hinge sequence).

[0058] Additionally, tag sequences such as glutathione-S-transferase (GST), which specifically binds to glutathione, and maltose-binding protein (MBP), which specifically binds to maltose, can also be used.

[0059] Furthermore, "epitope tags" utilizing antigen-antibody reactions can also be used. By attaching an antigenic peptide (epitope) as a tag sequence, antibodies against that epitope can be bound to it. Examples of epitope tags include HA (hemagglutinin peptide sequence of influenza virus) tags, myc tags, and FLAG tags. By using epitope tags, artificial fibroin can be easily purified with high specificity.

[0060] Furthermore, a version in which the tag sequence can be cleaved with a specific protease can also be used. By treating the protein adsorbed via the tag sequence with a protease, the artificial fibroin from which the tag sequence has been cleaved can be recovered.

[0061] Specific examples of artificial fibroin include the artificial fibroin shown in the table below.

[0062] [Table 2]

[0063] Artificial fibroin may be an artificial fibroin that possesses at least two of the characteristics of the first artificial fibroin, the second artificial fibroin, the third artificial fibroin, the fourth artificial fibroin, the fifth artificial fibroin, and the sixth artificial fibroin.

[0064] The molecular weight of the artificial fibroin is not particularly limited, but may be, for example, 2 kDa or more and 700 kDa or less. The molecular weight of the artificial fibroin according to this embodiment may 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 may 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.

[0065] An artificial protein having repeating sequence units (e.g., artificial fibroin) according to one embodiment may have an amino acid sequence with a reduced glutamine residue content compared to naturally derived fibroin. The glutamine residue content in the artificial protein is preferably 9% or less, more preferably 7% or less, even more preferably 4% or less, and particularly preferably 0%.

[0066] In this specification, the "glutamine residue content" is a value calculated by the following method. In an artificial protein containing a domain sequence represented by Equation 2:[(A)n motif-REP3]m or Equation 3:[(A)n motif-REP3]m-(A)n motif, the glutamine residue content is calculated as u / t when all REP3s contained in the sequence obtained by removing the sequence from the C-terminal (A)n motif to the C-terminus of the domain sequence (the sequence corresponding to "region A" in Figure 1) are excluded from the domain sequence, and the total number of amino acid residues of all REP3s obtained by removing the sequence from the C-terminal (A)n motif to the C-terminus of the domain sequence and then the (A)n motif is excluded is t.

[0067] In one embodiment, an artificial protein having a repeat sequence unit may have an amino acid sequence in which, compared to naturally occurring fibroin, one or more glutamine residues in REP3 of formulas 2 and 3 are deleted or substituted with other amino acid residues (resulting in a reduced glutamine residue content).

[0068] "Other amino acid residues" can be any amino acid residue other than glutamine residues, but it is preferable that they be amino acid residues with a higher hydrophobicity index than glutamine residues. The hydrophobicity indexes of amino acid residues are as shown in the table above.

[0069] As shown in the table above, amino acid residues with a higher hydrophobicity index than glutamine residues include amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P), and histidine (H). Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), and alanine (A) are more preferable, and amino acid residues selected from isoleucine (I), valine (V), leucine (L), and phenylalanine (F) are even more preferable.

[0070] The artificial protein containing the domain sequence represented by formula 2 or formula 3 preferably has a hydrophobicity of REP3 of -0.8 or higher, more preferably -0.7 or higher, even more preferably 0 or higher, even more preferably 0.3 or higher, and particularly preferably 0.4 or higher. There is no particular upper limit to the hydrophobicity of REP, and it may be 1.0 or lower, or 0.7 or lower.

[0071] In this specification, the "hydrophobicity of REP3" is a value calculated by the following method. In an artificial protein (e.g., artificial fibroin) containing a domain sequence represented by Equation 2: [(A)n motif-REP3]m or Equation 3: [(A)n motif-REP3]m-(A)n motif, the hydrophobicity of REP3 is calculated as v / t when the sum of the hydrophobicity indices of each amino acid residue in the sequence obtained by removing the sequence from the C-terminal (A)n motif to the C-terminus of the domain sequence from the domain sequence (the sequence corresponding to "region A" in Figure 1) is v, and the total number of amino acid residues in all REP3 after removing the sequence from the C-terminal (A)n motif to the C-terminus of the domain sequence and then removing the (A)n motif is t.

[0072] In one embodiment, an artificial protein having a repeat sequence unit may have a domain sequence that, compared to naturally occurring fibroin, is modified in a manner equivalent to the deletion of one or more glutamine residues in REP3 and / or the substitution of one or more glutamine residues in REP3 with other amino acid residues, as well as further modifications of the amino acid sequence equivalent to the substitution, deletion, insertion and / or addition of one or more amino acid residues.

[0073] Artificial proteins having repeat sequence units can be obtained, for example, by deleting one or more glutamine residues in REP3 from the gene sequence of cloned naturally occurring fibroin, and / or by substituting one or more glutamine residues in REP3 with other amino acid residues. Alternatively, they can be obtained by designing an amino acid sequence equivalent to deleting one or more glutamine residues in REP3 and / or substituting one or more glutamine residues in REP3 with other amino acid residues from the amino acid sequence of naturally occurring fibroin, and then chemically synthesizing the nucleic acid encoding the designed amino acid sequence.

[0074] A more specific example of an artificial protein having a repeating sequence unit according to one embodiment is (i) a modified fibroin containing the amino acid sequence shown in SEQ ID NO: 9 (Met-PRT888), SEQ ID NO: 10 (Met-PRT965), SEQ ID NO: 11 (Met-PRT889), SEQ ID NO: 12 (Met-PRT916), SEQ ID NO: 13 (Met-PRT918), SEQ ID NO: 14 (Met-PRT699), SEQ ID NO: 15 (Met-PRT698), SEQ ID NO: 16 (Met-PRT966), SEQ ID NO: 17 (Met-PRT917), or SEQ ID NO: 18 (Met-PRT1028), or (ii) a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0075] The artificial protein (i) is described below. The amino acid sequence shown in SEQ ID NO: 9 is obtained by replacing all QQ with VL in the amino acid sequence shown in SEQ ID NO: 19 (Met-PRT410). The amino acid sequence shown in SEQ ID NO: 10 is obtained by replacing all QQ with TS in the amino acid sequence shown in SEQ ID NO: 19, and replacing the remaining Q with A. The amino acid sequence shown in SEQ ID NO: 11 is obtained by replacing all QQ with VL in the amino acid sequence shown in SEQ ID NO: 19, and replacing the remaining Q with I. The amino acid sequence shown in SEQ ID NO: 12 is obtained by replacing all QQ with VI in the amino acid sequence shown in SEQ ID NO: 19, and replacing the remaining Q with L. The amino acid sequence shown in SEQ ID NO: 13 is obtained by replacing all QQ with VF in the amino acid sequence shown in SEQ ID NO: 19, and replacing the remaining Q with I.

[0076] The amino acid sequence shown in SEQ ID NO: 14 is obtained by replacing all QQs with VLs in the amino acid sequence shown in SEQ ID NO: 20 (Met-PRT525). The amino acid sequence shown in SEQ ID NO: 15 is obtained by replacing all QQs with VLs in the amino acid sequence shown in SEQ ID NO: 20, and replacing the remaining Qs with Is.

[0077] The amino acid sequence shown in Sequence ID No. 16 is obtained by replacing all QQs with VFs in the sequence that repeats twice the region of 20 domain sequences present in the amino acid sequence shown in Sequence ID No. 19 (Met-PRT410), and replacing the remaining Qs with Is.

[0078] The amino acid sequence shown in SEQ ID NO: 17 (Met-PRT917) is obtained by replacing all QQs in the amino acid sequence shown in SEQ ID NO: 19 with LIs, and replacing the remaining Qs with Vs. The amino acid sequence shown in SEQ ID NO: 18 (Met-PRT1028) is obtained by replacing all QQs in the amino acid sequence shown in SEQ ID NO: 19 with IFs, and replacing the remaining Qs with Ts.

[0079] The amino acid sequences shown in SEQ ID NOs. 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 all have a glutamine residue content of 9% or less (see table below).

[0080] [Table 3]

[0081] The artificial protein in (i) may consist of the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0082] The artificial protein in (ii) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. The artificial protein in (ii) is also a protein that contains a domain sequence represented by formula 2:[(A)n motif-REP3]m or formula 3:[(A)n motif-REP3]m-(A)n motif. The above sequence identity is preferably 95% or more.

[0083] The artificial protein in (ii) preferably has a glutamine residue content of 9% or less. Furthermore, the artificial protein in (ii) preferably has a GPGXX motif content of 10% or more.

[0084] In one embodiment, an artificial protein having a repeating sequence unit may include a tag sequence at either the N-terminus or the C-terminus, or both. This enables the isolation, immobilization, detection, and visualization of the artificial protein.

[0085] More specific examples of artificial proteins containing tag sequences include (iii) modified fibroin containing the amino acid sequence shown in SEQ ID NO: 21 (PRT888), SEQ ID NO: 22 (PRT965), SEQ ID NO: 23 (PRT889), SEQ ID NO: 24 (PRT916), SEQ ID NO: 3 (PRT918), SEQ ID NO: 25 (PRT699), SEQ ID NO: 26 (PRT698), SEQ ID NO: 2 (PRT966), SEQ ID NO: 27 (PRT917), or SEQ ID NO: 28 (PRT1028), or (iv) modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 3, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 2, SEQ ID NO: 27, or SEQ ID NO: 28.

[0086] The amino acid sequences shown in SEQ ID NOs. 21, 22, 23, 24, 3, 25, 26, 2, 27, and 28 are obtained by adding the amino acid sequence shown in SEQ ID NOs. 8 (including the His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NOs. 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, respectively. Because only the tag sequence is added to the N-terminus, there is no change in the glutamine residue content, and the amino acid sequences shown in SEQ ID NOs. 21, 22, 23, 24, 3, 25, 26, 2, 27, and 28 all have a glutamine residue content of 9% or less (see table below).

[0087] [Table 4]

[0088] The artificial protein in (iii) may consist of the amino acid sequence shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 3, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 2, SEQ ID NO: 27, or SEQ ID NO: 28.

[0089] The artificial protein of (iv) contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 3, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 2, SEQ ID NO: 27, or SEQ ID NO: 28. The artificial protein of (iv) is also a protein that contains a domain sequence represented by formula 2:[(A)n motif-REP3]m or formula 3:[(A)n motif-REP3]m-(A)n motif. The above sequence identity is preferably 95% or more.

[0090] The artificial protein in (iv) preferably has a glutamine residue content of 9% or less. Furthermore, the artificial protein in (iv) preferably has a GPGXX motif content of 10% or more.

[0091] In one embodiment, an artificial protein having a repeating sequence unit may include a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of host.

[0092] Examples of artificial proteins having repeating sequence units according to one embodiment include artificial collagen, artificial elastin, and artificial resilience.

[0093] Examples of artificial collagen include proteins containing the domain sequence represented by formula 4:[REP4]p (wherein formula 3, p is an integer from 5 to 300. REP4 represents an amino acid sequence composed of Gly-XY, where X and Y represent any amino acid residues other than Gly. Multiple REP4 sequences may have the same amino acid sequence or different amino acid sequences).

[0094] Examples of artificial elastins include proteins with amino acid sequences such as NCBl Genebank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine).

[0095] Examples of artificial resilience include proteins containing a domain sequence represented by formula 5:[REP5]q (wherein formula 5, q is an integer from 4 to 300. REP5 represents an amino acid sequence consisting of Ser-JJ-Tyr-Gly-U-Pro. J represents any amino acid residue, preferably selected from the group consisting of Asp, Ser, and Thr. U represents any amino acid residue, preferably selected from the group consisting of Pro, Ala, Thr, and Ser. Multiple REP5s may have the same amino acid sequence or different amino acid sequences).

[0096] Examples of artificial keratin include type I keratin from Capra hircus.

[0097] [Artificial protein derivatives] The artificial protein according to this embodiment may be an artificial protein derivative such as a block copolymer. For example, the artificial protein derivative may be a block copolymer comprising a first segment containing a polypeptide chain and a second segment bonded to the first segment.

[0098] The polypeptide chain constituting the first segment may be, for example, an artificial protein as described in detail in the sections on [artificial proteins], [hydrophobic artificial proteins], and [artificial proteins having repetitive sequences].

[0099] The second segment may be a molecular chain having a plasticizing function for polypeptide chains. A molecular chain having a plasticizing function for polypeptide chains means a molecular chain that enhances the flexibility of the fiber compared to a fiber containing only polypeptide chains. A block copolymer containing a second segment containing a molecular chain having a plasticizing function for polypeptide chains may have high flexibility suitable for fabrics used in clothing, etc. In addition, a block copolymer containing a second segment containing a molecular chain having a plasticizing function for polypeptide chains may be biodegradable.

[0100] The second segment may be a molecular chain containing polyoxyalkylene, polyester, polycarbonate, polyamide, polyol, or modified polysaccharide chain. These molecular chains may have a plasticizing function for polypeptide chains.

[0101] Examples of polyoxyalkylenes that may constitute the second segment include polyoxyethylene, polyoxypropylene, and polyoxytetramethylene. Examples of polyesters that may constitute the second segment include polylactic acid, poly(3-hydroxybutanoic acid), polyhydroxybutanoic acid / hydroxyvaleryl acid copolymer, polyhydroxybutanoic acid / 4-hydroxybutanoic acid copolymer, polyhydroxybutanoic acid / hydroxyhexanoic acid copolymer, polytrimethylene terephthalate, butanediol / long-chain dicarboxylic acid copolymer, polyethylene terephthalate, polybutylene succinate, polybutylene succinate-adipate copolymer, polybutylene adipate-terephthalate copolymer, polycaprolactone, and polytrimethylene frangipane carboxylate. Polycarbonates that can constitute the second segment may contain constituent units derived from aliphatic diols, and examples include 1,6-hexanediol polycarbonate, 1,5-pentanediol polycarbonate, and 1,10-decanediol carbonate. Examples of polyamides that can constitute the second segment include nylon 3, nylon 4, nylon 5, nylon 6, nylon 11, and nylon 610. Examples of polyols that can constitute the second segment include polyvinyl alcohol and ethylene-vinyl alcohol copolymers. Examples of modified polysaccharides that can constitute the second segment include chemically modified polysaccharides such as cellulose, starch, chitin, and chitosan. Specific examples of modified polysaccharides include cellulose acetate, ethylcellulose, starch acetate, hydroxypropylated starch, carboxymethyl chitin, and carboxymethyl chitosan.

[0102] [Modified artificial protein] The artificial protein according to this embodiment may be a chemically modified artificial protein. That is, for example, at least some or all of the lysine residues, serine residues, threonine residues, tyrosine residues, or cysteine ​​residues in the artificial protein may be modified to impart specific functions or properties.

[0103] [Protein production using microbiological methods] The artificial protein according to this embodiment may be produced by microbiological methods. The artificial protein may be produced by referring to the descriptions in International Publication Nos. 2017 / 188430, 2017 / 188434, 2017 / 222034, 2018 / 025886, 2019 / 022163, etc.

[0104] Artificial proteins can be produced, for example, by a method that includes the step of expressing nucleic acids in a host transformed with an expression vector. Expression methods include direct expression, secretory production, fusion protein expression, etc., in accordance with methods described in Molecular Cloning, 2nd Edition. When expressed in yeast, animal cells, or insect cells, proteins can be obtained as polypeptides to which sugars or sugar chains have been added.

[0105] <Inorganic carbonates> The method for producing polyamino acids in this embodiment uses inorganic carbonates. As the inorganic carbonate, alkali metal or alkaline earth metal carbonates are preferred, and alkali metal carbonates are more preferred. Examples of alkali metal carbonates include sodium carbonate, potassium carbonate, rubidium carbonate, and cesium carbonate, with potassium carbonate or cesium carbonate being preferred, and cesium carbonate being more preferred. Examples of alkaline earth metal carbonates include magnesium carbonate, calcium carbonate, strontium carbonate, and barium carbonate, with calcium carbonate being preferred. Among these, the inorganic carbonate preferably contains at least one compound selected from the group consisting of cesium carbonate and potassium carbonate, and is preferably the compound containing cesium carbonate.

[0106] In the second method for producing polyamino acids, the mass of the inorganic carbonate added to the reaction system is 60 to 100% by mass of the total mass of compound A and other compounds added to the reaction system. In the first method for producing polyamino acids or the third method for producing polyamino acids, the mass of the inorganic carbonate added to the reaction system is preferably 60 to 100% by mass of the total mass of compound A and compounds other than the solvent added to the reaction system. In the method for producing polyamino acids according to this embodiment, the mass of the inorganic carbonate is preferably 70 to 100% by mass, more preferably 80 to 100% by mass, even more preferably 90 to 100% by mass, particularly preferably 95 to 100% by mass, and even more preferably 99 to 100% by mass, relative to the total mass of compound A and compounds other than the solvent added to the reaction system. Here, an embodiment in which the mass of the inorganic carbonate is 100% by mass relative to the total mass of compound A and compounds other than the solvent added to the reaction system is also one of the preferred embodiments of the present invention. In the method for producing polyamino acids of this embodiment, one inorganic carbonate may be used alone, or two or more may be used in combination. When two or more are used in combination, it is preferable that the total amount is within the aforementioned range.

[0107] Furthermore, in the method for producing polyamino acids according to this embodiment, the amount of inorganic carbonate used is preferably 0.1 to 10 molar equivalents, more preferably 0.5 to 5 molar equivalents, and even more preferably 1 to 3 molar equivalents relative to compound A. In the method for producing polyamino acids of this embodiment, one inorganic carbonate may be used alone, or two or more may be used in combination. When two or more are used in combination, it is preferable that the total amount is within the aforementioned range.

[0108] <Compounds for forming derivatives> In the third method for producing polyamino acids, the mass of the derivative-forming compound added to the reaction system is 0 to 40% by mass relative to the mass of compound A added to the reaction system. In the first method for producing polyamino acids and the second method for producing polyamino acids, the mass of the derivative-forming compound added to the reaction system is preferably 0 to 40% by mass relative to the mass of compound A added to the reaction system.

[0109] A derivative-forming compound refers to a compound in which, in the reaction between compound A and an inorganic carbonate in this embodiment, compound A and the derivative-forming compound first react to form a derivative (intermediate), and then the inorganic carbonate and the derivative react to form a dehydroalanine-like structure. Examples of compounds used for derivative formation include tosylating agents such as toluenesulfonyl chloride, phosphonylating agents such as diphenylphosphonic acid chloride, alkyl halogenated compounds such as methyl iodide, and imide halogenated compounds such as 3-bromomaleimide.

[0110] In the method for producing polyamino acids of this embodiment, the mass of the derivative-forming compound added to the reaction system is preferably 0 to 30% by mass, more preferably 0 to 20% by mass, even more preferably 0 to 10% by mass, particularly preferably 0 to 5% by mass, and even more preferably 0 to 1% by mass, relative to the mass of compound A added to the reaction system. Hereinafter, an embodiment in which the mass of the derivative-forming compound added to the reaction system is 0.1% by mass or less, and furthermore, an embodiment in which the mass of the derivative-forming compound added to the reaction system is 0.01% by mass or less, relative to the mass of compound A added to the reaction system, is also one of the preferred embodiments of the present invention. In the method for producing polyamino acids according to this embodiment, if two or more derivative-forming compounds are included, it is preferable that the total amount be within the aforementioned range.

[0111] Furthermore, in the method for producing polyamino acids according to this embodiment, the molar amount of the derivative-forming compound added to the reaction system is preferably 40 or less, more preferably 30 or less, even more preferably 20 or less, particularly preferably 10 or less, even more preferably 5 or less, and even more preferably 1 or less, relative to the total molar amount of hydroxyl groups and thiol groups (preferably hydroxyl groups and thiol groups located at the β-position of the carbonyl group) contained in compound A added to the reaction system (i.e., the product of the mass of compound A added to the system and the total content of hydroxyl groups and thiol groups in compound A (mol / g)). When two or more derivative-forming compounds are included, it is preferable that the total amount be within the aforementioned range.

[0112] <Reaction> In the first method for producing polyamino acids, it is preferable that at least one substituent selected from the group consisting of hydroxyl groups and thiol groups is directly removed by the inorganic carbonate during the reaction between compound A and the inorganic carbonate. In the second method for producing polyamino acids and the third method for producing polyamino acids, it is preferable that at least one substituent selected from the group consisting of hydroxyl groups and thiol groups is directly removed by the inorganic carbonate during the reaction between compound A and the inorganic carbonate. Direct elimination means that a compound that is neither compound A nor an inorganic carbonate, such as a derivative-forming compound added to the reaction system, is eliminated in a single step without forming an intermediate.

[0113] The reaction may be carried out in an air atmosphere, or under conditions where part or all of the reaction atmosphere is replaced with an inert gas or other gas. However, it is preferable to carry out the reaction in an air-containing atmosphere, and more preferably in an air atmosphere.

[0114] The reaction between compound A and the inorganic carbonate may be carried out by a mechanochemical method or in a solvent, but it is preferable to carry it out by a mechanochemical method.

[0115] [Mechanochemical method] Mechanochemical processes are methods for carrying out chemical reactions caused by directly absorbing mechanical energy. This mechanical energy may be exerted, for example, by impact force or shear force. Specifically, such mechanochemical processes are carried out using rolling ball mills, media stirring mills, planetary mills, jet mills, mixer mills, extruders (twin-screw extruders), etc. Specifically, compound A and an inorganic carbonate are placed in a grinding jar, and media balls, etc., are added depending on the type of mill used. Then, the grinding jar is set in a mixer mill device and vibrated at a predetermined frequency and reaction time. At this time, solvents, etc., may be further added to the grinding jar. Alternatively, compound A and an inorganic carbonate are placed in a mixer mill, and the milling procedure is started to continuously knead the reaction product. At this time, solvents, etc., may be further added to the mixer mill. Alternatively, compound A and an inorganic carbonate are placed in an extruder and kneaded together. At this time, solvents, etc., may be further added to the extruder. Furthermore, by performing mechanochemical treatment using an extruder, it becomes possible to continuously produce polyamino acids having the desired dehydroalanine-like skeleton.

[0116] The frequency can be adjusted as appropriate by those skilled in the art depending on the reaction, and may be, for example, 10-50Hz, 10-45Hz, 10-40Hz, 10-35Hz, 15-50Hz, 15-45Hz, 15-40Hz, 15-35Hz, 20-50Hz, 20-45Hz, 20-40Hz, or 20-35Hz.

[0117] The reaction time can be defined as the point at which the starting compound A disappears or the target product is detected at a certain level or higher using infrared (IR) absorption spectroscopy, gel filtration chromatography (GPC), thin-layer chromatography, nuclear magnetic resonance (NMR), etc. The reaction time can be any time, for example, 5 to 240 minutes, 5 to 210 minutes, 5 to 180 minutes, 5 to 150 minutes, 5 to 120 minutes, 5 to 60 minutes, 5 to 50 minutes, 5 to 40 minutes, or 10 to 30 minutes.

[0118] The solvent can be any solvent that can swell compound A or dissolve at least one of compound A and the inorganic carbonate, but it must be a liquid at room temperature and pressure and must not chemically react with compound A. Mechanochemical treatment with the addition of such a solvent is known as liquid-assisted grinding (LAG), and is a method that allows the desired reaction to proceed more efficiently by adding a small amount of solvent to the grinding jar. Examples of such solvents include alcohol-based solvents such as methanol and ethanol, acetonitrile (MeCN), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpiperidone (NMP), tetrahydrofuran (THF), and dihydrolevoglucocenone. The amount of solvent used may be, for example, 0.01g to 1g per 1g of compound A, but may also be 0.01g to 0.8g, 0.01g to 0.6g, 0.01g to 0.4g, or 0.01g to 0.2g. In other words, the amount of solvent used may be, for example, 1 to 100% by mass relative to the protein, but may also be 1 to 80% by mass, 1 to 60% by mass, 1 to 40% by mass, or 1 to 20% by mass. It is thought that trace amounts of solvent form a microscopic reaction field by swelling compound A or locally dissolving it. Note that the addition of solvent is not always necessary, and there may be cases where the addition of solvent is not required.

[0119] After the reaction is complete, the mixture may be removed from the grinding jar and washed with a solvent. Washing can remove unreacted substances and excess by-products generated by the reaction. Suitable solvents for washing include water, methanol, ethanol, acetonitrile, acetone, tetrahydrofuran, ethyl acetate, and hexane. After washing, the solvent used for washing may be removed by distillation by drying the product. Drying may be carried out under reduced pressure.

[0120] The average particle size of the polyamino acid having a dehydroalanine-like skeleton according to this embodiment is preferably 1-80 μm, 1-50 μm, 2-50 μm, 2-40 μm, 4-25 μm, 5-25 μm, 8-25 μm, or 8-16 μm. When the average particle size is within the above range, it is easy to handle. The average particle size can be determined, for example, by the following method: After uniformly dispersing polyamino acid particles or powder on a glass plate by suction in a vacuum chamber, the particle size is measured five times each using a wet / dry image analysis particle size distribution analyzer (product name: DW-200nano, manufactured by JUSCO International Co., Ltd.), a projection image is captured with a 10-megapixel camera, and the obtained projection image is analyzed using image analysis software.

[0121] The polyamino acid having a dehydroalanine-like skeleton according to this embodiment is obtained as a powder with a smaller and more uniform average particle size by mechanochemically treating a mixture containing a protein and a molecule capable of plasticizing the protein, followed by freeze-drying. The average particle size of the powder of polyamino acid having a dehydroalanine-like skeleton obtained by freeze-drying is, for example, about 1-30 μm, 1-20 μm, 1-10 μm, or 2-8 μm. Such powdered polyamino acids having a dehydroalanine-like skeleton are also easier to handle, and the amount used can be finely adjusted. Moreover, freeze-dried polyamino acids having a dehydroalanine-like skeleton not only have a small average particle size, but the tertiary structure of the protein can also be expected to be loosened, so improved dispersibility in aqueous media (aqueous liquids) such as water, basic aqueous solutions, acidic aqueous solutions, and neutral aqueous solutions containing inorganic salts, as well as improved solubility in solvents, can be expected.

[0122] Polyamino acids produced by the mechanochemical method do not use organic solvents in the manufacturing process, or only use very small amounts (enough to swell proteins or molecules that can plasticize proteins), resulting in less residual organic solvents and easier purification as a powder. Furthermore, as described above, the compound polyamino acids according to this embodiment can also be purified into smaller particles with a uniform particle size distribution.

[0123] [Solvent method] The reaction between compound A and the inorganic carbonate may be carried out in a solvent. Examples of solvents that can be used include acetonitrile (MeCN), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpiperidone (NMP), tetrahydrofuran (THF), acetone, toluene, and dichloromethane. Among these, acetonitrile or acetone are preferred from the viewpoint of yield.

[0124] Here, the amount of solvent used is preferably such that the concentration of compound A is 0.01 to 3 mol / L, more preferably 0.05 to 1 mol / L, and even more preferably 0.1 to 0.5 mol / L. Two or more solvents may be used in combination, and if so, it is preferable that the total amount is within the above range.

[0125] The reaction time can be until the starting compound A disappears or the target product is detected at a certain level or higher using infrared (IR) absorption spectroscopy, gel filtration chromatography (GPC), etc. The reaction time can be any time, for example, 5 to 240 minutes, 5 to 210 minutes, 5 to 180 minutes, 5 to 150 minutes, 5 to 120 minutes, 5 to 60 minutes, 5 to 50 minutes, 5 to 40 minutes, or 10 to 30 minutes.

[0126] The reaction temperature may be any temperature, for example, 0-100°C, 5-80°C, 5-70°C, 5-60°C, 5-50°C, 10-40°C, 15-40°C, or 20-40°C.

[0127] After the reaction is complete, the product may be extracted and purified with a solvent such as ethyl acetate. Such extraction and purification methods can be carried out according to the information provided.

[0128] <Other processes> The method for producing polyamino acids according to this embodiment may further include other steps. Other steps include synthesizing compound A, and further purifying the obtained polyamino acid having a dehydroalanine-like skeleton according to its molecular weight, the amount of the dehydroalanine-like skeleton, etc.

[0129] <Application> The polyamino acids having a dehydroalanine-like skeleton obtained by the method for producing polyamino acids of this embodiment can utilize the dehydroalanine-like skeleton as a "structure for adding new functions to proteins." Specifically, as described in the method for producing polyamino acid derivatives of this embodiment below, the dehydroalanine-like skeleton introduced by the method for producing polyamino acids of this embodiment can be used as a reaction site for reactions with various nucleophiles. For example, it is possible to improve the physical properties and add functions to materials such as fibers, films, resins, artificial leather, adhesives, and coatings by modifying the structure of proteins. Furthermore, it can be applied to regulating specific enzyme activity on proteins and modifying protein-protein interactions. In addition, new biomaterials (e.g., gels, films, nanomaterials, etc.) can be designed and synthesized based on polyamino acids with a dehydroalanine-like skeleton. This opens up possibilities for diverse applications, such as medical materials, biosensors, and scaffolding materials for tissue engineering. Furthermore, by reacting polyamino acids with a dehydroalanine-like skeleton with nucleophiles to perform structural transformations, it is conceivable that drug delivery systems targeting specific cells or tissues could be constructed. As described above, the method for producing polyamino acids having a dehydroalanine-like skeleton, which can be produced atomically efficiently while suppressing steric changes, is considered to have applicability in various fields.

[0130] (Method for producing polyamino acid derivatives) The method for producing the polyamino acid derivative of this embodiment includes the method for producing a polyamino acid having a dehydroalanine-like skeleton of this embodiment, and comprises reacting the compound A after the reaction (i.e., the polyamino acid having a dehydroalanine-like skeleton) with a nucleophile.

[0131] The nucleophile used in the method for producing polyamino acid derivatives of this embodiment is not particularly limited and includes compounds having a hydroxyl group, compounds having a thiol group, compounds having an amino group, compounds having a selenol group, compounds having a phospho group (phosphorus compounds), and the like. The nucleophile may be a low molecular weight compound or a high molecular weight compound such as a resin. In other words, according to the method for producing polyamino acid derivatives of this embodiment, reactions such as attaching a low molecular weight functional structure to a polyamino acid having a dehydroalanine-like skeleton, or attaching a high molecular weight oligomer block, polymer block, etc., can be carried out. The method for producing polyamino acids having a dehydroalanine-like skeleton according to this embodiment is extremely useful because it allows for the production of polyamino acids having a dehydroalanine-like skeleton that react with such a wide range of nucleophiles. Furthermore, the method for producing polyamino acid derivatives according to this embodiment is extremely useful because, by using the polyamino acid obtained by the method for producing polyamino acids having a dehydroalanine-like skeleton according to this embodiment, a wide range of nucleophiles can be used, making it possible to easily produce various polyamino acids.

[0132] The reaction conditions with the nucleophile are not particularly limited and can be determined by referring to conventional methods. Furthermore, the reaction between the nucleophile and the polyamino acid having a dehydroalanine-like skeleton may be carried out by mechanochemical methods or in a solvent. The reaction conditions described above can be used as a reference for these methods.

[0133] <Other processes> The method for producing polyamino acid derivatives according to this embodiment may further include other steps. Other steps include further purifying the obtained polyamino acid derivative according to its molecular weight, the reaction rate of the nucleophile, and other factors.

[0134] <Application> The polyamino acid derivatives obtained by the method for producing polyamino acid derivatives of this embodiment can be used as fibers, films, resins, artificial leather, adhesives, coatings, and the like. Furthermore, it can be used without any particular restrictions in applications where artificial proteins are used. In other words, the method for producing polyamino acid derivatives of this embodiment allows the use of various nucleophiles and improves the design flexibility of artificial proteins. This makes it easier to improve the physical properties, modify or add functions to artificial proteins in applications where they are typically used.

[0135] (Method of manufacturing fibers) The method for producing fibers according to this embodiment includes the method for producing polyamino acid derivatives according to this embodiment, and includes using compound A after reaction with the nucleophile (i.e., the polyamino acid derivative obtained by the method for producing polyamino acid derivatives according to this embodiment) as fibers. Regarding the method of producing fibers, for example, a polyamino acid derivative can be dissolved in a solvent to form a dope solution, which is then spun using known spinning methods such as wet spinning, dry spinning, wet-dry spinning, or melt spinning. Examples of solvents capable of dissolving proteins include dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, and hexafluoroisopropanol (HFIP). Inorganic salts may be added to the solvent as dissolution accelerators. Furthermore, there are no particular limitations on the method of producing the fibers, and known methods can be used without any particular restrictions. In other words, according to the method for producing polyamino acid derivatives of this embodiment, various proteins can be easily synthesized using various nucleophiles, making it possible to easily improve the physical properties of the fibers, change or add functions, etc., while utilizing conventional methods for producing protein fibers. [Examples]

[0136] The present invention will be described in more detail below with reference to examples. The materials, amounts used, proportions, processing content, and processing procedures shown in the following examples can be modified as appropriate, as long as they do not depart from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the specific examples shown below.

[0137] <Synthesis Example: Synthesis of Dipeptide 1a> To a solution of Boc-protected alanine derivative 1a-A (0.19 g, 1.0 mmol) in CH2Cl2 (5 mL), 1-hydroxybenzotriazole (HOBt, 0.14 g, 1.0 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 0.19 g, 1.0 mmol) were added at 0°C. The reaction mixture was stirred at 0°C for 1.5 hours. After 1.5 hours, 4-methylmorpholine (0.12 g, 1.2 mmol) and cystine dimethyl ester dihydrochloride 1a-B (0.15 g, 0.45 mmol) were added to the reaction mixture at 0°C, and the resulting mixture was stirred at 0°C for 1.5 hours. The mixture was then warmed to room temperature and stirred for a further 15 hours. After 15 hours, H2O (5 mL) was added to the reaction mixture at room temperature. After removing CH2Cl2 by distillation, the reaction product was extracted three times with ethyl acetate (5 mL x 3 times). The combined ethyl acetate extract was washed with saturated sodium bicarbonate aqueous solution (5 mL x 3 times) and brine (5 mL x 1 time). The resulting ethyl acetate solution was dried over Na2SO4. After removing Na2SO4 by filtration, the filtrate was concentrated. The residue was purified by flash column chromatography using silica gel (hexane / ethyl acetate = 10:1~1:2 (eluate)) to obtain disulfide 1a-C (0.23 g, 0.38 mmol). To a solution of disulfide 1a-C (0.28 g, 0.30 mmol) in THF (2 mL), 1 mL of 0.1 M acetic acid (AcOH·AcONa) buffer [pH 4.7] and tributylphosphine (67 mg, 0.33 mmol) were added at room temperature. The reaction mixture was heated to 50°C and stirred at 50°C for 1 hour. After 1 hour, the reaction mixture was cooled to room temperature. 5 mL of water at room temperature was added to the reaction mixture, and the reaction product was extracted three times with ethyl acetate (5 mL x 3). The combined ethyl acetate extract was dried over Na₂SO₄. After filtering off the Na₂SO₄, the filtrate was concentrated. The residue was purified by flash column chromatography using silica gel (hexane / ethyl acetate = 10:1~1:2 (eluate)) to obtain dipeptide 1a. [ka]

[0138] (Example 1) <Production of polyamino acids with a dehydroalanine-like skeleton from dipeptides (examination of base species)> We investigated the reaction that converts a dipeptide containing a cysteine ​​residue into a polyamino acid with a dehydroalanine-like skeleton in the presence of various bases. Dipeptide 1a containing a cysteine ​​residue (the above-mentioned synthesized product, 0.3 mmol) was dissolved in acetonitrile (3.0 mL), warmed to 35°C, and stirred for 5 minutes. Then, the base was added to the warmed solution and stirred under air at 35°C for 2 hours. The type of base and the amount (equivalent) used in each example and comparative example are listed in the table. After 2 hours of stirring, H2O (5 mL) was added to the reaction mixture. The reaction product was first extracted three times with ethyl acetate (5 mL). The aqueous phase was acidified with 1N HCl and then extracted three more times with ethyl acetate (5 mL). The ethyl acetate extract was dried over anhydrous sodium sulfate. After filtering to remove anhydrous sodium sulfate, the filtrate was concentrated. The residue was purified on silica gel by flash column chromatography (using hexane / ethyl acetate as the eluent) to obtain dehydroalanine derivative 1. The yield of the target dehydroalanine derivative 1, the enantiomeric excess (ee), and the enantiomer ratio (er), as well as the yield of the monomer or dimer 2 of the substrate, and the yield of the hydrolyzed carboxylic acid compound 3 of substrate 1a are shown in the table below. [ka]

[0139] [Table 5]

[0140] In Examples 1-1 to 1-5, which used inorganic carbonates, the target dehydroalanine derivative 1 was obtained. Furthermore, the obtained target dehydroalanine derivative 1 had a very high enantiomeric excess (ee) and enantiomeric ratio (er), indicating that steric change was suppressed and the stereochemistry was maintained. In particular, from the viewpoint of yielding dehydroalanine derivative 1 and suppressing steric change, potassium carbonate or cesium carbonate is preferred, and cesium carbonate is more preferred. In Comparative Examples 1-1 to 1-3, inorganic hydroxides were used instead of inorganic carbonates. In these examples, dehydroalanine derivative 1 could not be detected in the ethyl acetate extract. In Comparative Examples 1-1 and 1-3, when the crude composition in the aqueous phase obtained by back-extraction during liquid-liquid separation was methyl esterified in the presence of iodomethane according to the following scheme, the target dehydroalanine derivative 1 was obtained in the extract. That is, it was confirmed that the methyl ester of the substrate was broken down and moved to the aqueous phase during extraction. [ka] [ka]

[0141] (Example 2) <Production of polyamino acids with a dehydroalanine-like skeleton from dipeptides (investigation of solvent types)> We investigated the reaction process that converts a dipeptide containing a cysteine ​​residue into a dehydroalanine derivative in various solvents. Dipeptide 1a containing a cysteine ​​residue (the above-mentioned synthesized product, 0.3 mmol) was dissolved in various solvents (3.0 mL), heated to 35°C, and stirred for 5 minutes. The solvents used in each example are listed in the table. Subsequently, cesium carbonate (3 equivalents) was added to the heated solution and stirred under air at 35°C for 2 hours. After 2 hours of stirring, H2O (5 mL) was added to the reaction mixture. The reaction product was first extracted three times with ethyl acetate (5 mL). The aqueous phase was acidified with 1N HCl and then extracted three more times with ethyl acetate (5 mL). The ethyl acetate extract was dried over anhydrous sodium sulfate. After filtering to remove anhydrous sodium sulfate, the filtrate was concentrated. The residue was purified on silica gel by flash column chromatography (using hexane / ethyl acetate as the eluent) to obtain dehydroalanine derivative 1. The yield of the target dehydroalanine derivative 1, the enantiomeric excess (ee), and the enantiomer ratio (er), as well as the yield of the monomer or dimer 2 of the substrate, and the yield of the hydrolyzed carboxylic acid compound 3 of substrate 1a are shown in the table below. [ka] [Table 6]

[0142] In Examples 2-1 to 2-6, the target dehydroalanine derivative 1 was obtained regardless of the solvent used. Furthermore, the obtained target dehydroalanine derivative 1 had a very high enantiomeric excess (ee) and enantiomeric ratio (er), indicating that stereostructural changes were suppressed and the stereochemistry was maintained. In particular, it was found that the yield was excellent when using acetonitrile or acetone.

[0143] (Example 3) <Production of polyamino acids with a dehydroalanine-like skeleton from dipeptides (substrate investigation)> We investigated the reaction process that converts a dipeptide containing a cysteine ​​or serine residue into a dehydroalanine derivative using a substrate (SM). Various dipeptides containing cysteine ​​residues (0.3 mmol) were dissolved in acetonitrile (3.0 mL), heated to 35°C, and stirred for 5 minutes. The substrate species used in each example (1a-1j) are listed in the table. Subsequently, cesium carbonate (1 equivalent or 3 equivalents) was added to the warmed solution and stirred under air at 35°C for 2 hours. After 2 hours of stirring, H2O (5 mL) was added to the reaction mixture. The reaction product was first extracted three times with ethyl acetate (5 mL). The aqueous phase was acidified with 1N HCl and then extracted three more times with ethyl acetate (5 mL). The ethyl acetate extract was dried over anhydrous sodium sulfate. After filtering to remove anhydrous sodium sulfate, the filtrate was concentrated. The residue was purified on silica gel by flash column chromatography (using hexane / ethyl acetate as the eluent) to obtain dehydroalanine derivative a. The yield of the target dehydroalanine derivative a, the enantiomeric excess (ee), and / or the enantiomer ratio (er), as well as the yield of the monomer or dimer b of the substrate, are shown in the table below. [ka] R 1 The relevant part of the structure described in 1a to 1j below is R 2 This indicates -OH or -SH.

[0144] [Table 7]

[0145] The structures of 1a to 1j in the table are as follows. [ka]

[0146] In Examples 3-1 to 3-12, regardless of the substrate used, the target dehydroalanine derivative a was obtained in a single step reaction without degrading the amino acid structure. Furthermore, in all examples, the obtained target dehydroalanine derivative a had a very high enantiomeric excess (ee) and enantiomeric ratio (er), indicating that steric change was suppressed and the stereochemistry was maintained.

[0147] (Reference example 1) <Production of amino acids with a dehydroalanine-like skeleton from serine (Boc-Ser-OMe) (mechanochemical method)> 200 mg of methyl ester of serine with amino groups protected by Boc (Boc-Ser-OMe) 10 and 3 equivalents of Cs2CO3 were placed in a 5 mL ball mill container. Acetonitrile (in an amount equivalent to 5% by mass relative to the amount of Boc-Ser-OMe) was then added dropwise as a swelling solvent to moisten the entire powder. Two 10mm diameter crushing balls (manufactured by Verder Scientific Co., Ltd.) were placed in the container and sealed. The crushing machine was then vibrated at a frequency of 30Hz and allowed to react for 20 minutes. The reaction residue was dissolved in ethyl acetate, and the organic layer was washed by liquid-liquid separation. Then, amino acid 11, which has a dehydroalanine-like skeleton and is the target compound, was isolated by flash column chromatography. The conversion to the target compound was confirmed by NMR. [ka]

[0148] The conversion of amino acids to those possessing a dehydroalanine-like skeleton was possible using a mechanochemical method. These results suggest that, similarly, polyamino acids can also be converted to those possessing a dehydroalanine-like skeleton using a mechanochemical method.

[0149] (Example 4) <Production of polyamino acids with a dehydroalanine-like skeleton from dipeptides (mechanochemical method)> [Example 4-1] 116 mg of substrate 1j (Boc-Ala-Ser-OMe), 1.5 equivalents of Cs2CO3, and acetonitrile (5% by mass relative to the amount of substrate 1j) were placed in a 10 mL hardened steel grinding jar (Letsch) along with stainless steel grinding media (2 × φ10 mm) and ground using a mixer mill type ball mill (Mixer Mill MM 400 Control, Letsch) (30 Hz, 10 min). The reaction mixture was filtered by vacuum filtration (Kiriyama No. 5A φ95 mm filter paper, Kiriyama Seisakusho; PG201 diaphragm vacuum pump, Yamato Scientific Co., Ltd.) (washed with ethyl acetate) to obtain a pale yellow solution. This solution was concentrated under reduced pressure using a rotary vaporizer, and the residue was separated by autocolumn (Yamazen Co, Smart Flash EPCLC W-Prep 2XY) (n-hexane:ethyl acetate = 87:13, Rf = 0.2) to obtain dehydroalanine derivative 1 (yield 7%). Furthermore, the enantiomeric excess (ee) of the obtained target dehydroalanine derivative 1 was 98%. [ka]

[0150] From the above, it was found that polyamino acids with a dehydroalanine-like skeleton can be obtained using the mechanochemical method while suppressing conformational changes.

[0151] (Example 5) <Production of polyamino acids with a dehydroalanine-like skeleton from dipeptides (environmental investigation)> We investigated the reaction process that converts a dipeptide containing a serine residue into a dehydroalanine derivative under various atmospheric conditions. Dipeptide 14 (0.3 mmol) containing serine residues was dissolved in various solvents, heated to 35°C, and stirred for 5 minutes. The solvents used in each example and the concentrations of the substrate after dissolution are shown in the table. Subsequently, cesium carbonate (3 equivalents) was added to the heated solution and stirred at 35°C for 2 hours under the atmospheric conditions shown in the table. After 2 hours of stirring, H2O (5 mL) was added to the reaction mixture. The reaction product was first extracted three times with ethyl acetate (5 mL). The aqueous phase was acidified with 1N HCl and then extracted three more times with ethyl acetate (5 mL). The ethyl acetate extract was dried over anhydrous sodium sulfate. After removing anhydrous sodium sulfate by filtration, the filtrate was concentrated. The residue was purified on silica gel by flash column chromatography (using hexane / ethyl acetate as the eluent) to obtain dehydroalanine derivative 15. The yields of the target dehydroalanine derivative 15, the monomer or dimer 16 of the substrate, and the hydrolyzed carboxylic acid compound 17 of the substrate 14 are shown in the table below. [ka]

[0152] [Table 8]

[0153] From the above results, it can be seen that under nitrogen displacement conditions (Example 5-3), the yield of the target dehydroalanine derivative 15 is significantly lower compared to when the reaction is carried out in an air atmosphere using the same solvent (Example 5-2). Based on the above, it can be said that the yield of the desired dehydroalanine derivative is excellent when the reaction is carried out in an air atmosphere.

[0154] (Reference example 2) <Investigation of aza-Michael addition reactions to amino acids with a dehydroalanine-like skeleton> Glutathione derivative 12 (1.1 equivalents, 0.11 mmol) was added to amino acid 11 (0.1 mL) having a dehydroalanine-like skeleton in acetone (0.5 mL) / 0.1 M phosphate buffer (0.5 mL), and the reaction was carried out at 35 °C. After 7 hours, 2 mL of H2O was added, and liquid-liquid extraction was performed three times using ethyl acetate. After drying with sodium sulfate, the solvent was removed to obtain the crude product. The target product 13 was isolated and purified by silica gel column chromatography (yield: 87%). [ka]

[0155] We were able to react an amino acid having a dehydroalanine-like skeleton with the nucleophile glutathione derivative 12 via a thio-Michael addition reaction in high yield. These results suggest that, similarly, polyamino acids having a dehydroalanine-like skeleton that can be produced by the present invention can also react with a nucleophile via a thio-Michael addition reaction.

[0156] (Example 6) <Production of proteins containing a dehydroalanine-like skeleton and confirmation of the presence of the skeleton> [Synthesis of DhaBp] Cesium carbonate (Tokyo Chemical Industries, Ltd.) (340 mg, 1.05 mmol), the protein indicated by Sequence ID No. 29 (500 mg, 0.046 mmol), and DMSO (40 μL, 5 wt%) were placed in a 10 mL hardened steel grinding jar (Letcher) together with stainless steel grinding media (2 × φ10 mm) and ground using a mixer mill type ball mill (Mixer Mill MM 400 Control, Letcher) (30 Hz, 90 min). The reaction mixture was filtered by vacuum filtration (Kiriyama No. 5A φ95 mm filter paper, Kiriyama Seisakusho; PG201 diaphragm vacuum pump, Yamato Scientific Co., Ltd.) to obtain a white powder. This crude powder was thoroughly washed with saturated (NH4)2SO4 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (2 x 50 mL), RO water (RO Pure Rainbow, manufactured by Unimat Life Co., Ltd.) (2 x 50 mL), and acetone (2 x 50 mL, manufactured by Junsei Chemical Co., Ltd.). Then, it was treated in a vacuum oven (VOS-310C vacuum oven, EYELA Tokyo Rikakikai Co., Ltd.; GCD-051X oil rotary vacuum pump, ULVAC, Inc.) (12 h, 80 °C) to obtain DhaBp as an off-white fine amorphous powder. [ka]

[0157] The previously synthesized DhaBP was subjected to ball milling conditions at 30 Hz for 30 minutes in the presence of 4-F-BnNH2. Specifically, 4-F-BnNH2 (manufactured by Tokyo Chemical Industry Co., Ltd.) (49 μL, 0.43 mmol) / 4-F-benzyl mercaptan (manufactured by Tokyo Chemical Industry Co., Ltd.) (52 μL, 0.43 mmol) / tris(2-carboxyethyl)phosphine) (manufactured by Tokyo Chemical Industry Co., Ltd.) (107 mg, 0.43 mmol) and DhaBP (100 mg, 0.01 mmol) were placed in a 1.5 mL hardened steel grinding jar (manufactured by Lechner) together with stainless steel grinding media (2 × φ5 mm), and ground using a mixer mill type ball mill (Mixer Mill MM 400 Control, manufactured by Lechner) (30 Hz, 30 min). The reaction mixture was poured from the jar into acetone (Junsei Kagaku Co., Ltd.) (20 mL) and stirred for 10 minutes, after which the white powder was filtered off. This crude powder was washed with RO water (RO Pure Rainbow, Unimat Life Co., Ltd.) (2 × 20 mL) and acetone (2 × 20 mL, Junsei Kagaku Co., Ltd.), and the product was recovered by vacuum filtration (Kiriyama No. 5A φ95 mm filter paper, Kiriyama Seisakusho Co., Ltd.; PG201 diaphragm vacuum pump, Yamato Kagaku Co., Ltd.). The mixture was then treated in a vacuum oven (VOS-310C vacuum oven, EYELA Tokyo Rikakikai Co., Ltd.; GCD-051X oil rotary vacuum pump, ULVAC, Inc.) (12 h, 80 °C) to obtain a fine off-white amorphous powder as the synthesized compound.

[0158] Figure 2 shows 4-F-BnNH2 and the synthetic compound (Product). 19The 19F NMR spectrum is shown. A borosilicate NMR tube (standard 5φmm, outer diameter (mm) φ4.90~4.96, wall thickness (mm) 0.38, length (mm) 178±1, manufactured by Sankyo Co., Ltd.) was used to record the 19F nuclear magnetic resonance (NMR) spectrum in deuterated dimethyl sulfoxide (DMSO-d6) (manufactured by Sigma-Aldrich Co., Ltd.) using a JEOL Ltd. ECX-400 400MHz NMR spectrometer. Each peak was observed at a different position. Furthermore, when 1 μL of 4-F-BnNH2 was added to the NMR sample of the synthetic compound and the NMR was measured again, two peaks appeared. Since the top of the right-hand peak is coupled, it can be seen that this is derived from 4-F-BnNH2, and the left-hand peak is derived from the protein. From these findings, it was confirmed that aza-Michael addition occurred to the dehydroalanine-like structure in BP, resulting in the desired transformation.

[0159] 19 By adding fluorobenzene (internal standard) using 1F NMR, it was also possible to determine the 7% NMR yield (over 2 steps) after the conversion to a dehydroalanine-like structure (dha) and the addition of aza-Michael (Figure 3). [Industrial applicability]

[0160] This disclosure provides a method for producing polyamino acids having a dehydroalanine-like skeleton under mild reaction conditions, and a method for producing polyamino acid derivatives from the obtained polyamino acids having a dehydroalanine-like skeleton. This is a useful technique in that it facilitates protein structural transformation and increases the variety of structural transformations, and therefore has industrial applicability.

Claims

1. The process involves reacting compound A, which is a compound having a peptide bond and at least one substituent selected from the group consisting of a hydroxyl group and a thiol group, with an inorganic carbonate. In the above reaction, at least one of the substituents is directly removed by the inorganic carbonate. A method for producing polyamino acids having a dehydroalanine-like skeleton.

2. The process involves reacting compound A, which is a compound having a peptide bond and at least one substituent selected from the group consisting of a hydroxyl group and a thiol group, with an inorganic carbonate. In the above reaction, the mass of the inorganic carbonate added to the reaction system is 60 to 100% by mass of the total mass of compound A and other compounds added to the reaction system. A method for producing polyamino acids having a dehydroalanine-like skeleton.

3. The process involves reacting compound A, which is a compound having a peptide bond and at least one substituent selected from the group consisting of a hydroxyl group and a thiol group, with an inorganic carbonate. In the above reaction, the mass of the derivative-forming compound added to the reaction system is 0 to 40% by mass relative to the mass of compound A added to the reaction system. A method for producing polyamino acids having a dehydroalanine-like skeleton.

4. A method for producing a polyamino acid having a dehydroalanine-like skeleton according to any one of claims 1 to 3, wherein the reaction is carried out by a mechanochemical method.

5. A method for producing a polyamino acid having a dehydroalanine-like skeleton according to any one of claims 1 to 3, wherein the inorganic carbonate comprises at least one compound selected from the group consisting of cesium carbonate and potassium carbonate.

6. A method for producing a polyamino acid having a dehydroalanine-like skeleton according to any one of claims 1 to 3, wherein the compound A is a structural protein.

7. The method for producing a polyamino acid having a dehydroalanine-like skeleton according to claim 6, wherein compound A is a hydrophobic structural protein.

8. A method for producing a polyamino acid having a dehydroalanine-like skeleton according to claim 7, wherein the average hydroxyl index of the hydrophobic structural protein is greater than 0.

9. A method for producing a polyamino acid having a dehydroalanine-like skeleton according to any one of claims 1 to 3, wherein the reaction is carried out in an atmosphere containing air.

10. The method for producing a polyamino acid having a dehydroalanine-like skeleton according to any one of claims 1 to 3, This includes reacting the compound A after the above reaction with a nucleophile. A method for producing polyamino acid derivatives.

11. The method for producing a polyamino acid derivative according to claim 10 is included, This includes forming the compound A, after reaction with the nucleophile, into fibers. A method for manufacturing fibers.

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