Cyclic diselenide compound, protein folding agent, and protein folding method

Abstract

To provide a cyclic diselenide compound that exhibits fast oxidative folding and aggregation inhibitory effects by small-quantity addition, a protein folding agent, and a protein folding method.SOLUTION: The present invention pertains to a cyclic diselenide compound represented by the formula (1) or a salt thereof (where R is a group represented by -L-X, or a group represented by -X, L is a C1-10 hydrocarbon group, X is an imidazole group, -NH2, -NH-C(=NH)-NH2, or -H).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cyclic diselenide compound having protein folding catalytic activity. The present invention further relates to a protein folding agent and a protein folding method. [Background technology]

[0002] The synthesized polypeptide chains express physiological activity by folding into the correct three-dimensional structure. Folding is a process in which disulfide (SS)-related reactions between cysteine ​​residues, i.e., SS formation reaction and SS isomerization reaction, are linked, and is also called oxidative folding. However, when oxidative folding reactions are performed in a test tube, protein molecules may become intricately entangled and precipitate as aggregates, or misfolded bodies may be generated due to mismatching of SS bonds, making it difficult to efficiently obtain the target protein. The following methods (1) to (7) have been reported as methods for solving such problems in protein folding.

[0003] (1) Folding method using a mixed solution of oxidized glutathione (GSSG) and reduced glutathione (GSH) (Non-Patent Documents 1 to 4) Glutathione is a redox compound that exists in living organisms and is inexpensively available, so it is widely used as a folding reagent. GSSG, which has oxidizing activity, promotes the reaction of converting thiol groups in polypeptide chains into disulfide bonds (i.e., SS formation reaction). On the other hand, GSH promotes the reaction of temporarily breaking SS bonds in misaligned protein molecules and replacing them with SS bonds in the correct position (i.e., SS isomerization reaction). Therefore, by dissolving a reduced protein (R) in which the SS bonds of a protein have been cleaved in a solution containing these components in an appropriate ratio, R gradually folds into the native structure (N).

[0004] (2) Folding method using water-soluble thiol reagents (Non-Patent Documents 5 to 8) As an alternative to GSH, which has a low SS isomerization ability, various organic thiol molecules have been developed. All of them are designed to improve the nucleophilicity of the thiol (SH) moiety for protein SS bonds, and the rate of oxidative folding can be improved by adding these reagents instead of GSH.

[0005] (3) Folding method using a water-soluble selenoxide reagent (Non-Patent Document 9) The low oxidative activity of GSSG is one of the decisive factors slowing down the rate of oxidative folding reactions. To rapidly complete the SS bond, water-soluble selenoxide reagents with high oxidative activity have been developed and applied to protein folding.

[0006] (4) Folding method using a water-soluble diselenide reagent (Non-Patent Documents 10 and 11) As an alternative molecule to GSSG, which has low SS-forming ability, reagents with diselenide (Se-Se) bonds instead of SS bonds have been developed. Se-Se bonds not only have high SS-forming ability, but also have highly nucleophilic selenol (SeH) groups generated as by-products of SS-forming reactions, so they can effectively promote reactions that temporarily cleave and recombine mismatched SS bonds in proteins (i.e., SS isomerization reactions).

[0007] (5) Coagulation inhibitors (Non-patent Document 12) In protein folding, a major problem is the loss of yield due to the formation of protein aggregates. For this reason, aggregation inhibitors such as urea and guanidine have been used in the past. However, these also cause the collapse of protein structure (unfolding), making them incompatible with the use of these agents to achieve protein structure formation (folding).

[0008] (6) Thiol molecules that have aggregation inhibitory properties (Non-Patent Document 13 and Patent Document 1) Urea-type thiol molecules are alternative molecules to GSH that have folding-promoting properties and also exhibit aggregation-inhibiting effects.

[0009] (7) Addition of Protein Disulfide Isomerase Catalyst (Non-Patent Document 14) Protein disulfide isomerase (PDI) is an enzyme that promotes the oxidative folding of proteins in vivo, and it shows high aggregation suppression effect and catalytic activity of oxidative folding even in a test tube. Therefore, PDI is sometimes used for protein synthesis. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] JP 2019-210258 A [Non-patent literature]

[0011] [Non-Patent Document 1] Lyles, MM, and Gilbert, HF (1991) Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer. Biochemistry 30, 613-619. [Non-Patent Document 2] Hevehan, DL, and Clark, EDB (1997) Oxidative renaturation of lysozyme at high concentrations. Biotechnol. Bioeng. 54, 221-230. [Non-Patent Document 3] Gurbhele-Tupkar, MC, Perez, LR, Silva, Y., and Lees, WJ (2008) Rate enhancement of the oxidative folding of lysozyme by the use of aromatic thiol containing redox buffers. Bioorg. Med. Chem. 16, 2579–2590.

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[0012] In the above-mentioned "(1) folding method using a mixed solution of oxidized glutathione (GSSG) and reduced glutathione (GSH) (Non-Patent Documents 1 to 4)," the SS formation ability of GSSG and the SS isomerization ability of GSH are not high, and it is necessary to add a large amount of reagent to the protein substrate, and it may take several hours to several tens of hours to achieve oxidative folding. The above-mentioned "(2) folding method using a water-soluble thiol reagent (Non-Patent Documents 5 to 8)" also requires the addition of a large amount of reagent to the protein substrate, which raises concerns about costs and inefficiencies in the purification process.

[0013] In the above-mentioned "(3) folding method using a water-soluble selenoxide reagent," when this reagent is used, a large amount of misfolded proteins in which S-S bonds are crosslinked at random positions are generated before the S-S isomerization reaction proceeds, making it impossible to obtain a physiologically active protein with the correct three-dimensional structure (native structure (N)). In the above-mentioned "(4) folding method using a water-soluble diselenide reagent (Non-Patent Documents 10 and 11)," a large amount of reagent needs to be added to the protein substrate, which raises concerns about the cost and inefficiency of the purification process. Furthermore, the conventional molecule does not have the aggregation-inhibiting activity described below.

[0014] As described above in "(5) Aggregation Inhibitors (Non-Patent Document 12)", many aggregation inhibitors that do not cause denaturation have been proposed, but they require the addition of large amounts of reagents (on the order of several mM to several M). In the above-mentioned "(6) Thiol molecules having aggregation-inhibiting ability (Non-Patent Document 13 and Patent Document 1)," it is necessary to add them at a high concentration of the order of several mM in order to achieve a sufficient folding-promoting effect and aggregation-inhibiting effect. Regarding the above-mentioned “(7) Addition of Protein Disulfide Isomerase catalyst (Non-Patent Document 14)”, PDI is too expensive for frequent use in the synthesis field and is therefore not a practical additive.

[0015] In the artificial preparation of proteins, it is important to fold the protein into the desired protein in high yield while suppressing the formation of aggregates. Furthermore, taking into consideration the purification process after folding and the cost, it is more ideal to have an additive that can achieve an effective folding reaction with a very small amount. However, as mentioned above, in order to fully promote folding and suppress aggregation, all conventional compounds require the addition of about 10 to 100 times the amount of reagent relative to the substrate protein. It is necessary to redesign the organic molecules to be added and develop additives that are sufficiently effective with smaller amounts.

[0016] In the present invention, it is an object of the present invention to synthesize a new additive that exhibits rapid oxidative folding and aggregation inhibition effects with smaller amounts added than conventional molecules, and to provide an efficient method for oxidative folding of proteins and a method for inhibiting aggregation. That is, an object of the present invention is to provide a cyclic diselenide compound, a protein folding agent, and a method for folding proteins that exhibit rapid oxidative folding with small amounts added. In addition, a non-essential secondary object of the present invention is to provide a cyclic diselenide compound, a protein folding agent, and a method for folding proteins that exhibit an aggregation inhibition effect. [Means for solving the problem]

[0017] As a result of intensive research by the present inventors to solve the above problems, the inventors have designed the following compound (1) by mimicking the amino acid sequence in the active center of PDI. This compound is a compound in which three basic amino acids (histidine (1a), lysine (1b), and arginine (1c)) are bonded to a cyclic diselenide site that has redox activity for the SS bond and SH group of a protein. The basic side chains (R) of these amino acid sites enhance the reactivity of the selenium atom site, thereby enhancing the catalytic activity for the SS formation reaction and the SS isomerization reaction. Furthermore, these basic side chains (R), which are highly polar functional groups, form complexes with protein molecules through ionic interactions, thereby inhibiting the association between protein molecules, and thus suppressing the formation of aggregates. The present invention was completed based on the above findings.

[0018] [ka]

[0019] That is, according to the present invention, the following inventions are provided. <1> A cyclic diselenide compound represented by the following formula (1) or a salt thereof: [ka] (In the formula, R represents a group represented by -LX or a group represented by -X, L represents a hydrocarbon group having 1 to 10 carbon atoms, and X represents an imidazole group, -NH2, -NH-C(=NH)-NH2, or -H.) <2> R [ka] Either <1> 2. The compound or a salt thereof according to claim 1 . <3> <1> or <2> 2. A protein-folding agent comprising the cyclic diselenide compound according to claim 1 or a salt thereof. <4> <1> or <2> 2. A method for folding a protein, comprising the step of treating an unstructured protein in the presence of the cyclic diselenide compound or a salt thereof according to claim 1. Effect of the Invention

[0020] The cyclic diselenide compound, protein-folding agent, and protein-folding method of the present invention can exert rapid oxidative folding and aggregation-inhibiting effects even when added in smaller amounts than conventional molecules. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 shows the results of a folding experiment of lysozyme (HEL). [Diagram 2] FIG. 2 shows HPLC chromatograms of samples obtained from the oxidative folding experiment of reduced lysozyme (RHEL) in the absence (a) and presence (b) of synthetic compound 1a and the refolding experiment of misfolded lysozyme (4SSHEL) in the absence (c) and presence (d) of synthetic compound 1a. [Diagram 3] FIG. 3 shows the results of a competition study of the aggregation and folding reactions of HEL. [Figure 4] FIG. 4 shows a comparison of the final enzyme activity recovery rates of HEL in samples obtained by oxidative folding of reduced lysozyme (RHEL) at high concentrations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention will now be described in further detail. The present invention relates to a cyclic diselenide compound represented by the following formula (1) or a salt thereof. The cyclic diselenide compound or a salt thereof of the present invention can be used as a protein folding agent. [ka] (In the formula, R represents a group represented by -LX or a group represented by -X, L represents a hydrocarbon group having 1 to 10 carbon atoms, and X represents an imidazole group, -NH2, -NH-C(=NH)-NH2, or -H.)

[0023] According to the cyclic diselenide compound or a salt thereof of the present invention, the oxidative folding rate can be improved by adding a very small amount of tens of μM. Furthermore, the aggregation of aggregating proteins can be suppressed and the proteins can be folded effectively by adding an amount of 1 mM or less. According to the present invention, the oxidative folding step, which tends to be the rate-limiting step in protein synthesis, can be achieved simply, quickly, and in high yield.

[0024] The hydrocarbon group having 1 to 10 carbon atoms represented by L may be either linear or branched, and may be either saturated or unsaturated. Examples of the hydrocarbon group having 1 to 10 carbon atoms include linear or branched alkylene groups having 1 to 10 carbon atoms, linear or branched alkenylene groups having 2 to 10 carbon atoms, and linear or branched alkynylene groups having 2 to 10 carbon atoms.

[0025] The alkylene group includes a linear or branched alkyl group having 1 to 10 carbon atoms, in which one hydrogen atom at any position of the carbon skeleton is replaced with one additional bonding site to form a divalent moiety. The number of carbon atoms in the alkylene group is preferably 1 to 5, more preferably 1 to 4. Examples of the linear or branched alkyl group having 1 to 10 carbon atoms include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl (sec-butyl), isobutyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and 1-ethylpropyl. Preferably, the linear or branched alkyl group having 1 to 5 carbon atoms is selected from the group consisting of methylene, ethylene, propylene, butylene, and pentylene.

[0026] The alkenylene group includes a divalent moiety formed by replacing one hydrogen atom at any position of the carbon skeleton of a linear or branched alkenyl having 2 to 10 carbon atoms with one further bond site, and the main chain has one or more double bonds. The number of carbon atoms in the alkenylene group is preferably 2 or more and 5 or less. Examples of the linear or branched alkenyl group having 2 or more and 5 or less carbon atoms include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, Examples of the alkenyl group include 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl, and positional isomers thereof. Preferably, the linear or branched alkenylene group having 2 to 5 carbon atoms is selected from the group consisting of ethenylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 3-butenylene, 1-pentenylene, 2-pentenylene, 3-pentenylene, and 4-pentenylene.

[0027] The alkynylene group includes a linear or branched alkynyl group having 2 to 10 carbon atoms, in which one hydrogen atom at any position of the carbon skeleton is replaced with one additional bonding site to form a divalent moiety, and the main chain may have one or more triple bonds. The number of carbon atoms in the alkynylene group is preferably 2 to 5. Examples of the linear or branched alkynyl group having 2 to 5 carbon atoms include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-2-butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, and 3-methyl-1-butynyl. Preferably, the linear or branched alkynylene group having 2 to 5 carbon atoms is selected from the group consisting of ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 3-butynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, and 4-pentynylene.

[0028] R is preferably [ka] Either:

[0029] The cyclic diselenide compound represented by formula (I) of the present invention or a salt thereof can be synthesized, for example, according to the method described in the following Examples, according to Scheme 1 below. That is, the cyclic diselenide compound represented by formula (I) of the present invention can be synthesized by mixing and reacting Boc-L-amino acid (3) dissolved in dehydrated DMF with (S)-1,2-diselenan-4-amine hydrochloride (2).

[0030] The cyclic diselenide compound represented by formula (I) may be in the form of a salt or a solvate. The salt is not particularly limited, and examples thereof include salts with inorganic acids such as hydrochloric acid, phosphoric acid, and sulfuric acid, and salts with organic acids such as acetic acid and trifluoroacetic acid. The cyclic diselenide compound represented by formula (I) can be isolated as an anhydride, but may also be in the form of a solvate, such as a hydrate.

[0031] The cyclic diselenide compound or a salt thereof of the present invention can be used as an active ingredient in a refolding agent that forms disulfide bonds in proteins. The refolding agent of the present invention contains at least one selected from the group consisting of cyclic diselenide compounds represented by formula (1) or salts thereof as an active ingredient, and two or more cyclic diselenide compounds represented by formula (1) or salts thereof may be used in combination, if desired.

[0032] According to the present invention, there is provided a method for folding a protein, which comprises the step of treating an unstructured protein in the presence of a cyclic diselenide compound or a salt thereof of the present invention. Unstructured means that the protein is not in a native structure that has activity. For example, in the case of lysozyme, it is reduced lysozyme (R HEL ) and 4SS lysozyme (4SS HEL ) meanings.

[0033] As used herein, "protein refolding" includes (1) a step of unfolding a protein, (2) a step of refolding the unfolded protein, and (3) a step of isolating the refolded protein. As used herein, the term "refolding reaction" refers to the above step (2).

[0034] The target proteins to be refolded using the cyclic diselenide compound or a salt thereof of the present invention include peptides, polypeptides, proteins, and complexes thereof, regardless of their origin, such as natural or artificial (chemical synthesis, fermentation, recombinant gene) or production method. The proteins may be of any type, and include, for example, intracellular proteins, extracellular proteins, membrane proteins, and nuclear proteins. The protein does not necessarily have to be a protein having a disulfide bond, but preferably includes a protein containing at least one disulfide bond. Specific examples include the following enzymes, recombinant proteins, and antibodies.

[0035] Examples of the enzymes include hydrolases, isomerases, oxidoreductases, transferases, synthases, and lyases. Examples of the hydrolases include proteases, serine proteases, amylases, lipases, cellulases, and glucoamylases. Examples of the isomerases include glucose isomerase. Examples of the oxidoreductases include protein disulfide isomerase and peroxidase. Examples of the transferases include acyltransferase and sulfotransferase. Examples of the synthases include fatty acid synthase, phosphate synthase, and citrate synthase. Examples of the lyases include pectin lyase.

[0036] Examples of recombinant proteins include protein preparations and vaccines. Recombinant proteins produced by genetic engineering using heterologous expression systems such as prokaryotes such as Escherichia coli, eukaryotes such as yeast, and cell-free extraction systems are often obtained as insoluble and inactive aggregates, so-called inclusion bodies, and therefore the refolding agent of the present invention can be suitably used. Examples of protein preparations include interferon α, interferon β, interleukin 1 to 12, growth hormone, erythropoietin, insulin, granulocyte-colony stimulating factor (G-CSF), tissue plasminogen activator (TPA), defensin, natriuretic peptides, blood coagulation factor 2, somatomedin, glucagon, growth hormone releasing factor, serum albumin, calcitonin, and the like. Examples of vaccines include hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine, and the like. Examples of antibodies include, but are not limited to, medical antibodies.

[0037] As used herein, the term "unfolded protein" refers to a protein unfolded by any method, but from the viewpoint of refolding effect, a protein unfolded by guanidine hydrochloride, urea, thiourea, or a combination of these is preferred. When the protein contains a disulfide bond in the molecule, the protein may be unfolded by adding a reducing agent such as β-mercaptoethanol, dithiothreitol, tris(2-carboxyethyl)phosphine (TCEP), or thiophenol in addition to the unfolding agent.

[0038] The molecular weight of the unfolded protein is not particularly limited, but may be about 1,000 to 10,000,000. From the viewpoint of refolding effect, the protein preferably has a molecular weight of 1,000 to 250,000. The cyclic diselenide compound or a salt thereof of the present invention can be applied to high molecular weight proteins since it can perform refolding with high efficiency.

[0039] In the refolding method of the present invention, the step of treating an unfolded protein in the presence of the refolding agent of the present invention (hereinafter also simply referred to as the treatment step) includes a step of contacting the unfolded protein with a refolding agent, and specifically includes a step of blending the unfolded protein and the refolding agent in a refolding buffer solution and mixing them by stirring or the like, and further includes a step of leaving the mixture to stand for a certain period of time after the step, if necessary, in order to proceed with refolding more sufficiently.

[0040] The treatment time for the above treatment step is not particularly limited as long as the refolding is sufficiently performed, but is, for example, 5 minutes to 24 hours, preferably 10 minutes to 3 hours. The temperature can be appropriately selected depending on the heat resistance of the target protein, and is, for example, in the range of 0 to 100°C, preferably in the range of 4 to 40°C.

[0041] The concentration of the cyclic diselenide compound or its salt in the refolding agent used in the above treatment step is not particularly limited, but is usually 0.001 to 100 mM, preferably 0.01 to 5 mM, relative to the solution containing the target protein (e.g., refolding buffer solution). The concentration of the unfolded protein contained in the solution containing the target protein (e.g., refolding buffer solution) is usually 0.01 to 100 μM, preferably 0.1 to 70 μM, more preferably 1 to 50 μM.

[0042] The refolding buffer is not particularly limited as long as it has a concentration and composition that does not cause the function of the target protein to be lost, and specific examples include amine buffers such as Tris buffer, MES buffer, HEPES buffer solution, and Tricine buffer, phosphate buffer, various Good's buffers, etc. The pH of the refolding buffer can be adjusted usually to a range of pH 4 to 10, preferably a range of pH 5 to 9, and more preferably a range of pH 7 to 9.

[0043] In addition to the refolding agent of the present invention, various additives can be added to the refolding buffer solution. Examples of such additives include salts such as sodium chloride and calcium chloride; buffer solutions such as citrates, phosphates, and acetates; bases such as sodium hydroxide; acids such as hydrochloric acid and acetic acid; and organic solvents such as methanol, ethanol, and propanol. In addition to the refolding agent of the present invention and the additives, the buffer solution can also contain a surfactant, a pH adjuster, or a protein stabilizer. Those skilled in the art can adjust the amount of use as appropriate.

[0044] Examples of pH adjusters include Tris (N-tris(hydroxymethyl)methylaminoethanesulfonic acid), HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), and phosphate buffers (e.g., disodium monohydrogen phosphate + hydrochloric acid aqueous solution, or monosodium dihydrogen phosphate + sodium hydroxide aqueous solution). When a pH adjuster is used, the content is adjusted to a pH range of 4 to 10, preferably a pH range of 5 to 9, and more preferably a pH range of 7 to 9, and is usually 0.01 to 200 mM, preferably 0.05 to 150 mM, and more preferably 0.1 to 100 mM, relative to a solution containing the target protein (e.g., a refolding buffer solution).

[0045] Examples of the protein stabilizer include reducing agents such as glutathione, β-mercaptoethanol, dithiothreitol, TCEP, ascorbic acid, and cysteine. The content of such a reducing agent in the solution (refolding buffer solution) containing the target protein is usually 0.01 to 100 mM, preferably 0.05 to 10 mM, and more preferably 0.1 to 5 mM. When such a reducing agent is contained in the refolding agent, the total amount of the reducing agent and the reducing agent contained therein is used. Examples of the protein stabilizer include polyols, metal ions, and chelating agents. Examples of polyols include glycerin, glucose, sucrose, ethylene glycol, polyethylene glycol, sorbitol, and mannitol. Examples of metal ions include divalent metal ions such as magnesium ions, manganese ions, and calcium ions. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA) and glycol ether diamine-N,N,N',N'-tetraacetic acid (EGTA). When a protein stabilizer is used, the content is usually 10% by mass or less, preferably 0.001 to 10% by mass, and more preferably 0.01 to 1% by mass, relative to the solution containing the target protein (refolding buffer solution).

[0046] The solution (refolding buffer) containing the target protein may contain an unfolding agent, i.e., a denaturing agent (e.g., guanidine hydrochloride or urea). In this case, the content of the unfolding agent is usually 0.01 to 100 mM, preferably The concentration is 0.05 to 10 mM, more preferably 0.1 to 5 mM. The concentration may be adjusted by adding the refolding agent of the present invention to an unfolded protein solution to dilute and reduce the concentration of the unfolding agent, or by dialyzing the unfolded protein suspension to dilute and reduce the concentration of the unfolding agent.

[0047] The present invention also relates to a method for regenerating a protein, comprising a step of treating and refolding a protein in the presence of the cyclic diselenide compound of the present invention or a salt thereof. The method for regenerating a protein of the present invention comprises a step of treating and refolding a protein in the presence of the cyclic diselenide compound of the present invention or a salt thereof (hereinafter also referred to as a refolding step), and further comprises a step of isolating the refolded protein after the step, if necessary (hereinafter also referred to as an isolation step).

[0048] An example of the isolation step is isolating the target normal protein (refolded protein) from the protein solution obtained in the above refolding step by using column chromatography or the like. Packing materials used in column chromatography include silica, dextran, agarose, cellulose, acrylamide, vinyl polymers, and the like. Commercially available products include the Sephadex series, Sephacryl series, and Sepharose series (all from Pharmacia), and the Bio-Gel series (Bio-Rad). The target normal protein (refolded protein) may also be isolated by dialysis.

[0049] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. EXAMPLES

[0050] [ka]

[0051] <Basic steps in compound synthesis> The synthesis was carried out according to Scheme 1 above. In a 10 mL glass eggplant flask, Boc-L-amino acid (3) (Watanabe Chemical, 0.113 mmol) was dissolved in dehydrated DMF (Fujifilm Wako Pure Chemical, 1.5 mL). The starting material (S)-1,2-diselenan-4-amine hydrochloride (2) (synthesized according to reference [K. Arai, et al., ChemBioChem 2018, 19, 207-211], 0.0150 g, 0.0565 mmol) was added and stirred. PyBOP (Watanabe Chemical, 0.0588 g, 0.113 mmol) and DIPEA (Fujifilm Wako Pure Chemical, 39.4 μL, 0.226 mmol) were added to the resulting solution, and the flask was replaced with argon gas, and then stirred at 25 °C for 15 hours. 15 mL of distilled water was added, and extraction was performed with dichloromethane (Kanto Chemical, 15 mL, 3 times). The obtained dichloromethane solution was washed with a saturated aqueous sodium chloride solution (20 mL, 1 time), and further dehydrated by passing through an ISOLUTE(R) Phase Separator. (Biotage Japan), and the solvent was distilled off under reduced pressure. The obtained yellow liquid was purified by silica gel column chromatography (neutral silica gel, Kanto Chemical, developing solvent: ethyl acetate / hexane) to obtain the Boc-protected compound of compound 1 as a yellow oil. To deprotect the Boc group, the obtained yellow oil was dissolved in dichloromethane (Kanto Chemical, 0.8 mL) in a 10 mL glass eggplant-shaped flask, trifluoroacetic acid (Fujifilm Wako Pure Chemical Industries, Ltd., 0.5 mL) was slowly added in an ice bath, and the mixture was stirred in the ice bath for 30 minutes, after which the temperature was raised to 25°C and further stirred for 4 hours. The solvent in the obtained solution was distilled off under reduced pressure to obtain compound 1.

[0052] <Synthesis of (S)-2-amino-N-((S)-1,2-diselenan-4-yl)-3-(1H-imidazol-4-yl)propenamide di-TFA salt (1a)> The starting compound 3 was synthesized according to the general procedure for the synthesis of the above compounds using Boc-L-His(Boc) dicyclohexylammonium salt (60.6 mg, 0.113 mmol). The crude Boc-protected product was purified by silica gel column chromatography using ethyl acetate / hexane, 8:1, as the developing solvent to obtain the final target product (1a) as a brown powder (31.0 mg, 91%).

[0053] <Identification of Synthetic Compound (1a)> Mp 122.1-125.8℃; 1 H NMR (500 MHz, D2O): δ 8.60 (d, J=1.3 Hz, 1H), 7.31 (d, J=0.5 Hz, 1H), 4.12-4.06 (m, 1H), 3.94-3.39 (m, 1H), 3.28-3.16 (m, 3H), 3.11-3.06 (m, 1H), 2.80-2.77 (m, 1H), 2.72-2.66 (m, 1H), 2.16-2.10 (m, 1H), 1.79-1.72 (m, 1H) ppm; 13 C NMR (125.8 MHz, D2O): δ 166.3, 134.2, 126.0, 118.3, 52.1, 48.7, 33.9, 26.0, 25.7, 23.3 ppm; 77 Se NMR (95.4 MHz, D2O): δ 269.3 ppm; HRMS (ESI-TOF) m / z: [M+H -2TFA] + calcd for C 10 H 17 N4OSe2 + , 368.9727; found, 368.9743.

[0054] <Synthesis of (S)-2,6-diamino-N-((S)-1,2-diselenan-4-yl)hexanamide di-TFA salt (1b)> The starting compound 3 was Boc-L-Lys(Boc) (39.1 mg, 0.113 mmol) and synthesized according to the general procedure for the synthesis of the above compounds. The crude Boc-protected product was purified by silica gel column chromatography using ethyl acetate / hexane (2:1) as the developing solvent to obtain the final target product (1b) as a yellow powder (26.7 mg, 81%).

[0055] <Identification of synthetic compound (1b)> Mp 110.1-113.8℃; 1 H NMR (500 MHz, D2O): δ 3.96-3.91 (m, 1H), 3.80 (t, J=6.7 Hz, 1H), 3.22-3.10 (m, 2H), 3.00-2.83 (m, 4H), 2.19-2.13 (m, 1H), 1.83-1.71 (m, 3H), 1.59-1.52 (m, 2H), 1.39-1.25 (m, 2H) ppm; 13 C NMR (125.8 MHz, D2O): δ 168.1, 52.9, 49.0, 38.9, 34.2, 30.3, 26.3, 25.9, 23.8, 21.3 ppm; 77 Se NMR (95.4 MHz, D2O): δ 267.2 ppm; HRMS (ESI-TOF) m / z: [M+H -2TFA] + calcd for C 10 H 22 N3OSe2 + , 360.0088; found, 360.0086.

[0056] <Synthesis of (S)-2-amino-N-((S)-1,2-diselenan-4-yl)-5-guanidinopentanamide di-TFA salt (1c)> The starting compound 3 was synthesized according to the general procedure for the synthesis of the above compound using Boc-L-Arg(Boc)2 (53.6 mg, 0.113 mmol). The crude Boc-protected product was purified by silica gel column chromatography using ethyl acetate / hexane, 2:1, as the developing solvent to obtain the final target product (1c) as a yellow solid (29.4 mg, 85%).

[0057] <Identification of synthetic compound (1c)> Mp 100.8-103.2℃; 1 H NMR (500 MHz, D2O): δ 3.99-3.94 (m, 1H), 3.85 (t, J=6.6 Hz, 1H), 3.25-3.21 (m, 1H), 3.16-3.08 (m, 3H), 2.97-2.89 (m, 2H), 2.21-2.16 (m, 1H), 1.87-1.77 (m, 3H), 1.56-1.48 (m, 2H) ppm; 13 C NMR (125.8 MHz, D2O): δ 167.9, 156.7, 52.8, 52.7, 48.9, 40.2, 34.1, 27.9, 26.0, 23.5 ppm; 77 Se NMR (95.4 MHz, D2O): δ 268.1 ppm; HRMS (ESI-TOF) m / z: [M+H -2TFA] + calcd for C 10 H 21 N5OSe2 + , 388.0149; found, 388.0152.

[0058] <Synthesis of (S)-2-amino-N-((S)-1,2-diselenan-4-yl)propanamide TFA (1d)> The starting compound 3 was synthesized according to the general procedure for the synthesis of the above compound using Boc-L-Ala (21.4 mg, 0.113 mmol). The crude Boc-protected product was purified by silica gel column chromatography using ethyl acetate / hexane, 2:1, as the developing solvent to obtain the final target product (1d) as a yellow powder (21.9 mg, 94%).

[0059] <Identification of synthetic compound (1d)> Mp 102.0-103.9℃; 1 H NMR (500 MHz, D2O): δ 3.97-3.88 (m, 2H), 3.24-3.13 (m, 2H), 2.98-2.89 (m, 2H), 2.21-2.16 (m, 1H), 1.85-1.77 (m, 1H), 1.39 (d, J=7.1 Hz 3H) ppm; 13 C NMR (125.8 MHz, D2O): δ 169.3, 49.1, 48.9, 34.3, 26.0, 23.9, 16.5 ppm; 77 Se NMR (95.4 MHz, D2O): δ 265.1 ppm; HRMS (ESITOF) m / z: [M+H -TFA] + Calculate for C7H 15 N2OSe2 + , 302.9509; found, 302.9497.

[0060] <Reduced denatured lysozyme (R HEL Preparation of The lysozyme was prepared according to the following procedure, as described in [K. Arai, et al., Int. J. Mol. Sci. 2013, 14, 13194-13212]. Commercially available hen egg white lysozyme (12.0 mg, Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 100 mM Tris-HCl buffer solution pH 8.0 (700 μL) containing 8 M urea and 1 mM EDTA, and the reducing agent (±)-dithiothreitol (DTT) was added. red) (Fujifilm Wako Pure Chemical Industries, Ltd., 12.0 mg) was added and dissolved, and the reaction was carried out at 40.0°C for 2 hours. The reaction was stopped by adding 0.1 M acetic acid aqueous solution (1.3 mL) to the obtained sample solution, and the sample was desalted and purified by gel filtration chromatography (Sephadex G-50 DNA Grade F, GE Healthcare Life Sciences, developing solvent: 0.1 M acetic acid aqueous solution). The obtained solution was lyophilized to obtain reduced denatured lysozyme (R HEL , 10.0 mg) was obtained.

[0061] <Misfolded Lysozyme (4SS HEL Preparation of The R prepared above HEL The powder (2 mg) was dissolved in 200 mM acetic acid-sodium acetate buffer solution (pH 4.0) (250 μL) containing 8 M urea and 1 mM EDTA, and then the same buffer solution (750 μL) without urea was added and mixed well. 280 = 33890 cm -1 M -1 ) to R HEL The concentration of the solution was determined. A 20-fold increase in the concentration of trans-3,4-dihydroxytetrahydroselenophene-1-oxide (DHS) was prepared in the same buffer solution without urea. ox (Synthesized according to the literature [M. Iwaoka, et al., Heteroatom Chem. 2001, 19, 293-299]) solution (1 mL) was HEL The mixture was added to the solution (1 mL) and mixed, and reacted at room temperature for 15 minutes. The obtained sample solution was desalted and purified by gel filtration chromatography (Sephadex G-50 DNA Grade F, GE Healthcare Life Sciences, developing solvent: 0.1 M aqueous acetic acid). The obtained solution was lyophilized to obtain misfolded lysozyme in which SS bonds are randomly crosslinked within the protein molecule (4SS HEL , 1.2 mg) was obtained.

[0062] <Reduced denatured lysozyme (R HEL) Oxidative folding The catalytic effect of the synthesized compounds on oxidative folding was confirmed by the following method. The synthetic compounds (1a to d and 2), GSH, and GSSG were prepared at 200 μM, 5.0 mM, and 1.0 mM, respectively, using a 100 mM Tris-HCl buffer solution, pH 7.5, containing 1 mM EDTA and bubbled with argon gas. HEL The white powder (1 mg) was dissolved in 100 mM Tris-HCl buffer solution (pH 7.5) containing 2 M urea and 1 mM EDTA. HEL The concentration was measured using a UV-visible absorption altimeter (ε 280 = 33890 cm -1 M -1 ) and R HEL The adjusted R was diluted with the same buffer solution to a concentration of 20 μM. HEL The solution (500 μL), synthetic compound solution (100 μL), GSH solution (200 μL), and GSSG solution (200 μL) were quickly mixed and reacted at 37.0°C. After a certain time, 48 μL of the sample solution was dispensed, and the reaction was stopped by adding 3 M hydrochloric acid aqueous solution (2 μL), and the solution was stored in a refrigerator. The amount of enzyme activity contained in the sample obtained at each time was estimated based on the literature (K. Arai, et al., Chem. Asian J. 2014, 9, 3464-3471), and the relative enzyme activity recovery rate was plotted against the reaction time.

[0063] The results are shown in Figure 1a and Table 1. Figure 1a shows the reduced lysozyme (R HEL ) shows the comparison of the enzyme activity recovery rate of HEL in the oxidative folding of [R HEL [GSH]0 = 10 μM, [GSSG]0 = 0.20 mM, [Catalyst] = 20 μM, 37 °C, pH 7.5, in the presence of 1 M urea. Data are presented as mean ± SEM (n = 3). Since the enzyme activity of protein enzymes is restored by folding, the rate of enzyme activity recovery can be considered as the folding rate. It can be seen that the folding reaction is greatly accelerated by adding synthetic compounds 1 or 2. Furthermore, the addition of a catalytic amount of the commonly used GSSG shows no effect. Furthermore, it can be seen that histidine-conjugated 1a promotes the reaction more than other catalysts. In fact, when the reaction rate constant (k) of this oxidative folding was estimated (Table 1), the rate of increase in the reaction rate was greatest when synthetic compound 1a was used.

[0064] <4SS HEL Refolding of > The catalytic effect of the synthesized compounds on the refolding of misfolded forms was confirmed by the following method. The synthetic compound (1a-d or 2) and GSH were prepared at 80 μM and 4.0 mM, respectively, in a 100 mM Tris-HCl buffer solution (pH 7.5) containing 1 mM EDTA and bubbled with argon gas. HEL (1 mg) of the white powder was dissolved in 100 mM Tris-HCl buffer solution, pH 7.5, containing 2 M urea and 1 mM EDTA. HEL The concentration was measured using a UV-visible absorption altimeter (ε 280 = 33890 cm -1 M -1 ) and 4SS HEL The 4SS solution was diluted with the same buffer solution to a concentration of 20 μM. HEL The solution (500 μL), synthetic compound solution (250 μL), and GSH solution (250 μL) were quickly mixed and reacted at 37.0°C. After a certain time, 48 μL of the sample solution was dispensed, and the reaction was stopped by adding 3 M hydrochloric acid aqueous solution (2 μL) and stored in a refrigerator. The amount of enzyme activity contained in the samples obtained at each time was estimated based on the literature (K. Arai, et al., Chem. Asian J. 2014, 9, 3464-3471), and the relative enzyme activity recovery rate was plotted against the reaction time.

[0065] The results are shown in FIG. Figure 1b shows the rate of recovery of enzyme activity of HEL upon refolding of misfolded lysozyme (4SS). Reaction conditions: [4SS]0 = 10 μM, [GSH]0 = 1.0 mM, [Catalyst] = 20 μM, 37°C, pH 7.5, in the presence of 1 M urea. Data are shown as mean ± standard error (n = 3). As in Figure 1a, the time required for the enzyme activity to recover by 50% t 50 Looking at [min], 1a conjugated with histidine showed the greatest increase in reaction rate (Table 1).

[0066] [Table 1]

[0067] The reaction rate constant k for oxidative folding is %HEL activity = A max (1-e -kt ) to the experimental values ​​(Fig. 1a), and A max is the maximum enzyme activity of HEL restored by folding. The time required for the enzyme activity to recover by 50% is t 50 [min] is 4SS HEL The time required for the enzyme activity of HEL to be restored by 50% in the refolding reaction of 100 μg / ml. All data in the table are shown as the mean ± standard error (n = 3).

[0068] <Evaluation of lysozyme folding yield by high performance liquid chromatography (HPLC)> R HEL Oxidative folding and 4SS of HELRefolding of lysozyme was started according to the method described previously. After a certain time, 200 μL of the reaction solution was dispensed and transferred to a 1.5 mL plastic tube containing 2-aminoethyl methanethiosulfonate (AEMTS; synthesized according to the literature [M. Iwaoka, et al., Heteroatom Chem.2001, 19, 293-299]) aqueous solution (7 mg / mL, 200 μL). The obtained sample solution was mixed with acetic acid (10 μL) and the pH of the solution was adjusted to 3.5, and then desalted and purified using column chromatography (Sephadex G-50 DNA Grade F, GE Healthcare Life Sciences, developing solvent: 0.1 M acetic acid aqueous solution). The obtained sample solution was lyophilized, and the resulting residue was dissolved in 0.1% trifluoroacetic acid aqueous solution (1100 μL), and the folding yield of lysozyme was estimated by high performance liquid chromatography (HPLC) analysis using a reversed phase column. The HPLC analysis conditions were based on those described in the literature (K. Arai, et al., Int. J. Mol. Sci. 2013, 14, 13194-13212).

[0069] Figure 2 shows the reduced lysozyme (R ) in the absence (a) and presence (b) of synthetic compound 1a. HEL ) and misfolded lysozyme (4SS) in the absence (c) and presence (d) of synthetic compound 1a. HEL 2 shows the HPLC chromatograms of the samples obtained from the refolding experiment of (A). The reaction conditions in FIG. 2 are the same as those in FIG. 1. In Figure 2, we used HPLC to analyze the components in the samples during the oxidative folding and refolding reactions of HEL. In both experiments, it can be seen that the N-isomer was produced more quickly in the presence of synthetic compound 1a. This result is consistent with the evaluation of the folding rate of HEL evaluated from the enzyme activity recovery rate.

[0070] <Evaluation of the inhibitory effect of synthetic compounds on the aggregation of heat-denatured lysozyme> The protein aggregation inhibitory effect of the synthetic compounds was confirmed by the following method. Commercially available hen egg white lysozyme (5.0 mg, Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 50 mM Tris-HCl buffer solution, pH 7.5 (1.0 mL), containing 0.5 mM EDTA and 0.28 M NaCl. 280 = 37600 cm -1 M -1 ) to determine the concentration of lysozyme, and diluted it to 200 μM using the same buffer solution. The synthetic compounds (1a-d and 2) were prepared to 1.0 mM using the same buffer solution. For synthetic compound 1a, 0.04, 0.1, 0.2, 0.4, 0.6, and 0.8 mM were also prepared using the same buffer solution to confirm the concentration dependency. The prepared lysozyme solution (400 μL) and 400 μL of the compound solution were added to a 1.5 mL plastic tube and incubated at 30, 50, 70, 80, and 90 °C for 5 minutes. The heated sample was immediately left at 23 °C for 30 minutes. The sample solution was centrifuged (14,000 rpm) for 3 minutes to sediment the insoluble precipitate, and the amount of enzyme activity in the obtained sample was estimated based on the literature (K. Arai, et al., Chem. Asian J. 2014, 9, 3464-3471).

[0071] The results are shown in Figure 3. Figure 3(a) shows a photograph of HEL solution (100 μM) in 50 mM Tris-HCl buffer solution pH 7.5 containing 0.28 M NaCl and 0.5 mM EDTA obtained by heating at 90 °C for 10 min and then cooling to 23 °C. Figure 3(b) shows the relative enzyme activity recovery rate of HEL in the sample solution obtained from the same experiment as (a). Data are shown as mean ± standard error (n = 3). Figure 3(c) shows the concentration dependence of compound 1a on the aggregation of HEL after heat denaturation and the enzyme activity recovery rate. The reaction conditions were the same as (a) except for the concentration conditions of compound 1a. HEL is denatured by heating, and intermolecular association occurs, resulting in aggregation (white turbidity). In the sample solution containing no compound (Blank), as well as compound 2, compounds in which compound 2 is conjugated with an amino acid other than histidine (1b-d), a mixture of compound 2 and the amino acid histidine (2+His), and sample solutions containing guanidine hydrochloride (Gdn-HCl), which is often used as an aggregation inhibitor, white precipitation due to aggregate formation was observed after thermal denaturation. On the other hand, no aggregate formation was confirmed by adding compound 1a (a). Furthermore, when the enzyme activity recovery rate was estimated, the enzyme activity was recovered to almost 100% in the presence of compound 1a (b). Furthermore, it was found that the aggregation inhibition effect of 1a was sufficient even at an extremely low concentration of 0.3 mM (c).

[0072] <High concentration R accompanied by agglutination reaction HEL Oxidative folding of The oxidative folding and aggregation inhibitory effects of the synthesized compounds were confirmed by the following methods. The synthetic compounds (1a-d and 2, or GSSG) and GSH were prepared to 2.67 mM and 533 μM, respectively, in a 100 mM Tris-HCl buffer solution, pH 7.5, containing 1 mM EDTA and bubbled with argon gas. HEL The powder (2 mg) was dissolved in 100 mM Tris-HCl buffer solution (pH 7.5) containing 1 mM EDTA and 4 M urea. HEL The concentration was measured using a UV-visible absorption altimeter (ε 280 = 33890 cm -1 M -1 ) and R HEL The adjusted R was diluted with the same buffer solution to a concentration of 200 μM. HELThe solution (50 μL), synthetic compound solution (75 μL), and GSSG solution (75 μL) were quickly mixed and reacted at 37.0°C. After 5 and 24 hours, 48 ​​μL of the sample solution was dispensed, and the reaction was stopped by adding 3 M hydrochloric acid aqueous solution (2 μL), and stored in a refrigerator. The amount of enzyme activity contained in the samples obtained at each time was estimated with reference to the literature (K. Arai, et al., Chem. Asian J.2014, 9, 3464-3471).

[0073] The results are shown in Figure 4. The reaction conditions in Figure 4 are [R HEL [GSH]0 = 50 μM, [GSH]0 = 1.0 mM, [Compound]0 = 0.20 mM, 37 °C, pH 7.5, in the presence of 1 M urea. The reaction time was 24 h when GSSG was used and 5 h for the others. Data are shown as the mean ± SEM (n = 3). Buffer solutions containing GSSG and GSH are widely used as solvents for oxidative folding of proteins. In Figure 4, instead of GSSG, one of the synthesized compounds 1a to 1d was added, and oxidative folding of HEL was carried out at a high concentration of 50 μM (0.7 mg / mL). Normally, under such high concentration conditions, R HEL However, the folding yield was not sufficient because of oligomerization and aggregation of HEL. In fact, when GSSG / GSH (0.2 / 1.0 mM) was used, the folding yield was only about 25% after 24 hours. On the other hand, when synthetic compound 1 / GSH (0.2 / 1.0 mM) was used, the folding yield increased to 55% after 5 hours of reaction. Considering that the folding yield was only about 37% when 2 / GSH (0.2 / 1.0 mM) or 2 / His / GSH (0.2 / 0.2 / 1.0 mM) were used, it can be considered that synthetic compound 1 (especially 1a) suppressed oligomerization and aggregation of HEL, thereby further improving the folding yield. [Industrial Applicability]

[0074] A folding step is essential to obtain a physiologically active protein from a polypeptide chain prepared by chemical synthesis or recombinant techniques. According to the present invention, protein folding is possible. The cyclic diselenide compound, protein folding agent, and protein folding method of the present invention are useful in the artificial preparation of novel proteins that are beneficial in the fields of structural biology and drug discovery.

Claims

1. A cyclic diselenide compound represented by the following formula (1) or a salt thereof: 【Chemistry 1】 (In the formula, R represents a group represented by -L-X or a group represented by -X, L represents a hydrocarbon group having 1 to 10 carbon atoms, and X represents an imidazole group, -NH ２ , -NH-C(=NH)-NH ２ , or -H.)

2. R 【Chemistry 2】 The compound or salt thereof according to claim 1,

3. A protein-folding agent comprising the cyclic diselenide compound or a salt thereof according to claim 1 or 2.

4. A method for folding a protein, comprising the step of treating an unstructured protein in the presence of the cyclic diselenide compound or a salt thereof according to claim 1 or 2.

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