JP7606207B2 - Cyclic diselenide compound, protein folding agent, and protein folding method - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act December 5, 2019 (Reiwa 1) Presented at the 46th Organic Representative Element Chemistry Conference Abstracts December 5-7, 2019 (Reiwa 1) Presented at the 46th Organic Representative Element Chemistry Conference July 13, 2020 Chem Asian J. Published on 2020, 15, 2646-2652 August 17, 2020 https://www. u-tokai. ac. jp/academics/undergraduate/science/news/detail/chemistry-an_asian_journal_important_paper.html

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.

The synthesized polypeptide chains express physiological activity by folding into the correct three-dimensional structure. Folding is a process in which disulfide (S-S)-related reactions between cysteine residues, i.e., S-S formation reactions and S-S isomerization reactions, 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 produced due to mismatched S-S bonds, making it often 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.

(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 the body 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., S-S formation reaction). On the other hand, GSH promotes the reaction of temporarily cleaving misaligned S-S bonds in protein molecules and replacing them with S-S bonds in the correct position (i.e., S-S isomerization reaction). Therefore, by dissolving a reduced protein (R) in which the S-S bonds of a protein have been cleaved in a solution in which these are mixed in an appropriate component ratio, R gradually folds into the native structure (N).

(2) Folding method using water-soluble thiol reagents (Non-Patent Documents 5 to 8)
As alternatives to GSH, which has a low S-S 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 S-S bonds, and the rate of oxidative folding can be improved by adding these reagents instead of GSH.

(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 the oxidative folding reaction. To rapidly complete the S-S bonds, water-soluble selenoxide reagents with high oxidative activity have been developed and applied to protein folding.

(4) Folding method using a water-soluble diselenide reagent (Non-Patent Documents 10 and 11)
As an alternative molecule to GSSG, which has a low S-S forming ability, a reagent that has a diselenide (Se-Se) bond instead of an S-S bond has been developed. Not only does the Se-Se bond have a high S-S forming ability, but the selenol (SeH) group generated as a by-product of the S-S forming reaction has a very high nucleophilicity, so it can effectively promote the reaction of temporarily cleaving and recombining mismatched S-S bonds in proteins (i.e., S-S isomerization reaction).

(5) Aggregation 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).

(6) Thiol molecules having aggregation inhibitory properties (Non-Patent Document 13 and Patent Document 1)
Urea-type thiol molecules are substitutes for GSH that have folding-promoting properties and also exhibit aggregation-inhibiting effects.

(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 exhibits high aggregation suppression effect and catalytic activity of oxidative folding even in a test tube. Therefore, PDI is sometimes used for protein synthesis.

JP 2019-210258 A

Lyles, M. M., and Gilbert, H. F. (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. Hevehan, D. L., and Clark, E. D. B. (1997) Oxidative renaturation of lysozyme at high concentrations. Biotechnol. Bioeng. 54, 221-230. Gurbhele-Tupkar, M. C., Perez, L. R., Silva, Y., and Lees, W. J. (2008) Rate enhancement of the oxidative folding of lysozyme by the use of aromatic thiol containing redox buffers. Bioorg. Med. Chem. 16, 2579- 2590. Kibria, F. M., and Lees, W. J. (2008) Balancing Conformational and Oxidative Kinetic Traps during the Folding of Bovine Pancreatic Trypsin Inhibitor (BPTI) with Glutathione and Glutathione Disulfide. J. Am. Chem. Soc. 130, 796-797. Lees, W. J. (2008) Small-molecule catalysts of oxidative protein folding. Curr. Opin. Chem. Biol. 12, 740-745. Madar, D. J., Patel, A. S., and Lees, W. J. (2009) Comparison of the oxidative folding of lysozyme at a high protein concentration using aromatic thiols versus glutathione. J. Biotechnol. 142, 214-219. Potempa, M., Hafner, M., and Frech, C. (2010) Mechanism of Gemini Disulfide Detergent Mediated Oxidative Refolding of Lysozyme in a New Artificial Chaperone System. Protein J. 29, 457-465. Iii, J. C. L., Andersen, K. A., Wallin, K. K., and Raines, R. T. (2014) Organocatalysts of oxidative protein folding inspired by protein disulfide isomerase. Org. Biomol. Chem. 12, 8598-8602. Arai, K., Dedachi, K., and Iwaoka, M. (2011) Rapid and Quantitative Disulfide Bond Formation for a Polypeptide Chain Using a Cyclic Selenoxide Reagent in an Aqueous Medium. Chem. Eur. J. 17, 481-485. Beld, J., Woycechowsky, K. J., and Hilvert, D. (2007) Selenoglutathione: Efficient Oxidative Protein Folding by a Diselenide. Biochemistry 46, 5382-5390. Reddy, P. S., and Metanis, N. (2016) Small molecule diselenide additives for in vitro oxidative protein folding. Chem. Commun. 52, 3336-3339. Hanpanich, O. and Maruyama, A. (2020) Artificial chaperones: From materials designs to applications. Biomaterials 254, 120150. Okada, S., Matsusaki, M., Arai, K., Hidaka, Y., Inaba, K., Okumura, M., and Muraoka, T. (2019) Coupling effects of thiol and urea-type groups for promotion of Oxidative protein folding. Chem. Commun. 55, 759-762. Arai, K., Takei, T., Shinozaki, R., Noguchi, M., Fujisawa, S., Katayama, H., Moroder, L., Ando, S., Okumura, M., Inaba, K., Hojo, H., and Iwaoka, M. (2018) Characterization and optimization of two-chain folding pathways of insulin via native chain assembly. Commun. Chem. 1, 1-11.

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 S-S forming ability of GSSG and the S-S 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 the cost and inefficiency of the purification process.

In the above-mentioned "(3) folding method using a water-soluble selenoxide reagent", when this reagent is used, a large amount of misfolded protein in which S-S bonds are crosslinked at random positions is generated before the S-S isomerization reaction proceeds, and therefore a physiologically active protein having a correct three-dimensional structure (native structure (N)) cannot be obtained.
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.

As described in the above "(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 molecule having aggregation inhibition ability (Non-Patent Document 13 and Patent Document 1)," it is necessary to add it at a high concentration of the order of several mM in order to achieve a sufficient folding promotion effect and aggregation inhibition 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 synthetic field, and therefore cannot be said to be a practical additive.

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 costs, an additive that can achieve an effective folding reaction with a very small amount would be ideal. However, as mentioned above, with all conventional compounds, in order to fully promote folding and suppress aggregation, it is necessary to add about 10 to 100 times the amount of reagent compared 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.

In the present invention, the aim is 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, the object of the present invention is to provide a cyclic diselenide compound, a protein folding agent, and a protein folding method 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 protein folding method that exhibit an aggregation inhibition effect.

As a result of intensive research conducted 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 S-S bonds and SH groups in proteins. The basic side chains (R) of these amino acid sites increase the reactivity of the selenium atom site, thereby enhancing the catalytic activity for S-S formation reactions and S-S isomerization reactions. Furthermore, these basic side chains (R), which are highly polar functional groups, form complexes with protein molecules through ionic interactions, thereby inhibiting association between protein molecules and thus suppressing the formation of aggregates. The present invention was completed based on the above findings.

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:

(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 is
The compound or salt thereof according to <1>,
<3> A protein-folding agent comprising the cyclic diselenide compound or a salt thereof according to <1> or <2>.
<4> A method for folding a protein, comprising a step of treating an unstructured protein in the presence of the cyclic diselenide compound or a salt thereof according to <1> or <2>.

The cyclic diselenide compound, protein folding agent, and protein folding method of the present invention can achieve rapid oxidative folding and aggregation inhibition effects with smaller addition amounts than conventional molecules.

FIG. 1 shows the results of a folding experiment of lysozyme (HEL). FIG. 2 shows HPLC chromatograms of samples obtained from oxidative folding experiments of reduced lysozyme (R ^HEL ) in the absence (a) and presence (b) of synthetic compound 1a, and refolding experiments of misfolded lysozyme (4SS ^HEL ) in the absence (c) and presence (d) of synthetic compound 1a. FIG. 3 shows the results of a competition test of the aggregation and folding reaction of HEL. FIG. 4 shows a comparison of the final enzyme activity recovery rates of HEL in samples obtained by oxidative folding of reduced lysozyme (R ^HEL ) at high concentrations.

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.
(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.)

The cyclic diselenide compound or salt thereof of the present invention can improve the oxidative folding rate with an extremely small amount of addition, such as several tens of μM. Furthermore, with an addition of 1 mM or less, it is possible to suppress aggregation of aggregating proteins and fold them effectively. According to the present invention, the oxidative folding step, which tends to be rate-limiting in protein synthesis, can be achieved simply, quickly, and with high yields.

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.

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.

The alkenylene group includes a linear or branched alkenyl group having 2 to 10 carbon atoms in which one hydrogen atom at any position in the carbon skeleton is replaced with one further bonding site to form a divalent moiety, and the main chain may have one or more double bonds. The number of carbon atoms in the alkenylene group is preferably 2 to 5. Examples of the linear or branched alkenyl group having 2 to 5 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.

The alkynylene group may be a linear or branched alkynyl group having 2 to 10 carbon atoms, in which one hydrogen atom at any position in 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.

R is preferably
Either:

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).

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.

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 may be used in combination of two or more cyclic diselenide compounds represented by formula (1) or salts thereof, if desired.

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.
The term "unstructured" means that the structure is not in a native structure having activity, and in the case of lysozyme, for example, it encompasses both reduced lysozyme (R ^HEL ) and 4SS lysozyme (4SS ^HEL ).

In this specification, "protein refolding" includes (1) the step of unfolding a protein, (2) the step of refolding the unfolded protein, and (3) the step of isolating the refolded protein. In this specification, the term "refolding reaction" refers to the above step (2).

The 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 or production method, such as natural or artificial (chemical synthesis, fermentation, or recombinant gene synthesis). 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 enzymes, recombinant proteins, and antibodies shown below.

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

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, interleukins 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.

In this specification, 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. If 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.

The molecular weight of the unfolded protein is not particularly limited, but typically ranges from 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 used for high molecular weight proteins because it can perform refolding with high efficiency.

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 the refolding agent, specifically, 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, to allow the refolding to proceed more fully.

The processing time for the above-mentioned processing 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.

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.

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

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 citrate, phosphate, and acetate; 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.

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 solution, or monosodium dihydrogen phosphate + sodium hydroxide 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 the solution containing the target protein (e.g., refolding buffer solution).

Examples of protein stabilizers include reducing agents such as glutathione, β-mercaptoethanol, dithiothreitol, TCEP, ascorbic acid, and cysteine. The content of such reducing agents when used is usually 0.01 to 100 mM, preferably 0.05 to 10 mM, and more preferably 0.1 to 5 mM, relative to the solution (refolding buffer) containing the target protein. When these reducing agents are contained in the refolding agent, the total amount of the reducing agent and the reducing agent contained therein is used. Examples of the protein stabilizers 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 chelating agents 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).

Furthermore, the solution containing the target protein (refolding buffer) may contain an unfolding agent, i.e., a denaturant (e.g., guanidine hydrochloride or urea). In this case, the content of the unfolding agent is usually 0.01 to 100 mM, preferably 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 the unfolded protein solution to dilute and reduce the unfolding agent concentration, or by dialyzing the unfolded protein suspension to dilute and reduce the unfolding agent concentration.

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, if necessary, after the step (hereinafter also referred to as an isolation step).

The isolation step may involve, for example, isolating the target normal protein (refolded protein) from the protein solution obtained in the 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.

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.

<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 saturated sodium chloride aqueous 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, 0.5 mL) was slowly added in an ice bath, and the mixture was stirred in the ice bath for 30 minutes, and then the temperature was raised to 25°C and stirred for another 4 hours. The solvent in the obtained solution was distilled off under reduced pressure to obtain compound 1.

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

<Identification of synthetic compound (1a)>
Mp 122.1~125.8℃; ¹ H NMR (500 MHz, D ₂ O): 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), ¹³ C NMR (125.8 MHz, D ₂ O): 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 N ₄ OSe ₂ ⁺ , 368.9727; found, 368.9743.

<Synthesis of (S)-2,6-diamino-N-((S)-1,2-diselenan-4-yl)hexanamide di-TFA salt (1b)>
The raw material compound 3 was Boc-L-Lys(Boc) (39.1 mg, 0.113 mmol), and the synthesis was carried out according to the general procedure for the synthesis of the above-mentioned compound. The crude product of the Boc-protected compound was obtained by the development solvent The crude product was purified by silica gel column chromatography using ethyl acetate/hexane (2:1) as a crude solvent to give the final product (1b) as a yellow powder (26.7 mg, 81%).

<Identification of synthetic compound (1b)>
Mp 110.1~113.8℃; ¹ H NMR (500 MHz, D ₂ O): 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), ¹³ C NMR (125.8 MHz, D ₂ O): 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, D ₂ O): δ 267.2 ppm; HRMS (ESI-TOF) m/z: [M+H -2TFA] ⁺ calcd for C ₁₀ H ₂₂ N ₃ OSe ₂ ⁺ , 360.0088; found, 360.0086.

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

<Identification of synthetic compound (1c)>
Mp 100.8~103.2℃; ¹ H NMR (500 MHz, D ₂ O): 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), ¹³ C NMR (125.8 MHz, D ₂ O): 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, D ₂ O): δ 268.1 ppm; HRMS (ESI-TOF) m/z: [M+H -2TFA] ⁺ calcd for C ₁₀ H ₂₁ N ₅ OSe ₂ ⁺ , 388.0149; found, 388.0152.

＜Synthesis of (S)-2-amino-N-((S)-1,2-diselenan-4-yl)propanamide TFA (1d)＞
The starting compound 3 was Boc-L-Ala (21.4 mg, 0.113 mmol) and synthesized according to the general procedure for the synthesis of the above compound. 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 compound (1d) as a yellow powder (21.9 mg, 94%).

<Identification of synthetic compound (1d)>
Mp 102.0~103.9℃; ¹ H NMR (500 MHz, D ₂ O): 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, D ₂ O): δ 169.3, 49.1, 48.9, 34.3, 26.0, 23.9, 16.5 ppm; ⁷⁷ Se NMR (95.4 MHz, D ₂ O): δ 265.1 ppm; HRMS (ESITOF) m/z: [M+H -TFA] ⁺ calcd for C ₇ H ₁₅ N ₂ OSe ₂ ⁺ , 302.9509; found, 302.9497.

<Preparation of reduced denatured lysozyme (R ^HEL )>
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 ^red ) (12.0 mg, Fujifilm Wako Pure Chemical Industries, Ltd.) was added and dissolved, and the mixture was reacted 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 resulting solution was freeze-dried to give reduced denatured lysozyme (R ^HEL , 10.0 mg) as a white solid.

<Preparation of misfolded lysozyme (4SS ^HEL )>
The powder of R- ^HEL (2 mg) prepared above 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 without urea (750 μL) was added and mixed well. The concentration of the R- ^HEL solution was determined using a UV-Vis spectrophotometer (ε ₂₈₀ = 33890 cm ^-1 M ^-1 ). A solution of trans-3,4-dihydroxytetrahydroselenophene-1-oxide (DHS ox; synthesized according to the literature [M. Iwaoka, et al., Heteroatom Chem. 2001, 19, 293-299] ⁾ (1 mL) prepared in the same buffer solution without urea to a concentration 20 times higher than the concentration of the R-HEL solution was added to the R- ^HEL solution (1 mL) and mixed, and the mixture was allowed to react 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, eluent: 0.1 M acetic acid aqueous solution). The obtained solution was lyophilized to obtain misfolded lysozyme (4SS ^HEL , 1.2 mg) in which SS bonds were randomly crosslinked within the protein molecule.

<Oxidative folding of reduced denatured lysozyme (R ^HEL )>
The catalytic effect of the synthesized compounds on oxidative folding was confirmed by the following method.
The synthetic compound (1a-d or 2), GSH, and GSSG were prepared in a 100 mM Tris-HCl buffer solution containing 1 mM EDTA, pH 7.5, and bubbled with argon gas to give concentrations of 200 μM, 5.0 mM, and 1.0 mM, respectively. The white powder of R ^HEL (1 mg) prepared above was dissolved in a 100 mM Tris-HCl buffer solution containing 2 M urea and 1 mM EDTA, pH 7.5. The R ^HEL concentration in the sample was determined using a UV-Vis spectrophotometer (ε ₂₈₀ = 33890 cm ^-1 M ^-1 ), and the sample was diluted with the same buffer solution to give a concentration of R ^HEL of 20 μM. The prepared R ^HEL 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 period of time, 48 μL of the sample solution was dispensed, and the reaction was stopped by adding 3 M hydrochloric acid (2 μL) and stored in a refrigerator. The amount of enzyme activity contained in the sample obtained at each time point 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.

The results are shown in Figure 1a and Table 1. Figure 1a shows the comparison of the enzyme activity recovery rate of HEL in the oxidative folding of reduced lysozyme (R ^HEL ). Reaction conditions: [R ^HEL ] ₀ =10 μM, [GSH] ₀ =1.0 mM, [GSSG] ₀ =0.20 mM, [Catalyst] = 20 μM, 37°C, pH 7.5, in the presence of 1 M urea. Data are shown as the mean ± standard error (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.

<Refolding of 4SS ^HEL >
The catalytic effect of the synthesized compounds on the refolding of misfolded forms was confirmed by the following method.
The synthetic compounds (1a-d or 2) and GSH were prepared in a 100 mM Tris-HCl buffer solution containing 1 mM EDTA, pH 7.5, with argon gas bubbling to give concentrations of 80 μM and 4.0 mM, respectively. The white powder of 4SS ^HEL (1 mg) prepared above was dissolved in a 100 mM Tris-HCl buffer solution containing 2 M urea and 1 mM EDTA, pH 7.5. The 4SS ^HEL concentration in the sample was determined using a UV-Vis spectrophotometer (ε ₂₈₀ = 33890 cm ^-1 M ^-1 ), and the sample was diluted with the same buffer solution to a concentration of 20 μM. The prepared 4SS ^HEL 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 period of time, 48 μL of the sample solution was dispensed, and the reaction was stopped by adding 3 M hydrochloric acid (2 μL) and stored in a refrigerator. The amount of enzyme activity contained in the sample obtained at each time point 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.

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 ± SEM (n = 3).
As in the case of FIG. 1a, when the time required for the enzyme activity to recover by 50%, t ₅₀ [min], was examined, histidine-conjugated 1a showed the greatest increase in reaction rate (Table 1).

The reaction rate constant k of oxidative folding is the apparent reaction rate constant obtained by fitting the experimental value (Fig. 1a) with %HEL activity = A _max (1-e ^-kt ), where A _max is the maximum enzyme activity of HEL recovered by folding. The time required for the enzyme activity to recover by 50%, t ₅₀ [min], is the time required for the enzyme activity of HEL to recover by 50% in the refolding reaction of 4SS ^HEL . All data in the table are shown as the mean ± standard error (n = 3).

<Evaluation of lysozyme folding yield by high performance liquid chromatography (HPLC)>
Oxidative folding of R ^HEL and refolding of 4SS ^HEL were initiated as previously described. 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 resulting sample solution was mixed with acetic acid (10 μL) and the pH of the solution was adjusted to 3.5. The sample was then desalted and purified using column chromatography (Sephadex G-50 DNA Grade F, GE Healthcare Life Sciences, eluent: 0.1 M acetic acid aqueous solution). The obtained sample solution was freeze-dried, and the 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 the literature (K. Arai, et al., Int. J. Mol. Sci. 2013, 14, 13194-13212).

Figure 2 shows HPLC chromatograms of samples obtained from the oxidative folding experiment of reduced lysozyme (R ^HEL ) in the absence (a) and presence (b) of synthetic compound 1a, and the refolding experiment of misfolded lysozyme (4SS ^HEL ) in the absence (c) and presence (d) of synthetic compound 1a. The reaction conditions in Figure 2 are the same as those in Figure 1.
In Figure 2, the components in the samples during the oxidative folding and refolding reactions of HEL were analyzed by HPLC. In both experiments, it was confirmed 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.

<Evaluation of the effect of synthetic compounds in inhibiting 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. The concentration of lysozyme was determined using a UV-Vis spectrophotometer (ε ₂₈₀ = 37600 cm ^-1 M ^-1 ) and diluted to 200 μM with the same buffer solution. Synthetic compounds (1a-d and 2) were prepared to 1.0 mM with 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 with the same buffer solution to confirm the concentration dependence. 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 samples were immediately left to stand at 23°C for 30 minutes. The sample solutions were centrifuged (14,000 rpm) for 3 minutes to sediment the insoluble precipitates, and the enzyme activity in the obtained samples was estimated with reference to the literature (K. Arai, et al., Chem. Asian J. 2014, 9, 3464-3471).

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 in (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 in (a) except for the concentration of compound 1a.
HEL is denatured by heating, and intermolecular association occurs, resulting in aggregation (white turbidity). After thermal denaturation, white precipitates due to aggregate formation were observed in the sample solutions containing no compound (Blank), as well as compound 2, compounds in which compound 2 was 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. On the other hand, no aggregate formation was observed when compound 1a was added (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).

<Oxidative folding of high-concentration R ^HEL accompanied by aggregation reaction>
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 in a 100 mM Tris-HCl buffer solution containing 1 mM EDTA, pH 7.5, with argon gas bubbling to give concentrations of 2.67 mM and 533 μM, respectively. The powder of R ^HEL (2 mg) prepared above was dissolved in a 100 mM Tris-HCl buffer solution containing 1 mM EDTA and 4 M urea, pH 7.5. The R ^HEL concentration in the sample was determined using a UV-Vis spectrophotometer (ε ₂₈₀ = 33890 ^{cm -1} M ^-1 ), and the sample was diluted with the same buffer solution to a concentration of 200 μM. The prepared R ^HEL 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, 3 M hydrochloric acid solution (2 μL) was added to stop the reaction, and the solution was stored in a refrigerator. The amount of enzyme activity contained in the sample obtained at each time point was estimated based on the literature (K. Arai, et al., Chem. Asian J.2014, 9, 3464-3471).

The results are shown in Figure 4.
The reaction conditions in Figure 4 were [R ^HEL ] ₀ =50 μM, [GSH] ₀ =1.0 mM, [Compound] ₀ =0.20 mM, 37°C, pH 7.5, and in the presence of 1 M urea. The reaction time was 24 hours when GSSG was used and 5 hours 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 Fig. 4, instead of GSSG, one of the synthesized compounds 1a-d was added and oxidative folding of HEL was carried out at a high concentration of 50 μM (0.7 mg/mL). Normally, such high concentration conditions cause R ^HEL to oligomerize and aggregate, making it impossible to achieve a sufficient folding yield. In fact, when GSSG/GSH (0.2/1.0 mM) was used, the folding yield was only about 25% after only 24 hours. On the other hand, when the synthetic compound 1/GSH (0.2/1.0 mM) was used, the folding yield increased to 55% after 5 hours of reaction time. 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) was used, it is considered that the synthetic compound 1 (especially 1a) suppressed the oligomerization and aggregate formation of HEL, thereby further improving the folding yield.

A folding step is essential to obtain a physiologically active protein from a polypeptide chain prepared by chemical synthesis or recombinant methods. 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 (4)

A cyclic diselenide compound represented by the following formula (1) or a salt thereof:
(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.) R

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