JP2022089310A - Cyclic diselenide compound, protein folding agent, and protein folding method - Google Patents

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

Application for application of Article 30, Paragraph 2 of the Patent Act December 5, 2019 (Reiwa 1st year) Announced in the abstracts of the 46th Organic Typical Element Chemistry Conference December 5, 2019 (Reiwa 1st year) 7th Announced at the 46th Debate on Typical Organic Element Chemistry July 13, 2020 (Reiwa 2nd year) Chem Asian J. et al. Announced at 2020, 15, 2646-2652 August 17, 2020 (Reiwa 2) https: // www. u-tokai. ac. jp / academics / undergraduate / science / news / data / chemistry-_an_asia_jonalvery_important_paper. Announced at html

The present invention relates to cyclic diselenide compounds having protein folding catalytic activity. Further, the present invention relates to a protein folding agent and a protein folding method.

The synthesized polypeptide chain expresses physiological activity by folding (folding) into the correct three-dimensional structure. Folding is a linkage of disulfide (SS) -related reactions between cysteine residues, that is, SS formation reaction and SS isomerization reaction, and is also called oxidative folding. However, when an oxidative folding reaction is carried out in vitro, protein molecules may be intricately entangled with each other and precipitate as an aggregate, or a misfolded form may be formed due to a misplaced SS bond. In many cases, it is difficult to obtain the target protein efficiently. As a method for solving such a problem in protein folding, the methods described in the following (1) to (7) have been reported.

(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 also exists in the living body and is widely used as a folding reagent because it can be obtained at low cost. GSSG having oxidative activity promotes a reaction for converting a thiol group in a polypeptide chain into a disulfide bond (that is, an SS formation reaction). On the other hand, GSH temporarily cleaves the SS bond in the wrong protein molecule and promotes a reaction (that is, an SS isomerization reaction) in which the SS bond is replaced with the SS bond at the correct position. Therefore, by dissolving the reduced protein (R) in which the SS bond of the protein is cleaved in a solution in which these are mixed at an appropriate component ratio, R gradually folds into the natural structure (N).

(2) Folding method using a water-soluble thiol reagent (Non-Patent Documents 5 to 8)
Various organic thiol molecules have been developed as alternative molecules for GSH having low SS isomerization ability. Both are molecularly designed to improve the nucleophilicity of the thiol (SH) moiety for protein SS binding, and the addition of these reagents in place of GSH improves the rate of oxidative folding. can do.

(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 that slow down the rate of oxidative folding reaction. In order to complete the SS bond rapidly, a water-soluble selenoxide reagent having a high oxidizing ability has 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 having a low ability to form SS, a reagent having a diselenide (Se—Se) bond instead of the SS bond has been developed. The Se-Se bond not only has a high ability to form SS, but also the selenol (SeH) group produced as a by-product of the SS formation reaction has a very high nucleophilicity, so that it is mistaken in the protein. It is possible to effectively promote a reaction (that is, an SS isomerization reaction) in which the SS bond is temporarily cleaved and recombined.

(5) Aggregation inhibitor (Non-Patent Document 12)
In protein folding, the decrease in yield due to the formation of protein aggregates becomes a major problem. Therefore, conventionally, aggregation inhibitors such as urea and guanidine have been used. However, these have contradictory properties as reagents for achieving structure formation (folding) because the structural collapse (unfolding) of the protein also exceeds the pull at the same time.

(6) Thiol molecule including aggregation inhibitory ability (Non-Patent Document 13 and Patent Document 1)
The urea-type thiol molecule is a substitute molecule for GSH having a folding promoting ability, and also exhibits an aggregation inhibitory effect.

(7) Addition of Protein Disulfide Isomerase catalyst (Non-Patent Document 14)
Protein Disulfide Isomerase (PDI) is an enzyme that promotes oxidative folding of proteins in vivo, and exhibits high aggregation inhibitory effect and catalytic activity of oxidative folding even in vitro. Therefore, PDI may be used for protein synthesis.

Japanese Unexamined Patent Publication No. 2019-210258

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 salts 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 "(1) Folding method using a mixed solution of oxidized glutathione (GSSG) and reduced glutathione (GSH) (Non-Patent Documents 1 to 4)", the SS forming ability of GSSG and the S of GSH The -S isomerization ability was not high, a large amount of reagent was required to be added to the protein substrate, and it sometimes took several hours to several tens of hours to achieve oxidative folding.
Also in the above-mentioned "(2) Folding method using a water-soluble thiol reagent (Non-Patent Documents 5 to 8)", it is necessary to add a large amount of reagent to the protein substrate, which is not cost effective and is not in the purification process. There is concern about efficiency.

In the above "(3) Folding method using a water-soluble selenoxide reagent", when this reagent is used, a mistake is made in which the SS bond is crosslinked at a random position before the SS isomerization reaction proceeds. Since a large amount of folded bodies are produced, it is not possible to obtain a physiologically active protein having a correct three-dimensional structure (natural structure (N)).
Also in the above-mentioned "(4) Folding method using water-soluble diselenide reagent (Non-Patent Documents 10 and 11)", it is necessary to add a large amount of reagent to the protein substrate, which is not cost effective and non-purifying step. There is concern about efficiency. Furthermore, conventional molecules do not have the aggregation inhibitory activity described later.

As described in the above "(5) Aggregation inhibitor (Non-Patent Document 12)", many aggregation inhibitors that do not cause denaturation have been proposed, but it is necessary to add a large amount (several mM to several M orders) of a reagent. Is.
In the above-mentioned "(6) Thiol molecule including aggregation inhibitory ability (Non-Patent Document 13 and Patent Document 1)", in order to achieve a sufficient folding promoting effect and aggregation suppressing effect, a high concentration on the order of several mM is used. Needs to be added.
Regarding the above-mentioned "(7) Addition of protein disulfide-isomerase catalyst (Non-Patent Document 14)", PDI is too expensive to be frequently used in the synthetic field, and therefore cannot be said to be a practical additive.

In the artificial preparation of proteins, it is important how to suppress the formation of aggregates and fold them into the target protein in high yield. Further, in consideration of the purification process after folding and the cost aspect, an additive capable of achieving an effective folding reaction with a very small amount is more ideal. However, as described above, all of the conventional compounds require the addition of a reagent about 10 to 100 times as much as that of the substrate protein in order to sufficiently achieve the promotion of folding and the suppression of aggregation. It is necessary to revise the design of the organic molecule to be added and develop an additive that exerts a sufficient effect with a smaller amount.

In the present invention, it is intended to synthesize a new additive that exhibits rapid oxidative folding and aggregation inhibitory effect with a smaller addition amount than conventional molecules, and to provide an efficient protein oxidative folding method and aggregation inhibitory method. I aimed. That is, an object of the present invention is to provide a cyclic disselenide compound, a protein folding agent, and a protein folding method that exhibit rapid oxidative folding with a small amount of addition. Further, as a non-essential secondary problem of the present invention, it is to provide a cyclic disselenide compound, a protein folding agent, and a protein folding method that exert an aggregation inhibitory effect.

As a result of diligent studies to solve the above problems, the present inventor designed the compound (1) shown below by mimicking the amino acid sequence in the active center of PDI. This compound contains three basic amino acids (histidine (1a), lysine (1b) and arginine (1c)) at the cyclic diselenide site that has redox activity for the SS bond and SH group of the protein. They are joined together. The basic side chain (R) of these amino acid moieties enhances the reactivity of the selenium atom moiety, 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, inhibit the association between protein molecules by forming a complex with the protein molecule by ionic interaction, and thus suppress the formation of aggregates. Can be done. The present invention has been completed based on the above findings.

（式中、Ｒは、－Ｌ－Ｘで示される基、または－Ｘで示される基を示し、Ｌは炭素数１～１０の炭化水素基を示し、Ｘはイミダゾール基、－ＮＨ_２、－ＮＨ－Ｃ（＝ＮＨ）－ＮＨ_２、または－Ｈを示す。）
＜２＞ Ｒが

の何れかである、＜１＞に記載の化合物又はその塩。
＜３＞ ＜１＞又は＜２＞に記載の環状ジセレニド化合物又はその塩を含む、タンパク質フォールディング剤。
＜４＞ ＜１＞又は＜２＞に記載の環状ジセレニド化合物又はその塩の存在下において、構造未形成タンパク質を処理する工程を含む、タンパク質のフォールディング方法。 That is, according to the present invention, the following invention is 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, X represents an imidazole group, -NH ₂ , -. NH-C (= NH) -NH ₂ or -H.)
<2> R is

The compound according to <1> or a salt thereof, which is any of the above.
<3> A protein folding agent containing the cyclic diselenide compound according to <1> or <2> or a salt thereof.
<4> A method for folding a protein, which comprises a step of treating a structure-unformed protein in the presence of the cyclic disselenide compound according to <1> or <2> or a salt thereof.

According to the cyclic disselenide compound, the protein folding agent, and the protein folding method of the present invention, rapid oxidative folding and aggregation inhibitory effect can be exhibited with a smaller amount of addition than conventional molecules.

FIG. 1 shows the results of a lysozyme (HEL) folding experiment. FIG. 2 shows an oxidative folding experiment of reduced lysoteam (R ^HEL ) in the absence (a) and presence (b) of synthetic compound 1a, and in the absence (c) and presence (d) of synthetic compound 1a. The HPLC chromatogram of the sample obtained from the refolding experiment of misfolded resoteam (4SS ^HEL ) in) is shown. FIG. 3 shows the results of a competitive test of HEL agglutination and folding reaction. FIG. 4 shows a comparison of the final HEL enzyme activity recovery rates in samples obtained by oxidative folding of high concentrations of reduced lysozyme (R ^HEL ).

（式中、Ｒは、－Ｌ－Ｘで示される基、または－Ｘで示される基を示し、Ｌは炭素数１～１０の炭化水素基を示し、Ｘはイミダゾール基、－ＮＨ_２、－ＮＨ－Ｃ（＝ＮＨ）－ＮＨ_２を、または－Ｈを示す。） Hereinafter, the present invention will be described in more detail.
The present invention relates to a cyclic disselenide compound represented by the following formula (1) or a salt thereof, and the cyclic disselenide 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, X represents an imidazole group, -NH ₂ , -. NH-C (= NH) -NH ₂ or -H.)

According to the cyclic diselenide compound of the present invention or a salt thereof, the oxidative folding rate can be improved with a very small amount of addition of several tens of μM. Further, with an addition amount of 1 mM or less, aggregation of aggregated proteins can be suppressed and effective folding can be achieved. According to the present invention, the oxidative folding step, which tends to be rate-determining in protein synthesis, can be easily, quickly, and in high yield.

The hydrocarbon group having 1 to 10 carbon atoms indicated by L may be either a straight chain or a branched chain, and may be saturated or unsaturated.
Hydrocarbon groups having 1 to 10 carbon atoms include an alkylene group having 1 to 10 carbon atoms in a straight chain or a branched chain, an alkaneylene group having 2 to 10 carbon atoms in a straight chain or a branched chain, and a linear or branched chain. Examples thereof include an alkynylene group having 2 or more and 10 or less carbon atoms.

As the alkylene group, one hydrogen atom at an arbitrary position in the carbon skeleton of a linear or branched alkyl group having 1 or more and 10 or less carbon atoms is replaced with one additional bond site to form a divalent moiety. What is forming is mentioned. The carbon number of the alkylene group is preferably 1 or more and 5 or less, and more preferably 1 or more and 4 or less. Examples of the linear or branched alkyl group having 1 or more and 10 or less carbon atoms are not limited to these, but 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, 1-ethylpropyl, etc. Can be mentioned. Preferably, the linear or branched alkylene group having 1 or more and 5 or less carbon atoms is selected from the group consisting of methylene, ethylene, propylene, butylene and pentylene.

As the alkenylene group, one hydrogen atom at an arbitrary position in the carbon skeleton of a linear or branched alkenyl having 2 or more and 10 or less carbon atoms is replaced with one additional bond site to form a divalent moiety. The main chain may have one or more double bonds. The carbon number of the alkenylene group is preferably 2 or more and 5 or less. Examples of the above-mentioned linear or branched alkenyl group having 2 or more and 5 or less carbon atoms are not limited to these, but are 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, 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 their positional isomers can be mentioned. Preferably, the linear or branched alkenylene group having 2 or more and 5 or less carbons is ethenylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 3-butenylene, 1-. It is selected from the group consisting of pentenylene, 2-pentenylene, 3-pentenylene and 4-pentenylene.

As the alkynylene group, one hydrogen atom at an arbitrary position in the carbon skeleton of a linear or branched alkynyl group having 2 or more and 10 or less carbon atoms is replaced with one additional bond site to form a divalent moiety. The main chain may have one or more triple bonds. The carbon number of the alkynylene group is preferably 2 or more and 5 or less. Examples of the linear or branched alkynyl group having 2 or more and 5 or less carbon atoms are not limited to these, but ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, and the like. 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- Examples thereof include methyl-1-butynyl. Preferably, the linear or branched alkynylene group having 2 or more and 5 or less carbon atoms is ethynylene, 1-propinylene, 2-propinylene, 1-butynylene, 2-butynylene, 3-butynylene, 1-pentynylene, 2 -Selected from the group consisting of pentinylene, 3-pentinylene and 4-pentinylene.

の何れかである。 R is preferably

Is either.

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

The cyclic diselenide compound represented by the 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 disselenide compound represented by the formula (I) can be isolated as an anhydrate, but may be a solvate. Examples of the solvate include hydrates.

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

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for folding a protein, which comprises a step of treating a structurally unformed protein in the presence of the cyclic disselenide compound of the present invention or a salt thereof.
Structural non-formation means that it does not have an active native structure, for example, in the case of lysozyme, it includes both reduced lysozyme (R ^HEL ) and 4SS lysozyme (4SS ^HEL ). ..

As used herein, "protein refolding" refers to (1) a step of unfolding a protein, (2) a step of refolding an unfolded protein, and (3) isolating the refolded protein. It shall include the steps to be performed. In the present specification, the term "refolding reaction" means the above step (2).

The protein to be refolded using the cyclic diselenide compound of the present invention or a salt thereof is a peptide or polypeptide regardless of the origin or production method of natural or artificial (chemical synthesis method, fermentation method, gene recombination method) or the like. , Proteins, and complexes thereof. The type of protein is not limited, and for example, intracellular protein, extracellular protein, membrane protein, and nuclear protein are all included. The protein does not necessarily have to be a protein having a disulfide bond, but preferably a protein containing at least one disulfide bond can be mentioned. Specific examples thereof include enzymes, recombinant proteins and antibodies shown below.

Examples of the enzyme include hydrolases, isomerizing enzymes, oxidoreductases, transferases, synthases and desorption enzymes. Examples of the hydrolase include protease, serine protease, amylase, lipase, cellulase, glucoamylase and the like. Examples of the isomerase include glucose isomerase. Examples of the redox enzyme include protein disulfide isomerase and peroxidase. Examples of the transferase include acyltransferase, sulfotransferase and the like. Examples of the synthase include fatty acid synthase, phosphate synthase, citrate synthase and the like. Examples of the desorbing enzyme include pectin lyase and the like.

Examples of recombinant proteins include protein preparations, vaccines and the like. Recombinant proteins produced genetically engineered using prokaryotes such as Escherichia coli, eukaryotes such as yeast, and heterologous expression systems such as cell-free extraction systems are often obtained as insoluble and inactive aggregates, so-called inclusion bodies. Therefore, the refolding agent of the present invention can be preferably used. Protein preparations include interferon α, interferon β, interleukins 1-12, growth hormone, erythropoetin, insulin, granular colony stimulating factor (G-CSF), tissue plasminogen activator (TPA), defensin, and sodium diuresis. Examples thereof include peptides, blood coagulation second factor, somatomedin, glucagon, growth hormone releasing factor, serum insulin, and calcitonin. Examples of the vaccine include hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine and the like. The antibody is not particularly limited, and examples thereof include medical antibodies.

As used herein, the "unfolded protein" may be a protein unfolded by any method, but from the viewpoint of refolding effect, it is unfolded with guanidine hydrochloride, urea, thiourea or a combination thereof. Protein is preferred. When the protein contains a disulfide bond in the molecule, in addition to the above unfolding agent, β-mercaptoethanol, dithiothreitol, tris (2-carboxyethyl) phosphine (TCEP), thiophenol, etc. It may be an unfolded protein with the addition of a reducing agent.

The unfolded protein does not particularly limit its molecular weight, but usually includes a protein of about 1,000 to 10,000,000. From the viewpoint of refolding effect, it is preferably a protein having a molecular weight of 1,000 to 250,000. According to the cyclic diselenide compound of the present invention or a salt thereof, refolding can be performed with high efficiency, so that it can be applied to a high molecular weight protein.

In the refolding method of the present invention, as a step of treating the unfolded protein in the presence of the refolding agent of the present invention (hereinafter, also simply referred to as a treatment step), the unfolded protein and the refolding agent are used. Specific examples thereof include a step of blending the unfolded protein and the refolding agent in a refolding buffer and mixing them by stirring or the like, and further, after the step, if necessary. It also includes a step of allowing it to stand for a certain period of time in order to proceed with refolding more sufficiently.

The processing time of the above 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 tolerance 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 disselenide compound or a salt thereof in the refolding agent used in the above treatment step is not particularly limited, but is usually 0.001 to 100 mM, preferably 0.001 to 100 mM with respect to a solution containing the target protein (for example, a refolding buffer solution). It is 0.01 to 5 mM. The concentration of the unfolding protein contained in the solution containing the target protein (for example, a refolding buffer solution) is usually 0.01 to 100 μM, preferably 0.1 to 70 μM, and more preferably 1 to 50 μM.

The refolding buffer is not particularly limited unless it has a concentration and composition that causes the function of the target protein to be lost, and specifically, amines such as Tris buffer, MES buffer, HEPES buffer, and Tricin buffer. Examples thereof include system buffers, phosphate buffers, and various Good's buffers. The pH of the refolding buffer can be adjusted usually in the range of pH 4 to 10, preferably in the range of pH 5 to 9, and more preferably in the range of pH 7 to 9.

In addition to the refolding agent of the present invention, various additives can be added to the refolding buffer solution. Such additives include salts such as sodium chloride and calcium chloride; buffers such as citrate, phosphate and acetate; basics such as sodium hydroxide; acids such as hydrochloric acid and acetic acid; methanol, Examples thereof include organic solvents such as ethanol and propanol. Further, in addition to the refolding agent and the additive of the present invention, the buffering agent may contain a surfactant, a pH adjuster, or a protein stabilizer. Those skilled in the art can appropriately adjust the amount used.

Examples of the pH adjusting agent include Tris (N-tris (hydroxymethyl) methylaminoethanesulfonic acid), HEPES (N-2-hydroxyethylpiperazin-N'-2-ethanesulfonic acid), and a phosphate buffering agent (N-2-hydroxyethylpiperazin-N'-2-ethanesulfonic acid). For example, 1 hydrogen phosphate 2 sodium + hydrochloric acid aqueous solution, or phosphoric acid 2 hydrogen 1 sodium + sodium hydroxide aqueous solution) and the like can be mentioned. When a pH regulator is used, the content is adjusted to be in the range of pH 4 to 10, preferably pH 5 to 9, more preferably pH 7 to 9, and a solution containing the target protein (for example, a refolding buffer solution). On the other hand, it is usually 0.01 to 200 mM, preferably 0.05 to 150 mM, and more preferably 0.1 to 100 mM.

Examples of the protein stabilizer include reducing agents such as glutathione, β-mercaptoethanol, dithiothreitol, TCEP, ascorbic acid and cysteine. When such a reducing agent is used, the content is usually 0.01 to 100 mM, preferably 0.05 to 10 mM, more preferably 0.1 to 0.1, based on the solution containing the target protein (refolding buffer). It is 5 mM. If these reducing agents are contained in the refolding agent, the total amount with the contained reducing agent shall be used. Examples of the protein stabilizer include polyols, metal ions, chelating reagents and the like. Examples of the polyols include glycerin, glucose, sucrose, ethylene glycol, polyethylene glycol, sorbitol, mannitol and the like. Examples of the metal ion include divalent metal ions such as magnesium ion, manganese ion and calcium ion. Examples of the chelating reagent include ethylenediaminetetraacetic acid (EDTA) and glycol etherdiamine-N, N, N', N'-4 acetic 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 0.01% by mass, based on the solution containing the target protein (refolding buffer). It is 1% by mass.

Further, the solution containing the target protein (refolding buffer) may contain an unfolding agent, that is, a denaturing agent (for example, guanidine hydrochloric acid or urea). In this case, the content of the unfolding agent is usually 0.01 to 100 mM, preferably 0.05 to 10 mM, and 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 lower the unfolding agent concentration, or dial the unfolded protein suspension. The unfolding agent concentration may be diluted and lowered to adjust the concentration.

The present invention also relates to a method for regenerating a protein, which comprises a step of treating and refolding the protein in the presence of the cyclic disselenide compound of the present invention or a salt thereof. The method for regenerating a protein of the present invention is a step of treating and refolding a protein in the presence of the cyclic disselenide compound of the present invention or a salt thereof (hereinafter, also referred to as a refolding step), and further, it is necessary after the step. A step of isolating the protein refolded by the above (hereinafter, also referred to as an isolation step) is included.

Examples of the isolation step include isolation of a target normal protein (refolding protein) from the protein solution obtained in the above refolding step by using column chromatography or the like. Examples of the filler used for column chromatography include silica, dextran, agarose, cellulose, acrylamide, vinyl polymer and the like. Examples of commercially available commercially available products include Sephadex series, Sephadex series, Sepharose series (hereinafter referred to as Pharmacia), Bio-Gel series (Bio-Rad) and the like. Further, the target normal protein (refolding protein) may be isolated by a dialysis method.

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

<Basic procedure in compound synthesis>
The synthesis was carried out according to Scheme 1 above. Boc-L-amino acid (3) (Watanabe Kagaku, 0.113 mmol) was dissolved in dehydrated DMF (Fuji Film Wako Pure Chemical Industries, Ltd. 1.5 mL) in a 10 mL glass eggplant-shaped flask. Starting material (S) -1,2-diselenan-4-amine hydrochloride (2) (synthesized by Ref. [K. Arai, et al., ChemBioChem 2018, 19, 207-211], 0.0150 g, 0.0565 mmol) Was added and stirred. PyBOP (Watanabe Kagaku, 0.0588 g, 0.113 mmol) and DIPEA (Fuji Film Wako Pure Chemical Industries, Ltd., 39.4 μL, 0.226 mmol) were added to the obtained solution, the inside of the flask was replaced with argon gas, and then the temperature was 25 ° C. The mixture was stirred 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 aqueous sodium chloride solution (20 mL, once), dehydrated by passing it through ISOLUTE (R) Phase Separator. (Biotage Japan), and then distilled off under reduced pressure. The obtained yellow liquid was produced by silica gel column chromatography (neutral silica gel, Kanto Chemical Co., Inc., developing solvent: ethyl acetate / hexane) to obtain a Boc-protected product of Compound 1 as a yellow oil. In order to deprotect the Boc group, the obtained yellow oil was dissolved in dichloromethane (Kanto Kagaku, 0.8 mL) in a 10 mL volumetric glass eggplant-shaped flask, and trifluoroacetic acid (Fuji Film 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, the temperature was raised to 25 ° C., and the mixture was further stirred for 4 hours. Compound 1 was obtained by distilling off the obtained solution under reduced pressure.

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

<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), 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, 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, D ₂ O): 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)>
Boc-L-Lys (Boc) (39.1 mg, 0.113 mmol) was used as the raw material compound 3, and the synthesis was carried out according to the above basic procedure for compound synthesis. The crude product of the Boc-protected product is purified by silica gel column chromatography using ethyl acetate / hexane, 2: 1 as the developing solvent, and the final target product (1b) is obtained as a yellow powder (26.7 mg, 81%). Obtained.

<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), 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, 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)>
Boc-L-Arg (Boc) ₂ (53.6 mg, 0.113 mmol) was used as the raw material compound 3, and the synthesis was carried out according to the basic procedure for synthesizing the above compound. The crude product of the Boc-protected product is purified by silica gel column chromatography using ethyl acetate / hexane, 2: 1 as the developing solvent, and the final target product (1c) is made into a yellow solid (29.4 mg, 85%). Obtained.

<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), 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, 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)>
Boc-L-Ala (21.4 mg, 0.113 mmol) was used as the raw material compound 3, and the synthesis was carried out according to the basic procedure for synthesizing the above compound. The crude product of the Boc-protected product is purified by silica gel column chromatography using ethyl acetate / hexane, 2: 1 as the developing solvent, and the final target product (1d) is obtained as a yellow powder (21.9 mg, 94%). Obtained.

<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 )>
It was prepared according to the following procedure according to the literature [K. Arai, et al., Int. J. Mol. Sci. 2013, 14, 13194-13212]. Commercially available chicken egg white lysozyme (Fuji Film Wako Pure Chemical Industries, Ltd. 12.0 mg) is dissolved in 100 mM Tris-HCl buffer solution pH 8.0 (700 μL) containing 8 M urea and 1 mM EDTA, and is a reducing agent (±). -Dithiothreitol (DTT ^red ) (Fuji Film Wako Pure Chemical Industries, Ltd. 12.0 mg) was added and dissolved, and then reacted at 40.0 ° C. for 2 hours. Add 0.1 M acetic acid aqueous solution (1.3 mL) to the obtained sample solution to stop the reaction, and use gel filtration chromatography (Sephadex G-50 DNA Grade F, GE Healthcare Life Sciences, developing solvent: 0.1 M acetic acid aqueous solution). Desalted and purified. The resulting solution was lyophilized to give a white solid, reduced denaturing lysozyme (R ^HEL , 10.0 mg).

<Preparation of misfolded lysozyme (4SS ^HEL )>
The powder of R ^HEL (2 mg) prepared above was dissolved in 200 mM acetate-sodium acetate buffer pH 4.0 (250 μL) containing 8 M urea and 1 mM EDTA, and then the urea-free buffer solution ( 750 μL) was added and mixed well. The concentration of the R ^HEL solution was determined using an ultraviolet-visible absorptiometer (ε ₂₈₀ = 33890 cm ^-1 M ^-1 ). Trans-3,4-dihydroxytetrahydroselenophene-1-oxide (DHS ^ox ; literature [M. Iwaoka, et al., Heteroatom Chem] prepared in the same buffer solution without urea so as to have a concentration 20 times higher than this concentration. . Synthesized by 2001, 19, 293-299] (1 mL) was added to the R ^HEL solution (1 mL), 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 solution). The resulting solution was lyophilized to give misfolded lysozyme (4SS ^HEL , 1.2 mg) with SS bonds randomly cross-linked within the protein molecule.

<Oxidative folding of reduced denatured lysozyme (R ^HEL )>
The effect of the synthetic compound as a catalyst on the oxidative folding was confirmed by the following method.
Using 100 mM Tris-HCl buffer pH 7.5 containing 1 mM EDTA bubbling synthetic compound (either 1a-d or 2), GSH, GSSG with argon gas to 200 μM, 5.0 mM, 1.0 mM, respectively. Prepared. The white powder of R ^HEL (1 mg) prepared above was dissolved in 100 mM Tris-HCl buffer pH 7.5 containing 2 M urea and 1 mM EDTA. The R ^HEL concentration in the sample was determined using an ultraviolet-visible absorptiometer (ε ₂₈₀ = 33890 cm ^-1 M ^-1 ) and diluted with the same buffer solution so that the R ^HEL concentration was 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 this sample solution was dispensed, 3 M aqueous hydrochloric acid solution (2 μL) was added to stop the reaction, and the mixture was stored in a refrigerator. With reference to the literature (K. Arai, et al., Chem. Asian J. 2014, 9, 3464-3471), estimate the amount of enzyme activity contained in the sample obtained at each time, and relative to the reaction time. Enzyme activity recovery rate was plotted.

The results are shown in Figure 1a and Table 1. FIG. 1a shows a comparison of the rate of recovery of HEL enzyme activity in 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, 1 M urea. The data are shown as mean ± standard error (n = 3).
Since the enzyme activity is restored by folding the protein enzyme, the rate of enzyme activity recovery rate can be considered to be the folding rate. It can be seen that the folding reaction is greatly accelerated by adding the synthetic compound 1 or 2. Furthermore, the addition of a commonly used catalytic amount of GSSG has no effect. Furthermore, it can be seen that 1a bonded with histidine further promotes the reaction than other catalysts. In fact, when the reaction rate constant (k) of this oxidative folding was estimated (Table 1), the rate of increase in reaction rate was maximum when synthetic compound 1a was used.

<Refolding of 4SS ^HEL >
The effect of the synthetic compound as a catalyst for refolding from a misfolded product was confirmed by the following method.
Prepared using 100 mM Tris-HCl buffer pH 7.5 containing 1 mM EDTA bubbling synthetic compounds (either 1a-d or 2) and GSH with argon gas to 80 μM and 4.0 mM, respectively. .. The white powder of 4SS ^HEL (1 mg) prepared above was dissolved in 100 mM Tris-HCl buffer pH 7.5 containing 2 M urea and 1 mM EDTA. The 4SS ^HEL concentration in the sample was determined using an ultraviolet visible absorbance altimeter (ε ₂₈₀ = 33890 cm ^-1 M ^-1 ) and diluted with the same buffer solution so that the 4SS ^HEL concentration was 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 this sample solution was dispensed, 3 M aqueous hydrochloric acid solution (2 μL) was added to stop the reaction, and the mixture was stored in a refrigerator. With reference to the literature (K. Arai, et al., Chem. Asian J. 2014, 9, 3464-3471), estimate the amount of enzyme activity contained in the sample obtained at each time, and relative to the reaction time. Enzyme activity recovery rate was plotted.

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

The oxidative folding reaction rate constant k is the apparent reaction rate constant obtained by fitting the experimental values (Fig. 1a) using% HEL activity = A _max (1-e ^-kt ), where A _max is the folding. The maximum enzymatic activity of HEL restored by. The time t ₅₀ [min] required for the enzyme activity to recover by 50% 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 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 according to the method described above. After a certain period of time, 200 μL was dispensed from the reaction solution and synthesized with 2-aminoethyl methanethiosulfonate (AEMTS; literature [M. Iwaoka, et al., Heteroatom Chem. 2001, 19, 293-299]) aqueous solution (7 mg /). Transferred to a 1.5 mL volume plastic tube containing mL, 200 μL). Acetic acid (10 μL) is added to the obtained sample solution and mixed to adjust the pH of the solution to 3.5, and then column chromatography (Sephadex G-50 DNA Grade F, GE Healthcare Life Sciences, developing solvent: 0.1 M). It was desalted and purified using an acetic acid aqueous solution). The obtained sample solution was freeze-dried, the obtained residue was dissolved in a 0.1% trifluoroacetic acid aqueous solution (1100 μL), and the folding yield of lysoteam was determined by high performance liquid chromatography (HPLC) analysis using a reverse phase column. I estimated. The HPLC analysis conditions were carried out with reference to the literature (K. Arai, et al., Int. J. Mol. Sci. 2013, 14, 13194-13212).

FIG. 2 shows an oxidative folding experiment of reduced lysoteam (R ^HEL ) in the absence (a) and presence (b) of synthetic compound 1a, and in the absence (c) and presence (d) of synthetic compound 1a. The HPLC chromatogram of the sample obtained from the refolding experiment of misfolded resoteam (4SS ^HEL ) in) is shown. Each reaction condition in FIG. 2 is the same as in FIG.
In FIG. 2, HPLC was used to perform component analysis in the sample during the oxidative folding and refolding reactions of HEL. In the presence of synthetic compound 1a, it can be confirmed that N-form is produced more quickly in any of the experiments. This result is consistent with the evaluation result of the folding rate of HEL evaluated from the recovery rate of enzyme activity.

<Evaluation of the effect of synthetic compounds on suppressing aggregation of heat-denatured lysozyme>
The protein aggregation inhibitory effect of the synthetic compound was confirmed by the following method.
Commercially available chicken egg white lysozyme (Fuji Film Wako Pure Chemical Industries, Ltd. 5.0 mg) was dissolved by adding 50 mM Tris-HCl buffer solution pH 7.5 (1.0 mL) containing 0.5 mM EDTA and 0.28 M NaCl. Using an ultraviolet-visible absorptiometer (ε ₂₈₀ = 37600 cm ^-1 M ^-1 ), the concentration of lysozyme was determined and diluted with the same buffer solution to 200 μM. Synthetic compounds (any of 1a-d and 2) were prepared using the same buffer solution to 1.0 mM. 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 in order to confirm the concentration dependence. Add 400 μL of lysozyme solution (400 μL) and compound solution prepared in a 1.5 mL plastic tube, incubate at 30, 50, 70, 80, 90 ° C temperatures for 5 minutes, and heat the sample immediately at 23 ° C. It was allowed to stand for 30 minutes. Centrifuge the sample solution for 3 minutes (14000 rpm) to settle the insoluble precipitate, and then refer to the literature (K. Arai, et al., Chem. Asian J. 2014, 9, 3464-3471). The amount of enzyme activity contained in the obtained sample was estimated.

The results are shown in FIG.
FIG. 3 (a) shows the HEL solution (100 μM) in 50 mM Tris-HCl buffer pH 7.5 containing 0.28 M NaCl and 0.5 mM EDTA obtained when heated to 90 ° C for 10 minutes and then cooled to 23 ° C. Shows a picture of. FIG. 3 (b) shows the relative enzyme activity recovery rate of HEL in the sample solution obtained from the same experiment as (a). The data are shown as mean ± standard error (n = 3). FIG. 3 (c) shows the concentration dependence of compound 1a on the aggregation of HEL after heat denaturation and the recovery rate of enzyme activity. The reaction conditions are the same as in (a) except for the concentration condition of compound 1a.
HEL is denatured by heating, and intramolecular association occurs and aggregates (white turbidity). Not only the sample without compound (Blank), but also compound 2, compound 2 bonded with amino acids other than histidine (1b ~ d), compound 2 mixed with amino acid histidine (2 + His), and aggregation inhibitor. After heat denaturation, a white precipitate due to aggregate formation was observed in the sample solution containing guanidine hydrochloride (Gdn-HCl), which is often used as a guanidine hydrochloride. On the other hand, the formation of aggregates was not confirmed by the addition of compound 1a (a). Furthermore, when the enzyme activity recovery rate was estimated, the enzyme activity was recovered by almost 100% in the coexistence of compound 1a (b). Furthermore, it was also found that the aggregation inhibitory effect of 1a exerts a sufficient effect even at an extremely low concentration of 0.3 mM (c).

<Oxidative folding of high-concentration R ^HEL with agglutination>
The oxidative folding and aggregation inhibitory effects of the synthetic compound were confirmed by the following methods.
Using 100 mM Tris-HCl buffer pH 7.5 containing 1 mM EDTA bubbling synthetic compounds (either 1a-d or 2 or GSSG) and GSH to 2.67 mM and 533 μM, respectively, with argon gas. Prepared. The powder of R ^HEL (2 mg) prepared above was dissolved in 100 mM Tris-HCl buffer pH 7.5 containing 1 mM EDTA and 4 M urea. The R ^HEL concentration in the sample was determined using an ultraviolet-visible absorptiometer (ε ₂₈₀ = 33890 cm ^-1 M ^-1 ) and diluted with the same buffer solution so that the R ^HEL concentration was 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 was dispensed from the sample solution, 3 M aqueous hydrochloric acid solution (2 μL) was added to stop the reaction, and the mixture was stored in the refrigerator. With reference to the literature (K. Arai, et al., Chem. Asian J. 2014, 9, 3464-3471), the amount of enzyme activity contained in the samples obtained at each time was estimated.

The results are shown in FIG.
The reaction conditions in FIG. 4 are [R ^HEL ] ₀ = 50 μM, [GSH] ₀ = 1.0 mM, [Compound] ₀ = 0.20 mM, 37 ° C, pH 7.5, and the presence of 1 M urea. The reaction time was 24 hours when GSSG was used and 5 hours in other cases. The data are shown as mean ± standard error (n = 3).
Buffer solutions containing GSSG and GSH are widely used as solvents for oxidative folding of proteins. In FIG. 4, instead of GSSG, any of the synthesized compounds 1a to d was added, and HEL oxidative folding was performed under the high concentration condition of 50 μM (0.7 mg / mL). Normally, under such high concentration conditions, R ^HEL is oligomerized and eventually aggregated, and sufficient folding yield cannot be achieved. In fact, when GSSG / GSH (0.2 / 1.0 mM) was used, the folding yield was about 25% after only 24 hours. On the other hand, when synthetic compound 1 / GSH (0.2 / 1.0 mM) was used, the folding yield increased to 55% with a reaction time of 5 hours. Synthetic compound 1 (especially 1a), 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. However, it can be considered that the folding yield was further improved by suppressing the oligomerization and aggregate formation of HEL.

A folding step is indispensable in order to obtain a polypeptide chain prepared by a chemical synthesis method or a recombinant method as a physiologically active protein. According to the present invention, protein folding is possible. The cyclic disselenide compounds, protein folding agents, and protein folding methods of the present invention are useful in the artificial preparation of novel proteins useful 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, X represents an imidazole group, -NH ₂ , -. NH-C (= NH) -NH ₂ or -H.) R is

The compound according to claim 1 or a salt thereof, which is any of the above. A protein folding agent comprising the cyclic diselenide compound according to claim 1 or 2 or a salt thereof. A method for folding a protein, which comprises a step of treating a structurally unformed protein in the presence of the cyclic disselenide compound according to claim 1 or 2 or a salt thereof.

Images