Protein folding agents

Abstract

To provide a protein folding agent capable of folding a protein with high efficiency.SOLUTION: The protein folding agent includes, as an active ingredient, at least one selected from among the compounds represented by formulas (1)-(14) and salts and solvates thereof.SELECTED DRAWING: Figure 2B

Description

[Technical Field]

[0001] The present invention relates to a protein folding agent. [Background technology]

[0002] Proteins are materials that are widely used in industry, primarily in pharmaceuticals. Since the formation of the native structure is essential for the function and activity of the enzyme, The folding technology is important for industrial use of proteins as it directly leads to cost reduction. Therefore, oxidative folding promoters to the native structure are of great importance for the development of protein formulations. It is useful for large-scale production of important protein pharmaceuticals such as insulin and immunoglobulins. The oxidative folding process involves the introduction of native disulfide bonds. The structure formation is the rate-determining reaction.

[0003] A typical method for controlling the oxidative folding process is shutting down disulfide bonds. Fling is a method to fold proteins by using both reducing and oxidizing agents in the folding process. This method involves repeating the formation and cleavage of the nucleotides to reach the most stable native structure. Typical reducing and oxidizing agents that can be used are glutathione (GSH), β-mercaptoethanol, and β-mercaptoethanol, respectively. ethanol (β-ME), dithiothreitol (DTT), and glutathione oxidized salt (GSSG) For example, in the case of RNase A, the recovery yield in the presence and absence (in air) of GSH / GSSG is The results show that the presence of GSH / GSSG results in a higher folding rate at the same time during the initial folding process. The enzyme activity of is about 2 times higher, and the speed of activity recovery (1 / 2 activity recovery time) is about 2.8 times faster (non Patent Document 1). In addition, small molecule reducing agents (and As the oxidizing agent to be combined with GSH / GSSG (Non-Patent Document 1), GSH+(±)-trans -1,2-bis(2-mercaptoacetamido)cyclohexane (BMC) / GSSG (non-patented Reference 2), Aromatic thiols and their disulfides ( Non-patent document 3), Cyclic selenoxide (Non-patent document 4), Cys-XX-Cy Peptides (non-patented) having the s structure (X is any amino acid, Cys is cysteine) Reference 5), selenoglutathione (Non-patent Document 6), and selenol However, cyclic selenoxide and seleno peptides are known. Regarding glutathione, there was a problem of toxicity due to the use of heavy metal selenium. C is used as a supplement to GSH, so it is added in larger quantities than the GSH / GSSG system. There was a problem in that an agent had to be added. Since it is a substance, nonspecific adsorption to proteins due to hydrophobic interactions may occur. For peptides with Cys-XX-Cys structure, protease contamination was a problem. The structural instability caused by decomposition due to cation and the high cost of synthesis were problems. In addition, low molecular weight thiols such as cysteamine have a strong unpleasant odor due to their high volatility. Furthermore, when these existing compounds were used, aggregation of oxidative folding intermediates was observed. The inhibitory effect was low, which was one of the reasons for the low yield.

[0004] Therefore, the present invention can fold proteins with high efficiency while avoiding the problems of the conventional technology. Therefore, there has been a demand for protein folding agents that can fold proteins. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] AK Ahmed et al., J. Biol. Chem., 1975, 250, 8477-8482 [Non-patent document 2] KJ Woycechowsky et al., Chem. Biol., 1999, 6, 871-879 [Non-patent document 3] DJ Madar et al., J. Biotech., 2009, 142, 214-219 [Non-patent document 4] K. Arai, K et al., Chem. Eur. J., 2011, 17, 481-485 [Non-Patent Document 5] WJ Lees et al., Curr. Opin. Chem. Biol., 2008, 12, 740-745 [Non-patent document 6] J. Beld et al., Biochemistry. 2007 May 8;46(18):5382-90 [Non-Patent Document 7] S. Tsukagoshi et al., Chem. Asian J. 2020 September 1;15(17):2646-52 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, the present invention provides a protein folding method capable of folding proteins with high efficiency. The present invention aims to provide a protein folding agent. [Means for solving the problem]

[0007] As a result of examining various means for solving the above problems, the present inventors have found a method for manufacturing a semiconductor device having a specific structure. By using the compounds, salts and solvates thereof, it is possible to obtain the compounds with high efficiency, i.e., high yields and and / or found that disulfide bonds can be introduced into proteins at high speed, The present invention has been completed.

[0008] That is, the gist of the present invention is as follows. [1] The following formula: [ka] TIFF0007722664000002.tif90111[In the formula, X1, X2 and X3 each independently contain 1 to 5 oxygen atoms in the molecular chain. an alkylene group having 1 to 10 carbon atoms, Y1 each independently represents a carbon number which may contain 1 to 5 oxygen atoms in the molecular chain. 1 to 10 alkylene groups, Z1, Z2, Z3, Z4 and Z5 each independently represent 1 to 5 oxygen atoms in the molecular chain. an alkylene group having 1 to 10 carbon atoms which may contain a group, R1 and R2 are each independently an alkyl group having 1 to 24 carbon atoms, M - are each independently a chloride ion, a bromide ion, or an iodide ion. and at least one compound selected from the group consisting of a compound represented by the formula (I) and a salt and solvate thereof as an active ingredient. A protein folding agent comprising: [2] A compound selected from the group consisting of compounds represented by formulas (1) to (7), and salts and solvates thereof. The fold-in protein according to the above [1], which contains at least one of the following as an active ingredient: Ginger. [3] A compound selected from the group consisting of compounds represented by formulas (8) to (14), and salts and solvates thereof: The protein fold change method according to the above [1], which contains at least one of the following as an active ingredient. Injecting agent. [4] A compound selected from the group consisting of compounds represented by formulas (1) to (7), and salts and solvates thereof. and compounds represented by formulas (8) to (14), and salts thereof, and The tan according to the above [1], which contains at least one selected from the group consisting of solvates as an active ingredient. Protein folding agent. [5] Compounds represented by formulas (1) to (14) defined in [1] above, and salts thereof and solvates thereof. A method for folding a protein, comprising the step of processing the protein. [6] Compounds represented by formulas (1) to (14) defined in [1] above, and salts thereof and solvates thereof. A method for regenerating a protein, comprising the step of treating the protein. [7] Compounds represented by formulas (1) to (14) defined in [1] above, and salts thereof and solvates thereof as an active ingredient. . [Effects of the Invention]

[0009] The protein folding agent of the present invention can fold proteins with high efficiency. It can be used for loading. [Brief explanation of the drawings]

[0010] [Figure 1A] FIG. 1A shows the structure of bovine pancreatic trypsin inhibitor (BPTI) and the disulfide bond bridge positions of each disulfide bond species in its folding pathway. [Figure 1B] FIG. 1B shows the relationship between HPLC retention time and each disulfide bond type when determining the disulfide bond crosslinking position of BPTI using reversed-phase HPLC. [Figure 2A] FIG. 2A shows the results of reverse-phase HPLC analysis when the folding reaction of reduced modified BPTI was carried out using GSH / GSSG (Comparative Example 1). [Figure 2B] FIG. 2B shows the results of reverse-phase HPLC analysis of the folding reaction of reduced modified BPTI using LDA-SH / LDA-SS (Example 1). [Figure 2C] FIG. 2C shows the results of reverse-phase HPLC analysis of the folding reaction of reduced modified BPTI using BDA-SH / BDA-SS (Example 2). [Figure 3] FIG. 3 is a graph showing the evaluation of activity recovery when the folding reaction of reduced and modified RNase A was carried out using LDA-SH / LDA-SS (Example 3), GSH / GSSG (Comparative Example 2), or GSSG. [Figure 4A] FIG. 4A shows the results of reverse-phase HPLC monitoring of the disulfide bond introduction reaction into reduced denatured β2m using GSH / GSSG (Comparative Example 3). [Figure 4B] FIG. 4B shows the reaction of introducing a disulfide bond into reduced denatured β2m using LDA-SH / LDA-SS (Example 4), followed by reversed-phase HPLC. [Figure 4C] FIG. 4C shows the reaction of introducing a disulfide bond into reduced denatured β2m using BDA-SH / BDA-SS (Example 5), followed by reverse-phase HPLC. [Figure 5] Figure 5 is a graph showing the quantification of oxidized β2m by disulfide bond introduction into reduced denatured β2m using GSH / GSSG (Comparative Example 3), LDA-SH / LDA-SS (Example 4), or BDA-SH / BDA-SS (Example 5). [Figure 6] FIG. 6 is a graph showing the evaluation of activity recovery when the folding reaction of reduced-denatured RNase A was performed using GSH / GSSG (Comparative Example 4), ImdM-SH / ImdM-SS (Example 6), or oPyM-SH / oPyM-SS (Example 7). [Figure 7] FIG. 7 is a graph showing the evaluation of activity recovery when the folding reaction of reduced-denatured RNase A was performed using GSH / GSSG (Comparative Example 5), pPyM-SH-NMe·Cl / SS (Example 8), pPyM-SH-NMe·I / SS (Example 9), or oPyM-SH-NMe·I / SS (Example 10). [Figure 8] FIG. 8 shows photographs of each sample solution (before centrifugation) obtained in the β2m aggregation experiment by heating. [Figure 9] FIG. 9 is a graph showing the remaining rate of native β2m in the supernatant obtained by centrifuging each sample solution in the heat-induced β2m aggregation experiment. [Figure 10] FIG. 10 is a graph showing the evaluation of activity recovery when the folding reaction of reduced-denatured RNase A was performed using GSH / GSSG (Comparative Example 4), ImdM-SH / ImdM-SS (Example 6), oPyM-SH / oPyM-SS (Example 7), or pPyM-SH / GSSG (Example 13). DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in detail below based on embodiments.

[0012] The present invention relates to a protein folding agent. The steroid is selected from the compounds represented by formulas (1) to (14), and salts and solvates thereof. The present invention relates to a method for folding proteins comprising the steps of: The agent can be used with higher efficiency, i.e., higher yield and / or Alternatively, disulfide bonds can be introduced into proteins at high speed, which improves the protein This allows the oxidative folding of proteins to proceed with high efficiency. This will lead to a reduction in the time required to produce proteins used in pharmaceuticals and an improvement in yield. This has the effect of enabling large-scale production of protein formulations and reducing production costs. The compounds represented by (1) to (7), and salts and solvates thereof are simply referred to as "thiol compounds." ", and compounds represented by formulas (8) to (14), and salts and solvates thereof. The substance is also simply called a "disulfide compound."

[0013] The thiol compound has the following formula: [ka] [In the formula, X1, X2 and X3 each independently contain 1 to 5 oxygen atoms in the molecular chain. an alkylene group having 1 to 10 carbon atoms, Y1 is an alkyl group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain. is a cyclohexene group, Z1, Z2, Z3, Z4 and Z5 each independently represent 1 to 5 oxygen atoms in the molecular chain. an alkylene group having 1 to 10 carbon atoms which may contain a group, R1 and R2 are each independently an alkyl group having 1 to 24 carbon atoms, M - are each independently a chloride ion, a bromide ion, or an iodide ion. Thiol compounds generally produce a foul odor. However, all of the above thiol compounds have a reduced odor, which is a useful point in their application. These thiol compounds have advantages over related compounds. Because the molecule is small enough, it can be easily separated by size exclusion chromatography after disulfide bond introduction reaction. Separation from proteins can be easily achieved using chromatography or dialysis.

[0014] The disulfide compound has the following formula: [ka] [In the formula, X1, X2 and X3 each independently contain 1 to 5 oxygen atoms in the molecular chain. an alkylene group having 1 to 10 carbon atoms, Y1 each independently represents a carbon number which may contain 1 to 5 oxygen atoms in the molecular chain. 1 to 10 alkylene groups, Z1, Z2, Z3, Z4 and Z5 each independently represent 1 to 5 oxygen atoms in the molecular chain. an alkylene group having 1 to 10 carbon atoms which may contain a group, R1 and R2 are each independently an alkyl group having 1 to 24 carbon atoms, M - are each independently a chloride ion, a bromide ion, or an iodide ion. The disulfide compound is a compound represented by the formula: Because it is a molecule that is sufficiently small compared to proteins, after the disulfide bond introduction reaction It can be easily separated from proteins using size exclusion chromatography or dialysis. can be done.

[0015] One embodiment of the protein folding agent of the present invention is represented by formulas (1) to (7): and at least one selected from the group consisting of compounds of the formula (8) and salts and solvates thereof. 1) to (14), and salts and solvates thereof. The present invention is characterized in that it contains at least one of the following as an active ingredient. The molar ratio of the thiol compound to the disulfide compound contained in the binder is set to 1 / 2000. As long as folding proceeds, there are no particular limitations, but for example, it is 1:1 to 20:1. The ratio is preferably 2:1 to 10:1, and more preferably 2:1 to 10:1. The folding agent of this quality is a mixture of the thiol compound and the disulfide compound. Even if the items are sold or distributed in a state, they may be sold separately as a kit or set, etc. It may be sold or distributed in a packaged state.

[0016] Another embodiment of the protein folding agent of the present invention is characterized by comprising, as an active ingredient, at least one compound selected from the compounds represented by formulas (1) to (7), and salts and solvates thereof. The protein folding agent of this embodiment can be used in combination with at least one compound selected from the compounds represented by formulas (8) to (14), and salts and solvates thereof. In this case, the molar ratio of the thiol compound contained in the protein folding agent of this embodiment to the disulfide compound used in combination is not particularly limited as long as folding proceeds efficiently. For example, a ratio of 1:1 to 20:1 is preferred, and a ratio of 2:1 to 10:1 is even more preferred. This protein folding agent containing the thiol compound can be used in a protein folding reaction together with the disulfide compound obtained separately. Furthermore, the disulfide compound used in combination may be a conventionally used compound such as GSSG. In this case, the preferred embodiment is based on the description of the disulfide compound, unless otherwise specified.

[0017] Another embodiment of the protein folding agent of the present invention is characterized by comprising, as an active ingredient, at least one selected from the compounds represented by formulas (8) to (14), and salts and solvates thereof. The protein folding agent of this embodiment can be used in combination with at least one selected from the compounds represented by formulas (1) to (7), and salts and solvates thereof. In this case, the molar ratio of the thiol compound used in combination with the disulfide compound contained in the protein folding agent of this embodiment is not particularly limited as long as folding proceeds efficiently. For example, a molar ratio of 1:1 to 20:1 is preferred, and a molar ratio of 2:1 to 10:1 is even more preferred. This protein folding agent containing the disulfide compound can be used in a protein folding reaction together with a separately obtained thiol compound. Furthermore, the thiol compound used in combination may be a conventional compound such as GSH. In this case, the preferred embodiment is based on the description of the thiol compound, unless otherwise specified.

[0018] In this specification, the term "active ingredient" refers to a compound that is produced during the folding of a protein. A substance that acts on a protein in a certain state in the process and a substance that acts on the substance, As used herein, the term "protein" refers to a substance that has the function of promoting protein folding. "Protein folding" involves (1) reducing and denaturing proteins to form unfolded proteins. (2) oxidatively treating the unfolded protein; The present invention also includes a step of folding the protein. The agent is a compound capable of reacting the thiol compound and the disulfide compound with each other, particularly in the oxidative formation of the compound in step (2). In the bonding reaction, they function as a reducing agent and an oxidizing agent, respectively, to dissociate proteins. Highly efficient protein folding by introducing sulfide bonds can be done.

[0019] In synthesizing the above thiol compounds and disulfide compounds, those skilled in the art will be able to Therefore, general organic synthesis methods can be appropriately adopted. Thiol compounds represented by formulas (1) to (7) and thiol compounds represented by formulas (8) to (14) The production method of the compound corresponding to the disulfide compound can be referred to.

[0020] The thiol compounds and disulfide compounds may be in the form of salts or solvates. The salt is not particularly limited, and examples thereof include sodium and potassium salts. salts with alkali metals; salts with alkaline earth metals such as magnesium and calcium; or salts with inorganic acids such as hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, sulfurous acid; formic acid, acetic acid, propyl acid onic acid, butyric acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, malic acid Acids such as acetic acid, mandelic acid, methanesulfonic acid, p-toluenesulfonic acid, and trifluoroacetic acid Examples of the solvates include hydrates, salts with alcohols (e.g., Methanol, ethanol, propanol, isopropanol), acetone, tetrahydrofuran Examples of solvates include those with solvents such as furan, dioxane, DMF, and DMSO.

[0021] The above "alkylene having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain" Specific examples of the "alkyl group" include a methylene group, an ethylene group, a propylene group, an isopropylene group, n-butylene group, isobutylene group, tert-butylene group, n-pentylene group, isopentene group ethylene group, neopentylene group, hexylene group, heptylene group, octylene group, nonylene group , decylene group, polyoxyalkylene group (wherein alkylene is, for example, an ethylene group, poly Examples of the polyoxyalkylene group include, but are not limited to, a propylene group. Examples of the olefin group include, but are not limited to, -(CH2CH2O)a- (wherein a is an integer of 1 to 5, and * indicates the bonding point with -NH2 for X1, X2, and X3. Y1 is attached to the -SH bond, Z1 is attached to the imidazole ring, Z2 is Z3, Z4 and Z5 indicate the points of attachment to the pyridine ring), *-(OCH2CH2) b- (wherein b is an integer of 1 to 5, and * represents -NH2 for X1, X2, and X3) the point of attachment to -SH for Y1; the point of attachment to the imidazole ring for Z1 Z2, Z3, Z4 and Z5 indicate the points of attachment to the pyridine ring), *-(CH2 CH2CH2O)c-(wherein c is an integer of 1 to 3, and * indicates that X1, X2, and X3 for Y1, the bonding point with -SH; for Z1, the bonding point with imidazoline; Z2, Z3, Z4, and Z5 indicate the points of attachment to the pyridine ring. ), *-(OCH2CH2CH2)d- (where d is an integer from 1 to 3, * represents X1 X2 and X3 are attached to -NH2, Y1 is attached to -SH, Z1 and Z2, Z3, Z4, and Z5 are the bonding points to the imidazole ring and the pyridine ring. (showing the point of attachment to the

[0022] In addition, the term "carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain" as used herein refers to a compound having a molecular chain which may contain 1 to 3 oxygen atoms. Specific examples of the alkylene group having 1 to 6 carbon atoms include a methylene group, an ethylene group, and a propylene group. , isopropylene group, n-butylene group, isobutylene group, tert-butylene group, n-pentene group ethylene group, isopentylene group, neopentylene group, hexylene group, polyoxyalkylene Examples of alkylene include ethylene and propylene groups. However, the polyoxyalkylene group is not limited to the above. However, *-(CH2CH2O)a- (where a is an integer between 1 and 3, and * is X1, X2, and X3 are attached to -NH2, and Y1 is attached to -SH. Z1 is the bonding point to the imidazole ring, Z2, Z3, Z4 and Z5 are the bonding points to the pyridinium ring. indicates the point of attachment to the vinyl ring), *-(OCH2CH2)b- (where b is an integer from 1 to 3) * indicates the bonding point with -NH2 for X1, X2 and X3, and -SH for Y1. for Z1, the point of attachment to the imidazole ring; for Z2, Z3, Z4, and Z5, the point of attachment to the imidazole ring; indicates the point of attachment to the pyridine ring), *-(CH2CH2CH2O)c- (where c is 1 or 2, and * indicates the point of attachment to -NH2 for X1, X2 and X3, and * indicates the point of attachment to -NH2 for Y1. For Z1, the bonding point to the imidazole ring is and Z5 indicates the point of attachment to the pyridine ring), *-(OCH2CH2CH2)d- (wherein d is 1 or 2, and * indicates a bond with —NH for X1, X2, and X3. Y1 is attached to the -SH bond, Z1 is attached to the imidazole ring, Z2 and Z3, Z4 and Z5 indicate the points of attachment to the pyridine ring).

[0023] In addition, the term "carbon which may contain one or two oxygen atoms in the molecular chain" as used herein is also used. Specific examples of the alkylene group having 1 to 4 prime numbers include a methylene group, an ethylene group, and a propylene group. group, isopropylene group, polyoxyalkylene group (wherein alkylene is, for example, ethylene Examples of the olefin group include, but are not limited to, olefin groups and propylene groups. Examples of the sialic alkylene group include, but are not limited to, -(CH2CH2O)a - (wherein a is an integer of 1 or 2, and * represents -NH2 for X1, X2, and X3) the point of attachment to -SH for Y1; the point of attachment to the imidazole ring for Z1 Z2, Z3, Z4 and Z5 indicate the points of attachment to the pyridine ring), *-(OCH 2CH2)b-(wherein b is an integer of 1 or 2, and * indicates the number of X1, X2, and X3 for Y1, the point of attachment to -SH; for Z1, the point of attachment to imidazoline; Z2, Z3, Z4 and Z5 indicate the points of attachment to the pyridine ring). *-(CH2CH2CH2O)c- (where c is 1 and * represents X1, X2 and X3 for the bonding point with -NH2, for Y1 the bonding point with -SH, and for Z1 the bonding point with imino group. Z2, Z3, Z4, and Z5 indicate the points of attachment to the pyridine ring. *-(OCH2CH2CH2)d- (where d is 1 and * indicates X1, X2 and and X3 are bonded to -NH2, Y1 is bonded to -SH, and Z1 is indicates the bonding point to the imidazole ring, and Z2, Z3, Z4, and Z5 indicate the bonding point to the pyridine ring. (showing points)

[0024] Specific examples of the above-mentioned "alkyl group having 1 to 24 carbon atoms" include a methyl group, an ethyl group, an n- Propyl group, n-butyl group, n-pentyl group, n-hexyl group, n-heptyl group, n-octyl group octyl group, n-nonyl group, n-decyl group, n-undecyl group, n-dodecyl group, n-tride decyl group, n-tetradecyl group, n-pentadecyl group, n-hexadecyl group, n-heptadecylic group Cyl, n-octadecyl group, n-nonadecyl group, n-icosyl group, n-henicosyl group, Examples include, but are not limited to, n-docosyl, n-tricosyl, and n-tetracosyl groups. It is not something that is done.

[0025] The thiol compound and the disulfide used in the protein folding agent of the present invention The combination of the peptide compounds is not particularly limited, but may be any of the following: In the disulfide bond shuffling, the thiol compound and the disulfide bond From the viewpoint of allowing the compound to act efficiently as a reducing agent and an oxidizing agent, respectively, A combination (for example, a thiol compound represented by formula (1) and its disulfide compound represented by formula (8) ) or a combination thereof. However, it is preferable that either a thiol compound or a disulfide compound is added to the fluorine-containing compound of the protein of the present invention. Even when the active ingredient of the folding agent is used, the folding process Both thiol compounds and disulfide compounds exist in equilibrium in the folding solution. and function as reducing and oxidizing agents in disulfide bond shuffling. It is also possible.

[0026] Generally, the reaction intermediates transiently generated during the oxidative folding process tend to aggregate. Therefore, the oxidative folding of proteins is From the viewpoint of improving the yield, the protein folding agent of the present invention is effective in condensing reaction intermediates. It is preferable that the present invention has a function of suppressing aggregation. Thiol compounds which are active ingredients of the protein folding agent or ingredients used in combination with the agent The disulfide compounds can be used as active ingredients of protein solubilizers.

[0027] Furthermore, the protein folding agent of the present invention can rapidly convert proteins into non-native structures in addition to native structures. From the viewpoint of rapid generation, it has high activity to introduce disulfide bonds extremely quickly. Proteins have completely different biochemical properties in their native and non-native structures. In the case of protein formulations, natural forms show the expected efficacy, while non-natural forms show low efficacy. Therefore, the protein of the present invention may have poor efficacy and may even cause serious side effects. Folding agents are used to investigate the biochemical properties of proteins with non-native structures and to aid in the development of protein formulations. Misfolded proteins, which may be present as impurities in From the perspective of comprehensively clarifying potential side effects of drug efficacy, we are investigating various proteins with non-native structures. It is preferable that the function be conferred at once. There have been no reports of compounds that can simultaneously generate a variety of non-natural structures for proteins that have non-natural structures. As a method to preferentially give proteins with native structure, some cysteine residues in the protein A method for replacing α-glucan with selenocysteine residues has been reported (N. Metanis et al., Angew. Chem. Int. Ed. 2012 51:5585-88). However, this method does not convert the structure of the protein itself. In addition, only a few types of selenoproteins containing selenium have been reported in vivo. The physiological function of selenoprotein is still unknown, and excessive selenium intake may have adverse effects on humans. Considering the toxicity of selenium, it is difficult to apply selenium-containing small molecules to pharmacological applications. It is thought that...

[0028] Furthermore, the protein folding agent of the present invention can suppress aggregation while forming non-native structures. Generally, proteins with non-native structures are agglutinating proteins. However, the above-mentioned functions have made research difficult. This is advantageous for investigating the biochemical properties and cytotoxicity of proteins with non-native structures in a simple system. Therefore, it will be useful for research into treatments for folding diseases caused by proteins with non-native structures. It is believed that the folding of the protein of the present invention having such properties will be useful. The thiol compounds and disulfide compounds that are the active ingredients of the anti-inflammatory agent or the ingredients used in combination with it It can be used as an active ingredient in a solubilizing agent for proteins with non-native structures.

[0029] Proteins to be folded using the protein-folding agent of the present invention The protein is not particularly limited as long as it contains at least one disulfide bond, and may be natural or Regardless of origin or manufacturing method (chemical synthesis, fermentation, genetic recombination, etc.) Proteins include peptides, polypeptides, proteins, and complexes thereof. Any type of protein can be used, including intracellular proteins, extracellular proteins, membrane proteins, and nuclear proteins. Specifically, the following enzymes, recombinant proteins, and antigens are included: Examples include the body.

[0030] Enzymes include hydrolases, isomerases, oxidoreductases, transferases, synthetases and dehydrogenases. Hydrolases include proteases, serine proteases, and enzymes. Examples of isomerases include amylase, lipase, cellulase, and glucoamylase. Examples of oxidoreductases include glucose isomerase. Examples of transferases include acyltransferase, amide isomerase, and peroxidase. Examples of synthetic enzymes include fatty acid synthases, sulfotransferases, etc. Examples of lyase enzymes include phosphate synthase, citrate synthase, etc. pectin lyase and the like.

[0031] Recombinant proteins include protein preparations and vaccines. They are produced by genetic engineering using heterologous expression systems such as eukaryotes (e.g., yeast) or cell-free extract systems. Recombinant proteins are often obtained as insoluble and inactive aggregates, so-called inclusion bodies. Therefore, the protein-folding agent of the present invention can be preferably used. Protein preparations include interferon alpha, interferon beta, and interleukin 1. ~12, growth hormone, erythropoietin, insulin, granulocyte colony-stimulating factor (G- CSF), tissue plasminogen activator (TPA), defensins, sodium utilization Urinary peptides, blood coagulation factor 2, somatomedin, glucagon, growth hormone-releasing factor, blood Examples of vaccines include hepatitis A vaccine, hepatitis B vaccine, and serum albumin and calcitonin. Examples of antibodies include, but are not limited to, hepatitis B vaccine, hepatitis C vaccine, etc. Examples include therapeutic antibodies.

[0032] When the protein folding agent of the present invention is used for oxidative folding, Unfolded proteins in the presence of the protein folding agent of the invention. A step of treating proteins (hereinafter also referred to simply as a treatment step), for example, The protein, the protein folding agent of the present invention, and optionally a thiol used in combination. The folding buffer solution is mixed with a diol compound or a disulfide compound and the mixture is stirred. This is done by mixing.

[0033] Protein unfolding can be carried out by appropriately employing a method commonly known to those skilled in the art. For example, guanidine hydrochloride, urea, thiourea, or a combination of these can be used to A folded protein can be obtained. Unfold the protein using the method described in "Creation of a Novel Protein" It is also possible.

[0034] The treatment time of the above treatment step is adjusted appropriately so that the target protein can be obtained with high efficiency. The temperature can be appropriately selected depending on the heat resistance of the target protein. For example, the temperature is in the range of 0 to 100°C, preferably in the range of 4 to 40°C.

[0035] The thiol compound and the disulfide compound ( The concentration of the target protein (total amount) is not particularly limited. For example, The concentration is usually 0.01 to 100 mM in the above target tank. Unfolding proteins contained in a solution (e.g., folding buffer) The protein concentration is usually 0.01 to 100 μM.

[0036] In the above treatment step, a solution containing the target protein (e.g., a folding buffer solution) In this case, the thiol compound (total amount) which is the reducing agent and the disulfide compound (total amount) which is the oxidizing agent are The molar ratio of the ATP (measured amount) is determined so that disulfide bonds can be efficiently introduced to obtain the target protein. There are no particular limitations as long as the reaction proceeds, but it is, for example, 1:1 to 20:1.

[0037] The folding buffer is used at a concentration and composition that will not cause the function of the target protein to be lost. Specifically, the buffer solution may be a Tris buffer solution, a MES buffer solution, or a Tricine buffer solution. Examples include amine buffers, phosphate buffers, and various Good's buffers. The pH of the folding buffer solution is usually 4 to 10, preferably 5 to 10. The pH can be adjusted to within a range of 7 to 9, more preferably within a range of 7 to 9.

[0038] The folding buffer contains a folding agent for the protein of the present invention and, if necessary, In addition to the thiol compound or disulfide compound used in combination, various additives are added. Such additives include salts such as sodium chloride and calcium chloride; Buffers such as citrate, phosphate, and acetate; bases such as sodium hydroxide; hydrochloric acid and vinegar and organic solvents such as methanol, ethanol, and propanol. The buffer may contain, in addition to the folding agent of the present invention and the additives, surfactants, p It is also possible to incorporate an H regulator or a protein stabilizer. Dosage can be adjusted.

[0039] The protein-folding agent of the present invention is an active ingredient or a component used in combination with the agent, and the active ingredient is a compound of the formula ( Thiol compounds represented by formulas (1) to (7) and disulfides represented by formulas (8) to (14) The compounds are described below.

[0040] The following thiol compound (1) is used as an active ingredient of the protein folding agent of the present invention: ) and the following disulfide compound (8): [ka] [In the formula, X1 each independently represents a carbon number which may contain 1 to 5 oxygen atoms in the molecular chain. 1 to 10 alkylene groups, Y1 each independently represents a carbon number which may contain 1 to 5 oxygen atoms in the molecular chain. 1 to 10 alkylene groups] and the above combinations as combined components, which are naturally occurring due to oxidative folding. This is preferable from the viewpoint of obtaining structural proteins in high yield. The protein is not particularly limited, and may be any of the above-mentioned proteins, as well as RNase A, Pancreatic trypsin inhibitor, β2-microglobulin, insulin, major histocompatibility complex In order to obtain the above effects, the antibody having multiple disulfide bonds is Proteins having four disulfide bonds, such as RNase A, are preferred. .

[0041] Furthermore, the above compounds or combinations inhibit the aggregation of reaction intermediates in oxidative folding. This is preferable from the viewpoint of obtaining a protein with a native structure in high yield. The target proteins are not particularly limited, and include the above-mentioned proteins and RNas e A, bovine pancreatic trypsin inhibitor, β2-microglobulin, insulin, major histocompatibility genes Examples of the compound include gene complexes, immunoglobulins, etc., and from the viewpoint of obtaining the above-mentioned effects, Proteins with disulfide bonds, such as bovine pancreatic trypsin inhibitor, have three disulfide bonds. It is preferable that the compound has the following structure.

[0042] Furthermore, the above compounds or combinations can be used to oxidatively fold native structural proteins. This is preferable from the viewpoint of obtaining the product in high yield and at high speed. The protein is not particularly limited, and may be any of the above-mentioned proteins, as well as RNase A, bovine pancreatic Trypsin inhibitor, β2-microglobulin, insulin, major histocompatibility complex, immunoglobulin From the viewpoint of obtaining the above effects, β2-microglobulin (β2m) and the like are preferred. Proteins having one disulfide bond are preferred.

[0043] From the viewpoint of obtaining the above-mentioned effect, X1 should contain 1 to 3 oxygen atoms in the molecular chain. It is preferably an alkylene group having 1 to 6 carbon atoms, and one or two It is more preferably an alkylene group having 1 to 4 carbon atoms which may contain an oxygen atom. In order to obtain the above-mentioned effects, Y1 contains 1 to 3 oxygen atoms in the molecular chain. Preferably, the alkylene group has 1 to 6 carbon atoms and may contain one or more alkylene groups in the molecular chain. It is more preferably an alkylene group having 1 to 4 carbon atoms which may contain two oxygen atoms. I wish.

[0044] The following thiol compound (2) is used as an active ingredient of the protein folding agent of the present invention. ) and the following disulfide compound (9): [ka] [In the formula, X2 and X3 may each independently contain 1 to 5 oxygen atoms in the molecular chain. an alkylene group having 1 to 10 carbon atoms] and the above combinations as combined components in oxidative folding, From the viewpoint of extremely fast introduction of disulfide bonds and simultaneous generation of various non-natural structures In this case, the protein to be folded is not particularly limited. In addition to the above proteins, RNase A, bovine pancreatic trypsin inhibitor, β2-microglobulin globulin, insulin, major histocompatibility complex, immunoglobulin, etc. From the viewpoint of obtaining the desired effect, proteins having multiple disulfide bonds, such as bovine pancreas, chicken Those having three disulfide bonds, such as psin inhibitors, are preferred.

[0045] The above compounds or combinations also inhibit aggregation during oxidative folding. In this case, the folding of the protein is preferably performed in order to generate a protein having a non-native structure. The target proteins are not particularly limited, and include the above-mentioned proteins and RNase. A, bovine pancreatic trypsin inhibitor, β2-microglobulin, insulin, major histocompatibility complex In order to obtain the above effects, a polymer having a plurality of disulfides is preferably used. Proteins with three disulfide bonds, such as bovine pancreatic trypsin inhibitor It is preferred that the compound has the following structure:

[0046] Furthermore, the above compounds or combinations can be used to oxidatively fold native structural proteins. This is preferable from the viewpoint of obtaining the product in high yield and at high speed. The protein is not particularly limited, and may be any of the above-mentioned proteins, as well as RNase A, bovine pancreatic Trypsin inhibitor, β2-microglobulin, insulin, major histocompatibility complex, immunoglobulin From the viewpoint of obtaining the above effects, β2-microglobulin (β2m) and the like are preferred. Proteins having one disulfide bond are preferred.

[0047] In order to obtain the above effects, X2 should contain 1 to 3 oxygen atoms in the molecular chain. It is preferably an alkylene group having 1 to 6 carbon atoms, and one or two It is more preferably an alkylene group having 1 to 4 carbon atoms which may contain an oxygen atom. In order to obtain the above-mentioned effects, X3 contains 1 to 3 oxygen atoms in the molecular chain. Preferably, the alkylene group has 1 to 6 carbon atoms and may contain one or more alkylene groups in the molecular chain. It is more preferably an alkylene group having 1 to 4 carbon atoms which may contain two oxygen atoms. Desirable.

[0048] The following thiol compound (3) is used as an active ingredient of the protein folding agent of the present invention. ) and the following disulfide compound (10): [ka] [In the formula, Z1 each independently represents a carbon number which may contain 1 to 5 oxygen atoms in the molecular chain. 1 to 10 alkylene groups] and the above combinations as combined components, which are naturally occurring due to oxidative folding. This is preferable from the viewpoint of obtaining structural proteins in high yield. The protein is not particularly limited, and may be any of the above-mentioned proteins, as well as RNase A, Pancreatic trypsin inhibitor, β2-microglobulin, insulin, major histocompatibility complex In order to obtain the above effects, the antibody having multiple disulfide bonds is Proteins having four disulfide bonds, such as RNase A, are preferred. .

[0049] From the viewpoint of obtaining the above-mentioned effect, Z1 should contain 1 to 3 oxygen atoms in the molecular chain. It is preferably an alkylene group having 1 to 6 carbon atoms, and one or two It is more preferably an alkylene group having 1 to 4 carbon atoms which may contain an oxygen atom. .

[0050] The following thiol compound (4) is used as an active ingredient of the protein folding agent of the present invention. ) and the following disulfide compound (11): [ka] [In the formula, Z2 each independently represents a carbon number which may contain 1 to 5 oxygen atoms in the molecular chain. 1 to 10 alkylene groups] and the above combinations as combined components promote oxidative folding. In this case, the protein to be folded is not particularly limited. The proteins mentioned above, as well as RNase A, bovine pancreatic trypsin inhibitor, and β2-microglobulin Examples include globulin, insulin, major histocompatibility complex, immunoglobulin, etc. In order to obtain the above effects, Z2 should contain 1 to 3 oxygen atoms in the molecular chain. It is preferably an alkylene group having 1 to 6 carbon atoms, and has one or two acids in the molecular chain. It is more preferably an alkylene group having 1 to 4 carbon atoms which may contain a hydrogen atom.

[0051] The following thiol compound (5) is used as an active ingredient of the protein folding agent of the present invention. ) and the following disulfide compound (12): [ka] [In the formula, Z3 each independently represents a carbon number which may contain 1 to 5 oxygen atoms in the molecular chain. 1 to 10 alkylene groups] and the above combinations as combined components, which are naturally occurring due to oxidative folding. This is preferable from the viewpoint of obtaining structural proteins in a short time and in high yield. The proteins to be subjected to the tagging are not particularly limited, and include the above-mentioned proteins and RNA. ase A, bovine pancreatic trypsin inhibitor, β2-microglobulin, insulin, major histocompatibility Examples of the compound include gene complexes, immunoglobulins, etc., and from the viewpoint of obtaining the above-mentioned effects, Proteins with disulfide bonds, such as RNase A, which has four disulfide bonds is preferred.

[0052] From the viewpoint of obtaining the above-mentioned effect, Z3 should contain 1 to 3 oxygen atoms in the molecular chain. It is preferably an alkylene group having 1 to 6 carbon atoms, and one or two It is more preferably an alkylene group having 1 to 4 carbon atoms which may contain an oxygen atom. .

[0053] The following thiol compound (6) is used as an active ingredient of the protein folding agent of the present invention. ) and the following disulfide compound (13): [ka] [In the formula, Z4 each independently represents a carbon number which may contain 1 to 5 oxygen atoms in the molecular chain. 1 to 10 alkylene groups, R1 is independently an alkyl group having 1 to 24 carbon atoms, M - are each independently a chloride ion, a bromide ion, or an iodide ion. and the above combination as a combined component inhibits oxidative folding of proteins. This is preferable from the viewpoint of obtaining a protein with a native structure in high yield by folding. The proteins to be subjected to the cloning are not particularly limited, and include the above-mentioned proteins and further RNase A, bovine pancreatic trypsin inhibitor, β2-microglobulin, insulin, major tissue-specific In order to obtain the above effects, a plurality of disulfides are used. Proteins with disulfide bonds, such as RNase A, which has four disulfide bonds It is preferable that:

[0054] From the viewpoint of obtaining the above-mentioned effects, Z4 should contain 1 to 3 oxygen atoms in the molecular chain. It is preferably an alkylene group having 1 to 6 carbon atoms, and one or two It is more preferably an alkylene group having 1 to 4 carbon atoms which may contain an oxygen atom. In order to obtain the above-mentioned effects, R1 is preferably an alkyl group having 1 to 6 carbon atoms. It is more preferable that the alkyl group is an alkyl group having 1 or 2 carbon atoms. From the viewpoint of obtaining such effects, - is preferably a chloride ion or an iodide ion .

[0055] The thiol compound (6) and the disulfide compound (13), as well as the combination thereof, The combination aggregates in water to form micelles due to the hydrophobic interaction of the R1 group, and the micelle The amphiphilic effect of the aqueous interior space and hydrophilic surface prevents denaturation and protein folding. Highly efficient introduction of disulfide bonds into proteins to suppress aggregation in the binding intermediate state In this case, R1 is Preferably, the alkyl group has 3 to 18 carbon atoms, and more preferably, the alkyl group has 4 to 12 carbon atoms. In this case, the target protein for folding is preferably The protein is not particularly limited, and may be any of the above-mentioned proteins, as well as RNase A and bovine pancreatic trypsin inhibitor. Intoxicants, β2-microglobulin, insulin, major histocompatibility complex, immunoglobulin etc.

[0056] The following thiol compound (7) is used as an active ingredient of the protein folding agent of the present invention. ) and the following disulfide compound (14): [ka] [In the formula, Z5 each independently represents a carbon number which may contain 1 to 5 oxygen atoms in the molecular chain. 1 to 10 alkylene groups, R2 is independently an alkyl group having 1 to 24 carbon atoms, M - are each independently a chloride ion, a bromide ion, or an iodide ion. and the above combinations as combined components, which are naturally occurring due to oxidative folding. This is preferable from the viewpoint of obtaining structural proteins in high yield. The protein is not particularly limited, and may be any of the above-mentioned proteins, as well as RNase A, Pancreatic trypsin inhibitor, β2-microglobulin, insulin, major histocompatibility complex In order to obtain the above effects, the antibody having multiple disulfide bonds is Examples of proteins that have four disulfide bonds include RNase A. do.

[0057] In order to obtain the above effects, Z5 should contain 1 to 3 oxygen atoms in the molecular chain. It is preferably an alkylene group having 1 to 6 carbon atoms, and one or two It is more preferably an alkylene group having 1 to 4 carbon atoms which may contain an oxygen atom. In order to obtain the above-mentioned effects, R2 is preferably an alkyl group having 1 to 6 carbon atoms. It is more preferable that the alkyl group is an alkyl group having 1 or 2 carbon atoms. From the viewpoint of obtaining such effects, - is preferably an iodide ion.

[0058] In addition, the thiol compound (7) and the disulfide compound (14), as well as the combination thereof, The combination forms micelles in water due to the hydrophobic interaction of the R2 groups. The amphiphilic effect of the aqueous interior space and hydrophilic surface prevents denaturation and protein folding. Highly efficient introduction of disulfide bonds into proteins to suppress aggregation in the binding intermediate state In this case, R2 is Preferably, the alkyl group has 3 to 18 carbon atoms, and more preferably, the alkyl group has 4 to 12 carbon atoms. In this case, the target protein for folding is preferably The protein is not particularly limited, and may be any of the above-mentioned proteins, as well as RNase A and bovine pancreatic trypsin inhibitor. Intoxicants, β2-microglobulin, insulin, major histocompatibility complex, immunoglobulin etc.

[0059] The present invention relates to compounds represented by formulas (1) to (14), and salts and solvates thereof. Treating the unfolded protein in the presence of at least one selected The present invention also relates to a protein folding method, which comprises the step of: In the method, the unfolded protein is treated in the presence of the compound. The step of carrying out the unfolded protein (hereinafter also referred to simply as the treatment step) is Specifically, the step of contacting the protein with the compound is The protein and the compound are mixed in a folding buffer solution and mixed by stirring or the like. and, if necessary, after the step, furthermore, in order to more fully proceed with folding. A preferred embodiment of the folding method of the present invention is Unless otherwise specified, the description of the folding agent of the present invention is referred to above. Let's say.

[0060] The present invention relates to compounds represented by formulas (1) to (14), and salts and solvates thereof. Treating the unfolded protein in the presence of at least one selected The present invention also relates to a method for refolding a protein, which comprises the step of folding the protein by using a protein refolding agent. The method for refolding proteins involves treating unfolded proteins in the presence of the above-mentioned compounds. a step of processing and folding the nucleic acid (hereinafter also referred to simply as a processing step), and After this, if necessary, a step of isolating the folded protein (hereinafter referred to as the isolation step) may be carried out. A preferred embodiment of the protein refolding method of the present invention is, unless otherwise specified, Unless otherwise specified, the above-mentioned folding agent of the present invention and the folding method of the present invention The description is to be cited.

[0061] The isolation step may be carried out by, for example, suspending the protein obtained in the folding step. The target normal protein (folded protein) is isolated from the Examples of methods for isolating the compound include using column chromatography. Fillers include silica, dextran, agarose, cellulose, acrylamide, vinyl Commercially available products include Sephadex silica. Sephacryl series, Sepharose series (all of which are Pharma Examples of suitable gelatin gels include those from Bio-Rad (Bio-Rad Laboratories) and Bio-Gel series (Bio-Rad Laboratories). Even if the target normal protein (folded protein) is isolated by dialysis, good.

[0062] The present invention also relates to compounds represented by formulas (1) to (14), and salts and solvates thereof. The present invention also relates to a protein solubilizer containing at least one selected from the following as an active ingredient. The solubilizer of the present invention inhibits aggregation of proteins in their native or non-native structures and solubilizes them. As explained above with respect to the protein folding agent of the present invention, The protein-folding agent of the present invention is an active ingredient or a component used in combination with the active ingredient of the protein-folding agent of the present invention. Thiol compounds represented by formulas (1) to (7) and dimers represented by formulas (8) to (14) Sulfide compounds have the function of inhibiting aggregation of proteins in native or non-native structures, particularly The oxidative folding of proteins is a process that involves the generation of intermediates and the formation of native structures. inhibits protein aggregation and generates one or more proteins with non-native structures It has the function of suppressing the aggregation of proteins with non-native structures in the oxidative folding reaction. The compounds represented by formulas (1) to (14) and their corresponding A preferred embodiment of the present invention for the protein folding agent of the present invention is Reference is made to the above description. [Example]

[0063] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is The present invention is not limited to these examples.

[0064] 1) Synthesis of LDA-SH (2-((2-aminoethyl)amino)ethane-1-thiol) and LDA-SS The above compounds (which correspond to the compounds represented by formulas (1) and (8) respectively) were classified into the following groups: It was synthesized by the team. [ka]

[0065] 1-1) Synthesis of Compound 2 Compound 1 (1.90 g, 18.3 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in dehydrated methyl chloride on an ice bath under a nitrogen atmosphere. The mixture was dissolved in ethylene (25 mL, Kanto Chemical). After adding ethanol (chemical), the mixture was warmed to room temperature. After 19 hours, the solvent was evaporated under reduced pressure, and water (25 mL) was added. The solution was extracted with ethyl acetate (25 mL, twice, Kishida Chemical). The collected ethyl acetate solution was washed with brine (25 mL). After adding the compound, the mixture was filtered. The filtrate was evaporated under reduced pressure and then purified by silica gel column chromatography ( Compound 2 was obtained by the method of Kanto Chemical Co., Ltd. Yield: 3.62 g, Yield: 65%.

[0066] 1-2) Synthesis of Compound 3 N-chlorosuccinimide (NCS, 0.849 g, 6.36 mmol, xylitol) was added to the flask at room temperature under a nitrogen atmosphere. Chemical) was dissolved in dehydrated tetrahydrofuran (THF, 36 mL, Kanto Chemical). A solution of 1.67 g (6.38 mmol) of 1,2-dichloro-2,4 ... After 11 minutes, a solution of compound 2 (1.75 g, 5.74 mmol) in dry THF (13 mL) was added dropwise. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Chemical). This gave compound 3. Yield: 0.60 g, yield: 32%.

[0067] 1-3) Synthesis of Compound 4 Compound 3 (0.597 g, 1.85 mmol) was dissolved in dehydrated N,N-dimethylformamide under a nitrogen atmosphere at room temperature. The solution was dissolved in DMF (3 mL, Kanto Chemical). Potassium thioacetate (AcSK, 0.285 g, 2. After adding 50 mmol of ethanol (Tokyo Chemical Industry Co., Ltd.), the temperature was raised to 90°C. After 12 hours, water (20 mL) was added. After that, it was extracted with ethyl acetate (25 mL, twice, Kishida Chemical). The collected ethyl acetate solution was washed with saturated saline (25 mL). ) was added, and the mixture was filtered. The filtrate was evaporated under reduced pressure. The resulting residue was heated at room temperature in a nitrogen atmosphere. Dehydrated methanol (5 mL, Kanto Chemical) was added under the conditions below, and sodium carbonate (0.757 g, 5.48 mmol, After 1 hour, hydrochloric acid (1 M, Kishida Chemical) was added until the pH reached 8 to 9. Water (25 mL) was added to the reaction mixture, and the mixture was extracted with methylene chloride (25 mL, 3 times, AGC). The extracted methylene chloride solution was washed with saturated saline (25 mL, twice). Sodium sulfate (Kishida Chemical) was added to the toluene solution, which was then filtered. The filtrate was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography (Kanto Chemical) to obtain compound 4. Yield: 0.257 g, yield: 43%.

[0068] 1-4) Synthesis of Compound 5 Compound 4 (0.257 g, 0.803 mmol) was dissolved in ethyl acetate (5.0 mL, Kanto Chemical) at room temperature in air. Sodium iodide (6.9 mg, 46 μmol, Kishida Chemical) was added thereto, and then the mixture was stirred for 30 minutes. % hydrogen peroxide solution (30 mL, Kishida Chemical) was added. After stirring for 1 hour, water (20 mL) was added and vinegar was added. The extracted ethyl acetate solution was diluted with saturated saline. (25 mL). Sodium sulfate (Kishida Chemical) was added to the collected ethyl acetate solution. The filtrate was evaporated under reduced pressure to give compound 5. Yield: 0.241 g, 94%.

[0069] 1-5) Synthesis of LDA-SS Compound 5 (256 mg, 0.401 mmol) was dissolved in methylene chloride (3 mL, AGC) at room temperature in air. Trifluoroacetic acid (2 mL, Kishida Chemical) was added thereto. After stirring for 1 hour, the solvent was reduced. The residue was added with methylene chloride (10 mL, AGC) and extracted with water (10 mL). The aqueous solution was evaporated under reduced pressure and purified by high performance liquid chromatography (apparatus: JASCO Corporation, PU-4086, UV- LDA-SS was obtained using a column (TA12S05-2520WX, column: 4075). Yield: 87 mg, yield: 60%.

[0070] 1-6) Synthesis of LDA-SH LDA-SS (76 mg, 0.632 mmol) was dissolved in water (2 mL) at room temperature under a nitrogen atmosphere. Dithiothreitol (112 mg, 0.726 mmol, Nacalai Tesque) was added to the mixture. After stirring for 24 hours, , high-performance liquid chromatography (apparatus: JASCO, PU-4086, UV-4075, column: TA12S05- LDA-SH was obtained by the method of (2520WX). Yield: 33 mg, yield: 43%.

[0071] 2) Synthesis of BDA-SH (1,3-diaminopropane-2-thiol) and BDA-SS The above compounds (which correspond to the compounds represented by formulas (2) and (9) respectively) were classified into the following groups: It was synthesized by the team. [ka]

[0072] 2-1) Synthesis of Compound 7 Compound 6 (0.539 g, 5.98 mmol) was dissolved in dehydrated methanol (30 mL, After heating to 45°C, (Boc)2O (2.84 g, 13.0 mmol, Kanto Chemical) and trimethylsilyl methylcellulose were added. Ethylamine (6.91 mL, Sigma) was added. After 20 hours, the solvent was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography (Kanto Chemical) to obtain compound 7. Yield: 1.72 g, yield: 99%.

[0073] 2-2) Synthesis of Compound 8 Compound 7 (1.57 g, 5.41 mmol) was dissolved in anhydrous methylene chloride (57 mL, After cooling to 0°C, triethylamine (2.20 mL, Sigma) and Mesilk After stirring for 5 hours, water (30 mL) was added. The mixture was extracted with methylene chloride (30 mL, twice, AGC). After washing with sodium bicarbonate solution (10 mL) and saturated saline solution (10 mL), sodium sulfate (Kishida Chemical) was added. The filtrate was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (Kanto Chemical Compound 8 was obtained by subjecting the resulting mixture to a 1.64 g, 82% yield.

[0074] 2-3) Synthesis of Compound 9 Compound 8 (0.756 g, 2.05 mmol) was dissolved in dehydrated N,N-dimethylformamide at room temperature under a nitrogen atmosphere. The solution was dissolved in DMF (13 mL, Kanto Chemical). Potassium thioacetate (0.392 g, 3.428 mmol, Add triethylamine (0.30 mL, Sigma) and stir at 60°C for 13 hours. After that, water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL, 3 times, Kishida Chemical). The ethyl acetate solution was washed with water (50 mL, twice) and saturated brine (10 mL), and then with sodium sulfate. (Kishida Chemical) was added and filtered. The filtrate was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography. The thioacetyl intermediate was obtained by chromatographing (Kanto Chemical) under a nitrogen atmosphere. The thioacetyl intermediate (0.346 g, 0.994 mmol) was dissolved in anhydrous methanol (3 mL, The solution was dissolved in 100 ml of ethanol (100 ml) and potassium carbonate (0.414 g, 2.998 mmol, Kishida Chemical) was added. After stirring, the solvent was evaporated under reduced pressure. Water (10 mL) was added, and the mixture was washed with methylene chloride (10 mL, 3 times, AGC). The recovered methylene chloride solution was diluted with saturated aqueous ammonium chloride (10 mL, After washing with saturated saline (10 mL), sodium sulfate (Kishida Chemical) was added and filtered. The filtrate was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Chemical). This gave compound 9. Yield: 0.275 g, yield: 43%.

[0075] 2-4) Synthesis of Compound 10 Compound 9 (0.279 g, 0.897 mmol) was dissolved in ethyl acetate (5 mL, Kanto Chemical) at room temperature. , sodium iodide (11.8 mg, 0.079 mmol, Kishida Chemical), 30% hydrogen peroxide solution (0.05 mL After stirring for 1 hour, water (30 mL) was added, and ethyl acetate (30 mL, twice) was added. The collected ethyl acetate solution was washed with saturated saline (10 mL) and then extracted with sodium sulfate. The filtrate was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography ( Compound 10 was obtained by subjecting the mixture to a chromatographic process (Kanto Chemical Co., Ltd.). Yield: 0.274 g, yield: 99%.

[0076] 2-5) Synthesis of BDA-SS Compound 10 (0.274 g, 0.449 mmol) was dissolved in methylene chloride (5 mL, AGC) and cooled to 0°C. The temperature was lowered, trifluoroacetic acid (1 mL, Kishida Chemical) was added, and the temperature was raised to room temperature. After stirring, the solvent was evaporated under reduced pressure. Chloroform (10 mL, Kishida Chemical) was added to the resulting residue. The resulting aqueous solution was evaporated under reduced pressure, and the residue was diluted with hydrochloric acid (1 M, 5 mL). mL, Kishida Chemical) was added. After stirring for 1 hour, the solvent was evaporated under reduced pressure to obtain BDA-SS. Yield: 0. 103 g, yield: 64%.

[0077] 2-6) Synthesis of BDA-SH Under a nitrogen atmosphere, BDA-SS (92.3 mg, 0.259 mmol) was dissolved in water (3 mL) at room temperature. Ostreitol (183 mg, 1.19 mmol, Nacalai Tesque) was added. After stirring for 24 hours, A mixed solvent of chloroform (Kishida Chemical) and 1-propanol (Kishida Chemical) (1:5, 5 mL) was added. The resulting solution was evaporated under reduced pressure to give BDA-SH. 85.5 mg, yield: 93%.

[0078] 3) Synthesis of ImdM-SH ((1H-imidazol-4-yl)methanethiol) and ImdM-SS The above compounds (which correspond to the compounds represented by formulas (3) and (10) respectively) were subjected to the following steps: It was synthesized according to the scheme. [ka]

[0079] 3-1) Synthesis of Compound 12 Compound 11 (5.512 g, 57.36 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in dehydrated ethanol at room temperature under a nitrogen atmosphere. The mixture was cooled to 0°C and dissolved in sodium borohydride (2.1 mL, Kanto Chemical). After stirring for 3 hours, water (10 mL) was added and the solvent was The obtained residue was subjected to silica gel column chromatography (Kanto Chemical). Compound 12 was obtained by the reaction of 4.10 g of the toluene with 2,000 ethanol and 1,000 ethanol. Yield: 73%.

[0080] 3-2) Synthesis of Compound 13 Compound 12 (0.565 g, 5.76 mmol) was dissolved in dehydrated tetrahydrofuran ( The solution was dissolved in 1.7 mL of (Boc)2O(1. A solution of 37 g (6.28 mmol) of 100 ml of HCl in dehydrated tetrahydrofuran (8.1 mL, Kanto Chemical) was added dropwise. After stirring for 13 hours, the solvent was evaporated under reduced pressure, water (15 mL) was added, and methylene chloride (20 mL, The recovered methylene chloride solution was extracted with sodium sulfate (Kishida Chemical Co., Ltd.) five times. The filtrate was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography. (Kanto Chemical) to obtain compound 13. Yield: 0.763 g, yield: 67%.

[0081] 3-3) Synthesis of Compound 14 Compound 13 (0.917 g, 4.63 mmol) was dissolved in anhydrous methylene chloride (8.4 mL) at room temperature under a nitrogen atmosphere. , Kanto Chemical) and dissolved in dehydrated N,N-dimethylformamide (DMF, 0.2 mL, Kanto Chemical). After cooling to 0°C, oxalyl chloride (1.22 g, 9.58 mmol, Kishida Chemical) was dissolved in dehydrated methylene chloride. After the temperature was raised to room temperature and stirring for 1.5 hours, water (15 mL) was added. The solution was extracted with methylene chloride (15 mL, twice, Kishida Chemical). Sodium sulfate (Kishida Chemical) was added and the mixture was filtered. The filtrate was evaporated under reduced pressure, and the residue was filtered with silica gel. Compound 14 was obtained by column chromatography (Kanto Chemical). 0.432 g, yield: 43%.

[0082] 3-4) Synthesis of Compound 15 Compound 14 (0.168 g, 0.774 mmol) was dissolved in dehydrated acetone (4.7 mL, The solution was dissolved in 100 ml of sodium iodide (0.233 g, 1.55 mmol, Kishida Chemical), thiol (0.233 g, 1.55 mmol, Kishida Chemical), and 100 ml of thiol (0.233 g, 1.55 mmol, Kishida Chemical). Potassium acetate (0.142 g, 1.25 mmol, Tokyo Chemical Industry Co., Ltd.) was added. After stirring for 21 hours, water (20 mL) was added and extracted with ethyl acetate (30 mL, twice, Kishida Chemical). The solution was washed with saturated saline (10 mL), sodium sulfate (Kishida Chemical) was added, and the mixture was filtered. The filtrate was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Chemical). This gave compound 15. Yield: 0.140 g, yield: 70%.

[0083] 3-5) Synthesis of ImdM-SS Compound 15 (0.911 g, 3.55 mmol) was dissolved in anhydrous methanol (10 mL, The solution was dissolved in 1.00 g of potassium carbonate (1.00 g, 7.24 mmol, Kishida Chemical Co., Ltd.) and the resulting solution was added to 1.00 g of potassium carbonate (1.00 g, 7.24 mmol, Kishida Chemical Co., Ltd.). After stirring for 1 hour, the solvent was evaporated under reduced pressure, water (10 mL) was added, and the mixture was neutralized with hydrochloric acid (1 M). Extraction was performed with a mixed solvent of ethanol (Kishida Chemical) and 1-propanol (Kishida Chemical) (3:1, 25 mL, 4 times). The solvent was evaporated under reduced pressure, and the resulting residue was purified by reversed-phase high-performance liquid chromatography (apparatus: The ImdM-SS was obtained by purifying the mixture using a spectrophotometer (PU-4086, UV-4075, column: TA12S05-2520WX). Yield: 27 mg, yield: 4%.

[0084] 3-6) Synthesis of ImdM-SH ImdM-SS (0.105 g, 0.464 mmol) was dissolved in water (7 mL) at room temperature under a nitrogen atmosphere. Osreitol (123 mg, 0.797 mmol, Nacalai Tesque) was added. After stirring for 7 hours, A mixture of chloroform (Kishida Chemical) and 1-propanol (Kishida Chemical) (9:1, 25 mL, 4 times) ) was added and extracted. Sodium sulfate was added, filtered, and the filtrate was evaporated under reduced pressure. The resultant was purified by reversed-phase high-performance liquid chromatography (apparatus: JASCO Corporation, PU-4086, UV-4075, column: TA12 S05-2520WX) to give ImdM-SH. Yield: 38 mg, yield: 18%.

[0085] 4) Synthesis of pPyM-SH (para-pyridin-4-ylmethanethiol) and pPyM-SS The above compounds (corresponding to the compounds represented by formulas (4) and (11) respectively) were subjected to the following steps: It was synthesized according to the scheme. [ka]

[0086] 4-1) Synthesis of pPyM-SH Compound 16 (2.175 g, 13.26 mmol) was dissolved in water and thiourea (1.450 g, 19.04 mmol) was added. After heating to 90°C, the mixture was stirred for 1.5 hours and then cooled to room temperature. After stirring for 22 hours, tert-butylmethyl The resulting aqueous solution was washed with diethyl ether (30 mL, twice, Kanto Chemical). The solution was neutralized with sodium sulfate (Kishida Chemical) and extracted with chloroform (30 mL). The filtrate was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography. The product was purified by chromatography (Kanto Chemical) to give pPyM-SH. Yield: 459 mg, yield: 14%.

[0087] 4-2) Synthesis of pPyM-SS pPyM-SH (51 mg, 0.41 mmol) was dissolved in water (1 mL) and 30% hydrogen peroxide solution (1 mL, xylene). After stirring for 14 hours, the mixture was extracted with methylene chloride (10 mL, 3 times, Kishida Chemical). Sodium sulfate was added and the mixture was filtered. The filtrate was evaporated under reduced pressure to give pPyM-SS. Yield: 46 mg. Yield: 91%.

[0088] 5) Synthesis of oPyM-SH (ortho-pyridin-2-ylmethanethiol) and oPyM-SS The above compounds (corresponding to the compounds represented by formulas (5) and (12) respectively) were subjected to the following steps: It was synthesized according to the scheme. [ka]

[0089] 5-1) Synthesis of oPyM-SH Compound 17 (1.035 g, 6.309 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in water and thiourea (0.713 g, 9.36 mmol, Kishida Chemical Co., Ltd.) was added. After heating to 90°C, the mixture was stirred for 1 hour and then cooled to room temperature. Sodium hydroxide (1.00 g, 25.0 mmol, Kishida Chemical) was added and stirred for 9 hours. The resulting aqueous solution was washed with methyl ether (40 mL, Kanto Chemical). The mixture was neutralized with methylene chloride (Kishida Chemical) and extracted with methylene chloride (30 mL, 3 times). Sodium sulfate (Kishida Chemical) was added to the polyethylene solution and filtered. The filtrate was evaporated under reduced pressure. The residue (oPyM-SH crude product) was purified by high performance liquid chromatography (apparatus: JASCO, PU-4086 The product was purified using a UV-4075 column (TA12S05-2520WX) to obtain oPyM-SH. Yield: 113 mg. 8%.

[0090] 5-2) Synthesis of oPyM-SS The crude oPyM-SH product obtained by the above method was dissolved in 30% hydrogen peroxide (8 mL, Kishida Chemical). The mixture was stirred at room temperature for 1 hour, extracted with chloroform (30 mL, 3 times, Kishida Chemical), and collected. Sodium sulfate (Kishida Chemical) was added to the chloroform solution, which was then filtered. The filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (Fuji Silysia Chemical). This gave oPyM-SS. Yield: 916 mg, yield: 58%.

[0091] 6) pPyM-SH-NMe·M ((4-(mercaptomethyl)-1-methylpyridin-1-ium) halide) and Synthesis of pPyM-SS-NMe·M The above compounds (corresponding to the compounds represented by formulas (6) and (13) respectively) were subjected to the following steps: It was synthesized according to the scheme. [ka]

[0092] 6-1) pPyM-SS-NMe·I(4,4'-(disulfanediylbis(methylene))bis(1-methylpyridine) Synthesis of di-1-ium diiodide The pPyM-SS (254 mg, 1.02 mmol) obtained by the above method was dissolved in acetonitrile (3.0 mL, Dissolve iodomethane (1.503 g, 10.59 mmO in acetone (3 mL, Kishida Chemical) and After heating to 60°C, the mixture was stirred for 16 hours and then cooled to room temperature. The residue was filtered to give pPyM-SS-NMe·I. Yield: 446 mg, yield: 82%.

[0093] 6-2) pPyM-SS-NMe·Cl(4,4'-(disulfanediylbis(methylene))bis(1-methylpiperidinyl) Synthesis of Lysin-1-ium Dichloride pPyM-SS-NMe·I (192 mg, 0.361 mmol) was dissolved in water (3.5 mL) and diluted with silver(I) chloride (115 mg, 0.805 mmol, Kishida Chemical Co., Ltd.) was added. After stirring for 4 hours at room temperature in the dark, the resulting precipitate The filtrate was evaporated under reduced pressure, and ethanol (2 mL, Kishida Chemical) was added to the residue. The residue was filtered. The filtrate was evaporated under reduced pressure to give pPyM-SS-NMe Cl. Yield: 80 mg, yield: 63%.

[0094] 6-3) pPyM-SH-NMe·Cl((4-(mercaptomethyl)-1-methylpyridin-1-ium) chloride) Synthesis of d) pPyM-SS-NMe Cl (39 mg, 0.141 mmol) was dissolved in degassed water (2.1 mL) and dithiothiazolinone was added. Leitol (DTT, 29 mg, 0.187 mmol, Nacalai Tesque) was added and the mixture was stirred at room temperature for 6 hours. After that, it was evaporated under reduced pressure, and the resulting residue (crude pPyM-SH-NMe·Cl product) was purified by high-performance liquid chromatography. The pPyM was purified using a filter (apparatus: JASCO Corporation, PU-4086, UV-4075, column: TA12S05-2520WX). -SH-NMe·Cl was obtained. Yield: 40 mg, yield: 81%.

[0095] 6-4) pPyM-SH-NMe·I((4-(mercaptomethyl)-1-methylpyridin-1-ium) iodide ) synthesis The pPyM-SS-NMe·I (104 mg, 0.195 mmol) obtained by the above method was dissolved in degassed water (3.1 mL). After dissolving, dithiothreitol (DTT, 125 mg, 0.809 mmol, Nacalai Tesque) was added. After stirring at room temperature for 3 hours, 2M hydrochloric acid (1 mL, Kishida Chemical) was added, and the mixture was evaporated under reduced pressure. The residue (crude pPyM-SH-NMe·I) was purified by high performance liquid chromatography (apparatus: JASCO, PU The product was purified using a TA12S05-2520WX column (UV-4086, UV-4075) to obtain pPyM-SH-NMe·I. Yield: 23 mg, yield: 22%.

[0096] 7) oPyM-SH-NMe·M ((2-(mercaptomethyl)-1-methylpyridin-1-ium) halide) and and oPyM-SS-NMe·M(2,2'-(disulfanediylbis(methylene))bis(1-methylpyridine-1 Synthesis of (-ium) halide The above compounds (corresponding to the compounds represented by formulas (7) and (14) respectively) were subjected to the following steps: It was synthesized according to the scheme. [ka]

[0097] 7-1)oPyM-SS-NMe·I(2,2'-(disulfanediylbis(methylene))bis(1-methylpyridine) Synthesis of di-1-ium diiodide The oPyM-SS (739 mg, 2.97 mmol) obtained by the above method was dissolved in acetonitrile (9.7 mL, The solution was dissolved in iodomethane (4.434 g, 31.24 mmHg) and acetone (10 mL, Kishida Chemical). After heating to 65°C, the mixture was stirred for 16 hours and then cooled to room temperature. The residue was filtered to give oPyM-SS-NMe·I. Yield: 1.286 g, 81%.

[0098] 7-2)oPyM-SH-NMe·I((2-(mercaptomethyl)-1-methylpyridin-1-ium) iodide ) synthesis oPyM-SS-NMe·I (108 mg, 0.203 mmol) was dissolved in degassed water (3 mL) and dithiothreitol was added. Dimethyl ether (DTT, 130 mg, 0.840 mmol, Nacalai Tesque) was added and the mixture was stirred at room temperature for 5 hours. After that, 2M hydrochloric acid (1 mL, Kishida Chemical) was added, and methylene chloride (20 mL, 5 times, Kishida Chemical) was added. The collected aqueous solution was evaporated under reduced pressure, and the resulting residue (crude oPyM-SH-NMe·I) was High-performance liquid chromatography (apparatus: JASCO, PU-4086, UV-4075, column: TA12S05-25 The product was purified by HPLC (20WX) to give oPyM-SH-NMe·I. Yield: 80 mg, yield: 74%.

[0099] 8) Preparation of reduced and denatured proteins Bovine pancreatic trypsin inhibitor (BP) was used as a model substrate. TI) (see References 1 and 2 below), RNase A (see Reference 3 below), and BPTI and RNase A were used. These proteins have three and four disulfide bonds, respectively, and are model substrates in the field. In addition, β2m was used as a model substrate with one disulfide bond. Ta.

[0100] Create a reduced denatured form of BPTI (Takara Bio Inc.) (a reduced form of three disulfide bonds) To determine the activity, 10 mg of BPTI was incubated in the presence of 8 M urea and 20 mM DTT at pH 8.0 at 50°C for 3 hours. The purified sample was purified by reversed-phase HPLC. All disulfide bonds in the purified sample were confirmed by MALDI-TOF / MS. After confirming that the fragment was cleaved, the fragment was freeze-dried and stored at -80°C (see Reference 1 below). did).

[0101] To prepare a reduced denatured form of RNase A (Sigma) (reduced form of four disulfide bonds) 8 mg of RNase A was incubated in the presence of 6 M guanidine hydrochloride and 100 mM DTT at pH 8.7 at 25°C for 2 hours. The solution was dialyzed against 10 mM HCl. Dialysis was further carried out twice, and the solution was diluted to 10 mM HCl after removing DTT and other components. (See Reference 3 below).

[0102] β2m was expressed as a recombinant in an E. coli expression system, and the collected cells were added to the lysate A (5 After suspending in 0 mM Tris-HCl (pH 8.1, 300 mM NaCl), the cells were sonicated. The fraction was incubated in the presence of 8 M urea and 20 mM DTT at pH 8.0 at 50°C for 3 hours, and then transferred to Cosmosil 5C. 18 The product was purified by reversed-phase column chromatography using an AR-II column (Hitachi).

[0103] Reference 1: M. Okumura, H. Kadokura, S. Hashimoto, K. Yutani, S. Kanemura, T. Hikima, Y. Hidaka, L. Ito, K. Shiba, S. Masui, D. Imai, S. Imaoka, H. Yamaguchi, K. Inaba, Inhibition of the functional interplay between endoplasmic reticulum (ER) oxidoreduclin-1α (Ero1α) and protein-disulfide isomerase (PDI) by the end ocrine disruptor bisphenol A. J Biol Chem. 2014 289(39):27004-27018. Reference 2: JS Weissman, PS Kim, Reexamination of the folding of BPTI: pre Dominance of native intermediates. Science 1991 253(5026):1386-93. Reference 3: MM Lyles, HF Gilbert Catalysis of the oxidative folding of rib onuclease A by protein disulfide isomerase: dependence of the rate on the compos ition of the redox buffer. Biochemistry. 1991 30(3):613-9.

[0104] 9) Evaluation of disulfide bond introduction ability into reduced denatured BPTI 30 μM of the reduced denatured BPTI prepared in 8) above was diluted with 1 mM of thiol compound / 0.2 mM disulfide. The following combinations of filo-compounds: Comparative example 1: GSH(nakalai) / GSSG(nakalai) Example 1: LDA-SH / LDA-SS Example 2: BDA-SH / BDA-SS A buffer solution containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl was incubated at 30°C and The reaction mixture was taken over time, and an equal volume of 1N HCl was added to remove the thiol groups. The disulfide bond exchange reaction was quenched with HCl. The molecular species in the reaction mixture (see Figure 1A) were To identify the compound, it was subjected to reversed-phase HPLC. The analysis using reversed-phase HPLC was performed as shown in Figure 1A and Figure 1B. As shown in Fig. 1, each disulfide bond species during BPTI folding can be separated and identified, and the time course of changes can be observed. The position of disulfide bonds in the ligation can be tracked.

[0105] The measurement and analysis conditions for reversed-phase HPLC are as follows: The column used was a TSKgel Protein C4-300 column (4.6 × 150 mm; Tosoh Bioscience). The absorbance at 29 nm was measured. Regarding the mixture of acetonitrile solution (liquid B) containing fluoroacetic acid, The volume increase rate was 1% / min from 0 to 15 min and 0.5% / min from 15 to 115 min in a linear gradient. did.

[0106] When GSH / GSSG was used (Comparative Example 1), the folding reaction time was 60 minutes. The yield was 26% (Fig. 2A). The yield of the native form (N) after 60 minutes of mixing was 31% (Figure 2B). It was shown that LDA-SH has a stronger folding-promoting effect than GSH.

[0107] When BDA-SH / BDA-SS was used (Example 2), the reduced and modified forms of BPTI (all thiol) were obtained within 1 minute of the reaction. The R group (all group) disappears, and the natural type (N) is generated, and the disulfide bond is formed very quickly. It can be seen that the non-native structure of BPTI (NonN in Figure 2C) has been introduced (Figure 2C). It can be seen that many types of disulfides (shown as The protein exhibits aggregation. However, when subjected to HPLC measurement in this way, Therefore, BDA-SH / BDA-SS inhibit the aggregation of non-native BPTI and solubilize it. It was found that the compound has properties as a drug.

[0108] 10) RNase A activity evaluation 1 To evaluate the folding promotion effect, we investigated the recovery of RNase A activity prepared in 8) above. Measurements were performed using the following combinations of 1 mM thiol compound / 0.2 mM disulfide compound: Comparative Example 2: GSH / GSSG Example 3: LDA-SH / LDA-SS In the presence of RNase A, a buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 8 μM RNase A was incubated at 30°C. The reaction mixture was then incubated at 200°C for 1 hour. The reaction mixture was then sampled over time and subjected to RNase A cleavage to identify cytidine 2':3 Dilute with the same buffer containing cytidine 2':3'-cyclic monophosphate (cCMP). The final concentration was 4 μM RNase A and 0.4 mM cCMP. The change was measured to quantify the nucleolytic activity of RNase A folded into its native structure.

[0109] As shown in Figure 3, the recovery of RNase A activity after 3 hours of incubation was 0.2 mM GSSG. (14%) and GSH / GSSG (Comparative Example 2) (33%), In the presence of Example 3, a high recovery activity of 42% was observed. It was shown to have a folding-promoting effect.

[0110] 11) Evaluation of disulfide bond introduction ability into reduced denatured β2m 10 μM of the reduced denatured β2m prepared in 8) above was diluted with 1 mM thiol compound / 0.2 mM disulfide. The following combinations of filo-compounds: Comparative Example 3: GSH / GSSG Example 4: LDA-SH / LDA-SS Example 5: BDA-SH / BDA-SS A buffer solution containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl was incubated at 30°C and The reaction mixture was collected over time and diluted with 6 M guanidine hydrochloride, 1.5 M HCl, and The disulfide bond introduction reaction was quenched by adding an equal amount of HCl. To investigate this, the oxidized (N) and reduced (R) forms of β2m were analyzed by reversed-phase HPLC (Figures 4A-C and Figure 5). The oxidized (N) and reduced (R) molecular species were identified by MALDI-TOF / MS.

[0111] The measurement and analysis conditions for reversed-phase HPLC are as follows: the column was a 5C18-AR2 column (4.6 × 150 mm; The absorbance was detected at 229 nm using a Nacalai tesque. Water containing 0.1% trifluoroacetic acid The mixture of solution A and acetonitrile solution B containing 0.1% trifluoroacetic acid was The volume increase rate of the proportion of solution B in the mixed solution was 1% / min from 0 to 10 minutes, and 0.5% / min from 10 to 35 minutes. The solution was developed with a linear concentration gradient of 1 min.

[0112] 4A to 4C and FIG. 5, when GSH / GSSG was used (Comparative Example 3), the folding reaction The yield after 60 minutes was 36%. The yield after 60 minutes of reaction was 95%. The yield after 60 minutes of heating reaction was 100%.

[0113] From these results, the disulfide bond introduction reaction into the reduced denatured β2m was assumed to be a pseudo-first-order reaction with a reaction rate of The coefficient of refraction was calculated as GSSG: 1.12 × 10 -4 , LDA-SS: 6.96 × 10 -4 , BDA-SS: 3.59 × 1 0 -2 As compared with GSH / GSSG (Comparative Example 3), LDA-SH / LDA-SS (Example 4) was 6.2 times as thick, and BDA -SH / BDA-SS (Example 5) was shown to have approximately 320 times higher disulfide bond introduction ability. β2m is one of the components involved in immunity, and its misfolded form is amyloid globulin. The major histocompatibility complex (MHC) is the foundation of the immune system. The disulfide bond of the present invention can be rapidly introduced into the light chain β2m. Sulfide bond-introducing agents are useful in the efficient production of protein formulations.

[0114] 12) RNase A activity evaluation 2 To evaluate the folding promotion effect, we investigated the recovery of RNase A activity prepared in 8) above. Measurements were performed using the following combinations of 1 mM thiol compound / 0.2 mM disulfide compound: Comparative Example 4: GSH / GSSG Example 6: ImdM-SH / ImdM-SS Example 7: oPyM-SH / oPyM-SS In the presence of RNase A, a buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 8 μM RNase A was incubated at 30°C. The reaction mixture was then incubated at 200°C for 1 hour. The reaction mixture was then sampled over time and subjected to RNase A cleavage to identify cytidine 2':3 Dilute with the same buffer containing cytidine 2':3'-cyclic monophosphate (cCMP). The final concentration was 4 μM RNase A and 0.4 mM cCMP. The change was measured to quantify the nucleolytic activity of RNase A folded into its native structure.

[0115] As shown in Figure 6, the recovery activity of RNase A after 6 hours of incubation was significantly higher than that of GSH / GSSG (Comparative Example 4). In the presence of ImdM-SH / ImdM-SS (Example 6), the recovery was as high as 45% compared to the case of ImdM-SH / ImdM-SS (Example 6) (38%). This indicates that ImdM-SH / ImdM-SS has a folding-promoting effect. Furthermore, oPyM-SH / oPyM-SS (Example 7) showed a significant reduction in RNase A activity after 3 hours of incubation. The recovery activity of GSH / GSSG (Comparative Example 4) was higher than that of GSH / GSSG (Comparative Example 4), and was high in the early stage up to 3 hours. It was shown that it has a strong folding-promoting effect.

[0116] 13) RNase A activity evaluation 3 To evaluate the folding promotion effect, we investigated the recovery of RNase A activity prepared in 8) above. Measurements were performed using the following combinations of 1 mM thiol compound / 0.2 mM disulfide compound: Comparative Example 5: GSH / GSSG Example 8: pPyM-SH-NMe Cl / pPyM-SS-NMe Cl Example 9: pPyM-SH-NMe·I / pPyM-SS-NMe·I Example 10: oPyM-SH-NMe·I / oPyM-SS-NMe·I In the presence of RNase A, a buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 8 μM RNase A was incubated at 30°C. The reaction mixture was then incubated at 200°C for 1 hour. The reaction mixture was then sampled over time and subjected to RNase A cleavage to identify cytidine 2':3 Dilute with the same buffer containing cytidine 2':3'-cyclic monophosphate (cCMP). The final concentration was 4 μM RNase A and 0.4 mM cCMP. The change was measured to quantify the nucleolytic activity of RNase A folded into its native structure.

[0117] As shown in Figure 7, the recovery activity of RNase A after 6 hours of incubation was significantly higher than that of GSH / GSSG (Comparative Example 5). ) (38%), in the presence of pPyM-SH-NMe·Cl / pPyM-SS-NMe·Cl (Example 8) 65% in the presence of pPyM-SH-NMe·I / pPyM-SS-NMe·I (Example 9), 54% in the presence of oPyM-SH-NMe·I / oPyM In the presence of -SS-NMe·I (Example 10), the recovery activity was as high as 43%. SH-NMe·Cl / pPyM-SS-NMe·Cl, pPyM-SH-NMe·I / pPyM-SS-NMe·I, and oPyM-SH-NMe·I / oP It was shown that yM-SS-NMe·I has a folding-promoting effect.

[0118] 14) Heat-induced aggregation of β2m (No additives) A buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 10 μM of native β2m was incubated at 25°C for 5 min. The mixture was heated to 0°C, and when it reached 50°C, it was equilibrated for 10 minutes, and then cooled to 25°C. The absorbance of the supernatant of the fraction obtained after separation was measured at 280 nm using a spectrophotometer. The residual rate of native β2m in the supernatant was measured.

[0119] (Additives included) The following was performed in a buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 10 μM of native β2m: 1 mM of the following thiol compounds: Comparative example 6; GSH Example 11: LDA-SH Example 12: BDA-SH The mixture was heated from 25 to 50°C, and equilibrated for 10 minutes at 50°C. The supernatant of the fraction obtained after centrifugation was measured for absorbance at 280 nm using a spectrophotometer. The remaining rate of native β2m in the supernatant was determined by measuring the concentration.

[0120] Figure 8 shows a photograph of the solution after cooling (before centrifugation). and BDA-SH (Example 12), compared with the case of no additive and GSH (Comparative Example 6), The result was that the solution was transparent, and aggregation of native β2m was suppressed. The residual rate of natural β2m in the supernatant was measured and the results are shown in Figure 9. In Example 11) and BDA-SH (Example 12), the results were compared with those without additives and GSH (Comparative Example 6). Therefore, the residual rate of native β2m is high, and therefore, LDA-SH and BDA-SH inhibit protein aggregation. It was found that the compound has a high solubilizing function.

[0121] 15) RNase A activity evaluation 4 To evaluate the folding-promoting effect, the activity recovery of the RNase A prepared in 8) above was measured. The following combinations of 1 mM thiol compound and 0.2 mM disulfide compound were used: Example 13: pPyM-SH / GSSG A buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 8 μM RNase A was incubated at 30°C in the presence of RNase A. Aliquots of the reaction mixture were collected over time and diluted with the same buffer solution containing cytidine 2′:3′-cyclic monophosphate (cCMP), a substrate for RNase A, to a final concentration of 4 μM RNase A and 0.4 mM cCMP. The change in absorbance at 284 nm was measured using a spectrophotometer to quantify the nucleolytic activity of RNase A folded into its native structure. The results are shown in Figure 10, along with those of Comparative Example 4 and Examples 6 and 7 in "12) Evaluation of RNase A Activity 2." [Industrial Applicability]

[0122] The protein folding agent of the present invention can be used for proteins such as antibodies having multiple disulfide bonds. The present invention can be used as an oxidative folding promoter for protein formulations. The protein folding agent has been shown to be highly effective in folding proteins with a single disulfide bond. The protein of the present invention can be used as a fast oxidative folding promoter. Protein folding agents are used to artificially store misfolded proteins in a soluble state. It can be used as a research reagent in basic research in the field of biochemistry. .

Claims

1. The following formula: 【Chemical 1】 【change】 [In the formula, X １ , X ２ and X ３ Each independently contains 1 to 5 oxygen atoms in the molecular chain. an alkylene group having 1 to 10 carbon atoms which may be Y １ each independently represents a carbon number that may contain 1 to 5 oxygen atoms in the molecular chain 1 to 10 alkylene groups, Z １ , Z ２ , Z ３ , Z ４ and Z ５ each independently contains 1 to 5 oxygen atoms in the molecular chain. an alkylene group having 1 to 10 carbon atoms, which may contain a group; R １ and R ２ are each independently an alkyl group having 1 to 24 carbon atoms, M － are each independently a chloride ion, a bromide ion, or an iodide ion. and at least one compound selected from the group consisting of a compound represented by the formula (I) and a salt and solvate thereof as an active ingredient. A protein folding agent comprising:

2. A compound selected from the group consisting of compounds represented by formulas (1) to (7), and salts and solvates thereof. The protein-folding agent according to claim 1, comprising at least one of the following as an active ingredient: 。

3. A compound selected from the group consisting of compounds represented by formulas (8) to (14), and salts and solvates thereof: The protein folding method according to claim 1, comprising at least one active ingredient. Agent.

4. A compound selected from the group consisting of compounds represented by formulas (1) to (7), and salts and solvates thereof. and compounds represented by formulas (8) to (14), and salts and solvents thereof.

2. The protein according to claim 1, comprising at least one compound selected from the group consisting of benzodiazepines, ... Folding agent.

5. Compounds represented by formulas (1) to (14) as defined in claim 1, and salts and solutions thereof. The unfolded protein is obtained in the presence of at least one selected from the group consisting of a soluble and a soluble protein. A method for folding a protein, comprising the step of treating the protein.

6. Compounds represented by formulas (1) to (14) as defined in claim 1, and salts and solutions thereof. The unfolded protein is obtained in the presence of at least one selected from the group consisting of a soluble and a soluble protein. A method for regenerating proteins, comprising the step of treating proteins.

7. Compounds represented by formulas (1) to (14) as defined in claim 1, and salts and solutions thereof. A protein solubilizer comprising at least one solvate selected from the group consisting of:

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