JP7722664B2 - Protein folding agents - Google Patents

Description

The present invention relates to a protein folding agent.

Proteins are materials widely used in industry, primarily in pharmaceuticals. Because the formation of a native structure is essential for proteins to exert their individual functions and activities, efficient folding techniques into the native structure are important for industrial protein applications, directly linked to cost reductions. Therefore, promoters of oxidative folding into the native structure are beneficial for the large-scale production of protein pharmaceuticals. For example, in the oxidative folding process of important protein pharmaceuticals such as insulin and immunoglobulin, the formation of the native structure, which involves the introduction of native disulfide bonds, is the rate-limiting step.

Disulfide bond shuffling is a typical method for controlling the oxidative folding process. By incorporating a reducing agent and an oxidizing agent into the folding process, disulfide bonds are repeatedly formed and broken, leading to the most stable native structure. Typical reducing and oxidizing agents used in this process are glutathione (GSH), β-mercaptoethanol (β-ME), dithiothreitol (DTT), and glutathione oxidized salt (GSSG), respectively. For example, in the case of RNase A, the recovery yield was compared between the presence and absence of GSH/GSSG (in air). The enzyme activity in the early folding process was approximately 2-fold higher in the presence of GSH/GSSG, and the recovery rate (half activity recovery time) was approximately 2.8-fold faster (Non-Patent Document 1). Furthermore, small molecule reducing agents (and oxidizing agents combined therewith) used in oxidative folding include GSH/GSSG (Non-Patent Document 1), GSH + (±)-trans-1,2-bis(2-mercaptoacetamido)cyclohexane (BMC)/GSSG (Non-Patent Document 2), and aromatic thiols and their disulfides (
Non-patent document 3), Cyclic selenoxide (Non-patent document 4), Cys-XX-Cy
Peptides with an s structure (X is any amino acid, Cys is cysteine) (Non-Patent Document 5), selenoglutathione (Non-Patent Document 6), and selenol-containing peptides (Non-Patent Document 7) are known. However, cyclic selenoxides and selenoglutathiones have the problem of toxicity due to the use of heavy metal selenium. In addition, BM
The problem with C is that it is used as an auxiliary agent for GSH, so a larger variety of additives must be added than with the GSH/GSSG system. Aromatic thiols, which contain a benzene ring, can be problematic because they can nonspecifically adsorb to proteins due to hydrophobic interactions. Peptides with Cys-XX-Cys structures have problems with structural instability, such as degradation due to protease contamination, and the high cost of their synthesis. Low-molecular-weight thiols, such as cysteamine, have a strong odor due to their high volatility. Furthermore, when these existing compounds are used, their inhibitory effect on the aggregation of oxidative folding intermediates is low, which contributes to reduced yields.

Therefore, there has been a demand for a protein folding agent that can fold proteins with high efficiency while avoiding the problems of the conventional techniques.

A. K. Ahmed et al., J. Biol. Chem., 1975, 250, 8477-8482 K. J. Woycechowsky et al., Chem. Biol., 1999, 6, 871-879 D. J. Madar et al., J. Biotech., 2009, 142, 214-219 K. Arai, K et al., Chem. Eur. J., 2011, 17, 481-485 W. J. Lees et al., Curr. Opin. Chem. Biol., 2008, 12, 740-745 J. Beld et al., Biochemistry. 2007 May 8;46(18):5382-90 S. Tsukagoshi et al., Chem. Asian J. 2020 September 1;15(17):2646-52

Therefore, an object of the present invention is to provide a protein folding agent that is capable of folding proteins with high efficiency.

As a result of investigating various means for solving the above problems, the present inventors have found that disulfide bonds can be introduced into proteins with high efficiency, i.e., high yield and/or high speed, by using a compound having a specific structure, and a salt and solvate thereof,
The present invention has been completed.

That is, the gist of the present invention is as follows.
[1] The following formula:
[In the formula,
X ₁ , X ₂ and X ₃ each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
_Y1 's each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
Z ₁ , Z ₂ , Z ₃ , Z ₄ and Z ₅ each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
R ₁ and R ₂ are each independently an alkyl group having 1 to 24 carbon atoms;
Each M ⁻ is independently a chloride ion, a bromide ion, or an iodide ion.
A protein-folding agent comprising, as an active ingredient, at least one compound selected from the group consisting of a compound represented by the formula (I) and a salt and solvate thereof.
[2] The protein-folding agent according to [1] above, comprising, as an active ingredient, at least one selected from the group consisting of compounds represented by formulae (1) to (7), and salts and solvates thereof:
[3] The protein-folding agent according to [1] above, comprising, as an active ingredient, at least one selected from the group consisting of compounds represented by formulae (8) to (14), and salts and solvates thereof:
[4] The protein-folding agent according to [1] above, comprising, as an active ingredient, at least one selected from the group consisting of compounds represented by formulas (1) to (7), and salts and solvates thereof; and at least one selected from the group consisting of compounds represented by formulas (8) to (14), and salts and solvates thereof.
[5] A method for folding a protein, comprising the step of treating an unfolded protein in the presence of at least one compound selected from the group consisting of compounds represented by formulae (1) to (14) defined in [1] above, and salts and solvates thereof.
[6] A method for refolding a protein, comprising the step of treating an unfolded protein in the presence of at least one compound selected from the group consisting of compounds represented by formulae (1) to (14) defined in [1] above, and salts and solvates thereof.
[7] A protein solubilizer comprising, as an active ingredient, at least one selected from the compounds represented by formulas (1) to (14) defined in [1] above, and salts and solvates thereof.

The protein folding agent of the present invention can fold proteins with high efficiency.

FIG. 1A shows the structure of bovine pancreatic trypsin inhibitor (BPTI) and the disulfide bond bridge locations of each disulfide bond species in its folding pathway. 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. FIG. 2A shows the results of reverse-phase HPLC analysis of the folding reaction of reduced modified BPTI using GSH/GSSG (Comparative Example 1). 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). 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). FIG. 3 is a graph showing the evaluation of activity recovery when the folding reaction of reduced and denatured RNase A was carried out using LDA-SH/LDA-SS (Example 3), GSH/GSSG (Comparative Example 2), or GSSG. 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). 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. FIG. 4C shows the reaction of introducing a disulfide bond into reduced denatured β2m using BDA-SH/BDA-SS (Example 5) followed by reversed-phase HPLC. Figure 5 is a graph showing the quantification of oxidized β2m by introducing a disulfide bond into reduced-denatured β2m using GSH/GSSG (Comparative Example 3), LDA-SH/LDA-SS (Example 4), or BDA-SH/BDA-SS (Example 5). 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). FIG. 7 is a graph showing the evaluation of activity recovery when the folding reaction of reduced-denatured RNase A was carried out 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). FIG. 8 shows photographs of each sample solution (before centrifugation) obtained in the β2m aggregation experiment by heating. 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. 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).

The present invention will be described in detail below based on the embodiments.

The present invention relates to a protein-folding agent. The protein-folding agent of the present invention contains, as an active ingredient, at least one compound selected from the group consisting of compounds represented by formulas (1) to (14) and salts and solvates thereof. The protein-folding agent of the present invention can fold proteins with higher efficiency, i.e., higher yield and/or activity, than conventional systems using glutathione.
Alternatively, disulfide bonds can be introduced into proteins at high speed, which allows oxidative folding of proteins to proceed with high efficiency.
This leads to a reduction in the time required to produce proteins used in formulations and an improvement in the yield, which results in the effect of large-scale production of protein formulations and a reduction in production costs.
The compounds represented by formulas (1) to (7), and their salts and solvates, are also referred to simply as "thiol compounds," and the compounds represented by formulas (8) to (14), and their salts and solvates, are also referred to simply as "disulfide compounds."

The thiol compound has the following formula:
[In the formula,
X ₁ , X ₂ and X ₃ each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
_Y1 is an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain,
Z ₁ , Z ₂ , Z ₃ , Z ₄ and Z ₅ each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
R ₁ and R ₂ are each independently an alkyl group having 1 to 24 carbon atoms;
Each M ⁻ is independently a chloride ion, a bromide ion, or an iodide ion.
The compounds are represented by the formula (I), as well as salts and solvates thereof. Generally, thiol compounds emit a foul odor, but the above thiol compounds all have a reduced foul odor, which gives them an advantage over conventional related compounds in terms of application. Furthermore, since the above thiol compounds are sufficiently small molecules compared to proteins, they can be easily separated from proteins using size exclusion chromatography or dialysis after the disulfide bond introduction reaction.

The disulfide compound has the following formula:
[In the formula,
X ₁ , X ₂ and X ₃ each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
_Y1 's each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
Z ₁ , Z ₂ , Z ₃ , Z ₄ and Z ₅ each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
R ₁ and R ₂ are each independently an alkyl group having 1 to 24 carbon atoms;
Each M ⁻ is independently a chloride ion, a bromide ion, or an iodide ion.
and salts and solvates thereof. Furthermore, since the disulfide compounds are sufficiently small molecules compared to proteins, they can be easily separated from proteins using size exclusion chromatography or dialysis after the disulfide bond introduction reaction.

One embodiment of the protein-folding agent of the present invention comprises at least one selected from the compounds represented by formulas (1) to (7) and salts and solvates thereof; and a compound represented by formula (8)
The protein-folding agent of this embodiment is characterized by comprising, as an active ingredient, at least one compound selected from the group consisting of compounds represented by formula (1) to (14), and salts and solvates thereof. The molar ratio of the thiol compound to the disulfide compound contained in the protein-folding agent of this embodiment is not particularly limited as long as folding proceeds efficiently, but is preferably 1:1 to 20:1, and more preferably 2:1 to 10:1, for example. The protein-folding agent of this embodiment may be sold or distributed in a state in which the thiol compound and the disulfide compound are mixed, or may be sold or distributed in a state in which each compound is packaged separately, such as as a kit or a set.

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 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 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 separately obtained disulfide compound. Furthermore, the disulfide compound used in combination may be a conventionally used compound such as GSSG. In this case, the preferred embodiment will be described with reference to the description of the disulfide compound unless otherwise specified.

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 group consisting of 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 compound selected from the group consisting of 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 will be described with reference to the description of the thiol compound unless otherwise specified.

As used herein, the term "active ingredient" refers to a substance that acts on a protein at a certain state during the process of folding the protein, or a substance that acts on the substance and has the function of promoting protein folding.
"Protein folding" includes (1) a step of unfolding a protein by reductive denaturation, and (2) a step of performing oxidative folding on the unfolded protein. The protein-folding agent of the present invention can fold proteins with high efficiency by introducing disulfide bonds into the protein, particularly in the oxidative folding reaction of step (2), where the thiol compound and the disulfide compound function as a reducing agent and an oxidizing agent, respectively.

The thiol compounds and disulfide compounds can be synthesized by appropriately employing organic synthesis methods commonly known to those skilled in the art. Specifically, reference can be made to the methods for producing the thiol compounds represented by formulas (1) to (7) and the disulfide compounds represented by formulas (8) to (14) shown in the following examples.

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

Specific examples of the above-mentioned "alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain" include a methylene group, an ethylene group, a propylene group, an isopropylene group,
Examples of the alkylene group include, but are not limited to, n-butylene, isobutylene, tert-butylene, n-pentylene, isopentylene, neopentylene, hexylene, heptylene, octylene, nonylene, decylene, and polyoxyalkylene groups (wherein alkylene is, for example, ethylene or propylene). Examples of the polyoxyalkylene group include, but are not limited to, *-(CH ₂ CH ₂ O) a - (where a is an integer of 1 to 5, * indicates the point of attachment of X ₁ , X ₂ , and X ₃ to —NH ₂ , the point of attachment of Y ₁ to —SH, the point of attachment of Z ₁ to the imidazole ring, Z ₂ ,
Z ₃ , Z ₄ and Z ₅ indicate the points of attachment to the pyridine ring), *—(OCH ₂ CH ₂ )
b- (wherein b is an integer of 1 to 5, and * represents —NH ₂ for X ₁ , X ₂ and X _{3)
indicates the} point _of attachment _{to — (} CH ₂
*-(CH ₂ CH ₂ O)c- (where c is an integer of 1 to 3, and * indicates the point of attachment to —NH ₂ for X ₁ , X ₂ , and X ₃ , the point of attachment to —SH for Y ₁ , the point of attachment to the imidazole ring for Z ₁ , and the point of attachment to the pyridine ring for Z ₂ , Z ₃ , Z ₄ , and Z ₅ ), *-(OCH ₂ CH ₂ CH ₂ )d- (where d is an integer of 1 to 3, and * indicates the point of attachment to X ₁
, _X2 and _X3 are attached to _-NH2 , _Y1 is attached to -SH, _Z1
indicates the point of attachment to the imidazole ring, and Z ₂ , Z ₃ , Z ₄ and Z ₅ indicate the point of attachment to the pyridine ring).

Specific examples of the "alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain" described in this specification include, but are not limited to, a methylene group, an ethylene group, a propylene group, an isopropylene group, an n-butylene group, an isobutylene group, a tert-butylene group, an n-pentylene group, an isopentylene group, a neopentylene group, a hexylene group, and a polyoxyalkylene group (wherein alkylene is, for example, an ethylene group or a propylene group). Examples of the polyoxyalkylene group include, but are not limited to, *-(CH ₂ CH ₂ O) a- (where a is an integer of 1 to 3, * indicates the point of attachment to —NH ₂ for X ₁ , X ₂ and X ₃ , the point of attachment to —SH for Y ₁ , the point of attachment to the imidazole ring for Z ₁ , and the point of attachment to the pyridine ring for Z ₂ , Z ₃ , Z ₄ and Z ₅ ), *-(OCH ₂ CH ₂ ) b- (where b is an integer of 1 to 3, * indicates the point of attachment to —NH ₂ for X ₁ , X ₂ and X ₃ , the point of attachment to —SH for Y ₁₎ ,
indicates the point of attachment to the imidazole ring for Z ₁ , and the point of attachment to the pyridine ring for Z ₂ , Z ₃ , Z ₄ and Z ₅ ), *-(CH ₂ CH ₂ CH ₂ O)c- (wherein c
is 1 or 2, * indicates the point of attachment to —NH ₂ for X ₁ , X ₂ and X ₃ , the point of attachment to —SH for Y ₁ , the point of attachment to the imidazole ring for Z ₁ , the point of attachment to Z ₂ , Z ₃ and Z ₄
and _Z5 indicates the point of attachment to the pyridine ring), *-(OCH ₂ CH ₂ CH ₂ )d-
(wherein d is 1 or 2, * indicates the point of attachment to —NH ₂ for X ₁ , X ₂ and X ₃ , the point of attachment to —SH for Y ₁ , the point of attachment to the imidazole ring for Z ₁ , the point of attachment to the imidazole ring for Z ₂
and Z ₃ , Z ₄ and Z ₅ indicate the points of attachment to the pyridine ring).

Specific examples of the "alkylene group having 1 to 4 carbon atoms which may contain one or two oxygen atoms in the molecular chain" described in this specification include, but are not limited to, methylene, ethylene, propylene, isopropylene, and polyoxyalkylene groups (wherein alkylene is, for example, an ethylene group or a propylene group). Examples of polyoxyalkylene groups include, but are not limited to, *-(CH ₂ CH ₂ O)a
- (wherein a is an integer of 1 or 2, and * represents -NH ₂ for X ₁ , X ₂ and X ₃₎
indicates the point of attachment to the imidazole ring for Y ₁ , and the point of attachment to the pyridine ring for Z ₂ , Z ₃ , Z ₄ and Z ₅ ), *- ₍ OCH
₂ CH ₂ )b- (wherein b is an integer of 1 or 2, and * indicates the point of attachment to —NH ₂ for X ₁ , X ₂ and X ₃ , the point of attachment to —SH for Y ₁ , the point of attachment to the imidazole ring for Z ₁ , and the point of attachment to the pyridine ring for Z ₂ , Z ₃ , Z ₄ and Z ₅ ),
*-(CH ₂ CH ₂ CH ₂ O)c- (where c is 1 and * is a substituted or unsubstituted group of X ₁ , X ₂ and X ₃
indicates the point of attachment to —NH2 for _X1 , the point of attachment to —SH for _Y1 , the point of attachment to the imidazole ring for _Z1 , and the point of attachment to the pyridine ring for _Z2 , _Z3 , _Z4 , _{and Z5} ), *-( _OCH2CH2CH2 )d- (where d is ₁ , * indicates the point of attachment to _—NH2 for _X1 , _X2 , and _X3 , the point of attachment to —SH for _Y1 , the point of attachment to the imidazole ring for _Z1 , and the point of attachment to the pyridine ring for _Z2 , _Z3 , _Z4 , and _Z5 ).

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, n-nonyl group, n-decyl, n-undecyl group, n-dodecyl group, n-tridecyl group, n-tetradecyl group, n-pentadecyl group, n-hexadecyl group, n-heptadecyl group, n-octadecyl group, n-nonadecyl group, n-icosyl group, n-henicosyl group,
Examples include, but are not limited to, an n-docosyl group, an n-tricosyl group, and an n-tetracosyl group.

The combination of the thiol compound and the disulfide compound used in the protein folding agent of the present invention is not particularly limited. However, from the viewpoint of allowing the thiol compound and the disulfide compound to act efficiently as a reducing agent and an oxidizing agent, respectively, in shuffling of disulfide bonds in an oxidative folding reaction, a combination in a corresponding relationship (for example, a thiol compound represented by formula (1) and its disulfide derivative represented by formula (8)) is preferred.
However, even when one of a thiol compound and a disulfide compound is used as the active ingredient of the protein-folding agent of the present invention, both the thiol compound and the disulfide compound can be present in equilibrium in the folding solution in the folding step, and can function as a reducing agent and an oxidizing agent in the shuffling of disulfide bonds.

Generally, reaction intermediates transiently generated in the oxidative folding process have a high tendency to aggregate, which reduces the yield. Therefore, from the viewpoint of improving the yield of protein oxidative folding, it is preferable that the protein folding agent of the present invention has the function of suppressing aggregation of reaction intermediates. Furthermore, thiol compounds and disulfide compounds, which are active ingredients of the protein folding agent of the present invention or ingredients used in combination with the agent, and which have such properties, can be used as active ingredients of protein solubilizers.

Furthermore, from the viewpoint of rapidly generating non-native structures in addition to native structures, the protein folding agent of the present invention preferably has high activity for extremely rapid introduction of disulfide bonds. Proteins exhibit completely different biochemical properties in their native and non-native structures. In the case of protein pharmaceuticals, while the native form exhibits the expected medicinal efficacy, the non-native form may exhibit low medicinal efficacy or even cause severe side effects. Therefore, from the viewpoint of investigating the biochemical properties of proteins with non-native structures and comprehensively elucidating potential side effects due to the medicinal efficacy of non-native structure proteins (misfolded proteins) that may be contained as impurities in protein pharmaceuticals, the protein folding agent of the present invention preferably has the ability to simultaneously generate multiple non-native structure proteins. Prior art has not reported compounds that can simultaneously generate multiple non-native structure proteins for proteins with multiple cysteine pairs. A method for preferentially generating non-native structure proteins has been reported in which some cysteine residues in a protein are replaced with selenocysteine residues (N. Metanis et al., Angew.
Chem.Int.Ed. 2012 51:5585-88). However, this method involves structural modification of the protein itself. Furthermore, only a few selenoproteins containing selenium have been reported in vivo. The physiological functions of these selenoproteins are still unknown. In addition, excessive selenium intake is harmful to humans. Considering this, it is thought that the pharmacological application of selenium-containing low-molecular-weight compounds will be difficult.

Furthermore, the protein-folding agent of the present invention preferably has the function of producing a protein with a non-native structure while suppressing aggregation. Generally, proteins with non-native structures have a high tendency to aggregate, significantly limiting the measurement system available, making research on them difficult. However, the above-mentioned function is advantageous for investigating the biochemical properties and cytotoxicity of proteins with non-native structures in a simple system, and is therefore thought to be useful in research into treatments for folding diseases caused by proteins with non-native structures. Furthermore, the thiol compounds and disulfide compounds that are active ingredients of the protein-folding agent of the present invention or ingredients used in combination with the agent can be used as active ingredients of solubilizing agents for proteins with non-native structures, which have such properties.

Proteins to be folded using the protein-folding agent of the present invention are not particularly limited as long as they contain at least one disulfide bond, and include peptides, polypeptides, proteins, and complexes thereof, regardless of their origin or production method, such as natural or artificial (chemical synthesis, fermentation, or genetic recombination). Proteins may be of any type, including intracellular proteins, extracellular proteins, membrane proteins, and nuclear proteins. Specific examples include the enzymes, recombinant proteins, and antibodies shown below.

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

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

When the protein-folding agent of the present invention is used for oxidative folding, a step of treating an unfolded protein in the presence of the protein-folding agent of the present invention (hereinafter also simply referred to as a treatment step) is carried out by, for example, blending the unfolded protein, the protein-folding agent of the present invention, and optionally a thiol compound or disulfide compound used in combination in a folding buffer solution and mixing them by stirring or the like.

Protein unfolding can be carried out by appropriately adopting methods commonly known to those skilled in the art. For example, unfolded proteins can be obtained using guanidine hydrochloride, urea, thiourea, or a combination of these. Protein unfolding can also be carried out by the method described in Example "8) Preparation of reduced and denatured proteins" below.

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

The thiol compound and the disulfide compound (which is an oxidizing agent) used in the above treatment step
The concentration of the unfolded protein (total amount) is not particularly limited, but is typically 0.01 to 100 mM relative to the solution containing the target protein (e.g., folding buffer solution). The concentration of the unfolded protein contained in the solution containing the target protein (e.g., folding buffer solution) is typically 0.01 to 100 μM.

In the solution containing the target protein in the treatment step (e.g., folding buffer solution), the molar ratio of the thiol compound (total amount) serving as the reducing agent to the disulfide compound (total amount) serving as the oxidizing agent is not particularly limited as long as introduction of disulfide bonds to obtain the target protein proceeds efficiently, and is, for example, 1:1 to 20:1.

The folding buffer is not particularly limited as long as it has a concentration and composition that do not cause the target protein to lose its function, and specific examples include amine buffers such as Tris buffer, MES buffer, and Tricine buffer, phosphate buffer, various Good's buffers, etc. The pH of the folding buffer 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.

In addition to the folding agent for the protein of the present invention and the thiol compound or disulfide compound used in combination as needed, various additives can be added to the folding buffer solution. Such additives include salts such as sodium chloride and calcium chloride;
Examples of the buffer include citrate, phosphate, acetate, and other buffers; bases such as sodium hydroxide; acids such as hydrochloric acid and acetic acid; and organic solvents such as methanol, ethanol, and propanol. In addition to the folding agent of the present invention and the additives described above, the buffer may also contain surfactants, p
An H regulator or a protein stabilizer may also be added. Those skilled in the art will be able to adjust the amount used as appropriate.

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

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):
[In the formula,
_X1 's each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
Each Y ₁ is independently an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain.
The above-mentioned combinations as components to be used together are preferred from the viewpoint of obtaining native structure proteins in high yields by oxidative folding. The proteins to be folded in this case are not particularly limited and include the above-mentioned proteins, as well as RNase A, bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, immunoglobulins, etc., and from the viewpoint of obtaining the above-mentioned effects, proteins with multiple disulfide bonds, such as those with four disulfide bonds such as RNase A, are preferred.

Furthermore, the above-mentioned compounds or combinations are preferred from the viewpoint of suppressing the aggregation of reaction intermediates in oxidative folding and obtaining native structure proteins in high yield. The proteins to be folded in this case are not particularly limited, and include the above-mentioned proteins, as well as RNas.
Examples of such proteins include eA, bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, immunoglobulin, etc., and from the viewpoint of achieving the above-mentioned effects, proteins having multiple disulfide bonds, such as those having three disulfide bonds such as bovine pancreatic trypsin inhibitor, are preferred.

The above compounds or combinations are also preferred from the viewpoint of obtaining native proteins in high yield and at high speed by oxidative folding. Proteins to be folded in this case are not particularly limited and include the above-mentioned proteins as well as RNase A, bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, immunoglobulins, etc., and from the viewpoint of achieving the above-mentioned effects, proteins with one disulfide bond, such as _β2 -microglobulin (β2m), are preferred.

From the viewpoint of obtaining the above-mentioned effects, _X1 is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain, and more preferably an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain. Also, from the viewpoint of obtaining the above-mentioned effects, _Y1 is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain, and more preferably an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain.

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):
[In the formula,
_X2 and _X3 each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain.
and the above combinations as combined components in oxidative folding,
This is preferred from the viewpoint of extremely rapid introduction of disulfide bonds and simultaneous generation of a variety of non-native structures. Proteins to be folded in this case are not particularly limited and include the above-mentioned proteins, as well as RNase A, bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, immunoglobulins, etc., and from the viewpoint of achieving the above-mentioned effects, proteins having multiple disulfide bonds, such as those having three disulfide bonds such as bovine pancreatic trypsin inhibitor, are preferred.

The above compounds or combinations are also preferred from the viewpoint of suppressing aggregation during oxidative folding and producing proteins with non-native structures. The proteins to be folded in this case are not particularly limited, and include the above proteins, as well as RNases.
Examples of such disulfide bonds include bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, and immunoglobulin. From the viewpoint of achieving the above-mentioned effects, proteins having multiple disulfide bonds, such as those having three disulfide bonds such as bovine pancreatic trypsin inhibitor, are preferred.

The above compounds or combinations are also preferred from the viewpoint of obtaining native proteins in high yield and at high speed by oxidative folding. Proteins to be folded in this case are not particularly limited and include the above-mentioned proteins as well as RNase A, bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, immunoglobulins, etc., and from the viewpoint of achieving the above-mentioned effects, proteins with one disulfide bond, such as _β2 -microglobulin (β2m), are preferred.

From the viewpoint of obtaining the above-mentioned effects, _X2 is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain, and more preferably an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain. Also, from the viewpoint of obtaining the above-mentioned effects, _X3 is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain, and more preferably an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain.

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):
[In the formula,
_Z1 's each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain.
The above-mentioned combinations as components to be used together are preferred from the viewpoint of obtaining native structure proteins in high yields by oxidative folding. The proteins to be folded in this case are not particularly limited and include the above-mentioned proteins, as well as RNase A, bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, immunoglobulins, etc., and from the viewpoint of obtaining the above-mentioned effects, proteins with multiple disulfide bonds, such as those with four disulfide bonds such as RNase A, are preferred.

From the viewpoint of obtaining the above-mentioned effects, _Z1 is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain, and more preferably an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain.

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):
[In the formula,
_Z2 each independently represents an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain.
The above-mentioned combinations as co-used components are preferred from the viewpoint of promoting oxidative folding. The proteins to be folded in this case are not particularly limited, and examples include the above-mentioned proteins, as well as RNase A, bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, immunoglobulins, etc.
From the viewpoint of obtaining the above-mentioned effects, _Z2 is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain, and more preferably an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain.

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):
[In the formula,
_Z3 each independently represents an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain.
The above combinations as components to be used in combination are preferred from the viewpoint of obtaining native structure proteins in a short time and in high yield by oxidative folding. The proteins to be folded in this case are not particularly limited, and include the above-mentioned proteins, as well as RNA.
Examples of suitable proteins include RNase A, bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, immunoglobulin, etc., and from the viewpoint of achieving the above-mentioned effects, proteins with multiple disulfide bonds, such as those with four disulfide bonds such as RNase A, are preferred.

From the viewpoint of obtaining the above-mentioned effects, _Z3 is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain, and more preferably an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain.

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):
[In the formula,
_Z4 's each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
Each R ₁ is independently an alkyl group having 1 to 24 carbon atoms,
Each M ⁻ is independently a chloride ion, a bromide ion, or an iodide ion.
The above combinations as components to be used in combination are preferred from the viewpoint of obtaining a protein with a native structure in high yield by oxidative folding of the protein. The protein to be folded in this case is not particularly limited, and may be any of the above-mentioned proteins, and further
Examples include RNase A, bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, immunoglobulin, etc., and from the viewpoint of achieving the above-mentioned effects, proteins with multiple disulfide bonds, such as those with four disulfide bonds such as RNase A, are preferred.

From the viewpoint of obtaining the above-mentioned effects, _Z4 is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain, and more preferably an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain. Also from the viewpoint of obtaining the above-mentioned effects, _R1 is preferably an alkyl group having 1 to 6 carbon atoms, and more preferably an alkyl group having 1 or 2 carbon atoms. Also from the viewpoint of obtaining the above-mentioned effects, ^M- is preferably a chloride ion or an iodide ion.

Furthermore, the thiol compound (6) and the disulfide compound (13), as well as combinations thereof, assemble in water to form micelles due to the hydrophobic interaction of the _R1 group. The amphiphilic effect of the hydrophobic interior space and hydrophilic surface of the micelles suppresses aggregation of denatured proteins and protein folding intermediates. Therefore, oxidative folding can be promoted by introducing disulfide bonds into proteins with high efficiency. In this case, _R1 is preferably an alkyl group having 3 to 18 carbon atoms, more preferably an alkyl group having 4 to 12 carbon atoms. Proteins to be folded in this case are not particularly limited and include the above-mentioned proteins, as well as RNase A, bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, immunoglobulins, etc.

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):
[In the formula,
_Z5 's each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
_R2 is independently an alkyl group having 1 to 24 carbon atoms,
Each M ⁻ is independently a chloride ion, a bromide ion, or an iodide ion.
The above-mentioned combinations as components to be used together are preferred from the viewpoint of obtaining native structure proteins in high yields by oxidative folding. Proteins to be folded in this case are not particularly limited and include the above-mentioned proteins as well as RNase A, bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, immunoglobulins, etc. From the viewpoint of obtaining the above-mentioned effects, proteins with multiple disulfide bonds, such as those with four disulfide bonds such as RNase A, are also preferred.

From the viewpoint of obtaining the above-mentioned effects, _Z5 is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain, and more preferably an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain. From the viewpoint of obtaining the above-mentioned effects, _R2 is preferably an alkyl group having 1 to 6 carbon atoms, and more preferably an alkyl group having 1 or 2 carbon atoms. From the viewpoint of obtaining the above-mentioned effects, ^M- is preferably an iodine ion.

Furthermore, the thiol compound (7) and the disulfide compound (14), as well as combinations thereof, assemble in water to form micelles due to the hydrophobic interaction of the _R2 group. The amphiphilic effect of the hydrophobic interior space and hydrophilic surface of the micelles suppresses aggregation of denatured proteins and protein folding intermediates. Therefore, oxidative folding can be promoted by introducing disulfide bonds into proteins with high efficiency. In this case, _R2 is preferably an alkyl group having 3 to 18 carbon atoms, more preferably an alkyl group having 4 to 12 carbon atoms. Proteins to be folded in this case are not particularly limited and include the above-mentioned proteins, as well as RNase A, bovine pancreatic trypsin inhibitor, _β2 -microglobulin, insulin, major histocompatibility complex, immunoglobulins, etc.

The present invention also relates to a protein folding method comprising the step of treating an unfolded protein in the presence of at least one compound selected from the group consisting of compounds represented by formulas (1) to (14) and salts and solvates thereof. The step of treating an unfolded protein in the presence of the compound (hereinafter also referred to simply as the treatment step) in the folding method of the present invention includes the step of contacting the unfolded protein with the compound, specifically the step of blending the unfolded protein and the compound in a folding buffer and mixing them by stirring or the like, and further including the step of allowing the mixture to stand for a certain period of time after the step, if necessary, to allow folding to proceed more fully. Unless otherwise specified, preferred embodiments of the folding method of the present invention are described above with reference to the description of the folding agent of the present invention.

The present invention also relates to a method for refolding a protein, comprising the step of treating and folding an unfolded protein in the presence of at least one compound selected from the group consisting of compounds represented by formulas (1) to (14) and salts and solvates thereof. The protein refolding method of the present invention also includes the step of treating and folding an unfolded protein in the presence of the compound (hereinafter also referred to simply as the treating step), and further includes the step of isolating the folded protein after the treating step, if necessary (hereinafter also referred to as the isolating step). Unless otherwise specified, preferred embodiments of the protein refolding method of the present invention are described above with reference to the descriptions of the folding agent of the present invention and the folding method of the present invention.

The isolation step may involve, for example, isolating the target normal protein (folded protein) from the protein suspension obtained in the folding step by column chromatography or the like. Packing materials used in column chromatography include silica, dextran, agarose, cellulose, acrylamide, vinyl polymers, etc. Commercially available products include the Sephadex series, Sephacryl series, and Sepharose series (all of which are available from Pharma, Inc.).
Examples of suitable dialysis gels include Bio-Gel series (Bio-Rad), Bio-Gel series (Bio-Rad), etc. Alternatively, the target normal protein (folded protein) may be isolated by dialysis.

The present invention also relates to a protein solubilizer containing, as an active ingredient, at least one selected from the compounds represented by formulas (1) to (14), and salts and solvates thereof.
The solubilizer of the present invention can inhibit aggregation of proteins in their native or non-native structures and solubilize them. As explained above about the protein-folding agent of the present invention, the active ingredient of the protein-folding agent of the present invention or an ingredient used in combination with the active ingredient of the protein-folding agent of the present invention is a compound represented by the formula (
The thiol compounds represented by formulas (1) to (7) and the disulfide compounds represented by formulas (8) to (14) function as solubilizing agents because they have the function of inhibiting the aggregation of proteins in native or non-native structures, particularly the function of inhibiting the aggregation of reaction intermediates produced in the oxidative folding of proteins and proteins in native structures, as well as the function of inhibiting the aggregation of proteins in non-native structures in oxidative folding reactions that produce one or more proteins in non-native structures. The preferred embodiments of the compounds represented by formulas (1) to (14) and the target proteins, etc., are described above with reference to the description of the protein-folding agent of the present invention.

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

1) Synthesis of LDA-SH (2-((2-aminoethyl)amino)ethane-1-thiol) and LDA-SS The above compounds (corresponding to the compounds represented by formulas (1) and (8), respectively) were synthesized according to the following scheme.

1-1) Synthesis of Compound 2 Under a nitrogen atmosphere, compound 1 (1.90 g, 18.3 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in dehydrated methylene chloride (25 mL, Kanto Chemical Co., Ltd.) on an ice bath. (Boc) _2O (5.22 g, 23.9 mmol, Kanto Chemical Co., Ltd.) was added, and the mixture was warmed to room temperature. After 19 hours, the solvent was distilled off under reduced pressure, water (25 mL) was added, and the mixture was extracted with ethyl acetate (25 mL, twice, Kishida Chemical Co., Ltd.). The extracted ethyl acetate solution was washed with saturated brine (25 mL). The collected ethyl acetate solution was diluted with sodium sulfate (Kishida Chemical Co., Ltd.).
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%.

1-2) Synthesis of Compound 3 At room temperature under a nitrogen atmosphere, N-chlorosuccinimide (NCS, 0.849 g, _6.36 mmol, Kishida 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 anhydrous THF (13 mL) was added dropwise. After 11 hours, the solvent was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography (Kanto Chemical) to obtain compound 3. Yield: 0.60 g, 32%.

1-3) Synthesis of Compound 4 Compound 3 (0.597 g, 1.85 mmol) was dissolved in dehydrated N,N-dimethylformamide (DMF, 3 mL, Kanto Chemical) at room temperature under a nitrogen atmosphere. Potassium thioacetate (AcSK, 0.285 g, 2.
After adding sodium hydroxide (0.757 g, 5.48 mmol, Tokyo Chemical Industry Co., Ltd.), the mixture was heated to 90°C. After 12 hours, water (20 mL) was added, followed by extraction with ethyl acetate (25 mL, twice, Kishida Chemical Co., Ltd.). The extracted ethyl acetate solution was washed with saturated brine (25 mL). Sodium sulfate (Kishida Chemical Co., Ltd.) was added to the collected ethyl acetate solution, followed by filtration. The filtrate was evaporated under reduced pressure. Anhydrous methanol (5 mL, Kanto Chemical Co., Ltd.) was added to the obtained residue at room temperature under a nitrogen atmosphere, 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, which was then extracted with methylene chloride (25 mL, 3 times, AGC).
The extracted methylene chloride solution was washed with saturated brine (25 mL, twice). Sodium sulfate (Kishida Chemical) was added to the recovered methylene chloride solution, followed by filtration. The filtrate was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Chemical) to obtain compound 4. Yield: 0.257 g, 43%.

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 the resulting 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 the mixture was extracted with ethyl acetate (50 mL, twice, Kishida Chemical). The extracted ethyl acetate solution was washed with saturated brine (25 mL). Sodium sulfate (Kishida Chemical) was added to the collected ethyl acetate solution, followed by filtration. The filtrate was evaporated under reduced pressure to give compound 5. Yield: 0.241 g, 94%.

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 to the solution. After stirring for 1 hour, the solvent was evaporated under reduced pressure. Methylene chloride (10 mL, AGC) was added to the residue, and the mixture was extracted with water (10 mL). The extracted aqueous solution was evaporated under reduced pressure and analyzed by high-performance liquid chromatography (apparatus: JASCO, PU-4086, UV-
LDA-SS was obtained using a column (HPLC: 4075, Column: TA12S05-2520WX). Yield: 87 mg, Yield: 60%.

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 solution. After stirring for 24 hours, the solution was analyzed by high-performance liquid chromatography (apparatus: JASCO Corporation, PU-4086, UV-4075, column: TA12S05-
LDA-SH was obtained by the procedure of (2520WX). Yield: 33 mg, yield: 43%.

2) Synthesis of BDA-SH (1,3-diaminopropane-2-thiol) and BDA-SS The above compounds (corresponding to the compounds represented by formulas (2) and (9), respectively) were synthesized according to the following scheme.

2-1) Synthesis of Compound 7 Compound 6 (0.539 g, 5.98 mmol) was dissolved in dehydrated methanol (30 mL, Kanto Chemical) at room temperature under a nitrogen atmosphere. After heating to 45°C, (Boc) _2O (2.84 g, 13.0 mmol, Kanto Chemical) and triethylamine (6.91 mL, Sigma) were added. After 20 hours, the solvent was evaporated under reduced pressure, and the resulting residue was subjected to silica gel column chromatography (Kanto Chemical) to obtain compound 7.
Yield: 1.72 g, yield: 99%.

2-2) Synthesis of Compound 8. Compound 7 (1.57 g, 5.41 mmol) was dissolved in dehydrated methylene chloride (57 mL, Kanto Chemical) at room temperature under a nitrogen atmosphere. After cooling to 0°C, triethylamine (2.20 mL, Sigma) and mesyl chloride (0.544 mL, 7.03 mmol, Tokyo Chemical Industry) were added. After stirring for 5 hours, water (30 mL) was added and the mixture was extracted with methylene chloride (30 mL, twice, AGC). The recovered methylene chloride solution was washed with saturated aqueous sodium bicarbonate (10 mL) and saturated brine (10 mL), then 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) to obtain compound 8. Yield: 1.64 g, 82%.

2-3) Synthesis of Compound 9 Compound 8 (0.756 g, 2.05 mmol) was dissolved in dehydrated N,N-dimethylformamide (DMF, 13 mL, Kanto Chemical) at room temperature under a nitrogen atmosphere. Potassium thioacetate (0.392 g, 3.428 mmol,
Tokyo Chemical Industry Co., Ltd.) and triethylamine (0.30 mL, Sigma) were added. After stirring at 60 °C for 13 hours, water (50 mL) was added and the mixture was extracted with ethyl acetate (50 mL, 3 times, Kishida Chemical). The collected ethyl acetate solution was washed with water (50 mL, 2 times) and saturated brine (10 mL), then 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) to obtain the thioacetyl intermediate. Under a nitrogen atmosphere, the thioacetyl intermediate (0.346 g, 0.994 mmol) was dissolved in anhydrous methanol (3 mL, Kanto Chemical) at room temperature, and potassium carbonate (0.414 g, 2.998 mmol, Kishida Chemical) was added. After stirring for 2 hours, the solvent was evaporated under reduced pressure. Water (10 mL) was added and the mixture was extracted with methylene chloride (10 mL, 3 times, AGC). The recovered methylene chloride solution was washed with saturated aqueous ammonium chloride (10 mL, Kishida Chemical) and saturated brine (10 mL), then 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) to obtain compound 9. Yield: 0.275 g, 43%.

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, and 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 the mixture was extracted with ethyl acetate (30 mL, twice). The collected ethyl acetate solution was washed with saturated brine (10 mL), sodium sulfate was added, and the mixture was filtered. 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%.

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. Trifluoroacetic acid (1 mL, Kishida Chemical) was added, and the temperature was raised to room temperature. After stirring for 2 hours, the solvent was evaporated under reduced pressure. Chloroform (10 mL, Kishida Chemical) was added to the resulting residue, and the mixture was extracted with water (10 mL). The solvent from the resulting aqueous solution was evaporated under reduced pressure, and the residue was diluted with hydrochloric acid (1 M, 5
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%.

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, and dithiothreitol (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, and the mixture was extracted with water (5 mL). The solvent from the resulting aqueous solution was evaporated under reduced pressure to obtain BDA-SH. Yield:
85.5 mg, yield: 93%.

3) Synthesis of ImdM-SH ((1H-imidazol-4-yl)methanethiol) and ImdM-SS The above compounds (corresponding to the compounds represented by formulas (3) and (10), respectively) were synthesized according to the following scheme.

3-1) Synthesis of Compound 12 Compound 11 (5.512 g, 57.36 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in dehydrated ethanol (100 mL, Kanto Chemical Co., Ltd.) at room temperature under a nitrogen atmosphere. After cooling to 0°C, sodium borohydride (2.1
The resulting mixture was stirred for 3 hours, and water (10 mL) was added, followed by evaporation of the solvent under reduced pressure. The resulting residue was subjected to silica gel column chromatography (Kanto Chemical) to give compound 12. Yield: 4.10 g, 73%.

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 the above-mentioned 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 mixture was extracted with hexane (Kishida Chemical) five times. Sodium sulfate (Kishida Chemical) was added to the recovered methylene chloride solution, and the mixture was filtered. The filtrate was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Chemical) to obtain compound 13. Yield: 0.763 g, 67%.

3-3) Synthesis of Compound 14 Compound 13 (0.917 g, 4.63 mmol) was dissolved in dehydrated methylene chloride (8.4 mL) at room temperature under a nitrogen atmosphere.
The compound was dissolved in dehydrated N,N-dimethylformamide (DMF, 0.2 mL, Kanto Chemical). After cooling to 0°C, a solution of oxalyl chloride (1.22 g, 9.58 mmol, Kishida Chemical) in dehydrated methylene chloride (7.0 mL, Kanto Chemical) was added dropwise. After warming to room temperature and stirring for 1.5 hours, water (15 mL) was added and the mixture was extracted with methylene chloride (15 mL twice, Kishida Chemical). Sodium sulfate (Kishida Chemical) was added to the recovered methylene chloride solution and the mixture was filtered. The filtrate was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Chemical) to obtain compound 14. Yield:
0.432 g, yield: 43%.

3-4) Synthesis of Compound 15 Compound 14 (0.168 g, 0.774 mmol) was dissolved in dehydrated acetone (4.7 mL,
The mixture was dissolved in water (20%), sodium iodide (0.233 g, 1.55 mmol, Kishida Chemical Co., Ltd.), and potassium thioacetate (0.142 g, 1.25 mmol, Tokyo Chemical Industry Co., Ltd.) were added. After stirring for 21 hours, the mixture was dissolved in water (20%).
The mixture was added with 10 mL of ethyl acetate (30 mL, twice, Kishida Chemical) and extracted with ethyl acetate. The collected ethyl acetate solution was washed with saturated brine (10 mL), then 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) to obtain compound 15. Yield: 0.140 g, 70%.

3-5) Synthesis of ImdM-SS Compound 15 (0.911 g, 3.55 mmol) was dissolved in dehydrated methanol (10 mL,
The mixture was dissolved in ethanol (Kanto Chemical) and potassium carbonate (1.00 g, 7.24 mmol, Kishida Chemical) was added. After stirring for 3 hours, the solvent was removed under reduced pressure, water (10 mL) was added, and the mixture was neutralized with hydrochloric acid (1 M). The mixture was extracted with a 3:1 mixture of chloroform (Kishida Chemical) and 1-propanol (Kishida Chemical) (25 mL, 4 times). The solvent was removed under reduced pressure, and the resulting residue was purified by reversed-phase high-performance liquid chromatography (apparatus: JASCO, PU-4086, UV-4075, column: TA12S05-2520WX) to obtain ImdM-SS. Yield: 27 mg, 4%.

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, and dithiothreitol (123 mg, 0.797 mmol, Nacalai Tesque) was added. After stirring for 7 hours, the mixture was extracted with a chloroform (Kishida Chemical) and 1-propanol (Kishida Chemical) mixed solvent (9:1, 25 mL, 4 times). Sodium sulfate was added, filtered, and the filtrate was evaporated under reduced pressure. The resulting residue was analyzed 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%.

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 synthesized according to the following scheme.

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.
Kishida Chemical Co., Ltd.) was added. The mixture was heated to 90°C, stirred for 1.5 hours, and then cooled to room temperature. Sodium hydroxide (2.058 g, 51.45 mmol, Kishida Chemical Co., Ltd.) was added, and the mixture was stirred for 22 hours. The mixture was then washed with tert-butyl methyl ether (30 mL, twice, Kanto Chemical Co., Ltd.). The resulting aqueous solution was neutralized with hydrochloric acid (1 M, Kishida Chemical Co., Ltd.) and extracted with chloroform (30 mL, Kishida Chemical Co., Ltd.). Sodium sulfate (Kishida Chemical Co., Ltd.) was added, and the mixture was filtered. The filtrate was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (Kanto Chemical Co., Ltd.) to give pPyM-SH. Yield: 459 mg, 14%.

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, Kishida Chemical) was added. 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%.

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 synthesized according to the following scheme.

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 the mixture was stirred for 9 hours. After that, it was washed with tert-butyl methyl ether (40 mL, Kanto Chemical). The resulting aqueous solution was neutralized with hydrochloric acid (1 M, Kishida Chemical) and extracted with methylene chloride (30 mL, 3 times, Kishida Chemical). Sodium sulfate (Kishida Chemical) was added to the recovered methylene chloride solution, and the mixture was filtered. The filtrate was evaporated under reduced pressure, and the resulting 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%.

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

6) pPyM-SH-NMe·M ((4-(mercaptomethyl)-1-methylpyridin-1-ium) halide) and
Synthesis of pPyM-SS-NMe·M and pPyM-SS-NMe·M The above compounds (corresponding to the compounds represented by formulas (6) and (13), respectively) were synthesized according to the following scheme.

6-1) Synthesis of pPyM-SS-NMe.I (4,4'-(disulfanediylbis(methylene))bis(1-methylpyridin-1-ium) diiodide) pPyM-SS (254 mg, 1.02 mmol) obtained by the above method was dissolved in acetonitrile (3.0 mL, Kishida Chemical) and acetone (3 mL, Kishida Chemical), and iodomethane (1.503 g, 10.59 mmol) was added.
pPyM-SS-NMe.I (Tokyo Chemical Industry Co., Ltd.) was added. The temperature was raised to 60°C, and the mixture was stirred for 16 hours, then cooled to room temperature. The resulting precipitate was filtered, and pPyM-SS-NMe.I was obtained from the residue. Yield: 446 mg, 82%.

6-2) Synthesis of pPyM-SS-NMe.Cl (4,4'-(disulfanediylbis(methylene))bis(1-methylpyridin-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
pPyM-SS-NMe·Cl was obtained by distilling off the filtrate under reduced pressure. After stirring at room temperature for 4 hours in the dark, the resulting precipitate was filtered. The filtrate was evaporated under reduced pressure, and ethanol (2 mL, Kishida Chemical) was added. The resulting precipitate was filtered. The filtrate was evaporated under reduced pressure to obtain pPyM-SS-NMe·Cl. Yield: 80 mg, 63%.

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

6-4) Synthesis of pPyM-SH-NMe.I ((4-(mercaptomethyl)-1-methylpyridin-1-ium) iodide) pPyM-SS-NMe.I (104 mg, 0.195 mmol) obtained by the above method was dissolved in degassed water (3.1 mL), and dithiothreitol (DTT, 125 mg, 0.809 mmol, Nacalai Tesque) was added. After stirring at room temperature for 3 hours, 2 M hydrochloric acid (1 mL, Kishida Chemical) was added and the mixture was evaporated under reduced pressure. The resulting residue (crude pPyM-SH-NMe.I product) 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%.

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
The above compounds (corresponding to the compounds represented by formulas (7) and (14) , respectively ) were synthesized according to the following scheme.

7-1) Synthesis of oPyM-SS-NMe.I (2,2'-(disulfanediylbis(methylene))bis(1-methylpyridin-1-ium) diiodide) oPyM-SS (739 mg, 2.97 mmol) obtained by the above method was dissolved in acetonitrile (9.7 mL, Kishida Chemical) and acetone (10 mL, Kishida Chemical), and iodomethane (4.434 g, 31.24 mm
ol, Tokyo Chemical Industry Co., Ltd.) was added. After heating to 65°C, the mixture was stirred for 16 hours and then cooled to room temperature. The resulting precipitate was filtered, and oPyM-SS-NMe.I was obtained from the resulting residue. Yield: 1.286 g, 81%.

7-2) Synthesis of oPyM-SH-NMe·I ((2-(mercaptomethyl)-1-methylpyridin-1-ium) iodide)
oPyM-SS-NMe·I (108 mg, 0.203 mmol) was dissolved in degassed water (3 mL), and dithiothreitol (DTT, 130 mg, 0.840 mmol, Nacalai Tesque) was added. After stirring at room temperature for 5 hours, 2 M 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 (oPyM-SH-NMe·I crude product) was purified by high performance liquid chromatography (apparatus: JASCO Corporation, 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%.

8) Preparation of reduced and denatured proteins. Bovine pancreatic trypsin inhibitor (BP) was used as a model substrate.
The enzymes used were BPTI (see References 1 and 2 below), RNase A (see Reference 3 below), and _β2 -microglobulin (β2m). BPTI and RNase A are proteins with three and _four disulfide bonds, respectively, and are commonly used as model substrates in the field. β2m was also used as a model substrate with one disulfide bond.

To prepare a reduced denatured form of BPTI (Takara Bio Inc.) (reduced form of three disulfide bonds), 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 and purified by reverse-phase HPLC. After confirming that all disulfide bonds in the purified sample had been cleaved by MALDI-TOF/MS, the sample was lyophilized and stored at -80°C (see Reference 1 below).

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, and then dialyzed against 10 mM HCl. After two more dialysis cycles, the solution was replaced with 10 mM HCl to remove DTT and other components (see Reference 3 below).

β2m was expressed as a recombinant in an E. coli expression system, and the cells were then incubated with lysate A (5
The cells were suspended in 0 mM Tris-HCl (pH 8.1, 300 mM NaCl) and then sonicated. The fraction obtained after centrifugation 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.
The product was purified by reversed-phase column chromatography using _{an 18} -AR-II column (Hitachi).

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.

9) Evaluation of disulfide bond introduction ability into reduced denatured BPTI 30 μM of reduced denatured BPTI prepared in 8) above was added to the following combination of 1 mM thiol compound/0.2 mM disulfide compound:
Comparative example 1: GSH (nakalai)/GSSG (nakalai)
Example 1: LDA-SH/LDA-SS
Example 2: BDA-SH/BDA-SS
The folding reaction was carried out by incubation at 30°C in a buffer solution containing BPTI (50 mM Tris-HCl pH 7.5, 300 mM NaCl). Aliquots of the reaction mixture were taken over time, and an equal volume of 1N HCl was added to quench the thiol group and disulfide bond exchange reaction. To identify the molecular species present in the reaction mixture (see Figure 1A), reverse-phase HPLC was performed. As shown in Figures 1A and 1B, reverse-phase HPLC analysis allowed for the separation and identification of each disulfide bond species during BPTI folding, and the position of disulfide bridges could be tracked over time.

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 detected. A mixture of an aqueous solution containing 0.05% trifluoroacetic acid (solution A) and an acetonitrile solution containing 0.05% trifluoroacetic acid (solution B) was developed with a linear gradient in which the volume increase rate of solution B in the mixture was 1%/min from 0 to 15 min and 0.5%/min from 15 to 115 min.

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

When BDA-SH/BDA-SS was used (Example 2), the reduced modified form of BPTI (all thiol groups), R, disappeared within 1 minute of the reaction, and the native form (N) was produced, demonstrating the extremely rapid introduction of disulfide bonds (Fig. 2C).
It can be seen that many types of disulfide bonds (shown as BPTI) are simultaneously produced. Proteins with non-native disulfide bonds usually exhibit aggregation. However, the fact that BDA-SH/BDA-SS can be used for HPLC analysis indicates that they suppress the aggregation of non-native BPTI and have the properties of a solubilizing agent.

10) RNase A activity evaluation 1
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:
Comparative Example 2: GSH/GSSG
Example 3: LDA-SH/LDA-SS
In the presence of 50 mM Tris-HCl pH 7.5, 300 mM NaCl, containing 8 μM RNase A, the
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
The RNase A was diluted with the same buffer containing cytidine 2':3'-cyclic monophosphate (cCMP) to a final concentration of 4 μM RNase A and 0.4 mM cCMP. The change in absorbance at 284 nm was measured spectrophotometrically to quantify the nucleolytic activity of RNase A folded to its native structure.

As shown in Figure 3, the recovery activity of RNase A after 3 hours of incubation was 42% in the presence of LDA-SH/LDA-SS (Example 3), a higher recovery activity than in the presence of 0.2 mM GSSG (14%) and GSH/GSSG (Comparative Example 2) (33%). This indicates that LDA-SH/LDA-SS has a folding-promoting effect.

11) Evaluation of disulfide bond introduction ability into reduced denatured β2m 10 μM of reduced denatured β2m prepared in 8) above was used in the following combination of 1 mM thiol compound/0.2 mM disulfide compound:
Comparative Example 3: GSH/GSSG
Example 4: LDA-SH/LDA-SS
Example 5: BDA-SH/BDA-SS
The folding reaction was carried out by incubating the reaction mixture at 30°C in a buffer containing 6 M guanidine hydrochloride, 1.5 M HCl, and 50 mM Tris-HCl pH 7.5, 300 mM NaCl.
The disulfide bond introduction reaction was quenched by adding an equal volume of β2m. To identify the molecular species in the reaction mixture, the oxidized (N) and reduced (R) forms of β2m were analyzed by reverse-phase HPLC (Figures 4A-C and 5). The oxidized (N) and reduced (R) forms were identified by MALDI-TOF/MS.

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 at 229 nm was detected using a Nacalai tesque. When an aqueous solution containing 0.1% trifluoroacetic acid (solution A) was mixed with an acetonitrile solution containing 0.1% trifluoroacetic acid (solution B), the volume increase rate of the proportion of solution B in the mixture was 1%/min from 0 to 10 min, and 0.5%/min from 10 to 35 min.
The solution was developed with a linear concentration gradient of 1 min.

4A-4C and 5, when GSH/GSSG was used (Comparative Example 3), the yield after a 60-minute folding reaction was 36%. When LDA-SH/LDA-SS was used (Example 4), the yield after a 60-minute folding reaction was 95%. When BDA-SH/BDA-SS was used (Example 5), the yield after a 60-minute folding reaction was 100%.

From these results, the reaction rate constants for the disulfide bond introduction reaction into reduced denatured β2m were calculated assuming a pseudo-first-order reaction. The results were 1.12 × 10 ^-4 for GSSG, 6.96 × 10 ^-4 for LDA-SS, and 3.59 × 10 -4 for BDA-SS.
0 ^-2 , and compared with GSH/GSSG (Comparative Example 3), LDA-SH/LDA-SS (Example 4) was 6.2 times higher, and BDA
It was shown that -SH/BDA-SS (Example 5) has approximately 320-fold higher disulfide bond introduction ability. β2m is one of the components involved in immunity, and its misfolded form is associated with the pathology of dialysis-related amyloidosis. The disulfide bond introduction agent of the present invention, which can rapidly introduce disulfide bonds into β2m, the light chain of the major histocompatibility complex that plays a central role in the immune system, is useful in the efficient production of protein formulations.

12) RNase A activity evaluation 2
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:
Comparative Example 4: GSH/GSSG
Example 6: ImdM-SH/ImdM-SS
Example 7: oPyM-SH/oPyM-SS
In the presence of 50 mM Tris-HCl pH 7.5, 300 mM NaCl, containing 8 μM RNase A, the
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
The RNase A was diluted with the same buffer containing cytidine 2':3'-cyclic monophosphate (cCMP) to a final concentration of 4 μM RNase A and 0.4 mM cCMP. The change in absorbance at 284 nm was measured spectrophotometrically to quantify the nucleolytic activity of RNase A folded to its native structure.

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).
The recovery activity in the presence of ImdM-SH/ImdM-SS (Example 6) was 45%, which was higher than that in the case of oPyM-SH/oPyM-SS (Example 7) (38%). This indicates that ImdM-SH/ImdM-SS has a folding-promoting effect. Furthermore, the recovery activity of oPyM-SH/oPyM-SS after 3 hours of incubation with RNase A
The recovery activity of the enzyme was higher than that of GSH/GSSG (Comparative Example 4), demonstrating that it had a high folding-promoting effect in the early stage up to 3 hours.

13) RNase A activity evaluation 3
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:
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 50 mM Tris-HCl pH 7.5, 300 mM NaCl, containing 8 μM RNase A, the
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
The RNase A was diluted with the same buffer containing cytidine 2':3'-cyclic monophosphate (cCMP) to a final concentration of 4 μM RNase A and 0.4 mM cCMP. The change in absorbance at 284 nm was measured spectrophotometrically to quantify the nucleolytic activity of RNase A folded to its native structure.

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.

14) Heat-induced aggregation experiment of β2m (without 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 allowed to equilibrate for 10 minutes, after which it was cooled to 25°C. The absorbance of the supernatant of the fraction obtained after centrifugation was measured at 280 nm using a spectrophotometer to determine the remaining rate of native β2m in the supernatant.

(Additives included)
10 μM of native β2m was added to a buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 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 to determine the remaining rate of native β2m in the supernatant.

Figure 8 shows a photograph of the solution after cooling (before centrifugation).
The results showed that the solutions containing LDA-SH (Example 11) and BDA-SH (Example 12) were clearer and inhibited aggregation of native β2m compared with the solutions containing no additive and GSH (Comparative Example 6). The results of measuring the residual rate of native β2m in the supernatant obtained by centrifugation are shown in Figure 9. Figure 9 shows that the residual rate of native β2m was higher with LDA-SH (Example 11) and BDA-SH (Example 12) compared with the solutions containing no additive and GSH (Comparative Example 6). Therefore, it was found that LDA-SH and BDA-SH have a strong ability to inhibit protein aggregation and solubilize proteins.

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), an RNase A substrate, 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."

The protein folding agent of the present invention can be used as an oxidative folding promoter for protein preparations such as antibodies having multiple disulfide bonds. The protein folding agent of the present invention can be used as a promoter of rapid oxidative folding of proteins having a single disulfide bond. Furthermore, the protein folding agent of the present invention can be used as a research reagent in basic research in the field of biochemistry, as a reagent for artificially accumulating misfolded proteins in a soluble state.

Claims (7)

The following formula:
[In the formula,
X ₁ , X ₂ and X ₃ each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
_Y1 's each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
Z ₁ , Z ₂ , Z ₃ , Z ₄ and Z ₅ each independently represent an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain;
R ₁ and R ₂ are each independently an alkyl group having 1 to 24 carbon atoms;
Each M ⁻ is independently a chloride ion, a bromide ion, or an iodide ion.
A protein-folding agent comprising, as an active ingredient, at least one compound selected from the group consisting of a compound represented by the formula (I) and a salt and solvate thereof. 2. The protein-folding agent according to claim 1, comprising, as an active ingredient, at least one selected from the group consisting of compounds represented by formulas (1) to (7), and salts and solvates thereof. 2. The protein-folding agent according to claim 1, comprising, as an active ingredient, at least one selected from the group consisting of compounds represented by formulas (8) to (14), and salts and solvates thereof. The protein-folding agent according to claim 1, comprising as an active ingredient at least one selected from the group consisting of compounds represented by formulas (1) to (7), and salts and solvates thereof; and at least one selected from the group consisting of compounds represented by formulas (8) to (14), and salts and solvates thereof. A method for folding a protein, comprising the step of treating an unfolded protein in the presence of at least one compound selected from the group consisting of compounds represented by formulas (1) to (14) as defined in claim 1, and salts and solvates thereof. A method for refolding a protein, comprising the step of treating an unfolded protein in the presence of at least one compound selected from the group consisting of compounds represented by formulas (1) to (14) as defined in claim 1, and salts and solvates thereof. A protein solubilizer comprising, as an active ingredient, at least one selected from the compounds represented by formulas (1) to (14) defined in claim 1, and salts and solvates thereof.