JP2022135954A - protein folding agent - Google Patents

Abstract

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

Description

The present invention relates to protein folding agents.

Proteins are materials that are widely used in industry, mainly for pharmaceuticals. Since the formation of a native structure is essential for proteins to exert their individual functions and activities, techniques for efficient folding into the native structure are important in the industrial use of proteins, directly linked to cost reduction and the like. Therefore, oxidative folding promoters to the native structure are beneficial for large-scale production of protein pharmaceuticals. For example, in the oxidative folding process of important protein pharmaceuticals such as insulin and immunoglobulins, native structure formation with the introduction of native disulfide bonds is the rate-limiting step reaction.

A typical control method for the oxidative folding process is the shuffling of disulfide bonds. In this method, a reducing agent and an oxidizing agent are allowed to coexist during the folding process, and the formation and cleavage of disulfide bonds are repeatedly caused, leading to the most stable natural structure. Typical reducing and oxidizing agents used therein are glutathione (GSH), β-mercaptoethanol (β-ME) and dithiothreitol (DTT), and oxidized glutathione (GSSG), respectively. For example, in the case of RNase A, the recovery yields in the presence and absence of GSH/GSSG (under air) were compared, and the enzyme activity at the same time in the initial folding process was higher in the presence of GSH/GSSG. is about 2 times higher, and the speed of activity recovery (1/2 activity recovery time) is about 2.8 times faster (Non-Patent Document 1). In addition, GSH/GSSG (Non-Patent Document 1), GSH + (±)-trans-1,2-bis(2-mercaptoacetamide) are used as small-molecule reducing agents (and oxidizing agents combined therewith) used in oxidative folding. Cyclohexane (BMC) / GSSG (Non-Patent Document 2), aromatic thiols and their disulfides (Aromatic thiols and their disulfides) (
Non-Patent Document 3), Cyclic selenoxide (Non-Patent Document 4), Cys-XX-Cy
Peptides having an s structure (X is an arbitrary amino acid and Cys is cysteine) (Non-Patent Document 5), selenoglutathione (Non-Patent Document 6), and selenol-containing peptides (Non-Patent Document 7) is known. However, cyclic selenoxide and selenoglutathione have had toxicity problems due to the use of heavy metal selenium. Also BM
Since C is used as an adjuvant for GSH, there was a problem in that it was necessary to add various and large amounts of additives compared to the GSH/GSSG system. Aromatic thiol is a substance with a benzene ring, so there is a problem that non-specific adsorption to proteins due to hydrophobic interaction may occur. Peptides with the Cys-XX-Cys structure have problems of structural instability such as degradation due to protease contamination and high cost for their synthesis. In addition, low-molecular-weight thiols such as cysteamine have a problem of strong odor associated with high volatility. In addition, when these existing compounds are used, the effect of suppressing the aggregation of oxidative folding intermediates is low, which has been a factor in lowering the yield.

Therefore, there has been a demand for a protein folding agent capable of folding proteins with high efficiency while avoiding the problems of the prior art.

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 capable of folding proteins with high efficiency.

As a result of various studies on means for solving the above problems, the present inventors have found that by using a compound having a specific structure, and salts and solvates thereof, high efficiency, that is, high yield and / or found that disulfide bonds can be introduced into proteins at high speed,
We have completed the present invention.

［式中、
Ｘ_１、Ｘ_２及びＸ_３は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでい
てもよい炭素数１～１０個のアルキレン基であり、
Ｙ_１は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでいてもよい炭素数
１～１０個のアルキレン基であり、
Ｚ_１、Ｚ_２、Ｚ_３、Ｚ_４及びＺ_５は、それぞれ独立して、分子鎖中に１～５個の酸素原
子を含んでいてもよい炭素数１～１０個のアルキレン基であり、
Ｒ_１及びＲ_２は、それぞれ独立して、炭素数１～２４個のアルキル基であり、
Ｍ^－は、それぞれ独立して、塩素イオン、臭素イオン又はヨウ素イオンである］
で表される化合物、並びにその塩及び溶媒和物から選択される少なくとも１種を有効成分
として含むタンパク質のフォールディング剤。
［２］式（１）ないし（７）で表される化合物、並びにその塩及び溶媒和物から選択され
る少なくとも１種を有効成分として含む、上記［１］に記載のタンパク質のフォールディ
ング剤。
［３］式（８）ないし（１４）で表される化合物、並びにその塩及び溶媒和物から選択さ
れる少なくとも１種を有効成分として含む、上記［１］に記載のタンパク質のフォールデ
ィング剤。
［４］式（１）ないし（７）で表される化合物、並びにその塩及び溶媒和物から選択され
る少なくとも１種；並びに式（８）ないし（１４）で表される化合物、並びにその塩及び
溶媒和物から選択される少なくとも１種を有効成分として含む、上記［１］に記載のタン
パク質のフォールディング剤。
［５］上記［１］に定義される式（１）ないし（１４）で表される化合物、並びにその塩
及び溶媒和物から選択される少なくとも１種の存在下で、アンフォールディングされたタ
ンパク質を処理する工程を含む、タンパク質のフォールディング方法。
［６］上記［１］に定義される式（１）ないし（１４）で表される化合物、並びにその塩
及び溶媒和物から選択される少なくとも１種の存在下で、アンフォールディングされたタ
ンパク質を処理する工程を含む、タンパク質の再生方法。
［７］上記［１］に定義される式（１）ないし（１４）で表される化合物、並びにその塩
及び溶媒和物から選択される少なくとも１種を有効成分として含むタンパク質の可溶化剤
。 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 are each independently an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain,
Y ₁ is each independently 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 are each independently 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 iodine ion]
A protein folding agent comprising, as an active ingredient, at least one selected from compounds represented by and salts and solvates thereof.
[2] The protein folding agent according to [1] above, which contains as an active ingredient at least one selected from compounds represented by formulas (1) to (7), and salts and solvates thereof.
[3] The protein folding agent according to [1] above, which contains as an active ingredient at least one selected from compounds represented by formulas (8) to (14), and salts and solvates thereof.
[4] at least one selected from compounds represented by formulas (1) to (7), and salts and solvates thereof; and compounds represented by formulas (8) to (14), and salts thereof and a solvate as an active ingredient, the protein folding agent according to [1] above.
[5] A protein unfolded in the presence of at least one selected from compounds represented by formulas (1) to (14) defined in [1] above, and salts and solvates thereof A protein folding method comprising the step of treating.
[6] A protein unfolded in the presence of at least one selected from compounds represented by formulas (1) to (14) defined in [1] above, and salts and solvates thereof A method for refolding a protein, comprising the step of treating.
[7] A protein solubilizer comprising, as an active ingredient, at least one selected from 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 position of each disulfide bond species in its folding pathway. FIG. 1B is a diagram showing the relationship between HPLC retention time and each disulfide bond species when determining disulfide bond cross-linking positions of BPTI using reversed-phase HPLC. FIG. 2A is a diagram showing the results of reverse-phase HPLC analysis when GSH/GSSG (Comparative Example 1) was used to carry out the folding reaction of reductively modified BPTI. FIG. 2B is a diagram showing the results of reverse-phase HPLC analysis when the folding reaction of reductive-denatured BPTI was carried out using LDA-SH/LDA-SS (Example 1). FIG. 2C is a diagram showing the results of reversed-phase HPLC analysis of the folding reaction of reductive-denatured BPTI using BDA-SH/BDA-SS (Example 2). FIG. 3 is a graph showing the activity recovery evaluation when the folding reaction of reductively denatured RNase A was performed using LDA-SH/LDA-SS (Example 3), GSH/GSSG (Comparative Example 2), or GSSG. . FIG. 4A is a diagram showing the reaction of disulfide bond introduction into reductively modified β2m using GSH/GSSG (Comparative Example 3) followed by reversed-phase HPLC. FIG. 4B is a diagram showing the reaction of disulfide bond introduction into reductively modified β2m using LDA-SH/LDA-SS (Example 4) followed by reversed-phase HPLC. FIG. 4C is a diagram showing the reaction of disulfide bond introduction into reductively modified β2m using BDA-SH/BDA-SS (Example 5) followed by reversed-phase HPLC. FIG. 5 shows oxidized forms by introduction of disulfide bonds into reductively modified β2m by GSH/GSSG (Comparative Example 3), LDA-SH/LDA-SS (Example 4), or BDA-SH/BDA-SS (Example 5). It is a graph quantifying β2m. FIG. 6 shows the folding reaction of reductively denatured RNase A using GSH/GSSG (Comparative Example 4), ImdM-SH/ImdM-SS (Example 6), or oPyM-SH/oPyM-SS (Example 7). It is a graph which shows the activity recovery evaluation in the case of. FIG. 7 shows GSH/GSSG (Comparative Example 5), pPyM-SH-NMe.Cl/SS (Example 8), pPyM-SH-NMe.I/SS (Example 9) or oPyM-SH-NMe.I/ Fig. 10 is a graph showing the activity recovery evaluation when a reductive denatured RNase A folding reaction was performed using SS (Example 10). FIG. 8 is a photograph of each sample solution (before centrifugation) obtained in the β2m aggregation experiment by heating. FIG. 9 is a graph showing the survival rate of native β2m in the supernatant obtained by centrifuging each sample solution in the β2m aggregation experiment by heating. Figure 10 shows GSH/GSSG (Comparative Example 4), ImdM-SH/ImdM-SS (Example 6), oPyM-SH/oPyM-SS (Example 7) or pPyM-SH/GSSG (Example 13). 2 is a graph showing evaluation of activity recovery when a reductive denatured RNase A folding reaction was carried out using .

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below based on embodiments.

The present invention relates to protein folding agents. The protein folding agent of the present invention contains, as an active ingredient, at least one selected from compounds represented by formulas (1) to (14), and salts and solvates thereof. According to the protein folding agent of the present invention, compared to the conventional system using glutathione, it is highly efficient, that is, high yield and/or
Alternatively, disulfide bonds can be introduced into proteins at high speed, allowing oxidative folding of proteins to proceed with high efficiency. This is
Since it leads to shortening of the time required for production of proteins used in formulations and the like and improvement in yield, effects of large-scale production of protein formulations and reduction in production cost can be obtained. Below, the formula (
1) The compounds represented by to (7) and salts and solvates thereof are also simply referred to as "thiol compounds", and the compounds represented by formulas (8) to (14) and salts and solvates thereof The substance is also simply called a "disulfide compound".

［式中、
Ｘ_１、Ｘ_２及びＸ_３は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでい
てもよい炭素数１～１０個のアルキレン基であり、
Ｙ_１は、分子鎖中に１～５個の酸素原子を含んでいてもよい炭素数１～１０個のアルキ
レン基であり、
Ｚ_１、Ｚ_２、Ｚ_３、Ｚ_４及びＺ_５は、それぞれ独立して、分子鎖中に１～５個の酸素原
子を含んでいてもよい炭素数１～１０個のアルキレン基であり、
Ｒ_１及びＲ_２は、それぞれ独立して、炭素数１～２４個のアルキル基であり、
Ｍ^－は、それぞれ独立して、塩素イオン、臭素イオン又はヨウ素イオンである］
で表される化合物、並びにその塩及び溶媒和物である。一般にチオール化合物は悪臭を発
するが、上記チオール化合物はいずれも悪臭が抑えられており、この点は応用する上で従
来の関連化合物に対して利点を有する。また、上記チオール化合物はタンパク質と比べて
十分に小さい分子であるため、ジスルフィド結合導入反応を行った後にサイズ排除クロマ
トグラフィーや透析を用いてタンパク質からの分離を容易に行うことができる。 The thiol compound has the following formula:

[In the formula,
X ₁ , X ₂ and X ₃ are each independently an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain,
Y ₁ 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 are each independently 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 iodine ion]
is a compound represented by and salts and solvates thereof. Generally, thiol compounds emit offensive odors, but all of the above thiol compounds have suppressed offensive odors, which is advantageous over conventional related compounds in terms of application. In addition, since the thiol compound is a sufficiently small molecule compared to proteins, it can be easily separated from proteins using size exclusion chromatography or dialysis after disulfide bond introduction reaction.

［式中、
Ｘ_１、Ｘ_２及びＸ_３は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでい
てもよい炭素数１～１０個のアルキレン基であり、
Ｙ_１は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでいてもよい炭素数
１～１０個のアルキレン基であり、
Ｚ_１、Ｚ_２、Ｚ_３、Ｚ_４及びＺ_５は、それぞれ独立して、分子鎖中に１～５個の酸素原
子を含んでいてもよい炭素数１～１０個のアルキレン基であり、
Ｒ_１及びＲ_２は、それぞれ独立して、炭素数１～２４個のアルキル基であり、
Ｍ^－は、それぞれ独立して、塩素イオン、臭素イオン又はヨウ素イオンである］
で表される化合物、並びにその塩及び溶媒和物である。また、上記ジスルフィド化合物は
タンパク質と比べて十分に小さい分子であるため、ジスルフィド結合導入反応を行った後
にサイズ排除クロマトグラフィーや透析を用いてタンパク質からの分離を容易に行うこと
ができる。 The disulfide compound has the formula:

[In the formula,
X ₁ , X ₂ and X ₃ are each independently an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain,
Y ₁ is each independently 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 are each independently 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 iodine ion]
A compound represented by and salts and solvates thereof. In addition, since the disulfide compound is a molecule that is sufficiently smaller than a protein, it can be easily separated from the protein using size exclusion chromatography or dialysis after the disulfide bond introduction reaction.

One embodiment of the protein folding agent of the present invention is at least one selected from compounds represented by formulas (1) to (7), and salts and solvates thereof;
) to (14), and at least one selected from salts and solvates thereof as an active ingredient. The molar ratio of the thiol compound and the disulfide compound contained in the protein folding agent of the present embodiment is not particularly limited as long as the folding proceeds efficiently, but is, for example, 1:1 to 20:1. Preferably, it is more preferably 2:1 to 10:1. The protein folding agent of the present 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 separately as a kit, set, or the like. It may be distributed.

Another embodiment of the protein folding agent of the present invention comprises, as an active ingredient, at least one selected from compounds represented by formulas (1) to (7), and salts and solvates thereof. Characterized by The protein folding agent of the present embodiment can be used in combination with at least one 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 the present embodiment and the disulfide compound used in combination is not particularly limited as long as the folding proceeds efficiently. 1 is preferred, and 2:1 to 10:1 is more preferred. The protein folding agent containing the above thiol compound can be used in a protein folding reaction together with the separately obtained disulfide compound. The disulfide compound to be used in combination may be a conventionally used one such as GSSG, and the preferred embodiment in this case refers to the above disulfide compound unless otherwise specified.

Another embodiment of the protein folding agent of the present invention comprises, as an active ingredient, at least one selected from compounds represented by formulas (8) to (14), and salts and solvates thereof. Characterized by The protein folding agent of the present embodiment can be used in combination with at least one selected from compounds represented by formulas (1) to (7), and salts and solvates thereof. In this case, the molar ratio between the thiol compound used in combination and the disulfide compound contained in the protein folding agent of the present embodiment is not particularly limited as long as the folding proceeds efficiently. 1 is preferred, and 2:1 to 10:1 is more preferred. The present protein folding agent containing the disulfide compound can be used in a protein folding reaction together with the separately obtained thiol compound. In addition, the thiol compound to be used in combination may be a conventionally used one such as GSH, and the preferred embodiment in this case shall refer to the description of the above thiol compound unless otherwise specified.

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

In synthesizing the thiol compound and the disulfide compound, a general organic synthesis method for those skilled in the art can be appropriately employed. Specifically, refer to the methods for producing compounds corresponding to thiol compounds represented by formulas (1) to (7) and disulfide compounds represented by formulas (8) to (14) shown in the examples below. be able to.

Both the thiol compound and the disulfide compound may be in the form of salts or solvates. The above salts are not particularly limited, and examples 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; Examples thereof include salts with organic acids such as fluoroacetic acid. Examples of the solvates include hydrates and alcohols (e.g.,
methanol, ethanol, propanol, isopropanol), acetone, tetrahydrofuran, dioxane, DMF, DMSO and the like.

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 methylene group, ethylene group, propylene group, isopropylene group,
n-butylene group, isobutylene group, tert-butylene group, n-pentylene group, isopentylene group, neopentylene group, hexylene group, heptylene group, octylene group, nonylene group, decylene group, polyoxyalkylene group (where alkylene is , for example, an ethylene group or a propylene group), but is not limited thereto. Examples of polyoxyalkylene groups include, but are not limited to, *-(CH ₂ CH ₂ O)a- (where a is an integer of 1 to 5, * is X ₁ , X ₂ and X ₃ is the point of attachment to —NH ₂ , Y ₁ is the point of attachment to —SH, Z ₁ is the point of attachment to the imidazole ring, Z ₂ ,
Z ₃ , Z ₄ and Z ₅ indicate the bonding point with the pyridine ring), *-(OCH ₂ CH ₂ )
b—where b is an integer from 1 to 5 and * is —NH ₂ for X ₁ , X ₂ and X ₃
, the point of bonding to —SH for Y ₁ , the point of bonding to the imidazole ring for Z ₁ , the point of bonding to the pyridine ring for Z ₂ , Z ₃ , Z ₄ and Z ₅ ), *-( _CH2
CH ₂ CH ₂ O)c— (where c is an integer from 1 to 3, * is the point of attachment to —NH ₂ for X ₁ , X ₂ and X ₃ and —SH for Y ₁ point of attachment to the imidazole ring for Z ₁ , 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 from 1 to 3 and * is X ₁
, the point of attachment with —NH ₂ for X ₂ and X ₃ , the point of attachment with —SH for Y ₁ , Z ₁
for Z ₂ , Z ₃ , Z ₄ and Z ₅ are binding points to the pyridine ring).

Further, 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 methylene group, ethylene group, propylene group, isopropylene group, n-butylene group, isobutylene group, tert-butylene group, n-pentylene group, isopentylene group, neopentylene group, hexylene group, polyoxyalkylene group (where alkylene is, for example, ethylene group, propylene group is), but is not limited to this. Examples of polyoxyalkylene groups include, but are not limited to, *-(CH ₂ CH ₂ O)a- (where a is an integer of 1 to 3, * is X ₁ , X ₂ and the point of bonding with —NH ₂ for X ₃ , the point of bonding with —SH for Y ₁ , the point of bonding with the imidazole ring for Z ₁ , the pyridine ring for Z ₂ , Z ₃ , Z ₄ and Z ₅ *—(OCH ₂ CH ₂ )b— (where b is an integer from 1 to 3 and * is for X ₁ , X ₂ and X ₃ to —NH ₂ point of attachment, -SH for Y ₁
, the point of attachment to the imidazole ring for Z ₁ , 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 or 2, * is 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 , _{Z2 , Z3} , Z4
and Z ₅ indicates the point of attachment to the pyridine ring), *-(OCH ₂ CH ₂ CH ₂ )d-
(where d is 1 or 2, * is 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 imidazole ring for Z ₁ The point of attachment with, Z ₂
, Z ₃ , Z ₄ and Z ₅ indicate the bonding point with the pyridine ring).

Further, specific examples of the "alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms in the molecular chain" described in this specification include methylene group, ethylene group, propylene group, isopropylene group, polyoxyalkylene group (where alkylene is, for example, ethylene group, propylene group), but is not limited thereto. Examples of polyoxyalkylene groups include, but are not limited to, *-(CH ₂ CH ₂ O)a
- (where a is an integer of 1 or 2 and * is -NH ₂ for X ₁ , X ₂ and X ₃
, the point of bonding to —SH for Y ₁ , the point of bonding to the imidazole ring for Z ₁ , the point of bonding 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 or 2, * is the point of attachment to —NH ₂ for X ₁ , X ₂ and X ₃ and —SH for Y ₁ point, the point of attachment to the imidazole ring for Z ₁ , 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 X ₁ , X ₂ and X _3
-NH2 for Y1, _-SH for Y1, imidazole ring for _Z1 , pyridine ring for _{Z2 , Z3} , Z4 and _Z5 points), *-(OCH ₂ CH ₂ CH ₂ )d- (where d is 1, * is the point of attachment to -NH ₂ for X ₁ , X ₂ and X ₃ , Y ₁ is a bonding point with —SH, Z ₁ is a bonding point with an imidazole ring, and Z ₂ , Z ₃ , Z ₄ and Z ₅ are bonding points with a pyridine ring).

Specific examples of the "alkyl group having 1 to 24 carbon atoms" include a methyl group, an ethyl group, 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, n-docosyl group, n-tricosyl group, n-tetracosyl group and the like.

The combination of the thiol compound and the disulfide compound used in the protein folding agent of the present invention is not particularly limited. From the viewpoint of efficiently acting as an agent, a combination having a corresponding relationship (for example, a thiol compound represented by formula (1) and its disulfide form of formula (8
) in combination with a disulfide compound represented by ), or is preferably used in combination. However, even when one of the thiol compound and the disulfide compound is used as an active ingredient of the protein folding agent of the present invention, both the thiol compound and the disulfide compound are present in equilibrium in the folding solution in the folding step. can also act as reducing and oxidizing agents in disulfide bond shuffling.

Reaction intermediates that are transiently produced in the oxidative folding process are generally highly cohesive and cause a decrease in yield. Therefore, from the viewpoint of improving the yield of protein oxidative folding, the protein folding agent of the present invention preferably has a function of suppressing aggregation of reaction intermediates. The thiol compound and disulfide compound, which are the active ingredients of the protein folding agent of the present invention having such properties or are used in combination, can be used as the active ingredients of the protein solubilizer.

In addition, the protein folding agent of the present invention preferably has high activity to introduce disulfide bonds very quickly, from the viewpoint of rapidly forming non-natural structures in addition to natural structures. Proteins exhibit quite different biochemical properties in native and non-native structures. In the case of protein formulations, while the natural type exhibits expected efficacy, the non-natural type may produce low efficacy and even serious side effects. Therefore, the protein folding agent of the present invention examines the biochemical properties of non-naturally occurring structural proteins, and investigates potential side effects due to the efficacy of non-naturally occurring structural proteins (misfolded proteins) that may be contained as impurities in protein preparations. From the viewpoint of comprehensive elucidation, it is preferable to have the function of providing various non-natural structural proteins at once. In the prior art, there is no report on a compound that simultaneously produces a variety of non-natural structures for proteins having multiple cysteine pairs. As a method for preferentially producing non-natural structural proteins, a method of replacing some cysteine residues in the protein with selenocysteine residues has been reported (N.Metanis et al., Angew.
Chem. Int. Ed. 2012 51:5585-88). However, this method converts the structure of the protein itself. In addition, only a few types of selenoproteins containing selenium in vivo have been reported, and the physiological functions of these selenoproteins are still unknown. Pharmacological application of low-molecular-weight compounds is considered difficult.

Furthermore, the protein folding agent of the present invention preferably has a function of suppressing aggregation while producing a non-natural structural protein. In general, non-natural structural proteins are highly agglutinative, which significantly limits measurement systems, making research difficult. This is advantageous for investigation, and is therefore expected to be useful in research on therapeutic methods for folding diseases caused by non-natural structural proteins. The thiol compound and disulfide compound, which are the active ingredients of the protein folding agent of the present invention having such properties or are used in combination, can be used as the active ingredients of the solubilizer for proteins with non-natural structures.

The target protein to be folded using the protein folding agent of the present invention is not particularly limited as long as it contains at least one disulfide bond. Peptides, polypeptides, proteins, and complexes thereof are included regardless of origin or production method. Proteins of any kind are included, for example, intracellular proteins, extracellular proteins, membrane proteins, and intranuclear proteins. Specifically, the following enzymes, recombinant proteins, antibodies, and the like are included.

Examples of enzymes include hydrolases, isomerases, oxidoreductases, transferases, synthases, and lyases. Hydrolytic enzymes include protease, serine protease, amylase, lipase, cellulase, glucoamylase and the like. Isomerases include glucose isomerase. The oxidoreductase includes protein disulfide isomerase, peroxidase and the like. Transferases include acyltransferase, sulfotransferase and the like. Synthetic enzymes include fatty acid synthase, phosphate synthase, citrate synthase, and the like. As a releasing enzyme,
pectin lyase and the like.

Recombinant proteins include protein formulations, vaccines, and the like. Recombinant proteins produced by genetic engineering using prokaryotes such as E. coli, eukaryotes such as yeast, or heterologous expression systems such as cell-free extraction systems are often obtained as insoluble and inert aggregates, so-called inclusion bodies. Therefore, the protein folding agent of the present invention can be preferably used.
As protein preparations, interferon α, interferon β, interleukin 1
~12, growth hormone, erythropoietin, insulin, granular colony stimulating factor (G-
CSF), tissue plasminogen activator (TPA), defensins, natriuretic peptides, blood coagulation factor 2, somatomedin, glucagon, growth hormone releasing factor, serum albumin, calcitonin and the like. Vaccines include hepatitis A vaccine, B
Examples include hepatitis vaccine, hepatitis C vaccine, and the like. Antibodies are not particularly limited, but
Medical antibodies are included.

When the protein folding agent of the present invention is used for oxidative folding, a step of treating the unfolded protein in the presence of the protein folding agent of the present invention (hereinafter simply referred to as a treatment step), for example, The protein, the folding agent for the protein of the present invention, and optionally a thiol compound or disulfide compound used in combination are blended in a folding buffer and mixed by stirring or the like.

Protein unfolding can be carried out by appropriately adopting a general method for those skilled in the art. For example, guanidine hydrochloride, urea, thiourea, or a combination thereof can be used to obtain unfolded proteins. In addition, protein unfolding can also be performed by the method described in the following example "8) Production of reduction-denatured protein".

The treatment time of the above treatment steps can be appropriately adjusted so that the target protein can be obtained with high efficiency. Moreover, the temperature can be appropriately selected according to the thermotolerance of the target protein, and is, for example, in the range of 0 to 100°C, preferably in the range of 4 to 40°C.

A thiol compound and an oxidizing agent disulfide compound (
Although the concentration of total amount) is not particularly limited, it is usually 0.01 to 100 mM in a solution containing the target protein (eg, folding buffer). In addition, the concentration of the unfolded protein contained in the solution containing the target protein (for example, folding buffer) is usually 0.01 to 100 μM.

In the solution containing the target protein in the above treatment step (e.g., folding buffer), the molar ratio of the thiol compound (total amount) as a reducing agent and the disulfide compound (total amount) as an oxidizing agent is adjusted to obtain the target protein. is not particularly limited as long as the introduction of the disulfide bond of is progressing efficiently, but it 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 impair the function of the target protein. Specifically, amine buffers such as Tris buffer, MES buffer and Tricine buffer Phosphate buffer, various Good's buffers, and the like are included. The pH of the folding buffer is usually pH 4 to 10, preferably pH 5 to
pH can be adjusted in the range of 9, more preferably in the range of pH 7-9.

Various additives can be added to the folding buffer, in addition to the protein folding agent of the present invention and, if necessary, the thiol compound or disulfide compound used in combination. Such additives include salts such as sodium chloride and calcium chloride;
buffers such as citrate, phosphate, and acetate; bases such as sodium hydroxide; acids such as hydrochloric acid and acetic acid; organic solvents such as methanol, ethanol, and propanol; In addition to the folding agent of the present invention and the above additives, the buffering agent includes a surfactant, p
H regulators or protein stabilizers can also be incorporated. A person skilled in the art can appropriately adjust the amount used.

Formula (
The thiol compounds represented by 1) to (7) and the disulfide compounds represented by formulas (8) to (14) are described below.

［式中、
Ｘ_１は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでいてもよい炭素数
１～１０個のアルキレン基であり、
Ｙ_１は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでいてもよい炭素数
１～１０個のアルキレン基である］
、並びに併用される成分としての上記組み合わせは、酸化的フォールディングにより天然
構造タンパク質を高収率で得る観点から好ましい。この場合のフォールディングの対象と
なるタンパク質としては、特に制限されず、上述したタンパク質、さらにはRNase A、ウ
シ膵臓トリプシン阻害剤、β₂-ミクログロブリン、インスリン、主要組織適合遺伝子複合
体、免疫グロブリン等が挙げられ、上記効果を得る観点から、複数のジスルフィド結合を
有するタンパク質、例えばRNase A等の４本のジスルフィド結合を有するものが好ましい
。 The following thiol compound (1
) and the following disulfide compound (8):

[In the formula,
each X ₁ is independently an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain,
Y ₁ is each independently 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 components used in combination are preferable from the viewpoint of obtaining a native structural protein at a high yield by oxidative folding. Proteins to be folded in this case are not particularly limited, and the proteins described above, as well as RNase A, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulin, etc. From the viewpoint of obtaining the above effects, proteins having multiple disulfide bonds, such as RNase A and the like, having four disulfide bonds are preferred.

Moreover, the above compound or combination is preferable from the viewpoint of suppressing aggregation of reaction intermediates of oxidative folding and obtaining a native structural protein at a high yield. The protein to be folded in this case is not particularly limited, and the proteins described above, as well as RNAs
eA, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulin, etc., and from the viewpoint of obtaining the above effects, proteins having multiple disulfide bonds, such as bovine pancreatic trypsin Those having three disulfide bonds, such as inhibitors, are preferred.

In addition, the above compounds or combinations are preferred from the viewpoint of obtaining the native structural protein at high yield and speed by oxidative folding. Proteins to be folded in this case are not particularly limited, and the proteins described above, as well as RNase A, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulin, etc. From the viewpoint of obtaining the above effects, proteins having one disulfide bond such as β ₂ -microglobulin (β2m) are preferred.

From the viewpoint of obtaining the effects described above, X ₁ 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, it is an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms. Further, from the viewpoint of obtaining the above effects, Y ₁ is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain, and the molecular chain More preferably, it is an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms therein.

［式中、
Ｘ_２及びＸ_３は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでいてもよ
い炭素数１～１０個のアルキレン基である］
、並びに併用される成分としての上記組み合わせは、酸化的フォールディングにおいて、
極めて速くジスルフィド結合を導入し、多種の非天然構造体を一度に生成させる観点から
好ましい。この場合のフォールディングの対象となるタンパク質としては、特に制限され
ず、上述したタンパク質、さらにはRNase A、ウシ膵臓トリプシン阻害剤、β₂-ミクログ
ロブリン、インスリン、主要組織適合遺伝子複合体、免疫グロブリン等が挙げられ、上記
効果を得る観点から、複数のジスルフィド結合を有するタンパク質、例えばウシ膵臓トリ
プシン阻害剤等の３本のジスルフィド結合を有するものが好ましい。 The following thiol compounds (2
) and the following disulfide compound (9):

[In the formula,
X ₂ and X ₃ are each independently an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain]
, and the above combination as a component used together, in oxidative folding,
It is preferable from the viewpoint of rapidly introducing disulfide bonds and generating a variety of non-natural structures at once. Proteins to be folded in this case are not particularly limited, and the proteins described above, as well as RNase A, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulin, etc. From the viewpoint of obtaining the above effects, proteins having multiple disulfide bonds, for example, those having three disulfide bonds, such as bovine pancreatic trypsin inhibitor, are preferred.

In addition, the above compound or combination is preferable from the viewpoint of producing a non-natural structural protein while suppressing aggregation in oxidative folding. The protein to be folded in this case is not particularly limited, and the proteins described above, as well as RNase
A, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulin and the like. Those having three disulfide bonds, such as agents, are preferred.

In addition, the above compounds or combinations are preferred from the viewpoint of obtaining the native structural protein at high yield and speed by oxidative folding. Proteins to be folded in this case are not particularly limited, and the proteins described above, as well as RNase A, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulin, etc. From the viewpoint of obtaining the above effects, proteins having one disulfide bond such as β ₂ -microglobulin (β2m) are preferred.

From the viewpoint of obtaining the effects described above, X ₂ is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain. More preferably, it is an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms. Further, from the viewpoint of obtaining the effects described above, X ₃ is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain. More preferably, it is an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms therein.

［式中、
Ｚ_１は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでいてもよい炭素数
１～１０個のアルキレン基である］
、並びに併用される成分としての上記組み合わせは、酸化的フォールディングにより天然
構造タンパク質を高収率で得る観点から好ましい。この場合のフォールディングの対象と
なるタンパク質としては、特に制限されず、上述したタンパク質、さらにはRNase A、ウ
シ膵臓トリプシン阻害剤、β₂-ミクログロブリン、インスリン、主要組織適合遺伝子複合
体、免疫グロブリン等が挙げられ、上記効果を得る観点から、複数のジスルフィド結合を
有するタンパク質、例えばRNase A等の４本のジスルフィド結合を有するものが好ましい
。 The following thiol compounds (3
) and the following disulfide compound (10):

[In the formula,
Z ₁ is each independently 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 components used in combination are preferable from the viewpoint of obtaining a native structural protein at a high yield by oxidative folding. Proteins to be folded in this case are not particularly limited, and the proteins described above, as well as RNase A, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulin, etc. From the viewpoint of obtaining the above effects, proteins having multiple disulfide bonds, such as RNase A and the like, having four disulfide bonds are preferred.

From the viewpoint of obtaining the effects described above, Z ₁ 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, it is an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms.

［式中、
Ｚ_２は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでいてもよい炭素数
１～１０個のアルキレン基である］
、並びに併用される成分としての上記組み合わせは、酸化的フォールディングを促進させ
る観点から好ましい。この場合のフォールディングの対象となるタンパク質は特に制限さ
れず、上述したタンパク質、さらにはRNase A、ウシ膵臓トリプシン阻害剤、β₂-ミクロ
グロブリン、インスリン、主要組織適合遺伝子複合体、免疫グロブリン等が挙げられる。
上記のような効果を得る観点から、Ｚ_２は、分子鎖中に１～３個の酸素原子を含んでいて
もよい炭素数１～６個のアルキレン基であることが好ましく、分子鎖中に１又は２個の酸
素原子を含んでいてもよい炭素数１～４個のアルキレン基であることがさらに好ましい。 The following thiol compounds (4
) and the following disulfide compound (11):

[In the formula,
Z ₂ is each independently 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 components used in combination are preferable from the viewpoint of promoting oxidative folding. The protein to be folded in this case is not particularly limited, and includes the above proteins, RNase A, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulin, and the like. be done.
From the viewpoint of obtaining the above effects, Z ₂ 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, it is an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms.

［式中、
Ｚ_３は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでいてもよい炭素数
１～１０個のアルキレン基である］
、並びに併用される成分としての上記組み合わせは、酸化的フォールディングにより天然
構造タンパク質を短時間かつ高収率で得る観点から好ましい。この場合のフォールディン
グの対象となるタンパク質としては、特に制限されず、上述したタンパク質、さらにはRN
ase A、ウシ膵臓トリプシン阻害剤、β₂-ミクログロブリン、インスリン、主要組織適合
遺伝子複合体、免疫グロブリン等が挙げられ、上記効果を得る観点から、複数のジスルフ
ィド結合を有するタンパク質、例えばRNase A等の４本のジスルフィド結合を有するもの
が好ましい。 The following thiol compounds (5
) and the following disulfide compound (12):

[In the formula,
Z ₃ is each independently an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain]
, and the above combination as components used in combination are preferable from the viewpoint of obtaining the native structural protein in a short time and at a high yield by oxidative folding. Proteins to be folded in this case are not particularly limited, and the proteins described above, as well as RN
ase A, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulins, etc., and from the viewpoint of obtaining the above effects, proteins having multiple disulfide bonds, such as RNase A, etc. Those having four disulfide bonds of are preferred.

From the viewpoint of obtaining the effects described above, Z ₃ is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain. More preferably, it is an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms.

［式中、
Ｚ_４は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでいてもよい炭素数
１～１０個のアルキレン基であり、
Ｒ_１は、それぞれ独立して、炭素数１～２４個のアルキル基であり、
Ｍ^－は、それぞれ独立して、塩素イオン、臭素イオン又はヨウ素イオンである］
、並びに併用される成分としての上記組み合わせは、タンパク質の酸化的フォールディン
グにより天然構造タンパク質を高収率で得る観点から好ましい。この場合のフォールディ
ングの対象となるタンパク質としては、特に制限されず、上述したタンパク質、さらには
RNase A、ウシ膵臓トリプシン阻害剤、β₂-ミクログロブリン、インスリン、主要組織適
合遺伝子複合体、免疫グロブリン等が挙げられ、上記効果を得る観点から、複数のジスル
フィド結合を有するタンパク質、例えばRNase A等の４本のジスルフィド結合を有するも
のが好ましい。 The following thiol compounds (6
) and the following disulfide compound (13):

[In the formula,
each Z ₄ is independently 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 iodine ion]
, and the above combinations as components used in combination are preferable from the viewpoint of obtaining a native structural protein at a high yield by oxidative folding of the protein. The protein to be folded in this case is not particularly limited, and the proteins described above, as well as
RNase A, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulins, etc., and from the viewpoint of obtaining the above effects, proteins having multiple disulfide bonds, such as RNase A, etc. Those having four disulfide bonds of are preferred.

From the viewpoint of obtaining the effects described above, Z ₄ is preferably an alkylene group having 1 to 6 carbon atoms which may contain 1 to 3 oxygen atoms in the molecular chain. More preferably, it is an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms. From the viewpoint of obtaining the above effects, R ₁ is preferably an alkyl group having 1 to 6 carbon atoms, more preferably an alkyl group having 1 or 2 carbon atoms. Moreover, from the viewpoint of obtaining the above effects, M ⁻ is preferably a chloride ion or an iodine ion.

In addition, the thiol compound (6) and the disulfide compound (13), as well as the combination thereof, aggregate in water to form micelles due to the hydrophobic interaction of the R ₁ groups, and the hydrophobic inner space and hydrophilic surface of the micelles Since the amphiphilic effect of β suppresses aggregation of denatured proteins and intermediate states of protein folding, oxidative folding can also be promoted by introducing disulfide bonds into proteins with high efficiency. In this case, R ₁ 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 the proteins described above, as well as RNase A, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulin, etc. are mentioned.

［式中、
Ｚ_５は、それぞれ独立して、分子鎖中に１～５個の酸素原子を含んでいてもよい炭素数
１～１０個のアルキレン基であり、
Ｒ_２は、それぞれ独立して、炭素数１～２４個のアルキル基であり、
Ｍ^－は、それぞれ独立して、塩素イオン、臭素イオン又はヨウ素イオンである］
、並びに併用される成分としての上記組み合わせは、酸化的フォールディングにより天然
構造タンパク質を高収率で得る観点から好ましい。この場合のフォールディングの対象と
なるタンパク質としては、特に制限されず、上述したタンパク質、さらにはRNase A、ウ
シ膵臓トリプシン阻害剤、β₂-ミクログロブリン、インスリン、主要組織適合遺伝子複合
体、免疫グロブリン等が挙げられ、上記効果を得る観点から、複数のジスルフィド結合を
有するタンパク質、例えばRNase A等の４本のジスルフィド結合を有するものが挙げられ
る。 The following thiol compounds (7
) and the following disulfide compound (14):

[In the formula,
each Z ₅ is independently an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain,
each R ₂ is independently an alkyl group having 1 to 24 carbon atoms,
each M ⁻ is independently a chloride ion, a bromide ion, or an iodine ion]
, and the above combinations as components used in combination are preferable from the viewpoint of obtaining a native structural protein at a high yield by oxidative folding. Proteins to be folded in this case are not particularly limited, and the proteins described above, as well as RNase A, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulin, etc. From the viewpoint of obtaining the above effects, proteins having multiple disulfide bonds, such as RNase A and the like, having four disulfide bonds are included.

From the viewpoint of obtaining the effects described above, Z ₅ 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, it is an alkylene group having 1 to 4 carbon atoms which may contain 1 or 2 oxygen atoms. From the viewpoint of obtaining the above effects, R ₂ is preferably an alkyl group having 1 to 6 carbon atoms, more preferably an alkyl group having 1 or 2 carbon atoms. Moreover, from the viewpoint of obtaining the above effects, M ⁻ is preferably an iodine ion.

In addition, the thiol compound (7) and the disulfide compound (14), as well as the combination thereof, aggregate in water to form micelles due to the hydrophobic interaction of the _R2 groups, and the hydrophobic inner space and hydrophilic surface of the micelles Since the amphiphilic effect of β suppresses aggregation of denatured proteins and intermediate states of protein folding, oxidative folding can also be promoted by introducing disulfide bonds into proteins with high efficiency. In this case, R ₂ 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 the proteins described above, as well as RNase A, bovine pancreatic trypsin inhibitor, β ₂ -microglobulin, insulin, major histocompatibility complex, immunoglobulin, etc. are mentioned.

The present invention provides a protein comprising a step of treating the unfolded protein in the presence of at least one selected from compounds represented by formulas (1) to (14), and salts and solvates thereof. It also relates to the folding method of In the folding method of the present invention, the step of treating the unfolded protein in the presence of the compound (hereinafter also simply referred to as the treatment step) includes a step of contacting the unfolded protein with the compound. Specifically, a step of blending the unfolded protein and the compound in a folding buffer and mixing by stirring or the like, furthermore, after this step, if necessary, in order to promote folding more sufficiently A step of standing still for a certain period of time is also included. Preferred embodiments of the folding method of the present invention refer to the description of the folding agent of the present invention, unless otherwise specified.

The present invention comprises a step of treating and folding an unfolded protein in the presence of at least one compound selected from compounds represented by formulas (1) to (14), and salts and solvates thereof. It also relates to methods for renaturing proteins, including: The method for refolding the protein of the present invention includes a step of treating the unfolded protein to fold it in the presence of the above compound (hereinafter, simply referred to as a treatment step), and further folding is performed after the step, if necessary. It also includes a step of isolating the protein (hereinafter also referred to as an isolation step). Preferred embodiments of the protein renaturation method of the present invention refer to the descriptions of the folding agent of the present invention and the folding method of the present invention, unless otherwise specified.

Examples of the isolation step include isolation of the target normal protein (folded protein) from the protein suspension obtained in the folding step, using column chromatography or the like. Fillers used in column chromatography include silica, dextran, agarose, cellulose, acrylamide, vinyl polymers and the like. Commercially available products include the Sephadex series, the Sephacryl series, and the Sepharose series (all of which are Pharma
cia), Bio-Gel series (Bio-Rad), and the like. 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 compounds represented by formulas (1) to (14), salts and solvates thereof.
According to the solubilizer of the present invention, it is possible to suppress the aggregation of proteins with natural or non-natural structures and solubilize them. As described above for the protein folding agent of the present invention, the formula (
The thiol compounds represented by 1) to (7) and the disulfide compounds represented by formulas (8) to (14) have the function of suppressing aggregation of proteins with natural or non-natural structures, particularly protein oxidation. A function of suppressing aggregation of reaction intermediates and proteins with native structures generated in oxidative folding, and a function of suppressing aggregation of proteins with non-natural structures in oxidative folding reactions that generate one or more proteins with non-natural structures. and thus functions as a solubilizer. For preferred embodiments of the compounds represented by formulas (1) to (14) and the target protein, etc., the above description of the protein folding agent of the present invention should be referred to.

EXAMPLES Hereinafter, the present invention will be described in more detail using examples. However, 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 compounds represented by formulas (1) and (8), respectively) was synthesized according to the following scheme.

1-1) Synthesis of Compound 2 Compound 1 (1.90 g, 18.3 mmol, Tokyo Chemical Industry) was dissolved in dehydrated methylene chloride (25 mL, Kanto Kagaku) on an ice bath under a nitrogen atmosphere. After adding (Boc) ₂ O (5.22 g, 23.9 mmol, Kanto Kagaku) there, the temperature was raised 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). The extracted ethyl acetate solution was washed with saturated brine (25 mL). Sodium sulfate (Kishida Chemical) was added to the recovered ethyl acetate solution.
was added and then filtered. After the filtrate was distilled off under reduced pressure, it was subjected to silica gel column chromatography (
Kanto Kagaku) gave compound 2. Yield: 3.62 g, Yield: 65%.

1-2) Synthesis of compound 3 N-chlorosuccinimide (NCS, 0.849 g, 6.36 mmol, Kishida Chemical) was dissolved in dehydrated tetrahydrofuran (THF, 36 mL, Kanto Chemical) at room temperature under a nitrogen atmosphere. There PPh ₃ (
A dehydrated THF solution (13 mL) of 1.67 g, 6.38 mmol, Fujifilm Wako Pure Chemical) was added dropwise. 20
After a minute, a dehydrated THF solution (13 mL) of compound 2 (1.75 g, 5.74 mmol) was added dropwise. After 11 hours, the solvent was distilled off under reduced pressure. Compound 3 was obtained by subjecting the residue to silica gel column chromatography (Kanto Kagaku). Yield: 0.60 g, Yield: 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 Kagaku) at room temperature under a nitrogen atmosphere. Potassium thioacetate (AcSK, 0.285 g, 2.
50 mmol, Tokyo Kasei Kogyo) was added, and then the temperature was raised to 90°C. After 12 hours, water (20 mL) was added, followed by extraction with ethyl acetate (25 mL, twice, Kishida Chemical). 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 and filtered. The filtrate was evaporated under reduced pressure. Dehydrated methanol (5 mL, Kanto Kagaku) was added to the resulting residue at room temperature under a nitrogen atmosphere, and sodium carbonate (0.757 g, 5.48 mmol,
Kishida Chemical) was added. After 1 hour, hydrochloric acid (1 M, Kishida Chemical) was added until the pH was 8-9. Water (25 mL) was added to the reaction mixture and 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 Co., Ltd.) was added to the recovered methylene chloride solution and filtered. The filtrate was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Kagaku) to obtain compound 4. Yield: 0.257 g, Yield: 43%.

1-4) Synthesis of Compound 5 Compound 4 (0.257 g, 0.803 mmol) was dissolved in ethyl acetate (5.0 mL, Kanto Kagaku) at room temperature in air. After adding sodium iodide (6.9 mg, 46 μmol, Kishida Chemical),
% 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). After sodium sulfate (Kishida Chemical Co., Ltd.) was added to the recovered ethyl acetate solution, the mixture was filtered. The filtrate was distilled off under reduced pressure to obtain compound 5. Yield: 0.241 g, Yield: 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 thereto. After stirring for 1 hour, the solvent was distilled off under reduced pressure. Methylene chloride (10 mL, AGC) was added to the residue and extracted with water (10 mL). The extracted aqueous solution was distilled off under reduced pressure and subjected to high-performance liquid chromatography (equipment: JASCO Corporation, PU-4086, UV-
4075, column: TA12S05-2520WX) to obtain LDA-SS. 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 nitrogen atmosphere. Dithiothreitol (112 mg, 0.726 mmol, Nacalai Tesque) was added thereto. After stirring for 24 hours, high-performance liquid chromatography (equipment: JASCO, PU-4086, UV-4075, column: TA12S05-
2520WX) to obtain LDA-SH. Yield: 33 mg, Yield: 43%.

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

2-1) Synthesis of Compound 7 Compound 6 (0.539 g, 5.98 mmol) was dissolved in dehydrated methanol (30 mL, Kanto Kagaku) at room temperature under a nitrogen atmosphere. After heating to 45°C, (Boc) ₂ O (2.84 g, 13.0 mmol, Kanto Kagaku) and triethylamine (6.91 mL, Sigma) were added. After 20 hours, the solvent was distilled off under reduced pressure, and the resulting residue was subjected to silica gel column chromatography (Kanto Kagaku) to obtain Compound 7.
got 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 Kagaku) 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 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), added with sodium sulfate (Kishida Chemical), and filtered. The filtrate was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Kagaku) to obtain compound 8. Yield: 1.64 g, Yield: 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 Kagaku) at room temperature under a nitrogen atmosphere. potassium thioacetate (0.392 g, 3.428 mmol,
Tokyo Kasei Kogyo) 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, twice) and saturated brine (10 mL), added with sodium sulfate (Kishida Chemical), and filtered. The filtrate was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Kagaku) to obtain a thioacetyl intermediate. The thioacetyl intermediate (0.346 g, 0.994 mmol) was dissolved in dehydrated methanol (3 mL, Kanto Kagaku) at room temperature under a nitrogen atmosphere, and potassium carbonate (0.414 g, 2.998 mmol, Kishida Chemical) was added. After stirring for 2 hours, the solvent was distilled off under reduced pressure. Water (10 mL) was added and extracted with methylene chloride (10 mL, 3 times, AGC). The recovered methylene chloride solution was washed with saturated aqueous ammonium chloride solution (10 mL, Kishida Chemical Co., Ltd.) and saturated brine (10 mL), added with sodium sulfate (Kishida Chemical Co., Ltd.), and filtered. The filtrate was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Kagaku) to obtain compound 9. Yield: 0.275 g, Yield: 43%.

2-4) Synthesis of compound 10 Compound 9 (0.279 g, 0.897 mmol) was dissolved in ethyl acetate (5 mL, Kanto Kagaku) at room temperature, sodium iodide (11.8 mg, 0.079 mmol, Kishida Chemical), 30% excess Oxidized water (0.05 mL
, Kishida Chemical) was added. After stirring for 1 hour, water (30 mL) was added and extracted with ethyl acetate (30 mL, twice). The collected ethyl acetate solution was washed with saturated brine (10 mL), added with sodium sulfate, and filtered. The filtrate was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (
Kanto Kagaku), compound 10 was obtained. 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. After adding trifluoroacetic acid (1 mL, Kishida Chemical), the temperature was raised to room temperature. After stirring for 2 hours, the solvent was distilled off 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 of the resulting aqueous solution was distilled off under reduced pressure, and the residue was treated with hydrochloric acid (1 M, 5
mL, Kishida Chemical) was added. After stirring for 1 hour, the solvent was distilled off 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. rice field. 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 of the obtained aqueous solution was distilled off 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 Synthesized.

3-1) Synthesis of Compound 12 Compound 11 (5.512 g, 57.36 mmol, Tokyo Kasei Kogyo) was dissolved in dehydrated ethanol (100 mL, Kanto Kagaku) at room temperature under a nitrogen atmosphere. After cooling to 0°C, sodium borohydride (2.1
65 g, 57.24 mmol, Tokyo Kasei Kogyo) was added. After stirring for 3 hours, water (10 mL) was added and the solvent was distilled off under reduced pressure. Compound 12 was obtained by subjecting the resulting residue to silica gel column chromatography (Kanto Kagaku). Yield: 4.10 g, Yield: 73%.

3-2) Synthesis of Compound 13 Compound 12 (0.565 g, 5.76 mmol) was added to dehydrated tetrahydrofuran (
1.7 mL, Kanto Kagaku) and triethylamine (0.85 mL, Sigma). (Boc) _2O (1.
37 g, 6.28 mmol, Kanto Kagaku) in dehydrated tetrahydrofuran (8.1 mL, Kanto Kagaku) was added dropwise. After stirring for 13 hours, the solvent was distilled off under reduced pressure, water (15 mL) was added, and methylene chloride (20 mL,
It was extracted 5 times with Kishida Chemical). Sodium sulfate (Kishida Chemical Co., Ltd.) was added to the recovered methylene chloride solution and filtered. The filtrate was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Kagaku) to obtain compound 13. Yield: 0.763 g, Yield: 67%.

3-3) Synthesis of Compound 14 Compound 13 (0.917 g, 4.63 mmol) was added to dehydrated methylene chloride (8.4 mL) at room temperature under a nitrogen atmosphere.
, Kanto Kagaku) and dehydrated N,N-dimethylformamide (DMF, 0.2 mL, Kanto Kagaku). After cooling to 0°C, a dehydrated methylene chloride solution (7.0 mL, Kanto Kagaku) of oxalyl chloride (1.22 g, 9.58 mmol, Kishida Chemical) was added dropwise. After raising the temperature 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 Co., Ltd.) was added to the recovered methylene chloride solution and filtered. The filtrate was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Kagaku) 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 added to dehydrated acetone (4.7 mL,
Kanto Kagaku). Sodium iodide (0.233 g, 1.55 mmol, Kishida Chemical) and potassium thioacetate (0.142 g, 1.25 mmol, Tokyo Chemical Industry) were added. After stirring for 21 hours, water (20
mL) was added and extracted with ethyl acetate (30 mL, twice, Kishida Chemical). The collected ethyl acetate solution was washed with saturated brine (10 mL), added with sodium sulfate (Kishida Chemical Co., Ltd.), and filtered. The filtrate was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (Kanto Kagaku) to obtain compound 15. Yield: 0.140 g, Yield: 70%.

3-5) Synthesis of ImdM-SS Compound 15 (0.911 g, 3.55 mmol) was added to dehydrated methanol (10 mL,
Kanto Kagaku), and potassium carbonate (1.00 g, 7.24 mmol, Kishida Kagaku) was added. After stirring for 3 hours, the solvent was distilled off under reduced pressure, water (10 mL) was added, and the mixture was neutralized with hydrochloric acid (1 M). Extraction was performed with a mixed solvent of chloroform (Kishida Chemical) and 1-propanol (Kishida Chemical) (3:1, 25 mL, 4 times). The solvent was distilled off under reduced pressure, and the resulting residue was purified by reverse-phase high-performance liquid chromatography (device: JASCO Corporation, PU-4086, UV-4075, column: TA12S05-2520WX) to obtain ImdM-SS. Yield: 27 mg, Yield: 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. rice field. After stirring for 7 hours, a mixed solvent of chloroform (Kishida Chemical) and 1-propanol (Kishida Chemical) (9:1, 25 mL, 4 times) was added for extraction. Sodium sulfate was added and filtered, the filtrate was distilled off under reduced pressure, and the resulting residue was subjected to reverse phase high performance liquid chromatography (device: 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 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)
, Kishida Chemical) was added. After heating to 90°C, the mixture was stirred for 1.5 hours and then cooled to room temperature. Sodium hydroxide (2.058 g, 51.45 mmol, Kishida Chemical) was added, and after stirring for 22 hours, washed with tert-butyl methyl ether (30 mL, twice, Kanto Chemical). The resulting aqueous solution was neutralized with hydrochloric acid (1 M, Kishida Chemical) and extracted with chloroform (30 mL, Kishida Chemical). Sodium sulfate (Kishida Chemical) was added and filtered. The filtrate was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (Kanto Kagaku) to obtain pPyM-SH. Yield: 459 mg, Yield: 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, it was extracted with methylene chloride (10 mL, 3 times, Kishida Chemical). Sodium sulfate was added and filtered. The filtrate was evaporated under reduced pressure to obtain pPyM-SS. Yield: 46 mg,
Yield: 91%.

5) Synthesis of oPyM-SH (ortho-pyridin-2-ylmethanethiol) and oPyM-SS The above compounds (corresponding to 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) was dissolved in water, and thiourea (0.713
g, 9.36 mmol, Kishida Chemical) 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 after stirring for 9 hours, 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 Co., Ltd.) was added to the recovered methylene chloride solution and filtered. The filtrate was evaporated under reduced pressure, and the resulting residue (oPyM-SH crude product) was subjected to high-performance liquid chromatography (device: JASCO Corporation, PU-4086
, UV-4075, column: TA12S05-2520WX) to obtain oPyM-SH. Yield: 113 mg, Yield:
8%.

5-2) Synthesis of oPyM-SS The oPyM-SH crude product obtained by the above method was dissolved in 30% hydrogen peroxide solution (8 mL, Kishida Chemical) and stirred at room temperature for 1 hour. Extraction was performed with chloroform (30 mL, 3 times, Kishida Chemical), sodium sulfate (Kishida Chemical) was added to the recovered chloroform solution, and the mixture was filtered. The filtrate was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography (Fuji Silysia Chemical) to obtain oPyM-SS. Yield: 916 mg, Yield: 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 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) was dissolved in acetonitrile (3.0 mL, Kishida Chemical) and acetone (3 mL, Kishida Chemical) and iodomethane (1.503 g, 10.59 mmol)
l, Tokyo Kasei) was added. After the temperature was raised to 60°C, the mixture was stirred for 16 hours and cooled to room temperature. The resulting precipitate was filtered, and pPyM-SS-NMe·I was obtained from the resulting residue. Yield: 446 mg, Yield: 82%.

6-2) Synthesis of pPyM-SS-NMe Cl (4,4'-(disulfanediylbis(methylene))bis(1-methylpyridin-1-ium)dichloride)
Dissolve pPyM-SS-NMe I (192 mg, 0.361 mmol) in water (3.5 mL) and add silver(I) chloride (115
mg, 0.805 mmol, Kishida Chemical) was added. After stirring for 4 hours at room temperature in the dark, the resulting precipitate was filtered. The filtrate was evaporated under reduced pressure, ethanol (2 mL, Kishida Chemical) was added, and the resulting precipitate was filtered. The filtrate was distilled off under reduced pressure to obtain pPyM-SS-NMe.Cl. Yield: 80 mg, Yield: 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, it was distilled off under reduced pressure, and the resulting residue (pPyM-SH-NMe·Cl crude product) was subjected to high-performance liquid chromatography (device: JASCO Corporation, PU-4086, UV-4075, column: TA12S05-2520WX) and 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) 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, 2M hydrochloric acid (1 mL, Kishida Chemical Co., Ltd.) was added and evaporated under reduced pressure. Spectroscopy, PU
-4086, UV-4075, column: TA12S05-2520WX) 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
-ium)halide) 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) 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 Kasei) was added. After the temperature was raised to 65°C, the mixture was stirred for 16 hours and cooled to room temperature. The resulting precipitate was filtered, and oPyM-SS-NMe·I was obtained from the obtained residue. Yield: 1.286 g, Yield: 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, 2M hydrochloric acid (1 mL, Kishida Chemical) was added, and methylene chloride (20 mL, 5 times, Kishida Chemical) was added.
washed with The collected aqueous solution was distilled off under reduced pressure, and the resulting residue (oPyM-SH-NMe/I crude product) was subjected to high-performance liquid chromatography (device: JASCO Corporation, PU-4086, UV-4075, column: TA12S05-25
20WX) to obtain oPyM-SH-NMe·I. Yield: 80 mg, Yield: 74%.

8) Bovine Pancreas Trypsin Inhibitor (BP) as a model substrate for preparation of reduced denatured protein
TI) (see references 1 and ₂ below), RNase A (see reference 3 below), and β ₂ -microglobulin (β2m) were employed. BPTI and RNase A are proteins with three and four disulfide bonds, respectively, and are popular 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.) (a reduced form of three disulfide bonds), 10 mg of BPTI was incubated in the presence of 8 M urea, 20 mM DTT, pH 8.0, 50°C for 3 hours, and reversed. Purified by phase HPLC. After confirming by MALDI-TOF/MS that all disulfide bonds in the purified preparation had been cleaved, the purified preparation was freeze-dried and stored at -80°C (see Reference 1 below).

In order to prepare a reduced 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 and 25°C for 2 hours. Dialyzed with 10 mM HCl. Dialysis was performed twice more, and the solution was exchanged to 10 mM HCl to remove DTT and the like (see reference 3 below).

β2m was expressed as a recombinant in an E. coli expression system, and lysate A (5
After being suspended in 0 mM Tris-HCl (pH 8.1, 300 mM NaCl), it was sonicated. The fractions obtained after centrifugation were incubated in the presence of 8 M urea, 20 mM DTT, pH 8.0, at 50°C for 3 hours, followed by Cosmosil 5C
It was purified by reversed-phase column chromatography (Hitachi) using an ₁₈ -AR-II column.

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 Ability to Introduce Disulfide Bonds into Reduction-denatured BPTI The following combinations of 1 mM thiol compound/0.2 mM disulfide compound were added to 30 μM reduction-denatured BPTI prepared in 8) above:
Comparative Example 1: GSH (nakalai)/GSSG (nakalai)
Example 1: LDA-SH/LDA-SS
Example 2: BDA-SH/BDA-SS
A buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl was incubated at 30°C to carry out the folding reaction. The reaction solution was sampled over time, and an equal volume of 1N HCl was added to quench the thiol group and disulfide bond exchange reaction. In order to identify the molecular species (see FIG. 1A) in the reaction solution, reverse phase HPLC was performed. As shown in FIGS. 1A and 1B, analysis using reversed-phase HPLC can separate and identify each disulfide bond species when BPTI folds, and track disulfide bond cross-linking positions over time.

Measurement and analysis conditions for reversed-phase HPLC are shown below:
A TSKgel Protein C4-300 column (4.6 x 150 mm; Tosoh Bioscience) was used.
Detected by absorbance at 29 nm. Regarding the mixing of an aqueous solution (A solution) containing 0.05% trifluoroacetic acid and an acetonitrile solution (B solution) containing 0.05% trifluoroacetic acid, the volume increase rate of the ratio of B solution in the mixed solution is 1% from 0 to 15 minutes. /min, developed with a linear concentration gradient of 0.5%/min from 15 to 115 min.

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

When BDA-SH/BDA-SS was used (Example 2), R, which is a reductive modified form of BPTI (all thiol groups), disappeared after 1 minute from the start of the reaction, and a natural form (N) was produced. It can be seen that the disulfide bond is introduced very quickly (Fig. 2C). In addition, the non-natural structure BPTI (NonN in Fig. 2C)
) are also generated at the same time. Generally, proteins with non-natural disulfide bonds show aggregation. However, it was found that BDA-SH/BDA-SS inhibits the aggregation of non-natural BPTI and has properties as a solubilizer, since it can be subjected to HPLC measurement.

10) RNase A activity evaluation 1
In order to evaluate the folding-promoting effect, the RNase A activity recovery assay prepared in 8) above was performed. The following combinations of 1 mM thiol compounds/0.2 mM disulfide compounds:
Comparative Example 2: GSH/GSSG
Example 3: LDA-SH/LDA-SS
buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 8 µM RNase A in the presence of
was incubated with Aliquot the reaction mixture over time and extract cytidine 2′:3, which is a substrate for RNase A.
′-Cytidine 2′:3′-cyclic monophosphate (cCMP) was diluted in the same buffer to a final concentration of 4 μM RNase A, 0.4 mM cCMP. The change in absorbance at 284 nm was measured by a spectrophotometer to quantify the nucleolytic activity of RNase A folded into its native structure.

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

11) Evaluation of Ability to Introduce Disulfide Bonds into Reduced-denatured β2m The following combinations of 1 mM thiol compound/0.2 mM disulfide compound were added to 10 μM reduced-denatured β2m prepared in 8) above:
Comparative Example 3: GSH/GSSG
Example 4: LDA-SH/LDA-SS
Example 5: BDA-SH/BDA-SS
A folding reaction was carried out by incubating a buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing The reaction solution was collected over time and added to 6 M guanidine hydrochloride and 1.5 M HCl.
was added to quench the disulfide bond introduction reaction. In order to identify the molecular species in the reaction mixture, the oxidized (N) and reduced (R) forms of β2m were analyzed by reverse-phase HPLC (Figs. 4A to 4C and Fig. 5). Oxidized (N) and reduced (R) molecular species were identified by MALDI-TOF/MS.

The measurement and analysis conditions for reversed-phase HPLC are shown below: The column is a 5C18-AR2 column (4.6 × 150 mm;
Nacalai tesque) was used and detected at absorbance at 229 nm. Regarding the mixing of an aqueous solution containing 0.1% trifluoroacetic acid (liquid A) and an acetonitrile solution containing 0.1% trifluoroacetic acid (liquid B), the volume increase rate of the proportion of liquid B in the mixed solution is 1% from 0 to 10 minutes. /min, 0.5% from 10 to 35 min/
It was developed with a linear concentration gradient of minutes.

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

Based on these results, the reaction rate constants were calculated assuming that the disulfide bond introduction reaction to reductively modified ^β2m was a ^pseudo -first-order reaction. 1
0 ^-2 , compared with GSH / GSSG (Comparative Example 3), LDA-SH / LDA-SS (Example 4) is 6.2 times, BDA
-SH/BDA-SS (Example 5) was shown to have about 320-fold higher ability to introduce disulfide bonds. β2m is one of the components involved in immunity, and its misfolded form is associated with the pathology of dialysis amyloidosis. The disulfide bond-introducing agent of the present invention, which is capable of rapidly introducing disulfide bonds into β2m, which is the light chain of the major histocompatibility complex, which is the basis of the immune system, is effective in the production of protein preparations. Useful.

12) RNase A activity evaluation 2
In order to evaluate the folding-promoting effect, the RNase A activity recovery assay prepared in 8) above was performed. The following combinations of 1 mM thiol compounds/0.2 mM disulfide compounds:
Comparative Example 4: GSH/GSSG
Example 6: ImdM-SH/ImdM-SS
Example 7: oPyM-SH/oPyM-SS
buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 8 µM RNase A in the presence of
was incubated with Aliquot the reaction mixture over time and extract cytidine 2′:3, which is a substrate for RNase A.
′-Cytidine 2′:3′-cyclic monophosphate (cCMP) was diluted in the same buffer to a final concentration of 4 μM RNase A, 0.4 mM cCMP. The change in absorbance at 284 nm was measured by a spectrophotometer to quantify the nucleolytic activity of RNase A folded into its native structure.

As shown in FIG. 6, the RNase A recovery activity after 6 hours of incubation was GSH/GSSG (Comparative Example 4
) showed a high recovery activity of 45% in the presence of ImdM-SH/ImdM-SS (Example 6), compared to 38%. This indicated that ImdM-SH/ImdM-SS has a folding promoting effect. In addition, oPyM-SH/oPyM-SS (Example 7) had RNase A
was higher than that of GSH/GSSG (Comparative Example 4), indicating that it had a high folding-promoting effect in the initial stage up to 3 hours.

13) RNase A activity evaluation 3
In order to evaluate the folding-promoting effect, the RNase A activity recovery assay prepared in 8) above was performed. The following combinations of 1 mM thiol compounds/0.2 mM disulfide compounds:
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
buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 8 µM RNase A in the presence of
was incubated with Aliquot the reaction mixture over time and extract cytidine 2′:3, which is a substrate for RNase A.
′-Cytidine 2′:3′-cyclic monophosphate (cCMP) was diluted in the same buffer to a final concentration of 4 μM RNase A, 0.4 mM cCMP. The change in absorbance at 284 nm was measured by a spectrophotometer to quantify the nucleolytic activity of RNase A folded into its native structure.

As shown in FIG. 7, the RNase A recovery activity after 6 hours of incubation was GSH/GSSG (Comparative Example 5
) in the presence of pPyM-SH-NMe Cl/pPyM-SS-NMe Cl (Example 8) compared to (38%)
65%, 54% in the presence of pPyM-SH-NMe.I/pPyM-SS-NMe.I (Example 9), oPyM-SH-NMe.I/oPyM
In the presence of -SS-NMe·I (Example 10), it exhibited a high recovery activity of 43%. From this, pPyM-
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) Aggregation experiment of β2m by heating (without additives)
A buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 10 μM native β2m was added at 25°C to 5
It was heated to 0°C, allowed to equilibrate for 10 minutes when it reached 50°C, and then cooled to 25°C. The absorbance at 280 nm of the supernatant of the fraction obtained after centrifugation was measured with a spectrophotometer to measure the residual rate of native β2m in the supernatant.

(with additives)
1 mM of the following thiol compounds in buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 10 μM native β2m:
Comparative Example 6; GSH
Example 11: LDA-SH
Example 12: BDA-SH
was added, heated from 25°C to 50°C, equilibrated for 10 minutes when 50°C was reached, then 25°C.
Cooled to °C. The absorbance at 280 nm of the supernatant of the fraction obtained after centrifugation was measured with a spectrophotometer to measure the residual rate of native β2m in the supernatant.

FIG. 8 shows a photograph of the solution after cooling (before centrifugation). From FIG. 8, LDA-SH (Example 11)
and BDA-SH (Example 12), compared to GSH (Comparative Example 6) with no additive, the solution was transparent, indicating that the aggregation of natural β2m was suppressed. FIG. 9 shows the results of measurement of the residual rate of native β2m in the supernatant obtained by centrifugation. From FIG. 9, LDA-SH (Example 11) and BDA-SH (Example 12) had a higher residual rate of natural β2m than GSH (Comparative Example 6) without additives. , LDA-SH and BDA-SH were found to have high ability to inhibit protein aggregation and solubilize.

15) RNase A activity evaluation 4
In order to evaluate the folding-promoting effect, the RNase A activity recovery assay prepared in 8) above was performed. The following combinations of 1 mM thiol compounds/0.2 mM disulfide compounds:
Example 13: pPyM-SH/GSSG
A buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 8 μM RNase A was incubated at 30 °C in the presence of . The reaction solution was collected over time and diluted with the same buffer containing cytidine 2′:3′-cyclic monophosphate (cCMP), which is a substrate for RNase A. The concentration was 4 µM RNase A and 0.4 mM cCMP. The change in absorbance at 284 nm was measured by a spectrophotometer to quantify the nucleolytic activity of RNase A folded into its native structure. The results are shown in FIG. 10 together with Comparative Example 4, Examples 6 and 7 of "12) RNase A activity evaluation 2".

The protein folding agent of the present invention can be used as an oxidative folding promoter for protein preparations such as antibodies having many disulfide bonds. The protein folding agent of the present invention can be used as an agent for promoting rapid oxidative folding of proteins having a single disulfide bond. In addition, 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 formula below:

[In the formula,
X ₁ , X ₂ and X ₃ are each independently an alkylene group having 1 to 10 carbon atoms which may contain 1 to 5 oxygen atoms in the molecular chain,
Y ₁ is each independently 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 are each independently 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 iodine ion]
A protein folding agent comprising, as an active ingredient, at least one selected from compounds represented by and salts and solvates thereof. 2. The protein folding agent according to claim 1, comprising as an active ingredient at least one selected from 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 compounds represented by formulas (8) to (14), and salts and solvates thereof. At least one selected from compounds represented by formulas (1) to (7), and salts and solvates thereof; and compounds represented by formulas (8) to (14), and salts and solvates thereof 2. The protein folding agent according to claim 1, comprising at least one selected from substances as an active ingredient. A step of treating the unfolded protein in the presence of at least one selected from compounds represented by formulas (1) to (14) defined in claim 1, and salts and solvates thereof methods of protein folding, including; A step of treating the unfolded protein in the presence of at least one selected from compounds represented by formulas (1) to (14) defined in claim 1, and salts and solvates thereof methods for refolding proteins, including; A protein solubilizer comprising, as an active ingredient, at least one selected from compounds represented by formulas (1) to (14) defined in claim 1, and salts and solvates thereof.

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