WO2017043653A1

WO2017043653A1 - Compound for stabilizing protein

Info

Publication number: WO2017043653A1
Application number: PCT/JP2016/076697
Authority: WO
Inventors: 哲志山口; 岡本　晃充; 英史塩田
Original assignee: 国立大学法人東京大学
Priority date: 2015-09-11
Filing date: 2016-09-09
Publication date: 2017-03-16
Also published as: JPWO2017043653A1

Abstract

The present invention pertains to a compound represented by formula (I) [in the formula, A¹ is a hydroxyl group or an active group, A² is a photodegradable group, A³ is an oxyethylene group, A⁴ is a partner group of a capture molecule, L¹ and L² are each a linker, k¹ and k² are each independently 0 or 1, and n indicates the average number of moles of the oxyethylene group added, and 50 ≤ n ≤ 450 is satisfied]. The compound can be used to stabilize a protein.

Description

Compounds for stabilizing proteins

The present invention relates to a compound for stabilizing a protein, and a modified protein obtained by reacting the compound with a protein.

In recent years, genes related to diseases and important life phenomena have been clarified one after another, and it is expected that proteins that are expression products of such genes will be used as protein drugs. However, the structure of proteins easily loosens in aqueous solution, which causes irreversible aggregation and protease degradation during the production, storage, and transportation of protein drugs. Aggregation of proteinaceous drugs has been reported to be extremely dangerous by inducing a strong biological response after administration. Moreover, degradation by protease causes a decrease in drug efficacy. Therefore, a technique for stabilizing a protein from the manufacturing process to the bedside is a technique that is eagerly desired in the field of proteinaceous pharmaceuticals.

The existing protein stabilization techniques can be roughly divided into the following three types.
(1) A technique of adding a saccharide, glycerol, or the like that stabilizes the three-dimensional structure of a protein, or an arginine hydrochloride, a surfactant, or the like that suppresses protein aggregation (Non-patent Document 1)
(2) Technology for producing a fusion protein with a highly stable protein such as an antibody Fc domain (Non-patent Documents 2 and 3)
(3) Technology for chemically modifying a hydrophilic polymer such as polyethylene glycol (hereinafter abbreviated as PEG) (Non-patent Documents 4 and 5)

The above (1) is most commonly used as an existing method, but high-concentration additives are required for protein stabilization, and toxicity or the like becomes a problem when administered directly into the body. In addition, when the protein concentration is high, there are many cases where aggregation cannot be suppressed.

In the above (2), there are problems such as a decrease in function due to fusion with other proteins, immunogenicity and undesirable activity after administration of the fusion partner protein.

In the above (3), it is known that the stabilizing effect is higher than other methods, and it can be used in combination with other methods. However, there is a problem that the function (activity) of the protein is lowered by chemically modifying a bulky polymer. In particular, when the number of modifications and the length of the polymer are increased in order to increase the stability, the activity is greatly reduced accordingly, and it is difficult to simultaneously realize high stability and high activity.

In recent years, a PEGylation method (Reversible PEGylation method) in which the modified PEG is detached from the protein surface and its activity is recovered by degradation of the linker under basic conditions has been reported (Non-cited references 6, 7).

In addition, although there is no mention of stabilization of protein pharmaceuticals, Non-cited Document 8 discloses that PEG can be modified through a photodegradable linker to control protein activity. Although the PEGylated protein is inactive, the photodegradable linker containing PEG is removed from the protein by light irradiation, and the activity of the protein is restored.

However, in the methods of Non-Patent Documents 6 and 7, it takes a long time to remove the PEG-containing site under weakly basic conditions that hardly affect the protein activity (several tens of hours to several hundreds of hours). time). Some proteins denature under basic conditions. In addition, it is desirable that the PEG-containing site can be quickly administered into the living body after removal from the proteinaceous drug, but it is not preferable to administer the basic aqueous solution directly into the living body. Therefore, there is a need for a PEGylation method that can quickly remove PEG-containing sites under conditions other than basic conditions.

In addition, after removing the PEG group-containing site from the proteinaceous drug, it is necessary to be able to quickly separate the produced PEG group-containing compound from the proteinous drug in order to be able to rapidly administer the proteinous drug into the living body. is there. The prior art does not disclose any such separation technique.

Therefore, the present invention can improve the stability of the protein, can be removed quickly under neutral conditions, does not impair the activity of the protein after being removed, and can be quickly removed after being removed. Development of a PEGylation reagent that can be separated from

As a result of intensive studies in view of the above problems, the present inventors can solve the above problems by using a compound having (1) a photodegradable group, (2) a PEG group, and (3) a capture molecule partner group. And found the present invention.

That is, this invention has the following aspects.
[1]
The following formula (I)

[Where:
A ¹ is a hydroxyl group or an active group,
A ² is a photodegradable group,
A ³ is an oxyethylene group,
A ⁴ is a partner group of the capture molecule,
L ¹ and L ² are each a linker,
k ¹ and k ² are each independently 0 or 1,
n represents the average number of moles added of the oxyethylene group and 50 ≦ n ≦ 450]
A compound represented by
[2]
A ¹ is the following substituent:

[Wherein the arrow indicates the connection to A ² ]
The compound according to [1], which is selected from the group consisting of:
[3]
A ² is a divalent group having a skeleton selected from the group consisting of a 2-nitrobenzyl skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton, and a 7-nitroindolinocarbonyl skeleton, [1] or The compound according to [2].
[4]
The combination of A ⁴ and the capture molecule is a combination of biotinyl group and streptavidin, a combination of maltosyl group and maltose binding protein, a combination of glutathion group and glutathione-S-transferase, HaloTag® ligand and HaloTag ( (Registered trademark) protein combination, guanylylmethylphenyl group and SNAP-tag (registered trademark) combination, cytosynylmethylphenyl group and CLIP-tag (registered trademark) combination, the following groups:

[Where:
The arrow indicates a connection to A ³ when k ² is 0, and a connection to L ² when k ² is 1]
And a combination of dihydrofolate reductase, a combination of Strep-tag (registered trademark) and Strep-tactin (registered trademark), a combination of an antigen and an antibody, and a combination of an azido group and dibenzocyclooctyne. The compound according to any one of [1] to [3].
[5]
The following formula (Ia)

[Where:
A ¹ , A ⁴ , and n are as defined in [1],
m ¹ , m ² and m ³ are each independently an integer of 1 to 10,
L ¹¹ is a C _1-20 alkylene group, wherein the methylene group in the C _1-20 alkylene group may be substituted with 1 to 5 oxo groups, and adjacent methylene groups are 1 1 to 10 methylene groups among the methylene groups in the alkylene group may be NH, N (C _1-10 alkyl), O, S, C _6-14 arylene, may be replaced with 5-10 membered heteroarylene]
The compound according to any one of [1] to [4], which is a compound represented by:
[6]
The following formula (II)

[Where:
A ¹ , A ² , A ³ , L ¹ , k ¹ and n are as defined in [1],
L ³ is a linker,
k ³ is 0 or 1,
B ¹ is represented by the following formula (III)

(Where
A ⁴ is as defined in [1],
L ⁴ is a linker,
k ⁴ is 0 or 1)
A substituent that can be linked to B ^{2 of the} compound represented by
A compound represented by
[7]
B ¹ and B ² are each independently an azide group or an alkynyl group, an azide group or a cyclooctynyl group, an azide group or a phosphinothioester group, a vinyl group or a thiol group, or A compound represented by the formula (II) according to [6], which is a C _1-10 alkyl group or hydrazino group substituted with an oxo group, but the two are not the same.
[8]
The following formula (II-a)

[Where:
A ¹ and n are as defined in [1],
m ⁴ , m ⁵ , m ⁶ and m ⁷ are each independently an integer of 1 to 10]
A compound represented by the formula (II) according to [6] or [7], which is a compound represented by:
[9]
The following formula (II-b)

[Where:
n represents the average number of moles added of the oxyethylene group and 50 ≦ n ≦ 450]
A compound represented by the formula (II) according to any one of [6] to [8], which is a compound represented by:
[10]
The compound represented by the formula (III) is represented by the following (III-a):

A compound represented by the formula (II) according to any one of [6] to [9], which is a compound represented by:
[11]
A method for producing a modified protein comprising reacting a protein with the compound represented by formula (I) according to any one of [1] to [5].
[12]
A method for producing a modified protein, comprising reacting a protein with a compound represented by the formula (II) according to any one of [6] to [10].
[13]
(1) preparing an intermediate of a modified protein by reacting the protein with the compound represented by the formula (II) described in any one of [6] to [10];
(2) The intermediate is represented by the formula (III)

(Where
A ⁴ , B ² , L ⁴ and k ⁴ are as defined in [6])
A method for producing a modified protein, which comprises reacting with a compound represented by the formula:
[14]
The following formula (IV):

[Where:
A ² , A ³ , A ⁴ , L ¹ , L ² , k ¹ , k ² and n are as defined in [1],
Arrow indicates linkage with hydroxyl, amino or thiol group in protein]
The modified protein which has a substituent represented by these.
[15]
The following formula (V):

[Where:
A ² , A ³ , B ¹ , L ¹ , L ³ , k ¹ , k ³ and n are as defined in [6],
Arrow indicates linkage with hydroxyl, amino or thiol group in protein]
The modified protein which has a substituent represented by these.

The stability of the protein can be improved by using the compound of the present invention. The modified protein obtained by using the compound of the present invention may have a decreased physiological activity compared to the unmodified protein, but the modified site can be removed by photoreaction, and the activity of the protein after the removal is removed. Recover. Furthermore, the modified moiety that has been removed can be separated from the protein by reacting with the capture molecule. Therefore, according to the present invention, a high concentration proteinaceous pharmaceutical can be transported and stored in a stable state, and the PEG group-containing part can be removed by a photoreaction immediately before administration to a patient, and the resulting PEG group By excluding the contained compounds, it is possible to quickly administer a high concentration proteinaceous drug in the active state to the patient.

FIG. 1 is a schematic diagram showing removal and separation of PEG-containing sites from the modified protein of the present invention. FIG. 2 is a diagram showing an analysis result by SDS-PAGE before and after removal of a PEG-containing site linked to lysozyme. FIG. 3 is a diagram showing absorption spectrum measurement results of the solution after the photoreaction and before the streptavidin treatment and the supernatant of the solution after the streptavidin treatment. FIG. 4 is a diagram showing an analysis result by SDS-PAGE of a product obtained by reacting lysozyme with a compound of the formula (X). FIG. 5 is a diagram showing the measurement results of aggregation in a solution after heating the modified lysozyme obtained by reacting the compound of formula (X). FIG. 6 is a diagram showing an analysis result by SDS-PAGE after light irradiation was performed on the modified lysozyme obtained by reacting the compound of formula (X). FIG. 7 is a diagram showing that the activity of the modified lysozyme obtained by reacting the compound of formula (X) is recovered by a photolysis reaction. FIG. 8 is a diagram showing an analysis result by SDS-PAGE of a product obtained by reacting transferrin with a compound of the formula (II-b). FIG. 9 is a graph showing the measurement results of the relative concentration of transferrin remaining in the solution after heating the modified transferrin obtained by reacting the compound of formula (II-b). FIG. 10 is a diagram showing an analysis result by SDS-PAGE after light irradiation was performed on the modified transferrin obtained by reacting the compound of formula (II-b).

In the present specification, the “C _1-10 alkyl group” is a linear or branched alkyl group having 1 to 10 carbon atoms. Examples of C _1-10 alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and isomers thereof.

In the present specification, the “C _1-40 alkylene group” means a linear or branched alkylene group having 1 to 40 carbon atoms. Specifically, for example, methylene group, ethylene group, propylene group, butylene group, pentylene group, hexylene group, heptylene group, octylene group, nonylene group, decylene group, undecylene group, dodecylene group, tridecylene group, tetradecylene group, pentadecylene. Group, hexadecylene group, heptadecylene group, octadecylene group, nonadecylene group, icosanylene group, heicosanylene group, docosanylene group, tricosanylene group, tetracosanylene group, pentacosanylene group, hexacosanylene group, heptacosanylene group, octacosanylene group, nonacosasanylene group, nonacosasanylene group, Triacontanilene, dotriacontanilene group, tritriacontanilene group, tetratriacontanilene group, pentatriacontanilene group, hexatriacontanilene group Hepta triacontyl Tani alkylene group, octamethylene triacontyl Tani alkylene group, nona triacontyl Tani alkylene group, tetra con Tani alkylene group and isomers thereof.

In the present specification, the “C _1-20 alkylene group” means a linear or branched alkylene group having 1 to 20 carbon atoms. Specifically, for example, methylene group, ethylene group, propylene group, butylene group, pentylene group, hexylene group, heptylene group, octylene group, nonylene group, decylene group, undecylene group, dodecylene group, tridecylene group, tetradecylene group, pentadecylene. Group, hexadecylene group, heptadecylene group, octadecylene group, nonadecylene group, icosanylene group and isomers thereof.

In the present specification, the “C _1-10 alkylene group” means a linear or branched alkylene group having 1 to 10 carbon atoms. Specific examples include a methylene group, ethylene group, propylene group, butylene group, pentylene group, hexylene group, heptylene group, octylene group, nonylene group, decalene group and isomers thereof.

In the present specification, the “C _6-14 aryl group” refers to an aromatic cyclic group having 6 to 14 carbon atoms, and specific examples thereof include a phenyl group, a naphthyl group, and an anthracenyl group.

In the present specification, the “C _6-14 arylene group” is a divalent group composed of an aromatic carbocyclic ring having 6 to 14 carbon atoms, and examples thereof include a phenylene group, a naphthylene group, and an anthracenylene group. .

In the present specification, the “5- to 10-membered heteroarylene group” is a divalent group consisting of a 5- to 10-membered aromatic heterocycle, and examples of the aromatic heterocycle include a pyrrole ring and an indole Ring, thiophene ring, benzothiophene ring, furan ring, benzofuran ring, pyridine ring, quinoline ring, isoquinoline ring, thiazole ring, benzothiazole ring, isothiazole ring, benzoisothiazole ring, pyrazole ring, indazole ring, oxazole ring, benzo Examples thereof include an oxazole ring, an isoxazole ring, a benzisoxazole ring, an imidazole ring, a benzimidazole ring, a triazole ring, a benzotriazole ring, a pyrimidine ring, a uridine ring, a pyrazine ring, and a pyridazine ring.

In the present specification, the “oxo group” refers to a group that forms a carbonyl group together with the carbon atom to which it is bonded.

One aspect of the present invention provides the following formula (I):

[Where:
A ¹ is a hydroxyl group or an active group,
A ² is a photodegradable group,
A ³ is an oxyethylene group,
A ⁴ is a partner group of the capture molecule,
L ¹ and L ² are each a linker,
k ¹ and k ² are each independently 0 or 1,
n represents the average number of moles added of the oxyethylene group and 50 ≦ n ≦ 450]
It is a compound represented by these.

In the present specification, the “active group” is a substituent capable of reacting with a hydroxyl group, amino group or thiol group in a protein to form a modified protein. Although it does not specifically limit as an active group, For example, the following substituents are mentioned.

[In the formula, the arrow indicates the connection with A ² . ]

In the present specification, the “photodegradable group” is a divalent group in which a bond in the photodegradable group is cleaved by a photoreaction. The photodegradable group is not particularly limited, and is, for example, a divalent group having a skeleton selected from the group consisting of a 2-nitrobenzyl skeleton, a coumarin-4-ylmethyl skeleton, and a phenylcarbonylmethyl skeleton.

In the present specification, the “divalent group having a 2-nitrobenzyl skeleton” is a divalent group having the following structure or a derivative structure thereof.

[Wherein L ⁵ is a C _1-10 alkylene group or is not present, wherein the carbon atom in the alkylene group may be substituted with 1 to 5 oxo groups, The atoms may be connected by 1 to 5 unsaturated bonds, and 1 to 4 carbon atoms in the alkylene group may be NH, N (C _1-10 alkyl), O Or it may be replaced by S. ]

As the divalent group having a 2-nitrobenzyl skeleton, the following are preferable.

[In the formula, the left arrow indicates the connection to A ¹ , the right arrow indicates the connection to A ³ when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. Indicates concatenation. ]

In the present specification, the “divalent group having a coumarin-4-ylmethyl skeleton” is a divalent group having the following structure or a derivative structure thereof.

[In the formula, the left arrow indicates the connection to A ¹ , the right arrow indicates the connection to A ³ when k ¹ is 0, and the connection to L ¹ when k ¹ is 1. L ⁶ is a C _1-10 alkylene group or absent, wherein the carbon atom in said alkylene group may be substituted with 1 to 5 oxo groups, and adjacent carbon The atoms may be connected by 1 to 5 unsaturated bonds, and 1 to 4 carbon atoms in the alkylene group may be NH, N (C _1-10 alkyl), O Or it may be replaced by S. ]

As the divalent group having a coumarin-4-ylmethyl skeleton, the following are preferable.

In the present specification, the “divalent group having a phenylcarbonylmethyl skeleton” is a divalent group having the following structure or a derivative structure thereof.

The following are preferable as the divalent group having a phenylcarbonylmethyl skeleton.

In the present specification, the “divalent group having a 7-nitroindolinocarbonyl skeleton” is a divalent group having the following structure or a derivative structure thereof.

As the divalent group having a 7-nitroindolinocarbonyl skeleton, the following are preferable.

In the present specification, the “oxyethylene group” means a — (CH ₂ CH ₂ O) — group or a — (OCH ₂ CH ₂ ) — group. A — (CH ₂ CH ₂ O) _n — group and a — (OCH ₂ CH ₂ ) _n — group (where n represents the average number of moles of oxyethylene group added and 50 ≦ n ≦ 450). In the present specification, it is also referred to as “polyoxyethylene group” or “PEG group”. n is preferably 50 ≦ n ≦ 400, and more preferably 50 ≦ n ≦ 350.

In the present specification, “capture molecule” refers to a molecule that captures a PEG group-containing compound removed from a modified protein by a photoreaction and allows separation of the compound from an unmodified protein. The capture molecule may be used by being immobilized on a carrier (for example, a bead or a filter).

In the present specification, the “partner group of the capture molecule” is a group to which the capture molecule is bound covalently or non-covalently.

The combination of the partner group and the capture molecule is not particularly limited, but can be linked by, for example, a combination of a substituent that can be used as a protein tag and a protein that specifically binds to the tag, and a Huisgen cyclization reaction. And a combination of a substituent and a compound. Preferably, a combination of biotinyl group and streptavidin, a combination of maltosyl group and maltose binding protein, a combination of glutathionyl group and glutathione-S-transferase, a combination of HaloTag® ligand and HaloTag® protein , A combination of guanylylmethylphenyl group and SNAP-tag (registered trademark), a combination of cytosynylmethylphenyl group and CLIP-tag (registered trademark),
The following groups:

[Where:
The arrow indicates a connection to A ³ when k ² is 0, and a connection to L ² when k ² is 1]
And a combination of dihydrofolate reductase, a combination of Strep-tag (registered trademark) and Strep-tactin (registered trademark), a combination of an antigen and an antibody, and a combination of an azido group and dibenzocyclooctyne.

In the present specification, the “linker” is a divalent group for connecting two functional groups. Although it does not specifically limit as a linker, For example, it is a _C1-40 alkylene group. Here, the methylene group in the C _1-40 alkylene group may be substituted with 1 to 10 oxo groups, and adjacent methylene groups may be connected with 1 to 10 unsaturated bonds. In the alkylene group, 1 to 20 methylene groups are replaced by NH, N (C _1-10 alkyl), O, S, C _6-14 arylene, 5-10 membered heteroarylene. It may be done.

As compounds of formula (I), the following formula (Ia):

[Where:
A ¹ , A ⁴ , and n are as defined above;
m ¹ , m ² and m ³ are each independently an integer of 1 to 10,
L ¹¹ is a C _1-20 alkylene group, wherein the methylene group in the C _1-20 alkylene group may be substituted with 1 to 5 oxo groups, and adjacent methylene groups are 1 1 to 10 methylene groups among the methylene groups in the alkylene group may be NH, N (C _1-10 alkyl), O, S, C _6-14 arylene, may be replaced with 5-10 membered heteroarylene]
The compound represented by these is preferable.

As compounds of formula (I), the following formula (Ib):

[Where:
n represents the average number of moles of oxyethylene group added and 150 ≦ n ≦ 450]
Is particularly preferred.

One aspect of the present invention is the following formula (II)

[Where:
A ¹ , A ² , A ³ , L ¹ , k ¹ and n are as defined in the above formula (I),
L ³ is a linker,
k ³ is 0 or 1,
B ¹ is represented by the following formula (III)

(Where
A ⁴ is as defined in the above formula (I),
L ⁴ is a linker,
k ⁴ is 0 or 1)
A substituent that can be linked to B ^{2 of the} compound represented by
To a compound of formula (II)

B ¹ and B ² are not particularly limited as long as both can be connected. For example, it may be a combination of substituents that can be condensed with each other (for example, a carboxyl group and an amino group), or a combination of substituents that are linked by a Huisgen cyclization reaction (for example, an azide group and an alkynyl group). There may be. B ¹ and B ² are preferably independently of each other an azide group or an alkynyl group, an azide group or a cyclooctynyl group, an azide group or a phosphinothioester group, a vinyl group or a thiol group. Or an alkyl group substituted with an oxo group or a hydrazino group provided that they are not the same.

As a compound of formula (II), the following formula (II-a):

[Where:
A ¹ and n are as defined in the above formula (I),
m ⁴ , m ⁵ , m ⁶ and m ⁷ are each independently an integer of 1 to 10]
The compound represented by these is preferable.

As a compound of formula (II), the following formula (II-b):

[Where:
n represents the average number of moles added of the oxyethylene group and 50 ≦ n ≦ 450]
The compound represented by these is more preferable.

As a compound of formula (III), the following (III-a):

The compound represented by these is preferable.

The compounds represented by formulas (I) and (II) are also referred to as “compounds of the invention” in the present specification.

The compounds represented by the formulas (I) to (III) can be synthesized by adapting various known synthesis methods using characteristics based on the basic skeleton or the type of substituent. Although the typical manufacturing method is illustrated below, it is not limited only to the method as described below. Depending on the type of functional group, it may be effective in terms of production technology to change the functional group to a suitable protecting group at the raw material or intermediate stage, that is, a group that can be easily converted to the functional group. Yes, the protecting group can be removed as necessary to obtain the desired compound. Examples of such functional groups include a hydroxyl group, a carboxyl group, an amino group, and the like, and examples of the protecting group include a protective group by Greene and Wutt, a group group, a group group, an organic group, a synthesis group, a third edition “ Protective groups described in "Protective" Groups "in" Organic "Synthesis" (third'edition) "can be given, and these may be used as appropriate according to the reaction conditions. The starting materials and the reaction reagents are known compounds, or can be easily produced from known compounds according to methods well known in the field of organic chemistry.

Compounds of formula (Ia) included in formula (I) can be prepared as outlined in Scheme 1 below. The symbols in the following scheme are the same as those described above.

Process 1
Compound 3, which is an N-hydroxysuccinimide ester, can be reacted with compound 2 to produce compound 3. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, diethyl ether, tetrahydrofuran, 1,4-dioxane, benzene, toluene, xylene, hexane, cyclohexane, N, N-dimethyl Examples include formamide, ethyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, dimethyl sulfoxide, acetone, acetonitrile, or a mixed solvent thereof. N, N-dimethylformamide, tetrahydrofuran and acetonitrile are preferred. While the reaction temperature in this step varies depending on the raw materials and solvent to be used, it is generally -78 to 200 ° C, preferably 0 to 100 ° C, and the reaction time is usually 1 minute to 7 days, preferably 5 minutes to 72 hours.

This step can also be performed using an activated ester obtained by using another activator instead of the N-hydroxysuccinimide ester. Such activators are not particularly limited, but include, for example, N-hydroxysulfosuccinimide ester, 4-nitrophenyl ester, 1-hydroxybenzotriazole ester and 1-hydroxy-7-azabenzotriazole ester, and N-carbonylimidazo -There are examples.

Process 2
Compound 5 can be produced by reacting compound 3 with compound 4 in the presence of a condensing agent. The condensing agent used in this step is not particularly limited. For example, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, benzotriazol-1-yloxy-trisdimethylamino Phosphonium salt, hexafluorophosphoric acid (benzotriazol-1-yloxy) tripyrrolidinophosphonium, 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride, etc. Is mentioned. 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is preferred. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, diethyl ether, tetrahydrofuran, 1,4-dioxane, benzene, toluene, xylene, hexane, cyclohexane, N, N-dimethyl Examples include formamide, ethyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, dimethyl sulfoxide, acetone, acetonitrile, or a mixed solvent thereof. Tetrahydrofuran or dichloromethane is preferred. While the reaction temperature in this step varies depending on the raw materials and solvent to be used, it is generally -78 to 200 ° C, preferably 0 to 100 ° C, and the reaction time is usually 1 minute to 7 days, preferably 5 minutes to 72 hours.

Process 3
A compound of formula (Ia) can be prepared by converting the hydroxyl group of compound 5 to an active group A ¹ . For example, when A ¹ is a 4-nitrophenyloxycarbonyloxy group, 4-nitrophenyl chloroformate is reacted with the hydroxyl group of compound 5. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, diethyl ether, tetrahydrofuran, 1,4-dioxane, benzene, toluene, xylene, hexane, cyclohexane, N, N-dimethyl Examples include formamide, ethyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, dimethyl sulfoxide, acetone, acetonitrile, or a mixed solvent thereof. N, N-dimethylformamide, tetrahydrofuran and acetonitrile are preferred. While the reaction temperature in this step varies depending on the raw materials and solvent to be used, it is generally -78 to 200 ° C, preferably 0 to 100 ° C, and the reaction time is usually 1 minute to 7 days, preferably 5 minutes to 72 hours.

Compounds of formula (II-a) included in formula (II) can be prepared as outlined in Scheme 2 below. The symbols in the following scheme are the same as those described above.

Process 4
Compound 7 can be produced by reacting the carboxyl group of Compound 3 obtained in Scheme 1 above with Compound 6 in the presence of a condensing agent. This step can be performed in the same manner as the above step 2.

Process 5
A compound of formula (II-a) can be prepared by converting the hydroxyl group of compound 7 to the active group A ¹ . For example, when A ¹ is a 4-nitrophenyloxycarbonyloxy group, 4-nitrophenyl chloroformate is reacted with the hydroxyl group of compound 7. This step can be performed in the same manner as the above step 3.

The compound represented by the formula (III) used in the present invention may be commercially available or may be prepared according to a known method.

In each reaction in the present specification, the reaction involving heating can be performed using a water bath, an oil bath, a sand bath, or a microwave, as will be apparent to those skilled in the art.

In each reaction in the present specification, a solid-phase-supported reagent supported on a polymer (eg, polystyrene, polyacrylamide, polypropylene, polyethylene glycol, etc.) may be used as appropriate.

The compounds and intermediates obtained as described above are isolated and purified by ordinary organic synthesis operations such as extraction, crystallization, recrystallization, dialysis, and various chromatography.

One embodiment of the present invention relates to a modified protein obtained by reacting a compound represented by the above formula (I) with a protein.

Further, one embodiment of the present invention relates to a method for producing a modified protein, which comprises reacting a protein with a compound represented by the above formula (I).

When A ^{1 of the} compound represented by the formula (I) is an active group, the active group reacts with a hydroxyl group, amino group or thiol group in the protein to form a modified protein. The solvent used in the reaction is not particularly limited as long as it does not inhibit the reaction. For example, water, buffer, N, N-dimethylformamide, dimethyl sulfoxide, acetonitrile, or a mixed solvent thereof can be used. Can be mentioned. A buffer having a pH of about 8 is preferable (for example, sodium borate buffer (pH 8.3)). While the reaction temperature in this reaction varies depending on the raw materials and solvent to be used, it is generally −78 to 100 ° C., preferably 0 to 40 ° C., and the reaction time is usually 1 minute to 7 days, preferably 5 minutes to 72 hours.

When A ^{1 of the} compound represented by the above formula (I) is a hydroxyl group, the hydroxyl group may be converted into an active group and then reacted with a hydroxyl group, amino group or thiol group in protein. This embodiment is included in “reacting a protein with a compound represented by the above formula (I)”.

The modified protein obtained by reacting the compound represented by the above formula (I) with a protein is separated and purified using dialysis, gel filtration chromatography, affinity chromatography, or the like.

One embodiment of the present invention relates to a modified protein obtained by reacting a compound represented by the above formula (II) with a protein.

Further, one embodiment of the present invention relates to a method for producing a modified protein, which comprises reacting a protein with a compound represented by the above formula (II).

When A ^{1 of the} compound represented by the formula (II) is an active group, the active group reacts with a hydroxyl group, amino group or thiol group in the protein to form a modified protein. The solvent used in the reaction is not particularly limited as long as it does not inhibit the reaction. For example, water, buffer, N, N-dimethylformamide, dimethyl sulfoxide, acetonitrile, or a mixed solvent thereof can be used. Can be mentioned. A buffer having a pH of about 8 is preferable (for example, sodium borate buffer (pH 8.3)). While the reaction temperature in this reaction varies depending on the raw materials and solvent to be used, it is generally −78 to 100 ° C., preferably 0 to 40 ° C., and the reaction time is usually 1 minute to 7 days, preferably 5 minutes to 72 hours.

When A ^{1 of the} compound represented by the above formula (II) is a hydroxyl group, the hydroxyl group may be converted into an active group and then reacted with a hydroxyl group, amino group or thiol group in protein. This embodiment is included in “reacting the protein with the compound represented by the above formula (II)”.

The modified protein obtained by reacting the compound represented by the above formula (II) with a protein is separated and purified using dialysis, gel filtration chromatography, affinity chromatography, or the like.

One embodiment of the present invention is an intermediate B ¹ of a modified protein obtained by reacting a compound represented by the above formula (II) with a protein, and B ^{2 of} a compound represented by the above formula (III). The present invention relates to a modified protein obtained by linking.

Another embodiment of the present invention is: (1) preparing an intermediate of a modified protein by reacting a protein with a compound represented by the above formula (II); (2) converting the intermediate into the above formula (III) It is related with the manufacturing method of a modified protein including reacting with the compound represented by this.

The connection condition between B ¹ and B ² varies depending on the combination. For example, when B ¹ is an alkynyl group and B ² is an azide group, the solvent used for the ligation reaction is not particularly limited as long as it does not inhibit the reaction. For example, water, buffer solution, N , N-dimethylformamide, dimethyl sulfoxide, acetonitrile, or a mixed solvent thereof. A buffer having a pH of 7 to 8 (for example, phosphate buffered saline (PBS)) is preferable. While the reaction temperature in this reaction varies depending on the raw materials and solvent to be used, it is generally −78 to 100 ° C., preferably 0 to 40 ° C., and the reaction time is usually 1 minute to 7 days, preferably 5 minutes to 72 hours. The resulting modified protein is separated and purified using dialysis, gel filtration chromatography, affinity chromatography, and the like.

One aspect of the present invention is the following formula (IV):

[Where:
A ² , A ³ , A ⁴ , L ¹ , L ² , k ¹ , k ² and n are as defined in the above formula (I),
Arrow indicates linkage with hydroxyl, amino or thiol group in protein]
The present invention relates to a modified protein having a substituent represented by: The modified protein can be obtained, for example, by reacting the protein with a compound represented by the above formula (I). Alternatively, it can be obtained by reacting a protein with a compound represented by the above formula (II) to prepare an intermediate of a modified protein and reacting the intermediate with a compound represented by the above formula (III). .

The modified protein having a substituent represented by the formula (IV) is preferably the following formula (IV-a):

[Where:
A ⁴ , n, m ¹ , m ² , m ³ and L ¹ are as defined in the above formula (Ia),
Arrow indicates linkage with hydroxyl, amino or thiol group in protein]
A modified protein having a substituent represented by:

As the modified protein having a substituent represented by the formula (IV), more preferably the following formula (IV-b):

[Where:
n represents the average number of added moles of oxyethylene groups, and 150 ≦ n ≦ 450,
Arrow indicates linkage with hydroxyl, amino or thiol group in protein]
A modified protein having a substituent represented by:

One embodiment of the present invention is the following formula (V):

[Where:
A ² , A ³ , B ¹ , L ¹ , L ³ , k ¹ , k ³ and n are as defined in the above formula (II),
Arrow indicates linkage with hydroxyl, amino or thiol group in protein]
The present invention relates to a modified protein having a substituent represented by: The modified protein can be obtained, for example, by reacting the protein with a compound represented by the above formula (II).

The modified protein having a substituent represented by the formula (V) is preferably the following formula (Va):

[Where:
n, m ⁴ , m ⁵ , m ⁶ and m ⁷ are as defined in the above formula (II-a);
Arrow indicates linkage with hydroxyl, amino or thiol group in protein]
A modified protein having a substituent represented by:

The modified protein having a substituent represented by the formula (V) is more preferably the following formula (Vb):

A modified protein obtained by reacting a protein with the compound represented by the above formula (I), an intermediate of the modified protein obtained by reacting the protein with a compound represented by the above formula (II), and the intermediate In the present specification, a modified protein obtained by reacting a compound represented by the above formula (III) with a substituent represented by the above formula (IV) is referred to as “modified protein of the present invention”. Also called.

In the modified protein of the present invention, the protein to be modified is not particularly limited, and examples thereof include protein drugs. Examples of protein pharmaceuticals include lysozyme, transferrin, insulin, interferon, erythropoietin, hormone, antibody and the like. The modified protein of the present invention that has been modified at the PEG-containing site has extremely high stability, but may have reduced physiological activity compared to the unmodified protein.

By irradiating the modified protein of the present invention with light, a photodegradation reaction occurs at A ² , thereby removing the PEG-containing site from the protein, resulting in a PEG group-containing compound. After the PEG group-containing site is removed, the protein recovers its original physiological activity.

The wavelength of the light to be irradiated may be determined according to the kind of the photodegradable group in the compound of the present invention. Usually, light having a wavelength in the range of 280 to 500 nm, preferably in the vicinity of 350 to 450 nm is irradiated. Light sources such as sunlight, electric light such as mercury lamps, laser light (semiconductor lasers, solid state lasers, gas lasers), light emission of light emitting diodes, light emission of electroluminescent elements can be used. The reaction temperature is not particularly limited, but is usually −78 to 200 ° C., preferably 0 to 100 ° C. The light irradiation energy is usually 0.5 to 100 J / cm ² , and preferably 1 to 10 J / cm ² . The solvent used in the photolysis reaction is not particularly limited as long as it does not inhibit the reaction. For example, water, buffer solution, N, N-dimethylformamide, dimethyl sulfoxide, acetonitrile, or a mixed solvent thereof, etc. Is mentioned. A buffer having a pH of about 7-8 (eg Dulbecco's phosphate buffer) is preferred. The light irradiation time may vary depending on the performance of the light irradiation device, the type of the photodegradable group in the compound of the present invention, the amount of the modified protein of the present invention, and the like. It is performed for ˜12 hours, preferably 30 seconds to 5 hours.

The PEG group-containing compound produced by the photoreaction can be captured by a capture molecule. Thereby, separation of the protein and the PEG group-containing compound becomes possible.

For example, by suspending a capture molecule-immobilized magnetic bead in a mixed solution of a PEG group-containing compound and unmodified protein removed by light irradiation, the partner group present at the end of the PEG group-containing compound is captured by the capture molecule. To do. Thereafter, the magnetic beads are collected on the bottom surface of the container using a magnet, and only the supernatant is collected, whereby the PEG group-containing compound and the protein can be separated.

In addition, for example, the capture molecule-immobilized gel is suspended in a mixed solution of the PEG group-containing compound and unmodified protein removed by light irradiation, and the partner group present at the end of the PEG group-containing compound is captured by the capture molecule. To do. Thereafter, the PEG group-containing compound and the protein can be separated by collecting the gel on the bottom of the container by centrifugation and collecting only the supernatant.

The present invention will be described in more detail with reference to the following examples and reference examples, but it goes without saying that the scope of the present invention is not limited by these examples.

Example 1
Production of compound of formula (II-b) (1) Production of compound of formula (iii)

Compound (i) in a two-necked eggplant flask (25 mL) (produced according to 60.0 mg, 307.6 mmol, 1.0 eq, ED Funder, et al., J. Org. Chem., 77, 3134-3142 (2012)) And dissolved in 2 mL of dry CH ₂ Cl ₂ . Therefore, N-Boc-ethylenediamine of formula (ii) (150 mg, 922.8 μmol, manufactured by Sigma-Aldrich) was added dropwise, and the mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The formation of the target compound was confirmed by TLC (DCM: MeOH = 20: 1, Rf = 0.5, colored by ninhydrin), and the solvent was distilled off by an evaporator, followed by purification by silica gel column chromatography. When the solvent was distilled off under reduced pressure with an evaporator, a colorless transparent oily compound of formula (iii) was obtained. Yield 74 mg, 99% yield.
1H-NMR (600 MHz, CD3OD, TMS): δ 3.27 (t, 2H), 3.17 (t, 2H), 2.49 (t, 2H), 2.41 (t, 2H), 2.29 (s, 1H), 1.46 ( s, 9H).

(2) Production of compound of formula (iv)

30 mL of 4 N HCl / AcOEt was added to the 50 mL eggplant flask containing the compound of formula (iii) (74 mg) prepared above, and the mixture was stirred at room temperature for 30 minutes. After confirming the disappearance of compound (iii) by TLC, the solvent was distilled off under reduced pressure and dried under vacuum overnight to obtain a colorless transparent oily compound of formula (iv). Yield 55.6 mg, 100% yield.
1H-NMR (600 MHz, CD3OD, TMS): δ 3.27 (t, 2H), 3.17 (t, 2H), 2.49 (t, 2H), 2.41 (t, 2H), 2.29 (s, 1H), 1.46 ( s, 9H).

(3) Production of compound of formula (vii)

[Wherein n is about 103. ]

Take PA-050HC of formula (v) (average molecular weight: 5000) (200 mg, 40 μmol, NOF Corporation) in a 25 mL two-necked eggplant flask, and dry CH ₂ Cl ₂ (3.0 mL) at room temperature under a nitrogen atmosphere. Dissolved in. At the same time, take the compound of formula (vi) (79.27 mg, 200 μmol, manufactured according to S. Takamori, et al., Chem. Commun., 49, 3013-3015 (2013)) in a 25 mL two-necked eggplant flask and add nitrogen. Dissolved in dry CH ₂ Cl ₂ (2.0 mL) at room temperature under atmosphere. The whole amount was added to a flask containing PA-050HC and stirred at room temperature. Et ₃ N (about 100 μL, excess amount) that had been distilled in advance was added thereto. After stirring for 24 hours, the disappearance of the raw materials was confirmed by TLC (coloration with ninhydrin), and the entire solution was dropped into 30 mL of ether, stirred well with a vortex mixer and then allowed to stand at -80 ° C for 10 minutes. I put it. This solution was centrifuged at 15000 G for 10 minutes, and the supernatant was quickly removed by decantation. Then, it dried under vacuum, diethyl ether was removed completely, about 6 mL of Tris / HCl buffer (pH 8.0) was added there, and it dialyzed using the dialysis membrane of the molecular weight fraction 3500. The solution after dialysis was freeze-dried to obtain a yellow solid. Yield: 199 mg.
1H-NMR (600 MHz, CDCl3, TMS): δ 7.57 (s, 1H), 7.33 (s, 1H), 6.42 (brs, 1H), 5.55 (q, 1H), 4.11 (t, 2H), 3.98 ( s, 3H), 3.50-3.80 (brm), 3.37 (q, 2H), 2.38 (t, 2H), 2.29 (s, 2H), 2.19 (quin, 2H), 1.76 (quin, 2H), 1.61 (m , 4H), 1.54 (d, 3H), 1.39 (m, 2H).

(4) Production of compound of formula (viii)

[Wherein n is about 103. ]

Compound (vii) (150 mg, 30 μmol), Compound (iv) (16.8 mg, 120 μmol), N-hydroxysuccinimide (12 mg, 104.3 μmol), N, N'- Dicyclohexylcarbodiimide (24 mg, 116.3 μmol) was taken and dissolved in dry DMF (3.0 mL) at room temperature under a nitrogen atmosphere. Triethylamine (about 100 μL, excess) previously distilled was added thereto. After stirring at 50 ° C. for 12 hours, the entire solution was dropped into 30 mL of ether, stirred well with a vortex mixer, and then allowed to stand at −80 ° C. for 10 minutes. This solution was centrifuged at 15000 G for 10 minutes, and the supernatant was quickly removed by decantation. Thereafter, the tube was dried under vacuum, diethyl ether was completely removed, dissolved in a small amount of milliQ, a precipitate was removed by applying a cosmony filter (aqueous system), and dialysis was performed using a dialysis membrane having a molecular weight fraction of 3500. The solution after dialysis was freeze-dried to obtain a yellow solid. Yield: 140 mg.
1H-NMR (600 MHz, CDCl3, TMS): δ 7.57 (s, 1H), 7.34 (s, 1H), 6.75 (brs, 1H), 6.56 (brs, 1H), 6.52 (brs, 1H), 5.55 ( q, 1H), 4.11 (t, 2H), 3.98 (s, 3H), 3.50-3.80 (brm), 3.37 (m, 2H), 2.52 (t, 2H), 2.41 (m, 4H), 2.19 (quin , 2H), 2.00 (s, 1H), 1.77 (quin, 2H), 1.63 (m, 4H), 1.54 (d, 3H), 1.39 (m, 2H).

(5) Production of compound of formula (II-b)

[Wherein n is about 103. ]

Compounds of formula (viii) in 10 mL two-neck eggplant-shaped flask, protected from light with aluminum foil (32 mg, 6.0 μmol) takes, under a nitrogen atmosphere, was dissolved in dry CH _₂ Cl ₂ (1.0 mL) at room temperature. At the same time, 4-nitrophenyl chloroformate (20 mg, 99.2 μmol) was placed in a 25 mL two-necked eggplant flask and dissolved in dry CH ₂ Cl ₂ (1.0 mL) at room temperature under a nitrogen atmosphere. The whole amount of this was added to a flask containing the compound of formula (viii) and stirred at room temperature. Triethylamine (about 100 μL, excess amount) previously distilled was added thereto. After stirring overnight, the solvent and triethylamine were distilled off under reduced pressure under a nitrogen atmosphere, and again dissolved in dry CH ₂ Cl ₂ , and then the compound of formula (II-b) was obtained by ether precipitation.
1H-NMR (600 MHz, CDCl3, TMS): δ8.25 (d, 2H), 7.61 (s, 1H), 7.38 (d, 2H), 7.10 (s, 1H), 6.75 (brs, 1H), 6.54 (brs, 2H), 4.14 (t, 2H), 4.00 (s, 3H), 3.50-3.80 (brm), 3.37 (m, 2H), 2.52 (t, 2H), 2.42 (t, 2H), 2.39 ( t, 2H), 2.20 (m, 2H), 2.00 (s, 1H), 1.78 (m, 5H), 1.63 (m, 4H), 1.39 (m, 2H).

Example 2
Chemical modification to lysozyme using the compound of formula (II-b) and subsequent biotinyl group modification From lysozyme powder (from chicken egg white, manufactured by Wako Pure Chemical Industries), lysozyme aqueous solution (3.21 mg / mL, 0.1 M boric acid Sodium buffer, pH 8.3) was prepared. Next, the compound of the formula (II-b) that was 25 equivalents with respect to lysozyme was mixed and allowed to stand overnight at room temperature. The modification was confirmed by SDS-PAGE in the Crude state, and then dialyzed in PBS using a dialysis cup having a molecular weight fraction of 12000. An aqueous solution (100 μL, 112.4 mM) of Azide-PEG3-biotin (manufactured by Sigma-Aldrich) and click reaction to a lysozyme aqueous solution (313 μL, approximately 3.2 mg / mL) modified with this PEG group-containing substituent A catalyst solution (80 μL) was added, stirred by pipetting, and allowed to stand overnight at room temperature. Then, it dialyzed using the dialysis cup of a 12000 molecular weight fraction, the density | concentration was determined by BCA assay, and was 3.22 mg / ml. The click reaction catalyst solution was prepared by dissolving 9.6 mg CuSO ₄ , 60 mg L-ascorbic acid in PBS (2 mL).

Example 3
Removal of PEG group-containing site and separation from protein The aqueous solution of lysozyme substituted with a group containing a biotinyl group and a PEG group (hereinafter also simply referred to as “modified lysozyme of Example 2”) obtained in Example 2 was used. It adjusted to 0.5 mg / mL, 40 microliters was taken to the microtube, and 360-nm ultraviolet light was irradiated to 32 J / cm < ² > using the light irradiation machine (MAX-302, the Asahi Spectroscope company). Irradiation was performed for 270 minutes (2 mW / cm ² ). On the other hand, streptavidin (SA) modified magnetic beads (MyOne (trademark) Streptavidin C1, manufactured by Life technologies) were suspended, and 1 mL (10 mg) was taken in another microtube. Using a neodymium magnet, only the supernatant of the SA-modified bead suspension was discarded, PBS was added for resuspension, and only the supernatant was discarded again 10 times (bead washing). Only the beads were collected on the bottom of the tube, and 20 μL of a light-irradiated PEGylated lysozyme aqueous solution was added and suspended therein, and then allowed to stand for 30 minutes. Using a neodymium magnet, only the supernatant was collected without sucking the beads. The analysis results by SDS-PAGE before and after removal of the PEG-containing site linked to lysozyme are shown in FIG. FIG. 2 shows that the PEG-containing site is removed by light irradiation, and that the PEG-containing site is separated from the protein by using streptavidin-modified beads, thereby obtaining highly pure lysozyme. Moreover, the absorption spectrum of the supernatant obtained after the light irradiation and before the streptavidin treatment and the supernatant obtained after the streptavidin treatment was measured. The results are shown in FIG. In the solution before the treatment, absorption derived from the removed residue was observed, but in the supernatant absorption spectrum, no absorption derived from the residue was observed, which coincided with the absorption spectrum of unmodified lysozyme.

Reference example 1
Production of compounds of formula (X)

[Wherein n is about 117. ]

(1) Production of compound of formula (x-ii)

[Wherein n is about 117. ]

25 mL two-necked eggplant flask MEPA-50H protected from light with aluminum foil (average molecular weight 5000) (0.99 mg, 30 [mu] mol, manufactured by NOF CORPORATION) takes, at room temperature under a nitrogen atmosphere in dry CH _₂ Cl ₂ (1.0 mL) Melted. At the same time, compound of formula (vi) in a 25 mL two-necked eggplant flask (produced according to 35.7 mg, 90 μmol, 3 eq., S. Takamori, et al., Chem. Commun., 49, 3013-3015 (2013)) Was dissolved in dry CH ₂ Cl ₂ (1.0 mL) at room temperature under a nitrogen atmosphere. The whole amount was added to a flask containing MEPA-50H and stirred at room temperature. Thereto was added Et ₃ N (about 50 μL, excess) that had been distilled in advance. After stirring for 24 hours, the disappearance of the raw materials was confirmed by TLC (coloration with ninhydrin), and the whole solution was dropped into 30 mL of ether, and the mixture was thoroughly stirred with a vortex mixer, and then at -80 ° C. Let stand for 10 minutes. This was centrifuged at 10 krpm for 10 minutes, and the supernatant was quickly removed by decantation. Further, the same amount of ether was added and the same operation was performed. Then, dry under vacuum to completely remove diethyl ether, add about 6 mL of Tris / HCl buffer (pH 8.0), centrifuge at 3.5 krpm for 5 minutes, and then use a dialysis membrane with a molecular weight fraction of 3500. Dialysis was performed. The solution after dialysis was freeze-dried to obtain a yellow powder. Yield: 150.5 mg.
¹ H-NMR (600 MHz, CD ₃ OD, TMS): δ 7.57 (s, 1H), 7.35 (s, 1H), 6.48 (bs, 1H), 5.55 (q, 1H), 4.11 (t, 2H) , 3.98 (s, 3H), 3.50-3.80 (brm), 3.39 (m, 5H), 2.39 (t, 2H), 2.19 (quin, 2H), 1.77 (quin, 2H), 1.53 (d, 3H).

(2) Production of compound of formula (X)

[Wherein n is about 117. ]

3 (30 mg, 6.0 μmol) was placed in a 10 mL two-necked eggplant flask protected from light with aluminum foil, and dissolved in dry CH ₂ Cl ₂ (1.0 mL) at room temperature under a nitrogen atmosphere. At the same time, 4-nitrophenyl chloroformate (48.4 mg, 240 μmol, 40 eq.) Was placed in a 25 mL two-necked eggplant flask and dissolved in dry CH ₂ Cl ₂ (1.0 mL) at room temperature under a nitrogen atmosphere. The whole amount was added to the flask containing the above, and stirred at room temperature. Et3N (about 50 μL, excess amount) previously distilled was added thereto. After 20 hours, the reaction was stopped by distilling off the solvent under reduced pressure using a vacuum pump. Dry CH ₂ Cl ₂ (3.0 mL) was added again to dissolve the product, and the entire solution was dropped into 80 mL of ether, and the mixture was sufficiently stirred with a vortex mixer and allowed to stand at −80 ° C. for 10 minutes. This was centrifuged at 10 krpm for 10 minutes, and the supernatant was quickly removed by decantation. Further, the same amount of ether was added and the same operation was performed. Then, it dried under vacuum and obtained white powder.
¹ H-NMR (600 MHz, CD ₃ OD, TMS): δ8.25 (d, 2H), 7.61 (s, 1H), 7.38 (d, 2H), 7.10 (s, 1H), 6.60 (brs, 1H ), 6.54 (m, 1H), 4.14 (t, 2H), 4.00 (s, 3H), 3.50-3.80 (brm), 3.39 (m, 5H), 2.39 (t, 2H), 2.19 (quin, 2H) , 1.80 (m, 5H).

Reference example 2
Chemical modification to lysozyme using compound of formula (X) Lysozyme powder (from chicken egg white, manufactured by Wako Pure Chemical Industries) is dissolved in sodium borate buffer (0.1 M, pH 8.3), and 450 μM lysozyme aqueous solution is dissolved. Prepared. Next, the compounds of the formula (X) corresponding to 1 equivalent, 5 equivalents, 10 equivalents or 25 equivalents were each taken in tubes. An aqueous lysozyme solution was added to this tube and pipetted well. The mixture was allowed to stand overnight at room temperature, and dialyzed in a phosphate buffer solution (PBS) under physiological conditions using a dialysis cup having a molecular weight fraction of 12000. The products obtained by reacting the compound of the formula (X) corresponding to 1 equivalent, 5 equivalents, 10 equivalents and 25 equivalents with lysozyme are respectively referred to as “modified lysozyme (1 eq)” and “modified lysozyme (5 eq)”, respectively. ”,“ Modified lysozyme (10 eq) ”, and“ modified lysozyme (25 eq) ”. The amount of PEG modification of each modified lysozyme was confirmed using SDS-PAGE. The analysis result by SDS-PAGE is shown in FIG.

Increasing the amount of the compound of formula (X) also increased the amount of PEG molecule modification to lysozyme. In the modified lysozyme (25 eq), almost no unmodified lysozyme was observed, and 1 to 7 PEG molecules were modified per lysozyme molecule.

Reference example 3
Heat resistance test of the modified lysozyme of Reference Example 2 A PBS solution of 1.0 mg / mL unmodified lysozyme and the modified lysozyme of Reference Example 2 was prepared, and 120 μL each was placed in a quartz cell and capped. A quartz cell was set in the measurement part of an absorption spectrometer (UV-2550, manufactured by Shimadzu Corporation), and turbidity was measured at an absorption wavelength of 600 nm. The temperature for denaturation aggregation was increased from 50 ° C. to 100 ° C. at a temperature increase rate of 0.1 ° C./min. The results are shown in FIG.

Unmodified lysozyme began to aggregate near 70 ° C., and turbidity increased. On the other hand, the increase in turbidity was suppressed by the modification of PEG to lysozyme. In particular, in the modified lysozyme (25 eq), no increase in turbidity was observed even when the temperature exceeded 90 ° C.

Reference example 4
Removal of the PEG group-containing moiety Take the modified lysozyme (25eq) (0.5 mg / mL, 40 μL) obtained in Reference Example 2 in an Eppendorf tube, and use a light irradiation machine (MAX-102, manufactured by Asahi Spectroscope) at 360 nm. Of light (2.0 mW / cm ² ) was irradiated at 4 J / cm ² (33 minutes), 16 J / cm ² (133 minutes) or 32 J / cm ² (266 minutes). After light irradiation, degradation of PEG modification was examined by SDS-PAGE (16% T). The results are shown in FIG. Photodegradable groups were decomposed by light irradiation, and PEG-containing sites were removed from lysozyme.

Reference Example 5
Measurement of the lytic activity of the modified lysozyme of Reference Example 2 Take 2.0 μL of the modified lysozyme (25 eq) solution obtained in Reference Example 2 into the cell, and 120 μL of Micrococcus lysodeikticus cells The suspension (1.0 mg / mL) was added and pipetted 3 times. The cell was quickly attached to an absorptiometer (UV-2550, manufactured by Shimadzu Corporation), covered, and the time change of turbidity at 450 nm was measured every 1 second at 25 ° C. The slope of the regression line by the least square method was calculated for turbidity data from 11 to 30 seconds after the start of measurement, and was used as the lysis reaction rate. Each sample was measured in triplicate, and the ratio of the unmodified lysozyme at the same concentration to the reaction rate was calculated as the relative activity. The results are shown in FIG. The activity of the modified lysozyme that had been inactivated by PEG group modification was recovered by removing the PEG-containing site by light irradiation.

Example 4
Chemical modification to transferrin using compound of formula (II-b) A 10.9 mg / mL (142 μM) transferrin solution (from human, using holo form, Sigma-Aldrich) was prepared. Next, compounds of formula (II-b) corresponding to 2 equivalents, 9 equivalents, 18 equivalents or 46 equivalents were taken in tubes, respectively. An aqueous transferrin solution was added to the tube, and pipetting was performed 10 times. The mixture was allowed to stand at room temperature for 20.5 hours under light-shielding conditions, 14 μL of 50 mM Tris / HCl buffer (pH 8.0) was added, and the mixture was allowed to stand at room temperature for one hour under light-shielding conditions. Thereafter, dialysis was performed using a dialysis cup having a molecular weight fraction of 12000. The analysis result by SDS-PAGE is shown in FIG. In FIG. 8, “Intact” refers to unmodified transferrin. In addition, “2 eq”, “9 eq”, “18 eq”, and “46 eq” respectively represent compounds of the formula (II-b) corresponding to 2 equivalent, 9 equivalent, 18 equivalent, and 46 equivalent with transferrin. `` Modified transferrin (2 eq) '', `` Modified transferrin (9 eq) '', `` Modified transferrin (18 eq) '', `` Modified transferrin (46 eq) '' Also called.

Increasing the amount of the compound of formula (II-b) also increased the amount of PEG molecule modification to transferrin. When 18 equivalents or more of the compound of formula (II-b) was added, no unmodified transferrin was observed.

Example 5
Thermostability test of the modified transferrin of Example 4 5.38 mg / mL unmodified transferrin and 40 mL of the modified transferrin solution of Example 4 were placed in a tube and incubated on a heat block set at 90 ° C. for 10 minutes. Centrifugation was performed at 14000 G for 10 minutes, and aggregates were dropped on the bottom of the tube. Aggregates were observed at the bottom of the tube with unmodified transferrin, whereas aggregates decreased with the modified transferrin (2 eq) solution and aggregates with the modified transferrin (9 eq, 18 eq, 46 eq) solution. Not observed.

After allowing to stand at room temperature for several minutes, the supernatant of each tube was measured three times in the UV-vis mode of nano-drop (Thermo-Fisher), and the absorbance at 280 nm was examined. However, the concentration of the solution was determined by calculating the absorbance derived from the aromatic ring of the photodegradable group at 280 nm from the absorbance at 365 nm, and using the value obtained by subtracting it. The relative concentration was calculated from the concentration of the solution before the heat denaturation operation and the concentration of the supernatant after the heat denaturation operation. The results are shown in FIG. In the modified transferrin, a decrease in the transferrin concentration in the supernatant after the heat denaturation operation was suppressed.

Example 6
Removal of the PEG group-containing site Take the modified transferrin (46eq) (1.67 mg / mL) obtained in Example 4 in a microtube and use a light irradiator (MAX-102, manufactured by Asahi Spectroscopy) to light at 360 nm ( 5.0 mW / cm ² ) was irradiated at 32 J / cm ² (106 minutes) and 87 J / cm ² (290 minutes). The solution before and after the light irradiation was electrophoresed by SDS-PAGE (6%). The results are shown in FIG. The photodegradable group was decomposed by light irradiation, and the PEG-containing site was removed from transferrin.

The compound of the present invention can be suitably used for stabilizing industrial and pharmaceutical proteins.

Claims

The following formula (I)

[Where:
A 1 is a hydroxyl group or an active group,
A 2 is a photodegradable group,
A 3 is an oxyethylene group,
A 4 is a partner group of the capture molecule,
L 1 and L 2 are each a linker,
k 1 and k 2 are each independently 0 or 1,
n represents the average number of moles added of the oxyethylene group and 50 ≦ n ≦ 450]
A compound represented by
A 1 is the following substituent:
[Wherein the arrow indicates the connection to A 2 ]
2. The compound of claim 1 selected from the group consisting of:
2. A 2 is a divalent group having a skeleton selected from the group consisting of a 2-nitrobenzyl skeleton, a coumarin-4-ylmethyl skeleton, a phenylcarbonylmethyl skeleton, and a 7-nitroindolinocarbonyl skeleton. 2. The compound according to 2.
The combination of A 4 and the capture molecule is a combination of biotinyl group and streptavidin, a combination of maltosyl group and maltose binding protein, a combination of glutathion group and glutathione-S-transferase, HaloTag® ligand and HaloTag ( (Registered trademark) protein combination, guanylylmethylphenyl group and SNAP-tag (registered trademark) combination, cytosynylmethylphenyl group and CLIP-tag (registered trademark) combination, the following groups:

[Where:
The arrow indicates a connection to A 3 when k 2 is 0, and a connection to L 2 when k 2 is 1]
And a combination of dihydrofolate reductase, a combination of Strep-tag (registered trademark) and Strep-tactin (registered trademark), a combination of an antigen and an antibody, and a combination of an azido group and dibenzocyclooctyne. The compound according to any one of claims 1 to 3.
The following formula (Ia)

[Where:
A 1 , A 4 , and n are as defined in claim 1;
m 1 , m 2 and m 3 are each independently an integer of 1 to 10,
L 11 is a C 1-20 alkylene group, wherein the methylene group in the C 1-20 alkylene group may be substituted with 1 to 5 oxo groups, and adjacent methylene groups are 1 1 to 10 methylene groups among the methylene groups in the alkylene group may be NH, N (C 1-10 alkyl), O, S, C 6-14 arylene, may be replaced with 5-10 membered heteroarylene]
The compound according to any one of claims 1 to 4, which is a compound represented by the formula:
The following formula (II)

[Where:
A 1 , A 2 , A 3 , L 1 , k 1 and n are as defined in claim 1;
L 3 is a linker,
k 3 is 0 or 1,
B 1 is represented by the following formula (III)

(Where
A 4 is as defined in claim 1;
L 4 is a linker,
k 4 is 0 or 1)
A substituent that can be linked to B 2 of the compound represented by
A compound represented by
B 1 and B 2 are each independently an azide group or an alkynyl group, an azide group or a cyclooctynyl group, an azide group or a phosphinothioester group, a vinyl group or a thiol group, or A compound represented by formula (II) according to claim 6, which is a C 1-10 alkyl group or hydrazino group substituted with an oxo group, but the two are not the same.
The following formula (II-a)

[Where:
A 1 and n are as defined in claim 1;
m 4 , m 5 , m 6 and m 7 are each independently an integer of 1 to 10]
The compound represented by Formula (II) of Claim 6 or 7 which is a compound represented by these.
The following formula (II-b)

[Where:
n represents the average number of moles added of the oxyethylene group and 50 ≦ n ≦ 450]
The compound represented by the formula (II) according to any one of claims 6 to 8, which is a compound represented by the formula:
The compound represented by the formula (III) is represented by the following (III-a):

A compound represented by the formula (II) according to any one of claims 6 to 9, which is a compound represented by the formula:
A method for producing a modified protein, comprising reacting a protein with the compound represented by the formula (I) according to any one of claims 1 to 5.
A method for producing a modified protein, which comprises reacting a protein with the compound represented by the formula (II) according to any one of claims 6 to 10.
(1) preparing a modified protein intermediate by reacting the protein with the compound represented by formula (II) according to any one of claims 6 to 10;
(2) The intermediate is represented by the formula (III)

(Where
A 4 , B 2 , L 4 and k 4 are as defined in claim 6)
A method for producing a modified protein, which comprises reacting with a compound represented by the formula:
The following formula (IV):

[Where:
A 2 , A 3 , A 4 , L 1 , L 2 , k 1 , k 2 and n are as defined in claim 1;
Arrow indicates linkage with hydroxyl, amino or thiol group in protein]
The modified protein which has a substituent represented by these.
The following formula (V):

[Where:
A 2 , A 3 , B 1 , L 1 , L 3 , k 1 , k 3 and n are as defined in claim 6;
Arrow indicates linkage with hydroxyl, amino or thiol group in protein]
The modified protein which has a substituent represented by these.