WO2010053115A1 - Novel affinity support - Google Patents

Abstract

Disclosed is a solid-phase support for affinity chromatography, said support being useful for isolating/purifying a membrane protein that specifically binds to a ligand. Specifically disclosed is a solid-phase support for affinity column chromatography, wherein a ligand and a hydrophobic capping agent, which is selected from among cholesterol derivatives, fatty acid derivatives and aliphatic amines, are immobilized to a hydrophilic resin.  The solid-phase support is characterized in that the ligand is covalently bonded to the hydrophilic resin via a spacer that contains a polyethylene glycol moiety.

Description

New affinity carrier

The present invention provides a hydrophilic solid phase carrier in which a ligand and a hydrophobic capping agent are immobilized, a method for concentrating, isolating or purifying a ligand-specific binding protein using the solid phase carrier.

In recent years, there have been many attempts to search for proteins having specific interactions with specific compounds using techniques based on intermolecular interactions, or to study the interactions in detail. Specifically, a ligand specific binding protein is isolated by immobilizing a specific low molecular compound ligand on a solid phase carrier, binding a protein that specifically interacts with the ligand, and then eluting the protein. -Affinity column chromatography to be purified is known. For example, in 1989, Shriver et al. Identified an immunosuppressive agent, FK506 binding protein (FK506: FK506 binding proteins) by using affinity column chromatography (see Non-Patent Document 1). As described above, by using affinity column chromatography, a protein to which a drug or drug candidate compound specifically binds in vivo can be isolated, purified, and identified.
However, affinity column chromatography has a problem that not only a substance that specifically binds to a ligand but also a substance that interacts with a carrier due to non-specific hydrophobic interaction is adsorbed.
Furthermore, in the case of membrane-bound proteins, unlike normal water-soluble proteins, they are incorporated in lipid bilayers in vivo, making them very difficult to handle, and their functions and structures are still unknown. There are many. When such a membrane protein is purified by affinity column chromatography, there is currently no affinity carrier suitable for the membrane protein or a versatile method.
By the way, the pamphlet of International Publication No. 2004/25297 (Patent Document 1) describes a hydrophilic spacer that can be introduced between a ligand for affinity column chromatography and the surface of a solid phase carrier such as polystyrene. JP 2005/37881 pamphlet (Patent Document 2) describes a resin for affinity chromatography having a hydrophilic property comparable to that of an agarose resin, in which a hydrophilic spacer is incorporated in a monomer of a methacrylate resin. Furthermore, International Publication No. 2006/18901 pamphlet (Patent Document 3) discloses that a hydrophilic spacer is introduced into a solid phase carrier of a metal to suppress the adsorption of non-specific proteins and the specific interaction between molecules. It is described to be enhanced.
In addition, International Publication No. 2005/10528 (Patent Document 4) discloses that a hydrophobic capping agent such as stearic acid is immobilized on a methacrylate resin to bind to a nonspecific protein that hinders target protein search. In the pamphlet of International Publication No. 2006/80559 (Patent Document 5), ketoprofen as a ligand and stearic acid as a capping agent are respectively immobilized, and hydrophobic properties of the surface of the solid phase carrier are disclosed. An agarose-based solid phase carrier for affinity in which is controlled is disclosed.
However, an affinity column capable of efficiently adsorbing a membrane-bound protein that specifically binds to a ligand, a capping agent immobilized on the resin, and a ligand immobilized on the resin via a hydrophilic polymer A solid phase carrier for chromatography has not been known.

Nature, Volume 341, p.758-760 (1989)

International Publication No. 2004/25297 Pamphlet International Publication No. 2005/37881 Pamphlet International Publication No. 2006/18901 Pamphlet International Publication No. 2005/10528 Pamphlet International Publication No. 2006/80559 Pamphlet

The problem to be solved by the present invention is that a hydrophilic solid phase carrier capable of adsorbing a membrane protein that specifically binds to a ligand, and isolating a membrane protein that specifically binds to a ligand using the hydrophilic solid phase carrier, It is to provide a method of purification.

（Ｘ¹は単結合、又は親水性樹脂における水酸基とポリエチレングリコール部分を共有結合で連結する二価基を表し、Ｚ¹はリガンドと前記ポリエチレングリコール部分を共有結合で連結する二価基を表し、Ｙ¹は１～５０個のエチレングリコールユニットを含むポリエチレングリコール部分を表す）
で表される部分構造を含む、〔１〕～〔５〕のいずれかに記載の固相担体；
〔７〕　固相担体が、以下の式（２）：

（ｓは０又は１を表し、Ｘ²は単結合、又は親水性樹脂における水酸基とポリエチレングリコール部分もしくは疎水性キャッピング剤を共有結合で連結する二価基を表し、Ｚ²は２つのポリエチレングリコール部分どうし、又は疎水性キャッピング剤と前記ポリエチレングリコール部分を共有結合で連結する二価基を表し、Ｙ²は１～５０個のエチレングリコールユニットを含むポリエチレングリコール部分を表す）
で表される部分構造を含む、〔１〕～〔６〕のいずれかに記載の固相担体；
〔８〕　式（２）において、ｓが１である、〔７〕に記載の固相担体；
〔９〕　式（１）におけるＹ¹及び式（２）におけるＹ²が以下の式（３）～（６）：

（ａ、ｃ、ｆ及びｊは独立して２～５の整数を表し、ｄ、ｇ、ｉ及びｌは独立して１～５の整数を表し、ｂ、ｅ、ｈ及びｋは独立して１～５０の整数を表す）
から独立して選択される二価基、又は前記式（３）～式（６）から独立して選択される同一もしくは異なる１又は複数の二価基がアミド結合を介して結合して形成される二価基である、〔６〕～〔８〕のいずれかに記載の固相担体；
〔１０〕　式（１）におけるＸ¹、及び式（２）におけるＸ²が、単結合及び式（７）～（１０）:
（７） -X³-(CH₂)_m-X⁴-
（８） -X³-(CH₂)_m-X⁵-(CH₂)_n-X⁴-
（９） -X³-(CH₂)_m-X⁵-(CH₂)_n-X⁶-(CH₂)_o-X⁴-
（１０）-X³-(CH₂)_m-X⁵-(CH₂)_n-X⁶-(CH₂)_o-X⁷-(CH₂)_p-X⁴-
（式中、Ｘ³は単結合又はＣＯを表し、Ｘ⁵、Ｘ⁶及びＸ⁷はＯ、Ｓ、ＮＲ¹、ＮＨＣＯ又はＣＯＮＨを表し、Ｘ⁴は酸素原子、ＮＨ又はＣＯを表し、Ｒ¹は水素原子又は炭素数１～３のアルキル基を表し、ｍ、ｎ、ｏ及びｐは独立して１～５の整数を表す）
のいずれかで表される二価基から独立して選択されることを特徴とする、〔６〕～〔９〕のいずれかに記載の固相担体；
〔１１〕　式（１）におけるＺ¹、及び式（２）におけるＺ²が、単結合、ＮＨ、ＣＯ、及び式（１１）：
（１１）　－Ｚ³－（ＣＨ₂）_t－Ｚ⁴－
（式中、Ｚ³はＮＨ又はＣＯを表し、Ｚ⁴はＮＨ、ＣＯ、ＮＨＣＯ、ＣＯＮＨ、ＣＯ－Ｏ又はＯ－ＣＯを表し、ｔは１～５の整数を表す）
で表される二価基から、独立して選択されることを特徴とする、〔６〕～〔１０〕のいずれかに記載の固相担体；
〔１２〕　Ｙ¹におけるエチレングリコールのユニット数が、Ｙ²におけるエチレングリコールのユニット数よりも２以上大きいことを特徴とする、〔６〕～〔１１〕のいずれかに記載の固相担体；
〔１３〕　Ｙ¹におけるエチレングリコールのユニット数が２～３６であり、Ｙ²におけるエチレングリコールのユニット数が０～６である、〔１２〕に記載の固相担体；
〔１４〕　以下の（ａ）～（ｂ）の工程を含む、リガンド特異的結合蛋白質の濃縮、単離又は精製方法：
（ａ）〔１〕～〔１３〕のいずれかに記載の固相担体及び試料を接触させる工程；及び
（ｂ）固相担体から、リガンド特異的結合蛋白質を溶出する工程；
〔１５〕　リガンド特異的結合蛋白質が、膜結合型蛋白質である、〔１４〕に記載の精製方法；
〔１６〕　〔１〕～〔１3〕のいずれかに記載の固相担体の、アフィニティーカラムクロマトグラフィー用担体としての使用；
に関する。 As a result of intensive investigations to obtain a solid phase support for affinity chromatography that is useful for efficiently isolating and purifying a membrane protein that specifically binds to a ligand, the present inventors have determined that a ligand has a hydrophilic spacer. Thus, a hydrophilic solid phase carrier immobilized thereon was obtained. That is, the present invention
[1] A hydrophobic capping agent selected from cholesterol derivatives, fatty acid derivatives and aliphatic amines, and a solid phase carrier for affinity column chromatography in which a ligand is immobilized on a hydrophilic resin, wherein the ligand is A solid phase carrier characterized by being covalently bonded to the hydrophilic resin via a spacer containing a polyethylene glycol moiety;
[2] The solid phase carrier according to [1], wherein the hydrophilic resin is a resin obtained by polymerizing a saccharide or a resin obtained by polymerizing a highly hydrophilic monomer;
[3] The solid phase carrier according to [2], wherein the hydrophilic resin obtained by polymerizing saccharides is a sepharose resin, a dextran resin, a cellulose resin, an amylose resin, or an agarose resin;
[4] The solid phase carrier according to [2], wherein the resin obtained by polymerizing a highly hydrophilic monomer is a methacrylate resin in which methacrylic acid having a polyethylene glycol group or a polyol group is polymerized;
[5] The solid phase carrier according to any one of [1] to [4], wherein the ligand is a substance that specifically adsorbs to a membrane-bound protein;
[6] The solid phase carrier is represented by the following formula (1):

(X ¹ represents a single bond or a divalent group that links a hydroxyl group and a polyethylene glycol moiety in a hydrophilic resin by a covalent bond, Z ¹ represents a divalent group that links a ligand and the polyethylene glycol moiety by a covalent bond, Y ¹ represents a polyethylene glycol moiety containing 1 to 50 ethylene glycol units)
A solid phase support according to any one of [1] to [5], comprising a partial structure represented by:
[7] The solid phase carrier is represented by the following formula (2):

(S represents 0 or 1, X ² represents a single bond, or a divalent group that covalently connects a hydroxyl group and a polyethylene glycol moiety or a hydrophobic capping agent in a hydrophilic resin, and Z ² represents two polyethylene glycol moieties. Or a divalent group for covalently linking the polyethylene glycol moiety with the hydrophobic capping agent, and Y ² represents a polyethylene glycol moiety containing 1 to 50 ethylene glycol units)
A solid phase support according to any one of [1] to [6], comprising a partial structure represented by:
[8] The solid phase carrier according to [7], wherein s is 1 in formula (2);
[9] Y ¹ in formula (1) and Y ² in formula (2) are the following formulas (3) to (6):

(A, c, f and j independently represent an integer of 2 to 5, d, g, i and l independently represent an integer of 1 to 5, and b, e, h and k independently Represents an integer from 1 to 50)
Or a divalent group independently selected from the above formulas (3) to (6), or one or more divalent groups independently selected from the above formulas (3) to (6) are bonded via an amide bond. A solid phase carrier according to any one of [6] to [8],
[10] X ¹ in formula (1) and X ² in formula (2) are a single bond and formulas (7) to (10):
^{(7) -X 3 - (CH} 2) m -X 4 -
^{(8) -X 3 - (CH} 2) m -X 5 - (CH 2) n -X 4 -
^{(9) -X 3 - (CH} 2) m -X 5 - (CH 2) n -X 6 - (CH 2) o -X 4 -
^{(10) -X 3 - (CH} 2) m -X 5 - (CH 2) n -X 6 - (CH 2) o -X 7 - (CH 2) p -X 4 -
(Wherein X ³ represents a single bond or CO, X ⁵ , X ⁶ and X ⁷ represent O, S, NR ¹ , NHCO or CONH, X ⁴ represents an oxygen atom, NH or CO, R ¹ Represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and m, n, o and p independently represent an integer of 1 to 5)
The solid phase carrier according to any one of [6] to [9], which is independently selected from divalent groups represented by any of the following:
[11] Z ¹ in formula (1) and Z ² in formula (2) are a single bond, NH, CO, and formula (11):
(11) —Z ³ — (CH ₂ ) _t —Z ⁴ —
(Wherein Z ³ represents NH or CO, Z ⁴ represents NH, CO, NHCO, CONH, CO—O or O—CO, and t represents an integer of 1 to 5)
The solid phase carrier according to any one of [6] to [10], wherein the solid phase carrier is independently selected from divalent groups represented by:
[12] The solid phase carrier according to any one of [6] to [11], wherein the number of ethylene glycol units in Y ¹ is 2 or more than the number of ethylene glycol units in Y ² ;
[13] The solid phase carrier according to [12], wherein the number of ethylene glycol units in Y ¹ is 2 to 36, and the number of ethylene glycol units in Y ² is 0 to 6;
[14] A method for concentrating, isolating or purifying a ligand-specific binding protein comprising the following steps (a) to (b):
(A) a step of bringing the solid phase carrier according to any one of [1] to [13] into contact with a sample; and (b) a step of eluting a ligand-specific binding protein from the solid phase carrier;
[15] The purification method according to [14], wherein the ligand-specific binding protein is a membrane-bound protein;
[16] Use of the solid phase carrier according to any one of [1] to [13] as a carrier for affinity column chromatography;
About.

According to the present invention, it has become possible to provide a hydrophilic solid phase carrier capable of adsorbing a membrane-bound protein that specifically binds to a ligand. The carrier is useful as a column filler for affinity chromatography, and can be used to isolate, purify, and identify a ligand-specific binding protein.

It is a figure which shows the typical solid-phase carrier for affinity chromatography of this invention. It is a band obtained by the binding experiment, SDS-PAGE and Western blotting experiment described in Example 5. It shows whether or not the solid phase carrier represented by the formula (1-13) obtained in Example 1 specifically binds γ-secretase as a resin for affinity chromatography. FIG. 3 is a view showing that the solid phase carrier represented by the formula (1-13) does not bind non-specifically to a large amount of protein. A solid support using Toyopearl, which is a hydrophobic resin instead of Affigel, corresponding to Formula (1-13) and Formula (1-19) was prepared as a control, and the presence or absence of nonspecific protein binding was determined. It is a figure which shows the result investigated. It is a band obtained by the binding experiment, SDS-PAGE and Western blotting experiment described in Example 8. The examination result of the affinity resin which each immobilized DAPT from which linker length differs is shown.

In the present specification, the “membrane-bound protein” is a protein attached to a biological membrane such as a cell or an organelle. These proteins are proteins that are partially contained in the biological membrane, proteins that penetrate the biological membrane, or proteins that are temporarily bound to an integral membrane protein. Examples of biological membranes include cell membranes, outer mitochondrial membranes, inner mitochondrial membranes, nuclear membranes, endoplasmic reticulum membranes, and Golgi membranes.
These membrane-bound proteins may form a complex by associating a plurality of proteins. Examples of membrane-bound proteins include receptors, channels, transporters, pumps and enzymes.
The cells are not particularly limited, and specific examples of cells used for the affinity column chromatography sample using the solid phase carrier of the present invention include any cells and cells prepared from rats, mice, and human tissues. Cell lines available from banks or the like may be cultured and used.
Examples of the organelle include nucleus, endoplasmic reticulum, Golgi apparatus, endosome, lysosome, mitochondria, peroxisome and the like.
The protein that can be purified by the solid phase carrier for affinity chromatography of the present invention is preferably a membrane-bound protein having a molecular weight of 5 kDa to 500 kDa.

“Ligand” is a compound that specifically binds to the membrane-bound protein, and represents a low-molecular compound that serves as a probe in affinity column chromatography. The low molecular compound is not particularly limited as long as it has at least one functional group capable of binding to a spacer or a partial structure that can be converted to the functional group, and preferably includes a compound having a molecular weight of 1,000 or less. It is done. Particularly preferred are compounds having a hydroxyl group, amino group, carboxy group, thiol group and / or formyl group, or a group having a molecular weight of 1,000 or less having a group convertible to these groups. For example, a γ-secretase inhibitor (DAPT, L -685458, etc.), histamine H1 receptor inhibitors (ebastine, Allegra, etc.), calcium antagonists (amlodipine, etc.), cambinoid CB1 receptor antagonists (eg rimonabant), cyclooxygenase-2 inhibitors (celecoxib, etc.), dopamine D2 inhibition Agents, serotonin 5-HT2 inhibitors (bronanserin, risperidone, etc.), MAO-B inhibitors (rasagiline, etc.), MAO-A inhibitors (moclobemide, etc.), potassium channel antagonists (amiodarone, dofetilide, etc.), sodium channel antagonists (Cibenzoline, flucainide, moracidin, etc.) Zojiazepin antagonists (flumazenil, etc.), adrenergic β-blockers (allotynol, amosulalol, carazolol, etc.), antiepileptic agents (zonisamide and the like) and the like, and these pharmaceutically equivalent derivatives thereof. Here, as a pharmaceutically equivalent derivative, a derivative in which a partial structure that is not essential for maintaining the biological activity of the ligand is chemically modified, or a partial structure that does not affect the biological activity of the ligand is added chemically. Derivatives and the like.

A `` hydrophobic capping agent '' is a compound having a hydrophobic structure that has an affinity for proteins present in membranes such as cell membrane, outer mitochondrial membrane, inner mitochondrial membrane, nuclear membrane, endoplasmic reticulum membrane, and Golgi membrane. And selected from cholesterol derivatives, fatty acid derivatives and aliphatic amines. The hydrophobic capping agent is not particularly limited as long as it is a compound having at least one functional group capable of binding to a spacer or a hydrophilic resin, or a partial structure that can be converted to the functional group, and preferably has a molecular weight of 1,000 or less. The compound of this is mentioned.
Examples of the functional group in the ligand or the hydrophobic capping agent include a hydroxyl group, an amino group, a carboxy group, a thiol group, and a formyl group. The partial structure that can be converted to the functional group is not particularly limited, and examples thereof include a carboxylic acid ester that can be converted to a carboxy group by hydrolysis, a carbon-carbon double bond that can be converted to a hydroxyl group by an oxidation reaction, and the like. A conversion reaction to a functional group may be appropriately performed by a synthesis method well known to those skilled in the art.
Moreover, as a method of introducing a functional group into a ligand, 1) a method of introducing a hydroxyl group into an aromatic compound using an oxidizing agent, 2) S9mix (a drug such as phenobarbital or the like in the abdominal cavity of a mammal such as a rat) A method in which a hydroxyl group is introduced using a metabolic enzyme such as a supernatant fraction S9 of liver homogenate in which metabolic enzymes are induced by administration) 3) a method of introducing a nitro group into an aromatic compound, and then a reducing agent And 4) a method of introducing a carboxylic acid using a palladium catalyst after introducing a halogen atom onto the aromatic ring of the aromatic compound.
Ligand and hydrophobic capping agents are hydrophilic when the functional group forms an ester bond, ether bond, thioether bond, disulfide bond, amine bond, amide bond, urea bond or urethane bond with a hydrophilic resin or spacer. Immobilized in resin. Therefore, in order to increase the reactivity, the functional group may be appropriately modified and immobilized. Specifically, the carboxy group may be an acid halide such as acid chloride or acid bromide, or the hydroxyl group may be methanesulfonylated. The reactivity of the functional group can be increased.

The cholesterol derivative is not particularly limited as long as it is a cholesterol derivative having a steroid skeleton, and specifically, cholesterol (cholesterol), 6-ketocholestanol, stigmastanol, 5α-cholestane. -3β-ol (5α-cholestan-3β-ol), 5β-cholestan-3α-ol (5β-cholestan-3α-ol), 5β-cholestan-3β-ol (5β-cholestan-3β-ol), α- Cholestanol (α-cholestanol), 5α-hydroxycholestanol (5α-hydroxycholestanol), 5α-cholestan-3β, 6α-diol (5α-cholestan-3β, 6α-diol), 5β-cholestan-3α, 7α, 12α- Triol (5β-cholestan-3α, 7α, 12α-triol), 5α-cholestan-24α-methyl-3β-ol (5α-cholestan-24α-methyl-3β-ol), 5α-cholestan-3β-ol-6- ON 3-acetate (5α-cholestan-3β-ol-6-one 3-acetate), 5α-cholestane-3β, 6β- All (5α-cholestan-3β, 6β-diol), 5α-cholestan-3β, 5α-diol-6-one (5α-cholestan-3β, 5α-diol-6-one), 5α-cholestan-3β, 5β, 6β-triol (5α-cholestan-3β, 5β, 6β-triol), 5β-cholestane-3β, 6β-diol (5β-cholestan-3β, 6β-diol), 5α-cholestan-3β, 7α-diol (5α- cholestan-3β, 7α-diol), 5α-cholestan-3β, 7β-diol, 5α-cholestan-3β, 7β-diol, 20α-hydroxycholesterol, β-sitosterol, 7 -Ketocholesterol (7-ketocholesterol), fucosterol (fucosterol), desmosterol (22-ketocholesterol), 25-hydroxycholesterol, 3β-hydroxy-δ5-cholenic acid ( 3β-hydroxy-δ5-cholenic acid), 22 (R) -hydroxycholesterol, epicholester Roll (epicholesterol), 5-cholenic acid-3β-ol methyl ether, 3- [3β-cholest-5-en-3-yloxy] -1-propanamine (3 -[3β-cholest-5-en-3-yloxy] -1-propanamine), 5-cholesten-24α-ethyl-3β-ol (5-cholesten-24α-ethyl-3β-ol), 24-ketocholesterol ( 24-ketocholesterol), or 7,5α-cholesten-24α-methyl-3β-ol (7,5α-cholesten-24α-methyl-3β-ol), and the like. It is linked to the spacer via a bond. Alternatively, a derivative in which a functional group such as an amino group, a hydroxyl group, or a carboxy group is introduced into the cholesterol derivative as appropriate so that the cholesterol derivative can be chemically bonded to a hydrophilic resin or a spacer via an amide bond, a urea bond, a urethane bond, or the like. It can also be used.

The cholesterol derivative can be led to an intermediate for producing the solid phase carrier of the present invention by appropriately modifying a functional group such as a hydroxyl group, an amino group or a carboxy group. Specifically, a compound having a carboxy group obtained by reacting a hydroxyl group of a cholesterol derivative with a dicarboxylic acid such as succinic acid, a compound obtained by converting the hydroxyl group of a cholesterol derivative into chloroformate, and the like can be mentioned. For example, 5α-cholestan-3α-ol hemisuccinate (5α-cholestan-3α-ol hemisuccinate), 5α-cholestan-3β-ol hemisuccinate (5α-cholestan-3β-ol hemisuccinate), 3β, 5α, 6β-trihydroxycholestane 3 -Hemisuccinate (3β, 5α, 6β-trihydroxycholestan 3-hemisuccinate), 5β-cholestan-3α-ol hemisuccinate (5β-cholestan-3α-ol hemisuccinate), 5β-cholestan-3β-ol hemisuccinate (5β-cholestan-3β- ol hemisuccinate), cholesteryl hydrogen succinate, 5α-cholestan-3α-ol hemisuccinate, 5α-cholestan-3α-ol hemisuccinate, 5α-cholestan-3β-ol hemisuccinate (5α-cholestan-3β-ol hemisuccinate ), 3β, 5α, 6β-trihydroxycholestane 3-hemisuccinate (3β, 5α, 6β-trihydroxycholestane 3-hemisucc inate), 5β-cholestan-3α-ol hemisuccinate (5β-cholestan-3α-ol hemisuccinate), 5β-cholestan-3β-ol hemisuccinate (5β-cholestan-3β-ol hemisuccinate), cholesteryl hydrogen succinate , 25-hydroxycholesterol 3-hemisuccinate (25-hydroxycholesterol 3-hemisuccinate), 25-hydroxycholesterol 3-hemisuccinate (25-hydroxycholesterol 3-hemisuccinate), 5-cholesten-24α-ethyl-3β-ol chloroformate (5- cholesten-24α-ethyl-3β-ol chloroformate), 5α-cholestan-3β-ol, chloroformate コ レ, cholesteryl chloroformate, 5-cholesten-24α-ethyl- 3β-ol chloroformate (5-cholesten-24α-ethyl-3β-ol chloroformate Etc. can be exemplified.

Examples of fatty acid derivatives include linear or branched carboxylic acids, phosphoric acids or sulfonic acids having 6 to 30 carbon atoms, which may have 1 to 6 unsaturated bonds (double or triple bonds), or these Of the ester. Specifically, linear saturated fatty acids (eg, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid), linear unsaturated fatty acids (oleic acid, linoleic acid, linolenic acid, DHA), branched saturated fatty acids (For example, 19-methyleicosanoic acid (19-methyleicosanoic acid), 12-methyltetradecanoic acid (12-methyltetradecanoic acid), isostearic acid, etc.), or branched unsaturated fatty acid ( Examples thereof include 2-propyl-2,4-pentadienic acid, 2-methylheptadienic acid, (6Z) -8-methylnonenoic acid, and the like. Preferred examples include saturated fatty acids having 8 to 24 carbon atoms, more preferably 14 to 20 carbon atoms.

Examples of the aliphatic amine include linear or branched aliphatic amines having 6 to 30 carbon atoms which may have 1 to 6 unsaturated bonds (double bonds or triple bonds). Specific examples include alkylamines (for example, stearylamine, 1-aminodecane, myristylamine, octadecylamine), alkenylamines (for example, oleylamine), and the like. Preferably, an alkylamine having 8 to 18 carbon atoms is used.

“Hydrophilic resin” represents a hydrophilic resin obtained by polymerizing a hydrophilic monomer having a plurality of hydroxyl groups or polyethylene glycol groups. Specifically, a hydrophilic resin formed by polymerizing saccharides or a resin formed by polymerizing highly hydrophilic monomers can be used.

Examples of the “hydrophilic resin obtained by polymerizing saccharides” include sepharose resins, dextran resins, amylose resins, cellulose resins, and agarose resins.
Examples of the sepharose resin include ECH Sepharose 4B and EAH-Sepharose 4B (registered trademark) manufactured by GE Healthcare. Examples of the dextran-based resin include Sephadex (registered trademark) manufactured by GE Healthcare.
Examples of the amylose resin include amylose resin (registered trademark), amylose sepharose resin, amylose agarose affinity resin, and the like manufactured by New England BioLab.
Examples of the cellulose resin include cellulose (trade name: Cellurose (cat.No; 22182), Cellurose, beaded (cat.No; C7079), Sodium carboxymethyl cellulose (cat.No; 419311)) manufactured by Sigma. .
Since the sepharose resin and amylose resin both have a hydroxyl group, they can form an ester bond with a compound having a carboxy group. Moreover, it can guide | induce to the manufacturing intermediate which has a carboxy group and an amino group by alkylating a hydroxyl group with the method shown by the following manufacturing method 2.

Examples of the agarose resin include Affi-gel (registered trademark) manufactured by Bio-Rad, and specific examples include Affigel 10, Affigel 102 and Affigel 15, and Agarose manufactured by Sigma (trade name: Iminodiaceticgaracid Agarose (cat No. I4758)), Low Density Aminoethyl 6 BCL, Amine Reactive Agarose の, etc. from Agarose Bead Technology (AGAROSE BEAD TECHNOLOGIES).

In addition, a via-core chip (GE Healthcare; Sensor Sensor Chip CM5; Cat. Mark No; BR-1000-12) in which a hydrophilic resin obtained by polymerizing the saccharide is supported on a gold film is also included in the present invention. Included in the category of hydrophilic resins.

The “highly hydrophilic monomer” in the “resin formed by polymerizing highly hydrophilic monomers” includes methacrylic acid having a polyethylene glycol group or a polyol group. Specifically, if a polyethylene glycol group is necessary for the carboxy group of methacrylic acid, a monomer covalently bonded via a spacer, or a monomer in which an alkyl group having two or more hydroxyl groups is ester-bonded to the carboxy group of methacrylic acid, etc. Can be mentioned. As the “resin formed by polymerizing a highly hydrophilic monomer”, a resin described in International Publication Pamphlet WO2007 / 142331 or a resin marketed as Aquafarm (trade name) can be used.

A ligand or hydrophobic capping agent is “immobilized” means that a functional group such as amino group, hydroxyl group, carboxy group, thiol group or formyl group (CHO) contained in the ligand or hydrophobic capping agent is a hydrophilic resin. This means that it is covalently bonded to the above hydroxyl group via a spacer if necessary. That is, the functional group of the ligand or the hydrophobic capping agent is shared with the functional group of the spacer or the hydrophilic resin through an ester bond, an ether bond, a thioether bond, a disulfide bond, an amine bond, an amide bond, a urea bond or a urethane bond. Are connected.
Here, the bonding rate of the ligand and the hydrophobic capping agent to the hydroxyl group on the surface of the hydrophilic resin (this is referred to as the immobilization rate) is not particularly limited. Are connected.
The ratio of the ligand and the hydrophobic capping agent is not particularly limited, and examples of the ligand: hydrophobic capping agent include 1: 100 to 100: 1.
Further, the solid phase carrier of the present invention may have a plurality of types of hydrophobic capping agents, and preferably may have 1 to 3 types of hydrophobic capping agents. Specific examples of the solid phase carrier having a plurality of types of hydrophobic capping agents include solid phase carriers on which cholesterol derivatives and fatty acid derivatives, cholesterol derivatives and phospholipid derivatives are immobilized.

In the present specification, the “spacer” represents a divalent group connecting a capping agent or a ligand and a hydrophilic resin, and the spacer for immobilizing the ligand on the hydrophilic resin is a chemical containing a polyethylene glycol moiety. As long as it is a divalent group capable of forming a stable covalent bond. The spacer for immobilizing the hydrophobic capping agent on the hydrophilic resin is not particularly limited as long as it is a divalent group capable of forming a chemically stable covalent bond, and may contain a polyethylene glycol moiety. Specifically, as a spacer for immobilizing the ligand on the hydrophilic resin, the divalent group represented by “—X ¹ —Y ¹ —Z ¹ —” in the formula (1) makes the hydrophobic capping agent hydrophilic. Specific examples of the spacer for immobilizing the conductive resin include a divalent group represented by “—X ² — (Y ² —Z ² ) s—” in the formula (2).
In the formula (1), X ¹ represents a single bond or a divalent group that links a hydroxyl group and a polyethylene glycol moiety in a hydrophilic resin by a covalent bond. The divalent group in X ¹ is not particularly limited as long as it is a divalent group that chemically and stably connects the functional group at the end of the polyethylene glycol moiety represented by Y ¹ and the hydroxyl group in the hydrophilic resin. That is, as the divalent group, any methylene group may be appropriately substituted with an oxygen atom, a sulfur atom, NR ¹ (R ¹ is as defined above), NHCO or CONH. There are 25 linear alkylenes. Specifically, a divalent group represented by any one of the formulas (7) to (10) can be given.
In the formula (2), X ² represents a single bond or a divalent group that links a hydroxyl group in a hydrophilic resin and a polyethylene glycol moiety or a hydrophobic capping agent by a covalent bond.
In the formula (2), when s represents 0, that is, when the spacer connecting the hydrophobic capping agent and the hydrophilic resin does not contain a polyethylene glycol moiety, X ² is stable with the hydroxyl group on the hydrophilic resin. Represents a divalent group for forming a covalent bond. When s represents 0 and the hydrophobic capping agent has a carboxy group, X ² may represent a single bond.
In the formula (2), when s represents 1, X ² represents a single bond or a divalent group that links a hydroxyl group and a polyethylene glycol moiety in a hydrophilic resin by a covalent bond. The divalent group in X ² is not particularly limited as long as it is a divalent group that chemically and stably connects the functional group at the end of the polyethylene glycol moiety represented by Y ² and the hydroxyl group in the hydrophilic resin. That is, as the divalent group, any methylene group may be appropriately substituted with an oxygen atom, a sulfur atom, NR ¹ (R ¹ is as defined above), NHCO or CONH. There are 25 linear alkylenes. Specifically, a divalent group represented by any one of the formulas (7) to (10) can be given.
Y ¹ in the formula (1) represents a polyethylene glycol moiety containing 1 to 50 ethylene glycol units (a divalent group represented by —O—CH ₂ CH ₂ —). Here, the polyethylene glycol moiety can form an ester bond, an ether bond, a thioether bond, a disulfide bond, an amine bond, an amide bond, a urea bond, or a urethane bond with X ¹ or a hydroxyl group in a hydrophilic resin and Z ^{1 at} both ends, respectively. It has a functional group. Further, as long as the polyethylene glycol part contains 1 to 50 ethylene glycol units, there is no particular limitation on the bonding mode between individual ethylene glycol units, and 1 to 50 ethylene glycols are polymerized to form polyethylene glycol. Or the same or different divalent having a functional group on the both ends of polyethylene glycol polymerized with 1 to 50, preferably 2 to 15 ethylene glycol via a linear alkylene having 1 to 5 carbon atoms as appropriate. One or a plurality of groups may be bonded to form a divalent group containing 1 to 50 ethylene glycol units as a whole. Specifically, a divalent group selected from the above formulas (3) to (6), or the same or different divalent group independently selected from the above formulas (3) to (6) is an amide bond. And a divalent group formed by bonding via a hydrogen atom.
The number of ethylene glycol units contained in Y ¹ is preferably 2 to 36, more preferably 5 to 24.

Y ² in formula (2) represents a polyethylene glycol moiety containing 1 to 50 ethylene glycol units. Here, the polyethylene glycol moiety can form an ester bond, an ether bond, a thioether bond, a disulfide bond, an amine bond, an amide bond, a urea bond, or a urethane bond with X ² or a hydroxyl group in a hydrophilic resin and Z ^{2 at} both ends. It has a functional group. Further, if the polyethylene glycol part contains 1 to 50 ethylene glycol units, there is no particular limitation on the bonding mode between the individual ethylene glycol units, and polyethylene glycol in which 1 to 50 ethylene glycols are polymerized is formed. Or the same or different divalent group having a functional group through a linear alkylene having 1 to 5 carbon atoms as appropriate at both ends of polyethylene glycol obtained by polymerizing 1 to 50, preferably 2 to 15 ethylene glycol. May be bonded to form a divalent group containing 1 to 50 ethylene glycol units as a whole. Specifically, a divalent group selected from the above formulas (3) to (6), or the same or different divalent group independently selected from the above formulas (3) to (6) is an amide bond. And a divalent group formed by bonding via the above.
The number of ethylene glycol units contained in Y ² is preferably 1 to 6, and more preferably 1 to 4. Preferably, the number of ethylene glycol units contained in Y ¹ is 2 or more larger than the number of ethylene glycol units contained in Y ² .

Z ¹ in Formula (1) represents a divalent group or a single bond that links an adjacent polyethylene glycol moiety and a ligand or a hydrophobic capping agent by a covalent bond. Z ² in Formula (2) represents a divalent group or a single bond that connects two adjacent polyethylene glycol moieties, or a polyethylene glycol moiety and a hydrophobic capping agent by a covalent bond. The divalent group in Z ¹ and Z ² is not particularly limited as long as it is a divalent group that chemically and stably connects the functional group at the end of the polyethylene glycol moiety and a ligand or a hydrophobic capping agent. That is, as the divalent group, NH, CO, or any methylene group may be appropriately substituted with an oxygen atom, a sulfur atom, NR ¹ (R ¹ is as defined above), NHCO, or CONH, chemically. Examples thereof include straight-chain alkylene having 1 to 10 carbon atoms, preferably 1 to 7 carbon atoms. Specific examples include a divalent group represented by the formula (11).

The condensation reaction between the functional group of the ligand or the hydrophobic capping agent and the functional group of the spacer can be carried out by a method well known to those skilled in the art. That is, a hydrophilic resin in which a functional group is appropriately introduced via a spacer and a ligand or a hydrophobic capping agent in which a functional group capable of forming a covalent bond with the functional group is appropriately introduced may be condensed. Here, the functional group of the ligand, the hydrophobic capping agent or the spacer may be appropriately converted into a functional group having high reactivity. For example, the carboxy group can be used as an active ester with N-hydroxysuccinimide ester or the like and used for dehydration condensation reaction with a hydroxyl group or an amino group. It can also be used to convert a thiol group to 2-thiopyridyl disulfide to form a disulfide bond. In addition, a compound having an aldehyde group as a functional group can be reacted in the presence of a compound having an amino group and a reducing agent (for example, a boron-based reducing agent such as sodium cyanoborohydride).
Specifically, for example, Affigel 102, which is an agarose resin, has a spacer capable of forming an amide bond with a compound having a carboxy group introduced into the hydroxyl group of the agarose resin, and has the following formula:

で表される官能基を有する。また、アフィゲル10は、アフィゲル102を無水コハク酸で処理しカルボキシ酸体とした後、N-ヒドロキシコハク酸イミドで活性エステルとした担体であり、アミノ基もしくは水酸基を有する化合物とアミド結合もしくはエステル結合形成可能なスペーサーが導入されて以下の式： 

It has a functional group represented by Affigel 10 is a carrier in which Affigel 102 is treated with succinic anhydride to form a carboxylic acid form, and then converted into an active ester with N-hydroxysuccinimide, and an amide bond or ester bond with a compound having an amino group or a hydroxyl group A formable spacer is introduced and the following formula:

で表される。
　また、アフィゲル15における活性型官能基は以下の式：

It is represented by
The active functional group in Affigel 15 has the following formula:

で表され、スペーサー内に4級アミンを含む担体であり、アミノ基もしくは水酸基を有する化合物とアミド結合もしくはエステル結合等を形成可能である。
　また、セファロース系樹脂にはN-ヒドロキシコハク酸イミドで活性エステル化されたカルボキシ基を有する6-Aminocaproic acid N-hydoxysuccinimide ester-activated-Sepharose 4B(登録商標）、6-Aminohexanoic acid N-hydroxysuccinimide ester-activated Sepharose 4B及びCH-activated Sepharose 4B、臭化シアンで活性化されたCNBr-activated Sepharose 4B等がアミノ基を有する化合物を混合するだけでそのまま共有結合を形成できる活性型担体として市販されている。また、アミノ基を有するEAH-Sepharose 4B、及びカルボキシ基を有するECH-Sepahroseが市販されている。更に、エポキシ基を有するEpoxy-activated Sepharose 6Bが、アミノ基、水酸基又はチオール基を有する化合物と共有結合を形成できる担体として市販されている。また、活性化されたチオール基を有するActivated Thiol Sepharose 4Bは、チオール基を有する化合物とジスルフィド結合を形成できる担体として市販されている。

It is a carrier containing a quaternary amine in the spacer, and can form an amide bond or an ester bond with a compound having an amino group or a hydroxyl group.
Sepharose resins also include 6-Aminocaproic acid N-hydoxysuccinimide ester-activated-Sepharose 4B (registered trademark), 6-Aminohexanoic acid N-hydroxysuccinimide ester- having a carboxy group activated with N-hydroxysuccinimide. Activated Sepharose 4B, CH-activated Sepharose 4B, CNBr-activated Sepharose 4B activated with cyanogen bromide, and the like are commercially available as active carriers that can form a covalent bond as they are by simply mixing a compound having an amino group. In addition, EAH-Sepharose 4B having an amino group and ECH-Sepahrose having a carboxy group are commercially available. Furthermore, Epoxy-activated Sepharose 6B having an epoxy group is commercially available as a carrier capable of forming a covalent bond with a compound having an amino group, a hydroxyl group or a thiol group. Activated Thiol Sepharose 4B having an activated thiol group is commercially available as a carrier capable of forming a disulfide bond with a compound having a thiol group.

The solid phase carrier of the present invention is preferably the following formula (13):

（式中、Ｘ¹、Ｘ²、Ｚ¹、Ｚ²、Ｙ¹、Ｙ²及びｓは前記と同義である）
で表される（図１）。ここで、疎水性キャッピング剤もしくはリガンドが固定化されている親水性樹脂上の水酸基の割合、又は、固定化されたキャッピング剤及び固定化されたリガンドの割合に特に限定は無いが、担体としてアフィゲルを用いる場合、水酸基を、アミノ基等を有する基に変換（官能基化）し、当該官能基化された水酸基に対して、リガンドは６０％から９０％程度、キャッピング剤は０．１％から４０％程度が固定化されていることが好ましい。
　式（１）のＹ¹や式（２）のＹ²におけるポリエチレングリコール部分として、以下の式（３）～（６）：

(Wherein X ¹ , X ² , Z ¹ , Z ² , Y ¹ , Y ² and s are as defined above)
(FIG. 1). Here, the ratio of the hydroxyl group on the hydrophilic resin on which the hydrophobic capping agent or the ligand is immobilized, or the ratio of the immobilized capping agent and the immobilized ligand is not particularly limited, but the carrier is Affigel. When a hydroxyl group is used, the hydroxyl group is converted into a group having an amino group or the like (functionalized), and the ligand is about 60% to 90% and the capping agent is about 0.1% with respect to the functionalized hydroxyl group. About 40% is preferably immobilized.
As the polyethylene glycol moiety in Y ^{1 of} formula (1) and Y ² of formula (2), the following formulas (3) to (6):

（ａ、ｂ、ｃ、ｄ、ｅ、ｆ、ｇ、ｈ、ｉ、ｊ、ｋ及びｌは前記と同義である）
で表される二価基が挙げられる。

(A, b, c, d, e, f, g, h, i, j, k and l are as defined above)
The bivalent group represented by these is mentioned.

The divalent group represented by the formula (3) is a divalent group having b ethylene glycol units, and has NH at both ends. Accordingly, the divalent group can be bonded to X ¹ in formula (1) or X ² in formula (2) by an amide bond. Therefore, when X ¹ or X ² is a divalent group represented by any one of the formulas (7) to (10), X ⁴ in the formulas (7) to (10) represents CO.
In addition, when the divalent group represented by the formula (3) binds to a capping agent or a ligand and the capping agent or ligand has a carboxy group, they can be bonded via an amide bond. In this case, Z ¹ in formula (1) or Z ² in formula (2) represents a single bond. Further, the divalent group represented by the formula (3) can be bonded through an NHCOO bond when the capping agent or the ligand has a hydroxyl group. In this case, Z ¹ in formula (1) or Z ² in formula (2) represents a carbonyl group. Further, the divalent group represented by the formula (3) can be bonded via an NHCONH bond when the capping agent or the ligand has an amino group. In this case, Z ¹ in formula (1) or Z ² in formula (2) represents a carbonyl group.
Further, the divalent group represented by the formula (3) may be bonded to a capping agent or a ligand via a spacer composed of the divalent group represented by the formula (11). In this case, the formula (1) Z ^{1 in the} formula or Z ² in the formula ( ² ) is represented by the formula (11), and in the formula (11), Z ³ represents a carbonyl group.

The divalent group represented by the formula (4) is a divalent group having e ethylene glycol units, and has a carbonyl group and NH at both ends. Accordingly, the divalent group can be bonded to X ¹ in formula (1) or X ² in formula (2) by an amide bond. Specifically, when X ¹ or X ² is a divalent group represented by any one of the formulas (7) to (10), X ⁴ in the formulas (7) to (10) represents NH. .
Further, when the divalent group represented by the formula (4) is bonded to a capping agent or a ligand, and the capping agent or ligand has a carboxy group, it can be bonded through an amide bond. In this case, Z ¹ in formula (1) or Z ² in formula (2) represents a single bond. Further, the divalent group represented by the formula (4) can be bonded through an NHCOO bond when the capping agent or the ligand has a hydroxyl group. In this case, Z ¹ in formula (1) or Z ² in formula (2) represents a carbonyl group. Further, the divalent group represented by the formula (4) can be bonded through an NHCONH bond when the capping agent or the ligand has an amino group. In this case, Z ¹ in formula (1) or Z ² in formula (2) represents a carbonyl group.
Further, the divalent group represented by the formula (4) may be bonded to a capping agent or a ligand via a spacer composed of the divalent group represented by the formula (11). In this case, the formula (1) Z ^{1 in the} formula or Z ² in the formula ( ² ) is represented by the formula (11), and in the formula (11), Z ³ represents a carbonyl group.

The divalent group represented by the formula (5) is a divalent group having h ethylene glycol units, and has carbonyl groups at both ends. Accordingly, the divalent group can be bonded to X ¹ in formula (1) or X ² in formula (2) by an amide bond. Specifically, when X ¹ or X ² is a divalent group represented by any one of formulas (7) to (10), X ⁴ represents NH.
Further, it is used when the divalent group represented by the formula (5) is bonded to a capping agent or a ligand, and the capping agent or ligand has an amino group or a hydroxyl group, and can be bonded through an amide bond or an ester bond. In this case, Z ¹ in formula (1) or Z ² in formula (2) represents a single bond.
Further, the divalent group represented by the formula (5) may be bonded to a capping agent or a ligand via a spacer composed of the divalent group represented by the formula (11). In this case, the formula (1) Z ^{1 in the} formula or Z ² in the formula ( ² ) is represented by the formula (11), and in the formula (11), Z ³ represents an NH group.

The divalent group represented by the formula (6) is a divalent group having k ethylene glycol units, and has NH and a carbonyl group at the terminal. Accordingly, the divalent group can be bonded to X ¹ in formula (1) or X ² in formula (2) by an amide bond. Specifically, when X ¹ or X ² is a divalent group represented by any one of the formulas (7) to (10), X ⁴ in the formulas (7) to (10) represents a carbonyl group. To express.
In addition, the divalent group represented by the formula (6) is bonded to a capping agent or a ligand, and the capping agent or the ligand has an amino group or a hydroxyl group, and can be bonded via an amide bond or an ester bond. In this case, Z ¹ in formula (1) or Z ² in formula (2) represents a single bond.
Further, the divalent group represented by the formula (6) may be bonded to a capping agent or a ligand through a spacer composed of the divalent group represented by the formula (11). In this case, the formula (1) Z ^{1 in the} formula or Z ² in the formula ( ² ) is represented by the formula (11), and in the formula (11), Z ³ represents an NH group.

The polyethylene glycol moiety is represented by any one of the above formulas (3) to (6) having a carboxy group or an amino group at both ends of an ethylene glycol oligomer obtained by polymerizing 1 to 15, preferably 1 to 6 ethylene glycol. In this case, the divalent groups may be the same or different and 1 to 5 polymerized ones may be used. In this case, the divalent groups are selected from any combination capable of forming an amide bond with each other. obtain.
When Z ¹ in the formula (1) and Z ² in the formula (2) are represented by the formula (11), when the capping agent or the ligand has a carboxy group, Z ⁴ in the formula (11) represents NH. To express. When the capping agent or ligand has a hydroxyl group, Z ⁴ in the formula (11) represents a carbonyl group or NHCO. When the capping agent or ligand has an amino group, Z ⁴ in the formula (11) represents a carbonyl group or NHCO.

As the binding site between the hydrophilic resin and the polyethylene glycol moiety, specifically, the following formula:

（ｑは２～６の整数を表し、ｒは１～６の整数を表す）
で表される部分構造等が挙げられる。
　本発明の固相担体の好ましい態様として、上記式（１３）で表される構造を有する固相担体が挙げられる。式（１３）において、Ｙ²は、好ましくは上記式（３）～式（６）から選択される、同一もしくは異なる１～５個の二価基がアミド結合で結合して形成される二価基を表し、この場合、Ｙ²に含まれるポリエチレングリコールのユニット数は好ましくは１～６である。好ましくは、Ｙ²が式（３）～（６）のいずれかを表し、当該式（３）～（６）においてｂ、ｅ、ｈ及びｋが１～５、好ましくは１～３を表す。
　式（１３）において、Ｙ¹としては、好ましくは上記式（３）～式（６）から選択される同一もしくは異なる１～５個の二価基がアミド結合で結合して形成される二価基が挙げられ、この場合、Ｙ¹に含まれるポリエチレングリコールのユニット数は好ましくは２～３６である。好ましくは、Ｙ¹が式（３）～（６）のいずれかから選択される同一もしくは異なる任意の基が２～５個結合して形成される二価基を表し、当該式（３）～（６）においてｂ、ｅ、ｈ及びｋが１～５、好ましくは２～３を表す。

(Q represents an integer of 2 to 6, and r represents an integer of 1 to 6)
A partial structure represented by
A preferred embodiment of the solid phase carrier of the present invention includes a solid phase carrier having a structure represented by the above formula (13). In Formula (13), Y ² is preferably a divalent group formed by combining the same or different 1 to 5 divalent groups selected from the above Formula (3) to Formula (6) with an amide bond. In this case, the number of units of polyethylene glycol contained in Y ² is preferably 1-6. Preferably, Y ² represents any one of formulas (3) to (6), and b, e, h and k in formulas (3) to (6) represent 1 to 5, preferably 1 to 3.
In Formula (13), Y ¹ is preferably a divalent group formed by combining the same or different 1 to 5 divalent groups selected from Formulas (3) to (6) above with an amide bond. In this case, the number of units of polyethylene glycol contained in Y ¹ is preferably 2 to 36. Preferably, Y ¹ represents a divalent group formed by combining 2 to 5 arbitrary groups identical or different selected from any ^one of the formulas (3) to (6), In (6), b, e, h and k each represent 1 to 5, preferably 2 to 3.

The solid phase carrier of the present invention is a method well known to those skilled in the art by condensing a hydrophilic resin optionally having a spacer with a ligand and a hydrophobic capping agent optionally having a spacer. Can be manufactured.
Hereinafter, the method for producing the solid phase carrier of the present invention is specifically exemplified.
[Production Method 1]
The solid phase carrier of the present invention can be produced by the following steps:

（式中、「－Ｘ¹”－ＮＨＣＯ－Ｘ¹’－」は式（１）における「－Ｘ¹－」に相当する二価基であり、「－Ｘ¹”－ＮＨＣＯ－Ｘ²’－」は式（２）における「－Ｘ²－」に相当する二価基であり、Ｙ¹、Ｙ²、Ｚ¹及びＺ²は前記と同義である）。
　すなわち、アミノ基を有する担体、カルボキシ基を有するキャッピング剤及びカルボキシ基を有するリガンドを適当な溶媒中で混合し、脱水縮合剤を用いてアミド化反応を行うことにより、本発明の固相担体を製造することができる。
　ここで原料となるアミノ基を有する担体は、以下の工程で製造することができる：

(Wherein “—X ¹ ” —NHCO—X ¹ ′ — ”is a divalent group corresponding to“ —X ¹ — ”in formula (1), and“ —X ¹ ”—NHCO—X ² ′ — Is a divalent group corresponding to “—X ² —” in the formula (2), and Y ¹ , Y ² , Z ¹ and Z ² are as defined above.
That is, by mixing a carrier having an amino group, a capping agent having a carboxy group and a ligand having a carboxy group in an appropriate solvent, and performing an amidation reaction using a dehydrating condensing agent, the solid phase carrier of the present invention is obtained. Can be manufactured.
Here, the carrier having an amino group as a raw material can be produced by the following steps:

（式中Ｘ¹”は前記と同義であり、Ｌｇは臭素原子等の脱離基を表し、Ｐｇは保護基を表す）。
　すなわち、１）アルキル化反応（Ｌｇで表される脱離基が臭素原子やメタンスルホニルオキシ基等であり、担体とＸ¹”がエーテル結合をしている場合）、又はジシクロヘキシルカルボジイミド、1-エチル-3-(3'-ジメチルアミノプロピル）カルボジイミド（WSCD）もしくは2-(1H-7-アザベンゾトリアゾール-1-イル)-1,1,3,3-テトラメチルウロニウムヘキサフルオロフォスフェート　メタナミニウム（ＨＡＴＵ）等の縮合剤による脱水縮合反応（Ｌｇで表される脱離基が水酸基であり、担体とＸ¹”がエステル結合をしている場合）、２）アミノ基の保護基を除去する脱保護反応を経て製造することができる。

(Wherein X ¹ ″ is as defined above, Lg represents a leaving group such as a bromine atom, and Pg represents a protecting group).
That is, 1) alkylation reaction (when the leaving group represented by Lg is a bromine atom, a methanesulfonyloxy group or the like, and the carrier and X ¹ ″ have an ether bond), or dicyclohexylcarbodiimide, 1-ethyl -3- (3'-dimethylaminopropyl) carbodiimide (WSCD) or 2- (1H-7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate metanaminium ( Dehydration condensation reaction with a condensing agent such as HATU) (in the case where the leaving group represented by Lg is a hydroxyl group and the carrier and X ¹ ″ have an ester bond), 2) a deprotection to remove the amino protecting group It can be produced via a protective reaction.

[Production Method 2]
The solid phase carrier of the present invention can also be produced by the following steps using a carrier having a carboxy group:

（式中、「－Ｘ¹”－ＣＯＮＨ－Ｘ¹’－」は式（１）における「－Ｘ¹－」に相当する二価基であり、「－Ｘ¹”－ＣＯＮＨ－Ｘ²’－」は式（２）における「－Ｘ²－」に相当する二価基であり、Ｙ¹、Ｙ²、Ｚ¹及びＺ²は前記と同義である）。
　カルボキシ基を有する担体は、以下の工程で製造することができる：

(Wherein “—X ¹ ” —CONH—X ¹ ′ — ”is a divalent group corresponding to“ —X ¹ — ”in formula (1), and“ —X ¹ ”—CONH—X ² ′ — Is a divalent group corresponding to “—X ² —” in the formula (2), and Y ¹ , Y ² , Z ¹ and Z ² are as defined above.
A carrier having a carboxy group can be produced by the following steps:

（式中Ｘ¹”は前記と同義であり、Ｌｇは臭素原子等の脱離基を表し、Ｐｇは保護基を表す）。
　すなわち、１）アルキル化反応（Ｌｇで表される脱離基が臭素原子やメタンスルホニルオキシ基等であり、担体とＸ¹”がエーテル結合をしている場合）、又はジシクロヘキシルカルボジイミド、1-エチル-3-(3'-ジメチルアミノプロピル）カルボジイミド(WSCD)、もしくは2-(1H-7-アザベンゾトリアゾール-1-イル)-1,1,3,3-テトラメチルウロニウムヘキサフルオロフォスフェート　メタナミニウム（ＨＡＴＵ）等の縮合剤による脱水縮合反応（Ｌｇで表される脱離基が水酸基であり、担体とＸ¹”がエステル結合をしている場合）、２）カルボキシ基の保護基を除去する脱保護反応を経て製造することができる。
　あるいは、Ｘ¹”が－ＣＯ（ＣＨ₂）₂－を表す場合、無水コハク酸を用いてカルボキシ基を有する担体を製造することもできる。

(Wherein X ¹ ″ is as defined above, Lg represents a leaving group such as a bromine atom, and Pg represents a protecting group).
That is, 1) alkylation reaction (when the leaving group represented by Lg is a bromine atom, a methanesulfonyloxy group or the like, and the carrier and X ¹ ″ have an ether bond), or dicyclohexylcarbodiimide, 1-ethyl -3- (3'-dimethylaminopropyl) carbodiimide (WSCD) or 2- (1H-7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate metanaminium Dehydration condensation reaction with a condensing agent such as (HATU) (when the leaving group represented by Lg is a hydroxyl group and the carrier and X ¹ ″ have an ester bond), 2) The protecting group for the carboxy group is removed It can be produced through a deprotection reaction.
Alternatively, when X ¹ ″ represents —CO (CH ₂ ) ₂ —, a carrier having a carboxy group can be produced using succinic anhydride.

[Production Method 3]
Next, a method for producing an intermediate in which a polyethylene glycol moiety is introduced into a capping agent or a ligand will be described.
When the capping agent has a hydroxyl group, an intermediate having a polyethylene glycol moiety introduced into the capping agent can be produced, for example, by the following steps.

（式中Ｙ²及びＰｇは前記と同義である）。
　すなわち、キャッピング剤上の水酸基を、トリホスゲン等の試薬を用いてクロロ蟻酸化し、次いで塩基の存在下にPg-Y²-Hで表される化合物のアミノ基もしくはヒドロキシ基と反応させた後保護基を脱保護する。
　リガンドが水酸基を有する場合、リガンドにポリエチレングリコール部分を導入した中間体は、前記と同様の方法で製造できる。
　キャッピング剤がカルボキシ基を有する場合、キャッピング剤にポリエチレングリコール部分を導入した中間体は、例えば以下の工程で製造できる：

(Wherein Y ² and Pg are as defined above).
That is, the hydroxyl group on the capping agent is subjected to chloroformate oxidation using a reagent such as triphosgene, and then reacted with the amino group or hydroxy group of the compound represented by Pg-Y ² -H in the presence of a base, followed by protection. Deprotect the group.
When the ligand has a hydroxyl group, an intermediate having a polyethylene glycol moiety introduced into the ligand can be produced by the same method as described above.
When the capping agent has a carboxy group, an intermediate having a polyethylene glycol moiety introduced into the capping agent can be produced, for example, by the following steps:

（式中Ｙ²及びＰｇは前記と同義である）。
　すなわち、キャッピング剤を、塩基及びジシクロヘキシルカルボジイミド、WSCDもしくはHATU等の脱水縮合剤の存在下にPg-Y²-Hで表される化合物のアミノ基もしくはヒドロキシ基と、反応させた後、保護基を脱保護する。
　リガンドがカルボキシ基を有する場合、リガンドにポリエチレングリコール部分を導入した中間体は前記と同様の方法で製造できる。
　キャッピング剤がアミノ基を有する場合、キャッピング剤にポリエチレングリコール部分を導入した中間体は、例えば以下の工程で製造できる：

(Wherein Y ² and Pg are as defined above).
That is, the capping agent is reacted with the amino group or hydroxy group of the compound represented by Pg-Y ² —H in the presence of a base and a dehydrating condensing agent such as dicyclohexylcarbodiimide, WSCD or HATU, and then the protective group is removed. Deprotect.
When the ligand has a carboxy group, an intermediate having a polyethylene glycol moiety introduced into the ligand can be produced by the same method as described above.
When the capping agent has an amino group, an intermediate having a polyethylene glycol moiety introduced into the capping agent can be produced, for example, by the following steps:

（式中Ｙ²及びＰｇは前記と同義である）。
　すなわち、キャッピング剤を、塩基及びジシクロヘキシルカルボジイミド、WSCDもしくはHATU等の脱水縮合剤の存在下にPg-Y²-OHで表される化合物のカルボキシ基と反応させた後、保護基を脱保護する。
　リガンドがアミノ基を有する場合、リガンドにポリエチレングリコール部分を導入した中間体は前記と同様の方法で製造できる。
　尚、上記製造方法３に記載された、アミド化（エステル化）反応及び脱保護反応を適宜繰り返すことにより、より高分子量のポリエチレングリコール部分を導入することができる。

(Wherein Y ² and Pg are as defined above).
That is, the capping agent is reacted with the carboxy group of the compound represented by Pg—Y ² —OH in the presence of a base and a dehydrating condensing agent such as dicyclohexylcarbodiimide, WSCD or HATU, and then the protective group is deprotected.
When the ligand has an amino group, an intermediate having a polyethylene glycol moiety introduced into the ligand can be produced by the same method as described above.
A higher molecular weight polyethylene glycol moiety can be introduced by appropriately repeating the amidation (esterification) reaction and deprotection reaction described in Production Method 3 above.

[Production Method 4]
The solid phase carrier of the present invention can also immobilize the capping agent and the ligand after introducing a polyethylene glycol moiety into the carrier. For example, the following formula:

（式中、Ｘ¹”、Ｘ¹’、Ｘ²’、Ｙ¹、Ｙ²、Ｚ¹、Ｚ²及びＬｇは前記と同義であり、Ｐｇ¹及びＰｇ²は異なる条件で脱保護可能な保護基を表す）
で示される方法で製造できる。
　すなわち、上記製造方法１～３に記載のアミド化反応及び脱保護反応を行うことにより、ポリエチレングリコール部分を有する固相担体を製造し、これにリガンド及びキャッピング剤を反応させることができる。
　また、カルボキシ基を有する担体から出発して、同様の方法で本発明の固相担体を製造することもできる。

(Wherein, X ¹ ″, X ¹ ′, X ² ′, Y ¹ , Y ² , Z ¹ , Z ² and Lg have the same meanings as described above, and Pg ¹ and Pg ² are protections that can be deprotected under different conditions. Represents a group)
Can be produced by the method shown in FIG.
That is, by performing the amidation reaction and the deprotection reaction described in the above production methods 1 to 3, a solid phase carrier having a polyethylene glycol moiety can be produced, and a ligand and a capping agent can be reacted therewith.
Further, starting from a carrier having a carboxy group, the solid phase carrier of the present invention can be produced by the same method.

Another embodiment of the present invention is a method for purifying a ligand-specific binding protein using the solid phase carrier of the present invention.
That is, the present invention provides a method for concentrating, isolating or purifying a ligand-specific binding protein comprising the following steps (a) to (b):
(A) contacting the solid phase carrier according to [1] and a sample; and (b) eluting the ligand-specific binding protein from the solid phase carrier.
The above purification method is suitable when the ligand-specific protein is a membrane-bound protein. That is, the “sample” used in the purification method is not limited in purity and the like as long as it is a sample containing a membrane-bound protein, but the purification method of the present invention is a membrane-bound protein bound to a cell membrane. It is particularly useful for purifying samples containing Accordingly, examples of the sample include human and animal-derived biological tissues and organ-derived cell membrane fractions (including both crude and unpurified samples).

Hereinafter, each step of the purification method of the present invention will be described in detail.
[Step (a)]
The solid phase carrier and the sample are usually contacted in a buffer solution (buffer). Examples of the buffer used here include MES buffer, HEPES buffer, Tris-hydrochloric acid buffer, MOPS buffer, and phosphoric acid (Phosphate) buffer.
The pH of the buffer is adjusted to 5.0 to 9.0, preferably 6.0 to 8.0.
The concentration of the sample at the time of contacting with the solid phase carrier is not particularly limited, but in the case of a protein mixture (lysate), that is, a protein mixture extracted from tissue or cells, 0.1 mg / ml to 10 mg / ml is preferable, and more preferable. Is 0.5 mg / ml to 5 mg / ml.
The time for contacting the sample and the solid phase carrier is not particularly limited, but the membrane-bound protein contained in the sample can be adsorbed to the solid phase carrier usually by contacting for 10 minutes to 24 hours. The contact is usually made at 4 to 37 ° C.
After the sample is adsorbed on the solid phase carrier, the solid phase carrier is washed 3 to 20 times with a washing buffer. As the washing buffer, the same one as that used when the sample is contacted is usually used.

[Step (b)]
For the elution of a membrane-bound protein that specifically binds to a ligand, an aqueous solution or buffer in which the ligand is usually dissolved at a high concentration can be used. If necessary, 1 to 70% of a hydrophilic organic solvent (including acetonitrile and isopropanol) may be added. Further, 1% to 5% SDS or 0.1% to 10% surfactant (for example, chaps, chapsoo, octyl glucoside, dodecyl maltoside, triton, etc.) may be added as appropriate. Specifically, a buffer having a composition comprising 0.1 M Tris-HCl (pH 6.8), 2% SDS, 20% Glycerol, 0.2% BPB, and 0.2M DTT can be exemplified.

Alternatively, the adsorbed membrane-bound protein can be eluted using an aqueous solution or buffer containing 1% to 5% SDS or an aqueous solution containing 0.1% to 10% surfactant. It is also possible to elute with 8M urea, 6M guanidine hydrochloride and the like.
The pH of the eluate is not particularly limited and may be any of acidic conditions (pH 2.0 to 5.0), alkaline conditions (pH 9.0-11.0), and neutral conditions (pH 5.0-9.0). It is preferably eluted under neutral conditions. The elution temperature is 4 ° C to 95 ° C.
The eluted membrane-bound protein can be detected by methods known to those skilled in the art, such as Western blotting. That is, the eluted protein solution is subjected to SDS-PAGE, the protein is transferred to a PVDF membrane or the like, and the band obtained using an antibody or the like is analyzed. Alternatively, the eluted protein solution may be subjected to SDS-PAGE, and the target band is cut out and analyzed. Specifically, the target band is cut out from the gel, and the peptide obtained by the in-gel trypsin digestion method can be analyzed by mass spectrometry.
Alternatively, a protein containing the peptide fragment obtained by treating with a digestive enzyme such as trypsin without eluting the protein bound to the resin can be subjected to mass spectral analysis.

As another aspect of the present invention, the solid phase carrier of the present invention can be used to search for a ligand compound that specifically binds to a target protein. That is, whether a specific membrane protein, a solid phase carrier of the present invention having a known ligand that specifically binds to the protein, and a test substance are contacted, and the test substance antagonizes specific binding between the protein and the known ligand. By examining whether or not, it is possible to evaluate whether or not the test substance is a ligand that specifically binds to the protein. Specifically, a specific ligand of the target protein can be identified by the following steps (a) to (b):
(A) contacting the solid phase carrier according to [1], a target protein, and a test substance having a known ligand that specifically binds to the target protein;
(B) eluting the target protein from the solid phase carrier;
(C) a step of detecting the eluted target protein; and (d) a step of determining a test substance having a small target protein detection amount as a target protein-specific ligand based on the detection result of (c). .

As another embodiment of the present invention, the formula (12):

（ａ、ｂ及びｃは、式（３）と同義である）
で表わされる化合物またはその塩が挙げられる。当該化合物またはその塩は、γ－セクレターゼを精製するためのアフィニティークロマトグラフィー用固相担体の製造中間体として有用である。すなわち式（１２）で表わされる化合物又はその塩は、γ－セクレターゼのリガンドとする本発明の固相担体を製造するためのスペーサーを導入した化合物に相当する。
　前記塩は、当業者に周知の塩であれば特に限定は無く、有機酸塩又は無機酸塩が用いられる。具体的には塩酸塩、臭化水素酸塩、硫酸塩、酢酸塩、トリフルオロ酢酸塩等を例示することができる。
　式（１２）で表わされる化合物は、実施例１に記載の方法に準じて製造することができる。

(A, b, and c are synonymous with Formula (3).)
Or a salt thereof. The compound or a salt thereof is useful as an intermediate for producing a solid phase carrier for affinity chromatography for purifying γ-secretase. That is, the compound represented by the formula (12) or a salt thereof corresponds to a compound into which a spacer for producing the solid phase carrier of the present invention as a ligand for γ-secretase is introduced.
The salt is not particularly limited as long as it is a salt well known to those skilled in the art, and an organic acid salt or an inorganic acid salt is used. Specific examples include hydrochloride, hydrobromide, sulfate, acetate, trifluoroacetate and the like.
The compound represented by the formula (12) can be produced according to the method described in Example 1.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not necessarily limited thereto.
The abbreviations used in this specification are as follows:
iPr ₂ NEt: Diisopropylethylamine
AcOEt: Ethyl acetate
DMF: Dimethylformamide
WSCD: Water-soluble carbodiimide (1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide)
HOBt: N-hydroxybenzotriazole
Boc: tert-butoxycarbonyl
DAPT: N- [N- (3,5-difluorophenylacetyl) -L-alanyl] -S-phenylglycine-t-butyl ester
HATU: 2- (1H-7-Azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate metanaminium (2- (1H-7-Azabenzotriazol-1-yl)- 1,1,3,3-tetramethyl uronium hexafluorophosphate Methanaminium)

Example 1

(1) Synthesis of cholesterol-PEG (1) -NH ₂ (1-4)

　クロロぎ酸コレステロール（１－１）（東京化成、Cat.no.C0694；637ｍｇ，1.42mmol)をジクロロメタン６０ｍｌに溶かし、氷冷下攪拌した。Mono-N-t-Boc-amido-dPEG3TM-amine（１－２） (QUANTA BIODESIGN製; 500mg,1.56mmol) をジクロロメタン１ｍｌに溶かし、ゆっくりと滴下した。ジイソプロピルエチルアミン（494μl, 2.84mmol) を加え、室温にて一昼夜攪拌した。減圧濃縮後、酢酸エチル２００ｍｌを加えた。酢酸エチル層を水１００ｍｌ、飽和食塩水にて洗浄した。無水硫酸マグネシウムにて乾燥後、減圧濃縮し、シリカゲルカラムクロマトグラフィーにて精製した。目的のBoc体（１－３）(770mg)を収率７４％にて得た。質量分析結果：ＭS（ｍ／ｚ）：　７３３（ＭＨ＋）。
　得られたBoc体（１－３）（770mg, 1.05mmol) を酢酸エチル６０ｍｌに溶かし、4N-HCl/AcOEt (国産化学製;５ｍｌ) を加え、室温にて５時間攪拌した。反応完結後、溶媒を留去し、クロロホルム５０ｍｌを加え、減圧下溶媒を留去した。この操作を３回繰り返した。真空ポンプにて溶媒を完全に留去し、目的のアミノ体塩酸塩（１－４）（703mg）を定量的に得た。質量分析結果：ＭS（ｍ／ｚ）：　６３３（ＭＨ＋）。
　
　以下に、製造した化合物の1H-NMRデータを示した。
式（１－３）の化合物：
¹H-NMR (CDCl₃) δ0.67(s, 3H), 0.86 (d, J=6.6Hz, 3H), 0.87 (d, J=6.6Hz, 3H), 0.91 (d, J=6.4Hz, 3H), 0.94-1.06 (m, 3H), 1.00 (s, 3H), 1.06-1.18 (m, 5H), 1.30-1.40 (m, 3H), 1.40-1.45 (m, 2H), 1.44 (s, 9H), 1.45-1.60 (m, 7H), 1.76 (m, 4H), 1.84 (m, 1H), 1.86 (m, 2H), 1.94 (m, 1H), 2.00 (m, 2H), 2.27 (br d, J=13.1, 5.1, 1.9Hz, 1H), 2.35 (ddd, J=13.1, 5.1, 1.9Hz, 1H), 3.22(br t, J=6.1Hz, 2H), 3.27 (m, 2H), 3.51-3.57(m, 4H), 3.57-3.62 (m, 4H), 3.62-3.66 (m, 4H), 4.48 (m, 1H), 5.36 (m, 1H), 4.95 (br s, 1H), 5.14 (br s, 1H); 
HRMS-ESI (m/z): [M+H]+calcd. for C₄₃H₇₇O₇N₂, 733.5725; found, 733.5724.
式（１－４）の化合物：
¹H-NMR (CDCl₃) δ0.67 (s, 3H), 0.86 (d, J=6.6Hz, 3H), 0.87 (d, J=6.6Hz, 3H), 0.88-0.94 (m, 1H), 0.91 (d, J=6.6Hz, 3H), 0.94-1.06 (m, 3H), 1.00 (s, 3H), 1.04-1.20 (m, 5H), 1.20-1.40 (m, 4H), 1.40-1.60 (m, 6H), 1.76-1.90 (m, 4H), 1.80 (m, 2H), 1.92-2.12 (m, 3H), 2.06 (m, 2H), 2.29 (br dd, J=13.1, 2.3Hz, 1H), 2.34 (ddd, J=13.1, 5.2, 1.7Hz, 1H), 3.20-3-30 (m, 4H), 3.58 (m, 2H), 3.64 (m, 6H), 3.73 (m, 4H), 4.44 (m, 1H), 5.35 (m, 1H), 8.05-8.30 (m, 3H);
HRMS-ESI (m/z): [M+H]+calcd. for C₃₈H₆₉O₅N₂, 633.5201; found, 633.5192

Cholesterol chloroformate (1-1) (Tokyo Kasei, Cat. No. C0694; 637 mg, 1.42 mmol) was dissolved in 60 ml of dichloromethane and stirred under ice cooling. Mono-Nt-Boc-amido-dPEG3TM-amine (1-2) (manufactured by QUANTA BIODESIGN; 500 mg, 1.56 mmol) was dissolved in 1 ml of dichloromethane and slowly added dropwise. Diisopropylethylamine (494 μl, 2.84 mmol) was added, and the mixture was stirred overnight at room temperature. After concentration under reduced pressure, 200 ml of ethyl acetate was added. The ethyl acetate layer was washed with 100 ml of water and saturated brine. The extract was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography. The desired Boc form (1-3) (770 mg) was obtained in a yield of 74%. Mass spectral result: MS (m / z): 733 (MH +).
The obtained Boc form (1-3) (770 mg, 1.05 mmol) was dissolved in 60 ml of ethyl acetate, 4N-HCl / AcOEt (made by Kokusan Chemical; 5 ml) was added, and the mixture was stirred at room temperature for 5 hours. After completion of the reaction, the solvent was distilled off, 50 ml of chloroform was added, and the solvent was distilled off under reduced pressure. This operation was repeated three times. The solvent was completely distilled off with a vacuum pump to quantitatively obtain the desired amino acid hydrochloride (1-4) (703 mg). Mass spectral result: MS (m / z): 633 (MH +).

The 1H-NMR data of the produced compound are shown below.
Compound of formula (1-3):
¹ H-NMR (CDCl ₃ ) δ0.67 (s, 3H), 0.86 (d, J = 6.6Hz, 3H), 0.87 (d, J = 6.6Hz, 3H), 0.91 (d, J = 6.4Hz, 3H), 0.94-1.06 (m, 3H), 1.00 (s, 3H), 1.06-1.18 (m, 5H), 1.30-1.40 (m, 3H), 1.40-1.45 (m, 2H), 1.44 (s, 9H), 1.45-1.60 (m, 7H), 1.76 (m, 4H), 1.84 (m, 1H), 1.86 (m, 2H), 1.94 (m, 1H), 2.00 (m, 2H), 2.27 (br d, J = 13.1, 5.1, 1.9Hz, 1H), 2.35 (ddd, J = 13.1, 5.1, 1.9Hz, 1H), 3.22 (br t, J = 6.1Hz, 2H), 3.27 (m, 2H), 3.51-3.57 (m, 4H), 3.57-3.62 (m, 4H), 3.62-3.66 (m, 4H), 4.48 (m, 1H), 5.36 (m, 1H), 4.95 (br s, 1H), 5.14 (br s, 1H);
HRMS-ESI (m / z): [M + H] + calcd.for C ₄₃ H ₇₇ O ₇ N ₂ , 733.5725; found, 733.5724.
Compound of formula (1-4):
¹ H-NMR (CDCl ₃ ) δ0.67 (s, 3H), 0.86 (d, J = 6.6Hz, 3H), 0.87 (d, J = 6.6Hz, 3H), 0.88-0.94 (m, 1H), 0.91 (d, J = 6.6Hz, 3H), 0.94-1.06 (m, 3H), 1.00 (s, 3H), 1.04-1.20 (m, 5H), 1.20-1.40 (m, 4H), 1.40-1.60 ( m, 6H), 1.76-1.90 (m, 4H), 1.80 (m, 2H), 1.92-2.12 (m, 3H), 2.06 (m, 2H), 2.29 (br dd, J = 13.1, 2.3Hz, 1H ), 2.34 (ddd, J = 13.1, 5.2, 1.7Hz, 1H), 3.20-3-30 (m, 4H), 3.58 (m, 2H), 3.64 (m, 6H), 3.73 (m, 4H), 4.44 (m, 1H), 5.35 (m, 1H), 8.05-8.30 (m, 3H);
HRMS-ESI (m / z): [M + H] + calcd. For C ₃₈ H ₆₉ O ₅ N ₂ , 633.5201; found, 633.5192

(2) Synthesis of DAPT-PEG (2) -NH ₂ (1-11)

　DAPT（１－５）（5.0ｇ、11.56mmol）をジクロロメタン１００ｍｌに溶かし室温にて攪拌した。TFA１０ｍｌをゆっくりと滴下し、室温にて一昼夜攪拌した。溶媒を留去し、クロロホルム５０ｍｌを加え、減圧下溶媒を留去した。この操作を２回繰り返した。析出した結晶をジエチルエーテルにて洗浄し、結晶を濾取した。減圧下乾燥し、目的のカルボン酸体（１－６）(4.28g)を９８．４％にて得た。
カルボン酸体（１－６）（978mg, 2.60mmol) をジクロロメタン４０ｍｌに溶かし、窒素気流下、４℃にて攪拌した。WSCD (546μl, 3.12mmol), HOBt (420mg, 3.12mmol) をゆっくりと加え、室温にて６０分間攪拌した。再度、４℃にて攪拌後、 Mono-N-t-Boc-amido-dPEG3TM-amine (QUANTA BIODESIGN; 1.0g, 3.12mmol) をジクロロメタン2ml に溶かし、ゆっくりと滴下した。ジイソプロピルエチルアミン（542μl, 3.12mmol) を加え、室温にて60分間攪拌した。溶媒を留去後、反応液を酢酸エチル１００ｍｌに溶かし、水１００ｍｌ、飽和食塩水１００ｍｌにて洗浄した。無水硫酸マグネシウムにて乾燥後、減圧濃縮した。
　シリカゲルカラムクロマトグラフィーにて精製し、目的のBoc体（１－７）(1.14g)を収率６４．６％にて得た。質量分析結果: ＭS（ｍ／ｚ）：　６７９（ＭＨ＋）。
　得られたBoc体（１－７）（1.14g, 1.68mmol) を酢酸エチル５０ｍｌ、クロロホルム５０ｍｌの混合溶媒に溶かし、4M(N)-HCl/AcOEt (国産化学製;10ml) を加え、室温にて一昼夜攪拌した。反応完結後、溶媒を留去し、クロロホルム３０ｍｌを加え、減圧下溶媒を留去した。この操作を３回繰り返した。真空ポンプにて溶媒を完全に留去し、目的のアミノ体塩酸塩（１－８）(1.0g)を収率９７％にて得た。質量分析結果：ＭS（ｍ／ｚ）：　５７９（ＭＨ＋）。
　N-t-Boc-amido-d-PEG4TM-acid (QUANTA BIODESIGN,; 715mg, 1.96mmol)（１－９）をジクロロメタン(70ml)及びクロロホルム(10ml)の混合溶媒に溶かし、窒素気流下４℃にて攪拌した。 WSCD (343μl, 1.96mmol), HOBt (264mg, 1.96mmol) をゆっくりと加え、室温にて６０分間攪拌した。再度、４℃にて攪拌後、アミノ体塩酸塩（１－８） （1.0g，1.63mmol) をジクロロメタン１０ｍｌ，クロロホルム５ｍｌの混合溶媒に溶かし、反応系中に滴下した。ジイソプロピルエチルアミン（681μl, 3.91mmol) を加え、室温にて60分間攪拌した。溶媒を留去後、クロロホルム１００ｍｌを加え、水５０ｍｌ、飽和食塩水５０ｍｌにて洗浄した。無水硫酸マグシウムにて乾燥し、減圧濃縮した。
　シリカゲルカラムクロマトグラフィーにて精製し、目的のBoc体（１－１０）(1.07g)を収率７０．９％にて得た。質量分析結果：ＭS(ｍ/z): ９２６（ＭＨ＋）。
　得られたBoc体（１－１０）（1.0g, 1.08mmol)をクロロホルム(60ml)－酢酸エチル(20ml)の混合溶媒に溶かし、4M(N)-HCl/AcOEt (国産化学製;10ml) を加え、室温にて１時間攪拌した。反応完結後、溶媒を留去し、クロロホルム(30ml)を加え、減圧下溶媒を留去した。この操作を３回繰り返した。真空ポンプにて溶媒を完全に留去し、目的のアミノ体塩酸塩（１－１１）(931mg)を定量的に得た。質量分析結果: MS (m/z): ８２６（ＭＨ＋）。
尚原料となるＤＡＰＴは、公知化合物であり、J. Neurochem. 2001, 76, p173、Org. Biomol. Chem., 2005, 3, p2450-2457、及びBioorganic & Medicinal Chemistry Letters., 2004, 14, p1983-1985に記載の方法で製造できる。また、市販品（シグマ社、Cat. No. D5942)を入手することもできる。

　以下に、製造した化合物の1H-NMRデータを示した。
式（１－７）の化合物：
. ¹H-NMR (DMSO-d₆)δ1.21 (d, J=7.1Hz, 3H), 1.36 (s, 9H), 1.52-1.62 (m, 4H), 2.94 (q, J=6.6Hz, 2H), 3.07 (m, 2H), 3.26-3.54 (m, 14H), 4.40 (quin, J=7.1Hz, 1H), 5.35 (d, J=8.1Hz, 1H), 6.73 (br t, J=6.6Hz, 1H), 6.92-7.00 (m, 1H), 7.06 (tt, J=9.5, 2.4Hz, 1H), 7.22-7.36 (m, 5H), 8.19 (t, J=5.6Hz, 1H), 8.35 (d, J=7.1Hz, 1H), 8.38 (d, J=8.1Hz, 1H);
HRMS-ESI (m/z): [M+H]+calcd. for C₃₄H₄₉O₈N₄F₂, 679.3513; found, 679.3519
式（１－８）の化合物：
 ¹H-NMR (DMSO-d₆) δ1.22 (d, J=7.1Hz, 3H), 1.58 (quin, J=6.7Hz, 2H), 1.76 (quin, J=6.7Hz, 2H), 2.83 (t, J=6.7Hz, 2H), 3.08 (m, 2H), 3.30 (t, J=6.7Hz, 2H), 3.38-3.54 (m, 12H), 4.40 (quin, J=7.1Hz, 1H), 5.36 (d, J=7.8Hz, 1H), 6.94-7.00 (m, 2H), 7.07 (tt, J=9.4, 2.4Hz, 1H), 7.22-7.38 (m, 5H), 7.69 (br s, 2H), 8.23 (t, J=5.6Hz, 1H), 8.381 (d, J=7.8Hz, 1H), 8.383 (d, J=7.1Hz, 1H);
HRMS-ESI (m/z): [M+H]+calcd. for C₂₉H₄₁O₆N₄F₂, 579.2989; found, 579.2983.
式(1-10)の化合物：
¹H-NMR (DMSO-d₆)δ1.22 (d, J=7.2Hz, 3H), 1.37 (s, 9H), 1.54-1.64 (m, 4H), 2.28 (t, J=6.5Hz, 2H), 3.02-3.10 (m, 10H), 3.26-3.54 (m, 28H), 3.57 (t, J=6.5Hz, 2H), 4.41 (quin, J=7.2Hz, 1H), 5.36 (d, J=7.8Hz, 1H), 6.74 (t, J=5.4Hz, 1H), 6.85-7.00 (m, 2H), 7.07 (tt, J=9.5, 2.4Hz, 1H), 7.20-7.40 (m, 5H), 7.78 (t, J=5.6Hz, 1H), 8.21 (t, J=5.6Hz, 1H), 8.36 (d, J=7.2Hz, 1H), 8.39 (d, J=7.8Hz, 1H);
HRMS-ESI (m/z): [M+H]+calcd. for C₄₅H₇₀O₁₃N₅F₂, 926.4933; found, 926.4945.
式(1-11)の化合物：
¹H-NMR (DMSO-d₆)δ1.22 (d, J=7.1Hz, 3H), 1.52-1.64 (m, 4H), 2.29 (t, J=6.5Hz, 2H), 2.97 (q, J=5.5Hz, 2H), 3.02-3.12 (m, 4H), 3.26-3.62 (m, 30H), 4.40 (quin, J=7.1Hz, 1H), 5.36 (d, J=7.8Hz, 1H), 6.94-7.00 (m, 2H), 7.07 (tt, J=9.5, 2.4Hz, 1H), 7.22-7.38 (m, 5H), 7.77 (br s, 2H), 7.82 (t, J=6.5Hz, 1H), 8.22 (t, J=5.5Hz, 1H), 8.37 (d, J=7.1Hz, 1H), 8.38 (d, J=7.8Hz, 1H);
HRMS-ESI (m/z): [M+H]+calcd. for C₄₀H₆₂O₁₁N₅F₂, 826.4408; found, 826.4414.

式(1-7)の化合物の製造例（別法）
カルボン酸体（１－６）（587mg, 1.56mmol) Mono-N-t-Boc-amido-dPEG3TM-amine (QUANTA BIODESIGN; 500mg, 1.56mmol) をDMF10ml に溶かし、ジイソプロピルエチルアミン（300μl, 1.72mmol) を加え、氷冷下攪拌した。HATU（渡辺化学工業, Cat, no, A01695; 652mg, 1.72mmol）を加え氷冷下３時間攪拌した。 クロロホルム１５０ｍｌを加え、有機層を水１００ｍｌにて２回、飽和食塩水１００ｍｌにて洗浄した。無水硫酸マグネシウムにて乾燥後、減圧濃縮した。
　シリカゲルカラムクロマトグラフィーにて精製し、目的のBoc体（１－７）(656mg)を収率６２％にて得た。
　同様に、縮合剤としてWSCD及びHOBTのかわりにHATUを用いて、式（１－１０）、式（１－１３）、式（１－１４）、式（１－１９）、式（１－２０）、式（１－２１）、式（１－２２）、式（６－３）、式（６－４）及び式（９－１）化合物を合成することができる。

DAPT (1-5) (5.0 g, 11.56 mmol) was dissolved in 100 ml of dichloromethane and stirred at room temperature. 10 ml of TFA was slowly added dropwise and stirred at room temperature for a whole day and night. The solvent was distilled off, 50 ml of chloroform was added, and the solvent was distilled off under reduced pressure. This operation was repeated twice. The precipitated crystals were washed with diethyl ether, and the crystals were collected by filtration. It dried under reduced pressure and obtained the target carboxylic acid compound (1-6) (4.28 g) at 98.4%.
Carboxylic acid compound (1-6) (978 mg, 2.60 mmol) was dissolved in 40 ml of dichloromethane and stirred at 4 ° C. under a nitrogen stream. WSCD (546 μl, 3.12 mmol) and HOBt (420 mg, 3.12 mmol) were slowly added and stirred at room temperature for 60 minutes. After stirring again at 4 ° C., Mono-Nt-Boc-amido-dPEG3TM-amine (QUANTA BIODESIGN; 1.0 g, 3.12 mmol) was dissolved in 2 ml of dichloromethane and slowly added dropwise. Diisopropylethylamine (542 μl, 3.12 mmol) was added, and the mixture was stirred at room temperature for 60 minutes. After the solvent was distilled off, the reaction solution was dissolved in 100 ml of ethyl acetate and washed with 100 ml of water and 100 ml of saturated brine. The extract was dried over anhydrous magnesium sulfate and concentrated under reduced pressure.
Purification by silica gel column chromatography gave the target Boc form (1-7) (1.14 g) in a yield of 64.6%. Mass spectral result: MS (m / z): 679 (MH +).
The obtained Boc isomer (1-7) (1.14 g, 1.68 mmol) was dissolved in a mixed solvent of 50 ml of ethyl acetate and 50 ml of chloroform, and 4M (N) -HCl / AcOEt (manufactured by Kokusan Chemical; 10 ml) was added, Stir all day and night. After completion of the reaction, the solvent was distilled off, 30 ml of chloroform was added, and the solvent was distilled off under reduced pressure. This operation was repeated three times. The solvent was completely distilled off with a vacuum pump to obtain the desired amino hydrochloride (1-8) (1.0 g) in a yield of 97%. Mass spectral result: MS (m / z): 579 (MH +).
Nt-Boc-amido-d-PEG4TM-acid (QUANTA BIODESIGN ,; 715mg, 1.96mmol) (1-9) is dissolved in a mixed solvent of dichloromethane (70ml) and chloroform (10ml) and stirred at 4 ° C under a nitrogen stream. did. WSCD (343 μl, 1.96 mmol) and HOBt (264 mg, 1.96 mmol) were slowly added and stirred at room temperature for 60 minutes. After stirring again at 4 ° C., amino hydrochloride (1-8) (1.0 g, 1.63 mmol) was dissolved in a mixed solvent of 10 ml of dichloromethane and 5 ml of chloroform, and dropped into the reaction system. Diisopropylethylamine (681 μl, 3.91 mmol) was added, and the mixture was stirred at room temperature for 60 minutes. After distilling off the solvent, 100 ml of chloroform was added, followed by washing with 50 ml of water and 50 ml of saturated saline. The extract was dried over anhydrous magnesium sulfate and concentrated under reduced pressure.
Purification by silica gel column chromatography gave the target Boc (1-10) (1.07 g) in a yield of 70.9%. Mass spectral result: MS (m / z): 926 (MH +).
The obtained Boc form (1-10) (1.0 g, 1.08 mmol) was dissolved in a mixed solvent of chloroform (60 ml) -ethyl acetate (20 ml), and 4M (N) -HCl / AcOEt (made by Kokusan Chemical; 10 ml) was added. The mixture was further stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off, chloroform (30 ml) was added, and the solvent was distilled off under reduced pressure. This operation was repeated three times. The solvent was completely distilled off with a vacuum pump to quantitatively obtain the desired amino hydrochloride (1-11) (931 mg). Mass spectral result: MS (m / z): 826 (MH +).
DAPT as a raw material is a known compound, and is described in J. Neurochem. 2001, 76, p173, Org. Biomol. Chem., 2005, 3, p2450-2457, and Bioorganic & Medicinal Chemistry Letters., 2004, 14, p1983. -Can be produced by the method described in 1985. Commercial products (Sigma, Cat. No. D5942) can also be obtained.

The 1H-NMR data of the produced compound are shown below.
Compound of formula (1-7):
¹ H-NMR (DMSO-d ₆ ) δ1.21 (d, J = 7.1Hz, 3H), 1.36 (s, 9H), 1.52-1.62 (m, 4H), 2.94 (q, J = 6.6Hz, 2H), 3.07 (m, 2H), 3.26-3.54 (m, 14H), 4.40 (quin, J = 7.1Hz, 1H), 5.35 (d, J = 8.1Hz, 1H), 6.73 (br t, J = 6.6Hz, 1H), 6.92-7.00 (m, 1H), 7.06 (tt, J = 9.5, 2.4Hz, 1H), 7.22-7.36 (m, 5H), 8.19 (t, J = 5.6Hz, 1H), 8.35 (d, J = 7.1Hz, 1H), 8.38 (d, J = 8.1Hz, 1H);
HRMS-ESI (m / z): [M + H] + calcd. For C ₃₄ H ₄₉ O ₈ N ₄ F ₂ , 679.3513; found, 679.3519
Compound of formula (1-8):
¹ H-NMR (DMSO-d ₆ ) δ1.22 (d, J = 7.1Hz, 3H), 1.58 (quin, J = 6.7Hz, 2H), 1.76 (quin, J = 6.7Hz, 2H), 2.83 ( t, J = 6.7Hz, 2H), 3.08 (m, 2H), 3.30 (t, J = 6.7Hz, 2H), 3.38-3.54 (m, 12H), 4.40 (quin, J = 7.1Hz, 1H), 5.36 (d, J = 7.8Hz, 1H), 6.94-7.00 (m, 2H), 7.07 (tt, J = 9.4, 2.4Hz, 1H), 7.22-7.38 (m, 5H), 7.69 (br s, 2H ), 8.23 (t, J = 5.6Hz, 1H), 8.381 (d, J = 7.8Hz, 1H), 8.383 (d, J = 7.1Hz, 1H);
HRMS-ESI (m / z): [M + H] + calcd.for C ₂₉ H ₄₁ O ₆ N ₄ F ₂ , 579.2989; found, 579.2983.
Compound of formula (1-10):
¹ H-NMR (DMSO-d ₆ ) δ1.22 (d, J = 7.2Hz, 3H), 1.37 (s, 9H), 1.54-1.64 (m, 4H), 2.28 (t, J = 6.5Hz, 2H ), 3.02-3.10 (m, 10H), 3.26-3.54 (m, 28H), 3.57 (t, J = 6.5Hz, 2H), 4.41 (quin, J = 7.2Hz, 1H), 5.36 (d, J = 7.8Hz, 1H), 6.74 (t, J = 5.4Hz, 1H), 6.85-7.00 (m, 2H), 7.07 (tt, J = 9.5, 2.4Hz, 1H), 7.20-7.40 (m, 5H), 7.78 (t, J = 5.6Hz, 1H), 8.21 (t, J = 5.6Hz, 1H), 8.36 (d, J = 7.2Hz, 1H), 8.39 (d, J = 7.8Hz, 1H);
HRMS-ESI (m / z): [M + H] + calcd. For C ₄₅ H ₇₀ O ₁₃ N ₅ F ₂ , 926.4933; found, 926.4945.
Compound of formula (1-11):
¹ H-NMR (DMSO-d ₆ ) δ1.22 (d, J = 7.1Hz, 3H), 1.52-1.64 (m, 4H), 2.29 (t, J = 6.5Hz, 2H), 2.97 (q, J = 5.5Hz, 2H), 3.02-3.12 (m, 4H), 3.26-3.62 (m, 30H), 4.40 (quin, J = 7.1Hz, 1H), 5.36 (d, J = 7.8Hz, 1H), 6.94 -7.00 (m, 2H), 7.07 (tt, J = 9.5, 2.4Hz, 1H), 7.22-7.38 (m, 5H), 7.77 (br s, 2H), 7.82 (t, J = 6.5Hz, 1H) , 8.22 (t, J = 5.5Hz, 1H), 8.37 (d, J = 7.1Hz, 1H), 8.38 (d, J = 7.8Hz, 1H);
HRMS-ESI (m / z): [M + H] + calcd. For C ₄₀ H ₆₂ O ₁₁ N ₅ F ₂ , 826.4408; found, 826.4414.

Production Example of Compound of Formula (1-7) (Alternative Method)
Carboxylic acid compound (1-6) (587 mg, 1.56 mmol) Mono-Nt-Boc-amido-dPEG3TM-amine (QUANTA BIODESIGN; 500 mg, 1.56 mmol) was dissolved in 10 ml of DMF, diisopropylethylamine (300 μl, 1.72 mmol) was added, The mixture was stirred under ice cooling. HATU (Watanabe Chemical Industries, Cat, no, A01695; 652 mg, 1.72 mmol) was added and stirred for 3 hours under ice cooling. 150 ml of chloroform was added, and the organic layer was washed twice with 100 ml of water and 100 ml of saturated brine. The extract was dried over anhydrous magnesium sulfate and concentrated under reduced pressure.
Purification by silica gel column chromatography gave the target Boc (1-7) (656 mg) in a yield of 62%.
Similarly, using HATU instead of WSCD and HOBT as a condensing agent, the formula (1-10), formula (1-13), formula (1-14), formula (1-19), formula (1-20) ), Formula (1-21), formula (1-22), formula (6-3), formula (6-4) and formula (9-1).

(3) Modification of Affi-gel (DAPT-PEG (2) + Chole-PEG (1))

　Affi-Gel 102Gel (BIO-RAD, cat. No; 153-2401) 12ml（144μmol）をDMFにて置換し、無水コハク酸（28.8mg, 288mol)、iPr2NEt (75μmol, 432μmol)を加え、室温にて一昼夜攪拌した。樹脂をDMFにて洗浄後、ニンヒドリンテストを行い、定量的に目的のカルボン酸体 (１－１２)（144μmol）が得られたのを確認した。引き続き、樹脂を２０％無水酢酸DMF溶液にて３０分間室温で攪拌した。得られたカルボン酸樹脂(１－１２)（36μmol)にDMF１0ｍｌを加え、WSCD（7.5μl, 43.2μmol), HOBt (5.8mg, 43.2μmol)を加え室温にて３０分間攪拌した。化合物（１－１１）（18.6mg, 21.6μmol), 化合物（１－４）(7.2mg, 10.8μmol), エタノールアミン(0.22mg, 3.6μmol), iPr2NEt (15μl, 86.4μmol)を加え、室温にて一昼夜攪拌した。樹脂をDMFにて洗浄後、引き続き２０％エタノール水溶液にて洗浄し、目的の固相担体：DAPT-PEG(2)+コレステロール-PEG(1)（１－１３）を得た。
　樹脂に結合しない化合物（１－１１），化合物（１－４）を、ＨＰＬＣを用い定量した結果、樹脂上のカルボン酸官能基に対して(１-１１)が５７％，(１-１２)が３０％固定化された樹脂：DAPT-PEG(2)+コレステロール-PEG(1)(１－１３)を得た。
　尚、リガンドもしくは疎水性キャッピング剤の固定化率が異なる固相担体は、目的とする固定化率を達成するために理論的に必要なモル比に応じたリガンドもしくは疎水性キャッピング剤を原料として反応させることにより、製造した。　　　

Replace Affi-Gel 102Gel (BIO-RAD, cat. No; 153-2401) with 12 ml (144 μmol) with DMF, add succinic anhydride (28.8 mg, 288 mol) and iPr2NEt (75 μmol, 432 μmol) at room temperature Stir all day and night. The resin was washed with DMF and then subjected to a ninhydrin test to confirm that the objective carboxylic acid compound (1-12) (144 μmol) was obtained quantitatively. Subsequently, the resin was stirred in a 20% acetic anhydride DMF solution for 30 minutes at room temperature. To the obtained carboxylic acid resin (1-12) (36 μmol), 10 ml of DMF was added, WSCD (7.5 μl, 43.2 μmol) and HOBt (5.8 mg, 43.2 μmol) were added, and the mixture was stirred at room temperature for 30 minutes. Add compound (1-11) (18.6 mg, 21.6 μmol), compound (1-4) (7.2 mg, 10.8 μmol), ethanolamine (0.22 mg, 3.6 μmol), iPr2NEt (15 μl, 86.4 μmol) and bring to room temperature. Stir all day and night. The resin was washed with DMF, and then washed with a 20% aqueous ethanol solution to obtain the target solid phase carrier: DAPT-PEG (2) + cholesterol-PEG (1) (1-13).
Compound (1-11) and compound (1-4) that do not bind to the resin were quantified using HPLC. As a result, (1-11) was 57% of the carboxylic acid functional group on the resin, and (1-12) Was fixed to 30%: DAPT-PEG (2) + cholesterol-PEG (1) (1-13) was obtained.
In addition, solid phase carriers with different immobilization rates of ligands or hydrophobic capping agents react with ligands or hydrophobic capping agents as raw materials according to the theoretically required molar ratio to achieve the desired immobilization rate. It was manufactured by making it.

Example 2
(1) Synthesis of DAPT-PEG (3) -NH2 (1-15)

　N-t-Boc-amido-d-PEG4TM-acid (化合物（１－９）、QUANTA BIODESIGN,;127mg, 0.348mmol)をジクロロメタン１０ｍｌ、クロロホルム４ｍｌの混合溶媒に溶かし、窒素気流下４℃にて攪拌した。 WSCD (343μl, 1.96mmol), HOBt (264mg, 1.96mmol) をゆっくりと加え、室温にて６０分間攪拌した。再度、４℃にて攪拌後、上記で得られたアミノ体塩酸塩（１－１１）(250mg, 0.29mmol）ジクロロメタン１ｍｌ，クロロホルム２．５ｍｌの混合溶媒に溶かし、反応系中に滴下した。ジイソプロピルエチルアミン（121μl, 0.696mmol) を加え、室温にて1昼夜攪拌した。
　溶媒を留去後、クロロホルム１００ｍｌを加え、水５０ｍｌ、飽和食塩水５０ｍｌにて洗浄した。無水硫酸マグシウムにて乾燥し、減圧濃縮した。
シリカゲルカラムクロマトグラフィーにて精製し、目的のBoc体（１－１４）(150mg)を収率４４％にて得た。質量分析結果:ＭS (m/z)：１１７３（ＭＨ＋）。得られたBoc体（１－１４）（150mg, 0.128mmol)をクロロホルム１０ｍｌ－酢酸エチル２ｍｌの混合溶媒に溶かし、4M(N)-HCl/AcOEt (国産化学製;２ml) を加え、室温にて２時間攪拌した。反応完結後、溶媒を留去し、クロロホルム３０ｍｌを加え、減圧下溶媒を留去した。この操作を３回繰り返した。真空ポンプにて溶媒を完全にとばし、目的のアミノ体塩酸塩（１－１５）(123mg)を定量的に得た。質量分析結果: ＭS (m/z):　1073(ＭＨ＋)。

Nt-Boc-amido-d-PEG4TM-acid (compound (1-9), QUANTA BIODESIGN ,; 127 mg, 0.348 mmol) was dissolved in a mixed solvent of 10 ml of dichloromethane and 4 ml of chloroform, and stirred at 4 ° C. under a nitrogen stream. WSCD (343 μl, 1.96 mmol) and HOBt (264 mg, 1.96 mmol) were slowly added and stirred at room temperature for 60 minutes. Again, after stirring at 4 ° C., the amino hydrochloride (1-11) (250 mg, 0.29 mmol) obtained above was dissolved in a mixed solvent of 1 ml of dichloromethane and 2.5 ml of chloroform and added dropwise to the reaction system. Diisopropylethylamine (121 μl, 0.696 mmol) was added, and the mixture was stirred overnight at room temperature.
After distilling off the solvent, 100 ml of chloroform was added, followed by washing with 50 ml of water and 50 ml of saturated saline. The extract was dried over anhydrous magnesium sulfate and concentrated under reduced pressure.
Purification by silica gel column chromatography gave the target Boc (1-14) (150 mg) in a yield of 44%. Mass spectral result: MS (m / z): 1173 (MH +). The obtained Boc form (1-14) (150 mg, 0.128 mmol) was dissolved in a mixed solvent of chloroform (10 ml) -ethyl acetate (2 ml), and 4M (N) -HCl / AcOEt (made by Kokusan Chemical; 2 ml) was added at room temperature. Stir for 2 hours. After completion of the reaction, the solvent was distilled off, 30 ml of chloroform was added, and the solvent was distilled off under reduced pressure. This operation was repeated three times. The solvent was completely removed using a vacuum pump to quantitatively obtain the desired amino acid hydrochloride (1-15) (123 mg). Mass spectral result: MS (m / z): 1073 (MH +).

(2) Synthesis of cholesterol-PEG (0) -NH ₂ (1-18)

　クロロぎ酸コレステロール（１－１）（東京化成；637ｍｇ，1.42mmol)をジクロロメタン６０ｍｌに溶かし、氷冷下攪拌した。N-(2-アミノエチル）カルバミン酸tert-ブチル (東京化成; 250mg,1.56mmol) をジクロロメタン１ｍｌに溶かし、ゆっくりと滴下した。ジイソプロピルエチルアミン（494μl, 2.84mmol) を加え、室温にて一昼夜攪拌した。減圧濃縮後、酢酸エチル２００ｍｌを加えた。酢酸エチル層を水１００ｍｌ、飽和食塩水にて洗浄した。無水硫酸マグネシウムにて乾燥後、減圧濃縮し、シリカゲルカラムクロマトグラフィーにて精製した。目的のBoc体（１－１７）(645mg, 1.12mmol)を収率７９％にて得た。質量分析結果：ＭS (ｍ／ｚ): 595(M+Na)　。
　得られたBoc体（１－１７）（645mg, 1.12mmol) を酢酸エチル１０ｍｌ－クロロホルム３０ｍｌの混合溶媒に溶かし、4M(N)-HCl/AcOEt (国産化学製;　3ml) を加え、室温にて３時間攪拌した。反応完結後、結晶を濾取し、結晶をクロロホルム２ｍｌにて洗浄した。減圧下乾燥し、目的のアミノ体塩酸塩（１－１８）(570mg)を定量的に得た。質量分析結果: ＭS (m/z)：473(ＭＨ+)。
　化合物（１－１３）を得た同様の方法で、化合物（１－４）、（１－１５）、（１－１１），（１－１８）を組み合わせることによりポリエチレングリコール部分の長さの異なるアフィニティー樹脂（１－１９）（DAPT-PEG(2)+Chole-PEG(0))： 

Cholesterol chloroformate (1-1) (Tokyo Kasei; 637 mg, 1.42 mmol) was dissolved in 60 ml of dichloromethane and stirred under ice cooling. Tert-Butyl N- (2-aminoethyl) carbamate (Tokyo Kasei; 250 mg, 1.56 mmol) was dissolved in 1 ml of dichloromethane and slowly added dropwise. Diisopropylethylamine (494 μl, 2.84 mmol) was added, and the mixture was stirred overnight at room temperature. After concentration under reduced pressure, 200 ml of ethyl acetate was added. The ethyl acetate layer was washed with 100 ml of water and saturated brine. The extract was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography. The target Boc form (1-17) (645 mg, 1.12 mmol) was obtained with a yield of 79%. Mass spectral result: MS (m / z): 595 (M + Na).
The obtained Boc form (1-17) (645 mg, 1.12 mmol) was dissolved in a mixed solvent of 10 ml of ethyl acetate and 30 ml of chloroform, and 4M (N) -HCl / AcOEt (manufactured by Kokusan Chemical; 3 ml) was added at room temperature. Stir for 3 hours. After completion of the reaction, the crystals were collected by filtration and washed with 2 ml of chloroform. The product was dried under reduced pressure to give the desired amino acid hydrochloride (1-18) (570 mg) quantitatively. Mass analysis result: MS (m / z): 473 (MH @ +).
The length of the polyethylene glycol moiety is different by combining the compounds (1-4), (1-15), (1-11), and (1-18) in the same manner as the compound (1-13). Affinity resin (1-19) (DAPT-PEG (2) + Chole-PEG (0)):

、式（１－２０）（DAPT-PEG(3)+Chole-PEG(0))：

Formula (1-20) (DAPT-PEG (3) + Chole-PEG (0)):

及び式（１－２１）（DAPT-PEG(3)+Chole-PEG(1))：

And Formula (1-21) (DAPT-PEG (3) + Chole-PEG (1)):

を合成した。

Was synthesized.

Example 3
(1) Preparation of rat brain extract
i) Rat whole brain (2 g) was homogenized in 20 ml of subcellular buffer A (20 mM Hepes pH 7.5, 50 mM KCl, 2 mM EGTA + proteaseinhibitor).
ii) Centrifugation (rotation speed 1000 x g, 10 minutes)
iii) The supernatant obtained in ii) was further centrifuged (rotation speed 10,000 x g, 15 minutes)
iv) The supernatant obtained in iii) was further centrifuged (rotation speed 100,000 × g, 60 minutes) to obtain a pellet (0.26 g).
v) The obtained pellet (0.26 g) was suspended by adding buffer A containing 1% Chapso (1% CHAPSO / buffer A; 2.6 ml), and incubated for 60 minutes under ice cooling. Centrifugation was performed at 4 ° C. (rotation speed 100,000 × g, 30 minutes), 2.6 ml of buffer A was added, and the final concentration was adjusted to 0.5% CHAPSO.
(2) Binding experiment The affinity resin (1-13) prepared in Example 1 (equivalent to 4.8 μmol) was accurately weighed and the binding conditions (temperature) indicated as rat brain lysate (200 μl) obtained in (1). • The binding experiment was conducted at time). Antagonism (+) was previously added to Lysate 0.2 μl of 100 mM DAPT (final concentration 100 μM), incubated at each experimental temperature for 30 minutes, and then used for binding experiments. After completion of the binding experiment, the resin was centrifuged at 12,000 × g, the supernatant was discarded, and the remaining resin was washed 3 times with 800 μl of buffer B (20 mM Hepse pH 7.5, 50 mM KCl, 2 mM EGTA + protease inhibitor, 0.5% CHAPSO). (15 minutes × 3) Washed. 40 μl of SDS loading buffer (nakalai cat. No; 30566-22, 2-ME (2-mercaptoethanol) -containing electrophoresis sample buffer solution (2 ×)) was added to the resin and incubated at 90 ° C. for 3 minutes. The sample solution thus obtained was separated with a commercially available SDS gel (BioRad ready Gel J, 5-20% SDS, cat. No; 161-J371V), and the SDS gel was analyzed.

(3) Western blotting Proteins separated by SDS-PAGE were transferred to PVDF Membrane Filter Paper Sandwich 0.2um Pore Size, 20 / pk. (Invitrogen cat. No; LC2002). The PVDF membrane was transferred to a tray, 50 ml of blocking agent Blocking One (nakalai) was added, and the mixture was shaken at room temperature for 1 hour. Washed 3 times with TBS-T (10 min x 3). The primary antibody was diluted with Can Get Signal Solution 1 (TOYOBO, cat. No; NKB-101) and shaken at room temperature for 1 hour. Washed 3 times with TBS-T (10 min x 3). The secondary antibody was diluted with Can Get Signal Solution 2 (TOYOBO, cat. No; NKB-101) and incubated at room temperature for 1 hour. Washed 3 times with TBS-T (10 min x 3). The detection reagent was ECL Plus western Blotting Detection System (GE Healthcare cat. No; RPN2132). Detection was performed using a Lumino Image Analyzer LAS-1000 (Fuji Film) as a detection device.
As a result, in the case where DAPT, which is a γ-secretase inhibitor, was not previously added to the brain extract, γ-secretase (Nicastrin (Nct), Presenilin 1 (PS1-NTF, PS1-CTF), Af-1 (Aph- 1), pen-2 (Pen-2) complex), but when the antagonist DAPT was added to the brain extract in advance, Nct, PS1, Aph-1, and Pen-2 bands were formed. Not detected. From the disappearance of the band, it was found that these four components were specifically bound to DAPT as a complex. Therefore, it was found that the binding between the solid phase carrier of the formula (1-13) and the constituent component of γ-secretase is specific to be antagonized by DAPT.

Example 4
In the same manner as in Example 1, a solid support (DAPT- (PEG) 1-Chol (PEG1)) represented by the following formula (1-22) was produced.
Formula (1-22):

　Affi-Gel 102Gel (BIO-RAD, cat. No; 153-2401) 12ml（144μmol) をDMFにて置換し、無水コハク酸（28.8mg, 288μmol)、iPr₂NEt (75μl, 432μmol)を加え、室温にて一昼夜攪拌した。樹脂をDMFにて洗浄後、ニンヒドリンテストを行い、定量的に目的のカルボン酸体 (１－１２)が得られたのを確認した。引き続き、樹脂を２０％無水酢酸DMF溶液にて３０分間室温で攪拌した。得られたカルボン酸樹脂(１－１２)（36μmol)にDMF１０ｍｌを加え、WSCD（7.5μl, 43.2μmol), HOBt (5.8mg, 43.2μmol)を加え室温にて３０分間攪拌した。化合物（１－８）（ 21.6μmol), 化合物（１－４）(7.2mg, 10.8μmol), エタノールアミン(0.22mg, 3.6μmol), iPr₂NEt (15μl, 86.4μmol)を加え、室温にて一昼夜攪拌した。樹脂をDMFにて洗浄後、引き続き２０％エタノール水溶液にて洗浄し、目的の固相担体：DAPT-PEG(1)+コレステロール-PEG(1)（式（１－２２））を得た。

Affi-Gel 102Gel (BIO-RAD, cat. No; 153-2401) Replace 12 ml (144 μmol) with DMF, add succinic anhydride (28.8 mg, 288 μmol), iPr ₂ NEt (75 μl, 432 μmol), room temperature And stirred for a whole day. The resin was washed with DMF and then subjected to a ninhydrin test, and it was confirmed quantitatively that the desired carboxylic acid compound (1-12) was obtained. Subsequently, the resin was stirred in a 20% acetic anhydride DMF solution for 30 minutes at room temperature. To the obtained carboxylic acid resin (1-12) (36 μmol), 10 ml of DMF was added, WSCD (7.5 μl, 43.2 μmol) and HOBt (5.8 mg, 43.2 μmol) were added, and the mixture was stirred at room temperature for 30 minutes. Add compound (1-8) (21.6 μmol), compound (1-4) (7.2 mg, 10.8 μmol), ethanolamine (0.22 mg, 3.6 μmol), iPr ₂ NEt (15 μl, 86.4 μmol) at room temperature Stir all day and night. The resin was washed with DMF and then washed with a 20% aqueous ethanol solution to obtain the target solid phase carrier: DAPT-PEG (1) + cholesterol-PEG (1) (formula (1-22)).

Example 5
Whether the solid phase carrier represented by the formula (1-13) obtained in Example 1 specifically binds γ-secretase as an affinity chromatography resin was examined. Specifically, bands obtained by binding experiments, SDS-PAGE, and Western blotting experiments were analyzed for three types of solid phase carriers having DAPT immobilization rates different from 10%, 30%, and 60%.
The affinity resin (1-13) (corresponding to 4.8 μmol) prepared in Example 1 was accurately weighed, and the binding conditions (temperature, temperature and rat lysate (200 μl) obtained by the same method as in Example 3 were used. Time). After completion of the binding experiment, the resin was centrifuged at 12,000 × g, the supernatant was discarded, and the remaining resin was washed three times with 800 μl of buffer B (20 mM Hepse pH 7.5, 50 mM KCl, 2 mM EGTA + protease inhibitor, 0.5% CHAPSO). (15 minutes × 3) Washed. 40 μl of SDS loading buffer (nakalai cat. No; 30566-22, 2-ME (2-mercaptoethanol) -containing electrophoresis sample buffer solution (2 ×)) was added to the resin and incubated at 90 ° C. for 3 minutes. The sample solution thus obtained was separated by a commercially available SDS gel (BioRad ready Gel J, 5-20% SDS, cat. No; 161-J371V).
Western Blotting Proteins separated by SDS-PAGE were transferred to PVDF Membrane Filter Paper Sandwich 0.2um Pore Size, 20 / pk. (Invitrogen cat. No; LC2002). The PVDF membrane was transferred to a tray, 50 ml of blocking agent Blocking One (nakalai) was added, and the mixture was shaken at room temperature for 1 hour. Washed 3 times with TBS-T (10 min x 3). The primary antibody was diluted with Can Get Signal Solution 1 (TOYOBO, cat. No; NKB-101) and shaken at room temperature for 1 hour. Washed 3 times with TBS-T (10 min x 3). The secondary antibody was diluted with Can Get Signal Solution 2 (TOYOBO, cat. No; NKB-101) and incubated at room temperature for 1 hour. Washed 3 times with TBS-T (10 min x 3). The detection reagent was ECL Plus western Blotting Detection System (GE Healthcare cat. No; RPN2132). Detection was performed using a Lumino Image Analyzer LAS-1000 (Fuji Film) as a detection device.
The results are shown in FIG.
As shown in FIG. 2, DAPT has a polyethylene glycol content of 10% to 100% with respect to the carboxyl group on the carboxylic acid resin (1-12) obtained by treating the amino group on the Affigel 102 gel with succinic anhydride. The solid phase support (lanes 1-4) immobilized on the solid phase and the solid phase support (lane 8) on which cholesterol is immobilized at 30% with respect to the carboxyl group (1-12) on the resin While γ-secretase cannot bind at all, DAPT is immobilized via polyethylene glycol in 10 to 60% of the carboxyl group (1-12) on the resin, and cholesterol is immobilized in 30%. Four components of γ-secretase were bound to the solid phase carrier (lanes 5-7).
Further, it was confirmed that the solid phase carrier represented by the formula (1-13) does not adsorb a large amount of protein present in the sample nonspecifically. After SDS-PAGE, the gel was shaken in 10% acetic acid / 40% methanol aqueous solution for 30 minutes to remove the solution, and then shaken in 50 ml of CBB staining solution (Nacalai Tesque) for 15 minutes. The band obtained by washing the gel several times with water was analyzed. The results are shown in FIG.
As shown in FIG. 3, it was confirmed that the solid phase carrier represented by the formula (1-13) does not bind non-specifically to a large amount of protein. On the other hand, a similar experiment was conducted with a solid support produced using Toyopearl, which is a hydrophobic resin, instead of Affigel. As a result, as shown in FIG. 4, the solid phase carrier (1) corresponding to the formula (1-13) and the solid phase carrier (2) corresponding to the formula (1-19) are both nonspecific. Protein binding was observed.

Example 6
(1) Synthesis of C18-PEG (1) -NH2 (6-2) Stearic acid (404 mg, 1.42 mmol) was dissolved in 60 ml of dichloromethane and stirred under ice cooling. WSCD (343 μl, 1.96 mmol) and HOBt (264 mg, 1.96 mmol) were slowly added and stirred at room temperature for 60 minutes. After stirring again at 4 ° C., Mono-Nt-boc-amido-dPEG3TM-amine (1-2) (manufactured by QUANTA BIODESIGN; 500 mg, 1.56 mmol) was dissolved in 1 ml of dichloromethane and slowly added dropwise. Diisopropylethylamine (494 μl, 2.84 mmol) was added, and the mixture was stirred overnight at room temperature. After concentration under reduced pressure, 200 ml of ethyl acetate was added. The ethyl acetate layer was washed with 100 ml of water and saturated brine. The extract was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography. The target Boc form (6-1) (730 mg) was obtained in a yield of 87%. Mass spectral results: HRMS-ESI (m / z): [M + H] + calcd. For C33H67O6N2, 587.4994; found, 587.4986.
The obtained Boc form (700 mg, 1.19 mmol) was dissolved in 60 ml of ethyl acetate, 4N-HCl / AcOEt (made by Kokusan Chemical; 5 ml) was added, and the mixture was stirred at room temperature for 5 hours. After completion of the reaction, the solvent was distilled off, 50 ml of chloroform was added, and the solvent was distilled off under reduced pressure. This operation was repeated three times. The solvent was completely distilled off with a vacuum pump to obtain the desired amino acid hydrochloric acid (6-2). Mass spectral results: HRMS-ESI (m / z): [M + H] + calcd. For C28H59O4N2, 487.4469; found, 487.4449.

(2) Modification of Affi-Gel with C18-PEG (1) 10 ml of DMF was added to carboxylic acid resin (1-12) (36 μmol) prepared by the method described in Example 1, and WSCD (7.5 μl, 43.2 μmol), HOBt (5.8 mg, 43.2 μmol) was added and stirred at room temperature for 30 minutes. Compound (1-11) (18.6 mg, 21.6 μmol), C18-PEG (1) -NH ₂ (5.7 mg, 10.8 μmol), ethanolamine (0.22 mg, 3.6 μmol), iPr ₂ NEt (15 μl, 86.4 μmol) And stirred at room temperature for a whole day and night. The resin was washed with DMF, and then washed with a 20% aqueous ethanol solution to obtain the target solid phase carrier: DAPT-PEG (2) + C18-PEG (1) (6-3).

（３）　C18-PEG(1)及びコレステロール-PEG(1)によるAffi-Gelの修飾
　上記（２）と同様の方法で、カルボン酸樹脂(１－１２)（36μmol)にDMF１0ｍｌを加え、WSCD（7.5μl, 43.2μmol), HOBt (5.8mg, 43.2μmol)を加え室温にて３０分間攪拌した。化合物（１－１１）（18.6mg, 21.6μmol), 化合物（１－４）（2.4mg，3.6μmol）,C18-PEG(1)-NH2（６－２） (3.8mg,7.2μmol), エタノールアミン(0.22mg, 3.6μmol), iPr2NEt (15μl, 86.4μmol)を加え、室温にて一昼夜攪拌した。樹脂をDMFにて洗浄後、引き続き２０％エタノール水溶液にて洗浄し、目的の固相担体：DAPT-PEG(2)+C18-PEG(1)+コレステロール-PEG(1)（６－４）を得た。

(3) Modification of Affi-Gel with C18-PEG (1) and Cholesterol-PEG (1) In the same manner as in (2) above, 10 ml of DMF was added to carboxylic acid resin (1-12) (36 μmol), and WSCD ( 7.5 μl, 43.2 μmol) and HOBt (5.8 mg, 43.2 μmol) were added and stirred at room temperature for 30 minutes. Compound (1-11) (18.6 mg, 21.6 μmol), Compound (1-4) (2.4 mg, 3.6 μmol), C18-PEG (1) -NH2 (6-2) (3.8 mg, 7.2 μmol), Ethanol Amine (0.22 mg, 3.6 μmol) and iPr2NEt (15 μl, 86.4 μmol) were added, and the mixture was stirred overnight at room temperature. The resin is washed with DMF and then with 20% ethanol aqueous solution, and the target solid phase carrier: DAPT-PEG (2) + C18-PEG (1) + cholesterol-PEG (1) (6-4) Obtained.

Example 7
The effect of capping agents other than cholesterol was examined.
That is, a resin in which C18-PEG (1) as a capping agent or cholesterol-PEG (1) and C18-PEG (1) was introduced into 60% DAPT-PEG (2) was prepared, and the amount of γ-secretase bound Was analyzed.
The affinity resin (6-3) (corresponding to 4.8 μmol) prepared in Example 6 was accurately weighed, and rat brain lysate (200 μl) obtained by the same method as in Example 3 and the binding conditions (temperature, Time). After completion of the binding experiment, the resin was centrifuged at 12,000 × g, the supernatant was discarded, and the remaining resin was washed three times with 800 μl of buffer B (20 mM Hepse pH 7.5, 50 mM KCl, 2 mM EGTA + protease inhibitor, 0.5% CHAPSO). (15 minutes × 3) Washed. 40 μl of SDS loading buffer (nakalai cat. No; 30566-22, 2-ME (2-mercaptoethanol) -containing electrophoresis sample buffer solution (2 ×)) was added to the resin and incubated at 90 ° C. for 3 minutes. The sample solution thus obtained was separated by a commercially available SDS gel (BioRad ready Gel J, 5-20% SDS, cat. No; 161-J371V).
Western Blotting Proteins separated by SDS-PAGE were transferred to PVDF Membrane Filter Paper Sandwich 0.2um Pore Size, 20 / pk. (Invitrogen cat. No; LC2002). The PVDF membrane was transferred to a tray, 50 ml of blocking agent Blocking One (nakalai) was added, and the mixture was shaken at room temperature for 1 hour. Washed 3 times with TBS-T (10 min x 3). The primary antibody was diluted with Can Get Signal Solution 1 (TOYOBO, cat. No; NKB-101) and shaken at room temperature for 1 hour. Washed 3 times with TBS-T (10 min x 3). The secondary antibody was diluted with Can Get Signal Solution 2 (TOYOBO, cat. No; NKB-101) and incubated at room temperature for 1 hour. Washed 3 times with TBS-T (10 min x 3). The detection reagent was ECL Plus western Blotting Detection System (GE Healthcare cat. No; RPN2132). Detection was performed using a Lumino Image Analyzer LAS-1000 (Fuji Film) as a detection device.
Solid phase carrier in which DAPT is immobilized via polyethylene glycol at 100% with respect to the carboxyl group on the carboxylic acid resin (1-12), 30% with respect to the carboxyl group on the carboxylic acid resin (1-12) Γ-secretase is completely bound to the solid phase carrier in which stearic acid is immobilized at 30% and the solid phase carrier in which cholesterol is immobilized at 30% to the carboxyl group on the carboxylic acid resin (1-12). could not. On the other hand, with respect to the carboxyl group on the carboxylic acid resin (1-12), a solid phase carrier in which DAPT is immobilized at 60% via polyethylene glycol and 30% or 20% cholesterol is immobilized, There are four types of γ-secretase (Nct, PS1 (PS1) for solid phase carriers with 10% or 20% stearic acid immobilized and solid phase carriers with 10% cholesterol and 10% stearic acid immobilized. -NTF, PS1-CTF), Aph-1, Pen2) are bound.

Example 8
Specific binding γ-secretase was analyzed using an affinity resin to which cholesterol-PEG (1) as a capping agent was immobilized and DAPT having different linker lengths was immobilized.
The affinity resin (1-21) (corresponding to 4.8 μmol) prepared in Example 2 was accurately weighed, and the binding conditions (temperature / temperature) indicated as rat brain lysate (200 μl) obtained in the same manner as in Example 3. Time). Antagonism (+) was previously added to Lysate in 0.2 μl of 100 mM DAPT (final concentration 100 μM), incubated at each experimental temperature for 30 minutes, and then used for binding experiments. After completion of the binding experiment, the resin was centrifuged at 12,000 × g, the supernatant was discarded, and the remaining resin was washed 3 times with 800 μl of buffer B (20 mM Hepse pH 7.5, 50 mM KCl, 2 mM EGTA + protease inhibitor, 0.5% CHAPSO). (15 minutes × 3) Washed. 40 μl of SDS loading buffer (nakalai cat. No; 30566-22, 2-ME (2-mercaptoethanol) -containing electrophoresis sample buffer solution (2 ×)) was added to the resin and incubated at 90 ° C. for 3 minutes. The sample solution thus obtained was separated with a commercially available SDS gel (BioRad ready Gel J, 5-20% SDS, cat. No; 161-J371V).
Western Blotting Proteins separated by SDS-PAGE were transferred to PVDF Membrane Filter Paper Sandwich 0.2um Pore Size, 20 / pk. (Invitrogen cat. No; LC2002). The PVDF membrane was transferred to a tray, 50 ml of blocking agent Blocking One (nakalai) was added, and the mixture was shaken at room temperature for 1 hour. Washed 3 times with TBS-T (10 min x 3). The primary antibody was diluted with Can Get Signal Solution 1 (TOYOBO, cat. No; NKB-101) and shaken at room temperature for 1 hour. Washed 3 times with TBS-T (10 min x 3). The secondary antibody was diluted with Can Get Signal Solution 2 (TOYOBO, cat. No; NKB-101) and incubated at room temperature for 1 hour. Washed 3 times with TBS-T (10 min x 3). The detection reagent was ECL Plus western Blotting Detection System (GE Healthcare cat. No; RPN2132). Detection was performed using a Lumino Image Analyzer LAS-1000 (Fuji Film) as a detection device.
As shown in FIG. 5, the longer the polyethylene glycol moiety between the hydrophilic resin and DAPT, the better the constituents of γ-secretase are bound (lanes 3, 5 and 7). On the other hand, in the solid phase carrier (lane 1) which does not contain polyethylene glycol between the hydrophilic resin and DAPT, the binding amount of PS-1 (detected with an antibody recognizing the C terminus) and Pen-2 is not sufficient. I understood.

Example 9
The following solid phase carriers were produced in the same manner as in Example 2.

をリガンドとして有するアフィニティークロマトグラフィー用担体を製造すべく、
式（１０－６）で表される化合物を製造した。

　４－グリシジルオキシカルバゾール（化合物（１０－１）；東京化成；Cat. No, G0296; 976mg, 4.08mmol)と（２－アミノ－２－メチルプロピル）－カルバミックアシッドｔブチルエステル（化合物（１０－２）；Prime Organics. Inc;852mg, 4.53mmol)を１－ブタノール２ｍｌに加え９０℃にて５時間加熱還流した。１－ブタノールを留去し、シリカゲルカラムクロマトグラフィーで精製した。目的のBoc体（化合物（１０－３）；１．２６３g）を収率７２．４％にて得た。HRMS-ESI(m/z): [M+H]+calcd.for C24H33N3O4, 428.2544; found, 428.2541.
　得られたBoc体（1.185g, 2.77mmol)をクロロホルム３０ｍｌに溶かし、4M(N)-HCl/AcOEt (国産化学製；５ｍｌ）を加え、室温にて６時間攪拌した。反応完結後、溶媒を留去し、クロロホルム３０ｍｌを加え、減圧下溶媒を留去した。この操作を３回繰り返した。真空ポンプにて溶媒を完全に留去し、目的のアミノ体塩酸塩(１０－４)を定量的に得た。
HRMS-ESI(m/z):[M+H]+calcd.for C19H25N3O2, 328.2020; found, 328.2018.
　N-t-Boc-amido-d-PEG4TM-acid (化合物（１－９）；QUANTA BIODESIGN; 500mg, 1.37mmol)と得られたアミノ体塩酸塩（化合物（１０－４）；548mg,1.37mmol)をDMF３ｍｌに加え窒素気流下４℃にて攪拌した。HATU（572mg, 1.51mmol), ジイソプロピルエチルアミン（107μl, 2.736mmol)を加え、窒素気流下、４℃にて１時間攪拌した。クロロホルム１００ｍｌを加え、水５０ｍｌ、飽和食塩水５０ｍｌにて有機層を洗浄した。無水硫酸マグネシウムにて有機層を乾燥し、減圧濃縮した。シリカゲルカラムクロマトグラフィーにて精製し、目的のBoc体（化合物（１０－５）；359mｇ）を収率38.9％にて得た。
HRMS-ESI(m/z): [M+H]+calcd.for C35H54N4O9, 675.3964; found, 675.3960.
　得られたBoc体（１０－５）（302mg, 0.45mmol)をクロロホルム３０ｍｌに溶かし、4M(N)-HCl/AcOEt (国産化学製；５ｍｌ）を加え、室温にて６時間攪拌した。反応完結後、溶媒を留去し、クロロホルム３０ｍｌを加え、減圧下溶媒を留去した。
この操作を３回繰り返した。真空ポンプにて溶媒を完全に留去し、目的のアミノ体塩酸塩（化合物（１０－６））を定量的に得た。HRMS-ESI(m/z): [M+H]+calcd.for C30H46N4O7, 575.3439; found, 575.3440.
実施例１１ Example 10
Instead of DAPT (compound represented by formula (1-5)), carazolol (see Carazolol; Life Sci. 1979, 24 (24), 2255-64):

In order to produce a carrier for affinity chromatography having a ligand as a ligand,
A compound represented by the formula (10-6) was produced.

4-glycidyloxycarbazole (compound (10-1); Tokyo Kasei; Cat. No, G0296; 976 mg, 4.08 mmol) and (2-amino-2-methylpropyl) -carbamic acid tbutyl ester (compound (10- 2); Prime Organics. Inc; 852 mg, 4.53 mmol) was added to 2 ml of 1-butanol, and the mixture was heated to reflux at 90 ° C. for 5 hours. 1-Butanol was distilled off and purified by silica gel column chromatography. The target Boc form (compound (10-3); 1.263 g) was obtained in a yield of 72.4%. HRMS-ESI (m / z): [M + H] + calcd.for C24H33N3O4, 428.2544; found, 428.2541.
The obtained Boc form (1.185 g, 2.77 mmol) was dissolved in 30 ml of chloroform, 4M (N) -HCl / AcOEt (made by Kokusan Chemical; 5 ml) was added, and the mixture was stirred at room temperature for 6 hours. After completion of the reaction, the solvent was distilled off, 30 ml of chloroform was added, and the solvent was distilled off under reduced pressure. This operation was repeated three times. The solvent was completely distilled off with a vacuum pump to quantitatively obtain the desired amino acid hydrochloride (10-4).
HRMS-ESI (m / z): [M + H] + calcd.for C19H25N3O2, 328.2020; found, 328.2018.
Nt-Boc-amido-d-PEG4TM-acid (compound (1-9); QUANTA BIODESIGN; 500 mg, 1.37 mmol) and the resulting amino hydrochloride (compound (10-4); 548 mg, 1.37 mmol) in 3 ml of DMF And stirred at 4 ° C. under a nitrogen stream. HATU (572 mg, 1.51 mmol) and diisopropylethylamine (107 μl, 2.736 mmol) were added, and the mixture was stirred at 4 ° C. for 1 hour under a nitrogen stream. 100 ml of chloroform was added, and the organic layer was washed with 50 ml of water and 50 ml of saturated saline. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Purification by silica gel column chromatography gave the target Boc compound (compound (10-5); 359 mg) in a yield of 38.9%.
HRMS-ESI (m / z): [M + H] + calcd.for C35H54N4O9, 675.3964; found, 675.3960.
The obtained Boc form (10-5) (302 mg, 0.45 mmol) was dissolved in 30 ml of chloroform, 4M (N) -HCl / AcOEt (made by Kokusan Chemical; 5 ml) was added, and the mixture was stirred at room temperature for 6 hours. After completion of the reaction, the solvent was distilled off, 30 ml of chloroform was added, and the solvent was distilled off under reduced pressure.
This operation was repeated three times. The solvent was completely distilled off with a vacuum pump to quantitatively obtain the desired amino hydrochloride (compound (10-6)). HRMS-ESI (m / z): [M + H] + calcd.for C30H46N4O7, 575.3439; found, 575.3440.
Example 11

A carrier for affinity column chromatography was produced in the same manner as in Example 4, except that compound (10-6) obtained in Example 10 was used instead of compound (1-8) in Example 4.
That is, a solid phase carrier (Carazolol-PEG (1) -Chole-PEG (1)) represented by the following formula (11-1) was produced.
Formula (11-1):

　Affi-Gel 102Gel (BIO-RAD, cat. No; 153-2401) 12ml（144μmol) をDMFにて置換し、無水コハク酸（28.8mg, 288μmol)、iPr2NEt (75μl, 432μmol)を加え、室温にて一昼夜攪拌した。樹脂をDMFにて洗浄後、ニンヒドリンテストを行い、定量的に目的のカルボン酸体 (１－１２)が得られたのを確認した。引き続き、樹脂を２０％無水酢酸DMF溶液にて３０分間室温で攪拌した。得られたカルボン酸樹脂(１－１２)（36μmol)にDMF１０ｍｌを加え、アミノ体塩酸塩（１０－６）（14mg, 21.6μmol）,（化合物（１－４）(7.2mg, 10.8μmol), エタノールアミン(0.22mg, 3.6μmol),HATU(15mg, 39.6μmol), iPr₂NEt (22.5μl, 129.6μmol)を加え、室温にて一昼夜攪拌した。樹脂をDMFにて洗浄後、引き続き２０％エタノール水溶液にて洗浄し、目的の固相担体：Carazolol-PEG(1)+コレステロール-PEG(1)（式(11-1)）を得た。

Replace Affi-Gel 102Gel (BIO-RAD, cat. No; 153-2401) with 12 ml (144 μmol) with DMF, add succinic anhydride (28.8 mg, 288 μmol) and iPr2NEt (75 μl, 432 μmol) at room temperature Stir all day and night. The resin was washed with DMF and then subjected to a ninhydrin test, and it was confirmed quantitatively that the desired carboxylic acid compound (1-12) was obtained. Subsequently, the resin was stirred in a 20% acetic anhydride DMF solution for 30 minutes at room temperature. To the obtained carboxylic acid resin (1-12) (36 μmol), 10 ml of DMF was added, and amino hydrochloride (10-6) (14 mg, 21.6 μmol), (compound (1-4) (7.2 mg, 10.8 μmol), Ethanolamine (0.22 mg, 3.6 μmol), HATU (15 mg, 39.6 μmol), iPr ₂ NEt (22.5 μl, 129.6 μmol) were added, and the mixture was stirred overnight at room temperature. Washing with an aqueous solution gave the target solid phase carrier: Carazolol-PEG (1) + cholesterol-PEG (1) (formula (11-1)).

The agarose-based solid phase carrier of the present invention isolates, purifies, and identifies specific binding proteins, specifically membrane-bound proteins such as receptors, which are important for elucidating the mechanism of action of drugs or drug candidate compounds. Useful for.

Claims (16)

A hydrophobic capping agent selected from cholesterol derivatives, fatty acid derivatives and aliphatic amines, and a solid phase carrier for affinity column chromatography in which a ligand is immobilized on a hydrophilic resin, wherein the ligand is polyethylene glycol A solid phase carrier which is covalently bonded to the hydrophilic resin through a spacer including a portion. The solid phase carrier according to claim 1, wherein the hydrophilic resin is a resin obtained by polymerizing sugars or a resin obtained by polymerizing a highly hydrophilic monomer. The solid phase carrier according to claim 2, wherein the hydrophilic resin obtained by polymerizing the saccharide is a sepharose resin, a dextran resin, a cellulose resin, an amylose resin or an agarose resin. The solid phase carrier according to claim 2, wherein the resin obtained by polymerizing a highly hydrophilic monomer is a methacrylate resin in which methacrylic acid having a polyethylene glycol group or a polyol group is polymerized. The solid phase carrier according to any one of claims 1 to 4, wherein the ligand is a substance that specifically adsorbs to a membrane-bound protein. The solid phase carrier has the following formula (1):

(X ¹ represents a single bond or a divalent group that covalently links a hydroxyl group and a polyethylene glycol moiety in a hydrophilic resin, Z ¹ represents a divalent group that covalently links a ligand and the polyethylene glycol moiety, Y ¹ represents a polyethylene glycol moiety containing 1 to 50 ethylene glycol units)
The solid phase carrier according to any one of claims 1 to 5, comprising a partial structure represented by: The solid phase carrier has the following formula (2):

(S represents 0 or 1, X ² represents a single bond, or a divalent group that covalently connects a hydroxyl group and a polyethylene glycol moiety or a hydrophobic capping agent in a hydrophilic resin, and Z ² represents two polyethylene glycol moieties. Or a divalent group for covalently linking the polyethylene glycol moiety with the hydrophobic capping agent, Y ² represents a polyethylene glycol moiety containing 1 to 50 ethylene glycol units)
The solid phase carrier according to any one of claims 1 to 6, comprising a partial structure represented by: The solid support according to claim 7, wherein s is 1 in formula (2). Y ¹ in formula (1) and Y ² in formula (2) are the following formulas (3) to (6):

(A, c, f and j independently represent an integer of 2 to 5, d, g, i and l independently represent an integer of 1 to 5, and b, e, h and k independently Represents an integer from 1 to 50)
Or a divalent group independently selected from the above formulas (3) to (6), or one or more divalent groups independently selected from the above formulas (3) to (6) are bonded via an amide bond. The solid phase carrier according to any one of claims 6 to 8, which is a divalent group. X ¹ in formula (1) and X ² in formula (2) are a single bond and formulas (7) to (10):
^{(7) -X 3 - (CH} 2) m -X 4 -
^{(8) -X 3 - (CH} 2) m -X 5 - (CH 2) n -X 4 -
^{(9) -X 3 - (CH} 2) m -X 5 - (CH 2) n -X 6 - (CH 2) o -X 4 -
^{(10) -X 3 - (CH} 2) m -X 5 - (CH 2) n -X 6 - (CH 2) o -X 7 - (CH 2) p -X 4 -
(Wherein X ³ represents a single bond or CO, X ⁵ , X ⁶ and X ⁷ represent O, S, NR ¹ , NHCO or CONH, X ⁴ represents an oxygen atom, NH or CO, R ¹ Represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and m, n, o and p independently represent an integer of 1 to 5)
10. The solid phase carrier according to claim 6, wherein the solid phase carrier is independently selected from divalent groups represented by any of the above: Z ¹ in formula (1) and Z ² in formula (2) are a single bond, NH, CO, and formula (11):
(11) —Z ³ — (CH ₂ ) _t —Z ⁴ —
(Wherein Z ³ represents NH or CO, Z ⁴ represents NH, CO, NHCO, CONH, CO—O or O—CO, and t represents an integer of 1 to 5)
11. The solid phase carrier according to claim 6, wherein the solid phase carrier is independently selected from divalent groups represented by the formula: The solid phase carrier according to any one of claims 6 to 11, wherein the number of ethylene glycol units in Y ¹ is 2 or more than the number of ethylene glycol units in Y ² . The solid phase carrier according to claim 12, wherein the number of ethylene glycol units in Y ¹ is 2 to 36, and the number of ethylene glycol units in Y ² is 0 to 6. A method for concentrating, isolating or purifying a ligand-specific binding protein comprising the following steps (a) to (b):
(A) a step of bringing the solid phase carrier according to any one of claims 1 to 13 into contact with a sample; and (b) a step of eluting a ligand-specific binding protein from the solid phase carrier. The purification method according to claim 14, wherein the ligand-specific binding protein is a membrane-bound protein. Use of the solid phase carrier according to any one of claims 1 to 13 as a carrier for affinity column chromatography.

Images