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WO2010053115A1 - Nouveau support d’affinité - Google Patents

Nouveau support d’affinité Download PDF

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Publication number
WO2010053115A1
WO2010053115A1 PCT/JP2009/068893 JP2009068893W WO2010053115A1 WO 2010053115 A1 WO2010053115 A1 WO 2010053115A1 JP 2009068893 W JP2009068893 W JP 2009068893W WO 2010053115 A1 WO2010053115 A1 WO 2010053115A1
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Prior art keywords
solid phase
formula
phase carrier
group
resin
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Ceased
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PCT/JP2009/068893
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English (en)
Japanese (ja)
Inventor
晃 山崎
貴義 山本
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Sumitomo Chemical Co Ltd
Sumitomo Pharma Co Ltd
Original Assignee
Sumitomo Dainippon Pharma Co Ltd
Sumitomo Chemical Co Ltd
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Priority to JP2010536782A priority Critical patent/JP5561484B2/ja
Publication of WO2010053115A1 publication Critical patent/WO2010053115A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/289—Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3225—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating involving a post-treatment of the coated or impregnated product
    • B01J20/3227—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating involving a post-treatment of the coated or impregnated product by end-capping, i.e. with or after the introduction of functional or ligand groups

Definitions

  • 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.
  • 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).
  • affinity column chromatography 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • the hydrophilic resin obtained by polymerizing saccharides is a sepharose resin, a dextran resin, a cellulose resin, an amylose resin, or an agarose resin;
  • the solid phase carrier according to [2], wherein the resin obtained by polymerizing a highly hydrophilic monomer is a meth
  • 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
  • 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.
  • 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.
  • 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.
  • a ⁇ -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 ⁇ -blockers (allotynol, amosulalol, carazolol
  • 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 is added chemically.
  • 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.
  • 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.
  • 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, 5 ⁇ -cholestane.
  • -3 ⁇ -ol (5 ⁇ -cholestan-3 ⁇ -ol), 5 ⁇ -cholestan-3 ⁇ -ol (5 ⁇ -cholestan-3 ⁇ -ol), 5 ⁇ -cholestan-3 ⁇ -ol (5 ⁇ -cholestan-3 ⁇ -ol), ⁇ - Cholestanol ( ⁇ -cholestanol), 5 ⁇ -hydroxycholestanol (5 ⁇ -hydroxycholestanol), 5 ⁇ -cholestan-3 ⁇ , 6 ⁇ -diol (5 ⁇ -cholestan-3 ⁇ , 6 ⁇ -diol), 5 ⁇ -cholestan-3 ⁇ , 7 ⁇ , 12 ⁇ - Triol (5 ⁇ -cholestan-3 ⁇ , 7 ⁇ , 12 ⁇ -triol), 5 ⁇ -cholestan-24 ⁇ -methyl-3 ⁇ -ol (5 ⁇ -cholestan-24 ⁇ -methyl-3 ⁇
  • 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.
  • a functional group such as a hydroxyl group, an amino group or a carboxy group.
  • 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.
  • 5 ⁇ -cholestan-3 ⁇ -ol hemisuccinate 5 ⁇ -cholestan-3 ⁇ -ol hemisuccinate
  • 5 ⁇ -cholestan-3 ⁇ -ol hemisuccinate 5 ⁇ -cholestan-3 ⁇ -ol hemisuccinate
  • 5 ⁇ -cholestan-3 ⁇ -ol hemisuccinate 5 ⁇ -cholestan-3 ⁇ -ol hemisuccinate
  • 5 ⁇ -cholestan-3 ⁇ -ol hemisuccinate 5 ⁇ -cholestan-3 ⁇ -ol hemisuccinate
  • cholesteryl hydrogen succinate 5 ⁇ -cholestan-3 ⁇ -ol hemisuccinate, 5 ⁇ -cholestan-3 ⁇ -ol hemisuccinate, 5 ⁇ -cholestan-3 ⁇ -ol hemisuccinate
  • 5 ⁇ -cholestan-3 ⁇ -ol hemisuccinate
  • 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.
  • 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.
  • 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
  • aliphatic amine examples 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).
  • alkylamines for example, stearylamine, 1-aminodecane, myristylamine, octadecylamine), alkenylamines (for example, oleylamine), and the like.
  • 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.
  • cellulose resin examples 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
  • agarose resin examples 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 ⁇ , etc. from Agarose Bead Technology (AGAROSE BEAD TECHNOLOGIES).
  • Affi-gel registered trademark
  • 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 ⁇ , etc. from Agarose Bead Technology (AGAROSE BEAD TECHNOLOGIES).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the “spacer” represents a divalent group connecting a capping agent or a ligand and a hydrophilic resin
  • the spacer for immobilizing the ligand on the hydrophilic resin is a chemical containing a polyethylene glycol moiety.
  • 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.
  • the divalent group represented by “—X 1 —Y 1 —Z 1 —” in the formula (1) makes the hydrophobic capping agent hydrophilic.
  • the spacer for immobilizing the conductive resin include a divalent group represented by “—X 2 — (Y 2 —Z 2 ) s—” in the formula (2).
  • 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.
  • 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.
  • s 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.
  • X 2 may represent a single bond.
  • 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.
  • 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 —).
  • 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.
  • the polyethylene glycol part contains 1 to 50 ethylene glycol units
  • 1 to 50 ethylene glycols are polymerized to form polyethylene glycol.
  • One or a plurality of groups may be bonded to form a divalent group containing 1 to 50 ethylene glycol units as a whole.
  • 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.
  • a divalent group formed by bonding via a hydrogen atom 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.
  • 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.
  • 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.
  • 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.
  • the number of ethylene glycol units contained in Y 2 is preferably 1 to 6, and more preferably 1 to 4.
  • 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.
  • 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.
  • the functional group of the ligand, the hydrophobic capping agent or the spacer may be appropriately converted into a functional group having high reactivity.
  • 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.
  • 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).
  • 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:
  • Affigel 10 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:
  • Affigel 15 It is represented by The active functional group in Affigel 15 has the following formula:
  • 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.
  • EAH-Sepharose 4B having an amino group and ECH-Sepahrose having a carboxy group are commercially available.
  • 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):
  • 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.
  • 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.
  • the polyethylene glycol moiety in Y 1 of formula (1) and Y 2 of formula (2) the following formulas (3) to (6):
  • 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.
  • 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.
  • Z 1 in formula (1) or Z 2 in formula (2) represents a carbonyl group.
  • 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.
  • Z 1 in formula (1) or Z 2 in formula (2) represents a carbonyl group.
  • 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).
  • 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.
  • Z 1 in formula (1) or Z 2 in formula (2) represents a single bond.
  • 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.
  • Z 1 in formula (1) or Z 2 in formula (2) represents a carbonyl group.
  • 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.
  • Z 1 in formula (1) or Z 2 in formula (2) represents a carbonyl group.
  • 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).
  • 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.
  • X 1 or X 2 is a divalent group represented by any one of formulas (7) to (10)
  • X 4 represents NH.
  • Z 1 in formula (1) or Z 2 in formula (2) represents a single bond.
  • 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).
  • 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.
  • 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.
  • Z 1 in formula (1) or Z 2 in formula (2) represents a single bond.
  • 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).
  • 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.
  • the divalent groups may be the same or different and 1 to 5 polymerized ones may be used.
  • the divalent groups are selected from any combination capable of forming an amide bond with each other. obtain.
  • Z 1 in the formula (1) and Z 2 in the formula (2) are represented by the formula (11)
  • Z 4 in the formula (11) represents NH.
  • Z 4 in the formula (11) represents a carbonyl group or NHCO.
  • Z 4 in the formula (11) represents a carbonyl group or NHCO.
  • 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).
  • 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.
  • the number of units of polyethylene glycol contained in Y 2 is preferably 1-6.
  • 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.
  • 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.
  • the number of units of polyethylene glycol contained in Y 1 is preferably 2 to 36.
  • 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.
  • the method for producing the solid phase carrier of the present invention is specifically exemplified.
  • the solid phase carrier of the present invention can be produced by the following steps:
  • Lg represents a leaving group such as a bromine atom
  • Pg represents a protecting group
  • the solid phase carrier of the present invention can also be produced by the following steps using a carrier having a carboxy group:
  • a carrier having a carboxy group can be produced by the following steps:
  • Lg represents a leaving group such as a bromine atom
  • Pg represents a protecting group
  • 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.
  • a reagent such as triphosgene
  • an intermediate having a polyethylene glycol moiety introduced into the ligand can be produced by the same method as described above.
  • an intermediate having a polyethylene glycol moiety introduced into the capping agent can be produced, for example, by the following steps:
  • 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.
  • a base a dehydrating condensing agent such as dicyclohexylcarbodiimide, WSCD or HATU
  • the protective group is removed. Deprotect.
  • 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.
  • an intermediate having a polyethylene glycol moiety introduced into the capping agent can be produced, for example, by the following steps:
  • 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.
  • a base a dehydrating condensing agent such as dicyclohexylcarbodiimide, WSCD or HATU
  • the protective group is deprotected.
  • 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.
  • 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.
  • 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.
  • 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).
  • Step (a) The solid phase carrier and the sample are usually contacted in a buffer solution (buffer).
  • buffer solution examples 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.
  • 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.
  • 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.
  • a buffer having a composition comprising 0.1 M Tris-HCl (pH 6.8), 2% SDS, 20% Glycerol, 0.2% BPB, and 0.2M D
  • 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.
  • 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.
  • the eluted protein solution may be subjected to SDS-PAGE, and the target band is cut out and analyzed.
  • 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.
  • 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.
  • 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.
  • 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). .
  • the compound or a salt thereof is useful as an intermediate for producing a solid phase carrier for affinity chromatography for purifying ⁇ -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 ⁇ -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.
  • 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.
  • 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 ⁇ l, 3.12 mmol)
  • HOBt 420 mg, 3.12 mmol
  • 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.
  • 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%.
  • 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 ⁇ l, 1.96 mmol) and HOBt (264 mg, 1.96 mmol) were slowly added and stirred at room temperature for 60 minutes.
  • 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 ⁇ l, 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.
  • 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%.
  • 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.
  • 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.
  • 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 ⁇ l, 1.96 mmol)
  • HOBt 264 mg, 1.96 mmol
  • 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 ⁇ l, 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.
  • 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 ⁇ l, 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.
  • 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)):
  • 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).
  • Antagonism (+) was previously added to Lysate 0.2 ⁇ l of 100 mM DAPT (final concentration 100 ⁇ M), 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 ⁇ l 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 ⁇ l of SDS loading buffer (nakalai cat.
  • 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.
  • 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.
  • DAPT- (PEG) 1-Chol (PEG1) represented by the following formula (1-22) was produced.
  • Affi-Gel 102Gel (BIO-RAD, cat. No; 153-2401) Replace 12 ml (144 ⁇ mol) with DMF, add succinic anhydride (28.8 mg, 288 ⁇ mol), iPr 2 NEt (75 ⁇ l, 432 ⁇ mol), 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.
  • Example 5 Whether the solid phase carrier represented by the formula (1-13) obtained in Example 1 specifically binds ⁇ -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 ⁇ mol) prepared in Example 1 was accurately weighed, and the binding conditions (temperature, temperature and rat lysate (200 ⁇ l) obtained by the same method as in Example 3 were used. Time).
  • the resin was centrifuged at 12,000 ⁇ g, the supernatant was discarded, and the remaining resin was washed three times with 800 ⁇ l 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 ⁇ l 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.
  • SDS loading buffer Nakalai cat. No; 30566-22, 2-ME (2-mercaptoethanol) -containing electrophoresis sample buffer solution (2 ⁇ )
  • 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.
  • 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.
  • 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 ⁇ -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 ⁇ -secretase were bound to the solid phase carrier (lanes 5-7).
  • the solid phase carrier represented by the formula (1-13) does not adsorb a large amount of protein present in the sample nonspecifically.
  • 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.
  • 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 ⁇ l, 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.
  • 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 ⁇ -secretase bound was analyzed.
  • the affinity resin (6-3) (corresponding to 4.8 ⁇ mol) prepared in Example 6 was accurately weighed, and rat brain lysate (200 ⁇ l) obtained by the same method as in Example 3 and the binding conditions (temperature, Time).
  • the resin was centrifuged at 12,000 ⁇ g, the supernatant was discarded, and the remaining resin was washed three times with 800 ⁇ l 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 ⁇ l 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.
  • SDS loading buffer Nakalai cat. No; 30566-22, 2-ME (2-mercaptoethanol) -containing electrophoresis sample buffer solution (2 ⁇ )
  • 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.
  • 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.
  • Example 8 Specific binding ⁇ -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 ⁇ mol) prepared in Example 2 was accurately weighed, and the binding conditions (temperature / temperature) indicated as rat brain lysate (200 ⁇ l) obtained in the same manner as in Example 3. Time).
  • Antagonism (+) was previously added to Lysate in 0.2 ⁇ l of 100 mM DAPT (final concentration 100 ⁇ M), incubated at each experimental temperature for 30 minutes, and then used for binding experiments.
  • the resin was centrifuged at 12,000 ⁇ g, the supernatant was discarded, and the remaining resin was washed 3 times with 800 ⁇ l 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 ⁇ l 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.
  • SDS loading buffer Nakalai cat. No; 30566-22, 2-ME (2-mercaptoethanol) -containing electrophoresis sample buffer solution (2 ⁇ )
  • 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.
  • 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 longer the polyethylene glycol moiety between the hydrophilic resin and DAPT the better the constituents of ⁇ -secretase are bound (lanes 3, 5 and 7).
  • 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.
  • 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.
  • 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 ⁇ l, 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.
  • 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.
  • 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.

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Abstract

Support en phase solide pour chromatographie d’affinité, ledit support permettant d’isoler/purifier une protéine membranaire qui se lie spécifiquement à un ligand. Cette invention concerne spécifiquement un support en phase solide pour chromatographie d’affinité en colonne, où un ligand et un agent de coiffage hydrophobe pris parmi des dérivés de cholestérol, des dérivés d’acide gras et des amines aliphatiques sont immobilisés sur une résine hydrophile. Le support en phase solide est caractérisé en ce que le ligand est lié de manière covalente à la résine hydrophile via un espaceur contenant une fraction de polyéthylène glycol.
PCT/JP2009/068893 2008-11-07 2009-11-05 Nouveau support d’affinité Ceased WO2010053115A1 (fr)

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JP2015094757A (ja) * 2013-11-14 2015-05-18 学校法人兵庫医科大学 新規固相担体

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WO2004040305A1 (fr) * 2002-10-31 2004-05-13 Reverse Proteomics Research Institute Co., Ltd. Procede d'immobilisation d'un compose sur un support en phase solide
WO2005037881A1 (fr) * 2003-10-17 2005-04-28 Reverse Proteomics Research Institute Co., Ltd. Resine d'affinite
JP2009292804A (ja) * 2007-11-28 2009-12-17 Canon Inc リガンド分子固定ポリマー、リガンド分子固定粒子、標的物質の検出方法および標的物質の分離方法

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JPS60239425A (ja) * 1984-05-14 1985-11-28 Teijin Ltd 脂質代謝調節用固相担体
JP2680552B2 (ja) * 1994-11-22 1997-11-19 株式会社蛋白工学研究所 修飾低密度リポ蛋白質に対する結合活性を有するペプチド誘導体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004040305A1 (fr) * 2002-10-31 2004-05-13 Reverse Proteomics Research Institute Co., Ltd. Procede d'immobilisation d'un compose sur un support en phase solide
WO2005037881A1 (fr) * 2003-10-17 2005-04-28 Reverse Proteomics Research Institute Co., Ltd. Resine d'affinite
JP2009292804A (ja) * 2007-11-28 2009-12-17 Canon Inc リガンド分子固定ポリマー、リガンド分子固定粒子、標的物質の検出方法および標的物質の分離方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015094757A (ja) * 2013-11-14 2015-05-18 学校法人兵庫医科大学 新規固相担体

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