JP2007154083A - Affinity carrier and method for producing the same - Google Patents
Affinity carrier and method for producing the same Download PDFInfo
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- UPTHZKIDNHJFKQ-UHFFFAOYSA-N 2-methylprop-2-enoic acid;propane-1,2,3-triol Chemical compound CC(=C)C(O)=O.CC(=C)C(O)=O.OCC(O)CO UPTHZKIDNHJFKQ-UHFFFAOYSA-N 0.000 description 3
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- XFCMNSHQOZQILR-UHFFFAOYSA-N 2-[2-(2-methylprop-2-enoyloxy)ethoxy]ethyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCCOCCOC(=O)C(C)=C XFCMNSHQOZQILR-UHFFFAOYSA-N 0.000 description 2
- HLGNMOUJXWELKK-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methylprop-2-enoyloxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCCOCCOCCOCCOCCOCCOCCOCCOCCOC(=O)C(C)=C HLGNMOUJXWELKK-UHFFFAOYSA-N 0.000 description 2
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- 230000005526 G1 to G0 transition Effects 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
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- 238000004220 aggregation Methods 0.000 description 2
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- UKMBKKFLJMFCSA-UHFFFAOYSA-N [3-hydroxy-2-(2-methylprop-2-enoyloxy)propyl] 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCC(CO)OC(=O)C(C)=C UKMBKKFLJMFCSA-UHFFFAOYSA-N 0.000 description 1
- 150000001252 acrylic acid derivatives Chemical class 0.000 description 1
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- Polymerisation Methods In General (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
Abstract
Description
本発明は、マイクロメートルサイズの平均直径を有し、三次元網目状に連続した細孔と有機物質に富む骨格相からなる共連続構造をもつ粒子凝集型でない有機高分子ゲル状のアフィニティー担体（担持機能性ポリマーモノリス）、及び当該アフィニティー担体の製造方法に関するものである。 The present invention relates to an affinity support in the form of an organic polymer gel that is not a particle-aggregated type having an average diameter of a micrometer size and having a co-continuous structure composed of pores continuous in a three-dimensional network and a skeleton phase rich in organic substances ( Support functional polymer monolith) and a method for producing the affinity carrier.
現在、製薬企業において“生理自社化合物（毒性・副作用を含む）”の最大活用の重要性が認識されつつある。一方、それらの直接作用するターゲットタンパク質（特異的タンパク質）、生理的メカニズムのほとんどは解明されていない。そのような生理活性物質を担持したアフィニティークロマトグラフィー樹脂はターゲットタンパクの同定に重要な役割を果たす。アフィニティークロマトグラフィーによるターゲットタンパク質の単離に関しては、いくつかの報告例がある（例えば、非特許文献１及び２）。
アフィニティ樹脂は生化学の分野において多用されてきた手法であるが、従来の手法がペプチドやＤＮＡといった親水性のリガンドを対象としてきたのに対し、細胞膜を透過する生理活性物質の多くは一定レベル以上の疎水性を示すことから、これらの化合物を固定化したアフィニティ樹脂においては疎水的要因に支配される非特異的蛋白吸着が大きな問題となってきた。特に存在量の多いtubulinやactinはターゲットの存在を覆い隠す結果となることから、これらの非特異的蛋白吸着が抑制されたアフィニティ樹脂用担体の開発が求められてきた。
アフィニティー樹脂に用いる固相担体として市販されているものがいくつかあり、その一つにアガロース系樹脂の Affigel（商標）がある。Affigel（商標）は固相担体自身が高い親水性を有するため非特異的タンパク質吸着が少ないという点で優れているが、有機合成を伴う実験条件下では容易に変性し化学的安定性が低く広範な適用が不可能である。また、メタクリレート系樹脂のToyopearl（商標）が代表的な担体として挙げられる。これは、種々の合成条件下で化学的安定が高く、効率の良いアフィニティー樹脂合成が可能であるが、ターゲットタンパク質とともに多くの非特異的タンパク質の吸着が見られ、選択性を求められる実験においては意味をなさない。したがって、メタクリレート系樹脂担体のように化学的安定性が高く、アガロース系樹脂のように非特異的吸着を十分に抑制する親水的特性を持つ新規アフィニティー樹脂が求められる。
また、上記担体が粒子状、破砕状、あるいはブロック状であり、このため、実験後の除去操作などが煩雑でスピードを求められる実験において改善が求められていた。
Affinity resin is a technique that has been widely used in the field of biochemistry, but the conventional technique has been directed to hydrophilic ligands such as peptides and DNA, whereas many physiologically active substances that permeate cell membranes exceed a certain level. In the affinity resin to which these compounds are immobilized, nonspecific protein adsorption, which is governed by hydrophobic factors, has become a serious problem. In particular, tubulin and actin, which are present in large amounts, obscure the presence of the target, and thus development of a carrier for an affinity resin in which adsorption of these nonspecific proteins is suppressed has been demanded.
There are several commercially available solid phase carriers used for affinity resins, one of which is Affigel (trademark), an agarose resin. Affigel (TM) is superior in that the solid support itself has high hydrophilicity and less nonspecific protein adsorption, but it is easily denatured under experimental conditions involving organic synthesis and has low chemical stability and widespread. Application is impossible. Further, a typical resin is Toyopearl (trademark), which is a methacrylate resin. This is highly chemically stable under various synthesis conditions and enables efficient synthesis of affinity resins. However, in experiments where many nonspecific proteins are adsorbed together with the target protein, selectivity is required. Does not make sense. Therefore, there is a need for a novel affinity resin having high chemical stability like a methacrylate resin carrier and having hydrophilic properties that sufficiently suppress nonspecific adsorption like an agarose resin.
Further, the carrier is in the form of particles, crushed particles, or blocks, and therefore, there has been a demand for improvement in experiments where the removal operation after the experiment is complicated and speed is required.
本発明は、従来のアフィニティー樹脂における上述の問題点を解決し、メタクリレート系樹脂担体のように化学的安定性が高く、アガロース系樹脂のように非特異的吸着を十分に抑制する親水的特性を持ち、充分に大きな細孔を持つ一体型の新規アフィニティー樹脂を提供することを課題とする。又、上述の特性を有したアフィニティー樹脂を製造するのに適した方法を提供することも、本発明の課題である。 The present invention solves the above-mentioned problems in conventional affinity resins, has high chemical stability like a methacrylate resin carrier, and has hydrophilic properties that sufficiently suppress nonspecific adsorption like an agarose resin. It is an object of the present invention to provide an integrated new affinity resin having a sufficiently large pore. It is also an object of the present invention to provide a method suitable for producing an affinity resin having the above characteristics.
上記の課題を解決可能な本発明のアフィニティー担体は、マイクロメートルサイズの平均直径を有し、三次元網目状に連続した細孔と有機物質に富む骨格相からなる共連続構造をもつ粒子凝集型でない有機高分子ゲル状物（いわゆる有機モノリス体）であって、当該アフィニティー担体が、架橋剤としての、二官能性ビニルモノマー化合物、メタクリレート化合物及びアクリレート化合物の少なくともいずれか１種と、一官能性親水性モノマーとの共重合体であり、しかも、前記アフィニティー担体における前記架橋剤と前記一官能性親水性モノマーの体積比率が１００〜１０：０〜９０であることを特徴とする。 The affinity carrier of the present invention capable of solving the above-mentioned problems is a particle aggregation type having a micrometer-sized average diameter and having a co-continuous structure composed of three-dimensional network-like pores and a skeleton phase rich in organic substances A non-organic polymer gel (so-called organic monolith), wherein the affinity carrier is monofunctional with at least one of a bifunctional vinyl monomer compound, a methacrylate compound and an acrylate compound as a crosslinking agent. It is a copolymer with a hydrophilic monomer, and the volume ratio of the crosslinking agent and the monofunctional hydrophilic monomer in the affinity carrier is 100 to 10: 0 to 90.
又、本発明は、上記の特徴を有したアフィニティー担体において、前記アフィニティー担体の内外表面が、乾燥状態で当該表面に水を滴下した際に１分以内に当該担体内部に滴下された水を吸収し得る親水性を有していることを特徴とするものでもある。 The present invention also provides an affinity carrier having the above-described features, wherein the inner and outer surfaces of the affinity carrier absorb water dropped inside the carrier within 1 minute when water is dropped on the surface in a dry state. It is also characterized by having a hydrophilic property.
更に、本発明は、上記の特徴を有したアフィニティー担体において、前記一官能性親水性モノマーが、アミノ基、アミド基、アンモニウム基，カルボキシル基、エステル基、カルボニル基、水酸基、スルホ基及びリン酸基あるいはその誘導体、保護体から成るグループより選ばれた官能性基を有するものであることを特徴とするものでもある。 Furthermore, the present invention provides the affinity carrier having the above-described characteristics, wherein the monofunctional hydrophilic monomer is an amino group, an amide group, an ammonium group, a carboxyl group, an ester group, a carbonyl group, a hydroxyl group, a sulfo group, or phosphoric acid. It is also characterized by having a functional group selected from the group consisting of a group, a derivative thereof, and a protector.
又、マイクロメートルサイズの平均直径を有し、三次元網目状に連続した細孔と有機物質に富む骨格相からなる共連続構造をもつ粒子凝集型でない有機高分子ゲル状のアフィニティー担体を製造するための本発明の方法は、架橋剤としての、二官能性ビニルモノマー化合物、メタクリレート化合物及びアクリレート化合物の少なくともいずれか１種と、一官能性親水性モノマーとを、前記架橋剤と前記一官能性親水性モノマーの体積比率が１００〜１０：０〜９０となるように配合し、水及び分子量５００以下の水溶性溶媒から成るグループより選ばれた少なくとも１種の溶媒中で、ラジカル重合開始剤の存在下で共重合させてゲル状体を得、その後、当該ゲル状体を水で浸漬して洗浄を行い、乾燥させることによっても共連続構造を保つことを特徴とする。 In addition, a non-particle-aggregated organic polymer gel affinity carrier having a micrometer-sized average diameter and a co-continuous structure consisting of pores continuous in a three-dimensional network and a skeleton phase rich in organic substances is produced. In the method of the present invention, a crosslinking agent and at least one of a bifunctional vinyl monomer compound, a methacrylate compound and an acrylate compound, a monofunctional hydrophilic monomer, the crosslinking agent and the monofunctional In the at least one solvent selected from the group consisting of water and a water-soluble solvent having a molecular weight of 500 or less, the volume ratio of the hydrophilic monomer is 100 to 10: 0 to 90. It is possible to maintain a co-continuous structure by copolymerization in the presence to obtain a gel, and then immersing the gel in water for washing and drying. The features.
又、本発明は、上記の特徴を有したアフィニティー担体の製造方法において、前記水溶性溶媒が、エチレングリコール、ジエチレングリコール、トリエチレングリコール、テトラエチレングリコール、ペンタエチレングリコール，ヘキサエチレングリコール、トリエチルアミン、ビニルピリジン、ホルムアミド、ジメチルホルムアミド、アセトニトリル、アセトン、メタノール及びエタノールから成るグループより選ばれたものであることを特徴とするものでもある。 In the method for producing an affinity carrier having the above characteristics, the water-soluble solvent may be ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, triethylamine, vinylpyridine. , Formamide, dimethylformamide, acetonitrile, acetone, methanol and ethanol.
本発明のアフィニティー担体は、マイクロメートルサイズの細孔が三次元網目状に連続した構造を有した有機高分子ゲルで、メタクリレート系樹脂担体のように化学的安定性が高く、アガロース系樹脂のように非特異的吸着を十分に抑制する親水的特性を持ち、本発明の製法を用いることによって、このような特性を有したアフィニティー担体が、比較的簡単な工程にて効率良く製造可能である。 The affinity carrier of the present invention is an organic polymer gel having a structure in which micrometer-sized pores are continuous in a three-dimensional network, and has a high chemical stability like a methacrylate resin carrier, like an agarose resin. By using the production method of the present invention, the affinity carrier having such properties can be efficiently produced in a relatively simple process.
本発明のアフィニティー担体は、二官能性ビニルモノマー架橋剤あるいはメタクリレート架橋剤あるいはアクリレート架橋剤の少なくともいずれか１種に、一官能性親水性モノマー（一官能性の親水的な機能性モノマー）を０〜９０体積％、好ましくは５〜５０体積％含ませた担持機能性ポリマーモノリス型担体で、これら化合物の共重合によって得られる。そして、このアフィニティー担体は、ゲル状の共重合体で、ポリマー粒子凝集型ではなく共連続構造を有し、化学的安定性が高く、アガロース系樹脂のように非特異的吸着を十分に抑制する親水的特性を有している。本発明のアフィニティー担体においては、共連続した細孔のサイズが０．１μｍから５０μｍで単分散であり、骨格サイズが０．１μｍから５０μｍで単分散であるものの組み合わせである。 In the affinity carrier of the present invention, a monofunctional hydrophilic monomer (monofunctional hydrophilic functional monomer) is added to at least one of a bifunctional vinyl monomer crosslinking agent, a methacrylate crosslinking agent, and an acrylate crosslinking agent. A supported functional polymer monolith type carrier contained in an amount of ˜90% by volume, preferably 5 to 50% by volume, is obtained by copolymerization of these compounds. This affinity carrier is a gel-like copolymer and has a co-continuous structure, not a polymer particle aggregation type, has high chemical stability, and sufficiently suppresses non-specific adsorption like an agarose resin. Has hydrophilic properties. The affinity carrier of the present invention is a combination of monodisperse particles having a co-continuous pore size of 0.1 μm to 50 μm and a skeleton size of 0.1 μm to 50 μm.
本発明のアフィニティー担体と構成する「二官能性ビニルモノマー架橋剤あるいはメタクリレート架橋剤あるいはアクリレート架橋剤」としては、ジビニルベンゼン、エチレングリコールジメタクリレート、ジエチレングリコールジメタクリレート等のオリゴ、ポリエチレングリコールジメタクリレートおよびそのアクリレート置換体、グリセリンジメタクリレート、ビニルメタクリレート、Ｎ，Ｎ’‐メチレンビスアクリルアミドなどが挙げられ、特に好ましい化合物としては、三次元網目状の細孔連続構造形成性の点から、９個のポリエチレングリコール単位を有するノナエチレングリコールジメタクリレートが挙げられる。この化合物は、例えば新中村化学工業株式会社から「ＮＫ ＥＳＴＥＲ ９Ｇ」という商品名で市販されており、本発明では、市販の二官能性架橋剤が利用できる。 Examples of the “bifunctional vinyl monomer crosslinking agent or methacrylate crosslinking agent or acrylate crosslinking agent” constituting the affinity carrier of the present invention include oligos such as divinylbenzene, ethylene glycol dimethacrylate, and diethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, and acrylates thereof. Substituents, glycerin dimethacrylate, vinyl methacrylate, N, N′-methylenebisacrylamide and the like are mentioned. Particularly preferred compounds include nine polyethylene glycol units from the viewpoint of three-dimensional network pore continuous structure formation. Nonaethylene glycol dimethacrylate having This compound is commercially available, for example, from Shin-Nakamura Chemical Co., Ltd. under the trade name “NK ESTER 9G”. In the present invention, a commercially available bifunctional crosslinking agent can be used.
一方、本発明のアフィニティー担体と構成する「一官能性親水性モノマー」としては、アミノ基、アミド基、カルボキシル基、エステル基、カルボニル基、水酸基、スルホ基、リン酸基、およびその誘導体を含む親水的モノマー（一官能性の担持機能性モノマー）が挙げられ、例えば下記の化１の反応にて合成されるｐＯＨ型モノマーやＰＥＧ型モノマーが好適である。本発明において、上記の官能性基は、ゲルの疎水性に基づく非特異的吸着を排除する役割を果たす。 On the other hand, the “monofunctional hydrophilic monomer” constituting the affinity carrier of the present invention includes amino groups, amide groups, carboxyl groups, ester groups, carbonyl groups, hydroxyl groups, sulfo groups, phosphate groups, and derivatives thereof. Examples thereof include hydrophilic monomers (monofunctional supported functional monomers). For example, pOH type monomers and PEG type monomers synthesized by the following reaction of Chemical Formula 1 are suitable. In the present invention, the above functional group serves to eliminate non-specific adsorption based on the hydrophobicity of the gel.
また、親水基を導入可能なハロゲン化アルキル基を有するモノマー（例えばクロロメチルスチレン）や、エポキシ基を持つモノマー（例えばグリシジルメタクリレート）、フェニル基を有するモノマー（例えばスチレン）等も挙げられる。
選択的で効率良くターゲットタンパク質が吸着するアフィニティー樹脂として好適な本発明のアフィニティー担体においては、前記メタクリレートモノマーと前記一官能性モノマーの構成重量比率が２０〜１００：８０〜０であり、この際、５０〜９５：５０〜５が好ましく、３０〜８０：７０〜２０が特に好ましい。本発明では、前記メタクリレートモノマーの構成重量割合が１０以下になると、モノリス構造（三次元網目構造）が安定に維持できなくなる。
In addition, a monomer having a halogenated alkyl group capable of introducing a hydrophilic group (for example, chloromethylstyrene), a monomer having an epoxy group (for example, glycidyl methacrylate), a monomer having a phenyl group (for example, styrene), and the like are also included.
In the affinity carrier of the present invention suitable as an affinity resin that selectively and efficiently adsorbs the target protein, the constituent weight ratio of the methacrylate monomer and the monofunctional monomer is 20 to 100: 80 to 0, 50-95: 50-5 are preferable and 30-80: 70-20 are especially preferable. In the present invention, when the constituent weight ratio of the methacrylate monomer is 10 or less, the monolith structure (three-dimensional network structure) cannot be stably maintained.
上述の構成モノマーより成る本発明のアフィニティー担体は、乾燥状態で水を担体表面に滴下した際に１分以内、好ましくは１０秒以内、特に好ましくは１秒以内に担体内に水を収着するに充分な親水性を有しており、このような親水性は、細孔が三次元網目状に連続した構造と、構成モノマー中の親水性基に起因するものである。
本発明のアフィニティー担体の用途としては、アフィニティ担体、担持担体などの生化学用の固相体の他、イオン交換クロマトグラフィー担体、キラルクロマトグラフィー担体、フィリッククロマトグラフィー担体、逆相クロマトグラフィー担体などのクロマトグラフィー固定相、気体や液体などの環境毒に対する捕捉担体や、水浄化用の固定相や電気泳動などの担体、細胞培養担体などが挙げられる。
The affinity carrier of the present invention comprising the above-mentioned constituent monomers sorbs water within the carrier within 1 minute, preferably within 10 seconds, particularly preferably within 1 second when water is dropped onto the carrier surface in a dry state. The hydrophilicity is caused by the structure in which the pores are continuous in a three-dimensional network and the hydrophilic group in the constituent monomer.
The use of the affinity carrier of the present invention includes a solid phase body for biochemistry such as an affinity carrier and a carrier carrier, ion exchange chromatography carrier, chiral chromatography carrier, phyllic chromatography carrier, reverse phase chromatography carrier and the like. Examples include chromatographic stationary phases, capture carriers for environmental toxins such as gases and liquids, stationary phases for water purification, carriers for electrophoresis, cell culture carriers, and the like.
次に、上記のアフィニティー担体を効率良く製造するのに適した本発明の製造方法について説明する。本発明の製法では、ポリエチレングリコール単位を主鎖に含むメタクリレートモノマーと、重合可能な官能性基を一つ有した一官能性モノマーとを準備した後、前記メタクリレートモノマーと前記一官能性モノマーとを、重量比率（混合比率）が１０〜１００：９０〜０となるように配合して水溶性溶媒の中に添加、混合し、ラジカル重合開始剤を添加して加熱を行い、両モノマーを共重合させる。この際、前記水溶性溶媒としては、水に自由に溶解することが可能な低分子量（分子量５００以下）の溶媒が使用でき、好ましい溶媒としては、エチレングリコール、ジエチレングリコール、トリエチレングリコールなどのエチレングリコール縮合系溶媒、トリエチルアミン、ビニルピリジン等のアミノ基を有する溶媒、ホルムアミド、ジメチルホルムアミドなどのアミド基を有する溶媒、アセトニトリルのようなシアノ基を有する溶媒、アセトンなどのカルボニル基を有する溶媒、メタノールやエタノールなどの水酸基を有する溶媒などが挙げられる。本発明においては、共重合を行う際の溶媒として、水に溶解しない溶媒（例えばパラフィン類、芳香族炭化水素、ハロゲン化炭化水素など）を使用することはできず、この場合には、ナノメートルサイズの細孔が三次元網目状に連続した構造を有した有機高分子ゲルは得られない。尚、上記水溶性溶媒に添加されるモノマーの総量は、溶媒１００重量部に対して１０〜１２０重量部であることが好ましい。 Next, the production method of the present invention suitable for producing the above affinity carrier efficiently will be described. In the production method of the present invention, after preparing a methacrylate monomer having a polyethylene glycol unit in the main chain and a monofunctional monomer having one polymerizable functional group, the methacrylate monomer and the monofunctional monomer are , Blended so that the weight ratio (mixing ratio) is 10-100: 90-0, added and mixed in water-soluble solvent, added radical polymerization initiator and heated, copolymerized both monomers Let At this time, as the water-soluble solvent, a solvent having a low molecular weight (molecular weight of 500 or less) that can be freely dissolved in water can be used. Preferred solvents include ethylene glycol such as ethylene glycol, diethylene glycol, and triethylene glycol. Condensation solvents, solvents having amino groups such as triethylamine and vinylpyridine, solvents having amide groups such as formamide and dimethylformamide, solvents having cyano groups such as acetonitrile, solvents having carbonyl groups such as acetone, methanol and ethanol And a solvent having a hydroxyl group such as In the present invention, a solvent that does not dissolve in water (for example, paraffins, aromatic hydrocarbons, halogenated hydrocarbons, etc.) cannot be used as a solvent for copolymerization. An organic polymer gel having a structure in which pores of a size are continuous in a three-dimensional network cannot be obtained. In addition, it is preferable that the total amount of the monomer added to the said water-soluble solvent is 10-120 weight part with respect to 100 weight part of solvents.
本発明では、共重合に使用されるラジカル重合開始剤の種類が特に限定されるものではなく、加熱によって重合が開始可能な一般的なラジカル重合開始剤が種々使用でき、重合条件（重合温度や重合時間）についても特殊な条件を必要とせず、一般的な条件でラジカル重合が行える。本発明において適した開始剤としては、過酸化系ラジカル開始剤（過酸化ベンゾイル、過硫酸アンモニウム等）あるいは、アゾ系ラジカル開始剤（アゾビスイソブチロニトリル（ＡＩＢＮ）、アゾビス２，４‐ジメチルバレロニトリル（ＡＤＶＮ）等）および水溶性、あるいは油溶性のレドックス系ラジカル開始剤（ジメチルアニリン＋過酸化ベンゾイル）が挙げられ、０．２部〜１０部、好ましくは０．５部〜３部添加される。 In the present invention, the type of radical polymerization initiator used for copolymerization is not particularly limited, and various general radical polymerization initiators that can start polymerization by heating can be used, and polymerization conditions (polymerization temperature, The polymerization time) does not require special conditions, and radical polymerization can be performed under general conditions. Suitable initiators in the present invention include peroxide radical initiators (benzoyl peroxide, ammonium persulfate, etc.), azo radical initiators (azobisisobutyronitrile (AIBN), azobis 2,4-dimethylvalero). Nitrile (ADVN) etc.) and water-soluble or oil-soluble redox radical initiators (dimethylaniline + benzoyl peroxide) are included, and 0.2 to 10 parts, preferably 0.5 to 3 parts are added. The
前記化１に示したｐＯＨ型モノマーを用いてｐＯＨ型溶液重合樹脂を製造する際の合成スキームの一例を、以下の化２に示す。又、化３は、化２で得られたｐＯＨ型溶液重合樹脂の脱保護反応を示すスキームである。 An example of a synthesis scheme for producing a pOH type solution polymerization resin using the pOH type monomer shown in Chemical Formula 1 is shown in Chemical Formula 2 below. Chemical formula 3 is a scheme showing the deprotection reaction of the pOH type solution polymerization resin obtained in chemical formula 2.
更に、前記化１に示したＰＥＧ型モノマーを用いてＰＥＧ型溶液重合樹脂を製造する際の合成スキームの一例を、以下の化４に示す。又、化５は、化４で得られたＰＥＧ型溶液重合樹脂の脱保護反応を示すスキームである。 Furthermore, an example of a synthetic scheme for producing a PEG type solution polymerization resin using the PEG type monomer shown in Chemical Formula 1 is shown in Chemical Formula 4 below. Chemical formula 5 is a scheme showing the deprotection reaction of the PEG type solution polymerization resin obtained in chemical formula 4.
本発明では、上記の共重合を行った後、得られたゲル状体を水で浸漬して洗浄を行い、乾燥させる。この際、洗浄を２〜３回繰り返し、加熱乾燥を行うのが一般的である。
このようにして得られた重合体の表面を走査型電子顕微鏡（ＳＥＭ）にて観察すると、表面に１０ｎｍ〜１０μｍ程度の細孔が三次元網目状に連続していることがわかる。そして、このような三次元網目状の細孔構造を有するアフィニティー担体は、化学的安定性が高く、アガロース系樹脂のように非特異的吸着を十分に抑制する親水的特性を有している。
In this invention, after performing said copolymerization, the obtained gel-like body is immersed in water, washed, and dried. At this time, the washing is generally repeated 2 to 3 times and heat drying is performed.
When the surface of the polymer thus obtained is observed with a scanning electron microscope (SEM), it can be seen that pores of about 10 nm to 10 μm are continuous on the surface in a three-dimensional network. And the affinity support | carrier which has such a three-dimensional network-like pore structure has high chemical stability, and has the hydrophilic characteristic which suppresses nonspecific adsorption | suction fully like an agarose-type resin.
以下の化６には、上記化４により得られたＰＥＧ型樹脂を用いてＦＫ５０６担持樹脂を合成する際のスキームが示されており、化７は、ｐＯＨ型モノマー及びＰＥＧ型モノマーの合成法と担持法をまとめたスキームである。 The following chemical formula 6 shows a scheme for synthesizing the FK506-supported resin using the PEG type resin obtained by the above chemical formula 4, and chemical formula 7 is a method for synthesizing the pOH type monomer and the PEG type monomer. It is the scheme which put together the loading method.
以下、本発明の実施例を挙げて本発明を説明するが、本発明はこれらに限定されるものではない。 EXAMPLES Hereinafter, although an Example of this invention is given and this invention is demonstrated, this invention is not limited to these.
実施例１：本発明のアフィニティー担体の製造例（溶媒としてジエチレングリコールを使用した場合）
［試薬及び溶媒］
ポリエチレングリコール単位を主鎖に含むメタクリレートモノマーとしては、市販されているノナエチレングリコールジメタクリレート（商品名：ＮＫ ＥＳＴＥＲ ９Ｇ、新中村化学工業株式会社製）をそのまま用いた。
又、一つの官能性基を有する一官能性モノマーとしては、合成したＰＥＧ型モノマーの１種である化１に示されているもの（アミンの保護体）を用いた。
を用いた。尚、ラジカル重合開始剤としては、2,2’-アゾビス-2,4-ジメチルバレロニトリル（ＡＤＶＮ）（和光純薬工業、和光一級）をそのまま使用し、溶媒としては、ジエチレングリコール（和光純薬工業、和光一級）をそのまま使用した。
Example 1: Production example of affinity carrier of the present invention (when diethylene glycol is used as a solvent)
[Reagents and solvents]
As a methacrylate monomer containing a polyethylene glycol unit in the main chain, commercially available nonaethylene glycol dimethacrylate (trade name: NK ESTER 9G, manufactured by Shin-Nakamura Chemical Co., Ltd.) was used as it was.
Further, as the monofunctional monomer having one functional group, the one shown in Chemical Formula 1, which is one of the synthesized PEG type monomers (protector of amine), was used.
Was used. In addition, 2,2'-azobis-2,4-dimethylvaleronitrile (ADVN) (Wako Pure Chemical Industries, Wako First Grade) is used as the radical polymerization initiator, and diethylene glycol (Wako Pure Chemical Industries, Ltd.) is used as the solvent. , Wako first grade) was used as it was.
［ゲルの作製］
６０μｌのＮＫ ＥＳＴＥＲ ９Ｇに４ｍｇのＡＤＶＮを溶解し、下記の表１に記載される量のＰＥＧ型モノマーと、０．３７ｍｌのジエチレングリコールを加えて均一に混合し、６０℃で１時間加熱重合させた。得られたゲル体を水で浸漬して、超音波振動により３０分洗浄し、洗浄は三回繰り返した。尚、操作はエッペンドルフチューブ内で行った。得られたゲル体を６０℃の乾燥機内で乾燥させ、ＳＥＭ観察を行った。
実験に用いた組成を表１に示す。
[Production of gel]
4 mg of ADVN was dissolved in 60 μl of NK ESTER 9G, and the amount of PEG-type monomer described in Table 1 below and 0.37 ml of diethylene glycol were added and mixed uniformly, followed by heat polymerization at 60 ° C. for 1 hour. . The obtained gel body was immersed in water and washed with ultrasonic vibration for 30 minutes, and washing was repeated three times. The operation was performed in an Eppendorf tube. The obtained gel was dried in a dryer at 60 ° C., and SEM observation was performed.
The composition used in the experiment is shown in Table 1.
上記の重合によって製造された本発明のアフィニティー担体におけるモノリス構造（ナノメートルサイズの細孔が三次元網目状に連続した構造）を、図１及び図２の電子顕微鏡写真により示す。尚、縮尺は各写真右下にスケールで提示している。
図１及び図２に示されるように、本発明のアフィニティー担体（組成Ａ〜Ｈ）においては、ＰＥＧ型モノマーの含有率を変えた場合でも、ナノメートルサイズの細孔の共連続構造が出来ていることが分かる。
又、上記の本発明のアフィニティー担体（組成Ａ〜Ｈ）の表面にスポイトを用いて水を滴下したところ、いずれも担体の場合も１０秒以内に担体内部に水が吸収されることが確認された。
The monolith structure (structure in which nanometer-sized pores are continuous in a three-dimensional network) in the affinity carrier of the present invention produced by the above polymerization is shown by the electron micrographs of FIGS. The scale is shown in the scale at the lower right of each photo.
As shown in FIG. 1 and FIG. 2, the affinity carrier of the present invention (compositions A to H) has a nanometer-sized pore co-continuous structure even when the content of the PEG-type monomer is changed. I understand that.
Further, when water was dropped onto the surface of the above-described affinity carrier (compositions A to H) of the present invention using a dropper, it was confirmed that water was absorbed within the carrier within 10 seconds in both cases. It was.
実施例２：重合時に使用される溶媒としてトリエチルアミンを使用した場合のアフィニティー担体の製造
前記実施例１におけるジエチレングリコール（分子量１０６）の代わりに、分子量が類似した溶媒としてトリエチルアミン（分子量１０１）を使用し、前記の共重合条件と同じ条件下にて共重合を行い、得られた重合物の構造（形成される細孔の状態）を調べた。
図３に、この実施例にて得られたゲル状担体の電子顕微鏡写真を示す。
図３に示されるように、溶媒としてトリエチルアミンを使用した場合においても、モノリス構造が確認できた。
又、実施例１と同様にして、上記の本発明のアフィニティー担体の表面にスポイトを用いて水を滴下したところ、１０秒以内に担体内部に水が吸収されることが確認された。
Example 2: Production of affinity carrier when triethylamine was used as a solvent used in polymerization Instead of diethylene glycol (molecular weight 106) in Example 1, triethylamine (molecular weight 101) was used as a solvent having a similar molecular weight. Copolymerization was performed under the same conditions as those described above, and the structure of the resulting polymer (the state of the pores formed) was examined.
FIG. 3 shows an electron micrograph of the gel carrier obtained in this example.
As shown in FIG. 3, a monolith structure was confirmed even when triethylamine was used as the solvent.
Similarly to Example 1, when water was dropped onto the surface of the above-described affinity carrier of the present invention using a dropper, it was confirmed that water was absorbed inside the carrier within 10 seconds.
比較例１：重合時に使用される溶媒としてクロロペンタンを使用した場合の共重合生成物の構造観察
前記実施例１におけるジエチレングリコール（分子量１０６）の代わりに、分子量が類似した溶媒として、水に溶解しないクロロペンタン（分子量１０７）を使用し、前記の共重合条件と同じ条件下にて共重合を行い、得られた重合物の構造（形成される細孔の状態）を調べた。
図４（ａ）に、この比較例１にて得られたゲル状担体の電子顕微鏡写真を示す。
図４（ａ）に示されるように、溶媒としてクロロペンタンを使用した場合には、細孔（モノリス構造）が形成されないことがわかった。
又、この共重合物の表面にスポイトを用いて水を滴下したところ、１分以上が経過しても共重合物の内部に水が吸収されないことが確認された。
Comparative Example 1: Observation of structure of copolymerized product when chloropentane was used as a solvent used in polymerization In place of diethylene glycol (molecular weight 106) in Example 1, a solvent having a similar molecular weight did not dissolve in water Using chloropentane (molecular weight 107), copolymerization was performed under the same conditions as those described above, and the structure of the resulting polymer (the state of the pores formed) was examined.
FIG. 4A shows an electron micrograph of the gel carrier obtained in Comparative Example 1.
As shown in FIG. 4A, it was found that when chloropentane was used as the solvent, pores (monolith structure) were not formed.
Moreover, when water was dropped onto the surface of the copolymer using a dropper, it was confirmed that water was not absorbed into the copolymer even after 1 minute or more had elapsed.
比較例２：重合時に使用される溶媒としてキシレンを使用した場合の共重合生成物の構造観察
前記実施例１におけるジエチレングリコール（分子量１０６）の代わりに、分子量が類似した溶媒として、水に溶解しないキシレン（分子量１０６）を使用し、前記の共重合条件と同じ条件下にて共重合を行い、得られた重合物の構造（形成される細孔の状態）を調べた。
図４（ｂ）に、この比較例２にて得られたゲル状担体の電子顕微鏡写真を示す。
図４（ｂ）に示されるように、溶媒としてキシレンを使用した場合にも、細孔（モノリス構造）が形成されないことがわかった。
又、この共重合物の表面にスポイトを用いて水を滴下したところ、５分以上が経過しても共重合物の内部に水が吸収されないことが確認された。
Comparative Example 2: Structure observation of copolymerized product when xylene is used as a solvent used in polymerization As a solvent having a similar molecular weight instead of diethylene glycol (molecular weight 106) in Example 1, xylene that does not dissolve in water (Molecular weight 106) was used for copolymerization under the same conditions as those described above, and the structure of the resulting polymer (the state of the pores formed) was examined.
FIG. 4B shows an electron micrograph of the gel carrier obtained in Comparative Example 2.
As shown in FIG. 4B, it was found that even when xylene was used as the solvent, pores (monolith structure) were not formed.
Further, when water was dropped onto the surface of the copolymer using a dropper, it was confirmed that water was not absorbed into the copolymer even after 5 minutes or more had elapsed.
実施例３：本発明のアフィニティー担体による蛋白質捕捉実験
前記実施例１で得たアフィニティー担体（組成Ａ，Ｂ，Ｄ，Ｆ，Ｇ）の性能を検定するために、ＦＫ５０６（３２位からリンカー）を共通のリガンドとして選択し、従来条件と同じ方法で固定化した樹脂を合成し、これまで同様ラットの脳から調整したライゼートを蛋白混合物として用い、各アフィニティー樹脂の比較を行った。尚、ラットの脳はこれまでの研究からＦＫ５０６のターゲット蛋白質であるＦＫＢＰ１２及び非特異的吸着蛋白質として有名なtubulin, actinが多く含まれていることから本研究目的として適していることが知られている。図５に、樹脂上のアミノ基に対して約０．１等量のＦＫ５０６を固定化した結果を示す。尚、対照担体としては、市販のメタクリレート系樹脂担体（Toyopearl（商標）、東ソー製）を用いた。
Example 3: Protein capture experiment using the affinity carrier of the present invention In order to test the performance of the affinity carrier (compositions A, B, D, F, G) obtained in Example 1, FK506 (linker from position 32) was used. Resins selected as a common ligand and immobilized by the same method as in the conventional conditions were synthesized, and lysates prepared from rat brain were used as protein mixtures, and the affinity resins were compared. In addition, it is known that rat brain is suitable for the purpose of this research because it contains a lot of FKBP12 which is a target protein of FK506 and tubulin and actin which are famous as non-specific adsorption proteins. Yes. FIG. 5 shows the result of immobilizing about 0.1 equivalent of FK506 with respect to the amino group on the resin. As a control carrier, a commercially available methacrylate resin carrier (Toyopearl (trademark), manufactured by Tosoh Corporation) was used.
図５に示される実験結果から、市販のメタクリレート系樹脂担体を用いた場合には、アフィニティークロマトグラフィーによるターゲットタンパク質の単離において、非特異的なタンパク質吸着が見られたのに対して、本発明のアフィニティー担体を用いた場合には、従来のメタクリレート系樹脂担体を用いた場合の欠点が解消され、アフィニティー樹脂への非特異的タンパク質の吸着が見られず、選択的で効率良くターゲットタンパク質が吸着されることが確認された。 From the experimental results shown in FIG. 5, when a commercially available methacrylate resin carrier was used, nonspecific protein adsorption was observed in the isolation of the target protein by affinity chromatography. When using this affinity carrier, the disadvantages of using a conventional methacrylate resin carrier are eliminated, and nonspecific protein adsorption to the affinity resin is not observed, and the target protein is selectively and efficiently adsorbed. It was confirmed that
実施例４：二官能性モノマーのみの製造例
二官能性モノマーとして、グリセリンジメタクリレートを用い、溶媒としてジエチレングリコールとホルムアミドの混合溶媒、開始剤としてＡＤＶＮを用いた場合。
ジエチレングリコール１ｇとホルムアミド１ｍｌの混合溶媒に、グリセリンジメタクリレート１ｇとＡＤＶＮ０．０１ｇを溶解して撹拌し、均一溶液とした。その後、容器を密閉し、６０℃条件で２４時間ゲル化を行った。さらに得られたゲルをアルコールで洗浄し乾燥後、ポリマーモノリスを得た。
図６に、この実施例４にて得られたポリマーモノリスの電子顕微鏡写真を示す。
図６の電子顕微鏡写真から、二官能性モノマーであるグリセリンジメタクリレートの単独重合によって得られる重合物の場合も、マイクロメートルサイズの平均直径を有し、三次元網目状に連続した細孔と有機物質に富む骨格相からなる共連続構造が形成されていることが確認された。
Example 4 Production Example of Only Bifunctional Monomer When glycerin dimethacrylate is used as a bifunctional monomer, a mixed solvent of diethylene glycol and formamide is used as a solvent, and ADVN is used as an initiator.
In a mixed solvent of 1 g of diethylene glycol and 1 ml of formamide, 1 g of glycerol dimethacrylate and 0.01 g of ADVN were dissolved and stirred to obtain a uniform solution. Then, the container was sealed and gelled for 24 hours at 60 ° C. Furthermore, the obtained gel was washed with alcohol and dried to obtain a polymer monolith.
FIG. 6 shows an electron micrograph of the polymer monolith obtained in Example 4.
From the electron micrograph shown in FIG. 6, the polymer obtained by homopolymerization of glycerin dimethacrylate, which is a bifunctional monomer, also has a micrometer-sized average diameter, a continuous pore in a three-dimensional network, and organic It was confirmed that a co-continuous structure composed of a skeleton phase rich in substances was formed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010070774A1 (en) | 2008-12-18 | 2010-06-24 | オルガノ株式会社 | Monolithic organic porous body, monolithic organic porous ion exchanger, and process for producing the monolithic organic porous body and the monolithic organic porous ion exchanger |
| JP5354094B2 (en) * | 2010-03-31 | 2013-11-27 | Ｊｓｒ株式会社 | Affinity chromatography packing |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010070774A1 (en) | 2008-12-18 | 2010-06-24 | オルガノ株式会社 | Monolithic organic porous body, monolithic organic porous ion exchanger, and process for producing the monolithic organic porous body and the monolithic organic porous ion exchanger |
| KR20110112289A (en) | 2008-12-18 | 2011-10-12 | 오르가노 가부시키가이샤 | Monolithic organic porous body, Monolithic organic porous ion exchanger and their manufacturing method |
| US9346895B2 (en) | 2008-12-18 | 2016-05-24 | Organo Corporation | Monolithic organic porous body, monolithic organic porous ion exchanger, and process for producing the monolithic organic porous body and the monolithic organic porous ion exchanger |
| JP5354094B2 (en) * | 2010-03-31 | 2013-11-27 | Ｊｓｒ株式会社 | Affinity chromatography packing |
| US9090665B2 (en) | 2010-03-31 | 2015-07-28 | Jsr Corporation | Filler for affinity chromatography |
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