JP4518804B2 - 6-amino-6-deoxychitosan, process for producing the same and nucleic acid introduction agent comprising the same - Google Patents
6-amino-6-deoxychitosan, process for producing the same and nucleic acid introduction agent comprising the same Download PDFInfo
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本発明は、新規なキトサン誘導体とその製造方法およびその遺伝子工学の分野への用途に関するものである。 The present invention relates to a novel chitosan derivative, a method for producing the same, and use thereof in the field of genetic engineering.
キトサンはカニやエビの甲殻、昆虫の外骨格等の構成成分であるキチンをアルカリ加水分解処理して得られる多糖類で、豊富な生物資源として注目されている。キトサンは一般の有機溶媒には不溶であるが、有機酸水溶液中ではアミノ基がカチオン化して可溶となり、高分子材料としての利用度はキチンに比較して格段に高く、さらに生体親和性および生分解性を有し、かつ低毒性であることから、医用、化粧品、食品、その他の幅広い分野で使用できる材料であることが認められてきた（非特許文献１，２，３および４参照）。 Chitosan is a polysaccharide obtained by alkaline hydrolysis of chitin, which is a component of crabs, shrimp shells, and insect exoskeletons, and is attracting attention as an abundant biological resource. Chitosan is insoluble in common organic solvents, but in an organic acid aqueous solution, the amino group becomes cationized and becomes soluble, and its utilization as a polymer material is much higher than chitin. Since it has biodegradability and low toxicity, it has been recognized that it is a material that can be used in a wide range of fields such as medical, cosmetics, food, and others (see Non-Patent Documents 1, 2, 3, and 4). .
キトサンは３種類の官能基、すなわちＣ−２位に１級アミノ基、Ｃ−６位に１級水酸基、Ｃ−３位に２級水酸基を有する多官能性の高分子であり、化学修飾による機能化も多く行われてきた。糖化学において水酸基を他の官能基で置換した構造であるデオキシ化誘導体は新規化合物の合成前駆体として、あるいはそれ自身が機能性を示す物質として重要な位置を占めているが、上記のようにキトサンは有機溶媒中では溶解性を示さず、キトサンのデオキシ化誘導体を得る上で大きな障害であった。デオキシ化キトサン誘導体はこれまでに発明者らが学術論文として公表した下記の化学構造式（II）で表される繰り返し単位（構成単位）を有する６−デオキシ−６−ハロ−キトサン誘導体（６−デオキシ−６−ハロ−Ｎ−フタロイル化キトサン）のみしか知られていない（非特許文献５）。 Chitosan is a polyfunctional polymer having three types of functional groups, a primary amino group at the C-2 position, a primary hydroxyl group at the C-6 position, and a secondary hydroxyl group at the C-3 position. Many functionalizations have been made. In sugar chemistry, deoxygenated derivatives with a structure in which a hydroxyl group is substituted with another functional group occupy an important position as a precursor for the synthesis of a new compound or as a substance exhibiting functionality by itself. Chitosan did not exhibit solubility in organic solvents, and was a major obstacle in obtaining deoxygenated derivatives of chitosan. The deoxylated chitosan derivative is a 6-deoxy-6-halo-chitosan derivative having a repeating unit (constituent unit) represented by the following chemical structural formula (II) published by the inventors as an academic paper so far (6- Only deoxy-6-halo-N-phthaloylated chitosan) is known (Non-patent Document 5).
本発明の目的は、デオキシ化された新規なキトサン誘導体を提供し、その用途を開発することにある。 An object of the present invention is to provide a deoxylated novel chitosan derivative and develop its use.
本発明者らは、本発明者ら自身が見出した上記式（II）で表される繰り返し単位（構成単位）を有する６−デオキシ−６−ハロゲン化キトサン誘導体を原料に使用し、求核置換反応を行うことによって、容易に種々のデオキシ化キトサン誘導体が得られることを見出し、本発明を完成するに至った。
かくして、本発明に従えば、下記の化学構造式（Ｉ）で表される繰り返し単位（構成単位）を有することを特徴とする６−アミノ−６−デオキシキトサンが提供される。
The present inventors use a 6-deoxy-6-halogenated chitosan derivative having a repeating unit (structural unit) represented by the above formula (II) found by the present inventors as a raw material, and nucleophilic substitution. By carrying out the reaction, it was found that various deoxylated chitosan derivatives can be easily obtained, and the present invention has been completed.
Thus, according to the present invention, there is provided 6-amino-6-deoxychitosan having a repeating unit (structural unit) represented by the following chemical structural formula (I).
すなわち本発明は、キトサンのβ−(１−４)−２−アミノ−２−デオキシ−Ｄ−グルカン構造が上記式(Ｉ)で表される構造で置換された新規なキトサン誘導体、該キトサン誘導体の製造方法、該キトサン誘導体を用いる細胞への核酸導入剤としての用途に関するものである。 That is, the present invention relates to a novel chitosan derivative in which the β- (1-4) -2-amino-2-deoxy-D-glucan structure of chitosan is substituted with the structure represented by the above formula (I), the chitosan derivative And a use as a nucleic acid introduction agent into cells using the chitosan derivative.
本発明は、上記の式（II）の繰り返し単位を有する６−デオキシ−６−ハロゲン化キトサン誘導体を原料として利用することにより遺伝子導入剤をはじめとして種々の用途が期待される新規なキトサン誘導体の提供を可能にした。 The present invention provides a novel chitosan derivative that is expected to be used in various applications including a gene introduction agent by using a 6-deoxy-6-halogenated chitosan derivative having a repeating unit of the above formula (II) as a raw material. Made it possible.
本発明のキトサン誘導体は新規物質である。本発明のキトサン誘導体は、如何なる方法によって製造されたものであっても構わないが、好適には式（II）で表される繰り返し単位を有する６−デオキシ−６−ハロ−キトサン誘導体を原料にして図２に示す化学反応により容易に製造できる。 The chitosan derivative of the present invention is a novel substance. The chitosan derivative of the present invention may be produced by any method, but preferably a 6-deoxy-6-halo-chitosan derivative having a repeating unit represented by the formula (II) is used as a raw material. 2 can be easily manufactured by the chemical reaction shown in FIG.
なお、原料となる式（II）の繰り返し単位を有する６−デオキシ−６−ハロ−Ｎ−フタロイル化キトサンは、例えば、図１に示すように、式（Ａ）を繰り返し単位とするキトサンから式（Ｂ）を繰り返し単位とするＮ−フタロイルキトサンを経由して容易に得ることができる（非特許文献６参照）。ただし、原料となる６−デオキシ−６−ハロ−Ｎ−フタロイル化キトサンの製造方法は特にこのような方法に限定されるものではない。
図２に示す好適な方法に従い、上記の化学構造式（Ｉ）で表される繰り返し単位を有する６−アミノ−６−デオキシキトサンを製造するには、先ず、図２に示す化学構造式（II）で表される繰り返し単位を有する６−デオキシ−６−ハロ−キトサン誘導体をアジド化することにより、化学構造式（III）で表される繰り返し単位を有する６−アジド−６−デオキシキトサン誘導体を生成する。ここで、式（II）で表される繰り返し単位を有する６−デオキシ−６−ハロ−キトサン誘導体は非プロトン性極性有機溶媒に可溶である。一般に、非プロトン性極性有機溶媒はハロゲン化炭素への求核反応を行うための溶媒として優れているので、６−デオキシ−６−ハロ−キトサン誘導体とアジド化剤との反応は非プロトン性極性有機溶媒中、均一状態で良好に行うことができる。６−デオキシ−６−ハロ−キトサン誘導体を溶解させる非プロトン性極性有機溶媒としては、ジメチルスルホキシド、Ｎ，Ｎ−ジメチルホルムアミド、Ｎ−メチル−２−ピロリドン等を例示できる。また、アジド化剤としてはアジ化ナトリウムやアジ化リチウム等が例示でき、より好ましくはアジ化ナトリウムを用いる。 In order to produce 6-amino-6-deoxychitosan having the repeating unit represented by the chemical structural formula (I) according to the preferred method shown in FIG. 2, first, the chemical structural formula (II) shown in FIG. 6) -deoxy-6-halo-chitosan derivative having a repeating unit represented by formula (III) to form a 6-azido-6-deoxychitosan derivative having a repeating unit represented by chemical structural formula (III) Generate. Here, the 6-deoxy-6-halo-chitosan derivative having a repeating unit represented by the formula (II) is soluble in an aprotic polar organic solvent. In general, since an aprotic polar organic solvent is excellent as a solvent for performing a nucleophilic reaction to a halogenated carbon, the reaction between a 6-deoxy-6-halo-chitosan derivative and an azidating agent is aprotic polarity. It can be carried out satisfactorily in an organic solvent in a uniform state. Examples of the aprotic polar organic solvent in which the 6-deoxy-6-halo-chitosan derivative is dissolved include dimethyl sulfoxide, N, N-dimethylformamide, N-methyl-2-pyrrolidone and the like. Examples of the azidating agent include sodium azide and lithium azide. More preferably, sodium azide is used.
天然キチンより得たキトサンは一般にＣ−２位アミノ基の一部にアセトアミド基を有しており、また化学修飾によりキトサンのアミノ基にアセトアミド基を導入することも可能であるが、本発明において原料となる６−デオキシ−６−ハロ−キトサン誘導体、及び本発明で製造される６−アミノ−６−デオキシキトサンはこのようにＣ−２位の一部にアセトアミド基を有する構造のものを含む。 Chitosan obtained from natural chitin generally has an acetamide group as part of the amino group at the C-2 position, and it is also possible to introduce an acetamide group into the amino group of chitosan by chemical modification. The 6-deoxy-6-halo-chitosan derivative used as a raw material and the 6-amino-6-deoxychitosan produced in the present invention include those having a structure having an acetamide group at part of the C-2 position. .
本発明において原料となる６−デオキシ−６−ハロ−キトサン誘導体のハロゲン導入率は限定されるものではない。ハロゲン導入率が低い場合には非プロトン性極性有機溶媒への溶解性が減少するが、膨潤状態で反応を行うことが可能であり、本発明の修飾反応に特に支障となるものではない。 The halogen introduction rate of the 6-deoxy-6-halo-chitosan derivative used as a raw material in the present invention is not limited. When the halogen introduction rate is low, the solubility in an aprotic polar organic solvent is reduced, but the reaction can be performed in a swollen state and does not particularly hinder the modification reaction of the present invention.
以上のようにして一般式（III）で表される繰り返し単位を有するアジド化キトサン誘導体を製造するための反応条件は、原料の種類、反応性、生産性などを考慮して任意に設定できるが、一般に反応温度は室温〜120℃の範囲で行うことが好ましく、更に好ましくは60〜120℃である。原料として塩素化誘導体を使用する場合は反応率の観点から120℃での反応が好ましい。臭素化誘導体では80℃で十分に反応が進行し、ヨウ素化誘導体ではより低温での反応が可能である。反応時間は特に反応温度により制御されるが、一般的には4時間〜10時間の範囲で適宜選択できる。反応時間が短過ぎると原料の反応率が十分でない場合があり好ましくなく、長過ぎることは生産性の観点から好ましくない。 As described above, the reaction conditions for producing the azido chitosan derivative having the repeating unit represented by the general formula (III) can be arbitrarily set in consideration of the type of raw material, reactivity, productivity, and the like. In general, the reaction temperature is preferably room temperature to 120 ° C, more preferably 60 to 120 ° C. When a chlorinated derivative is used as a raw material, a reaction at 120 ° C. is preferable from the viewpoint of reaction rate. The reaction proceeds sufficiently at 80 ° C. with the brominated derivative, and the reaction at a lower temperature is possible with the iodinated derivative. The reaction time is particularly controlled by the reaction temperature, but can generally be appropriately selected within the range of 4 hours to 10 hours. If the reaction time is too short, the reaction rate of the raw materials may not be sufficient, which is not preferable, and too long is not preferable from the viewpoint of productivity.
アジド化反応後、生成した６−アジド−６−デオキシキトサン誘導体は濾別、遠心分離、水懸濁液の凍結乾燥等、一般の単離方法を適宜選択して単離することが出来る。一般には反応混合物をアルコール等に加えて沈澱したアジド化キトサン誘導体を濾過により単離し、アルコール等の溶媒及び水で洗浄後乾燥することにより高純度のアジド化キトサン誘導体を得ることが出来る。 After the azidation reaction, the produced 6-azido-6-deoxychitosan derivative can be isolated by appropriately selecting a general isolation method such as filtration, centrifugation, and freeze-drying of an aqueous suspension. In general, the azido chitosan derivative precipitated by adding the reaction mixture to alcohol or the like is isolated by filtration, washed with a solvent such as alcohol and water, and then dried to obtain a high-purity azido chitosan derivative.
６−アジド−６−デオキシキトサン誘導体から６−アミノ−６−デオキシキトサンへの変換における反応は２工程で行う。１工程目の反応はアジド基のホスフィンイミドへの変換である。すなわち、６−アジド−６−デオキシキトサン誘導体をホスフィンイミド化することにより、図２に示す化学構造式（IV）で表される繰り返し単位を有する６−ホスフィンイミド−６−デオキシキトサン誘導体を生成する。この工程は、６−アジド−６−デオキシキトサン誘導体にトリフェニルホスフィンを反応させて室温で十分に進行し、8〜12時間で完了する。 The reaction for converting 6-azido-6-deoxychitosan derivative to 6-amino-6-deoxychitosan is carried out in two steps. The reaction in the first step is conversion of an azide group to phosphine imide. That is, by phosphinimilating a 6-azido-6-deoxychitosan derivative, a 6-phosphinimide-6-deoxychitosan derivative having a repeating unit represented by the chemical structural formula (IV) shown in FIG. 2 is generated. . This process is sufficiently accomplished at room temperature by reacting 6-azido-6-deoxychitosan derivative with triphenylphosphine and is completed in 8-12 hours.
２工程目の反応は、得られた６−ホスフィンイミド−６−デオキシキトサン誘導体をヒドラジン（ヒドラジン一水和物）で処理することである。これにより、生成したホスフィンイミドの加水分解によるアミノ基への変換と、Ｃ−２位のアミノ基の保護基の除去が起こり、所望の６−アミノ−６−デオキシキトサンが得られる。この反応は反応率の観点から80〜100℃の範囲で行うことが好ましい。反応は4時間〜12時間の範囲で適宜選択できる。この２工程からなる反応は同一反応容器内で連続して行うことが可能である。 The reaction in the second step is to treat the obtained 6-phosphinimide-6-deoxychitosan derivative with hydrazine (hydrazine monohydrate). Thereby, conversion to the amino group by hydrolysis of the produced phosphine imide and removal of the protecting group of the amino group at the C-2 position occur, and the desired 6-amino-6-deoxychitosan is obtained. This reaction is preferably performed in the range of 80 to 100 ° C. from the viewpoint of the reaction rate. The reaction can be appropriately selected within a range of 4 hours to 12 hours. The reaction consisting of these two steps can be carried out continuously in the same reaction vessel.
反応後、生成した６−アミノ−６−デオキシキトサンは限外濾過、水溶液の凍結乾燥等、一般の単離方法を適宜選択して単離することが出来る。一般には反応混合物を水に加えて生成した６−アミノ−６−デオキシキトサンを限外濾過により単離し、アルコールと水の混合溶媒及び水で洗浄後乾燥することにより高純度の本発明の６−アミノ−６−デオキシキトサンを得ることが出来る。 After the reaction, the produced 6-amino-6-deoxychitosan can be isolated by appropriately selecting a general isolation method such as ultrafiltration or lyophilization of an aqueous solution. In general, 6-amino-6-deoxychitosan formed by adding the reaction mixture to water is isolated by ultrafiltration, washed with a mixed solvent of alcohol and water and water, and then dried to obtain the 6-amino acid of the present invention having high purity. Amino-6-deoxychitosan can be obtained.
なお、図１および図２において、ＮＭＰおよびＤＭＦは、好ましい溶媒として例示しているＮ−メチル−２−ピロリドンおよびＮ，Ｎ−ジメチルホルムアミドを表し、また、Ｐｈ３ＰまたはＰＰｈ３はトリフェニルホスフィンを表している。 In FIGS. 1 and 2, NMP and DMF represent N-methyl-2-pyrrolidone and N, N-dimethylformamide exemplified as preferable solvents, and Ph 3 P or PPh 3 represents triphenylphosphine. Represents.
本発明で得られる式（Ｉ）の繰り返し単位から成る６−アミノ−６−デオキシキトサンは酸性および中性条件下で水溶性を示すが、高濃度の条件では酸性条件でより良好な水溶性を示す。また、本発明の６−アミノ−６−デオキシキトサンは精製条件によって、あるいは溶解等の目的で酢酸、塩酸等の酸性化合物と混合した場合等に、アミノ基の一部あるいは全部が４級塩の状態になるが、本発明でいう６−アミノ−６−デオキシキトサンはそれらの状態の構造のものを含む。 The 6-amino-6-deoxychitosan comprising the repeating unit of the formula (I) obtained in the present invention shows water solubility under acidic and neutral conditions, but better water solubility under acidic conditions under high concentration conditions. Show. The 6-amino-6-deoxychitosan of the present invention is a quaternary salt in which a part or all of the amino group is a quaternary salt depending on purification conditions or when mixed with an acidic compound such as acetic acid or hydrochloric acid for the purpose of dissolution or the like. However, 6-amino-6-deoxychitosan as referred to in the present invention includes those in the structure of those states.
本発明の６−アミノ−６−デオキシキトサンならびにその出発原料および中間体となるキトサン誘導体の分子量は特に限定されないが、一般に分子量の低いものの方が高いものより溶液の粘度が低く、取扱いの上で好適である。本発明の６−アミノ−６−デオキシキトサンの好ましい分子量は10,000〜500,000であり、したがって、その出発原料や中間体となる式（II）、（III）、または（IV）で表される繰り返し単位を有するキトサン誘導体も上記分子量に対応する分子量を有するものが好ましい。本発明の６−アミノ−６−デオキシキトサンの如上の分子量は、一般に、酢酸塩として静的光散乱法により重量平均分子量として求めることができる。 The molecular weight of the 6-amino-6-deoxychitosan of the present invention and the chitosan derivative serving as a starting material and an intermediate thereof is not particularly limited, but in general, the lower molecular weight has a lower viscosity of the solution than the higher molecular weight. Is preferred. The preferred molecular weight of the 6-amino-6-deoxychitosan of the present invention is 10,000 to 500,000. Therefore, the repeating unit represented by the formula (II), (III), or (IV) serving as a starting material or intermediate thereof A chitosan derivative having a molecular weight corresponding to the molecular weight is preferred. The molecular weight of the 6-amino-6-deoxychitosan of the present invention can be generally determined as a weight average molecular weight by the static light scattering method as an acetate salt.
本発明で得られる６−アミノ−６−デオキシキトサンはカチオン性官能基であるアミノ基を高密度で有することから、アニオン性である核酸と高分子電解質複合体を形成することが可能であり、細胞への遺伝子（核酸）導入剤として有用である。すなわち、本発明の６−アミノ−６−デオキシキトサンと導入すべき核酸とを適当な緩衝液中で混合することにより、該キトサンを該核酸と複合体化して、細胞（一般には動物細胞）に添加するだけで、該細胞内に所望の核酸を効率的に導入することができる。本発明の６−アミノ−６−デオキシキトサンは、そのままでも遺伝子導入剤として用いることはできるが、好ましくは、酢酸塩として使用し、この場合、分子量は30,000〜40,000程度とするのが好ましい。
キトサンを使用した遺伝子導入の方法が特許に開示されているが、本発明で得られる６−アミノ−６−デオキシキトサンは、上記の方法と同様の条件でキトサンより高い遺伝子導入活性を示す。
A method for gene transfer using chitosan is disclosed in patents, but 6-amino-6-deoxychitosan obtained in the present invention exhibits higher gene transfer activity than chitosan under the same conditions as in the above method.
なお、本願発明において、細胞に導入される核酸類は特に限定されるものではなくＲＮＡ、ｃＤＮＡ、ＤＮＡ、プラスミドＤＮＡ、ベクターＤＮＡ、アンチセンスＤＮＡ、アンチジーンＤＮＡ等の核酸類が含まれる。 In the present invention, the nucleic acid introduced into the cell is not particularly limited, and includes nucleic acids such as RNA, cDNA, DNA, plasmid DNA, vector DNA, antisense DNA, and antigene DNA.
そのほかにも、本願発明の６−アミノ−６−デオキシキトサンは、重金属イオンの吸着（回収）剤、細胞組織培養用スキャホールド、抗菌剤、光学分割剤、液晶等の広範囲な用途に利用可能である。
以下に、実施例に沿って本発明の特徴をさらに具体的に説明するが本発明はこれらの実施例によって制限されるものではない。
In addition, the 6-amino-6-deoxychitosan of the present invention can be used for a wide range of applications such as heavy metal ion adsorption (recovery) agent, scaffold for cell tissue culture, antibacterial agent, optical resolution agent, and liquid crystal. is there.
Hereinafter, the features of the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
６−アジド−６−デオキシキトサン誘導体の製造 非特許文献１に記載の方法で得られた６−ブロモ−６−デオキシ−Ｎ−フタロイル化キトサン誘導体（フタロイル化率100％、ブロモ化率98％）0.50ｇを、Ｎ−メチル−２−ピロリドン50mlに添加し攪拌溶解させ、アジ化ナトリウム1.05ｇ（Ｃ−６位臭素の10モル当量に相当）を加えた。窒素雰囲気下、80℃で4時間撹拌した後、濾過により塩類を除去し、ろ液をエタノールに加えて生成物を沈澱させた。遠心分離法により沈殿物を分離し、エタノール次いで水で十分に洗浄後、凍結乾燥により淡褐色粉末0.39ｇを得た。 6. Preparation of 6-azido-6-deoxychitosan derivative 6-bromo-6-deoxy-N-phthaloylated chitosan derivative obtained by the method described in Non-Patent Document 1 (phthaloylation rate 100%, bromination rate 98%) 0.50 g was added to 50 ml of N-methyl-2-pyrrolidone and dissolved by stirring, and 1.05 g of sodium azide (corresponding to 10 molar equivalents of C-6 position bromine) was added. After stirring at 80 ° C. for 4 hours under a nitrogen atmosphere, salts were removed by filtration, and the filtrate was added to ethanol to precipitate the product. The precipitate was separated by centrifugation, washed thoroughly with ethanol and then with water, and then freeze-dried to obtain 0.39 g of a light brown powder.
この化合物のＩＲ分析の結果、2100cm-1にアジド基の特性吸収が確認された（図３参照）。また、重ジメチルスルホキシド溶液中での13Ｃ ＮＭＲ分析結果から、δ50.1、57.2、68.0、72.5、77.9、96.5(以上糖骨格炭素)および122.8、131.1、134.3、167.5ppm(以上フタロイル基炭素)が検出され、原料と生成物のＣ−６位炭素の化学シフトから、アジド化反応がＣ−６位選択的かつ完全に進行したことが示された（一般式（III）および図4参照、×：内部標準（ジメチルスルホキシド溶媒ピーク））。 As a result of IR analysis of this compound, characteristic absorption of the azide group was confirmed at 2100 cm −1 (see FIG. 3). From the results of 13 C NMR analysis in deuterated dimethyl sulfoxide solution, δ 50.1, 57.2, 68.0, 72.5, 77.9, 96.5 (above sugar skeleton carbon) and 122.8, 131.1, 134.3, 167.5 ppm (more than phthaloyl group carbon) The chemical shift of the C-6 carbon of the raw material and the product showed that the azidation reaction proceeded selectively and completely at the C-6 position (see general formula (III) and FIG. 4). X: Internal standard (dimethylsulfoxide solvent peak)).
この化合物の元素分析結果（表１参照）も、アジド基が95％以上導入されていることを支持する結果となった。 The results of elemental analysis of this compound (see Table 1) also supported the fact that 95% or more of the azide groups were introduced.
６−アミノ−６−デオキシキトサンの製造 実施例１で得た６−アジド−６−デオキシキトサン誘導体0.2ｇをＮ−メチル−２−ピロリドン20mlに溶解し、トリフェニルホスフィン0.5gを添加して窒素雰囲気下、室温で12時間で攪拌した。この反応溶液に水50mlおよびヒドラジン一水和物水溶液(4mol/L)20mlを加え、窒素雰囲気下、100℃で4時間加熱した。水を減圧留去して生じた懸濁液にエタノールを加えた後、遠心分離により沈殿物を分離回収し、新たに水を加えて沈殿物を溶解して限外濾過で十分に洗浄した。この水溶液を凍結乾燥して淡褐色粉末0.8gを得た。 Preparation of 6-amino- 6-deoxychitosan 0.2 g of the 6-azido-6-deoxychitosan derivative obtained in Example 1 was dissolved in 20 ml of N-methyl-2-pyrrolidone, 0.5 g of triphenylphosphine was added, and nitrogen was added. The mixture was stirred at room temperature for 12 hours under an atmosphere. To this reaction solution, 50 ml of water and 20 ml of a hydrazine monohydrate aqueous solution (4 mol / L) were added and heated at 100 ° C. for 4 hours under a nitrogen atmosphere. After ethanol was added to the suspension obtained by distilling off water under reduced pressure, the precipitate was separated and recovered by centrifugation, and water was newly added to dissolve the precipitate and washed thoroughly by ultrafiltration. This aqueous solution was lyophilized to obtain 0.8 g of a light brown powder.
得られた化合物のＩＲ分析の結果（図５参照）および5%重酢酸重水溶液中での13Ｃ ＮＭＲ分析結果：δ42.6、58.3、72.9、73.7、80.3、100.6ppm（以上糖骨格炭素）（図６参照）から、生成物が式（Ｉ）で示される繰り返し単位（構成単位）を有する物質であることを確かめた。 Results of IR analysis of the obtained compound (see FIG. 5) and 13 C NMR analysis results in a 5% aqueous solution of heavy acetic acid: δ42.6, 58.3, 72.9, 73.7, 80.3, 100.6 ppm (above sugar skeleton carbon) From FIG. 6, it was confirmed that the product was a substance having a repeating unit (structural unit) represented by the formula (I).
得られた６−アミノ−６−デオキシキトサンは水溶性を向上させるために酢酸水溶液に溶解させた後、Amicon社製の限外ろ過フィルター（Centricon-20、分画分子量10,000）を使用し、限外濃縮を繰り返し、凍結乾燥して酢酸塩へと変換した。 The obtained 6-amino-6-deoxychitosan was dissolved in an acetic acid aqueous solution in order to improve the water solubility, and then an ultrafiltration filter (Centricon-20, molecular weight cut off 10,000) manufactured by Amicon was used. Outer concentration was repeated, lyophilized and converted to acetate.
酢酸塩は更にAmicon社製の限外ろ過フィルター（Centricon-20）を使用し、分子量50,000以上と50,000〜10,000の２画分に分画した。以後、それぞれの画分を6-ACT(50k)と6-ACT(30k)と表記する。それぞれの画分の元素分析を行った結果、酢酸塩の構造を支持する結果（表２参照）が得られた。なお、組成式は、元素分析の結果から化合物中に含まれる酢酸および水の量を算出し、その計算値に基づいて求めた。 The acetate was further fractionated into two fractions having a molecular weight of 50,000 or more and 50,000 to 10,000 using an ultrafiltration filter (Centricon-20) manufactured by Amicon. Henceforth, each fraction is described as 6-ACT (50k) and 6-ACT (30k). As a result of elemental analysis of each fraction, results supporting the acetate structure (see Table 2) were obtained. In addition, the composition formula calculated | required the quantity of the acetic acid and water which are contained in a compound from the result of an elemental analysis, and calculated | required based on the calculated value.
リン酸緩衝液中での静的光散乱法（MALVERN社製NanoZS）による分子量測定の結果、6-ACT(30k)の重量平均分子量は36,000、また、6-ACT（50k）の重量平均分子量は55,000と決定された。
６−アミノ−６−デオキシキトサンによる遺伝子導入 実験材料としては、下記のものを用いた。細胞：アフリカミドリザル腎臓上皮がん細胞COS-1（American Type Culture Collection製）、培地：DMEM培地（日水製薬製）+10%ウシ胎児血清、６−アミノ−６−デオキシキトサン酢酸塩（分子量36,000）、非修飾キトサン塩酸塩（分子量10,000〜30,000）、遺伝子：ホタルルシフェラーゼ遺伝子を導入したプラスミドＤＮＡ（以下pGL3と略記し、塩基組成を配列表に配列番号１として示した）。 The following materials were used as experimental materials for gene transfer using 6-amino-6-deoxychitosan . Cells: African green monkey kidney epithelial cancer cell COS-1 (manufactured by American Type Culture Collection), medium: DMEM medium (manufactured by Nissui Pharmaceutical) + 10% fetal bovine serum, 6-amino-6-deoxychitosan acetate (molecular weight 36,000) ), Unmodified chitosan hydrochloride (molecular weight 10,000 to 30,000), gene: plasmid DNA introduced with a firefly luciferase gene (hereinafter abbreviated as pGL3, the base composition is shown as SEQ ID NO: 1 in the sequence listing).
６−アミノ−６−デオキシキトサンのpGL3と複合体形成能はゲル電気泳動法により確認した（図７参照）。６−アミノ−６−デオキシキトサンがキトサンよりもＤＮＡとの親和性が高いことが解る。 The ability of 6-amino-6-deoxychitosan to form a complex with pGL3 was confirmed by gel electrophoresis (see FIG. 7). It can be seen that 6-amino-6-deoxychitosan has higher affinity for DNA than chitosan.
遺伝子導入試験の実験方法について以下に説明する。まず、pGL3のTris-エチレンジアミン緩衝液（pH7.0）87.5μlと６−アミノ−６−デオキシキトサン酢酸塩のリン酸緩衝液（pH7.0）87.5μlを混合し、室温で30分以上静置してプラスミド−キトサン複合体を形成させた。混合溶液中のpGL3濃度は20μg/ml、６−アミノ−６−デオキシキトサンはpGL3の１塩基：６−アミノ−６−デオキシキトサンの糖単位＝1:7.5の割合になるようにした。 The experimental method for the gene transfer test is described below. First, 87.5 μl of pGL3 Tris-ethylenediamine buffer (pH 7.0) and 87.5 μl of 6-amino-6-deoxychitosan acetate phosphate buffer (pH 7.0) were mixed and allowed to stand at room temperature for 30 minutes or more. Thus, a plasmid-chitosan complex was formed. The concentration of pGL3 in the mixed solution was 20 μg / ml, and 6-amino-6-deoxychitosan was adjusted to a ratio of 1 base of pGL3: 6-amino-6-deoxychitosan sugar unit = 1: 7.5.
次に、COS-1細胞5万個を培地1ml中、二酸化炭素濃度5%、37℃で24時間培養後、培地を通常の1.25倍濃度のもの200μlに交換し、上記のpGL3と６−アミノ−６−デオキシキトサン複合体溶液を50μl加えた。このとき、培地のpHは7.0に調整しておいた。複合体存在下で3時間培養した後、上記複合体の入った培地を新たな培地に交換して48時間培養し、pGL3を細胞内に導入した。導入されたpGL3量はルシフェラーゼ活性試験により評価し、遺伝子導入剤としてポリエチレンイミン（分子量25,000）を使用したときのルシフェラーゼ活性を10,000としてその相対値を求めた（図８参照）。
比較例１
比較試験として６−アミノ−６−デオキシキトサン酢酸塩の代わりに非修飾のキトサン塩酸塩を用いて同様にpGL3の導入を行った。キトサンとpGL3の複合体は遺伝子発現効率が最も良好なpGL3の１塩基：キトサンの糖単位＝1:2.5の割合になるように調製した。また、複合体の調製はpH6.5の条件で行った。試験の結果、本発明で得られた６−アミノ−６−デオキシキトサン酢酸塩を用いて行った系で導入効率が上回ることが示された（図８参照）。
Comparative Example 1
As a comparative test, pGL3 was similarly introduced using unmodified chitosan hydrochloride instead of 6-amino-6-deoxychitosan acetate. The complex of chitosan and pGL3 was prepared so that the ratio of 1 base of pGL3: chitosan sugar unit = 1: 2.5 had the best gene expression efficiency. The complex was prepared at pH 6.5. As a result of the test, it was shown that the introduction efficiency exceeded in the system conducted using 6-amino-6-deoxychitosan acetate obtained in the present invention (see FIG. 8).
本発明の６−アミノ−６−デオキシキトサンはバイオテクノロジー分野における素材として有用であり、中でも、遺伝子治療剤の非ウイルスベクターとして利用することが可能である。更に、重金属イオン吸着（回収）剤や細胞組織培養用スキャホールド、抗菌剤、光学分割剤、液晶等への応用も期待される。 The 6-amino-6-deoxychitosan of the present invention is useful as a material in the field of biotechnology, and can be used as a non-viral vector for a gene therapy agent. Furthermore, application to heavy metal ion adsorption (recovery) agents, scaffolds for cell tissue culture, antibacterial agents, optical resolution agents, liquid crystals and the like is also expected.
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