JP4457554B2 - Polyuronic acid - Google Patents

Description

【０００９】
請求項２の発明は、Ｎ−アセチル化して、下記化学式（２）で表されるｍ：ｎの比率が８：２から１０：０の範囲にしたキトサンを調製する工程と、前記キトサンと、Ｎ−オキシル化合物と、水とを含む溶液を調製する工程と、前記溶液の温度を５℃以下にし、前記溶液のｐＨを１０から１１．５の範囲にし、前記溶液に酸化反応の進行具合に応じて量を調整しながら酸化剤を滴下する工程とを具備することを特徴とするポリウロン酸の製造方法である。
【００１０】
【化８】

【００１１】
請求項３の発明は、前記溶液は臭化アルカリ金属を含み、前記Ｎ−オキシル化合物が２，２，６，６−テトラメチル−１−ピペリジン−Ｎ−オキシルであり、前記酸化剤が次亜塩素酸ナトリウムであることを特徴とする請求項１または２に記載のポリウロン酸の製造方法である。
【００１５】
【発明の実施の形態】
以下、本発明を詳細に説明する。
本発明におけるポリウロン酸は、下記化学式（１）に示す構造からなるもので、式中のＸ、Ｙは、水素またはアルカリ金属を示すものである。また、Ｎ−アセチル−Ｄ−グルコサミヌロン酸（或いはＮ−アセチル−Ｄ−グルコサミヌロン酸のアルカリ金属塩）とＤ−グルコサミヌロン酸（或いはＤ−グルコサミヌロン酸のアルカリ金属塩）がβ−（１，４）−結合したもので、化学構造が明確、均一であり、二次修飾する場合の合成原料として特に好ましい。また、化学式（１）中のＸ、Ｙが、水素またはナトリウムであれば、２５℃の蒸留水に対して、１０％以上の溶解性を示すため、水系のコーティング材料として、及び、水系での反応原料として有用である。
【００１６】
【化３】

【００１７】
また、本発明におけるポリウロン酸は、前記化学式（１）中のｍ：ｎの比率、つまりＮ−アセチル−Ｄ−グルコサミヌロン酸（或いはそのアルカリ金属塩）とＤ−グルコサミヌロン酸（或いはそのアルカリ金属塩）の比率が、８：２から１０：０の範囲であることを特徴とするものである。Ｄ−グルコサミヌロン酸（或いはそのアルカリ金属塩）は、カチオン性のアミノ基を有し、アニオン性のカルボキシル基とイオン結合を形成し易いことから、Ｄ−グルコサミヌロン酸（或いはそのアルカリ金属塩）が全体の２０％以上では、安定性の低下、及び水溶性の低下を招くため好ましくない。
【００１８】
特に、本発明のポリウロン酸をアニオン性材料として、キトサン等のカチオン性多糖類とポリイオンコンプレックスを形成する際には、Ｎ−アセチル−Ｄ−グルコサミヌロン酸１００％からなるポリウロン酸を用いるよりも、１０％程度のＤ−グルコサミヌロン酸を含有する方が、生成したポリイオンコンプレックスの強度物性が向上する傾向も有する。
【００１９】
さらに本発明のポリウロン酸は、その重量平均分子量が３０，０００以上、より好ましくは５０，０００以上であることを特徴とし、上記したように、化学構造が明確、均一であり、且つ高分子量である点が大きな特徴である。そのため、本発明のポリウロン酸水溶液をコーティング材料として用いる場合には、塗工性が向上し、塗膜の耐湿性や膜物性の向上も期待できる。さらに、前記のようにポリイオンコンプレックスとした際も、その物性や安定性を向上させる。さらに本発明のポリウロン酸を原料に化学修飾する場合も、生成物の物性向上、及び物性の安定化に寄与し得る。
【００２０】
ここに記載した重量平均分子量とは、標準物質としてプルランを用いて、サイズ排除クロマトグラフィー法により測定した、プルラン換算重量平均分子量である。
【００２１】
さらに本発明のポリウロン酸は、キチン等を原料として、Ｎ−オキシル化合物触媒による酸化手法を用いることにより得られるが、本発明の特徴である均一な構造を有して且つ高分子量のポリウロン酸を得るためには、基質のアクセシビリティーを充分に高め、Ｃ６位の１級水酸基を選択的に全てカルボキシル基に変換するとともに、穏やかな条件で酸化することで分子量の低下やＣ３位の酸化などの副酸化をおさえることが重要となる。つまり、アルカリで充分に膨潤または溶解処理したキチンを原料として、又は、キトサンをＮ−アセチル化したものを原料として、Ｎ−オキシル化合物の触媒の存在下、臭化アルカリ金属と酸化剤を用いて、５℃以下の低温、水系で、ｐＨを１０〜１１．５の範囲で一定に保ちながら酸化することにより、本発明のポリウロン酸が得られる。ここでＮ−オキシル化合物としては、２，２，６，６−テトラメチル−１−ピペリジン−Ｎ−オキシル（ＴＥＭＰＯ）が、臭化アルカリ金属としては臭化ナトリウムが、酸化剤としては次亜塩素酸ナトリウムが特に好ましい。
【００２２】
前記のキチン、及びキトサンは、下記化学式（２）に示す構造を有するもので、キチンは、カニやエビなどの甲殻類、また昆虫類の骨格物質として存在し、一般的には、ほぼＮ−アセチル−Ｄ−グルコサミンから成るものであるが、精製過程における脱アセチル化等により、Ｄ−グルコサミンも含むものである。キチンは結晶性が高く、水には不溶であるが、高濃度のアルカリに浸漬後、氷を加えながら低温下で希釈していくことにより、粘調な液体となる。ここに塩酸を加えて中和すると、フレーク状のキチンが析出するが、ほぼ非晶質化したキチンが得られ、これを充分に水洗して乾燥させずに上記酸化反応に供することにより、ほぼ全てのＣ６位の１級水酸基が選択性高く酸化され、分子量低下は抑えられる。但し、アルカリ雰囲気下では、キチンの脱アセチル化の反応も進行することから、アルカリによる溶解および中和処理、さらに酸化反応中の系内の温度は５℃以下の低温に維持することが重要である。
【００２３】
【化４】

【００２４】
さらにキトサンは、キチンを脱アセチル化することにより得られる物質で、一般的には前記化学式（２）中のｎの比率の方が高い物質であり、酸に対して溶解する。キトサン溶液に無水酢酸を添加すると容易にＮ−アセチル化され、再びキチンの化学構造に戻すことが可能である。この操作を経て、充分に水洗したものを乾燥させずに上記酸化反応に供することにより、前記同様にＣ６位の１級水酸基のみ選択性高く酸化して、高分子量の本発明のポリウロン酸を得ることができる。また、この場合は、ｍ／ｎの比率を任意に設定することが可能である。
【００２５】
ここで上記酸化手法は、例えばＴＥＭＰＯと臭化ナトリウムを溶解した水溶液に、上記の湿潤原料を加えて均一に分散させ、系内を５℃以下に冷却、ｐＨを１０に調整し、酸化剤の次亜塩素酸ナトリウム溶液を反応の進行に応じて添加するとともに、水酸化ナトリウム水溶液を滴下して、系内のｐＨを１０〜１１．５の範囲で一定に保つ。また反応中は系内の温度を５℃以下に維持する。酸化が進むにつれ、系内は溶解し、均一な溶液となる。この反応条件においては、添加される水酸化ナトリウムの量は、ほぼ酸化により導入されたカルボキシル基の量に対応しており、原料のＣ６位の１級水酸基量と当モルの添加量に達した時点で、エタノールを添加して過剰の酸化剤を失活させ、過剰量のエタノール中で再沈させる。生成物はアセトンと水の混合溶液を用いて十分洗浄後、アセトンで脱水してから減圧乾燥することにより、本発明のポリウロン酸のナトリウム塩が得られる。
【００２６】
なお上記により得られたポリウロン酸のナトリウム塩を水溶後酸処理し、上記のエタノールで再沈、洗浄、乾燥の操作を繰り返すことで脱塩したポリウロン酸を得ることができる。
【００２７】
さらに、本発明のポリウロン酸は、構造が均一な、β−（１、４）−ポリウロン酸であるため、重水に溶解させて¹³Ｃ−ＮＭＲを測定すると、ピラノース環Ｃ６位の水酸基を持つ炭素に由来するピーク（δ＝６０〜６５ｐｐｍ付近）は見られず、カルボキシル基に由来するピーク（δ＝１７０〜１８０ｐｐｍ付近）を有し、さらに、Ｃ３位の２級水酸基の酸化により生じるケトンなどのピーク（δ＝２００〜２１０ｐｐｍ付近）は検出されないことを特徴とする。
【００２８】
【実施例】
以下実施例により、本発明についてさらに詳しく説明する。
【００２９】
＜実施例１＞
キチン（和光純薬工業（株）製）を５ｇ、４５％水酸化ナトリウム水溶液５０ｇに浸漬し、容器の周囲を氷水で冷却しながら、２時間攪拌した。これに、砕いた氷１７５ｇを、攪拌しながら添加した。このアルカリ処理によりキチンはほぼ溶解する。低温に維持したまま、塩酸で中和し、十分に水洗した後、乾燥させずに、５％のアルカリ処理キチン懸濁液とした。ここに、ＴＥＭＰＯ ７５ｍｇ、臭化ナトリウム １．０ｇを溶解させた水溶液を加え、キチンの固形分濃度が約２ｗｔ％になるよう調製した。反応系を冷却し、１１％次亜塩素酸ナトリウム水溶液１０ｇを添加し、酸化反応を開始する。反応温度は常に５℃以下に維持した。反応中は系内のｐＨが低下するが、０．５Ｎ−ＮａＯＨ水溶液を逐次添加し、ｐＨ１０．８付近に調整するとともに、さらに１１％次亜塩素酸ナトリウム水溶液３５ｇを反応の進行に応じて調整しながら滴下した。６位の１級水酸基の全モル数に対し、１００％のモル数に対応するアルカリ添加量に近づくと、次亜塩素酸ナトリウム水溶液の滴下に関係なく、アルカリの添加速度（反応速度）は遅くなり、系内は完全に溶解してくる。アルカリ添加量が前記の１００％（４９．２ｍｌ）に達した時点で、エタノールを添加して反応を停止させた。反応時間は２時間であった。この反応溶液は、濾過により不溶の不純物を除いてから、過剰量のエタノール中に投入して、生成物を再沈させた。さらに水：アセトン＝１：７の溶液により充分洗浄した後、アセトンで脱水して、４０℃減圧乾燥して、白色粉末状のポリウロン酸のナトリウム塩５．３ｇを得た。
【００３０】
＜実施例２＞
脱アセチル化度１００％のキトサン（大日精化工業（株）製）を５ｇ、１０％酢酸９５ｇに溶解した。濾過により不溶分を除去し、メタノール５００ｍｌで希釈して、攪拌しながら無水酢酸４．７６ｇを添加すると数分でゲル化した。１晩静置後、ホモジナイザーで多量のメタノール中に分散させた。メタノール及び水：アセトン＝１：７の溶液により充分洗浄し、さらに水洗してメタノールおよびアセトンを完全に除き、Ｎ−アセチル化キトサンの５％濃度の懸濁液とした。
このＮ−アセチル化キトサンの懸濁液の一部を凍結乾燥して、元素分析によりＮ−アセチル化度を求めたところ、９５％であった（前記化学式（２）におけるｍ：ｎ＝９５：５）。
さらに前記Ｎ−アセチル化キトサンの５％濃度の懸濁液１００ｇに対し、実施例１と同様の酸化処理を行った。反応時間は１時間１０分であり、白色粉末状のポリウロン酸のナトリウム塩５．３ｇが得られた。
【００３１】
＜実施例３＞
実施例１のポリウロン酸のナトリウム塩２ｇを４０ｍｌの蒸留水に溶解し、攪拌しながら、ｐＨ１になるまで２Ｎ−塩酸を添加した。溶液は透明な溶液のままであった。この溶液を過剰量のエタノール中に投入し、生成物を再沈させた。さらに水：アセトン＝１：７の溶液により充分洗浄した後、アセトンで脱水して、４０℃減圧乾燥して、白色粉末状の脱塩したポリウロン酸１．６ｇを得た。
【００３２】
得られた実施例１〜３のポリウロン酸を以下の方法で評価した。
（水溶性）
実施例１〜３のポリウロン酸１．０ｇを、２５℃の蒸留水１０ｍｌに溶解させた。いずれも完全に溶解し、さらに高濃度での溶解も可能であった。
【００３３】
（ＮＭＲによる構造分析）
実施例１〜３のサンプルを重水に溶解させ、１３Ｃ−ＮＭＲを測定した。その結果を図１〜３に示す。ＮＭＲスペクトルから、キチンのピラノース環Ｃ６位の水酸基をもつ炭素に由来するピーク（δ＝６０〜６５ｐｐｍ付近）が完全に消えて、カルボキシル基（δ＝１７０〜１８０ｐｐｍ付近）に変換しており、２位、３位の炭素に由来するピークは変化せず、ケトンなどのピーク（δ＝２００〜２１０ｐｐｍ付近）は確認されなかった。従って、本発明のポリウロン酸は、ほぼ構造が均一な、β−（１，４）−Ｎ−アセチル−Ｄ−グルコサミヌロン酸であると言える。
【００３４】
（重量平均分子量の測定）
実施例１〜３のポリウロン酸の重量平均分子量（Ｍｗ）を、ＧＰＣ法により測定した。カラムはＴＳＫ−ｇｅｌＧ６０００ＰＷＸＬ、ＴＳＫ−ｇｅｌＧ３０００ＰＷＸＬを用い、０．１Ｍ−ＮａＣｌを溶離液とし、ＲＩ検出器を用い測定した。分子量既知の標準プルランから検量線を作成し、プルラン換算の重量平均分子量を算出した。その結果、実施例１から順にＭｗ＝６２，０００、７８，０００、６５，０００といずれも分子量３０，０００以上のポリウロン酸であった。さらに数平均分子量（Ｍｎ）との比（Ｍｗ／Ｍｎ）は、いずれも３以下で、分子量分散としては決して広くはなかった。従って分子量分散が広いために、計算上の重量平均分子量が高くなった訳ではないと言える。
【００３５】
（生分解性の測定）
実施例１のポリウロン酸の生分解性を以下の手法で評価したところ、試験１０日後の生分解度は４３％、試験２０日後の生分解度は６２％であった。なお対照として用いた微結晶セルロースは試験１０日後の生分解度は３９％、試験２０日後の生分解度は６０％であり、セルロースと同等以上の高い生分解性を有することが分かる。
（生分解性の評価方法）
八幡物産（株）製の微生物酸化分解測定装置（ＭＯＤＡ）を用い、試験土壌として、水分６０％に調整した標準コンポスト（八幡物産（株）製 ＹＫ−２）２５０ｃｃと、水分１８％に調整した海砂２５０ｃｃを混合したものを用いた。試料１０ｇを試験土壌と均一に混合して、カラム状の反応筒に充填し、反応筒内の温度を３５℃で一定に保持した。さらに反応筒下方より水蒸気を飽和した脱炭酸空気を４０ｍｌ／分で通気し、反応筒上部からはガス漏れなく配管されて、アンモニアガスを除くために硫酸水浴中を通り、水分を除くためにシリカゲルと塩化カルシウムを充填した吸湿筒を通り、さらにソーダタルク及びソーダライムを充填した吸収筒に導かれる。試料が好気的に生分解して発生する二酸化炭素は全て、吸収筒に吸収されるため、吸収筒の重量変化から生分解により発生した二酸化炭素量を定量できるものである。なお試料を入れない試験土壌のみの空試験を同時に行い、空試験で発生した二酸化炭素量を差し引いて、分解により発生した二酸化炭素量を求めた。試料１０ｇ中の炭素含量から理論的に発生する二酸化炭素量を算出し、理論量に対する発生二酸化炭素量の割合を生分解度とした。試料としては、実施例１のポリウロン酸と、対照として、微結晶セルロース（Ａｖｉｃｅｌ）を用いた。
【００３６】
【発明の効果】
以上より、本発明のポリウロン酸は（化１）の構造よりなる、β−（１，４）−ポリウロン酸であり、その重量平均分子量は３０，０００以上を有し、高い水溶性を示す。また生分解性も良好で、安全性に優れる。従って、構造が明確ゆえに、各種機能と化学構造の関係を解析していく上で、極めて有効な材料であり、材料設計のし易い合成原料となり得る。また、高分子量で水溶性が良好なことから、水系のコーティング材料として、及び水系の反応原料として好ましく用いることができる。さらには、ポリウロン酸単体、或いは二次修飾や複合化した材料の物性の向上にも寄与し、食品、医療・医薬、化粧品等、様々な機能性材料として応用されることが期待できる。
【００３７】
【図面の簡単な説明】
【図１】実施例１のポリウロン酸ナトリウム塩を重水に溶解して測定した１３Ｃ−ＮＭＲスペクトルである。
【図２】実施例２のポリウロン酸ナトリウム塩を重水に溶解して測定した１３Ｃ−ＮＭＲスペクトルである。
【図３】実施例３のポリウロン酸を重水に溶解して測定した１３Ｃ−ＮＭＲスペクトルである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to the provision of water-soluble polyuronic acid having a high molecular weight and a uniform structure.
[0002]
[Prior art]
Naturally occurring polyuronic acids include alginic acid and pectin, and these are industrially used as food additives, thickeners, stabilizers and the like because of their high safety. Further, some mucopolysaccharides (glycosaminoglycans) existing in the body include uronic acid (β-D-glucuronic acid) such as hyaluronic acid and chondroitin sulfate as constituent monosaccharides. Polyuronic acids are said to be excellent in biocompatibility and biodegradability, and some of them have been industrialized and studied for application as cosmetic materials, medical / pharmaceutical materials and the like.
At present, highly water-degradable polyuronic acids with excellent safety, biocompatibility, and biodegradability are used as raw materials, and they are highly biodegradable through secondary modifications such as chemical and physical modification, derivatization, and complexation with other materials. Studies are also underway to develop functional materials. However, most of the naturally occurring polyuronic acids are heteropolysaccharides, which makes the material design difficult due to the heterogeneous structure.
[0003]
On the other hand, attempts have been made to obtain polyuronic acids by oxidizing inexpensive natural polysaccharide materials. In particular, chitin, which exists as a skeletal material of crustaceans and insects, becomes hyaluronic acid-like structure when it is oxidized by uron, so it is considered to be highly useful and has been actively studied. However, there are few methods for selectively oxidizing only the primary hydroxyl group at the C6 position of the pyranose ring, and it has been difficult to selectively oxidize chitin with a particularly small effective solvent. Currently proposed oxidation methods include oxidation with nitrogen dioxide and N-oxyl compound catalysts such as 2,2,6,6-tetramethyl-1-piperidine-N-oxyl (hereinafter sometimes referred to as TEMPO). Oxidation is mentioned. However, in the oxidation with nitrogen dioxide, it has been reported that when all the primary hydroxyl groups at the C6 position of the pyranose ring are oxidized, secondary hydroxyl groups such as the C3 position are also oxidized, and the degree of polymerization is greatly reduced. In the oxidation with a TEMPO catalyst, the primary hydroxyl group at the C6 position can be oxidized with high selectivity by controlling the reaction conditions to obtain a polyuronic acid having a uniform structure, but the weight average molecular weight is 10,000 or less. Only those having a low degree of polymerization have been obtained (for example, see Non-Patent Document 1).
[0004]
As described above, when chemically modifying water-soluble polyuronic acids to synthesize biodegradable high-performance materials, there is a case where low molecular weight in the modification reaction is unavoidable, and the raw material polyuronic acid is as high as possible. A molecular weight is desirable. In addition, it has been studied to obtain a polyion complex material by combining water-soluble polyuronic acids with an anionic material and a cationic polymer. In this case, the strength properties of the generated polyion complex include the raw material polyuronic acid. The degree of polymerization is greatly affected. Polyuronic acid aqueous solution is also used as a coating agent, but the molecular weight of polyuronic acid greatly affects the physical properties of the coating film.
[0005]
[Non-Patent Document 1]
Carbohydrate Polymers 39 (1999) 361-367
[0006]
[Problems to be solved by the invention]
An object of the present invention is a water-soluble polyuronic acid having a uniform structure and a high molecular weight, which is excellent in safety, biocompatibility and biodegradability, and is also useful as a raw material for synthesizing various functional materials such as food, medicine / pharmaceuticals and cosmetics. Is to provide.
[0007]
[Means for Solving the Problems]
The invention of claim 1 is a step of preparing chitin which is swollen or dissolved in an alkali and has a ratio of m: n represented by the following chemical formula (2) in the range of 8: 2 to 10: 0, And a step of preparing a solution containing an N-oxyl compound and water, the temperature of the solution is set to 5 ° C. or less, the pH of the solution is set to a range of 10 to 11.5, and an oxidation reaction proceeds in the solution. And a step of dropping the oxidizing agent while adjusting the amount according to the condition.
[0008]
[Chemical 7]

[0009]
The invention of claim 2 is a step of N-acetylating to prepare chitosan having a ratio of m: n represented by the following chemical formula (2) in the range of 8: 2 to 10: 0, and the chitosan, A step of preparing a solution containing an N-oxyl compound and water; a temperature of the solution is set to 5 ° C. or less; a pH of the solution is set to a range of 10 to 11.5; And a step of dropping the oxidant while adjusting the amount accordingly, and a method for producing polyuronic acid.
[0010]
[Chemical 8]

[0011]
According to a third aspect of the present invention, the solution contains an alkali metal bromide, the N-oxyl compound is 2,2,6,6-tetramethyl-1-piperidine-N-oxyl, and the oxidizing agent is hypochlorous acid. 3. The method for producing polyuronic acid according to claim 1, wherein the polyuronic acid is sodium chlorate.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The polyuronic acid in the present invention has a structure represented by the following chemical formula (1), and X and Y in the formula represent hydrogen or an alkali metal. In addition, N-acetyl-D-glucosaminouronic acid (or an alkali metal salt of N-acetyl-D-glucosamineuronic acid) and D-glucosaminouronic acid (or an alkali metal salt of D-glucosamineuronic acid) are β- (1,4)- It is bonded, has a clear and uniform chemical structure, and is particularly preferable as a synthetic raw material in the case of secondary modification. Further, when X and Y in the chemical formula (1) are hydrogen or sodium, they exhibit a solubility of 10% or more with respect to distilled water at 25 ° C. Therefore, as an aqueous coating material and in an aqueous system It is useful as a reaction raw material.
[0016]
[Chemical 3]

[0017]
In addition, the polyuronic acid in the present invention has a ratio of m: n in the chemical formula (1), that is, N-acetyl-D-glucosaminouronic acid (or an alkali metal salt thereof) and D-glucosaminouronic acid (or an alkali metal salt thereof). The ratio is in the range of 8: 2 to 10: 0. Since D-glucosaminouronic acid (or its alkali metal salt) has a cationic amino group and easily forms an ionic bond with an anionic carboxyl group, D-glucosamineuronic acid (or its alkali metal salt) is entirely If it is 20% or more, the stability is lowered and the water solubility is lowered.
[0018]
In particular, when the polyuronic acid of the present invention is used as an anionic material to form a polyion complex with a cationic polysaccharide such as chitosan, the polyuronic acid composed of 100% N-acetyl-D-glucosaminouronic acid is used in an amount of 10%. The one containing about 1% of D-glucosamineuronic acid also tends to improve the strength properties of the produced polyion complex.
[0019]
Furthermore, the polyuronic acid of the present invention has a weight average molecular weight of 30,000 or more, more preferably 50,000 or more, and as described above, the chemical structure is clear and uniform, and has a high molecular weight. A certain point is a major feature. Therefore, when the polyuronic acid aqueous solution of the present invention is used as a coating material, coating properties are improved, and improvement in moisture resistance and film properties of the coating film can be expected. Furthermore, the physical properties and stability of the polyion complex are improved as described above. Further, when the polyuronic acid of the present invention is chemically modified as a raw material, it can contribute to improvement of physical properties of the product and stabilization of physical properties.
[0020]
The weight average molecular weight described here is a weight average molecular weight in terms of pullulan measured by size exclusion chromatography using pullulan as a standard substance.
[0021]
Furthermore, the polyuronic acid of the present invention can be obtained by using chitin or the like as a raw material and using an oxidation method with an N-oxyl compound catalyst. In order to obtain it, the accessibility of the substrate is sufficiently increased, and all primary hydroxyl groups at the C6 position are selectively converted to carboxyl groups, and oxidation is performed under mild conditions to reduce molecular weight and oxidation at the C3 position. It is important to suppress suboxidation. That is, using chitin sufficiently swollen or dissolved with alkali as a raw material, or using N-acetylated chitosan as a raw material, an alkali metal bromide and an oxidizing agent in the presence of an N-oxyl compound catalyst. The polyuronic acid of the present invention can be obtained by oxidizing at a low temperature of 5 ° C. or lower and an aqueous system while keeping the pH constant within a range of 10 to 11.5. Here, the N-oxyl compound is 2,2,6,6-tetramethyl-1-piperidine-N-oxyl (TEMPO), the alkali metal bromide is sodium bromide, and the oxidizing agent is hypochlorous acid. Sodium acid is particularly preferred.
[0022]
The chitin and chitosan have a structure represented by the following chemical formula (2), and chitin exists as a crustacean such as crabs and shrimps, and insect skeletons. Although it consists of acetyl-D-glucosamine, it also contains D-glucosamine by deacetylation or the like in the purification process. Chitin has high crystallinity and is insoluble in water, but after being immersed in a high-concentration alkali, it becomes a viscous liquid by diluting at a low temperature while adding ice. When hydrochloric acid is added thereto and neutralized, flaky chitin precipitates, but an almost amorphous chitin is obtained. By subjecting the chitin to the above oxidation reaction without sufficiently washing and drying, All primary hydroxyl groups at the C6 position are oxidized with high selectivity, and molecular weight reduction is suppressed. However, since the reaction of deacetylation of chitin also proceeds in an alkaline atmosphere, it is important to maintain the temperature in the system during the dissolution and neutralization treatment with alkali and the oxidation reaction at a low temperature of 5 ° C. or less. is there.
[0023]
[Formula 4]

[0024]
Further, chitosan is a substance obtained by deacetylating chitin, and is generally a substance having a higher ratio of n in the chemical formula (2) and is soluble in acid. When acetic anhydride is added to the chitosan solution, it is easily N-acetylated and can be returned to the chemical structure of chitin again. Through this operation, by washing the well-washed product without subjecting it to drying, only the primary hydroxyl group at the C6 position is oxidized with high selectivity as described above to obtain a high molecular weight polyuronic acid of the present invention. be able to. In this case, the m / n ratio can be arbitrarily set.
[0025]
Here, for example, the oxidation method includes, for example, adding the wet raw material to an aqueous solution in which TEMPO and sodium bromide are dissolved, uniformly dispersing the system, cooling the system to 5 ° C. or lower, adjusting the pH to 10, and A sodium hypochlorite solution is added as the reaction proceeds, and an aqueous sodium hydroxide solution is added dropwise to keep the pH of the system in the range of 10 to 11.5. During the reaction, the temperature in the system is maintained at 5 ° C. or lower. As the oxidation proceeds, the system dissolves and becomes a uniform solution. Under these reaction conditions, the amount of sodium hydroxide added almost corresponds to the amount of carboxyl groups introduced by oxidation, and reached the amount of primary hydroxyl group at the C6 position of the raw material and an equivalent amount. At this point, ethanol is added to deactivate excess oxidant and reprecipitate in excess ethanol. The product is sufficiently washed with a mixed solution of acetone and water, dehydrated with acetone, and then dried under reduced pressure to obtain the sodium salt of polyuronic acid of the present invention.
[0026]
The sodium salt of polyuronic acid obtained as described above is subjected to acid treatment after water treatment, and desalted polyuronic acid can be obtained by repeating the reprecipitation, washing and drying operations with the above ethanol.
[0027]
Furthermore, since the polyuronic acid of the present invention is β- (1,4) -polyuronic acid having a uniform structure, it is a carbon having a hydroxyl group at the C6-position of the pyranose ring when ¹³ C-NMR is measured by dissolving in heavy water. There is no peak derived from (δ = 60 to 65 ppm), there is a peak derived from a carboxyl group (δ = 170 to 180 ppm), and a ketone produced by oxidation of the secondary hydroxyl group at the C3 position A peak (around δ = 200 to 210 ppm) is not detected.
[0028]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples.
[0029]
<Example 1>
Chitin (manufactured by Wako Pure Chemical Industries, Ltd.) was immersed in 5 g of a 45% sodium hydroxide aqueous solution 50 g, and stirred for 2 hours while cooling the periphery of the container with ice water. To this, 175 g of crushed ice was added with stirring. Chitin is almost dissolved by this alkali treatment. While maintaining a low temperature, the solution was neutralized with hydrochloric acid, sufficiently washed with water, and then dried to obtain a 5% alkali-treated chitin suspension. An aqueous solution in which 75 mg of TEMPO and 1.0 g of sodium bromide were dissolved was added thereto to prepare a chitin solid content concentration of about 2 wt%. The reaction system is cooled and 10 g of an 11% aqueous sodium hypochlorite solution is added to initiate the oxidation reaction. The reaction temperature was always kept below 5 ° C. During the reaction, the pH in the system decreases, but 0.5N-NaOH aqueous solution is sequentially added to adjust the pH to around 10.8, and 35 g of 11% sodium hypochlorite aqueous solution is adjusted according to the progress of the reaction. While dripping. When the alkali addition amount corresponding to 100% of the total number of moles of the primary hydroxyl group at the 6-position is approached, the alkali addition rate (reaction rate) is slow regardless of the dropping of the sodium hypochlorite aqueous solution. Thus, the system is completely dissolved. When the amount of alkali added reached 100% (49.2 ml), ethanol was added to stop the reaction. The reaction time was 2 hours. This reaction solution was filtered to remove insoluble impurities, and then poured into an excess amount of ethanol to reprecipitate the product. Further, after thoroughly washing with a solution of water: acetone = 1: 7, it was dehydrated with acetone and dried under reduced pressure at 40 ° C. to obtain 5.3 g of sodium salt of polyuronic acid in the form of white powder.
[0030]
<Example 2>
Chitosan having a deacetylation degree of 100% (manufactured by Dainichi Seika Kogyo Co., Ltd.) was dissolved in 5 g and 95 g of 10% acetic acid. The insoluble matter was removed by filtration, diluted with 500 ml of methanol, and 4.76 g of acetic anhydride was added with stirring to gel in a few minutes. After standing overnight, it was dispersed in a large amount of methanol with a homogenizer. The solution was thoroughly washed with a solution of methanol and water: acetone = 1: 7, and further washed with water to completely remove methanol and acetone to obtain a 5% concentration suspension of N-acetylated chitosan.
A part of the suspension of N-acetylated chitosan was freeze-dried, and the degree of N-acetylation was determined by elemental analysis. As a result, it was 95% (m: n = 95 in the chemical formula (2): 5).
Further, 100 g of the N-acetylated chitosan suspension having a concentration of 5% was subjected to the same oxidation treatment as in Example 1. The reaction time was 1 hour and 10 minutes, and 5.3 g of sodium salt of polyuronic acid in the form of a white powder was obtained.
[0031]
<Example 3>
2 g of sodium polyuronic acid of Example 1 was dissolved in 40 ml of distilled water, and 2N hydrochloric acid was added to pH 1 while stirring. The solution remained a clear solution. This solution was poured into an excess amount of ethanol to reprecipitate the product. Further, it was sufficiently washed with a solution of water: acetone = 1: 7, dehydrated with acetone, and dried under reduced pressure at 40 ° C. to obtain 1.6 g of desalted polyuronic acid as a white powder.
[0032]
The obtained polyuronic acids of Examples 1 to 3 were evaluated by the following methods.
(Water soluble)
The polyuronic acid 1.0g of Examples 1-3 was dissolved in 10 ml of 25 degreeC distilled water. All were completely dissolved, and dissolution at a higher concentration was possible.
[0033]
(Structural analysis by NMR)
The samples of Examples 1 to 3 were dissolved in heavy water, and 13C-NMR was measured. The results are shown in FIGS. From the NMR spectrum, a peak derived from carbon having a hydroxyl group at the C6 position of the pyranose ring of chitin (δ = 60 to 65 ppm) completely disappears and converted to a carboxyl group (δ = 170 to 180 ppm). The peak derived from carbon at the 3rd and 3rd positions was not changed, and a peak such as ketone (δ = 200 to 210 ppm) was not confirmed. Therefore, it can be said that the polyuronic acid of the present invention is β- (1,4) -N-acetyl-D-glucosamineuronic acid having a substantially uniform structure.
[0034]
(Measurement of weight average molecular weight)
The weight average molecular weights (Mw) of the polyuronic acids of Examples 1 to 3 were measured by the GPC method. TSK-gelG6000PWXL and TSK-gelG3000PWXL were used as columns, and 0.1M NaCl was used as an eluent, and measurement was performed using an RI detector. A calibration curve was prepared from a standard pullulan having a known molecular weight, and a weight average molecular weight in terms of pullulan was calculated. As a result, in order from Example 1, Mw = 62,000, 78,000, 65,000 and all were polyuronic acids having a molecular weight of 30,000 or more. Furthermore, the ratio (Mw / Mn) to the number average molecular weight (Mn) was 3 or less, and the molecular weight dispersion was never wide. Therefore, it can be said that the calculated weight average molecular weight is not high due to the wide molecular weight dispersion.
[0035]
(Measurement of biodegradability)
When the biodegradability of the polyuronic acid of Example 1 was evaluated by the following method, the biodegradation degree after 10 days of the test was 43%, and the biodegradation degree after 20 days of the test was 62%. The microcrystalline cellulose used as a control has a biodegradability of 39% after 10 days of the test and a biodegradability of 60% after the 20 days of the test.
(Evaluation method for biodegradability)
Using a microbial oxidative degradation measuring device (MODA) manufactured by Yawata Bussan Co., Ltd., standard compost (YK-2 manufactured by Yawata Bussan Co., Ltd.) 250 cc adjusted to 60% moisture and adjusted to 18% moisture as test soil. A mixture of 250 cc of sea sand was used. 10 g of the sample was uniformly mixed with the test soil, filled in a column-shaped reaction cylinder, and the temperature in the reaction cylinder was kept constant at 35 ° C. Further, decarboxylated air saturated with water vapor is vented at 40 ml / min from the bottom of the reaction cylinder, and the gas is not leaked from the top of the reaction cylinder, passes through a sulfuric acid bath to remove ammonia gas, and silica gel to remove moisture. And the moisture absorption cylinder filled with calcium chloride, and further led to the absorption cylinder filled with soda talc and soda lime. Since all the carbon dioxide generated by aerobic biodegradation of the sample is absorbed by the absorption cylinder, the amount of carbon dioxide generated by biodegradation can be quantified from the weight change of the absorption cylinder. In addition, the blank test of only the test soil which does not put a sample was performed simultaneously, and the amount of carbon dioxide generated by decomposition was calculated by subtracting the amount of carbon dioxide generated in the blank test. The theoretically generated carbon dioxide amount was calculated from the carbon content in 10 g of the sample, and the ratio of the generated carbon dioxide amount to the theoretical amount was defined as the degree of biodegradation. As a sample, polyuronic acid of Example 1 and as a control, microcrystalline cellulose (Avicel) were used.
[0036]
【The invention's effect】
As mentioned above, the polyuronic acid of this invention is (beta)-(1,4) -polyuronic acid which consists of a structure of (Chemical Formula 1), and the weight average molecular weight has 30,000 or more, and shows high water solubility. In addition, biodegradability is good and safety is excellent. Therefore, since the structure is clear, it is an extremely effective material for analyzing the relationship between various functions and the chemical structure, and can be a synthetic raw material that can be easily designed. Further, since it has a high molecular weight and good water solubility, it can be preferably used as an aqueous coating material and an aqueous reaction raw material. Furthermore, it contributes to the improvement of physical properties of polyuronic acid alone, or secondary modified or compounded materials, and can be expected to be applied as various functional materials such as foods, medical / pharmaceuticals and cosmetics.
[0037]
[Brief description of the drawings]
1 is a 13C-NMR spectrum measured by dissolving polyuronic acid sodium salt of Example 1 in heavy water. FIG.
2 is a 13C-NMR spectrum measured by dissolving polyuronic acid sodium salt of Example 2 in heavy water. FIG.
FIG. 3 is a 13C-NMR spectrum measured by dissolving the polyuronic acid of Example 3 in heavy water.

Claims (3)

A step of preparing chitin which is swollen or dissolved with an alkali and the ratio of m: n represented by the following chemical formula (2) is in the range of 8: 2 to 10: 0;
Wherein the chitin, the method comprising the steps of: preparing a N- oxyl compound, a solution containing water,
The temperature of the solution is 5 ° C. or less, the pH of the solution is in the range of 10 to 11.5, and an oxidizing agent is dropped into the solution while adjusting the amount according to the progress of the oxidation reaction. A method for producing polyuronic acid, which is characterized by the above.

A step of N-acetylating to prepare chitosan having a ratio of m: n represented by the following chemical formula (2) in the range of 8: 2 to 10: 0;
The chitosan, and preparing the N- oxyl compound, a solution containing water,
The temperature of the solution is 5 ° C. or less, the pH of the solution is in the range of 10 to 11.5, and an oxidizing agent is dropped into the solution while adjusting the amount according to the progress of the oxidation reaction. A method for producing polyuronic acid, which is characterized by the above.

  The solution contains an alkali metal bromide, the N-oxyl compound is 2,2,6,6-tetramethyl-1-piperidine-N-oxyl, and the oxidizing agent is sodium hypochlorite. The method for producing polyuronic acid according to claim 1 or 2, characterized in that

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