JP2004189923A - Polyuronic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a water-soluble polyuronic acid having uniform structure and high molecular weight, exhibiting excellent safety, biocompatibility and biodegradability and useful as a synthetic raw material for various functional materials such as foods, medical/pharmaceutical agents and cosmetics. <P>SOLUTION: The polyuronic acid is composed of N-acetyl-D-glucosaminuronic acid (or N-acetyl-D-glucosaminuronic acid alkali metal salt) and D-glucosaminuronic acid (or D-glucosaminuronic acid alkali metal salt) bonded with each other through β-(1-4) bond and has a weight average molecular weight of ≥30,000. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【０００９】
（式中、Ｘ、Ｙは水素またはアルカリ金属を示す。）
【００１０】
請求項２の発明は、前記重量平均分子量が５０，０００以上である、請求項１記載のポリウロン酸である。
【００１１】
請求項３の発明は、前記化学式（１）中のｍ：ｎの比率が、８：２から１０：０の範囲である請求項１または２記載のポリウロン酸である。
【００１２】
請求項４の発明は、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に、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の範囲で酸化することにより得られた、請求項１乃至３のいずれかに記載ポリウロン酸である。
【００１３】
請求項５の発明は、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の範囲で酸化することにより得られた、請求項１乃至３のいずれかに記載のポリウロン酸である。
【００１４】
請求項６の発明は、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、前記臭化アルカリ金属が臭化ナトリウムであり、前記酸化剤が次亜塩素酸ナトリウムである請求項４乃至５のいずれかに記載のポリウロン酸である。
【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]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to providing a water-soluble polyuronic acid having a high molecular weight and a uniform structure.
[0002]
[Prior art]
Examples of naturally occurring polyuronic acids include alginic acid and pectin, which are industrially used as food additives, thickeners, stabilizers, etc. due to their high safety. Further, among the mucopolysaccharides (glycosaminoglycans) present in the living body, there are also those having uronic acid (β-D-glucuronic acid) as a constituent monosaccharide, such as hyaluronic acid and chondroitin sulfate. It is said that polyuronic acids are also excellent in biocompatibility and biodegradability, and their application as cosmetic materials, medical / medical materials, and the like have been studied, and some have been industrialized.
Currently, high biodegradability is achieved by using water-soluble polyuronic acids with excellent safety, biocompatibility, and biodegradability as raw materials, and performing secondary modifications such as chemical and physical modification, derivatization, and compounding with other materials. There are also studies to develop functional materials. However, the above-mentioned naturally occurring polyuronic acids are mostly heteropolysaccharides, and their heterogeneous structures make material design difficult.
[0003]
On the other hand, attempts have been made to obtain polyuronic acids by oxidizing inexpensive natural polysaccharide materials. Particularly, chitin, which is present as a skeletal substance of crustaceans and insects, has a structure similar to hyaluronic acid when subjected to uronation, and is considered to be highly useful, and has been actively studied. However, there are few techniques for selectively oxidizing only the primary hydroxyl group at the C6 position of the pyranose ring, and it has been particularly difficult to selectively oxidize chitin, which has a small effective solvent. Oxidation techniques currently proposed include oxidation with nitrogen dioxide and an N-oxyl compound catalyst such as 2,2,6,6-tetramethyl-1-piperidine-N-oxyl (hereinafter sometimes referred to as TEMPO). Oxidation. 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 to be oxidized, the secondary hydroxyl groups at the C3 position and the like are also oxidized, or the degree of polymerization is greatly reduced. In the oxidation using 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 are obtained (for example, see Non-Patent Document 1).
[0004]
As described above, when a water-soluble polyuronic acid is chemically modified to synthesize a biodegradable high-functional material, a reduction in the molecular weight in the modification reaction may be inevitable, and the raw material polyuronic acid may be as high as possible. It is desirable to have a molecular weight. It has also been considered to obtain a polyion complex material by combining water-soluble polyuronic acids as an anionic material with a cationic polymer, but in this case, the strength physical properties of the resulting polyion complex include the raw material polyuronic acid. Greatly affects the degree of polymerization. The aqueous solution of polyuronic acid is also used as a coating agent, but the molecular weight of the 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 to provide a water-soluble polyuronic acid having a uniform structure and a high molecular weight, which is excellent in safety, biocompatibility, and biodegradability, and is useful as a raw material for synthesizing various functional materials such as food, medicine, medicine, and cosmetics. Is to provide.
[0007]
[Means for Solving the Problems]
The invention of claim 1 is a polyuronic acid having a weight average molecular weight of 30,000 or more and having a structure represented by the following chemical formula (1).
[0008]
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[0009]
(In the formula, X and Y represent hydrogen or an alkali metal.)
[0010]
The invention according to claim 2 is the polyuronic acid according to claim 1, wherein the weight average molecular weight is 50,000 or more.
[0011]
The invention of claim 3 is the polyuronic acid according to claim 1 or 2, wherein the ratio of m: n in the chemical formula (1) is in the range of 8: 2 to 10: 0.
[0012]
The invention of claim 4 is based on chitin sufficiently swelled or dissolved with an alkali, and using an alkali metal bromide and an oxidizing agent in the presence of an N-oxyl compound catalyst at a low temperature of 5 ° C. or lower and in an aqueous system. The polyuronic acid according to any one of claims 1 to 3, obtained by oxidizing the solution in a pH range of 10 to 11.5.
[0013]
The invention according to claim 5 is a method for producing an N-acetylated chitosan as a raw material, using an alkali metal bromide and an oxidizing agent in the presence of an N-oxyl compound catalyst, at a low temperature of 5 ° C. or lower, in an aqueous system, The polyuronic acid according to any one of claims 1 to 3, obtained by oxidizing the polyuronic acid in the range of 10 to 11.5.
[0014]
The invention according to claim 6, wherein the N-oxyl compound is 2,2,6,6-tetramethyl-1-piperidine-N-oxyl, the alkali metal bromide is sodium bromide, and the oxidizing agent Is polyuronic acid according to any one of claims 4 to 5, wherein is sodium hypochlorite.
[0015]
BEST MODE FOR CARRYING OUT 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), wherein X and Y in the formula represent hydrogen or an alkali metal. In addition, N-acetyl-D-glucosamineuronic acid (or an alkali metal salt of N-acetyl-D-glucosamineuronic acid) and D-glucosamineuronic 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 raw material for synthesis in the case of secondary modification. Further, if X and Y in the chemical formula (1) are hydrogen or sodium, they exhibit a solubility of 10% or more in distilled water at 25 ° C., so that they can be used as an aqueous coating material and Useful as a reaction raw material.
[0016]
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[0017]
In addition, the polyuronic acid in the present invention is the ratio of m: n in the chemical formula (1), that is, N-acetyl-D-glucosamineuronic acid (or an alkali metal salt thereof) and D-glucosamineuronic acid (or an alkali metal salt thereof). Is in the range of 8: 2 to 10: 0. D-glucosaminonuronic acid (or an alkali metal salt thereof) has a cationic amino group and easily forms an ionic bond with an anionic carboxyl group. If not less than 20%, the stability and the water solubility are undesirably reduced.
[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, a polyuronic acid composed of 100% of N-acetyl-D-glucosaminouronic acid is used more than 10%. % Of D-glucosaminonuronic acid also tends to improve the strength physical properties of the resulting polyion complex.
[0019]
Further, the polyuronic acid of the present invention is characterized in that its weight average molecular weight is 30,000 or more, more preferably 50,000 or more, and as described above, the chemical structure is clear, uniform, and high in molecular weight. One point is a major feature. Therefore, when the aqueous polyuronic acid solution of the present invention is used as a coating material, coatability is improved, and improvement in moisture resistance and physical properties of a coating film can be expected. Further, when the polyion complex is formed as described above, the physical properties and stability are improved. Further, when the polyuronic acid of the present invention is chemically modified as a raw material, it can also contribute to improving the physical properties of the product and stabilizing the physical properties.
[0020]
The weight average molecular weight described herein is a weight average molecular weight in terms of pullulan measured by size exclusion chromatography using pullulan as a standard substance.
[0021]
Further, the polyuronic acid of the present invention can be obtained by using an oxidation method using an N-oxyl compound catalyst using chitin or the like as a raw material, and has a uniform structure and a high molecular weight polyuronic acid which is a feature of the present invention. In order to obtain the compound, the accessibility of the substrate is sufficiently increased, the primary hydroxyl group at the C6 position is selectively converted to a carboxyl group, and oxidation is performed under mild conditions to reduce the molecular weight or oxidize at the C3 position. It is important to suppress secondary oxidation. That is, using chitin sufficiently swollen or dissolved with alkali as a raw material, or chitosan obtained by N-acetylation as a raw material, using 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 in an aqueous system while keeping the pH constant in the range of 10 to 11.5. Here, 2,2,6,6-tetramethyl-1-piperidine-N-oxyl (TEMPO) is used as the N-oxyl compound, sodium bromide is used as the alkali metal bromide, and hypochlorite is used as the oxidizing agent. Sodium acid is particularly preferred.
[0022]
The above-mentioned chitin and chitosan have a structure represented by the following chemical formula (2). Chitin is present as a skeletal substance of crustaceans such as crabs and shrimps, and insects. It is composed of acetyl-D-glucosamine, but also contains D-glucosamine due to deacetylation in the purification process. Chitin has high crystallinity and is insoluble in water, but becomes a viscous liquid by being immersed in a high-concentration alkali and then diluted at a low temperature while adding ice. When neutralized by adding hydrochloric acid, flake-like chitin is precipitated, but almost amorphous chitin is obtained, which is sufficiently washed with water and dried to be subjected to the above oxidation reaction. All the primary hydroxyl groups at the C6 position are oxidized with high selectivity, and a decrease in molecular weight is suppressed. However, since the deacetylation reaction of chitin proceeds in an alkaline atmosphere, it is important to maintain the temperature in the system during the dissolution and neutralization treatment with the alkali and the oxidation reaction at a low temperature of 5 ° C. or lower. is there.
[0023]
Embedded image

[0024]
Further, chitosan is a substance obtained by deacetylating chitin, and generally has a higher ratio of n in the chemical formula (2), and dissolves in an acid. When acetic anhydride is added to the chitosan solution, it is easily N-acetylated, and it is possible to return to the chitin chemical structure again. Through this operation, a sufficiently washed product is subjected to the above-mentioned oxidation reaction without drying, whereby only the primary hydroxyl group at the C6 position is oxidized with high selectivity in the same manner as described above to obtain a high molecular weight polyuronic acid of the present invention. be able to. In this case, the ratio of m / n can be set arbitrarily.
[0025]
Here, the oxidizing method is, for example, adding the above-mentioned wet raw material to an aqueous solution in which TEMPO and sodium bromide are dissolved and uniformly dispersing it, cooling the system to 5 ° C. or lower, adjusting the pH to 10, and adding an oxidizing agent. A sodium hypochlorite solution is added as the reaction proceeds, and an aqueous solution of sodium hydroxide is added dropwise to keep the pH in the system constant within a 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 into a homogeneous solution. Under these reaction conditions, the amount of sodium hydroxide added almost corresponded to the amount of carboxyl groups introduced by oxidation, and reached an amount equivalent to the amount of the primary hydroxyl group at the C6-position of the raw material. At this point, excess oxidant is deactivated by adding ethanol and reprecipitated 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 an acid treatment after being dissolved in water, and the above-mentioned operation of reprecipitation, washing and drying with ethanol is repeated to obtain a desalted polyuronic acid.
[0027]
Furthermore, since the polyuronic acid of the present invention is a β- (1,4) -polyuronic acid having a uniform structure, when it is dissolved in heavy water and measured by ¹³ C-NMR, a carbon having a hydroxyl group at the C6 position of the pyranose ring is obtained. No peak derived from (= about 60 to 65 ppm), a peak derived from a carboxyl group (about δ = about 170 to 180 ppm), and further, a ketone or the like generated by oxidation of a secondary hydroxyl group at the C3 position. It is characterized in that 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>
5 g of chitin (manufactured by Wako Pure Chemical Industries, Ltd.) was immersed in 50 g of a 45% aqueous sodium hydroxide solution, and the mixture was 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 substantially dissolved by this alkali treatment. While maintaining the temperature at a low temperature, the solution was neutralized with hydrochloric acid, washed sufficiently with water, and then dried to obtain a 5% alkali-treated chitin suspension. To this was added an aqueous solution in which 75 mg of TEMPO and 1.0 g of sodium bromide were dissolved, and the solid content of chitin was adjusted to about 2 wt%. The reaction system is cooled, and 10 g of 11% aqueous sodium hypochlorite solution is added to start 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 further, 35 g of 11% aqueous sodium hypochlorite solution is adjusted according to the progress of the reaction. While dripping. When approaching the alkali addition amount corresponding to 100% of the number of moles of the primary hydroxyl group at the 6-position, the alkali addition rate (reaction rate) becomes slow regardless of the dropping of the aqueous solution of sodium hypochlorite. And the system completely dissolves. When the amount of alkali added reached 100% (49.2 ml), ethanol was added to stop the reaction. The reaction time was 2 hours. The reaction solution was filtered to remove insoluble impurities, and then poured into an excessive amount of ethanol to reprecipitate the product. Further, after sufficiently washing with a solution of water: acetone = 1: 7, dehydration with acetone and drying under reduced pressure at 40 ° C. were obtained to obtain 5.3 g of sodium salt of polyuronic acid as a white powder.
[0030]
<Example 2>
5 g of chitosan (manufactured by Dainichi Seika Kogyo Co., Ltd.) having a degree of deacetylation of 100% was dissolved in 95 g of 10% acetic acid. The insoluble matter was removed by filtration, the mixture was 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, the mixture was dispersed in a large amount of methanol with a homogenizer. The mixture 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% suspension of N-acetylated chitosan.
A part of this suspension of N-acetylated chitosan was freeze-dried, and the degree of N-acetylation was determined by elemental analysis. The result was 95% (m: n = 95 in the chemical formula (2): 5).
Further, 100 g of the 5% concentration suspension of the N-acetylated chitosan 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 was obtained as a white powder.
[0031]
<Example 3>
2 g of the sodium salt of polyuronic acid of Example 1 was dissolved in 40 ml of distilled water, and 2N-hydrochloric acid was added with stirring until the pH reached 1. The solution remained a clear solution. This solution was poured into an excessive amount of ethanol to reprecipitate the product. Further, after sufficiently washing with a solution of water: acetone = 1: 7, the mixture was 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)
1.0 g of the polyuronic acid of Examples 1 to 3 was dissolved in 10 ml of distilled water at 25 ° C. All were completely dissolved, and dissolution at a higher concentration was also 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, the peak derived from carbon having a hydroxyl group at the C6 position of the pyranose ring of chitin (δ = about 60 to 65 ppm) completely disappeared, and was converted to a carboxyl group (δ = about 170 to 180 ppm). The peaks derived from carbon at the third and third positions did not change, and peaks such as ketones (δ = around 200 to 210 ppm) were 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. The column was measured using TSK-gelG6000PWXL and TSK-gelG3000PWXL using 0.1M-NaCl as an eluent and an RI detector. A calibration curve was prepared from standard pullulan having a known molecular weight, and a weight average molecular weight in terms of pullulan was calculated. As a result, Mw was 62,000, 78,000, and 65,000 in this order from Example 1 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 in each case, and the molecular weight dispersion was not wide. Therefore, it can be said that the calculated weight average molecular weight did not necessarily increase 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 biodegradability after 10 days from the test was 43%, and the biodegradability after 20 days from the test was 62%. The microcrystalline cellulose used as a control had a biodegradability of 39% after 10 days of the test and a biodegradability of 60% after 20 days of the test, indicating that it had high biodegradability equal to or higher than that of cellulose.
(Method of evaluating biodegradability)
Using a microbial oxidative decomposition analyzer (MODA) manufactured by Yawata Bussan Co., Ltd., the test soil was adjusted to 250 cc of standard compost (YK-2 manufactured by Yawata Bussan Co., Ltd.) adjusted to 60% moisture and 18% water. What mixed 250cc of sea sand was used. 10 g of the sample was uniformly mixed with the test soil, filled in a column-shaped reaction tube, and the temperature in the reaction tube was kept constant at 35 ° C. In addition, decarbonated air saturated with water vapor is passed from the bottom of the reaction tube at 40 ml / min. The piping is connected without gas leakage from the top of the reaction tube, passes through a sulfuric acid water bath to remove ammonia gas, and silica gel to remove water. And calcium chloride, and then into an absorption cylinder filled with soda talc and soda lime. Since all 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 without a sample was performed at the same time, and the amount of carbon dioxide generated by the decomposition was obtained by subtracting the amount of carbon dioxide generated in the blank test. The amount of carbon dioxide theoretically generated was calculated from the carbon content in 10 g of the sample, and the ratio of the amount of generated carbon dioxide to the theoretical amount was defined as the degree of biodegradation. As a sample, the polyuronic acid of Example 1 and microcrystalline cellulose (Avicel) were used as a control.
[0036]
【The invention's effect】
As described above, the polyuronic acid of the present invention is β- (1,4) -polyuronic acid having the structure of Chemical Formula 1, having a weight average molecular weight of 30,000 or more, and showing high water solubility. It also has good biodegradability and is excellent in safety. 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 for which material design is easy. Further, since it has a high molecular weight and good water solubility, it can be preferably used as an aqueous coating material and as an aqueous reaction raw material. Furthermore, it contributes to the improvement of the physical properties of polyuronic acid alone or the material of secondary modification or compounding, and can be expected to be applied as various functional materials such as food, medicine / medicine, and cosmetics.
[0037]
[Brief description of the drawings]
FIG. 1 is a 13 C-NMR spectrum measured by dissolving sodium polyuronate of Example 1 in heavy water.
FIG. 2 is a 13 C-NMR spectrum measured by dissolving sodium polyuronic acid salt of Example 2 in heavy water.
FIG. 3 is a 13 C-NMR spectrum measured by dissolving the polyuronic acid of Example 3 in heavy water.

Claims (6)

A polyuronic acid having a weight average molecular weight of 30,000 or more and having the structure of the following chemical formula (1).

(In the formula, X and Y represent hydrogen or an alkali metal.) The polyuronic acid according to claim 1, wherein the weight average molecular weight is 50,000 or more. The polyuronic acid according to claim 1 or 2, wherein the ratio of m: n in the chemical formula (1) is in the range of 8: 2 to 10: 0. Using chitin sufficiently swelled or dissolved with an alkali as a raw material, a pH of 10 to 11 at a low temperature of 5 ° C. or lower and in an aqueous system using an alkali metal bromide and an oxidizing agent in the presence of an N-oxyl compound catalyst. The polyuronic acid according to any one of claims 1 to 3, which is obtained by oxidizing in the range of 5. Using N-acetylated chitosan as a raw material, an alkali metal bromide and an oxidizing agent are used in the presence of an N-oxyl compound catalyst, at a low temperature of 5 ° C. or lower, in an aqueous system, at a pH of 10 to 11.5. The polyuronic acid according to any one of claims 1 to 3, obtained by oxidizing in a range. The N-oxyl compound is 2,2,6,6-tetramethyl-1-piperidine-N-oxyl, the alkali metal bromide is sodium bromide, and the oxidizing agent is sodium hypochlorite. The polyuronic acid according to any one of claims 4 to 5.

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