JP2006232779A - Two-headed type glycoside and method for producing the same - Google Patents
Two-headed type glycoside and method for producing the same Download PDFInfo
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本発明は、両末端に糖又は糖鎖を有する新規な双頭型配糖体、及びその製造方法に関する。 The present invention relates to a novel double-headed glycoside having sugars or sugar chains at both ends, and a method for producing the same.
糖質は、タンパク質や核酸等とともに、微生物から高等植物、高等動物に至る生物にとって生命活動に欠くことのできない構成要素の一つであり、単なるエネルギー源として利用されるだけではなく、生体内において糖タンパク質、糖脂質、プロテオグリカン等として多種多様な形で存在している。 Carbohydrate is one of the indispensable components of living activities for organisms ranging from microorganisms to higher plants and higher animals, as well as proteins and nucleic acids, and is not only used as a source of energy but also in vivo. It exists in various forms as glycoproteins, glycolipids, proteoglycans and the like.
例えば、糖タンパク質には、タンパク質ペプチド上のアスパラギン残基のγアミノ基にＮ−アセチルグルコサミンが結合したＮ−グリコシド結合糖鎖や、セリン、スレオニン残基の水酸基にＮ−アセチルガラクトサミンが結合したＯ−グリコシド結合糖鎖があり、これらの糖を原点として非還元末端方向に、糖が伸長と分岐を繰り返すことにより、一方向に拡散した扇形構造を形成している。 For example, glycoproteins include N-glycoside-linked sugar chains in which N-acetylglucosamine is bound to the γ-amino group of asparagine residues on protein peptides, and O-groups in which N-acetylgalactosamine is bound to the hydroxyl groups of serine and threonine residues. -There are glycoside-linked sugar chains, and these sugars are used as the origin to form a fan-shaped structure in which sugars are diffused in one direction by repeating extension and branching toward the non-reducing end.
また、糖脂質には、スフィンゴ糖脂質とグリセロ糖脂質があり、これらの糖鎖は比較的短く単純で、１〜１５残基程度の糖鎖部位とセラミドやグリセロールからなる脂質部位が結合した構造を形成している。 Glycolipids include glycosphingolipids and glyceroglycolipids. These sugar chains are relatively short and simple, and have a structure in which a sugar chain part of about 1 to 15 residues and a lipid part composed of ceramide or glycerol are combined. Is forming.
また、コンドロイチン硫酸やヒアルロン酸のように２糖単位の繰り返しからなるグリコサミノグリカン、あるいは構造多糖であるセルロース、キチン、キトサン等のように非常に長い一本鎖構造の糖鎖も存在している。 There are also glycosaminoglycans consisting of repeating disaccharide units such as chondroitin sulfate and hyaluronic acid, or very long single-chain sugar chains such as cellulose, chitin and chitosan, which are structural polysaccharides. Yes.
上記のような糖鎖は、細胞内外の情報を司り、細胞接着、細胞認識、炎症、感染、免疫、分化、癌等といった我々の生命現象に複雑かつ奥深く関わっていることが知られており、糖や糖鎖を利用した新規な機能性素材や医薬品等の開発も期待されている。 Sugar chains such as those described above are responsible for information inside and outside the cell, and are known to be involved in our life phenomena such as cell adhesion, cell recognition, inflammation, infection, immunity, differentiation, cancer, etc. Development of new functional materials and pharmaceuticals using sugars and sugar chains is also expected.
例えば、Lehmannらは、糖タンパク質の糖鎖モデルとして双頭型の糖鎖を合成し、それらがガラクトース転移酵素のよい基質になることを報告している（下記非特許文献１、２）。 For example, Lehmann et al. Have reported that double-headed sugar chains are synthesized as sugar chain models of glycoproteins and that they become good substrates for galactose transferase (Non-Patent Documents 1 and 2 below).
Hansenらは、１,３−ジヒドロキシメチルベンゼンや１,３−ジメルカプトメチルベンゼンに、二つのガラクトースがα１−４結合したガラクトビオース鎖を２本結合させた化合物の合成を報告している（下記非特許文献３）。 Hansen et al. Have reported the synthesis of a compound in which two galactobiose chains in which two galactoses are α1-4 bonded to 1,3-dihydroxymethylbenzene or 1,3-dimercaptomethylbenzene. Non-patent document 3) below.
Leeらは、糖タンパク糖鎖を模倣した１〜４価の糖鎖化合物を合成し、価数が増えるほど肝レクチンの結合阻害活性が増大することを報告している（下記非特許文献４）。 Lee et al. Synthesize 1 to 4 valent sugar chain compounds that mimic glycoprotein sugar chains, and report that liver lectin binding inhibitory activity increases as the valence increases (Non-patent Document 4 below). .
下記非特許文献５には、２価の糖質による凝集効果で、格子様ネットワークが形成されることについて触れられている。 Non-Patent Document 5 mentioned below mentions that a lattice-like network is formed due to the aggregation effect of divalent carbohydrates.
下記特許文献１、２には、重合性官能基を有する双頭型の糖脂質、またその重合体及びその製造方法について開示されており、それらが安定な微細繊維状凝集体を形成することが記載されている。 Patent Documents 1 and 2 below disclose double-headed glycolipids having a polymerizable functional group, a polymer thereof and a production method thereof, and describe that they form a stable fine fibrous aggregate. Has been.
下記特許文献３には、自己集積性を有し、機能性材料として有用な双頭型脂質及びその製造方法が開示されている。
しかしながら、上記のような非天然型の配糖体は、水酸基の保護、脱保護、アノマーの活性化等の多段階の工程が必要な有機合成的手法により合成されており、工業的に合成するには問題があった。 However, the non-natural glycosides as described above are synthesized by an organic synthetic method that requires multi-step processes such as hydroxyl group protection, deprotection, and anomer activation, and are synthesized industrially. Had a problem.
したがって、本発明の目的は、酵素反応によって容易に合成することができる新規な双頭型配糖体を提供するとともに、該双頭型配糖体の製造方法を提供することにある。 Accordingly, an object of the present invention is to provide a novel double-headed glycoside that can be easily synthesized by an enzymatic reaction and to provide a method for producing the double-headed glycoside.
本発明者らは、アミコラトプシス・オリエンタリス（Amycolatopsis orientalis）のキチン培養上清中に見出される粗酵素を用いることにより、温和な条件下で、一段階の反応により、Ｎ−アセチルグルコサミンをアルカンジオールやオリゴエチレングリコールに転移でき、Ｎ−アセチルグルコサミンを含む双頭型配糖体を合成できることを見出し、本発明を完成するに至った。 By using the crude enzyme found in the supernatant of chitin culture of Amycolatopsis orientalis , the present inventors have converted N-acetylglucosamine into alkanediol in a one-step reaction under mild conditions. It has been found that a double-headed glycoside comprising N-acetylglucosamine can be synthesized and transferred to oligoethylene glycol, and the present invention has been completed.
すなわち、本発明による双頭型配糖体は、両末端に単糖又は糖鎖を有する化合物であって、下記一般式（１）又は（２）で表される化学構造を有することを特徴とする。
Ｒ−Ｏ−（ＣＨ２）ｎ−Ｏ−Ｒ１ …（１）
Ｒ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−Ｒ１ …（２）
（上記式中、Ｒ及びＲ１は還元末端１位水酸基を有さない単糖又は２〜８糖からなる糖鎖を表し、Ｒ、Ｒ１は同じでも異なっていてもよい。ｎは２〜９の整数を表す。）
That is, the double-headed glycoside according to the present invention is a compound having a monosaccharide or a sugar chain at both ends, and has a chemical structure represented by the following general formula (1) or (2). .
R—O— (CH 2 ) n —O—R 1 (1)
R—O— (CH 2 —CH 2 —O) n —R 1 (2)
(In the above formula, R and R 1 represent a sugar chain composed of a monosaccharide or 2 to 8 sugars having no hydroxyl group at the 1-position of the reducing end, and R and R 1 may be the same or different. Represents an integer of 9.)
上記双頭型配糖体は、前記Ｒが、Ｎ−アセチルグルコサミンを還元末端とする糖鎖からなり、下記一般式（３）又は（４）で表される化学構造を有することが好ましい。
Ｒ２−Ｏ−ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）ｎ−Ｏ−Ｒ１ …（３）
Ｒ２−Ｏ−ＧｌｃＮＡｃ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−Ｏ−Ｒ１ …（４）
（上記式中、Ｒ１は還元末端１位水酸基を有さない単糖又は２〜８糖からなる糖鎖を表し、Ｒ２は還元末端１位水酸基を有さない単糖又は２〜７糖からなる糖鎖を表し、Ｒ１、Ｒ２は同じでも異なっていてもよい。ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表す。ｎは２〜９の整数を表す。）
In the double-headed glycoside, R preferably comprises a sugar chain having N-acetylglucosamine as a reducing end and has a chemical structure represented by the following general formula (3) or (4).
R 2 —O—GlcNAc—O— (CH 2 ) n —O—R 1 (3)
R 2 —O—GlcNAc—O— (CH 2 —CH 2 —O) n —O—R 1 (4)
(In the above formula, R 1 represents a monosaccharide having no hydroxyl group at the reducing end 1-position or a sugar chain consisting of 2 to 8 sugars, and R 2 is a monosaccharide having no hydroxyl group at the reducing end 1-position or 2 to 7 sugars. And R 1 and R 2 may be the same or different, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
また、前記一般式（１）〜（４）におけるＲ、Ｒ１、Ｒ２が、還元末端１位水酸基のないラクトース、Ｎ−アセチルグルコサミン、Ｎ−アセチルラクトサミン、Ｎ−アセチルキトビオースから選ばれた１種であることが好ましい。 In the general formulas (1) to (4), R, R 1 and R 2 are selected from lactose having no hydroxyl group at the 1-position of the reducing end, N-acetylglucosamine, N-acetyllactosamine, and N-acetylchitobiose. It is preferable that it is 1 type.
本発明の双頭型配糖体は、アルカンジオール又はオリゴエチレングリコールからなるリンカー部を介して、還元末端を結合点として双方向に糖、あるいは糖鎖が結合しており、一つの分子内において双方向に非還元末端糖鎖部分が露出する構造をとっている。そのため、従来の糖鎖とは異なる相互作用や反応性を示す可能性が考えられ、全く新しい機構による薬剤や生理活性物質としての利用が期待できる。 In the double-headed glycoside of the present invention, a sugar or sugar chain is bound in both directions with a reducing end as a bonding point via a linker portion composed of alkanediol or oligoethylene glycol. The non-reducing terminal sugar chain part is exposed in the direction. For this reason, there is a possibility that it exhibits an interaction and reactivity different from those of conventional sugar chains, and it can be expected to be used as a drug or physiologically active substance by a completely new mechanism.
一方、本発明による双頭型配糖体の製造方法の一つは、アルカンジオール（炭素数２〜９）と、キチンオリゴ糖（重合度２〜６）又はキチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）に、キチナーゼ又はＮ−アセチルヘキソサミニダーゼを作用させ、得られた生成物から下記式（５）で示される化合物を採取することを特徴とする。
ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）ｎ−Ｏ−ＧｌｃＮＡｃ …（５）
（上記式中、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。）
On the other hand, one of the methods for producing a double-headed glycoside according to the present invention is an alkanediol (2 to 9 carbon atoms) and a chitin oligosaccharide (degree of polymerization 2 to 6) or a paranitrophenyl derivative of a chitin oligosaccharide (polymerization). The compound represented by the following formula (5) is collected from the resulting product by allowing chitinase or N-acetylhexosaminidase to act at a degree of 1 to 6).
GlcNAc-O- (CH 2) n -O-GlcNAc ... (5)
(In the above formula, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
本発明による双頭型配糖体の製造方法のもう一つは、アルカンジオールの片側に１〜８糖の糖鎖が結合したアルカノール誘導体と、キチンオリゴ糖（重合度２〜６）又はキチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）に、キチナーゼ又はＮ−アセチルヘキソサミニダーゼを作用させ、得られた生成物から下記一般式（６）で示される化合物を採取することを特徴とする。
Ｒ−Ｏ−（ＣＨ２）ｎ−Ｏ−ＧｌｃＮＡｃ …（６）
（上記式中、Ｒは還元末端１位水酸基を有さない単糖又は２〜８糖からなる糖鎖を表し、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。）
Another method for producing a double-headed glycoside according to the present invention is an alkanol derivative in which a sugar chain of 1 to 8 sugars is bonded to one side of alkanediol, and a chitin oligosaccharide (polymerization degree 2 to 6) or chitin oligosaccharide. Characterized in that chitinase or N-acetylhexosaminidase is allowed to act on the paranitrophenyl derivative (polymerization degree 1 to 6), and a compound represented by the following general formula (6) is collected from the obtained product. To do.
R—O— (CH 2 ) n —O—GlcNAc (6)
(In the above formula, R represents a sugar chain composed of a monosaccharide or 2 to 8 sugars having no hydroxyl group at the reducing end, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9).
上記一般式（６）におけるＲは、Ｎ−アセチルグルコサミンを還元末端とする糖鎖からなり、該還元末端において前記アルカンジオールの片側に結合していることが好ましい。 R in the general formula (6) is composed of a sugar chain having N-acetylglucosamine as a reducing end, and is preferably bonded to one side of the alkanediol at the reducing end.
本発明による双頭型配糖体の製造方法の更にもう一つは、オリゴエチレングリコール（エチレン数２〜９）と、キチンオリゴ糖（重合度２〜６）又はキチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）に、キチナーゼ及び／又はＮ−アセチルヘキソサミニダーゼを作用させ、得られた生成物から下記式（７）で示される化合物を採取することを特徴とする。
ＧｌｃＮＡｃ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−ＧｌｃＮＡｃ…（７）
（上記式中、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。）
Still another method for producing a double-headed glycoside according to the present invention includes oligoethylene glycol (ethylene number 2 to 9) and chitin oligosaccharide (polymerization degree 2 to 6) or paranitrophenyl derivative of chitin oligosaccharide ( A chitinase and / or N-acetylhexosaminidase is allowed to act on the degree of polymerization 1 to 6), and a compound represented by the following formula (7) is collected from the obtained product.
GlcNAc-O- (CH 2 -CH 2 -O) n -GlcNAc ... (7)
(In the above formula, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
本発明による双頭型配糖体の製造方法の更にもう一つは、オリゴエチレングリコールの片側に１〜８糖の糖鎖が結合したオリゴエチレングルコール誘導体と、キチンオリゴ糖（重合度２〜６）又はキチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）に、キチナーゼ及び／又はＮ−アセチルヘキソサミニダーゼを作用させ、得られた生成物から下記一般式（８）で示される化合物を採取することを特徴とする。
Ｒ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−ＧｌｃＮＡｃ…（８）
（上記式中、Ｒは還元末端１位水酸基を有さない単糖又は２〜８糖からなる糖鎖を表し、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。）
Still another method for producing a double-headed glycoside according to the present invention is an oligoethylene glycol derivative in which a sugar chain of 1 to 8 sugars is bonded to one side of oligoethylene glycol and a chitin oligosaccharide (degree of polymerization of 2 to 6). ) Or a paranitrophenyl derivative of chitin oligosaccharide (degree of polymerization 1 to 6), and a compound represented by the following general formula (8) from the product obtained by allowing chitinase and / or N-acetylhexosaminidase to act. It is characterized by collecting.
R—O— (CH 2 —CH 2 —O) n —GlcNAc (8)
(In the above formula, R represents a sugar chain composed of a monosaccharide or 2 to 8 sugars having no hydroxyl group at the reducing end, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9).
上記一般式（８）におけるＲは、Ｎ−アセチルグルコサミンを還元末端とする糖鎖からなり、該還元末端において前記オリゴエチレングリコールの片側に結合していることが好ましい。 R in the general formula (8) is composed of a sugar chain having N-acetylglucosamine as a reducing end, and is preferably bonded to one side of the oligoethylene glycol at the reducing end.
本発明による双頭型配糖体の製造方法の更にもう一つは、アルカンジオール（炭素数２〜９）と、キチンオリゴ糖（重合度２〜６）又はキチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）に、キチナーゼ又はＮ−アセチルヘキソサミニダーゼを作用させ、得られた生成物から下記式（９）で示される化合物を採取し、この化合物に糖転移によって他の糖又は糖鎖を結合させることにより、前記一般式（６）で示される化合物を得ることを特徴とする。
ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）ｎ−ＯＨ …（９）
（上記式中、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。）
Still another method for producing a double-headed glycoside according to the present invention is an alkanediol (2 to 9 carbon atoms) and a chitin oligosaccharide (degree of polymerization 2 to 6) or a paranitrophenyl derivative of a chitin oligosaccharide (polymerization). A compound represented by the following formula (9) is collected from the obtained product by allowing chitinase or N-acetylhexosaminidase to act at a degree of 1 to 6), and other saccharides or A compound represented by the general formula (6) is obtained by combining chains.
GlcNAc-O- (CH 2) n -OH ... (9)
(In the above formula, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
本発明による双頭型配糖体の製造方法の更にもう一つは、オリゴエチレングリコール（エチレン数２〜９）と、キチンオリゴ糖（重合度２〜６）又はキチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）に、キチナーゼ及び／又はＮ−アセチルヘキソサミニダーゼを作用させ、得られた生成物から下記式（１２）で示される化合物を採取し、この化合物に糖転移によって他の糖又は糖鎖を結合させることにより、前記一般式（８）で示される化合物を得ることを特徴とする。
ＧｌｃＮＡｃ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−ＯＨ …（１２）
（上記式中、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。）
Still another method for producing a double-headed glycoside according to the present invention includes oligoethylene glycol (ethylene number 2 to 9) and chitin oligosaccharide (polymerization degree 2 to 6) or paranitrophenyl derivative of chitin oligosaccharide ( Chitinase and / or N-acetylhexosaminidase is allowed to act on the degree of polymerization 1 to 6), and a compound represented by the following formula (12) is collected from the obtained product. A compound represented by the general formula (8) is obtained by binding a sugar or a sugar chain.
GlcNAc-O- (CH 2 -CH 2 -O) n -OH ... (12)
(In the above formula, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
本発明による双頭型配糖体の製造方法においては、前記いずれかの方法により得られた双頭型配糖体に結合した糖又は糖鎖を、糖転移によって他の糖又は糖鎖と置換することにより、前記一般式（１）〜（４）で示される化合物を生成させることもできる。 In the method for producing a double-headed glycoside according to the present invention, the sugar or sugar chain bound to the double-headed glycoside obtained by any of the above methods is replaced with another sugar or sugar chain by sugar transfer. Thus, the compounds represented by the general formulas (1) to (4) can also be produced.
また、本発明による双頭型配糖体の製造方法においては、前記キチナーゼ及び／又はＮ−アセチルヘキソサミニダーゼとしては、アミコラトプシス・オリエンタリス（Amycolatopsis orientalis）の培養上清、又は該培養上清から調製された粗酵素若しくは精製酵素を用いることが好ましい。 In the method for producing a double-headed glycoside according to the present invention, the chitinase and / or N-acetylhexosaminidase may be a culture supernatant of Amycolatopsis orientalis or the culture supernatant. It is preferable to use a crude enzyme or a purified enzyme prepared from
本発明の双頭型配糖体の製造方法によれば、機能性素材や医薬品等への利用が期待できる新規な非天然型の双頭型配糖体を酵素反応により容易に得ることができる。 According to the method for producing a double-headed glycoside of the present invention, a novel unnatural double-headed glycoside that can be expected to be used for a functional material or a pharmaceutical can be easily obtained by an enzymatic reaction.
本発明によれば、機能性素材や医薬品等への利用が期待できる新規な非天然型の双頭型配糖体、及びその製造方法を提供することができる。本双頭型配糖体は、双方向に糖鎖部位を有するため、そのまま、あるいは該双頭型配糖体を基本骨格として酵素反応等により糖鎖を伸張することにより、タンパク質、受容体、レクチン、細胞、白血球、リンパ球、ホルモン、微生物、ウイルス等を１分子で両方向につなぎとめたり、異種のものをつなぎとめることも可能であるので、凝集剤や阻害剤、接着剤、ＤＤＳの輸送体等への利用が期待できる。また、その分子構造の特性上、保湿効果、安定化効果、界面活性効果、自己凝集効果等も期待でき、化粧品、医薬品、食品、農薬、電子等の様々な分野で応用できる可能性がある。 ADVANTAGE OF THE INVENTION According to this invention, the novel non-natural type | mold double-headed glycoside which can anticipate utilization to a functional raw material, a pharmaceutical, etc., and its manufacturing method can be provided. Since this double-headed glycoside has a sugar chain site in both directions, the sugar chain is extended as it is or by enzymatic reaction using the double-headed glycoside as a basic skeleton, thereby allowing the protein, receptor, lectin, Since cells, leukocytes, lymphocytes, hormones, microorganisms, viruses, etc. can be connected in both directions with one molecule, or dissimilar ones can be connected, it can be used as an aggregating agent, inhibitor, adhesive, DDS transporter, etc. Can be used. In addition, due to the characteristics of its molecular structure, it can be expected to have a moisturizing effect, a stabilizing effect, a surface active effect, a self-aggregating effect, and the like, and may be applied in various fields such as cosmetics, pharmaceuticals, foods, agricultural chemicals, and electronics.
本発明の双頭型配糖体は、両末端に単糖又は糖鎖を有する化合物であって、下記一般式（１）又は（２）で表される化学構造を有するものである。
Ｒ−Ｏ−（ＣＨ２）ｎ−Ｏ−Ｒ１ …（１）
Ｒ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−Ｒ１ …（２）
（上記式中、Ｒ及びＲ１は還元末端１位水酸基を有さない単糖又は２〜８糖からなる糖鎖を表し、Ｒ、Ｒ１は同じでも異なっていてもよい。ｎは２〜９の整数を表す。）
The double-headed glycoside of the present invention is a compound having a monosaccharide or a sugar chain at both ends, and has a chemical structure represented by the following general formula (1) or (2).
R—O— (CH 2 ) n —O—R 1 (1)
R—O— (CH 2 —CH 2 —O) n —R 1 (2)
(In the above formula, R and R 1 represent a sugar chain composed of a monosaccharide or 2 to 8 sugars having no hydroxyl group at the 1-position of the reducing end, and R and R 1 may be the same or different. Represents an integer of 9.)
また、上記双頭型配糖体は、前記Ｒが、Ｎ−アセチルグルコサミンを還元末端とする糖鎖からなり、下記一般式（３）又は（４）で表される化学構造を有することが好ましい。
Ｒ２−Ｏ−ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）ｎ−Ｏ−Ｒ１ …（３）
Ｒ２−Ｏ−ＧｌｃＮＡｃ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−Ｏ−Ｒ１ …（４）
（上記式中、Ｒ１は還元末端１位水酸基を有さない単糖又は２〜８糖からなる糖鎖を表し、Ｒ２は還元末端１位水酸基を有さない単糖又は２〜７糖からなる糖鎖を表し、Ｒ１、Ｒ２は同じでも異なっていてもよい。ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表す。ｎは２〜９の整数を表す。）
In the double-headed glycoside, R preferably comprises a sugar chain having N-acetylglucosamine as the reducing end and has a chemical structure represented by the following general formula (3) or (4).
R 2 —O—GlcNAc—O— (CH 2 ) n —O—R 1 (3)
R 2 —O—GlcNAc—O— (CH 2 —CH 2 —O) n —O—R 1 (4)
(In the above formula, R 1 represents a monosaccharide having no hydroxyl group at the reducing end 1-position or a sugar chain consisting of 2 to 8 sugars, and R 2 is a monosaccharide having no hydroxyl group at the reducing end 1-position or 2 to 7 sugars. And R 1 and R 2 may be the same or different, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
また、前記一般式（１）〜（４）におけるＲ、Ｒ１、Ｒ２が、還元末端１位水酸基のないラクトース、Ｎ−アセチルグルコサミン、Ｎ−アセチルラクトサミン、Ｎ−アセチルキトビオースから選ばれた１種であることが好ましい。 In the general formulas (1) to (4), R, R 1 and R 2 are selected from lactose having no hydroxyl group at the 1-position of the reducing end, N-acetylglucosamine, N-acetyllactosamine, and N-acetylchitobiose. It is preferable that it is 1 type.
ラクトースは、ガラクトースとグルコースがβ１−４結合した二糖であり、Ｎ−アセチルラクトサミンは、ガラクトースとＮ−アセチルグルコサミンがβ１−４結合した二糖であり、Ｎ−アセチルキトビオースは、Ｎ−アセチルグルコサミンとＮ−アセチルグルコサミンがβ１−４結合した二糖である。 Lactose is a disaccharide in which galactose and glucose are β1-4 linked, N-acetyllactosamine is a disaccharide in which galactose and N-acetylglucosamine are β1-4 linked, and N-acetylchitobiose is N -A disaccharide in which acetylglucosamine and N-acetylglucosamine are β1-4 linked.
本発明の双頭型配糖体の好ましい具体例としては、上記の他、例えば、ｉ）創傷作用や免疫作用が期待されるキチンオリゴ糖、キトサンオリゴ糖、ｉｉ）細菌毒素の中和作用が期待されるミルクオリゴ糖、ｉｉｉ）インフルエンザ阻害作用が期待されるシアル酸、ｉｖ）抗炎症作用が期待されるシアリルルイスＸ、ｖ）保湿作用が期待されるヒアルロン酸オリゴ糖等の糖鎖が結合した双頭型配糖体が挙げられる。 Preferable specific examples of the double-headed glycoside of the present invention include, in addition to the above, for example, i) chitin oligosaccharides and chitosan oligosaccharides that are expected to have wound action and immune action, and ii) neutralization action of bacterial toxins. Milk oligosaccharides, iii) sialic acid expected to have influenza inhibitory action, iv) sialyl Lewis X expected to have anti-inflammatory action, v) double-headed with sugar chains such as hyaluronic acid oligosaccharide expected to have moisturizing action Type glycosides.
次に、本発明の双頭型配糖体の製造方法について説明する。 Next, the manufacturing method of the double-headed glycoside of this invention is demonstrated.
本発明の双頭型配糖体の製造方法は、リンカー部になる化合物（受容体基質）と、キチンオリゴ糖又はその誘導体（供与体基質）を用い、これらの反応基質にキチナーゼ及び／又はＮ−アセチルヘキソサミニダーゼを作用させることにより、前記リンカー部となる化合物分子の末端にＮ−アセチルグルコサミンを転移させるものである。 The method for producing a double-headed glycoside of the present invention uses a compound (acceptor substrate) that becomes a linker moiety and a chitin oligosaccharide or a derivative thereof (donor substrate), and chitinase and / or N- as the reaction substrate. By acting on acetylhexosaminidase, N-acetylglucosamine is transferred to the end of the compound molecule serving as the linker moiety.
上記キチナーゼ及び／又はＮ−アセチルへキソサミニダーゼは、例えば、アミコラトプシス・オリエンタリス（Amycolatopsis orientalis）（ＩＦＯ１２８０６、ＡＴＣＣ１９７９５）、バチルス・サーキュランス（Bacillus circulans）（ＩＦＯ１３６２６、ＡＴＣＣ２４）、トリコデルマ・リーセイ（Trichoderma reesei）、卵白リゾチーム等に由来するものを用いることができる。本発明においては、アミコラトプシス・オリエンタリス（Amycolatopsis orientalis）由来のものを用いることが好ましい。アミコラトプシス・オリエンタリス（Amycolatopsis orientalis）をコロイダルキチンとともに培養して得られる培養上清には、キチナーゼとＮ−アセチルへキソサミニダーゼが分泌され、これらの酵素が強い転移活性を持っていることが知られている（Biochimica et Biophysica Acta923, 302 (1987)、Agric. Biol. Chem., 54 (4), 899 (1990)、Agric. Biol. Chem., 53 (8), 2189 (1989)）。本発明においては、上記の培養上清をそのまま用いてもよく、該培養上清から硫安分画等により粗精製してから用いてもよい。また、カラムクロマトグラフィーや高速液体クロマトグラフィー等により精製してから用いることもできる。 The chitinase and / or N-acetylhexosaminidase are, for example, Amycolatopsis orientalis (IFO12806, ATCC19795), Bacillus circulans (IFO13626, ATCC24), Trichoderma reesei ( Trichoderma reesei ) Those derived from egg white lysozyme and the like can be used. In the present invention, it is preferable to use those derived from Amycolatopsis orientalis . Chitinase and N-acetylhexosaminidase are secreted in the culture supernatant obtained by culturing Amycolatopsis orientalis with colloidal chitin, and it is known that these enzymes have strong transfer activity. (Biochimica et Biophysica Acta923, 302 (1987), Agric. Biol. Chem., 54 (4), 899 (1990), Agric. Biol. Chem., 53 (8), 2189 (1989)). In the present invention, the culture supernatant described above may be used as it is, or may be used after being roughly purified from the culture supernatant by an ammonium sulfate fraction or the like. It can also be used after being purified by column chromatography, high performance liquid chromatography or the like.
受容体基質として用いられる上記リンカー部になる化合物としては、アルカンジオール（好ましくは炭素数２〜９）やオリゴエチレングリコール（好ましくはエチレン数２〜９）が例示でき、更には、アルカンジオールの片側に１〜８糖の糖鎖が結合したアルカノール誘導体やオリゴエチレングリコールの片側に１〜８糖の糖鎖が結合したオリゴエチレングリコール誘導体等が例示できる。 Examples of the compound serving as the linker moiety used as the acceptor substrate include alkanediol (preferably having 2 to 9 carbon atoms) and oligoethylene glycol (preferably having 2 to 9 ethylene), and one side of alkanediol. Examples thereof include alkanol derivatives in which 1 to 8 sugar chains are bonded to each other, oligoethylene glycol derivatives in which 1 to 8 sugar chains are bonded to one side of oligoethylene glycol, and the like.
アルカンジオール又はオリゴエチレングリコールの片側に結合させる上記１〜８糖の糖鎖としては、例えば、グルコース、ガラクトース、Ｎ−アセチルグルコサミン、グルコサミン、Ｎ−アセチルガラクトサミン、ガラクトサミン、マンノース、フコース、キシロース、シアル酸、ウロン酸を構成糖とするものが好ましく、具体的には、キチンオリゴ糖、キトサンオリゴ糖、ポリラクトサミンオリゴ糖、ミルクオリゴ糖、コンドロイチン硫酸、ヒアルロン酸オリゴ糖、マンノースをコアとした糖タンパク、シアリルルイスＸ、ＨＮＫ−１抗原糖鎖、α−Ｇａｌ抗原糖鎖等が挙げられる。 Examples of the sugar chain of 1 to 8 sugars bonded to one side of alkanediol or oligoethylene glycol include, for example, glucose, galactose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine, galactosamine, mannose, fucose, xylose, sialic acid In particular, those having uronic acid as a constituent sugar are preferable. Specifically, chitin oligosaccharide, chitosan oligosaccharide, polylactosamine oligosaccharide, milk oligosaccharide, chondroitin sulfate, hyaluronic acid oligosaccharide, glycoprotein having mannose as a core Sialyl Lewis X, HNK-1 antigen sugar chain, α-Gal antigen sugar chain and the like.
上記アルカンジオールやオリゴエチレングリコールは、市販のものを用いることができる。また、上記アルカノール誘導体やオリゴエチレングルコール誘導体の製造方法については、例えば、トリコデルマ・リーセイ（Trichoderma reesei）由来の市販セルラーゼを用いたラクトース、若しくはＮ−アセチルラクトサミンの結合した誘導体の製造方法が知られている（Archires of Biochemistry and Biophysics, vol.385, No.1, January 1, pp70-77 (2001)）。 Commercially available alkanediol and oligoethylene glycol can be used. In addition, with regard to a method for producing the alkanol derivative or oligoethylene glycol derivative, for example, a method for producing lactose using a commercially available cellulase derived from Trichoderma reesei or a derivative to which N-acetyllactosamine is bound is known. (Archires of Biochemistry and Biophysics, vol. 385, No. 1, January 1, pp70-77 (2001)).
一方、供与体基質として用いられる上記キチンオリゴ糖又はその誘導体としては、好ましくはキチンオリゴ糖（重合度２〜６）や、キチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）を用いることができる。 On the other hand, the chitin oligosaccharide or derivative thereof used as a donor substrate is preferably chitin oligosaccharide (polymerization degree 2 to 6) or a paranitrophenyl derivative of chitin oligosaccharide (polymerization degree 1 to 6). Can do.
上記キチンオリゴ糖は、公知の方法により、キチンを加水分解することにより調製することもできるが、例えば、商品名「ＮＡＣＯＳ−Ｙ」（焼津水産化学工業株式会社製）等の市販のものを用いることもできる。また、キチンオリゴ糖のパラニトロフェニル誘導体は、例えば、文献［J Biochem, 104, 255 (1988)］に記載された方法により調製することができる。 The chitin oligosaccharide can be prepared by hydrolyzing chitin by a known method. For example, a commercially available product such as “NACOS-Y” (manufactured by Yaizu Suisan Chemical Co., Ltd.) is used. You can also. Moreover, the para nitrophenyl derivative of chitin oligosaccharide can be prepared by the method described in literature [J Biochem, 104, 255 (1988)], for example.
以下、本発明の双頭型配糖体の製造方法について、より具体的に説明する。 Hereinafter, the method for producing the double-headed glycoside of the present invention will be described more specifically.
リンカー部となる化合物と、キチンオリゴ糖又はキチンオリゴ糖のパラニトロフェニル誘導体との配合比（リンカー部となる化合物：キチンオリゴ糖又はキチンオリゴ糖のパラニトロフェニル誘導体）は、モル比で０．０５〜２：０．０５〜２となるようにすることが好ましい。 The compounding ratio of the compound serving as the linker part and the chitin oligosaccharide or the paranitrophenyl derivative of chitin oligosaccharide (compound serving as the linker part: chitin oligosaccharide or the paranitrophenyl derivative of chitin oligosaccharide) is 0. 05-2: It is preferable to be 0.05-2.
これらの化合物を、例えば、酢酸、リン酸、フタル酸緩衝液等の緩衝液に溶解し、アミコラトプシス・オリエンタリス（Amycolatopsis orientalis）の培養上清、あるいは該培養上清から調製された粗酵素若しくは精製酵素を加えて酵素反応を行う。 These compounds are dissolved in, for example, a buffer solution such as acetic acid, phosphoric acid, and phthalic acid buffer, and the culture supernatant of Amycolatopsis orientalis or a crude enzyme prepared from the culture supernatant or Enzymatic reaction is performed by adding purified enzyme.
酵素の添加量は、反応基質の量によって適宜設定すればよいが、反応基質の質量（受容体基質と供与体基質の合計）１ｇに対して、０．０２〜１０Ｕ添加することが好ましい。なお、本発明でいう「１Ｕ」とは、パラニトロフェニル−Ｎ−アセチルグルコサミンを基質として１分間に１μｍｏｌのパラニトロフェノールを遊離する酵素量を意味する。 The addition amount of the enzyme may be appropriately set depending on the amount of the reaction substrate, but it is preferable to add 0.02 to 10 U with respect to 1 g of the reaction substrate mass (the total of the acceptor substrate and the donor substrate). In the present invention, “1U” means the amount of enzyme that liberates 1 μmol of paranitrophenol per minute using paranitrophenyl-N-acetylglucosamine as a substrate.
酵素反応の条件は、ｐＨ３〜７、好ましくはｐＨ４〜６で、２０〜７０℃、好ましくは３０〜５０℃である。なお、反応の状態はＨＰＬＣ等で経時的に分析して、反応が平衡状態に達したことを確認してから、反応液を加熱するなどして反応を停止させればよい。 The conditions for the enzyme reaction are pH 3-7, preferably pH 4-6, 20-70 ° C, preferably 30-50 ° C. The reaction state may be analyzed over time by HPLC or the like, and after confirming that the reaction has reached an equilibrium state, the reaction may be stopped by heating the reaction solution.
反応液は、そのまま、あるいは濃縮して、活性炭カラムクロマトグラフィー、シリカゲルカラムクロマトグラフィーの順に二段階のカラム操作を行うことにより、生成した双頭型配糖体を分離精製することができる。 The resulting double-headed glycoside can be separated and purified by performing the two-stage column operation in the order of activated carbon column chromatography and silica gel column chromatography as it is or after concentrating the reaction solution.
例えば、活性炭クロマトグラフィーは、移動相として０〜６０％のエタノール又はイソプロピルアルコールを用い、グラジエント溶出することが好ましい。また、シリカゲルカラムクロマトグラフィーは、移動相としてクロロホルム／メタノール／水（容量）＝６／４／１や、７／３／０．５の混合溶媒を用いることができる。 For example, in activated carbon chromatography, it is preferable to use 0 to 60% ethanol or isopropyl alcohol as a mobile phase and perform gradient elution. Silica gel column chromatography can use a mixed solvent of chloroform / methanol / water (volume) = 6/4/1 or 7/3 / 0.5 as a mobile phase.
上記クロマトグラフィーにおいて、生成した双頭型配糖体の検出は、ＧｌｃＮＡｃのＮ−アセチル基に由来する２１０ｎｍでの吸収を観察したり、オルシノール硫酸法によって行うことができる。また、分離精製した双頭型配糖体は、１Ｈ−ＮＭＲ、１３Ｃ−ＮＭＲ、ＦＡＢ−ＭＳ等により構造解析することができる。 In the above chromatography, the produced double-headed glycoside can be detected by observing absorption at 210 nm derived from the N-acetyl group of GlcNAc or by the orcinol sulfate method. Further, the separated and purified double-headed glycoside can be structurally analyzed by 1 H-NMR, 13 C-NMR, FAB-MS and the like.
上記製造方法においては、アルカンジオール（炭素数２〜９）と、キチンオリゴ糖（重合度２〜６）又はキチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）に、キチナーゼ又はＮ−アセチルヘキソサミニダーゼを作用させた場合には、下記式（５）及び下記式（９）で示される化合物が生成されるので、この生成物から下記式（５）で示される化合物を分離生成することができる。
ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）ｎ−Ｏ−ＧｌｃＮＡｃ …（５）
ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）ｎ−ＯＨ …（９）
（上記式（５）、（９）中、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。）
In the above production method, alkanediol (2 to 9 carbon atoms) and chitin oligosaccharide (degree of polymerization 2 to 6) or paranitrophenyl derivative of chitin oligosaccharide (degree of polymerization 1 to 6) are mixed with chitinase or N-acetyl. When hexosaminidase is allowed to act, a compound represented by the following formula (5) and the following formula (9) is produced. Therefore, a compound represented by the following formula (5) is separated and produced from this product. be able to.
GlcNAc-O- (CH 2) n -O-GlcNAc ... (5)
GlcNAc-O- (CH 2) n -OH ... (9)
(In the above formulas (5) and (9), GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
また、アルカンジオールの片側に１〜８糖の糖鎖が結合したアルカノール誘導体と、キチンオリゴ糖（重合度２〜６）又はキチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）に、キチナーゼ又はＮ−アセチルヘキソサミニダーゼを作用させることにより、下記一般式（６）で示される化合物が生成されるので、この生成物から下記式（６）で示される化合物を分離精製することができる。
Ｒ−Ｏ−（ＣＨ２）ｎ−Ｏ−ＧｌｃＮＡｃ …（６）
（上記式中、Ｒは還元末端１位水酸基を有さない単糖又は２〜８糖からなる糖鎖を表し、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。）
In addition, an alkanol derivative in which a sugar chain of 1 to 8 sugars is bonded to one side of alkanediol and chitin oligosaccharide (polymerization degree 2 to 6) or a paranitrophenyl derivative of chitin oligosaccharide (polymerization degree 1 to 6) Alternatively, the compound represented by the following general formula (6) is produced by the action of N-acetylhexosaminidase, so that the compound represented by the following formula (6) can be separated and purified from this product. .
R—O— (CH 2 ) n —O—GlcNAc (6)
(In the above formula, R represents a sugar chain composed of a monosaccharide or 2 to 8 sugars having no hydroxyl group at the reducing end, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9).
この場合、上記一般式（６）におけるＲは、Ｎ−アセチルグルコサミンを還元末端とする糖鎖からなり、該還元末端において前記アルカンジオールの片側に結合していることが好ましく、それによって下記一般式（１０）で示される化合物を生成することができる。
Ｒ２−Ｏ−ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）ｎ−Ｏ−ＧｌｃＮＡｃ …（１０）
（上記式中、Ｒ２は還元末端１位水酸基を有さない単糖又は２〜７糖からなる糖鎖を表し、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表す。ｎは２〜９の整数を表す。
In this case, R in the general formula (6) is composed of a sugar chain having N-acetylglucosamine as a reducing end, and is preferably bonded to one side of the alkanediol at the reducing end. The compound represented by (10) can be produced.
R 2 -O-GlcNAc-O- ( CH 2) n -O-GlcNAc ... (10)
(In the above formula, R 2 represents a sugar chain composed of a monosaccharide having no hydroxyl group at the 1-position of the reducing end or 2 to 7 sugars, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.
更に、オリゴエチレングリコール（エチレン数２〜９）と、キチンオリゴ糖（重合度２〜６）又はキチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）に、キチナーゼ及び／又はＮ−アセチルヘキソサミニダーゼを作用させることにより、下記式（７）、（１２）で示される化合物が生成されるので、これらの生成物から下記式（７）で示される化合物を分離精製することができる。
ＧｌｃＮＡｃ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−ＧｌｃＮＡｃ …（７）
ＧｌｃＮＡｃ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−ＯＨ …（１２）
（上記式中、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。）
Further, oligoethylene glycol (ethylene number 2 to 9), chitin oligosaccharide (polymerization degree 2 to 6) or paranitrophenyl derivative of chitin oligosaccharide (polymerization degree 1 to 6), chitinase and / or N-acetylhexyl. By allowing sosaminidase to act, the compounds represented by the following formulas (7) and (12) are produced, and therefore the compound represented by the following formula (7) can be separated and purified from these products.
GlcNAc-O- (CH 2 -CH 2 -O) n -GlcNAc ... (7)
GlcNAc-O- (CH 2 -CH 2 -O) n -OH ... (12)
(In the above formula, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
更にまた、オリゴエチレングリコールの片側に１〜８糖の糖鎖が結合したオリゴエチレングルコール誘導体と、キチンオリゴ糖（重合度２〜６）又はキチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）に、キチナーゼ及び／又はＮ−アセチルヘキソサミニダーゼを作用させことにより、下記式（８）で示される化合物が生成されるので、この生成物から下記式（８）で示される化合物を分離生成することができる。
Ｒ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−ＧｌｃＮＡｃ…（８）
（上記式中、Ｒは還元末端１位水酸基を有さない単糖又は２〜８糖からなる糖鎖を表し、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。）
Furthermore, oligoethylene glycol derivatives in which sugar chains of 1 to 8 sugars are bonded to one side of oligoethylene glycol, chitin oligosaccharides (polymerization degree 2 to 6), or paranitrophenyl derivatives of chitin oligosaccharides (polymerization degree 1 to Since a compound represented by the following formula (8) is produced by allowing chitinase and / or N-acetylhexosaminidase to act on 6), a compound represented by the following formula (8) is produced from this product. Can be produced separately.
R—O— (CH 2 —CH 2 —O) n —GlcNAc (8)
(In the above formula, R represents a sugar chain composed of a monosaccharide or 2 to 8 sugars having no hydroxyl group at the reducing end, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9).
この場合、上記一般式（８）におけるＲは、Ｎ−アセチルグルコサミンを還元末端とする糖鎖からなり、該還元末端において前記オリゴエチレングリコールの片側に結合していることが好ましく、それによって下記一般式（１１）で示される化合物を生成することができる。
Ｒ２−Ｏ−ＧｌｃＮＡｃ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−Ｏ−ＧｌｃＮＡｃ …（１１）
（上記式中、Ｒ２は還元末端１位水酸基を有さない単糖又は２〜７糖からなる糖鎖を表し、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表す。ｎは２〜９の整数を表す。）
In this case, R in the general formula (8) is composed of a sugar chain having N-acetylglucosamine as a reducing end, and is preferably bonded to one side of the oligoethylene glycol at the reducing end. A compound represented by formula (11) can be produced.
R 2 -O-GlcNAc-O- ( CH 2 -CH 2 -O) n -O-GlcNAc ... (11)
(In the above formula, R 2 represents a sugar chain composed of a monosaccharide having no hydroxyl group at the 1-position of the reducing terminal or 2 to 7 sugars, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9. )
また、アルカンジオール（炭素数２〜９）と、キチンオリゴ糖（重合度２〜６）又はキチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）に、キチナーゼ又はＮ−アセチルヘキソサミニダーゼを作用させて得られる前記式（９）で示される化合物を採取し、この化合物に糖転移によって他の糖又は糖鎖を結合させることにより、前記一般式（６）で示される化合物を得ることもできる。 Moreover, chitinase or N-acetylhexosaminidase is added to alkanediol (2-9 carbon atoms) and chitin oligosaccharide (degree of polymerization 2-6) or paranitrophenyl derivative of chitin oligosaccharide (degree of polymerization 1-6). The compound represented by the formula (9) obtained by acting is collected, and the compound represented by the general formula (6) is obtained by binding another sugar or sugar chain to this compound by sugar transfer. You can also.
更に、オリゴエチレングリコール（エチレン数２〜９）と、キチンオリゴ糖（重合度２〜６）又はキチンオリゴ糖のパラニトロフェニル誘導体（重合度１〜６）に、キチナーゼ及び／又はＮ−アセチルヘキソサミニダーゼを作用させて得られる前記式（１２）で示される化合物を採取し、この化合物に糖転移によって他の糖又は糖鎖を結合させることにより、前記一般式（８）で示される化合物を得ることもできる。 Further, oligoethylene glycol (ethylene number 2 to 9), chitin oligosaccharide (polymerization degree 2 to 6) or paranitrophenyl derivative of chitin oligosaccharide (polymerization degree 1 to 6), chitinase and / or N-acetylhexyl. The compound represented by the general formula (8) is obtained by collecting the compound represented by the formula (12) obtained by the action of sosaminidase and linking another saccharide or sugar chain to the compound by sugar transfer. You can also get
上記糖転移反応は、α−ガラクトシダーゼ、β−ガラクトシダーゼ、Ｎ−アセチルヘキソサミニダーゼ、マンノシダーゼ、フコシダーゼ、ガラクトシルトランスフェラーゼ、Ｎ−アセチルグルコサミニルトランスフェラーゼ、フコシルトランスフェラーゼ等の加水分解酵素や糖転移酵素を用いることにより行うことができる。 The glycosyltransferase uses a hydrolase or glycosyltransferase such as α-galactosidase, β-galactosidase, N-acetylhexosaminidase, mannosidase, fucosidase, galactosyltransferase, N-acetylglucosaminyltransferase, fucosyltransferase, etc. Can be done.
また、上記で得られた式（５）〜（８）及び（１０）、（１１）で示される双頭型配糖体の片側あるいは両末端に、上記と同様な加水分解酵素や糖転移酵素を用いて、糖、オリゴ糖、糖鎖等を転移させることにより、前記一般式（１）〜（４）で示される化合物であって、前記式（５）〜（８）及び（１０）、（１１）で示される双頭型配糖体に該当しないものも、製造することができる。 In addition, a hydrolase or glycosyltransferase similar to the above is applied to one side or both ends of the double-headed glycoside represented by the formulas (5) to (8) and (10) and (11) obtained above. The compounds represented by the general formulas (1) to (4) by transferring sugars, oligosaccharides, sugar chains and the like, wherein the formulas (5) to (8) and (10), ( Those not corresponding to the double-headed glycoside shown in 11) can also be produced.
上記各反応において、糖転移させる糖、オリゴ糖、糖鎖としては、グルコース、ガラクトース、マンノース、フコース、キシロース、シアル酸、グルクロン酸、イズロン酸、ガラクツロン酸、Ｎ−アセチルグルコサミン、Ｎ−アセチルガラクトサミン、グルコサミン、ガラクトサミン及びこれらの硫酸化物、リン酸化物、硝酸化物等、天然に存在する糖鎖であるアミロース、セルロース、キチン、キトサン等、コンドロイチン硫酸、ケラタン硫酸、ヘパリン、ヘパラン硫酸、ヒアルロン酸等のグリコサミノグリカンやβ−グルカン、ポリラクトサミン、フコイダン、キシログルカンのような糖鎖構造を有するオリゴ糖が例示できる。 In each of the above reactions, the sugar, oligosaccharide, and sugar chain to be transferred are glucose, galactose, mannose, fucose, xylose, sialic acid, glucuronic acid, iduronic acid, galacturonic acid, N-acetylglucosamine, N-acetylgalactosamine, Glucosamine, galactosamine and their sulfated oxides, phosphorous oxides, nitrates, etc., naturally occurring sugar chains such as amylose, cellulose, chitin, chitosan, etc., chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronic acid, etc. Examples include oligosaccharides having a sugar chain structure such as saminoglycan, β-glucan, polylactosamine, fucoidan, and xyloglucan.
（１）アミコラトプシス・オリエンタリス（Amycolatopsis orientalis）培養液の調製
アミコラトプシス・オリエンタリス（Amycolatopsis orientalis）（ＩＦＯ１２８０６）株を、３０℃で３０時間培養した前培養液を用い、下記組成の培地により、３０℃で４８時間培養した。
(1) Preparation of Amycolatopsis orientalis ( Amycolatopsis orientalis ) Culture Solution Using a preculture solution obtained by culturing Amycolatopsis orientalis (IFO12806) strain at 30 ° C. for 30 hours, The cells were cultured at 30 ° C. for 48 hours.
培地 （ｇ／Ｌ）
コロイダルキチン ５．０
ＧｌｃＮＡｃ ５．０
ペプトン ２．０
酵母エキス ０．１
Ｋ２ＨＰＯ４ ０．７
ＫＨ２ＰＯ４ ０．３
Ｍｇ２ＳＯ４・７Ｈ２Ｏ ０．５
Medium (g / L)
Colloidal chitin 5.0
GlcNAc 5.0
Peptone 2.0
Yeast extract 0.1
K 2 HPO 4 0.7
KH 2 PO 4 0.3
Mg 2 SO 4 · 7H 2 O 0.5
培養終了後、培養液を遠心分離して菌体を除去し、固形硫安８０％飽和となるように加えて４℃で一夜放置してタンパク質を塩析させた。これを遠心分離により上清を回収し、純水中で一昼夜透析した後、凍結乾燥して粗酵素乾燥粉末１６０ｍｇを得た。以下の製造にはこの粗酵素を用いた。 After completion of the culture, the culture broth was centrifuged to remove the cells, added to a solid ammonium sulfate 80% saturation, and left overnight at 4 ° C. to salt out the protein. The supernatant was collected by centrifugation, dialyzed overnight in pure water, and then freeze-dried to obtain 160 mg of crude enzyme dry powder. This crude enzyme was used for the following production.
（２）ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）６−ＯＨ及びＧｌｃＮＡｃ−Ｏ−（ＣＨ２）６−Ｏ−ＧｌｃＮＡｃの製造例
供与体基質として（ＧｌｃＮＡｃ）４（１．７ｇ）、受容体基質として１,６−へキサンジオール（４．７ｇ）をモル比０．１：２となるように１０ｍＭ酢酸ナトリウム緩衝液（ｐＨ５．５）２５ｍｌに溶解した後、上記（１）で得られた粗酵素（２５６ｍＵ）を添加し、４０℃で４０時間振とう反応を行った。反応の経時変化はＨＰＬＣにより分析し、反応が平衡状態に達したことを確認後、反応液を１０分間煮沸して反応を停止した。
(2) GlcNAc-O- (CH 2) a 6 -OH and GlcNAc-O-Preparation donor substrate (CH 2) 6 -O-GlcNAc (GlcNAc) 4 (1.7g), 1 as a receptor substrate , 6-hexanediol (4.7 g) was dissolved in 25 ml of 10 mM sodium acetate buffer (pH 5.5) in a molar ratio of 0.1: 2, and then the crude enzyme (1) obtained above (1) 256 mU) was added and the reaction was shaken at 40 ° C. for 40 hours. The time course of the reaction was analyzed by HPLC, and after confirming that the reaction reached an equilibrium state, the reaction solution was boiled for 10 minutes to stop the reaction.
この反応液を、予め水で平衡化した活性炭セライトカラム（φ４．５×８５ｃｍ）に供し、エタノール濃度０−５０％直線濃度勾配法により溶出させた。転移生成物の検出は、ＧｌｃＮＡｃのＮ−アセチル基に由来する２１０ｎｍでの吸収を観察することにより行った。 This reaction solution was applied to an activated carbon celite column (φ4.5 × 85 cm) previously equilibrated with water, and eluted by an ethanol concentration 0-50% linear concentration gradient method. The detection of the transfer product was performed by observing the absorption at 210 nm derived from the N-acetyl group of GlcNAc.
転移生成物のフラクション（４８ｍｌ／ｔｕｂｅ）を回収して濃縮乾固し、これをさらにシリカゲル６０Ｎカラム（φ１．５×２５ｃｍ）に供した。移動相としてクロロホルム：メタノール：水（容量）＝７：３：０．５を用いてフラクションを分取し、各フラクション（１０ｍｌ／ｔｕｂｅ）についてオルシノール硫酸法によって生成物を検出後、各目的物のフラクションをそれぞれ回収、濃縮し、凍結乾燥することにより、ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）６−ＯＨ（６−ヒドロキシヘキシル−２−アセトアミド−２−デオキシ−Ｏ−β−Ｄ−グルコピラノシド）、及びＧｌｃＮＡｃ−Ｏ−（ＣＨ２）６−Ｏ−ＧｌｃＮＡｃ（β−Ｄ−２−アセトアミド−２−デオキシ−Ｏ−β−Ｄ−グルコピラノシル−１,６−ジオキシヘキシル−２−アセトアミド−２−デオキシ−Ｏ−β−Ｄ−グルコピラノシド）の乾燥粉末をそれぞれ３１４ｍｇ（収率４９％）、２３ｍｇ（収率２．２％）を得た。 The transfer product fraction (48 ml / tube) was collected and concentrated to dryness, and this was further applied to a silica gel 60N column (φ1.5 × 25 cm). Fraction was fractionated using chloroform: methanol: water (volume) = 7: 3: 0.5 as a mobile phase, and the product was detected for each fraction (10 ml / tube) by the orcinol sulfuric acid method. Each fraction was collected, concentrated, and lyophilized to give GlcNAc-O— (CH 2 ) 6 —OH (6-hydroxyhexyl-2-acetamido-2-deoxy-O-β-D-glucopyranoside), and GlcNAc -O- (CH 2) 6 -O- GlcNAc (β-D-2- acetamido-2-deoxy -O-beta-D-glucopyranosyl-1,6-dioxy-hexyl-2-acetamido-2-deoxy -O -Β-D-glucopyranoside) was obtained as 314 mg (yield 49%) and 23 mg (yield 2.2%), respectively.
そして、ＦＡＢ−ＭＳ、１Ｈ及び１３Ｃ−ＮＭＲスペクトルにより、得られた各化合物の構造を確認した。図１に、ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）６−ＯＨの１３Ｃ−ＮＭＲの測定スペクトルデータ、図２にＧｌｃＮＡｃ−Ｏ−（ＣＨ２）６−Ｏ−ＧｌｃＮＡｃの１３Ｃ−ＮＭＲの測定スペクトルデータを示す。 And the structure of each obtained compound was confirmed by FAB-MS, < 1 > H, and < 13 > C-NMR spectrum. Figure 1, GlcNAc-O- (CH 2 ) 6 -OH in 13 C-NMR measured spectrum data, the measured spectrum data of GlcNAc-O- (CH 2) a 6 -O-GlcNAc 13 C-NMR in Fig. 2 Indicates.
（３）ＬａｃＮＡｃ−Ｏ−（ＣＨ２）６−Ｏ−ＧｌｃＮＡｃの製造例
供与体基質としてＬａｃＮＡｃ（１．３ｇ）、受容体基質として１,６−へキサンジオール（３．４ｇ）をモル比０．５：４．０となるように５０ｍＭ酢酸ナトリウム緩衝液（ｐＨ５．０）７．１ｍｌ溶解した後、トリコデルマ・リーサイ（Trichoderma reesei）由来セルラーゼ製剤（商品名「セルラーゼＸＬ−５２２」、ナガセケムテック社製）（２１６Ｕ）を添加し、４０℃で７５時間振とう反応を行った。反応の経時変化はＨＰＬＣにより分析し、反応の転移生成物が飽和に達したことを確認後、反応液を１０分間煮沸して反応を停止した。
(3) Production Example of LacNAc-O— (CH 2 ) 6 —O-GlcNAc A molar ratio of LacNAc (1.3 g) as a donor substrate and 1,6-hexanediol (3.4 g) as an acceptor substrate was 0. .5: After dissolving 7.1 ml of 50 mM sodium acetate buffer (pH 5.0) to 4.0, cellulase preparation derived from Trichoderma reesei (trade name “Cellulase XL-522”, Nagase Chemtech) (216U) was added, and the reaction was shaken at 40 ° C. for 75 hours. The time course of the reaction was analyzed by HPLC, and after confirming that the transfer product of the reaction reached saturation, the reaction solution was boiled for 10 minutes to stop the reaction.
これを、予め純水で平衡化した活性炭セライトカラム（φ４．５×６５ｃｍ）に供し、充分にカラムを洗浄後、吸着物をエタノール濃度０−５０％直線濃度勾配法により溶出させた。転移生成物の検出は、ＧｌｃＮＡｃのＮ−アセチル基に由来する２１０ｎｍの吸収を観察することにより行い、転移生成物のピークを全て回収し濃縮乾固した。 This was applied to an activated carbon celite column (φ4.5 × 65 cm) previously equilibrated with pure water, and the column was sufficiently washed, and then the adsorbate was eluted by the ethanol concentration 0-50% linear concentration gradient method. The detection of the transfer product was performed by observing the absorption at 210 nm derived from the N-acetyl group of GlcNAc, and all the peaks of the transfer product were collected and concentrated to dryness.
次に、得られた転移生成物をシリカゲル６０Ｎカラム（φ１．５×２５ｃｍ）に供し、移動相クロロホルム：メタノール：水（容量）＝７：３：０．５によりフラクション（１０ｍｌ／ｔｕｂｅ）を分取した。各フラクションについてオルシノール硫酸法による生成物の検出を行った後、目的物が含まれる画分を回収、濃縮し、凍結乾燥することにより６−ヒドロキシヘキシル−Ｏ−β−Ｄ−ガラクトピラノシル（１−４）−２−アセトアミド−２−デオキシ−Ｏ−β−Ｄ−グルコピラノシドの乾燥粉末１００ｍｇ（収率６％）を得た。そして、ＦＡＢ−ＭＳ、１Ｈ及び１３Ｃ−ＮＭＲにより、得られた化合物の構造を確認した。 Next, the obtained transfer product was applied to a silica gel 60N column (φ1.5 × 25 cm), and the fraction (10 ml / tube) was separated by mobile phase chloroform: methanol: water (volume) = 7: 3: 0.5. I took it. After detecting the product by the orcinol-sulfuric acid method for each fraction, the fraction containing the target product was collected, concentrated, and lyophilized to give 6-hydroxyhexyl-O-β-D-galactopyranosyl ( 1-4) 100 mg (yield 6%) of dry powder of 2-acetamido-2-deoxy-O-β-D-glucopyranoside was obtained. And the structure of the obtained compound was confirmed by FAB-MS, < 1 > H, and < 13 > C-NMR.
得られた６−ヒドロキシヘキシル−Ｏ−β−Ｄ−ガラクトピラノシル（１−４）−２−アセトアミド−２−デオキシ−Ｏ−β−Ｄ−グルコピラノシドを次の反応に用いた。 The obtained 6-hydroxyhexyl-O-β-D-galactopyranosyl (1-4) -2-acetamido-2-deoxy-O-β-D-glucopyranoside was used for the next reaction.
供与体基質として（ＧｌｃＮＡｃ）４（１４１ｍｇ）、受容体基質として６−ヒドロキシヘキシル−Ｏ−β−Ｄ−ガラクトピラノシル（１−４）−２−アセトアミド−２−デオキシ−Ｏ−β−Ｄ−グルコピラノシド（９０ｍｇ）をモル比１：１となるように１０ｍＭ酢酸ナトリウム緩衝液（ｐＨ５．５）２．１ｍｌに溶解した後、上記（１）で得られた粗酵素（３５ｍＵ）を添加し、４０℃で２４時間振とう反応を行った。反応の経時変化はＨＰＬＣにより分析し、反応の転移生成物が平衡に達したことを確認後、反応液を１０分間煮沸して反応を停止した。 (GlcNAc) 4 (141 mg) as the donor substrate and 6-hydroxyhexyl-O-β-D-galactopyranosyl (1-4) -2-acetamido-2-deoxy-O-β-D as the acceptor substrate -Glucopyranoside (90 mg) was dissolved in 2.1 ml of 10 mM sodium acetate buffer (pH 5.5) so that the molar ratio was 1: 1, and then the crude enzyme (35 mU) obtained in (1) above was added, The reaction was shaken at 40 ° C. for 24 hours. The time course of the reaction was analyzed by HPLC, and after confirming that the transfer product of the reaction reached equilibrium, the reaction was boiled for 10 minutes to stop the reaction.
これを、予め水で平衡化した活性炭セライトカラム（φ２．５×６５ｃｍ）に供し、充分にカラムを水で洗浄後、吸着物をエタノール濃度０−５０％直線濃度勾配法により溶出させた。転移生成物の検出は、ＧｌｃＮＡｃのＮ−アセチル基に由来する２１０ｎｍの吸収と、オルシノール硫酸法により行った。転移生成物のピークを全て回収し濃縮乾固した。 This was applied to an activated carbon celite column (φ2.5 × 65 cm) previously equilibrated with water, and the column was sufficiently washed with water, and then the adsorbate was eluted by a linear concentration gradient method with an ethanol concentration of 0-50%. The transfer product was detected by absorption at 210 nm derived from the N-acetyl group of GlcNAc and the orcinol-sulfuric acid method. All peaks of the transfer product were collected and concentrated to dryness.
次に、これをシリカゲル６０Ｎカラム（φ１．５×２５ｃｍ）に供し、移動相クロロホルム：メタノール：水（容量）＝６：４：１により各フラクション（１０ｍｌ／ｔｕｂｅ）を分取した。各フラクションについてオルシノール硫酸法による生成物の検出を行った後、目的物が含まれる画分を回収し濃縮し、凍結乾燥することにより、ＬａｃＮＡｃ−Ｏ−（ＣＨ２）６−Ｏ−ＧｌｃＮＡｃ（β−Ｄ−２−アセトアミド−２−デオキシ−Ｏ−β−Ｄ−グルコピラノシル−１,６−ジオキシヘキシル−Ｏ−β−Ｄ−ガラクトピラノシル（１−４）−２−アセトアミド−２−デオキシ−Ｏ−β−Ｄ−グルコピラノシド）の乾燥粉末３４ｍｇ（収率２７％）を得た。 Next, this was applied to a silica gel 60N column (φ1.5 × 25 cm), and each fraction (10 ml / tube) was fractionated by mobile phase chloroform: methanol: water (volume) = 6: 4: 1. After detecting the product by the orcinol-sulfuric acid method for each fraction, the fraction containing the target product was collected, concentrated, and lyophilized to obtain LacNAc-O— (CH 2 ) 6 —O-GlcNAc (β -D-2-acetamido-2-deoxy-O-β-D-glucopyranosyl-1,6-dioxyhexyl-O-β-D-galactopyranosyl (1-4) -2-acetamido-2-deoxy 34 mg (yield 27%) of a dry powder of -O-β-D-glucopyranoside) was obtained.
そして、ＦＡＢ−ＭＳ、１Ｈ及び１３Ｃ−ＮＭＲにより、得られた化合物の構造を確認した。図３に、ＬａｃＮＡｃ−Ｏ−（ＣＨ２）６−Ｏ−ＧｌｃＮＡｃの１３Ｃ−ＮＭＲの測定スペクトルデータを示す。 And the structure of the obtained compound was confirmed by FAB-MS, < 1 > H, and < 13 > C-NMR. FIG. 3 shows 13 C-NMR measurement spectrum data of LacNAc—O— (CH 2 ) 6 —O—GlcNAc.
（４）ＧｌｃＮＡｃ−Ｏ−（ＣＨ２ＣＨ２Ｏ）４−ＧｌｃＮＡｃの製造例
供与体基質として（ＧｌｃＮＡｃ）４（８７９ｍｇ）、受容体基質としてテトラエチレングリコール（１９４μｌ）をモル比１：１となるように１０ｍＭ酢酸ナトリウム緩衝液（ｐＨ５．５）８．８ｍｌに溶解した後、上記（１）で得られた粗酵素（２３９ｍＵ）を添加し、４０℃で４８時間振とう反応を行った。反応の経時変化はＨＰＬＣにより分析し、反応が平衡状態に達したことを確認後、反応液を１０分間煮沸して反応を停止した。
(4) Production Example of GlcNAc-O— (CH 2 CH 2 O) 4 -GlcNAc (GlcNAc) 4 (879 mg) as a donor substrate and tetraethylene glycol (194 μl) as an acceptor substrate in a molar ratio of 1: 1. Thus, after dissolving in 8.8 ml of 10 mM sodium acetate buffer (pH 5.5), the crude enzyme (239 mU) obtained in (1) above was added, and the reaction was shaken at 40 ° C. for 48 hours. The time course of the reaction was analyzed by HPLC, and after confirming that the reaction reached an equilibrium state, the reaction solution was boiled for 10 minutes to stop the reaction.
この反応液を予め水で平衡化した活性炭セライトカラム（φ５．０×４５ｃｍ）に供し、エタノール濃度１０−５０％直線濃度勾配法により溶出させた。転移生成物の検出は、ＧｌｃＮＡｃのＮ−アセチル基に由来する２１０ｎｍでの吸収を観察することにより行った。 This reaction solution was applied to an activated carbon celite column (φ5.0 × 45 cm) previously equilibrated with water, and eluted by an ethanol concentration 10-50% linear concentration gradient method. The detection of the transfer product was performed by observing the absorption at 210 nm derived from the N-acetyl group of GlcNAc.
転移生成物のフラクション（５０ｍｌ／ｔｕｂｅ）を回収、濃縮乾固し、これをさらにシリカゲル６０Ｎカラム（φ１．５×２７ｃｍ）に供した。移動相としてクロロホルム：メタノール：水（容量）＝６：４：１を用いてフラクションを分取し、各フラクション（１０ｍｌ／ｔｕｂｅ）についてオルシノール硫酸法による生成物を検出後、目的物のフラクションを回収、濃縮し、凍結乾燥することによりＧｌｃＮＡｃ−Ｏ−（ＣＨ２ＣＨ２Ｏ）４−ＧｌｃＮＡｃ（ビス−Ｏ−１，８−テトラエチレングリコール−２−アセトアミド−２−デオキシ−Ｏ−β−Ｄ−グルコピラノシド）の乾燥粉末を１２．５ｍｇ（収率２．１％)を得た。 A fraction (50 ml / tube) of the transfer product was collected and concentrated to dryness, and this was further applied to a silica gel 60N column (φ1.5 × 27 cm). Fraction was fractionated using chloroform: methanol: water (volume) = 6: 4: 1 as the mobile phase, and the fraction of the desired product was recovered after detecting the product by the orcinol sulfuric acid method for each fraction (10 ml / tube). , concentrated, GlcNAc-O- (CH 2 CH 2 O) by lyophilization 4 -GlcNAc (bis -O-1,8-tetraethylene glycol-2-acetamido-2-deoxy -O-beta-D- 12.5 mg (yield 2.1%) of dry powder of glucopyranoside) was obtained.
そして、ＦＡＢ−ＭＳ、１Ｈ及び１３Ｃ−ＮＭＲにより、得られた化合物の構造を確認した。図４に該化合物の１３Ｃ−ＮＭＲの測定スペクトルデータを示す。 And the structure of the obtained compound was confirmed by FAB-MS, < 1 > H, and < 13 > C-NMR. FIG. 4 shows 13 C-NMR measurement spectrum data of the compound.
本発明の双頭型配糖体は、凝集剤や阻害剤、接着剤、ＤＤＳの輸送体等への利用が期待できるほか、保湿効果、安定化効果、界面活性効果、自己凝集効果等も期待でき、化粧品、医薬品、食品、農薬、電子等の様々な分野で応用できる可能性がある。 The double-headed glycoside of the present invention can be expected to be used as a flocculant, an inhibitor, an adhesive, a DDS transporter, and the like, and can also be expected to have a moisturizing effect, a stabilizing effect, a surface active effect, a self-aggregating effect, and the like. There is a possibility that it can be applied in various fields such as cosmetics, pharmaceuticals, foods, agricultural chemicals, and electronics.
Claims (13)
Ｒ−Ｏ−（ＣＨ２）ｎ−Ｏ−Ｒ１ …（１）
Ｒ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−Ｒ１ …（２）
（上記式中、Ｒ及びＲ１は還元末端１位水酸基を有さない単糖又は２〜８糖からなる糖鎖を表し、Ｒ、Ｒ１は同じでも異なっていてもよい。ｎは２〜９の整数を表す。） A double-headed glycoside, which is a compound having a monosaccharide or a sugar chain at both ends and having a chemical structure represented by the following general formula (1) or (2).
R—O— (CH 2 ) n —O—R 1 (1)
R—O— (CH 2 —CH 2 —O) n —R 1 (2)
(In the above formula, R and R 1 represent a sugar chain composed of a monosaccharide or 2 to 8 sugars having no hydroxyl group at the 1-position of the reducing end, and R and R 1 may be the same or different. Represents an integer of 9.)
Ｒ２−Ｏ−ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）ｎ−Ｏ−Ｒ１ …（３）
Ｒ２−Ｏ−ＧｌｃＮＡｃ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−Ｏ−Ｒ１ …（４）
（上記式中、Ｒ１は還元末端１位水酸基を有さない単糖又は２〜８糖からなる糖鎖を表し、Ｒ２は還元末端１位水酸基を有さない単糖又は２〜７糖からなる糖鎖を表し、Ｒ１、Ｒ２は同じでも異なっていてもよい。ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表す。ｎは２〜９の整数を表す。） The double-headed glycoside according to claim 1, wherein the R is composed of a sugar chain having N-acetylglucosamine as a reducing end and has a chemical structure represented by the following general formula (3) or (4).
R 2 —O—GlcNAc—O— (CH 2 ) n —O—R 1 (3)
R 2 —O—GlcNAc—O— (CH 2 —CH 2 —O) n —O—R 1 (4)
(In the above formula, R 1 represents a monosaccharide having no hydroxyl group at the reducing end 1-position or a sugar chain consisting of 2 to 8 sugars, and R 2 is a monosaccharide having no hydroxyl group at the reducing end 1-position or 2 to 7 sugars. And R 1 and R 2 may be the same or different, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）ｎ−Ｏ−ＧｌｃＮＡｃ …（５）
（上記式中、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。） Chitinase or N-acetylhexosaminidase acts on alkanediol (2-9 carbon atoms) and chitin oligosaccharides (degree of polymerization 2-6) or paranitrophenyl derivatives of chitin oligosaccharides (degree of polymerization 1-6) And a compound represented by the following formula (5) is collected from the obtained product: A method for producing a double-headed glycoside,
GlcNAc-O- (CH 2) n -O-GlcNAc ... (5)
(In the above formula, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
Ｒ−Ｏ−（ＣＨ２）ｎ−Ｏ−ＧｌｃＮＡｃ …（６）
（上記式中、Ｒは還元末端１位水酸基を有さない単糖又は２〜８糖からなる糖鎖を表し、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。） An alkanol derivative in which a sugar chain of 1 to 8 sugars is bonded to one side of alkanediol, chitin oligosaccharide (polymerization degree 2 to 6) or paranitrophenyl derivative of chitin oligosaccharide (polymerization degree 1 to 6), chitinase or N -A method for producing a double-headed glycoside, which comprises reacting acetylhexosaminidase and collecting a compound represented by the following general formula (6) from the obtained product.
R—O— (CH 2 ) n —O—GlcNAc (6)
(In the above formula, R represents a sugar chain composed of a monosaccharide or 2 to 8 sugars having no hydroxyl group at the reducing end, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9).
ＧｌｃＮＡｃ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−ＧｌｃＮＡｃ …（７）
（上記式中、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。） Oligoethylene glycol (ethylene number 2-9), chitin oligosaccharide (polymerization degree 2-6) or paranitrophenyl derivative of chitin oligosaccharide (polymerization degree 1-6), chitinase and / or N-acetylhexosamini A method for producing a double-headed glycoside, characterized in that a compound represented by the following formula (7) is collected from a product obtained by acting a dase.
GlcNAc-O- (CH 2 -CH 2 -O) n -GlcNAc ... (7)
(In the above formula, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
Ｒ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−ＧｌｃＮＡｃ …（８）
（上記式中、Ｒは還元末端１位水酸基を有さない単糖又は２〜８糖からなる糖鎖を表し、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。） Oligoethylene glycol derivative with sugar chain of 1-8 sugar bonded to one side of oligoethylene glycol and chitin oligosaccharide (polymerization degree 2-6) or paranitrophenyl derivative of chitin oligosaccharide (polymerization degree 1-6) A method for producing a double-headed glycoside, which comprises reacting chitinase and / or N-acetylhexosaminidase and collecting a compound represented by the following general formula (8) from the obtained product.
R—O— (CH 2 —CH 2 —O) n —GlcNAc (8)
(In the above formula, R represents a sugar chain composed of a monosaccharide or 2 to 8 sugars having no hydroxyl group at the reducing end, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9).
ＧｌｃＮＡｃ−Ｏ−（ＣＨ２）ｎ−ＯＨ …（９）
（上記式中、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。） Chitinase or N-acetylhexosaminidase acts on alkanediol (2-9 carbon atoms) and chitin oligosaccharides (degree of polymerization 2-6) or paranitrophenyl derivatives of chitin oligosaccharides (degree of polymerization 1-6) Then, a compound represented by the following formula (9) is collected from the obtained product, and a compound represented by the general formula (6) is obtained by binding another sugar or sugar chain to this compound by sugar transfer. A process for producing a double-headed glycoside characterized in that it is obtained.
GlcNAc-O- (CH 2) n -OH ... (9)
(In the above formula, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
ＧｌｃＮＡｃ−Ｏ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−ＯＨ …（１２）
（上記式中、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを表し、ｎは２〜９の整数を表す。） Oligoethylene glycol (ethylene number 2-9), chitin oligosaccharide (polymerization degree 2-6) or paranitrophenyl derivative of chitin oligosaccharide (polymerization degree 1-6), chitinase and / or N-acetylhexosamini A compound represented by the following formula (12) is collected from the resulting product by reacting with a oxidase, and another saccharide or sugar chain is bound to this compound by glycosyl transfer, whereby the compound represented by the above general formula (8) is obtained. A method for producing a double-headed glycoside, characterized in that the compound is obtained.
GlcNAc-O- (CH 2 -CH 2 -O) n -OH ... (12)
(In the above formula, GlcNAc represents N-acetylglucosamine, and n represents an integer of 2 to 9.)
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| JP2008214256A (en) * | 2007-03-02 | 2008-09-18 | Yaizu Suisankagaku Industry Co Ltd | Method for producing double-headed type glycoside compound, double-headed type glycoside compound and sugar chain-recognition protein binder containing the double-headed type glycoside compound |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02242692A (en) * | 1989-03-16 | 1990-09-27 | Kao Corp | Production of glycoside |
| JPH05244975A (en) * | 1992-03-04 | 1993-09-24 | Kuraray Co Ltd | Production of alkylglycoside |
| JP2003212890A (en) * | 2002-01-21 | 2003-07-30 | Yaizu Suisankagaku Industry Co Ltd | N-acetyllactosaminyl-cellooligosaccharide derivative and method for producing the same |
| JP2003212891A (en) * | 2002-01-21 | 2003-07-30 | Yaizu Suisankagaku Industry Co Ltd | N-acetylglucosaminyl-cellooligosaccharide derivative and method for producing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02242692A (en) * | 1989-03-16 | 1990-09-27 | Kao Corp | Production of glycoside |
| JPH05244975A (en) * | 1992-03-04 | 1993-09-24 | Kuraray Co Ltd | Production of alkylglycoside |
| JP2003212890A (en) * | 2002-01-21 | 2003-07-30 | Yaizu Suisankagaku Industry Co Ltd | N-acetyllactosaminyl-cellooligosaccharide derivative and method for producing the same |
| JP2003212891A (en) * | 2002-01-21 | 2003-07-30 | Yaizu Suisankagaku Industry Co Ltd | N-acetylglucosaminyl-cellooligosaccharide derivative and method for producing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008214256A (en) * | 2007-03-02 | 2008-09-18 | Yaizu Suisankagaku Industry Co Ltd | Method for producing double-headed type glycoside compound, double-headed type glycoside compound and sugar chain-recognition protein binder containing the double-headed type glycoside compound |
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