JP2004043782A - Water soluble polymer primer for synthesizing oligosaccharide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a water soluble polymer primer for efficiently synthesizing various oligosaccharides, applicable to an automatic synthesis and provide a method for producing the oligosaccharides by using the water soluble polymer primer. <P>SOLUTION: The water soluble polymer primer for synthesizing the oligosaccharides is a copolymer comprising an acrylamide derivative expressed by general formula (I) (wherein R<SP>1</SP>expresses a monosaccharide residue or an oligosaccharide residue; R<SP>2</SP>expresses a linker having length of 4-20 methylene groups) with at least one species of vinyl monomer. The acrylamide derivative expressed by general formula (I) is included in the whole vinyl copolymer in ≤10 mole%. <P>COPYRIGHT: (C)2004,JPO

Description

【００１０】
また、Ｃ．−Ｈ．　Ｗｏｎｇらは、固相担体であるアミノ化シリカに結合させる基を下記化３（式中、Ａｃはアセチル基を示す）に改良し、糖転移酵素により糖鎖を伸長させた後、ヒドラジン分解により糖鎖を遊離させる方法を報告しており、酵素による糖転移反応をほぼ定量的に行うことができたとも報告している（例えば、非特許文献４参照）。この方法によれば、伸長した糖鎖は６−カルボヒドラジドヘキサノール配糖体として遊離される。
【００１１】
【化３】

【００１２】
さらに、Ｃ．−Ｈ．　Ｗｏｎｇらは、アクリルアミド−アクリル酸−Ｎ−イソプロピルアクリルアミド共重合体のアクリルアミド残基に下記化４（式中、Ａｃはアセチル基を示す）の基を結合させた水溶性のポリマーをプライマーとし、糖転移酵素により糖鎖を伸長させた後、硝酸二アンモニウムセリウム（ＩＶ）により遊離させる方法を報告している（例えば、非特許文献５参照）。この方法によれば、酵素による糖転移反応は８０〜９０％の収率で進行し、伸長した糖鎖はｐ−ホルミルフェノール配糖体として遊離される。
【００１３】
【化４】

【００１４】
また、Ｍ．　Ｍｅｌｄａｌ　らは、ジアミノ化ポリエチレングリコールのモノおよびジアクリロイル化体の重合体ゲルに、下記化５（式中、Ａｃはアセチル基を示す）の基を結合させたものをプライマーとし、糖転移酵素を用いて糖鎖を伸長させた後、トリフロロ酢酸により糖鎖を糖ペプチドとして遊離させる方法を報告しており、糖転移反応もほぼ定量的に進行したと報告している（例えば、非特許文献６参照）　。この方法で得られる糖ペプチドのペプチド鎖はＡｓｎ（アスパラギン）−Ｇｌｙ（グリシン）であり、Ｃ末側のグリシン残基はグリシンアミド残基となっており、通常の糖ペプチドとは異なる。
【００１５】
【化５】

【００１６】
さらに、本発明者らがポリアクリルアミドのアミド態窒素原子に、下記化６（式中、Ａｃはアセチル基を示す）に示した基を結合させたものをプライマーとし、糖転移酵素を用いて糖鎖を伸長させた後、α−キモトリプシンの加水分解作用を利用して伸長させた糖鎖を遊離させる方法を報告している（例えば、非特許文献７参照）。本方法によれば、酵素による糖転移反応は定量的に進行し、伸長した糖鎖は６−アミノヘキサノール配糖体として得られる。
【００１７】
【化６】

【００１８】
上述したこれらの方法は、いずれも得られる糖鎖化合物がオリゴ糖ではないこと、必ずしも糖転移反応の収率が十分でないことが欠点として挙げられる。また、遺伝子組換えにより大量生産が可能になってきたとはいえ、まだまだ糖転移酵素は非常に高価であり、繰り返して使用できる固定化糖転移酵素の利用が望まれるが、上記の方法のうちのいくつかは不溶性担体上で糖鎖伸長反応を行うため、固定化糖転移酵素を利用できないという欠点もある。固定化糖転移酵素を利用するためには、不溶性担体ではなく、水溶性担体上で糖鎖伸長反応を行う必要がある。
【００１９】
Ｓ．　Ｒｏｔｈらは、まず、糖転移酵素の糖受容体を固相担体に結合させ、これをアフィニティ吸着体とし、この糖受容体と結合することのできる糖転移酵素を含む組織抽出液を接触させることにより、糖転移酵素をアフィニティ吸着体に結合させる。次いで、この糖転移酵素が結合したアフィニティ吸着体をこの糖転移酵素が糖供与体として利用できる糖ヌクレオチドを含む溶液と接触させることにより、糖転移酵素をアフィニティ吸着体から遊離させるとともに糖受容体に糖残基を一つ伸長させ、さらに、この糖残基が一つ伸長した糖受容体と結合することのできる糖転移酵素を含む組織抽出液を接触させ、同様のことを繰り返し所望の糖鎖を固相担体上に合成する方法を開示している（例えば、特許文献１参照）。しかしながら、この方法の有用性を示す具体的なデータは示されておらず、得られた糖鎖を固相担体から遊離させる方法も開示されていない。
【００２０】
糖転移酵素を用いた糖鎖の自動合成であり、得られる糖鎖化合物がオリゴ糖である例としては、Ｔ．　ＮｏｒｂｅｒｇらがＳｅｐｈａｒｏｓｅ　６Ｂ（アマシャムファルマシア社製）に下記化７に示した基を結合させたものをプライマーとし、糖転移酵素を用いて糖鎖を伸長させた後、臭素あるいはアンモニア／硼酸アンモニウムにより伸長させた糖鎖を遊離させる方法を報告している（例えば、非特許文献８参照）。酵素による糖転移反応は定量的に進行しており、収率的には問題はないが、プライマーを製造に高価な３，４−ジエトキシ−３−シクロブテン−１，２−ジオンを用いており、経済的ではない。また、不溶性担体上での糖鎖伸長反応を行うため、固定化糖転移酵素を利用できないという欠点もある。
【００２１】
【化７】

【００２２】
また、本発明者らはポリアクリルアミド中のアミド態窒素原子の５個に１個の割合で、下記化８（式中、Ａｃはアセチル基を示す）に示した基を結合させたものをプライマーとし、糖転移酵素を用いて糖鎖を伸長させた後、水素化分解により伸長させた糖鎖を遊離させる方法を報告している（例えば、非特許文献９、１０参照）。本方法によれば、酵素による糖転移反応は定量的に進行するが、後述するようにそれは遊離酵素の場合であり、固定化酵素では必ずしもそのようには進行しない。
【００２３】
【化８】

【００２４】
【特許文献１】
特表平５−５００９０５号公報
【００２５】
【非特許文献１】
Ｃａｒｂｏｈｙｄｒ．　Ｒｅｓ．，　１２４，　２３　（１９８３），　Ｃａｒｂｏｈｙｄｒ．　Ｒｅｓ．，　２２８，　２５５　（１９９２）
【００２６】
【非特許文献２】
Ｒｅａｃｔ．　Ｐｏｌｙｍ．，　２２，　１７１　（１９９４），　Ｃａｒｂｏｈｙｄｒ．　Ｒｅｓ．，　２６５，　１６１　（１９９４）
【００２７】
【非特許文献３】
Ｊ．　Ａｍ．　Ｃｈｅｍ．　Ｓｏｃ．，　１１６，　１１３６　（１９９４）
【００２８】
【非特許文献４】
Ｊ．　Ａｍ．　Ｃｈｅｍ．　Ｓｏｃ．，　１１６，　１１３１５　（１９９４）
【００２９】
【非特許文献５】
Ａｄｖ．　Ｓｙｎｔｈ．　Ｃａｔａｌ．，　３４３，　６７５　（２００１）
【００３０】
【非特許文献６】
Ｊ．　Ｃｈｅｍ．　Ｓｏｃ．，　Ｃｈｅｍ．　Ｃｏｍｍｕｎ．，　１８４９　（１９９４）
【００３１】
【非特許文献７】
Ｔｅｔｒａｈｅｄｒｏｎ　Ｌｅｔｔ．，　３６，　９４９３　（１９９５）　、Ｃａｒｂｏｈｙｄｒ．　Ｒｅｓ．，　３０５，　４４３　（１９９８）
【００３２】
【非特許文献８】
Ｃａｒｂｏｈｙｄｒ．　Ｒｅｓ．，　３１９，　８０　（１９９９）
【００３３】
【非特許文献９】
Ｔｅｔｒａｈｅｄｒｏｎ　Ｌｅｔｔ．，　３５，　５６５７　（１９９４）
【００３４】
【非特許文献１０】
Ｃａｒｂｏｈｙｄｒ．　Ｒｅｓ．，　３０５，　４４３　（１９９８
【００３５】
【発明が解決しようとする課題】
本発明の目的は、オリゴ糖の自動合成に適したプライマーを提供することにある。
【００３６】
【課題を解決するための手段】
本発明者らは前記問題点を解決するために鋭意検討した結果、一般式（Ｉ）（式中、Ｒ^１は単糖残基あるいはオリゴ糖残基、Ｒ^２はメチレン基４〜２０個分の長さを有するリンカーを示す）で表されるアクリルアミド誘導体と少なくとも１種類のビニル系単量体とを該アクリルアミド誘導体の含有率が１０％以下になるように共重合することにより、上記問題点を解決できることを見出し、本発明を完成するに至った。
【００３７】
すなわち、本発明は以下の発明を包含する。
【００３８】
（１）一般式（Ｉ）（式中、Ｒ^１は単糖残基あるいはオリゴ糖残基、Ｒ^２はメチレン基４〜２０個分の長さを有するリンカーを示す）で表されるアクリルアミド誘導体と少なくとも１種類のビニル系単量体とからなる共重合体で、一般式（Ｉ）で表されるアクリルアミド誘導体が全ビニル系共重合体中１０モル％以下であるオリゴ糖合成用水溶性高分子プライマー。
【００３９】
【化９】

【００４０】
（２）Ｒ^１がＮ−アセチルグルコサミン残基、グルコース残基、ラクトース残基である（１）のオリゴ糖合成用水溶性高分子プライマー
【００４１】
（３）Ｒ^２がペンチレン基である（１）又は（２）のオリゴ糖合成用水溶性高分子プライマー。
【００４２】
（４）ビニル系単量体がアクリルアミド類、メタクリルアミド類、アクリル酸、メタクリル酸、アクリル酸エステル類、メタクリル酸エステル類、スチレン類、脂肪酸ビニルエステル類からなる群より選ばれる（１）乃至（３）のオリゴ糖合成用水溶性高分子プライマー。
【００４３】
（５）オリゴ糖を製造する方法であって、
（工程１）（１）の水溶性高分子プライマーに糖ヌクレオチドの存在下に糖転移酵素と接触させることにより、糖ヌクレオチドより糖残基を該水溶性高分子プライマーに転移させる工程、
（工程２）工程１を１回または２回以上繰り返して糖鎖を伸長させる工程、
（工程３）必要に応じて、副生したヌクレオチド類や未反応の糖ヌクレオチド類を除去する工程、および、
（工程４）工程１〜工程３を複数回繰り返した後、複数の糖残基が転移して糖鎖が伸長した水溶性高分子プライマーからオリゴ糖を遊離させる工程、を含むことを特徴とするオリゴ糖を製造する方法。
【００４４】
（６）水溶性高分子プライマーより伸長した糖鎖を遊離させる工程が接触水素化分解あるいは２，３−ジクロロ−５，６−ジシアノベンゾキノンによる酸化工程である（５）のオリゴ糖を製造する方法。
【００４５】
【発明の実施の形態】
本発明のオリゴ糖合成用水溶性高分子プライマーは、一般式（Ｉ）で表されるアクリルアミド誘導体と少なくとも１種類のビニル系単量体とからなる共重合体で、一般式（Ｉ）で表されるアクリルアミド誘導体が全ビニル系共重合体中１０モル％以下のものであり、式中Ｒ^１は単糖残基あるいはオリゴ糖残基、Ｒ^２はメチレン基４〜２０個分の長さを有するリンカーを示す。
【００４６】
Ｒ^１の単糖残基あるいはオリゴ糖残基としては、特に制限はなく、グルコース残基、ガラクトース残基、マンノース残基、Ｎ−アセチルグルコサミン残基、Ｎ−アセチルガラクトサミン残基、キシロース残基、ラクトース残基、Ｎ−アセチルラクトサミン残基、キトビオース残基などが挙げられる。
【００４７】
Ｒ^２のリンカーとしては、例えばブチレン基、ペンチレン基、ヘプチレン基、ドデシレン基などが挙げられる。
【００４８】
本発明のオリゴ糖合成用水溶性高分子プライマーは、一般式（Ｉ）で表されるアクリルアミド誘導体少なくとも１種類のビニル系単量体との共重合により得ることができ、そのときの一般式（Ｉ）で表されるアクリルアミド誘導体の全ビニル系共重合体中に占める割合は１０％以下であり、好ましくは１〜１０％であり、さらに好ましくは２〜５％である。
【００４９】
上記の共重合は、特に限定されるものではなく、例えばラジカル重合、カチオン重合、アニオン重合などの手法を用いることにより行うことができ、通常ペルオキソ二硫酸アンモニウムなどを触媒とするラジカル重合を用いることにより好適に行うことができる。
【００５０】
上記のビニル系単量体は、特に限定されるものではなく、例えばアクリルアミド類、メタクリルアミド類、アクリル酸、メタクリル酸、アクリル酸エステル類、メタクリル酸エステル類、スチレン類、脂肪酸ビニルエステル類が挙げられる。アクリルアミド類としては、アクリルアミドやＮ−メチルアクリルアミド、Ｎ−エチルアクリルアミド、Ｎ−イソプロピルアクリルアミドなどのＮ−アルキルアクリルアミド類などが例示される。メタクリルアミド類としてはメタクリルアミド、Ｎ−メチルメタクリルアミドやＮ−イソプロピルメタクリルアミドなどのＮ−アルキルメタクリルアミド類などが例示される。アクリル酸エステル類としてはアクリル酸メチル、アクリル酸エチル、アクリル酸ヒドロキシエチル、アクリル酸ジメチルアミノエチルなどが例示される。メタクリル酸エステル類としてはメタクリル酸メチル、メタクリル酸エチル、メタクリル酸ヒドロキシエチル、メタクリル酸ジメチルアミノエチルなどが例示される。スチレン類としてはスチレン、ｐ−ヒドロキシスチレンなどが例示される。脂肪酸ビニルエステル類としては酢酸ビニル、酪酸ビニルなどが好適に用いられる。
【００５１】
共重合体の一般的な分子量は約１００００〜約５００００００であり、好ましくは２００００〜２００００００、より好ましくは５００００〜１００００００である。
【００５２】
前記一般式（Ｉ）で表されるアクリルアミド誘導体は、通常用いられている各種有機合成化学的な手法により合成することができる。例えば、一般式（ＩＩ）で表される糖オキサゾリン誘導体、一般式（ＩＩＩ）で表されるハロゲン化糖あるいはトリクロロアセトイミデート誘導体とｐ−ニトロベンジルアルコールとを適当な触媒存在下で縮合させた後、ニトロ基を還元してアミノ基へと変換する。その後、一般式（ＩＶ）で表されるω−アミノ脂肪酸と塩化アクリロイルとの縮合により得られる一般式（Ｖ）で表されるアクリルアミド誘導体と上記化合物とを適当な縮合剤存在下、縮合させることにより得られる。
【００５３】
【化１０】

【００５４】
【化１１】

【００５５】
【化１２】

【００５６】
【化１３】

【００５７】
本発明のオリゴ糖製造方法の第１工程は、水溶性高分子プライマーに糖ヌクレオチドの存在下に糖転移酵素と接触させることにより、糖ヌクレオチドより糖残基を該水溶性高分子プライマーに転移させる。
【００５８】
糖ヌクレオチドより水溶性高分子プライマーへの糖の転移は、通常水溶性高分子プライマーと糖ヌクレオチドとを含む中性の緩衝液中で、１０〜６０℃、好ましくは２０〜４０℃で、１〜１２０時間、好ましくは２〜７２時間、糖転移酵素と接触させることにより行われる。
【００５９】
また、反応液中には必要に応じて金属塩を添加してもよい。添加できる金属イオンとしては、例えば、マグネシウム、マンガン、コバルト、ニッケル、銅、亜鉛などがあり、通常塩化物等の形で添加することができる。
【００６０】
本発明で用いる糖転移酵素は、糖ヌクレオチド類を糖供与体として利用できるものであればよく特に限定されない。このような酵素としてＬｅｌｏｉｒ経路の糖転移酵素類を挙げることができる。例えば、ガラクトース転移酵素、Ｎ−アセチルグルコサミン転移酵素、Ｎ−アセチルガラクトサミン転移酵素、フコース転移酵素、シアル酸転移酵素、マンノース転移酵素、キシロース転移酵素、グルクロン酸転移酵素などが挙げられる。なお、これらの酵素は遊離酵素であっても、固定化酵素であっても構わないが、固定化酵素が好ましい。
【００６１】
本発明で用いる糖ヌクレオチド類は、上記酵素が利用できるものであれば特に限定されない。例えば、ウリジン−５’−ジリン酸ガラクトース、ウリジン−５’−ジリン酸−Ｎ−アセチルグルコサミン、ウリジン−５’−ジリン酸−Ｎ−アセチルガラクトサミン、ウリジン−５’−ジリン酸グルクロン酸、ウリジン−５’−ジリン酸キシロース、グアノシン−５’−ジリン酸フコース、グアノシン−５’−ジリン酸マンノース、シチジン−５’−モノリン酸−Ｎ−アセチルノイラミン酸およびこれらのナトリウム塩などが挙げられる。
【００６２】
本発明のオリゴ糖製造方法の第２工程は、工程１を１回または２回以上繰り返して、複数の糖残基を転移させることにより、糖鎖を伸長させる。
【００６３】
本発明のオリゴ糖製造方法の第３工程は、必要に応じて、副生したヌクレオチド類や未反応の糖ヌクレオチド類を除去する。副生したヌクレオチド類や未反応の糖ヌクレオチド類などを除去する方法は、水溶性高分子プライマーとヌクレオチド類および糖ヌクレオチド類などとを分離できる方法であれば特に限定されない。例えば、ゲルろ過クロマトグラフィー、イオン交換クロマトグラフィー、透析、限外ろ過などにより除去することができる。
【００６４】
本発明のオリゴ糖製造方法の第４工程は、工程１〜工程３を複数回繰り返した後、複数の糖残基が転移して糖鎖が伸長した水溶性高分子プライマーからオリゴ糖を遊離させる。本発明の水溶性高分子プライマーより、伸長した糖鎖を遊離させる方法としては、伸長した糖鎖を分解することなく、オリゴ糖して遊離させることのできる方法であれば、特に制限はないが、例えば接触水素化分解や２，３−ジクロロ−５，６−ジシアノベンゾキノンによる酸化反応などが挙げられる。
【００６５】
【実施例】
以下に、実施例を挙げて本発明をさらに詳細に説明するが、これらは単なる例示であって、本発明の範囲はかかる実施例に何ら限定されるものではない。
【００６６】
参考例１　２−メチル−（３，４，６−トリ−Ｏ−アセチル−１，２−ジデオキシ−α−Ｄ−グルコピラノ）−［２，１−ｄ］−２−オキサゾリンの合成
２−アセトアミド−１，３，４，６−テトラ−Ｏ−アセチル−２−デオキシ−Ｄ−グルコピラノシド６．０ｇを１，２−ジクロロエタン４０ｍｌに溶かし、ここにトリメチルシリルトリフロロメタンスルホン酸３．２ｍｌを加え、５０℃で７時間撹拌しながら反応させた。反応後、室温まで冷却した後、トリエチルアミン１０．８ｍｌを加えた。反応液を減圧濃縮した後、シリカゲルカラムクロマトグラフィー（溶出液；トルエン：酢酸エチル：トリエチルアミン＝１００：２００：１）で目的物を分離し、目的物を５．０ｇ得た。
【００６７】
参考例２　ｐ−ニトロベンジル−２−アセトアミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−Ｄ−グルコピラノシドの合成
参考例１で得た２−メチル−（３，４，６−トリ−Ｏ−アセチル−１，２−ジデオキシ−α−Ｄ−グルコピラノ）−［２，１−ｄ］−２−オキサゾリン２．８ｇをジクロロエタン４０ｍｌに溶解し、ここにｐ−ニトロベンジルアルコール１０．４ｇとＤ−カンファー−１０−スルホン酸０．２ｇを加え、８０℃で２時間撹拌しながら反応させた。反応後、室温まで冷却し、トリエチルアミン４．０ｍｌを加えた。反応液を減圧濃縮した後、シリカゲルカラムクロマトグラフィー（溶出液；クロロホルム：酢酸エチル：メタノール＝２００：４０：５）で目的物を分離し、目的物を３．７ｇ得た。
【００６８】
参考例３　６−アクリロイルアミノカプロン酸の合成
６−アミノカプロン酸１０．０ｇを１．２７Ｍ水酸化ナトリウム水溶液６０ｍｌに溶解し、塩化アクリロイル７．８ｍｌを２０ｍｌのテトラヒドロフランに溶かしたものを氷冷下で滴下した。このとき、ｐＨ８〜９になるように４Ｎ水酸化ナトリウム水溶液を用いて調整した。滴下後、徐々に室温に戻しながら２時間撹拌した。次いで、反応液に１Ｎ塩酸をｐＨ３になるまで加えた後、酢酸エチルで生成物を抽出した。抽出液を水で洗浄し、無水硫酸マグネシウムで乾燥させた。
硫酸マグネシウムをろ別し、ろ液を減圧濃縮した。残渣を少量の酢酸エチルに溶かし、ヘキサンで再結晶し、目的物９．６ｇを得た。
【００６９】
参考例４　ｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）アミノ−ベンジル−２−アセトアミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−Ｄ−グルコピラノシドの合成
参考例２で得たｐ−ニトロベンジル−２−アセトアミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−Ｄ−グルコピラノシド１．６ｇをメタノール５０ｍｌに溶解し、ここにギ酸アンモニウム２．１ｇおよび１０％パラジウム−炭素１７０ｍｇを加えた。室温で５分間撹拌した後、触媒をろ別し、ろ液を減圧濃縮した。残渣をクロロホルムで溶解した。蒸留水で有機層を洗浄し、無水硫酸ナトリウムで乾燥した。乾燥後、硫酸ナトリウムをろ別した後、ろ液を減圧濃縮した。残渣をジクロロエタン：Ｎ，Ｎ−ジメチルホルムアミド＝１０：１の混合溶媒４４ｍｌで溶かし、ここに参考例３で得た６−アクリロイルアミノカプロン酸０．６ｇを加えた。さらに、トリエチルアミン０．４６ｍｌと１−（３−ジメチルアミノプロピル）−３−エチルカルボジイミド塩酸塩６５ｍｇを０℃で撹拌しながら加えた。撹拌しながら室温まで戻し、２２時間反応させた。反応液にクロロホルム６０ｍｌを加え、１Ｎ水酸化ナトリウム水溶液、飽和炭酸水素ナトリウム水溶液、飽和食塩水の順で洗浄し、無水硫酸ナトリウムで乾燥した。硫酸ナトリウムをろ別し、ろ液を減圧濃縮した後、シリカゲルカラムクロマトグラフィー（溶出液；クロロホルム：エタノール＝３０：１）で目的物を分離し、目的物を１．１ｇ得た。
【００７０】
実施例１　ｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）アミノ−ベンジル−２−アセトアミド−２−デオキシ−Ｄ−グルコピラノシドの合成
参考例４で得たｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）アミノ−ベンジル−２−アセトアミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−Ｄ−グルコピラノシド６６０ｍｇをメタノール７０ｍｌに溶解し、ここにナトリウムメトキシド５０ｍｇを加え、室温で撹拌しながら１５時間反応させた。反応後、Ｈ^＋型の陽イオン交換樹脂Ｄｏｗｅｘ５０ＷＸ−８（ダウケミカル社製）をｐＨ７になるまで加えた。イオン交換樹脂をろ別し、ろ液を減圧濃縮し、目的物を５２０ｍｇ得た。
【００７１】
実施例２　ｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）アミノ−ベンジル−２−アセトアミド−２−デオキシ−Ｄ−グルコピラノシド−アクリルアミド共重合体（共重合比１：１０、プライマーＡ）の合成
実施例１で得たｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）アミノ−ベンジル−２−アセトアミド−２−デオキシ−Ｄ−グルコピラノシド６２ｍｇとアクリルアミド８９ｍｇをジメチルスルホキシド：蒸留水＝３：１の混合溶媒１ｍｌに溶解し、アスピレーターで十分に脱気した後、Ｎ，Ｎ，Ｎ’，Ｎ’−テトラメチルエチレンジアミン（以下、ＴＥＭＥＤと略する）１３．８μｌと過硫酸アンモニウム８．４ｍｇを加え、室温で２４時間撹拌し重合させた。反応後、反応液に蒸留水２ｍｌを加え、Ｓｅｐｈａｄｅｘ　Ｇ−５０（アマシャムファルマシア社製）を用いたカラムクロマトグラフィー（溶出液：５０ｍＭ酢酸アンモニウム）により目的物を分離した。得られた目的物画分を凍結乾燥し、目的物１２０ｍｇを得た。
【００７２】
実施例３　ｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）アミノ−ベンジル−２−アセトアミド−２−デオキシ−Ｄ−グルコピラノシド−アクリルアミド共重合体（共重合比１：２０、プライマーＢ）の合成
アクリルアミド１７８ｍｇ、ＴＥＭＥＤ２６．３μｌ、過硫酸アンモニウム１６．１ｍｇを用いて実施例２と同様の方法で目的物１９４ｍｇを得た。
【００７３】
実施例４　ｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）アミノ−ベンジル−２−アセトアミド−２−デオキシ−Ｄ−グルコピラノシド−アクリルアミド共重合体（共重合比１：５０、プライマーＣ）の合成
アクリルアミド４４６ｍｇ、ＴＥＭＥＤ６３．８μｌ、過硫酸アンモニウム３９．１ｍｇを用いて実施例２と同様の方法で目的物４１０ｍｇを得た。
【００７４】
参考例５　ｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）アミノ−ベンジル−２−アセトアミド−２−デオキシ−Ｄ−グルコピラノシド−アクリルアミド共重合体（共重合比１：５、プライマーＤ）の合成
アクリルアミド４５ｍｇ、ＴＥＭＥＤ７．５μｌ、過硫酸アンモニウム４．６ｍｇを用いて実施例２と同様の方法で目的物８９ｍｇを得た。
【００７５】
実施例５　β１，４−ガラクトース転移酵素による各種プライマーへのガラクトース転移
β１，４−ガラクトース転移酵素（東洋紡績社製）１Ｕ、ウリジン−５’−ジホスホガラクトース１０ｍＭ、塩化マンガン１０ｍＭ、α−ラクトアルブミン０．２６ｍｇ／ｍｌを含む５０ｍＭＨＥＰＥＳ緩衝液（ｐＨ７．５）２．０ｍｌに実施例２〜４および参考例５で得たプライマーＡ〜Ｄをそれぞれ２０ｍｇ加え、３７℃で２４時間反応させた。反応後、１００℃で３分間加熱することにより反応を停止させた。反応液を遠心分離して得られた上清より、Ｓｅｐｈａｄｅｘ　Ｇ−２５カラムクロマトグラフィー（溶出液：蒸留水）で生成物画分を分離し、凍結乾燥することにより生成物１９ｍｇを得た。得られた生成物の^１Ｈ−ＮＭＲスペクトルを測定し、そのスペクトルよりガラクトースの転移がいずれも定量的に進行していることを確認した。
【００７６】
参考例６　固定化α２，３−シアル酸転移酵素の調製
ＮＨＳ活性化Ｓｅｐｈａｒｏｓｅ（アマシャムファルマシア社製）０．５ｇをとり、１ｍＭ塩酸１００ｍｌを３回に分けて洗浄した。これにブタ肝臓由来α２，３−シアル酸転移酵素１Ｕ、シチジン−５’−ジホスフェート１ｍＭを含む５０ｍＭＨＥＰＥＳ緩衝液（ｐＨ７．５）５ｍｌを加え、４℃で一晩、穏やかに振とうした。固定化α２，３−シアル酸転移酵素をガラスフィルターでろ別し、α２，３−シアル酸転移酵素を除く上記緩衝液５ｍｌで洗浄した。０．１ＭＴｒｉｓ−ＨＣｌ緩衝液（ｐＨ８．０）５ｍｌを加え、担体中の未反応の活性化基をブロックし、さらに洗浄した後、α２，３−シアル酸転移酵素をシチジン−５’−モノホスホ−Ｎ−アセチルノイラミン酸（以下、ＣＭＰ−ＮｅｕＡｃと略する）１ｍＭを含む２５ｍＭカコジル酸緩衝液（ｐＨ７．４）中に浸漬し、４℃で保存した。
【００７７】
実施例６　固定化α２，３−シアル酸転移酵素による各種プライマーへのシアル酸転移
参考例６で得た固定化α２，３−シアル酸転移酵素０．２ｍｌ、ＣＭＰ−ＮｅｕＡｃ５０ｍＭ、塩化マンガン１０ｍＭを含む５０ｍＭＨＥＰＥＳ緩衝液（ｐＨ７．０）１．０ｍｌに実施例５で得たガラクトシル化されたプライマーＡ〜Ｄをそれぞれ６．８ｍｇ、１０．４ｍｇ、２１．０ｍｇ、５．１ｍｇ（Ｎ−アセチルラクトサミン残基で５μｍｏｌｅに相当）加え、３０℃で２４時間振とうしながら反応させた。反応後、遠心分離することにより反応液を上清として得た後、実施例５と同様の方法で生成物を分離し、生成物をそれぞれ５ｍｇ、８ｍｇ、１７ｍｇ、４ｍｇ得た。生成物の^１Ｈ−ＮＭＲスペクトルを測定し、そのスペクトルよりシアル酸転移反応の収率を求めたところ、プライマーＡは８４％、プライマーＢは９８％、プライマーＣは９７％、プライマーＤは５６％であった。その結果、プライマー中の糖残基の密度が高すぎると、反応収率が低下することが確認された。
【００７８】
実施例７　プライマーからの糖鎖の遊離
実施例６でシアル酸を転移させたプライマーＡ５ｍｇを蒸留水：エタノール＝３：１の混合溶媒１ｍｌに溶かし、ここに１０％パラジウム−炭素１ｍｇを加え、常圧下水素ガスにより室温で２４時間接触還元した。触媒をろ別した後、ろ液を限外ろ過ユニットウルトラフリーＭＣ（分画分子量１０，０００、ミリポア社製）でろ過し、通過液画分として遊離させたオリゴ糖を集めた。通過液画分を凍結乾燥し、オリゴ糖１．７ｍｇを得た。得られたオリゴ糖の^１Ｈ−ＮＭＲスペクトルを測定し、そのスペクトルより得られたオリゴ糖が目的であるα２，３−シアリル−Ｎ−アセチルラクトサミンであることを確認した。
【００７９】
参考例７　ｐ−ニトロベンジル２，３，４，６−テトラ−Ｏ−アセチル−Ｄ−ガラクトシル−２，３，６−トリ−Ｏ−アセチル−Ｄ−グルコピラノシドの合成
１−ブロモ−２，３，４，６−テトラ−Ｏ−アセチル−Ｄ−ガラクトシル−２，３，６−トリ−Ｏ−アセチル−Ｄ−グルコピラノシド５．０ｇをジクロロエタン５０ｍｌに溶解し、ここにｐ−ニトロベンジルアルコール２３．５ｇとモレキュラーシーブス４Ａを５．０ｇ加え０℃で撹拌した。窒素気流下で銀トリフレートを２．９ｇ加えで徐々に室温に戻しながら１２時間撹拌しながら反応させた。
反応液をクロロホルムで希釈してセライトパッド上で濾過したのち、ろ液を飽和食塩水で洗浄し、有機層を無水硫酸マグネシウム上で乾燥させた。ろ過により硫酸マグネシウムを除去し、ろ液を減圧下で濃縮したのち、シリカゲルクロマトグラフィー（溶出液；クロロホルム：メタノール＝５０：１）で目的物を分離し、目的物を５．６ｇ得た。
【００８０】
参考例８　ｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）−ベンジル−２，３，４，６−テトラ−Ｏ−アセチル−Ｄ−ガラクトシル−２，３，６−トリ−Ｏ−アセチル−Ｄ−グルコピラノシドの合成
参考例７で得たｐ−ニトロベンジル２，３，４，６−テトラ−Ｏ−アセチル−Ｄ−ガラクトシル−２，３，６−トリ−Ｏ−アセチル−Ｄ−グルコピラノシ３．０ｇをメタノール５０ｍｌに溶解し、ここにギ酸アンモニウムを１．８ｇおよび１０％パラジウム−炭素を２００ｍｇを加えた。室温で５分間撹拌した後、触媒をろ別し、ろ液を減圧濃縮した。残渣をクロロホルムで溶解した。蒸留水で有機層を洗浄し、無水硫酸マグネシウムで乾燥した。乾燥後、硫酸マグネシウムをろ別した後、ろ液を減圧濃縮した。残渣をジクロロエタン：Ｎ，Ｎ，−ジメチルホルムアミド＝１０：１の混合溶媒４０ｍｌで溶かし、ここで参考例３で得た６−アクリロイルアミノカプロン酸を０．８５ｇ加えた。続いてトリエチルアミン６３４μｌと１−（３−ジメチルアミノプロピル）−３−エチルカルボジイミド塩酸塩８７０ｍｇを加えて氷冷から室温に戻しながら２２時間撹拌した。反応液をクロロホルムで希釈し、１Ｎ硫酸および飽和重曹水、飽和食塩水で順次洗浄し、有機層を無水硫酸マグネシウム上で乾燥させた。濾過により硫酸マグネシウムを除去し、ろ液を減圧下で濃縮したのち、シリカゲルクロマトグラフィー（溶出液；クロロホルム：エタノール＝２０：１）で目的物を分離し、目的物を１．１ｇ得た。
【００８１】
実施例８　ｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）−ベンジル−Ｄ−ガラクトシル−Ｄ−グルコピラノシドの合成
参考例８で得たｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）−ベンジル−２，３，４，６−テトラ−Ｏ−アセチル−Ｄ−ガラクトシル−２，３，６−トリ−Ｏ−アセチル−Ｄ−グルコピラノシド１．０ｇをメタノール１０ｍＬに溶解させた。そこにナトリウムメトキシド２４ｍｇを加え室温で１５時間撹拌した。
反応終了後、反応液をイオン交換樹脂ダウエックス５０Ｗ−Ｘ８（Ｈ＋）で中和した。樹脂をろ別し、ろ液を減圧濃縮し、目的物を６５０ｍｇ得た。
【００８２】
実施例９　ｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）−ベンジル−Ｄ−ガラクトシル−Ｄ−グルコピラノシド−アクリルアミド共重合体（共重合比１：２０、プライマーＥ）の合成
実施例８で得たｐ−Ｎ−（６−アクリロイルアミノヘキサノイル）−ベンジル−Ｄ−ガラクトシル−Ｄ−グルコピラノシド６１．５ｍｇ、アクリルアミド１４２．２ｍｇをジメチルスルホキシド：蒸留水＝３：１の混合溶媒２ｍｌに溶解し、ＴＥＭＥＤ１９．５μｌと過硫酸アンモニウム１５．３ｍｇを用いて、実施例２と同様に共重合させ、目的物２００ｍｇを得た。
【００８３】
実施例１０　固定化α２，３−シアル酸転移酵素によるプライマーＥへのシアル酸転移
参考例６で得た固定化α２，３−シアル酸転移酵素０．５ｍｌ、ＣＭＰ−ＮｅｕＡｃ５０ｍＭ、塩化マンガン１０ｍＭを含む５０ｍＭＨＥＰＥＳ緩衝液（ｐＨ７．０）１．０ｍｌに実施例９で得たプライマーＥを１０．３ｍｇ（ラクトース残基で５μｍｏｌｅに相当）加え、３０℃で２４時間振とうしながら反応させた。反応後、遠心分離することにより反応液を上清として得た後、実施例５と同様の方法で生成物を分離し、生成物をそれぞれ８．５ｍｇ得た。生成物の^１Ｈ−ＮＭＲスペクトルを測定し、そのスペクトルよりシアル酸転移反応の収率を求めたところ、８９％であった。
【００８４】
【発明の効果】
本発明のオリゴ糖合成用高分子プライマーを用いることにより、種々のオリゴ糖を効率よく合成でき、自動合成にも適用することができる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a water-soluble polymer primer useful for oligosaccharide production and a method for producing an oligosaccharide using the water-soluble polymer primer.
[0002]
[Prior art]
Sugar is a major component of living organisms along with nucleic acids and proteins, but its structure or function is not well understood compared to nucleic acids and proteins. Sugars form polymers, usually called sugar chains, and they combine with proteins and lipids to form extremely complex complex molecules collectively called glycoproteins, glycolipids, or proteoglycans. Furthermore, while nucleic acids or proteins are macromolecules in which their constituent units, nucleotides or amino acids, are linearly linked, sugar chains are not only units that have multiple branch points in the molecule, but also their constituent units. The structure of monosaccharides is so complex that it is incomparable to nucleic acids and proteins due to the variety of bonding modes. The complexity of these structures is one of the major factors that has slowed research in this area.
[0003]
However, in recent years, as it has been gradually found that sugar chains are involved in cell recognition, immunity, differentiation, fertilization, aging, canceration, and the like, it has become a very attractive research field. Under such circumstances, attempts to synthesize a sugar chain having a natural structure or a novel sugar chain have been actively made. Under such circumstances, attempts to synthesize a sugar chain having a natural structure or a novel sugar chain have been actively made. Automated synthesis technology has been established for nucleic acids and proteins, and everyone has acknowledged that research in this field has made remarkable progress.Therefore, the establishment of automated synthesis technology for sugar chains has been awaited. I have.
[0004]
There have been several reports on attempts to synthesize sugar chains automatically, and there are roughly two methods. One is by chemical synthesis, but the method of stereoselectively linking sugar residues to sugar residues is not well established, and the steps of further binding or elimination of protecting groups are required. There is a problem that it is complicated.
[0005]
The other is by enzymatic synthesis, which does not require a protecting group, and is capable of stereoselectively linking sugar residues to each other. Such a method has been proposed. This is due to the fact that genes for various glycosyltransferases have recently been isolated, and mass production of glycosyltransferases by genetic recombination technology has become possible. In the automatic synthesis, a compound in which a sugar residue serving as a reaction starting point is bonded to a specific carrier via a linker that can be cleaved under specific conditions (also referred to as a primer) is used as a starting material. Depending on the type, it is released from the carrier in various forms such as oligosaccharides, glycosides and glycopeptides. However, considering that the synthesized sugar chain compound is used for various uses, an oligosaccharide is still most preferable.
[0006]
Examples of automatic synthesis of sugar chains using glycosyltransferases are described in US Pat. Zehavi et al. Report solid-phase synthesis using a glycosyltransferase using a polyacrylamide gel to which aminoethyl groups or aminohexyl groups are bonded as a solid-phase carrier (for example, see Non-Patent Document 1). In this method, an appropriate monosaccharide is converted into 4-carboxy-2-nitrobenzylglycoside, and a saccharide coupled with an amino group of the above carrier is used as a primer to carry out a sugar chain extension reaction with a glycosyltransferase, followed by photolysis. Is released as oligosaccharides. However, the yield of glycosyltransferase by glycosyltransferase is low, and is less than 10%.
[0007]
Until now, glycosyltransferases did not react very much with sugars or oligosaccharides bound on a solid support, and it has been said that it is difficult to efficiently carry out a sugar chain extension reaction. Zehavi et al. Improve the transglycosylation yield by up to 51% by linking 4-carboxy-2-nitrobenzylglycoside to a solid support with a long chain linker such as a hexamethylene or octamethylene group. (For example, see Non-Patent Document 2). However, even in this method, the yield is not at a sufficient level.
[0008]
As another example, C.I. -H. Wong et al. Use a primer obtained by bonding a group of the following formula 2 (wherein Ac represents an acetyl group and Boc represents a t-butoxycarbonyl group) to aminated silica and use a glycosyltransferase to form a sugar chain. A method has been reported in which, after elongation, the sugar chain that has been elongated is cut out in the form of a glycopeptide utilizing the hydrolysis action of α-chymotrypsin (see, for example, Non-Patent Document 3). However, the yield of a sugar chain elongation reaction by a glycosyltransferase is 55 to 65%, which cannot be said to be very satisfactory.
[0009]
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[0010]
C.I. -H. Wong et al. Improved the group to be bonded to the aminated silica as the solid support to the following formula (wherein Ac represents an acetyl group), extended the sugar chain with a glycosyltransferase, and then decomposed by hydrazine decomposition. A method for releasing a sugar chain has been reported, and it has been reported that a glycosyltransfer reaction by an enzyme could be performed almost quantitatively (for example, see Non-Patent Document 4). According to this method, the extended sugar chain is released as a 6-carbohydrazide hexanol glycoside.
[0011]
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[0012]
Further, C.I. -H. Wong et al. Use a water-soluble polymer in which an acrylamide residue of an acrylamide-acrylic acid-N-isopropylacrylamide copolymer is bonded to a group represented by the following formula (wherein Ac represents an acetyl group) as a primer, A method has been reported in which a sugar chain is extended with a transferase and then released with cerium (IV) diammonium nitrate (for example, see Non-Patent Document 5). According to this method, the enzymatic glycosyltransfer reaction proceeds with a yield of 80 to 90%, and the extended sugar chain is released as a p-formylphenol glycoside.
[0013]
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[0014]
Further, M. Meldal et al. Used a primer in which a group of the following formula (5) (wherein Ac represents an acetyl group) was bonded to a polymer gel of a mono- or diacryloylated product of diaminated polyethylene glycol, and used a glycosyltransferase as a primer. A method has been reported in which a sugar chain is released as a glycopeptide using trifluoroacetic acid after elongating the sugar chain using the method, and it has been reported that the sugar transfer reaction has progressed almost quantitatively (for example, Non-Patent Document 6). See). The peptide chain of the glycopeptide obtained by this method is Asn (asparagine) -Gly (glycine), and the C-terminal glycine residue is a glycinamide residue, which is different from a normal glycopeptide.
[0015]
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[0016]
Further, the present inventors used a primer obtained by bonding a group represented by the following formula 6 (wherein Ac represents an acetyl group) to an amide nitrogen atom of polyacrylamide as a primer, and using a glycosyltransferase to prepare a saccharide. A method has been reported in which, after elongating a chain, a sugar chain extended by utilizing the hydrolysis action of α-chymotrypsin is released (for example, see Non-Patent Document 7). According to this method, the glycosyltransfer reaction by the enzyme proceeds quantitatively, and the extended sugar chain is obtained as a 6-aminohexanol glycoside.
[0017]
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[0018]
These methods mentioned above have drawbacks in that the resulting sugar chain compound is not an oligosaccharide and that the yield of the sugar transfer reaction is not always sufficient. In addition, although gene production has made mass production possible, glycosyltransferases are still very expensive, and it is desired to use immobilized glycosyltransferases that can be used repeatedly. Some of them have a drawback that immobilized glycosyltransferase cannot be used because a sugar chain elongation reaction is performed on an insoluble carrier. In order to use immobilized glycosyltransferase, it is necessary to carry out a sugar chain elongation reaction on a water-soluble carrier, not on an insoluble carrier.
[0019]
S. Roth et al. First bind a glycosyltransferase sugar receptor to a solid support, use this as an affinity adsorbent, and contact a tissue extract containing a glycosyltransferase capable of binding to the sugar receptor. Thereby binds the glycosyltransferase to the affinity adsorbent. Next, by contacting the affinity adsorbent to which the glycosyltransferase is bound with a solution containing sugar nucleotides that can be used as a sugar donor by the glycosyltransferase, the glycosyltransferase is released from the affinity adsorbent and is converted to a sugar acceptor. One sugar residue is extended, and further, a tissue extract containing a glycosyltransferase capable of binding to the sugar receptor having one sugar residue extended is contacted, and the same process is repeated. (See, for example, Patent Document 1). However, no specific data showing the usefulness of this method is shown, and no method for releasing the obtained sugar chain from the solid support is disclosed.
[0020]
This is an automatic synthesis of sugar chains using a glycosyltransferase. Norberg et al. Used a primer obtained by bonding Sepharose # 6B (manufactured by Amersham Pharmacia) to a group shown in Chemical Formula 7 below to extend a sugar chain using a glycosyltransferase, and then extended it with bromine or ammonia / ammonium borate. A method for releasing a sugar chain that has been made has been reported (for example, see Non-Patent Document 8). The glycosyltransfer reaction by the enzyme has progressed quantitatively, and there is no problem in terms of yield. However, expensive 3,4-diethoxy-3-cyclobutene-1,2-dione is used for producing a primer, Not economic. There is also a drawback that immobilized glycosyltransferase cannot be used because a sugar chain elongation reaction is performed on an insoluble carrier.
[0021]
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[0022]
In addition, the present inventors have developed a primer in which a group represented by the following formula 8 (wherein Ac represents an acetyl group) is bonded at a ratio of one to five of the amide nitrogen atoms in polyacrylamide as a primer. A method has been reported in which a sugar chain is extended using a glycosyltransferase and then the extended sugar chain is released by hydrogenolysis (for example, see Non-Patent Documents 9 and 10). According to this method, the enzyme-mediated glycosyltransfer reaction proceeds quantitatively, but as described below, it is the case of a free enzyme, and does not necessarily proceed as such with an immobilized enzyme.
[0023]
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[0024]
[Patent Document 1]
Japanese Patent Publication No. 5-500905
[0025]
[Non-patent document 1]
Carbohydr. Res. , {124, {23} (1983), {Carbohydr. Res. , {228, {255} (1992)
[0026]
[Non-patent document 2]
React. Polym. , {22, {171} (1994), {Carbohydr. Res. , {265, 161} (1994)
[0027]
[Non-Patent Document 3]
J. {Am. {Chem. {Soc. , {116, {1136} (1994)
[0028]
[Non-patent document 4]
J. {Am. {Chem. {Soc. , {116, {11315} (1994)
[0029]
[Non-Patent Document 5]
Adv. {Synth. {Catal. , {343, 675} (2001)
[0030]
[Non-Patent Document 6]
J. {Chem. {Soc. , {Chem. {Commun. , {1849} (1994)
[0031]
[Non-Patent Document 7]
Tetrahedron @ Lett. , {36, {9493} (1995)}, Carbohydr. Res. , {305, {443} (1998)
[0032]
[Non-Patent Document 8]
Carbohydr. Res. , {319, {80} (1999)
[0033]
[Non-Patent Document 9]
Tetrahedron @ Lett. , {35, {5657} (1994)
[0034]
[Non-Patent Document 10]
Carbohydr. Res. , {305, {443} (1998
[0035]
[Problems to be solved by the invention]
An object of the present invention is to provide a primer suitable for automatic synthesis of an oligosaccharide.
[0036]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the general formula (I) (where R¹Is a monosaccharide or oligosaccharide residue, R²Represents a linker having a length of 4 to 20 methylene groups) and at least one vinyl monomer so that the content of the acrylamide derivative is 10% or less. It has been found that the above problems can be solved by polymerization, and the present invention has been completed.
[0037]
That is, the present invention includes the following inventions.
[0038]
(1) Formula (I) (wherein R¹Is a monosaccharide or oligosaccharide residue, R²Is a linker having a length of 4 to 20 methylene groups) and a copolymer comprising at least one vinyl monomer and represented by the general formula (I) A water-soluble polymer primer for synthesizing oligosaccharides, wherein the acrylamide derivative is 10 mol% or less in the total vinyl copolymer.
[0039]
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[0040]
(2) R¹Is a N-acetylglucosamine residue, a glucose residue, or a lactose residue.
[0041]
(3) R²Is a pentylene group. (1) or (2), a water-soluble polymer primer for oligosaccharide synthesis.
[0042]
(4) The vinyl monomer is selected from the group consisting of acrylamides, methacrylamides, acrylic acid, methacrylic acid, acrylates, methacrylates, styrenes, and fatty acid vinyl esters (1) to ( 3) A water-soluble polymer primer for oligosaccharide synthesis.
[0043]
(5) A method for producing an oligosaccharide, comprising:
(Step 1) a step of transferring a sugar residue from a sugar nucleotide to the water-soluble polymer primer by contacting the water-soluble polymer primer of (1) with a glycosyltransferase in the presence of a sugar nucleotide;
(Step 2) a step of repeating the step 1 one or more times to extend the sugar chain,
(Step 3) a step of removing by-product nucleotides and unreacted sugar nucleotides, if necessary, and
(Step 4) repeating steps 1 to 3 a plurality of times, and then releasing an oligosaccharide from the water-soluble polymer primer in which a plurality of sugar residues are transferred and the sugar chain is extended. A method for producing an oligosaccharide.
[0044]
(6) The method for producing an oligosaccharide according to (5), wherein the step of releasing the sugar chain extended from the water-soluble polymer primer is catalytic hydrogenolysis or an oxidation step with 2,3-dichloro-5,6-dicyanobenzoquinone. .
[0045]
BEST MODE FOR CARRYING OUT THE INVENTION
The water-soluble polymer primer for oligosaccharide synthesis of the present invention is a copolymer comprising an acrylamide derivative represented by the general formula (I) and at least one vinyl monomer, and is represented by the general formula (I). The acrylamide derivative is less than 10 mol% of the total vinyl copolymer,¹Is a monosaccharide or oligosaccharide residue, R²Represents a linker having a length of 4 to 20 methylene groups.
[0046]
R¹The monosaccharide residue or oligosaccharide residue of is not particularly limited, and includes a glucose residue, a galactose residue, a mannose residue, an N-acetylglucosamine residue, an N-acetylgalactosamine residue, a xylose residue, and a lactose residue. Group, N-acetyllactosamine residue, chitobiose residue and the like.
[0047]
R²Examples of the linker include a butylene group, a pentylene group, a heptylene group, and a dodecylene group.
[0048]
The water-soluble polymer primer for oligosaccharide synthesis of the present invention can be obtained by copolymerization with at least one vinyl monomer of the acrylamide derivative represented by the general formula (I). The proportion of the acrylamide derivative represented by the formula (1) in the total vinyl copolymer is 10% or less, preferably 1 to 10%, and more preferably 2 to 5%.
[0049]
The copolymerization is not particularly limited, for example, can be performed by using a method such as radical polymerization, cationic polymerization, anionic polymerization, and usually by using radical polymerization using ammonium peroxodisulfate as a catalyst. It can be suitably performed.
[0050]
The vinyl monomer is not particularly limited, and includes, for example, acrylamides, methacrylamides, acrylic acid, methacrylic acid, acrylic esters, methacrylic esters, styrenes, and fatty acid vinyl esters. Can be Examples of the acrylamides include N-alkylacrylamides such as acrylamide, N-methylacrylamide, N-ethylacrylamide, and N-isopropylacrylamide. Examples of the methacrylamides include N-alkyl methacrylamides such as methacrylamide, N-methyl methacrylamide and N-isopropyl methacrylamide. Examples of the acrylates include methyl acrylate, ethyl acrylate, hydroxyethyl acrylate, and dimethylaminoethyl acrylate. Examples of methacrylates include methyl methacrylate, ethyl methacrylate, hydroxyethyl methacrylate, dimethylaminoethyl methacrylate, and the like. Examples of styrenes include styrene and p-hydroxystyrene. As fatty acid vinyl esters, vinyl acetate, vinyl butyrate and the like are preferably used.
[0051]
Typical molecular weights of the copolymer are from about 10,000 to about 5,000,000, preferably from 20,000 to 2,000,000, more preferably from 50,000 to 1,000,000.
[0052]
The acrylamide derivative represented by the general formula (I) can be synthesized by various commonly used organic synthetic chemistry techniques. For example, a sugar oxazoline derivative represented by the general formula (II), a halogenated sugar represented by the general formula (III) or a trichloroacetimidate derivative is condensed with p-nitrobenzyl alcohol in the presence of a suitable catalyst. Thereafter, the nitro group is reduced and converted to an amino group. Thereafter, the acrylamide derivative represented by the general formula (V) obtained by condensation of the ω-amino fatty acid represented by the general formula (IV) with acryloyl chloride is condensed with the above compound in the presence of a suitable condensing agent. Is obtained by
[0053]
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[0054]
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[0055]
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[0056]
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[0057]
In the first step of the method for producing an oligosaccharide of the present invention, a sugar residue is transferred from a sugar nucleotide to the water-soluble polymer primer by contacting the water-soluble polymer primer with a glycosyltransferase in the presence of a sugar nucleotide. .
[0058]
The transfer of the sugar from the sugar nucleotide to the water-soluble polymer primer is usually performed in a neutral buffer containing the water-soluble polymer primer and the sugar nucleotide at 10 to 60 ° C., preferably at 20 to 40 ° C. It is performed by contacting with a glycosyltransferase for 120 hours, preferably 2 to 72 hours.
[0059]
Further, a metal salt may be added to the reaction solution as needed. Examples of metal ions that can be added include magnesium, manganese, cobalt, nickel, copper, zinc, and the like, and can be usually added in the form of chloride or the like.
[0060]
The glycosyltransferase used in the present invention is not particularly limited as long as sugar nucleotides can be used as a sugar donor. Such enzymes include the Leylir pathway glycosyltransferases. For example, galactose transferase, N-acetylglucosamine transferase, N-acetylgalactosamine transferase, fucose transferase, sialyltransferase, mannose transferase, xylose transferase, glucuronyltransferase and the like can be mentioned. These enzymes may be free enzymes or immobilized enzymes, but immobilized enzymes are preferred.
[0061]
The sugar nucleotide used in the present invention is not particularly limited as long as the above-mentioned enzyme can be used. For example, uridine-5'-diphosphate galactose, uridine-5'-diphosphate-N-acetylglucosamine, uridine-5'-diphosphate-N-acetylgalactosamine, uridine-5'-diphosphate glucuronic acid, uridine-5 '-Xylose diphosphate, fucose guanosine-5'-diphosphate, mannose guanosine-5'-diphosphate, cytidine-5'-monophosphate-N-acetylneuraminic acid and sodium salts thereof.
[0062]
In the second step of the oligosaccharide production method of the present invention, the sugar chain is extended by repeating step 1 one or more times to transfer a plurality of sugar residues.
[0063]
In the third step of the method for producing an oligosaccharide of the present invention, by-product nucleotides and unreacted sugar nucleotides are removed as necessary. The method for removing by-product nucleotides, unreacted sugar nucleotides, and the like is not particularly limited as long as it can separate the water-soluble polymer primer from nucleotides, sugar nucleotides, and the like. For example, it can be removed by gel filtration chromatography, ion exchange chromatography, dialysis, ultrafiltration and the like.
[0064]
In the fourth step of the method for producing an oligosaccharide of the present invention, after repeating steps 1 to 3 a plurality of times, a plurality of sugar residues are transferred to release the oligosaccharide from the water-soluble polymer primer having an extended sugar chain. . The method for releasing the extended sugar chain from the water-soluble polymer primer of the present invention is not particularly limited as long as it is a method capable of releasing the extended sugar chain by oligosaccharide without decomposing the extended sugar chain. Examples thereof include catalytic hydrogenolysis and an oxidation reaction with 2,3-dichloro-5,6-dicyanobenzoquinone.
[0065]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples. However, these are merely examples, and the scope of the present invention is not limited to such Examples.
[0066]
Reference Example 1 Synthesis of 2-methyl- (3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-glucopyrano)-[2,1-d] -2-oxazoline
6.0 g of 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranoside was dissolved in 40 ml of 1,2-dichloroethane, and 3.2 ml of trimethylsilyltrifluoromethanesulfonic acid was added thereto. The reaction was carried out while stirring at 50 ° C. for 7 hours. After the reaction, the mixture was cooled to room temperature, and 10.8 ml of triethylamine was added. After the reaction solution was concentrated under reduced pressure, the target product was separated by silica gel column chromatography (eluent; toluene: ethyl acetate: triethylamine = 100: 200: 1) to obtain 5.0 g of the target product.
[0067]
Reference Example 2 Synthesis of p-nitrobenzyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranoside
2.8 g of 2-methyl- (3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-glucopyrano)-[2,1-d] -2-oxazoline obtained in Reference Example 1. Was dissolved in 40 ml of dichloroethane, and 10.4 g of p-nitrobenzyl alcohol and 0.2 g of D-camphor-10-sulfonic acid were added thereto, and reacted at 80 ° C. for 2 hours with stirring. After the reaction, the mixture was cooled to room temperature, and 4.0 ml of triethylamine was added. After the reaction solution was concentrated under reduced pressure, the target product was separated by silica gel column chromatography (eluent; chloroform: ethyl acetate: methanol = 200: 40: 5) to obtain 3.7 g of the target product.
[0068]
Reference Example 3 Synthesis of 6-acryloylaminocaproic acid
A solution of 10.0 g of 6-aminocaproic acid in 60 ml of a 1.27 M aqueous sodium hydroxide solution and 7.8 ml of acryloyl chloride dissolved in 20 ml of tetrahydrofuran was added dropwise under ice cooling. At this time, the pH was adjusted to 8 to 9 using a 4N aqueous sodium hydroxide solution. After the dropwise addition, the mixture was stirred for 2 hours while gradually returning to room temperature. Next, 1N hydrochloric acid was added to the reaction solution until the pH became 3, and the product was extracted with ethyl acetate. The extract was washed with water and dried over anhydrous magnesium sulfate.
The magnesium sulfate was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in a small amount of ethyl acetate and recrystallized from hexane to obtain 9.6 g of the desired product.
[0069]
Reference Example 4 Synthesis of pN- (6-acryloylaminohexanoyl) amino-benzyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranoside
1.6 g of p-nitrobenzyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranoside obtained in Reference Example 2 was dissolved in 50 ml of methanol. 1 g and 170 mg of 10% palladium-carbon were added. After stirring at room temperature for 5 minutes, the catalyst was filtered off and the filtrate was concentrated under reduced pressure. The residue was dissolved with chloroform. The organic layer was washed with distilled water and dried over anhydrous sodium sulfate. After drying, the sodium sulfate was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in a mixed solvent of dichloroethane: N, N-dimethylformamide = 10: 1 (44 ml), and thereto was added 6 g of 6-acryloylaminocaproic acid obtained in Reference Example 3. Further, 0.46 ml of triethylamine and 65 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were added at 0 ° C with stirring. The mixture was returned to room temperature with stirring, and reacted for 22 hours. 60 ml of chloroform was added to the reaction solution, and the mixture was washed with a 1N aqueous solution of sodium hydroxide, a saturated aqueous solution of sodium hydrogen carbonate, and saturated saline in this order, and dried over anhydrous sodium sulfate. After sodium sulfate was filtered off and the filtrate was concentrated under reduced pressure, the desired product was separated by silica gel column chromatography (eluent; chloroform: ethanol = 30: 1) to obtain 1.1 g of the desired product.
[0070]
Example 1 Synthesis of p-N- (6-acryloylaminohexanoyl) amino-benzyl-2-acetamido-2-deoxy-D-glucopyranoside
660 mg of p-N- (6-acryloylaminohexanoyl) amino-benzyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranoside obtained in Reference Example 4 was added to 70 mL of methanol. After dissolution, 50 mg of sodium methoxide was added, and the mixture was reacted at room temperature with stirring for 15 hours. After the reaction, H⁺A type of cation exchange resin Dowex50WX-8 (manufactured by Dow Chemical Company) was added until the pH reached 7. The ion exchange resin was filtered off, and the filtrate was concentrated under reduced pressure to obtain 520 mg of the desired product.
[0071]
Example 2 Synthesis of pN- (6-acryloylaminohexanoyl) amino-benzyl-2-acetamido-2-deoxy-D-glucopyranoside-acrylamide copolymer (copolymerization ratio 1:10, primer A)
62 mg of p-N- (6-acryloylaminohexanoyl) amino-benzyl-2-acetamido-2-deoxy-D-glucopyranoside obtained in Example 1 and 89 mg of acrylamide were mixed solvents of dimethyl sulfoxide: distilled water = 3: 1. After dissolving in 1 ml and degassing sufficiently with an aspirator, 13.8 μl of N, N, N ′, N′-tetramethylethylenediamine (hereinafter abbreviated as TEMED) and 8.4 mg of ammonium persulfate were added, and the mixture was added at room temperature for 24 hours. After stirring for an hour, polymerization was carried out. After the reaction, 2 ml of distilled water was added to the reaction solution, and the target product was separated by column chromatography (eluent: 50 mM ammonium acetate) using Sephadex # G-50 (manufactured by Amersham Pharmacia). The obtained target fraction was freeze-dried to obtain 120 mg of the target product.
[0072]
Example 3 Synthesis of pN- (6-acryloylaminohexanoyl) amino-benzyl-2-acetamido-2-deoxy-D-glucopyranoside-acrylamide copolymer (copolymerization ratio 1:20, primer B)
Using 178 mg of acrylamide, 26.3 μl of TEMED, and 16.1 mg of ammonium persulfate, 194 mg of the desired product was obtained in the same manner as in Example 2.
[0073]
Example 4 Synthesis of pN- (6-acryloylaminohexanoyl) amino-benzyl-2-acetamido-2-deoxy-D-glucopyranoside-acrylamide copolymer (copolymerization ratio 1:50, primer C)
Using 446 mg of acrylamide, 63.8 μl of TEMED, and 39.1 mg of ammonium persulfate, 410 mg of the desired product was obtained in the same manner as in Example 2.
[0074]
Reference Example 5 Synthesis of p-N- (6-acryloylaminohexanoyl) amino-benzyl-2-acetamido-2-deoxy-D-glucopyranoside-acrylamide copolymer (copolymerization ratio 1: 5, primer D)
89 mg of the desired product was obtained in the same manner as in Example 2 using 45 mg of acrylamide, 7.5 μl of TEMED and 4.6 mg of ammonium persulfate.
[0075]
Example 5 Galactose transfer to various primers by β1,4-galactosyltransferase
1. 50 mM HEPES buffer (pH 7.5) containing 1 U of β1,4-galactose transferase (manufactured by Toyobo Co., Ltd.), 10 mM of uridine-5′-diphosphogalactose, 10 mM of manganese chloride, and 0.26 mg / ml of α-lactalbumin. 20 mg of each of the primers A to D obtained in Examples 2 to 4 and Reference Example 5 was added to 0 ml, and reacted at 37 ° C. for 24 hours. After the reaction, the reaction was stopped by heating at 100 ° C. for 3 minutes. The product fraction was separated from the supernatant obtained by centrifuging the reaction solution by Sephadex® G-25 column chromatography (eluent: distilled water), and lyophilized to obtain 19 mg of the product. Of the resulting product¹An H-NMR spectrum was measured, and it was confirmed from the spectrum that the galactose transfer had all proceeded quantitatively.
[0076]
Reference Example 6 Preparation of immobilized α2,3-sialyltransferase
0.5 g of NHS-activated Sepharose (manufactured by Amersham Pharmacia) was taken and washed with 100 ml of 1 mM hydrochloric acid in three portions. To this was added 5 ml of 50 mM HEPES buffer (pH 7.5) containing 1 U of pig liver-derived α2,3-sialyltransferase and 1 mM of cytidine-5'-diphosphate, and the mixture was gently shaken at 4 ° C overnight. The immobilized α2,3-sialyltransferase was filtered off with a glass filter, and washed with 5 ml of the above-mentioned buffer except for α2,3-sialyltransferase. After adding 5 ml of 0.1 M Tris-HCl buffer (pH 8.0) to block unreacted activating groups in the carrier and further washing, α2,3-sialyltransferase was converted to cytidine-5′-monophospho-phosphate. It was immersed in a 25 mM cacodylate buffer (pH 7.4) containing 1 mM of N-acetylneuraminic acid (hereinafter abbreviated as CMP-NeuAc) and stored at 4 ° C.
[0077]
Example 6 Sialyl transfer to various primers by immobilized α2,3-sialyltransferase
Galactosylation obtained in Example 5 was carried out in 1.0 ml of 50 mM HEPES buffer (pH 7.0) containing 0.2 ml of the immobilized α2,3-sialyltransferase obtained in Reference Example 6, 50 mM of CMP-NeuAc, and 10 mM of manganese chloride. Then, 6.8 mg, 10.4 mg, 21.0 mg, and 5.1 mg (equivalent to 5 μmole of N-acetyllactosamine residue) were added to each of the primers A to D, and reacted at 30 ° C. for 24 hours with shaking. After the reaction, the reaction solution was obtained as a supernatant by centrifugation, and the product was separated in the same manner as in Example 5 to obtain 5 mg, 8 mg, 17 mg, and 4 mg of the product, respectively. Product¹The H-NMR spectrum was measured, and the yield of the sialic acid transfer reaction was determined from the spectrum. The yield was 84% for primer A, 98% for primer B, 97% for primer C, and 56% for primer D. As a result, it was confirmed that when the density of the sugar residues in the primer was too high, the reaction yield was reduced.
[0078]
Example 7 Release of sugar chain from primer
5 mg of primer A to which sialic acid was transferred in Example 6 was dissolved in 1 ml of a mixed solvent of distilled water: ethanol = 3: 1, and 1 mg of 10% palladium-carbon was added thereto, followed by catalytic reduction with hydrogen gas at normal pressure at room temperature for 24 hours. did. After the catalyst was filtered off, the filtrate was filtered with an ultrafiltration unit Ultrafree MC (fraction molecular weight: 10,000, manufactured by Millipore) to collect the released oligosaccharide as a flow-through fraction. The flow-through fraction was freeze-dried to obtain 1.7 mg of oligosaccharide. Of the resulting oligosaccharide¹An H-NMR spectrum was measured, and it was confirmed from the spectrum that the obtained oligosaccharide was α2,3-sialyl-N-acetyllactosamine.
[0079]
Reference Example 7 Synthesis of p-nitrobenzyl 2,3,4,6-tetra-O-acetyl-D-galactosyl-2,3,6-tri-O-acetyl-D-glucopyranoside
5.0 g of 1-bromo-2,3,4,6-tetra-O-acetyl-D-galactosyl-2,3,6-tri-O-acetyl-D-glucopyranoside is dissolved in 50 ml of dichloroethane, and p 23.5 g of nitrobenzyl alcohol and 5.0 g of molecular sieves 4A were added, and the mixture was stirred at 0 ° C. Under a nitrogen stream, 2.9 g of silver triflate was added and reacted while stirring for 12 hours while gradually returning to room temperature.
After the reaction solution was diluted with chloroform and filtered on a celite pad, the filtrate was washed with a saturated saline solution, and the organic layer was dried over anhydrous magnesium sulfate. After magnesium sulfate was removed by filtration and the filtrate was concentrated under reduced pressure, the desired product was separated by silica gel chromatography (eluent; chloroform: methanol = 50: 1) to obtain 5.6 g of the desired product.
[0080]
Reference Example 8 p-N- (6-acryloylaminohexanoyl) -benzyl-2,3,4,6-tetra-O-acetyl-D-galactosyl-2,3,6-tri-O-acetyl-D- Synthesis of glucopyranosides
3.0 g of p-nitrobenzyl 2,3,4,6-tetra-O-acetyl-D-galactosyl-2,3,6-tri-O-acetyl-D-glucopyranosi obtained in Reference Example 7 was added to 50 ml of methanol. After dissolution, 1.8 g of ammonium formate and 200 mg of 10% palladium-carbon were added. After stirring at room temperature for 5 minutes, the catalyst was filtered off and the filtrate was concentrated under reduced pressure. The residue was dissolved with chloroform. The organic layer was washed with distilled water and dried over anhydrous magnesium sulfate. After drying, magnesium sulfate was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in 40 ml of a mixed solvent of dichloroethane: N, N, -dimethylformamide = 10: 1, and 0.85 g of 6-acryloylaminocaproic acid obtained in Reference Example 3 was added. Subsequently, 634 μl of triethylamine and 870 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were added, and the mixture was stirred for 22 hours while returning from ice cooling to room temperature. The reaction solution was diluted with chloroform, washed sequentially with 1N sulfuric acid, saturated aqueous sodium hydrogen carbonate and saturated brine, and the organic layer was dried over anhydrous magnesium sulfate. After magnesium sulfate was removed by filtration and the filtrate was concentrated under reduced pressure, the target product was separated by silica gel chromatography (eluent; chloroform: ethanol = 20: 1) to obtain 1.1 g of the target product.
[0081]
Example 8 Synthesis of p-N- (6-acryloylaminohexanoyl) -benzyl-D-galactosyl-D-glucopyranoside
P-N- (6-acryloylaminohexanoyl) -benzyl-2,3,4,6-tetra-O-acetyl-D-galactosyl-2,3,6-tri-O-acetyl obtained in Reference Example 8 1.0 g of -D-glucopyranoside was dissolved in 10 mL of methanol. Thereto was added 24 mg of sodium methoxide, and the mixture was stirred at room temperature for 15 hours.
After completion of the reaction, the reaction solution was neutralized with ion exchange resin Dowex 50W-X8 (H +). The resin was filtered off, and the filtrate was concentrated under reduced pressure to obtain 650 mg of the desired product.
[0082]
Example 9 Synthesis of pN- (6-acryloylaminohexanoyl) -benzyl-D-galactosyl-D-glucopyranoside-acrylamide copolymer (copolymerization ratio 1:20, primer E)
61.5 mg of p-N- (6-acryloylaminohexanoyl) -benzyl-D-galactosyl-D-glucopyranoside obtained in Example 8 and 142.2 mg of acrylamide were mixed with 2 ml of a mixed solvent of dimethyl sulfoxide: distilled water = 3: 1. And copolymerized with 19.5 μl of TEMED and 15.3 mg of ammonium persulfate in the same manner as in Example 2 to obtain 200 mg of the desired product.
[0083]
Example 10 Sialyl transfer to primer E by immobilized α2,3-sialyltransferase
The primer E obtained in Example 9 was added to 1.0 ml of 50 mM HEPES buffer (pH 7.0) containing 0.5 ml of the immobilized α2,3-sialyltransferase obtained in Reference Example 6, 50 mM of CMP-NeuAc, and 10 mM of manganese chloride. 10.3 mg (equivalent to 5 μmole of lactose residue) was added, and the mixture was reacted at 30 ° C. with shaking for 24 hours. After the reaction, the reaction solution was obtained as a supernatant by centrifugation, and then the products were separated in the same manner as in Example 5 to obtain 8.5 mg of each product. Product¹An H-NMR spectrum was measured, and the yield of the sialic acid transfer reaction was determined from the spectrum to be 89%.
[0084]
【The invention's effect】
By using the polymer primer for oligosaccharide synthesis of the present invention, various oligosaccharides can be efficiently synthesized and can be applied to automatic synthesis.

Claims (6)

An acrylamide derivative represented by the general formula (I) (wherein R ¹ represents a monosaccharide residue or an oligosaccharide residue, R ² represents a linker having a length of 4 to 20 methylene groups) and at least one A water-soluble polymer primer for synthesizing oligosaccharides, which is a copolymer composed of various kinds of vinyl monomers, wherein the acrylamide derivative represented by the general formula (I) accounts for 10 mol% or less of all vinyl copolymers.

The water-soluble polymer primer for oligosaccharide synthesis according to claim 1, wherein R ^{1 in the} general formula (1) is an N-acetylglucosamine residue, a glucose residue or a lactose residue. The water-soluble polymer primer for oligosaccharide synthesis according to claim 1 or 2, wherein R ^{2 in the} general formula (1) is a pentylene group. The vinyl monomer is selected from the group consisting of acrylamides, methacrylamides, acrylic acid, methacrylic acid, acrylic esters, methacrylic esters, styrenes, and fatty acid vinyl esters. Water-soluble polymer primer for oligosaccharide synthesis. A method for producing an oligosaccharide, comprising:
(Step 1) a step of transferring a sugar residue from a sugar nucleotide to the water-soluble polymer primer by contacting the water-soluble polymer primer according to claim 1 with a glycosyltransferase in the presence of a sugar nucleotide;
(Step 2) a step of repeating the step 1 one or more times to extend the sugar chain,
(Step 3) a step of removing by-product nucleotides and unreacted sugar nucleotides, if necessary, and
(Step 4) After repeating Step 1 to Step 3 a plurality of times, releasing oligosaccharides from the water-soluble polymer primer in which a plurality of sugar residues are transferred and sugar chains are extended,
A method for producing an oligosaccharide, comprising: The method for producing an oligosaccharide according to claim 5, wherein the step of releasing the sugar chain extended from the water-soluble polymer primer is a catalytic hydrogenolysis or an oxidation step with 2,3-dichloro-5,6-dicyanobenzoquinone.