JP4105873B2 - N-acetylglucosaminyl-cellooligosaccharide derivative and method for producing the same - Google Patents
N-acetylglucosaminyl-cellooligosaccharide derivative and method for producing the same Download PDFInfo
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Description
【0001】
【発明の属する技術分野】
本発明は、新規なN−アセチルグルコサミニル−セロオリゴ糖誘導体及びその製造方法に関する。
【0002】
【従来の技術】
分離精製技術や解析技術の進歩によって、複雑な糖鎖の構造やその機能が解明されつつある。糖鎖は、様々な生命現象に関与しており、糖鎖を利用した新たな医薬品等の開発が期待されている。
【0003】
糖鎖の持つ情報量は、構成単位である糖の種類、数、結合位置、及び立体等に依存しており、とてつもなく膨大となるため、未だ知られていない配列や結合等を有する糖鎖が存在する可能性は否定できない。
【0004】
糖鎖を利用した新たな医薬品等を開発研究する上で、非天然型糖鎖を構築することは、新たな発見をもたらす可能性を秘めていると共に、糖鎖の機能を向上させたり、新たな機能を付与したりできる可能性もある。
【0005】
これまで、糖鎖を合成する方法としては、有機合成化学的な方法と、酵素法によるトランスフェラーゼを用いた方法が知られている。
【0006】
【発明が解決しようとする課題】
しかし、有機合成化学的な合成方法は、官能基の保護・脱保護等の繰り返しによる多段階の反応工程及び複数回の精製操作が必要であり、工業的規模での実施は極めて困難であった。
【0007】
一方、トランスフェラーゼを用いた糖鎖合成方法は、上記のような化学合成法に比べて非常に簡便であり、また、異性体の生成を伴わないので、構造の明確な糖鎖を効率良く得ることができるが、トランスフェラーゼを大量に入手することが困難であり、また、基質となる糖ヌクレオチドも不安定で高価であることから、工業的規模での実施はコスト的に難しかった。また、トランスフェラーゼは、糖の認識が極めて厳密で、ある決まった糖鎖しか合成できないという弱点があり、修飾された糖鎖や非天然型糖鎖の合成には適さないという問題があった。
【0008】
したがって、本発明の目的は、非天然型の新規なセロオリゴ糖誘導体及びその製造方法を提供することにある。
【0009】
【課題を解決するための手段】
本発明者らは、糖加水分解酵素を用いて糖鎖を構築する研究の過程で、アルカリ性条件下において、キチンを分解する酵素であるキチナーゼの受容体特異性が、比較的寛容であり、糖受容体として二位のアセトアミド基が存在しないセロビオースやセロトリオースを認識することを見出し、この知見に基いて本発明を完成するに至った。
【0010】
すなわち、本発明の一つは、下記一般式(1)で表されるN−アセチルグルコサミニル−セロオリゴ糖誘導体である。
【0011】
【化3】
【0012】
また、本発明のもう一つは、糖受容体となる下記一般式(2)で表されるセロオリゴ糖又はセロオリゴ糖誘導体と、糖供与体となるN−アセチルラクトサミンのオキサゾリン誘導体に、アルカリ性条件下でファミリー18に属するキチナーゼを作用させた後、更にβ−D−ガラクトシダーゼを作用させることを特徴とするN−アセチルグルコサミニル−セロオリゴ糖誘導体の製造方法である。
【化4】
【0013】
上記製造方法においては、前記糖受容体と前記糖供与体に、pH8〜10の条件下で前記キチナーゼを作用させることが好ましい。
また、前記キチナーゼが、Bacillus属由来のキチナーゼであることが好ましい。
【0014】
本発明によれば、糖受容体としてセロオリゴ糖又はセロオリゴ糖誘導体、糖供与体としてN−アセチルラクトサミンのオキサゾリン誘導体を用い、これらにキチナーゼをアルカリ性条件下で作用させて、セロオリゴ糖又はセロオリゴ糖誘導体の非還元末端にN−アセチルラクトサミンがβ−1,4結合で転移した非天然型のN−アセチルラクトサミニル−セロオリゴ糖誘導体を得、これにβ−D−ガラクトシダーゼを作用させることにより、セロオリゴ糖又はセロオリゴ糖誘導体の非還元末端にN−アセチルグルコサミンがβ−1,4結合で転移した非天然型のセロオリゴ糖誘導体(N−アセチルグルコサミニル−セロオリゴ糖誘導体)を簡単かつ安価に調製することができる。このN−アセチルグルコサミニル−セロオリゴ糖誘導体は、新規生理活性物質として、医薬品、診断薬、化粧品、食品、農薬、肥料等としての利用が期待できる。また、上記セロオリゴ糖誘導体の還元末端には、固定化のための重合基や検出のための官能基等を付与することができるので、例えば、セルラーゼの活性測定用の基質やカラム充填剤としての利用も期待できる。
【0015】
【発明の実施の形態】
本発明のN−アセチルグルコサミニル−セロオリゴ糖誘導体の合成方法の基本は、糖供与体であるN−アセチルラクトサミンのオキサゾリン誘導体に、アルカリ性条件下でキチナーゼを作用させることにより、糖受容体となるセロオリゴ糖又はセロオリゴ糖誘導体の非還元末端側へβ−1,4結合で転移させた後、更にβ−D−ガラクトシダーゼを作用させて、非還元末端のガラクトース残基を切断するものである。
【0016】
本発明において、N−アセチルラクトサミンのオキサゾリン誘導体(以下、単にオキサゾリン誘導体という)とは、N−アセチルラクトサミンから誘導された下記式(3)に示される構造を有する化合物をいう。
【0017】
【化5】
【0018】
上記オキサゾリン誘導体は、例えばN−アセチルラクトサミンに塩化アセチルを作用させてアノマー位の塩素化及び水酸基の保護を行い、塩化テトラエチルアンモニウムと炭酸水素ナトリウムによりオキサゾリン環を形成し、最後にナトリウムメトキシドによる脱アセチル化を行なうことにより得ることができる(K. Sasaki, S. Ahlfors, T. Frejd, J. Kihlberg, G. Magnusson, J. Org. Chem., 53, 5629 (1988)、S. Nishimura, H. Kuzuhara, Y. Takiguchi, K. Shimahara, Carbohydr. Res., 194, 223 (1989))。
【0019】
糖受容体となるセロオリゴ糖又はセロオリゴ糖誘導体としては、二糖から六糖のセロオリゴ糖及びそれらの還元末端をメチル基、アクリルアミド基、パラニトロフェニル基等で修飾したもの等が好ましく用いられる。
【0020】
キチナーゼとしては、Henrissatらによる糖質加水分解酵素の分類(Henrissat, B., Biochem. J., 280, 309 (1991)、Henrissat, B., A. Bairoch., Biochem. J., 293, 781 (1993))において、ファミリー18に属するキチナーゼが用いられ、特に、Bacillus属由来のキチナーゼが好ましく用いられる。
【0021】
キチナーゼは、Bacillus属の菌体(例えばBacillus circulans(IFO13627)等)の培養液から、例えば硫安沈殿により粗酵素を調製し、必要に応じて更にキチンカラム等を用いて精製することにより調製できる(T. Watanabe, K. Suzuki, W. Oyanagi, K. Ohnishi, H. Tanaka, J. Biol. Chem., 265, 15659 (1990))。なお、キチナーゼは、精製されたものを用いてもよく、粗酵素を用いてもよい。また、市販のキチナーゼを用いてもよく、例えば商品名「キチナーゼ」(Bacillus sp.由来、和光純薬工業社製)等を用いることができる。
【0022】
本発明においては、安価に入手できることから、Bacillus sp.由来のキチナーゼが特に好ましく用いられる。
【0023】
β−D−ガラクトシダーゼとしては、特に制限はないが、例えばストレプトコッカス ニューモニア(Streptococcus pneumoniae(ATCC6305))、Bacillus circulans(IFO13627)等由来のものを使用することができる。
【0024】
β−D−ガラクトシダーゼは、これらの菌体の培養液から、例えば硫安沈殿(Green, A. A. et al. : Methods in Enzymology, vol. 1, p76, 1955)や、ゲル濾過クロマトグラフィー(Miyazaki et al. : Agric Bio Chem, 52, 625-631, 1988)により調製することができる。また、市販のβ−D−ガラクトシダーゼを用いてもよく、例えば商品名「乳糖分解酵素製剤Biolacta FN5」(Bacillus circulans由来、大和化成社製)等を用いることができる。なお、β−D−ガラクトシダーゼは、N−アセチルヘキソサミニダーゼ活性を除いておくことが好ましい。
【0025】
本発明においては、ストレプトコッカス ニューモニア(Streptococcus pneumoniae)由来のβ−D−ガラクトシダーゼが特に好ましく用いられる。
【0026】
本発明において一般式(1)で表されるN−アセチルグルコサミニル−セロオリゴ糖誘導体は、例えば以下のようにして得ることができる。
【0027】
すなわち、上記オキサゾリン誘導体と、セロオリゴ糖又はセロオリゴ糖誘導体を、アルカリ性(好ましくはpH7〜11、より好ましくはpH8〜10)に調整した緩衝液に溶解し、これにキチナーゼを添加して20〜40℃で反応を行なう。反応中は、反応液を経時的にサンプリングして高速液体クロマトグラフィーでオキサゾリン誘導体のピークの消失を確認した後、酵素を失活させ、高速液体クロマトグラフィー等により目的物(N−アセチルラクトサミニル−セロオリゴ糖誘導体)を単離する。
【0028】
そして、得られたN−アセチルラクトサミニル−セロオリゴ糖誘導体に、緩衝液(好ましくはpH5〜7、より好ましくはpH6〜6.5)中でβ−D−ガラクトシダーゼを作用させて、非還元末端のガラクトース残基を切断し、高速液体クロマトグラフィー等により精製することにより、一般式(1)で表されるN−アセチルグルコサミニル−セロオリゴ糖誘導体を得ることができる。
【0029】
本発明においては、キチナーゼを用いた酵素反応を、上記のようなアルカリ性条件下で行なうことにより、糖受容体となるセロオリゴ糖又はセロオリゴ糖誘導体の非還元末端側へ糖供与体であるN−アセチルラクトサミンのオキサゾリン誘導体をβ−1,4結合で効率よく転移させることができる。
【0030】
本発明においては、上記オキサゾリン誘導体と、セロオリゴ糖又はセロオリゴ糖誘導体をモル比で0.3〜3:1となるように用いることが好ましい。
【0031】
また、キチナーゼの添加量は、転移反応が充分に進行する量であれば特に制限はないが、例えば上記オキサゾリン誘導体を0.1mol/L含む反応系においては、キチナーゼの添加量は50〜500mU/mLが好ましく、70〜150mU/mLがより好ましい。なお、本発明において、キチナーゼ1U(ユニット)とは、pH6.8、37℃の条件下で酵素をN−アセチルキトサンに作用させた際に、1分間にN−アセチルキトサンから1μmolのN−アセチルグルコサミンに相当する還元糖を生成する酵素量を意味する。
【0032】
また、β−D−ガラクトシダーゼの添加量は、反応が充分に進行する量であれば特に制限はないが、例えば、基質(N−アセチルラクトサミニル−セロオリゴ糖誘導体)を5〜10mmol/L含む反応系においては、β−D−ガラクトシダーゼの添加量は5〜250mU/mLが好ましく、25〜125mU/mLがより好ましい。なお、本発明において、β−D−ガラクトシダーゼ1U(ユニット)とは、pH6.0、37℃の条件下で酵素をパラニトロフェニル−β−D−ガラクトシドに作用させた際に、1分間にパラニトロフェニル−β−D−ガラクトシドから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。
【0033】
上記の各酵素反応を行なう際に用いられる緩衝液としては、キチナーゼを用いた酵素反応の場合は、例えばトリス緩衝液、リン酸緩衝液、炭酸緩衝液等が挙げられる。また、β−D−ガラクトシダーゼを用いた酵素反応の場合は、例えばSodium cacodylate緩衝液、リン酸緩衝液等が挙げられる。
【0034】
また、高速液体クロマトグラフィーの条件は、例えば「TSK-gel Amide-80」(商品名、トーソー社製)、「Asahipak NH2P-50 4E」(商品名、昭和電工社製)等のカラムを用い、溶媒としてアセトニトリル/水混合溶媒(アセトニトリル/水(v/v)=80/20〜60/40)等を用いて行なうことができる。
【0035】
【実施例】
以下、本発明を実施例を挙げてさらに具体的に説明する。
製造例
N−アセチルラクトサミン4.0gに塩化アセチル20mLを加え、撹拌しながら室温で4日間反応を行なった。なお、反応中は、TLC(酢酸エチル/ヘキサン(v/v)=4/1)により反応を追跡した。
【0036】
この反応液をエバポレートによって濃縮し、過剰量の塩化メチレンで希釈後、pHが中性になるまで冷水及び飽和炭酸水素ナトリウム溶液で分液し、得られた有機溶媒相を無水硫酸ナトリウムで乾燥後、濾過して硫酸ナトリウムを除去した。
【0037】
得られた濾液をエバポレートにより濃縮し、更に減圧乾燥して溶媒を完全に除去して、塩化N−アセチル−3,6,2',3',4',6'−ヘキサ−O−アセチル−α−ラクトサミニル(以下、化合物(I)という)6.80gを得た。精製は行なわずにそのまま次の反応を行った。
【0038】
上記化合物(I)3.01gをアセトニトリル25mLに溶解し、この溶液を、塩化カルシウムによって一時間半乾燥させた塩化テトラエチルアンモニウム1.0gと炭酸水素ナトリウム1.0gに加え、55℃で1時間40分還流させた。TLC(酢酸エチル/ヘキサン(v/v)=4/1)にて反応終了を確認し、ガラスフィルターG4(商品名、柴田科学社製)で固形物を除去し、得られた濾液を減圧下で濃縮した後、過剰量の塩化メチレンで希釈し、水相のpHが中性になるまで冷水及び飽和炭酸水素ナトリウム溶液で分液し、得られた有機溶媒相を無水硫酸ナトリウムにより乾燥後、濾過して硫酸ナトリウムを除去した。
【0039】
得られた溶液をエバポレートによって濃縮した後、シリカゲルフラッシュカラムクロマトグラフィー(Gel:商品名「Silica Gel 60」、Merck社製、particle size:0.04−0.063mm、展開溶媒:酢酸エチル/ヘキサン(v/v)=4/1)により精製して、固体状の2−メチル{3,6ジ−O−アセチル−4−O−(2,3,4,6−テトラ−O−アセチル−β−D−ガラクトピラノシル)1,2−ジデオキシ−α−グルコピラノ}[2,1−d]−2−オキサゾリン(以下、化合物(II)という)1.40gを得た。
【0040】
アルゴン雰囲気下、上記化合物(II)1.40gを無水メタノール270mLに溶解させ、0.01Mの濃度になるようにナトリウムメトキシドのメタノール溶液を添加し、室温で2時間反応させた。TLC(酢酸エチル/ヘキサン(v/v)=4/1)により反応終了を確認した後、反応溶液が中性になるまでイオン交換樹脂Amberlite IR-120(H+)(商品名、オルガノ社製)を加えた後、濾過してイオン交換樹脂を除去した。
【0041】
得られた濾液をエバポレートによって濃縮し、更に減圧乾燥して、2−メチル{4−O−(β−D−ガラクトピラノシル)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(以下、オキサゾリン誘導体(I)という)0.80gを得た。なお、オキサゾリン誘導体(I)は、NMRにより構造を確認した。
【0042】
実施例1
上記オキサゾリン誘導体(I)36.5mgをエッペンドルフチューブに入れ、(N−アクリルアミドメチル)−アミノカルボニルエチル−1−チオ−β−D−セロビオシド16.9mgと市販キチナーゼ325mUを溶解した50mMトリス緩衝液(pH9.0)1mLを添加し、40℃の恒温槽に入れて反応を行なった。
【0043】
反応中、高速液体クロマトグラフィーにより反応を追跡し、オキサゾリン誘導体のピークが完全に消失したのを確認した後、90℃で20分間熱失活を行なった。なお、高速液体クロマトグラフィーは、以下の条件(1)で行なった。
【0044】
・条件(1)
カラム:「TSK-gel Amide-80 (4.6×25mm)」(商品名、トーソー社製)
溶媒:アセトニトリル/水(v/v)=75/25
温度:40℃
流速:1.0mL/min
検出:RI
次いで、得られた反応液を用いて、以下の条件(2)で高速液体クロマトグラフィーを行ない、目的物の画分を回収した。
【0045】
・条件(2)
カラム:「Inertsil ODS-3 (10.0×25mm)」(商品名、GL Sciences社製)
溶媒:水/メタノール(v/v)=900/70
温度:室温
流速:3.0mL/min
検出:UV(210nm)
そして、回収した画分をエバポレートで濃縮、凍結乾燥して、下記式で表されるN−アセチルラクトサミニル−セロオリゴ糖誘導体4.7mgを得た。
【0046】
【化6】
【0047】
得られたN−アセチルラクトサミニル−セロオリゴ糖誘導体4.5mgに、20mMリン酸緩衝液(pH6.0)730μL、及び市販のβ−D−ガラクトシダーゼ(Streptococcus pneumoniae由来、商品名「β1,4−ガラクトシダーゼ」、CALBIOCHEM社製)10.7mUを添加し、37℃の恒温槽中で反応を行なった。
【0048】
反応中、高速液体クロマトグラフィーにより反応を追跡し、N−アセチルラクトサミニルオリゴ糖誘導体のピークが完全に消失したのを確認した後、90℃で20分間熱失活を行なった。なお、高速液体クロマトグラフィーは、上記の条件(1)で行なった。
【0049】
次いで、得られた反応液を用いて、以下の条件(3)で高速液体クロマトグラフィーを行ない、目的物の画分を回収した。
【0050】
・条件(3)
カラム:「Inertsil ODS-3」(商品名、GL Sciences社製)
溶媒:水/メタノール(v/v)=900/70
温度:室温
流速:5.0mL/min
検出:UV(210nm)
そして、回収した画分をエバポレートで濃縮、凍結乾燥して、下記式で表されるN−アセチルグルコサミニル−セロオリゴ糖誘導体2.3mgを得た。
【0051】
【化7】
【0052】
なお、上記のN−アセチルラクトサミニル−セロオリゴ糖誘導体、及びN−アセチルグルコサミニル−セロオリゴ糖誘導体は、1H、13CNMR及びMALDI TOF MASSにより、その構造を確認した。
【0053】
【発明の効果】
以上説明したように、本発明によれば、糖受容体としてセロオリゴ糖又はセロオリゴ糖誘導体、糖供与体としてN−アセチルラクトサミンのオキサゾリン誘導体を用い、これらにキチナーゼをアルカリ性条件下で作用させて、セロオリゴ糖又はセロオリゴ糖誘導体の非還元末端にN−アセチルラクトサミンがβ−1,4結合した非天然型のN−アセチルラクトサミニル−セロオリゴ糖誘導体を得、これにβ−D−ガラクトシダーゼを作用させることにより、セロオリゴ糖又はセロオリゴ糖誘導体の非還元末端にN−アセチルグルコサミンがβ−1,4結合した非天然型のN−アセチルグルコサミニル−セロオリゴ糖誘導体を簡単かつ安価に調製することができる。このN−アセチルグルコサミニル−セロオリゴ糖誘導体は、新規生理活性物質として、医薬品、診断薬、化粧品、食品、農薬、肥料等としての利用が期待できる。また、上記セロオリゴ糖誘導体の還元末端には、固定化のための重合基や検出のための官能基等を付与することができるので、例えば、セルラーゼの活性測定用の基質やカラム充填剤としての利用も期待できる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel N-acetylglucosaminyl-cellooligosaccharide derivative and a method for producing the same.
[0002]
[Prior art]
Advances in separation and purification techniques and analysis techniques are elucidating the structures and functions of complex sugar chains. Sugar chains are involved in various life phenomena, and the development of new drugs using sugar chains is expected.
[0003]
The amount of information possessed by sugar chains depends on the type, number, binding position, and configuration of the sugars that are constituent units, and is extremely enormous. Therefore, sugar chains having sequences and bonds that are not yet known. The possibility of existence cannot be denied.
[0004]
In developing and researching new drugs using sugar chains, constructing non-natural sugar chains has the potential to bring new discoveries, improve the functions of sugar chains, There is a possibility that it can be given a new function.
[0005]
Until now, as a method for synthesizing a sugar chain, an organic synthetic chemistry method and a method using an enzyme-based transferase are known.
[0006]
[Problems to be solved by the invention]
However, the organic synthetic chemical synthesis method requires a multi-step reaction process by repeated functional group protection / deprotection and a plurality of purification operations, and it was extremely difficult to implement on an industrial scale. .
[0007]
On the other hand, the sugar chain synthesis method using transferase is much simpler than the above-described chemical synthesis method and does not involve the generation of isomers, so that a sugar chain with a clear structure can be obtained efficiently. However, it is difficult to obtain a large amount of transferase, and sugar nucleotides as substrates are unstable and expensive, so that implementation on an industrial scale is difficult in terms of cost. In addition, transferase has a weak point that sugar recognition is extremely strict and only certain sugar chains can be synthesized, and there is a problem that it is not suitable for the synthesis of modified sugar chains or unnatural sugar chains.
[0008]
Accordingly, an object of the present invention is to provide a novel non-natural cellooligosaccharide derivative and a method for producing the same.
[0009]
[Means for Solving the Problems]
In the course of research for constructing a sugar chain using a sugar hydrolase, the present inventors have a relatively tolerant receptor specificity for chitinase, an enzyme that degrades chitin under alkaline conditions. It has been found that cellobiose and cellotriose without a 2-position acetamide group as a receptor are recognized, and the present invention has been completed based on this finding.
[0010]
That is, one of the present invention is an N-acetylglucosaminyl-cellooligosaccharide derivative represented by the following general formula (1).
[0011]
[Chemical 3]
[0012]
Another aspect of the present invention is that alkaline conditions are applied to cellooligosaccharide or cellooligosaccharide derivative represented by the following general formula (2) serving as a sugar acceptor and N-acetyllactosamine oxazoline derivative serving as a sugar donor. A method for producing an N-acetylglucosaminyl-cellooligosaccharide derivative characterized in that a chitinase belonging to family 18 is allowed to act below and then β-D-galactosidase is allowed to act.
[Formula 4]
[0013]
In the said manufacturing method, it is preferable to make the said chitinase act on the said sugar acceptor and the said sugar donor on the conditions of pH 8-10.
The chitinase is preferably a chitinase derived from the genus Bacillus.
[0014]
According to the present invention, a cellooligosaccharide or cellooligosaccharide derivative is used as a sugar acceptor, an oxazoline derivative of N-acetyllactosamine is used as a sugar donor, and chitinase is allowed to act on these under alkaline conditions. To obtain a non-natural N-acetyllactosaminyl-cellooligosaccharide derivative in which N-acetyllactosamine is transferred to the non-reducing end of the N-acetyllactosamine by a β-1,4 bond, and β-D-galactosidase acts on this derivative Simple and inexpensive preparation of non-natural cellooligosaccharide derivatives (N-acetylglucosaminyl-cellooligosaccharide derivatives) in which N-acetylglucosamine is transferred to the non-reducing end of cellooligosaccharides or cellooligosaccharide derivatives by β-1,4 bonds can do. This N-acetylglucosaminyl-cellooligosaccharide derivative can be expected to be used as a new physiologically active substance, such as pharmaceuticals, diagnostics, cosmetics, foods, agricultural chemicals, and fertilizers. In addition, since the reducing end of the cellooligosaccharide derivative can be provided with a polymerization group for immobilization, a functional group for detection, or the like, for example, as a substrate for cellulase activity measurement or as a column filler Use is also expected.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
The basic method for synthesizing the N-acetylglucosaminyl-cellooligosaccharide derivative of the present invention is to allow a sugar acceptor to react with an oxazoline derivative of N-acetyllactosamine, which is a sugar donor, under alkaline conditions. After transferring to the non-reducing end side of the resulting cellooligosaccharide or cellooligosaccharide derivative by β-1,4 linkage, β-D-galactosidase is further acted on to cleave the non-reducing end galactose residue.
[0016]
In the present invention, an oxazoline derivative of N-acetyllactosamine (hereinafter simply referred to as an oxazoline derivative) refers to a compound having a structure represented by the following formula ( 3 ) derived from N-acetyllactosamine.
[0017]
[Chemical formula 5]
[0018]
The oxazoline derivative is prepared by, for example, reacting N-acetyllactosamine with acetyl chloride to chlorinate the anomeric position and protect the hydroxyl group, form an oxazoline ring with tetraethylammonium chloride and sodium bicarbonate, and finally with sodium methoxide. It can be obtained by performing deacetylation (K. Sasaki, S. Ahlfors, T. Frejd, J. Kihlberg, G. Magnusson, J. Org. Chem., 53, 5629 (1988), S. Nishimura, H. Kuzuhara, Y. Takiguchi, K. Shimahara, Carbohydr. Res., 194, 223 (1989)).
[0019]
As the cellooligosaccharide or cellooligosaccharide derivative serving as a sugar receptor, those obtained by modifying celluligosaccharides from disaccharide to hexasaccharide and their reducing ends with a methyl group, an acrylamide group, a paranitrophenyl group, or the like are preferably used.
[0020]
Chitinases include the classification of carbohydrate hydrolases by Henrissat et al. (Henrissat, B., Biochem. J., 280, 309 (1991), Henrissat, B., A. Bairoch., Biochem. J., 293, 781 (1993)), chitinases belonging to family 18 are used, and in particular, chitinases derived from the genus Bacillus are preferably used.
[0021]
The chitinase can be prepared by preparing a crude enzyme from, for example, ammonium sulfate precipitation from a culture solution of Bacillus genus cells (for example, Bacillus circulans (IFO 13627), etc.) and further purifying it using a chitin column or the like as necessary ( T. Watanabe, K. Suzuki, W. Oyanagi, K. Ohnishi, H. Tanaka, J. Biol. Chem., 265, 15659 (1990)). As the chitinase, a purified one or a crude enzyme may be used. Commercially available chitinase may also be used, for example, trade name “chitinase” (derived from Bacillus sp., Manufactured by Wako Pure Chemical Industries, Ltd.) and the like.
[0022]
In the present invention, Bacillus sp. The chitinase derived from is particularly preferably used.
[0023]
The β-D-galactosidase is not particularly limited, and for example, those derived from Streptococcus pneumoniae (ATCC 6305), Bacillus circulans (IFO 13627) and the like can be used.
[0024]
β-D-galactosidase is obtained from a culture solution of these cells by, for example, ammonium sulfate precipitation (Green, AA et al .: Methods in Enzymology, vol. 1, p76, 1955) or gel filtration chromatography (Miyazaki et al. : Agric Bio Chem, 52 , 625-631, 1988). Commercially available β-D-galactosidase may also be used. For example, the trade name “Lactose-degrading enzyme preparation Biolacta FN5” (derived from Bacillus circulans , manufactured by Daiwa Kasei Co., Ltd.) can be used. In addition, it is preferable that (beta) -D-galactosidase removes N-acetylhexosaminidase activity.
[0025]
In the present invention, β-D-galactosidase derived from Streptococcus pneumoniae is particularly preferably used.
[0026]
In the present invention, the N-acetylglucosaminyl-cellooligosaccharide derivative represented by the general formula (1) can be obtained, for example, as follows.
[0027]
That is, the above oxazoline derivative and cellooligosaccharide or cellooligosaccharide derivative are dissolved in a buffer solution adjusted to be alkaline (preferably pH 7-11, more preferably pH 8-10), and chitinase is added thereto to add 20-40 ° C. Perform the reaction at. During the reaction, the reaction solution is sampled over time and the disappearance of the peak of the oxazoline derivative is confirmed by high performance liquid chromatography. Then, the enzyme is deactivated and the target product (N-acetyllactosaminyl is obtained by high performance liquid chromatography or the like. -Cellooligosaccharide derivatives) are isolated.
[0028]
Then, β-D-galactosidase is allowed to act on the obtained N-acetyllactosaminyl-cellooligosaccharide derivative in a buffer solution (preferably pH 5 to 7, more preferably pH 6 to 6.5), so that the non-reducing end. The N-acetylglucosaminyl-cellooligosaccharide derivative represented by the general formula (1) can be obtained by cleaving the galactose residue.
[0029]
In the present invention, N-acetyl, which is a sugar donor toward the non-reducing terminal side of the cellooligosaccharide or cellooligosaccharide derivative serving as a sugar acceptor, by carrying out an enzymatic reaction using chitinase under the alkaline conditions as described above. An oxazoline derivative of lactosamine can be efficiently transferred with a β-1,4 bond.
[0030]
In the present invention, the oxazoline derivative and the cellooligosaccharide or cellooligosaccharide derivative are preferably used in a molar ratio of 0.3 to 3: 1.
[0031]
The amount of chitinase added is not particularly limited as long as the transfer reaction proceeds sufficiently. For example, in a reaction system containing 0.1 mol / L of the oxazoline derivative, the amount of chitinase added is 50 to 500 mU / mL is preferable, and 70 to 150 mU / mL is more preferable. In the present invention, chitinase 1U (unit) means 1 μmol of N-acetyl from N-acetylchitosan per minute when the enzyme is allowed to act on N-acetylchitosan under the conditions of pH 6.8 and 37 ° C. It means the amount of enzyme that produces a reducing sugar corresponding to glucosamine.
[0032]
The amount of β-D-galactosidase added is not particularly limited as long as the reaction proceeds sufficiently. For example, the substrate (N-acetyllactosaminyl-cellooligosaccharide derivative) is contained in an amount of 5 to 10 mmol / L. In the reaction system, the amount of β-D-galactosidase added is preferably 5 to 250 mU / mL, and more preferably 25 to 125 mU / mL. In the present invention, β-D-galactosidase 1U (unit) means that when enzyme is allowed to act on paranitrophenyl-β-D-galactoside under the conditions of pH 6.0 and 37 ° C. It means the amount of enzyme that liberates 1 μmol of paranitrophenol from nitrophenyl-β-D-galactoside.
[0033]
In the case of the enzyme reaction using chitinase, for example, Tris buffer solution, phosphate buffer solution, carbonate buffer solution and the like can be used as the buffer solution used when performing each enzyme reaction described above. Moreover, in the case of the enzyme reaction using (beta) -D-galactosidase, a sodium cacodylate buffer solution, a phosphate buffer solution, etc. are mentioned, for example.
[0034]
The conditions of high performance liquid chromatography are, for example, using columns such as “TSK-gel Amide-80” (trade name, manufactured by Tosoh Corporation), “Asahipak NH2P-50 4E” (trade name, manufactured by Showa Denko KK), etc. As the solvent, an acetonitrile / water mixed solvent (acetonitrile / water (v / v) = 80/20 to 60/40) or the like can be used.
[0035]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
Production Example N-acetyllactosamine (4.0 g) was added with 20 mL of acetyl chloride and reacted at room temperature for 4 days with stirring. During the reaction, the reaction was followed by TLC (ethyl acetate / hexane (v / v) = 4/1).
[0036]
The reaction solution is concentrated by evaporation, diluted with an excess amount of methylene chloride, and then separated with cold water and saturated sodium bicarbonate solution until the pH is neutral. The obtained organic solvent phase is dried over anhydrous sodium sulfate. And filtered to remove sodium sulfate.
[0037]
The obtained filtrate was concentrated by evaporation, further dried under reduced pressure to completely remove the solvent, and N-acetyl chloride-3,6,2 ', 3', 4 ', 6'-hexa- O -acetyl- 6.80 g of α-lactosaminyl (hereinafter referred to as compound (I)) was obtained. The next reaction was carried out without purification.
[0038]
3.01 g of the above compound (I) was dissolved in 25 mL of acetonitrile, and this solution was added to 1.0 g of tetraethylammonium chloride and 1.0 g of sodium bicarbonate dried for one and a half hours with calcium chloride. Reflux for minutes. The completion of the reaction was confirmed with TLC (ethyl acetate / hexane (v / v) = 4/1), solids were removed with a glass filter G4 (trade name, manufactured by Shibata Kagaku Co., Ltd.), and the obtained filtrate was reduced under reduced pressure. After concentration with, diluted with an excess amount of methylene chloride, partitioned with cold water and saturated sodium bicarbonate solution until the pH of the aqueous phase becomes neutral, and the resulting organic solvent phase was dried over anhydrous sodium sulfate, Filter to remove sodium sulfate.
[0039]
After the obtained solution was concentrated by evaporation, silica gel flash column chromatography (Gel: trade name “Silica Gel 60”, manufactured by Merck, particle size: 0.04-0.063 mm, developing solvent: ethyl acetate / hexane (v / v ) = 4/1) to give solid 2-methyl {3,6-di - O - acetyl-4-O - (2,3,4,6-tetra - O - acetyl-beta-D- 1.40 g of galactopyranosyl) 1,2-dideoxy-α-glucopyrano} [2,1-d] -2-oxazoline (hereinafter referred to as compound (II)) was obtained.
[0040]
Under an argon atmosphere, 1.40 g of the compound (II) was dissolved in 270 mL of anhydrous methanol, a methanol solution of sodium methoxide was added to a concentration of 0.01 M, and the mixture was reacted at room temperature for 2 hours. After confirming the completion of the reaction by TLC (ethyl acetate / hexane (v / v) = 4/1), ion exchange resin Amberlite IR-120 (H + ) (trade name, manufactured by Organo Corporation) until the reaction solution becomes neutral ) And then filtered to remove the ion exchange resin.
[0041]
The obtained filtrate was concentrated by evaporation and further dried under reduced pressure to give 2-methyl {4- O- (β-D-galactopyranosyl) 1,2-dideoxy-α-D-glucopyrano} [2,1 -D] -2-oxazoline (hereinafter referred to as oxazoline derivative (I)) 0.80 g was obtained. The structure of the oxazoline derivative (I) was confirmed by NMR.
[0042]
Example 1
36.5 mg of the above oxazoline derivative (I) was put in an Eppendorf tube, and 50 mM Tris buffer solution (1N mg of (N-acrylamidomethyl) -aminocarbonylethyl-1-thio-β-D-cellobioside and 325 mU of commercially available chitinase dissolved therein) ( 1 mL of pH 9.0) was added and the reaction was performed in a constant temperature bath at 40 ° C.
[0043]
During the reaction, the reaction was monitored by high performance liquid chromatography, and after confirming that the peak of the oxazoline derivative completely disappeared, heat inactivation was performed at 90 ° C. for 20 minutes. The high performance liquid chromatography was performed under the following condition (1).
[0044]
・ Condition (1)
Column: “TSK-gel Amide-80 (4.6 × 25mm)” (trade name, manufactured by Tosoh Corporation)
Solvent: acetonitrile / water (v / v) = 75/25
Temperature: 40 ° C
Flow rate: 1.0 mL / min
Detection: RI
Subsequently, using the obtained reaction solution, high performance liquid chromatography was performed under the following conditions (2) to collect a fraction of the target product.
[0045]
・ Condition (2)
Column: “Inertsil ODS-3 (10.0 × 25mm)” (trade name, manufactured by GL Sciences)
Solvent: water / methanol (v / v) = 900/70
Temperature: Room temperature Flow rate: 3.0 mL / min
Detection: UV (210 nm)
And the collect | recovered fraction was concentrated and lyophilized | freeze-dried by evaporation, and 4.7 mg of N-acetyllactosaminyl-cellooligosaccharide derivatives represented by a following formula were obtained.
[0046]
[Chemical 6]
[0047]
4.5 mg of the obtained N-acetyllactosaminyl-cellooligosaccharide derivative, 730 μL of 20 mM phosphate buffer (pH 6.0), and commercially available β-D-galactosidase (derived from Streptococcus pneumoniae , trade name “β1,4- 10.7 mU of “Galactosidase” (manufactured by CALBIOCHEM) was added, and the reaction was carried out in a constant temperature bath at 37 ° C.
[0048]
During the reaction, the reaction was followed by high performance liquid chromatography. After confirming that the peak of the N-acetyllactosaminyl oligosaccharide derivative completely disappeared, heat inactivation was performed at 90 ° C. for 20 minutes. The high performance liquid chromatography was performed under the above condition (1).
[0049]
Subsequently, using the obtained reaction solution, high performance liquid chromatography was performed under the following conditions (3) to collect a fraction of the target product.
[0050]
・ Condition (3)
Column: “Inertsil ODS-3” (trade name, manufactured by GL Sciences)
Solvent: water / methanol (v / v) = 900/70
Temperature: Room temperature Flow rate: 5.0 mL / min
Detection: UV (210 nm)
Then, the collected fraction was concentrated by evaporation and freeze-dried to obtain 2.3 mg of an N-acetylglucosaminyl-cellooligosaccharide derivative represented by the following formula.
[0051]
[Chemical 7]
[0052]
The structures of the N-acetyllactosaminyl-cellooligosaccharide derivatives and the N-acetylglucosaminyl-cellooligosaccharide derivatives were confirmed by 1 H, 13 CNMR and MALDI TOF MASS.
[0053]
【The invention's effect】
As described above, according to the present invention, cellooligosaccharide or cellooligosaccharide derivative is used as a sugar acceptor, and an oxazoline derivative of N-acetyllactosamine is used as a sugar donor, and chitinase is allowed to act on these under alkaline conditions, A non-natural N-acetyllactosaminyl-cellooligosaccharide derivative in which N-acetyllactosamine is β-1,4 linked to the non-reducing end of cellooligosaccharide or cellooligosaccharide derivative is obtained, and β-D-galactosidase acts on this. Can be used to easily and inexpensively prepare a non-natural N-acetylglucosaminyl-cellooligosaccharide derivative in which N-acetylglucosamine is β-1,4 linked to the non-reducing end of the cellooligosaccharide or cellooligosaccharide derivative. it can. This N-acetylglucosaminyl-cellooligosaccharide derivative can be expected to be used as a new physiologically active substance, such as pharmaceuticals, diagnostics, cosmetics, foods, agricultural chemicals, and fertilizers. In addition, since the reducing end of the cellooligosaccharide derivative can be provided with a polymerization group for immobilization, a functional group for detection, or the like, for example, as a substrate for cellulase activity measurement or as a column filler Use is also expected.
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