JPH1045788A - Production of new glycoconjugate - Google Patents
Production of new glycoconjugateInfo
- Publication number
- JPH1045788A JPH1045788A JP8223105A JP22310596A JPH1045788A JP H1045788 A JPH1045788 A JP H1045788A JP 8223105 A JP8223105 A JP 8223105A JP 22310596 A JP22310596 A JP 22310596A JP H1045788 A JPH1045788 A JP H1045788A
- Authority
- JP
- Japan
- Prior art keywords
- sugar chain
- glcnac
- sugar
- complex
- reaction
- Prior art date
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Compounds Of Unknown Constitution (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、酵素の糖鎖転移反
応を利用した新規な複合型糖鎖を持つ複合糖質およびそ
の製造方法に関する。医薬分野に応用される。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel complex saccharide having a complex type sugar chain utilizing a sugar chain transfer reaction of an enzyme and a method for producing the same. Applied in the pharmaceutical field.
【０００２】[0002]
【従来の技術】糖質および複合糖質は生物の細胞、体液
等に存在し、細胞の基質認識や細胞−細胞間の認識等に
深く関わっている。中でもシアル酸を含む複合型糖鎖を
有する複合糖質は細胞の認識機能に最も深く関わり、ま
た生体内物質の吸収、分解等の代謝の速度に関係してい
る。タンパク質には糖鎖を持つものが知られ、例えばエ
リスロポエチンの場合、糖鎖末端のシアル酸をはずすと
生体内での活性が速やかに消失し、シアロ複合型糖鎖が
生理的に特に重要な役割を果たしている。これらの生理
的機能に着目して、例えば動物細胞等を用いて遺伝子工
学的に作られたエリスロポエチンやティシュープラスミ
ノーゲンアクチベーター等の糖タンパク質が医薬として
利用されている。また、ヒト絨毛性性腺刺激ホルモン
（ｈＣＧ）等ペプチドホルモンの中にも糖鎖を持つもの
が知られている。一方、タンパク質、生理活性ペプチド
あるいはセラミド等に糖鎖を付けたりあるいは今ある糖
鎖を別の糖鎖に換えることにより、生理機能の強化や生
理活性の改変に役立つことが期待される。2. Description of the Related Art Carbohydrates and complex carbohydrates are present in living cells, body fluids and the like, and are deeply involved in cell substrate recognition and cell-cell recognition. Among them, complex carbohydrates having complex sugar chains containing sialic acid are most deeply involved in the recognition function of cells, and are related to the rate of metabolism such as absorption and decomposition of biological substances. Proteins with sugar chains are known.For example, in the case of erythropoietin, removal of the sialic acid at the sugar chain terminus causes rapid loss of activity in vivo, and sialo-complex sugar chains play a particularly important physiological role. Plays. Focusing on these physiological functions, for example, glycoproteins such as erythropoietin and tissue plasminogen activator, which have been genetically engineered using animal cells and the like, have been used as pharmaceuticals. Also, peptide hormones such as human chorionic gonadotropin (hCG) having a sugar chain are known. On the other hand, by attaching a sugar chain to a protein, a physiologically active peptide, ceramide, or the like, or replacing an existing sugar chain with another sugar chain, it is expected to be useful for enhancement of physiological functions and modification of biological activity.
【０００３】糖鎖を酵素的に改変する方法としては、
１）転移酵素あるいはエキソグリコシダーゼによる方法
と、２）エンドグリコシダーゼによる方法が考えられ
る。[0003] Methods for enzymatically modifying sugar chains include:
1) a method using a transferase or exoglycosidase, and 2) a method using an endoglycosidase.
【０００４】１）の方法としては、例えばＤ．Ｈ．ジョ
ジアッセ（D. H. Joziasse）ら［ヨーロピアン ジャー
ナル オブ バイオケミストリー（Eur. J. Bioche
m.)、 第１９１巻、第７５〜８３頁（１９９０）］があ
るが、これは糖鎖の非還元末端からの逐次反応である。
また、最近、Ｍ．シャスター（M. Schuster）ら［ジャ
ーナル オブ アメリカン ケミカル ソサエテイ（J.
Amer. Chem. Soc.）、第１１６巻、第１１３５〜１１
３６頁（１９９４）］は数種のグリコシルトランスフェ
ラーゼを組み合わせた糖鎖の固相合成法を報告してい
る。しかしこれらエキソグリコシダーゼまたはグリコシ
ルトランスフェラーゼを用いた糖鎖合成反応は糖残基一
つ一つについてその酵素反応を逐次的に行わねばなら
ず、反応ステップが多く、大変煩雑である。[0004] As the method 1), for example, D. H. DH Joziasse et al. [European Journal of Biochemistry (Eur. J. Bioche)
m.), 191: 75-83 (1990)], which is a sequential reaction from the non-reducing end of the sugar chain.
Recently, M.S. M. Schuster et al. [Journal of American Chemical Society (J.
Amer. Chem. Soc.), Vol. 116, Nos. 1135-11
36 (1994)] report a solid-phase synthesis method of a sugar chain using several kinds of glycosyltransferases. However, the sugar chain synthesis reaction using these exoglycosidases or glycosyltransferases requires that the enzymatic reaction be carried out sequentially for each sugar residue, which involves many reaction steps and is very complicated.
【０００５】一方、２）のエンドグリコシダーゼを用い
た糖転移反応としては、Ｒ．Ｂ．トリムブル（R. B. Tr
imble）ら［ジャーナル オブ バイオロジカル ケミ
ストリー（J. Biol. Chem.）、第２６１巻、第１２００
０〜１２００５頁（１９８６）］のフラボバクテリウム
メニンゴセプチカム（Flavobacterium meningoseptic
um）由来のエンド−β−Ｎ−アセチルグルコサミニダー
ゼ（エンド−Ｆ）に関するもの、Ｒ．Ｍ．バーデールス
（R. M. Bardales）ら［ジャーナル オブ バイオロジ
カル ケミストリー（J. Biol. Chem.）、第２６４巻、
第１９８９３〜１９８９７頁（１９８９）］のディプロ
コッカス ニューモニエ（Diprococcuspneumoniae）由
来のエンド−α−Ｎ−アセチルガラクトサミニダーゼに
関するものがあり、前者はグリセロールが受容体に、ま
た後者はグリセロール、ｐ−ニトロフェノール、セリ
ン、スレオニン等が受容体になるという報告である。そ
の後、竹川ら［特開平５−６４５９４号（１９９３）］
がアルスロバクター プロトホルミエ（Arthrobacter p
rotophormiae）由来のエンド−β−Ｎ−アセチルグルコ
サミニダーゼ（エンド−Ａ）による糖質への高マンノー
ス型糖鎖の転移反応を、また、Ｋ．ヤマモト（K. Yamam
oto） ら［バイオケミカル バイオフィジカルリサーチ
コミュニケーション（Biochem. Biophys. Res. Commu
n.）、第２０３巻、第２４４〜２５２頁（１９９４）］
はムコール ヒエマリス（Mucor hiemalis）由来のエン
ド−Ｍによる糖質への糖鎖転移反応を報告した。On the other hand, the glycosyltransfer reaction using endoglycosidase of 2) is described in B. Trim Bull (RB Tr
imble) et al. [Journal of Biological Chemistry (J. Biol. Chem.), Vol. 261, 1200]
0 to 12005 (1986)], Flavobacterium meningoseptic.
um) -derived endo-β-N-acetylglucosaminidase (endo-F); M. RM Bardales et al. [Journal of Biological Chemistry (J. Biol. Chem.), Vol.
198993-19897 (1989)], which relates to endo-α-N-acetylgalactosaminidase derived from Diprococcus pneumoniae. , Serine, threonine, etc. are reported to be receptors. Thereafter, Takekawa et al. [JP-A-5-64594 (1993)]
Is Arthrobacter p.
transfer reaction of high mannose type sugar chains to carbohydrates by endo-β-N-acetylglucosaminidase (endo-A) derived from K. rotophormiae). Yamamoto (K. Yamam
oto) et al. [Biochemical. Biophys. Res. Commu.
n.), Volume 203, Pages 244-252 (1994)].
Reported a transglycosylation reaction to a carbohydrate by endo-M derived from Mucor hiemalis.
【０００６】[0006]
【発明が解決しようとする課題】糖質に糖鎖を新たに付
与したりあるいは他の糖鎖と入れ換えたりする、いわゆ
る糖鎖の改変（リモデリング）により複合糖質の生体内
での安定性や生物活性が天然の複合糖質に比べて増強さ
れたり、天然にない生物機能が付加されれば医薬品に応
用した場合に有用である。また、複合糖質における糖鎖
のもつ生理的機能は今まで糖鎖改変の有効な手段がなか
ったために、十分には解明されていないが、その役割の
解析のための重要な手段を提供する。The stability of complex carbohydrates in vivo by so-called sugar chain modification (remodeling) by adding new sugar chains to sugars or replacing them with other sugar chains. It is useful when applied to pharmaceuticals if its biological activity is enhanced compared to natural glycoconjugates, or if biological functions not found in nature are added. In addition, the physiological functions of sugar chains in glycoconjugates have not been fully elucidated because there has been no effective means of sugar chain modification, but they provide important means for analyzing their roles. .
【０００７】エンドグリコシダーゼの糖鎖転移反応によ
る方法は、天然の糖鎖がブロックのまま糖鎖受容体へ転
移付加されて複合糖質が合成される点で新しい方法を提
供するものであった。[0007] The method based on the transglycosylation reaction of endoglycosidase has provided a new method in that a natural sugar chain is transferred and added to a sugar chain receptor in the form of a block to synthesize a complex saccharide.
【０００８】複合型糖鎖の転移活性を有するエンドグリ
コシダーゼとしてムコール ヒエマリス由来のエンド−
β−Ｎ−アセチルグルコサミニダーゼ（エンド−Ｍ）
は、糖鎖受容体のＡｓｎにβ配置で結合したＮ−アセチ
ル−Ｄ−グルコサミン残基（ＧｌｃＮＡｃ）に糖鎖を転
移することが知られているが、本発明者らは糖鎖受容体
の糖残基がＧｌｃＮＡｃ以外の糖残基の場合にも酵素が
認識して糖鎖転移が起きることを見出し、本発明を考案
した。As an endoglycosidase having a complex type sugar chain transfer activity, an endoglycosidase derived from Mucor hemaris
β-N-acetylglucosaminidase (End-M)
Is known to transfer a sugar chain to an N-acetyl-D-glucosamine residue (GlcNAc) bound to Asn of a sugar chain receptor in a β-configuration. The present inventors have found that the enzyme recognizes and causes sugar chain transfer even when the sugar residue is a sugar residue other than GlcNAc, and devised the present invention.
【０００９】[0009]
【課題を解決するための手段】本発明は、β−ＧｌｃＮ
Ａｃ以外の糖残基を有する糖鎖受容体にエンドグリコシ
ダーゼを用いて複合型糖鎖を転移付加させることにより
合成される天然にはない配列の複合型糖鎖を有する複合
糖質とその製造方法を提供する。DISCLOSURE OF THE INVENTION The present invention provides a β-GlcN
Complex carbohydrate having complex type sugar chain of non-natural sequence synthesized by transferring and adding complex type sugar chain to sugar chain receptor having sugar residue other than Ac using endoglycosidase and method for producing the same I will provide a.
【００１０】[0010]
【発明の実施の形態】本発明を概説すれば、本発明は天
然にはない配列の複合型糖鎖を有する複合糖質の製造法
に関する。エンドグリコシダーゼの存在下、下記式（式
１）： Ｘ−ＧｌｃＮＡｃ−β−ＧｌｃＮＡｃ−Ｙ ＋ Ｒ−Ｚ → Ｘ−ＧｌｃＮＡｃ −Ｒ−Ｚ ＋ β−ＧｌｃＮＡｃ−Ｙ （式１） （式中、Ｘは複合型糖鎖、ＧｌｃＮＡｃはＮ−アセチル
−Ｄ−グルコサミン、Ｙは糖質、複合糖質、ペプチドあ
るいはタンパク質、Ｒはβ−ＧｌｃＮＡｃ以外の糖残
基、Ｚは糖質、複合糖質あるいはペプチド等）で表され
る転移反応を行うことを特徴とする。BEST MODE FOR CARRYING OUT THE INVENTION In general, the present invention relates to a method for producing a complex saccharide having a complex type sugar chain having a sequence not found in nature. In the presence of endoglycosidase, the following formula (Formula 1): X-GlcNAc-β-GlcNAc-Y + R−Z → X-GlcNAc-R-Z + β-GlcNAc-Y (Formula 1) Complex sugar chain, GlcNAc is N-acetyl-D-glucosamine, Y is carbohydrate, complex carbohydrate, peptide or protein, R is sugar residue other than β-GlcNAc, Z is carbohydrate, complex carbohydrate or peptide, etc. ) Is carried out.
【００１１】本発明に用いるエンドグリコシダーゼとし
ては、例えばＳ．カドワキ（S. Kadowaki ）ら［アグリ
カルチュラル アンド バイオロジカル ケミストリー
（Agr. Biol. Chem.）、第５４巻、第９７〜１０６頁
（１９９０）］により報告されたムコール ヒエマリス
（Mucor hiemalis）により生産されるエンド−β−Ｎ−
アセチルグルコサミニダーゼ（ＥＣ３．２．１．９
６）、エンド−Ｍがある。The endoglycosidase used in the present invention includes, for example, S. cerevisiae. Produced by Mucor hiemalis reported by S. Kadowaki et al. [Agricultural and Biological Chemistry, Vol. 54, 97-106 (1990)]. End-β-N-
Acetylglucosaminidase (EC 3.2.1.9)
6), there is End-M.
【００１２】該酵素は下記式（式２）： Ｘ−ＧｌｃＮＡｃ−β−ＧｌｃＮＡｃ−Ａｓｎ−（ペプチド） （式２） （式中Ｘは複合糖鎖を示す）のアスパラギン（Ａｓｎ）
結合型糖鎖のキトビオース（ＧｌｃＮＡｃ−β−Ｇｌｃ
ＮＡｃ）部分のグリコシド結合を加水分解する。The enzyme is asparagine (Asn) of the following formula (Formula 2): X-GlcNAc-β-GlcNAc-Asn- (peptide) (Formula 2) (where X represents a complex sugar chain)
Chitobiose with a linked sugar chain (GlcNAc-β-Glc
Hydrolyze the glycosidic bonds of the NAc) moiety.
【００１３】（式２）のＸ−ＧｌｃＮＡｃ部分が水の水
酸基（この時、加水分解）でなく、糖鎖受容体の水酸基
に移れば糖鎖の転移反応が成立する。酵素エンド−Ｍは
高マンノース型糖鎖よりもシアル酸のついた複合型糖鎖
をより効率よく転移させることが出来る。If the X-GlcNAc moiety of the formula (2) is transferred to a hydroxyl group of a sugar chain receptor instead of a hydroxyl group of water (at this time, hydrolysis), a sugar chain transfer reaction is established. The enzyme endo-M can transfer a complex type sugar chain with sialic acid more efficiently than a high mannose type sugar chain.
【００１４】エンドグリコシダーゼによる糖鎖転移反応
の糖鎖受容体の糖部分は、通常、元の糖鎖配列にあった
のと同一構造の糖である。例えば酵素エンド−Ｍによる
複合型糖鎖の転移を例にとると、エンド−Ｍは（式２）
で示される複合型糖鎖のキトビオース（ＧｌｃＮＡｃ−
β−ＧｌｃＮＡｃ）部分に作用して切り取った糖鎖を受
容体のβ−ＧｌｃＮＡｃ部分に転移させる。即ち、（式
１）Ｒがβ−ＧｌｃＮＡｃである。 Ｘ−ＧｌｃＮＡｃ−β−ＧｌｃＮＡｃ−Ｙ ＋ β−ＧｌｃＮＡｃ−Ｚ → Ｘ−ＧｌｃＮＡｃ−β−ＧｌｃＮＡｃ−Ｚ ＋ β−ＧｌｃＮＡｃ−Ｙ （式３ ）The sugar moiety of the sugar chain receptor in the sugar chain transfer reaction by endoglycosidase is usually a sugar having the same structure as that in the original sugar chain sequence. For example, taking the transfer of a complex type sugar chain by the enzyme End-M as an example, End-M is represented by the following formula (2).
The complex type sugar chain chitobiose (GlcNAc-
Acting on the (β-GlcNAc) portion, the cut sugar chain is transferred to the β-GlcNAc portion of the receptor. That is, (Formula 1) R is β-GlcNAc. X-GlcNAc-β-GlcNAc-Y + β-GlcNAc-Z → X-GlcNAc-β-GlcNAc-Z + β-GlcNAc-Y (Formula 3)
【００１５】本発明者らは、酵素エンドグリコシダーゼ
による糖鎖転移反応の糖鎖受容体の糖残基の認識の特異
性を調べたところ、β−ＧｌｃＮＡｃ以外の糖残基の場
合にも複合型糖鎖の転移反応が起きることを見出し、本
発明を完成した。The present inventors examined the specificity of the recognition of sugar residues of sugar chain receptors in the sugar chain transfer reaction by the enzyme endoglycosidase, and found that complex types other than β-GlcNAc could be used as complex type. The present inventors have found that a sugar chain transfer reaction occurs and completed the present invention.
【００１６】即ち、この方法によればβ−ＧｌｃＮＡｃ
に類縁の糖残基を有する糖鎖受容体を合成して用いるこ
とにより、これに酵素的に糖鎖が転移付加して天然には
ない新しい配列の糖鎖を有する複合糖質を合成すること
が可能となる。That is, according to this method, β-GlcNAc
Synthesizing and using a sugar chain receptor having a sugar residue analogous to glycan to synthesize glycoconjugates having a new sequence of sugar chains that are not naturally occurring due to enzymatic transfer of sugar chains Becomes possible.
【００１７】β−ＧｌｃＮＡｃに代わる糖残基として
は、例えばα−ＧｌｃＮＡｃ（ＧｌｃＮＡｃのαアノマ
ー）、α及びβ−Ｄ−グルコース（Ｇｌｃ）、Ｄ−マン
ノース（Ｍａｎ）であり、キトビオース（ＧｌｃＮＡ
ｃ）2 や１−Ｓ−置換−Ｄ−グルコースあるいはＤ−キ
シロース（Ｘｙｌ）等も用いられる。これらはいずれも
β−ＧｌｃＮＡｃと同じ４位の水酸基がエクアトリアル
な立体配置をとるヘキソピラノースあるいはペントピラ
ノースである。Examples of sugar residues that can substitute for β-GlcNAc include α-GlcNAc (α anomer of GlcNAc), α and β-D-glucose (Glc), D-mannose (Man), and chitobiose (GlcNAc).
c) 2 or 1-S-substituted-D-glucose or D-xylose (Xyl) is also used. All of them are hexopyranose or pentopyranose in which the hydroxyl group at the 4-position same as β-GlcNAc has an equatorial configuration.
【００１８】糖鎖受容体として糖の１位のパラ−ニトロ
フェニル（ＰＮＰ）誘導体を用いて酵素エンド−Ｍによ
る複合型糖鎖の転移反応における糖の認識の特異性を調
べた結果を表１に、またその構造を図１（化１）に示
す。これら複合型糖鎖の受容体となる糖残基の構造はい
ずれも４位水酸基の立体配置がβ−ＧｌｃＮＡｃと同じ
エクアトリアルなヘキソピラノースあるいはペントピラ
ノースであり、この構造を有する糖に複合型糖鎖が転移
する。 表１。各種糖−ＰＮＰ誘導体への糖鎖転移反応収率 ──────────────────────────────────── Ｎｏ． 糖鎖受容体 相対転移反応収率（％） ──────────────────────────────────── １ ＰＮＰ−β−Ｄ−ＧｌｃＮＡｃ １００ ２ ＰＮＰ−α−Ｄ−ＧｌｃＮＡｃ ８６ ３ ＰＮＰ−β−Ｄ−キトビオース ３５ ４ ＰＮＰ−β−Ｄ−グルコース １１２ ５ ＰＮＰ−α−Ｄ−グルコース ６０ ６ ＰＮＰ−１ーＳ−β−Ｄ−グルコース ９３ ７ ＰＮＰ−β−Ｄ−マンノース ９６ ８ ＰＮＰ−β−Ｄ−キシロピラノース １５ ──────────────────────────────────── 糖鎖転移反応収率は、ＰＮＰ−β−Ｄ−ＧｌｃＮＡｃを
糖鎖受容体とした時の反応収率を１００として、これに
対する相対値（％）で表示した。Using the para-nitrophenyl (PNP) derivative at position 1 of the sugar as a sugar chain acceptor, the results of examining the specificity of sugar recognition in the complex sugar chain transfer reaction by the enzyme endo-M are shown in Table 1. And its structure is shown in FIG. The structure of the sugar residue serving as an acceptor of these complex type sugar chains is the equatorial hexopyranose or pentopyranose in which the configuration of the 4-hydroxyl group is the same as that of β-GlcNAc, and the complex type sugar chain is a sugar having this structure. Transfer. Table 1. Sugar chain transfer reaction yield to various sugar-PNP derivatives ──────────────────────────────────── No . Sugar chain receptor Relative transfer reaction yield (%) ──────────────────────────────────── 1 PNP -Β-D-GlcNAc 100 2 PNP-α-D-GlcNAc 86 3 PNP-β-D-chitobiose 354 PNP-β-D-glucose 112 5 PNP-α-D-glucose 606 PNP-1-S- β-D-glucose 937 PNP-β-D-mannose 968 PNP-β-D-xylopyranose 15 ─────────────────────────糖 The sugar chain transfer reaction yield is defined as a relative value (%) with respect to 100 as the reaction yield when PNP-β-D-GlcNAc is used as the sugar chain receptor. displayed.
【化１】 （式中、ＰＮＰはパラ−ニトロフェニル基を示し、化合
物１〜８は表１に示した糖鎖受容体に対応する）。Embedded image (Wherein PNP represents a para-nitrophenyl group, and compounds 1 to 8 correspond to the sugar chain receptors shown in Table 1).
【００１９】糖鎖受容体となるのはβ−ＧｌｃＮＡｃ以
外の糖残基を有する糖質あるいは複合糖質であり、糖残
基は４位水酸基の立体配置がエクアトリアルな構造をと
るヘキソピラノースあるいはペントピラノース誘導体で
ある。また、糖の１位水酸基はβでなくα−アノマーで
もよい。例えば、Ｄ−グルコース（Ｇｌｃ）残基を有す
る糖ペプチド誘導体等が用いられる。これらは化学合成
して用いられる。The sugar chain receptor is a carbohydrate or complex carbohydrate having a sugar residue other than β-GlcNAc, and the sugar residue is hexopyranose or pentopyranose having an equatorial structure of the 4-position hydroxyl group. It is a derivative. In addition, the 1-position hydroxyl group of the sugar may be an α-anomer instead of β. For example, a glycopeptide derivative having a D-glucose (Glc) residue is used. These are used after being chemically synthesized.
【００２０】例えば、糖−Ａｓｎ残基を有するペプチド
（糖−Ａｓｎ−ペプチド）およびその誘導体は、例えば
Ｔ．イナヅ（T. Inazu）ら［シンレット、第８６９〜８
７０頁（１９９３）］に述べた方法に準じて合成された
糖−Ａｓｎ誘導体を用い、同じくＴ．イナヅ（T. Inaz
u）ら［ペプチド ケミストリー １９９３（PeptideCh
emistry １９９３）、第１０１〜１０４頁（１９９
４）］の方法により固相合成法を用いて合成される。誘
導体はＮ末端アミノ酸のαアミノ基が無保護あるいは９
−フルオレニルメチルオキシカルボニル（Ｆｍｏｃ）誘
導体［ＧｌｃＮＡｃ−Ａｓｎ−（ペプチド）−Ｆｍｏ
ｃ］あるいは第３ブチルオキシカルボニル（ＢＯＣ）誘
導体［ＧｌｃＮＡｃ−Ａｓｎ−（ペプチド）−ＢＯＣ］
等である。保護基を付けたまま（この時は転移反応後に
常法により保護基を外す）、あるいは遊離型で糖鎖受容
体として反応に供する。For example, peptides having a sugar-Asn residue (sugar-Asn-peptide) and derivatives thereof are described in T. Inazu et al. [Shinletto, 869-8]
70 (1993)], using a sugar-Asn derivative synthesized according to the method described in Ina (T. Inaz
u) et al. [Peptide Chemistry 1993 (PeptideCh
emistry 1993), pp. 101-104 (199)
4)], using a solid phase synthesis method. Derivatives have the unprotected α-amino group of the N-terminal amino acid or 9
-Fluorenylmethyloxycarbonyl (Fmoc) derivative [GlcNAc-Asn- (peptide) -Fmo
c] or tert-butyloxycarbonyl (BOC) derivative [GlcNAc-Asn- (peptide) -BOC]
And so on. The reaction may be carried out with the protecting group attached (in this case, the protecting group is removed by a conventional method after the transfer reaction) or in a free form as a sugar chain receptor.
【００２１】本発明の転移反応において、糖鎖供与体と
してはシアル酸を含有するシアロ複合型糖鎖あるいはシ
アル酸のないアシアロ複合型糖鎖を有する複合糖質が用
いられる。例えば、ヒトトランスフェリンや牛フェツイ
ン等のシアロ複合糖質であり、そのまま用いてもよい
が、プロナーゼ等のタンパク質加水分解酵素によりタン
パク質部分をアスパラギン（Ａｓｎ）残基のみにまで小
さくした複合型糖質がより適している。In the transfer reaction of the present invention, a sialic acid-containing sialo complex-type sugar chain or a sialic acid-free asialo complex-type sugar chain-containing complex saccharide is used as a sugar chain donor. For example, it is a sialoconjugated carbohydrate such as human transferrin or bovine fetuin and may be used as it is. However, a complexed carbohydrate in which the protein portion is reduced to only asparagine (Asn) residues by a protease such as pronase is used. More suitable.
【００２２】このようにして天然の複合型糖鎖が受容体
の糖残基に転移して生じた糖鎖は、天然にはなかった全
く新しい配列の糖鎖となる。例えば、（化２）に示した
ようにＧｌｃ残基を有する糖質に２本鎖シアロ複合型糖
鎖を転移させると、生じた糖鎖は新しい配列の複合型糖
鎖になる。即ち、トランスフェリン由来のシアロ２本鎖
複合型糖鎖は（ＮｅｕＡｃ−Ｇａｌ−ＧｌｃＮＡｃ−Ｍ
ａｎ）2−Ｍａｎ−ＧｌｃＮＡｃ−ＧｌｃＮＡｃ−の構
造であるが、転移生成した糖鎖は（ＮｅｕＡｃ−Ｇａｌ
−ＧｌｃＮＡｃ−Ｍａｎ）2−Ｍａｎ−ＧｌｃＮＡｃ−
Ｇｌｃ−と、エンドグリコシダーゼにより認識され加水
分解されるＧｌｃＮＡｃ−ＧｌｃＮＡｃ部分がＧｌｃＮ
Ａｃ−Ｇｌｃに変わった全く新しい配列の糖鎖である。The sugar chain formed by the transfer of the natural complex-type sugar chain to the sugar residue of the receptor in this way becomes a sugar chain having a completely new sequence that does not exist in nature. For example, when a double-chain sialo complex type sugar chain is transferred to a sugar having a Glc residue as shown in (Chemical formula 2), the resulting sugar chain becomes a complex type sugar chain having a new sequence. That is, transferrin-derived sialo double-stranded complex type sugar chain is (NeuAc-Gal-GlcNAc-M
an) has a structure of 2- Man-GlcNAc-GlcNAc-, and the sugar chain formed by the transfer is (NeuAc-Gal).
-GlcNAc-Man) 2 -Man-GlcNAc-
Glc- and the GlcNAc-GlcNAc portion recognized and hydrolyzed by endoglycosidase are GlcN
It is a sugar chain with a completely new sequence that has been changed to Ac-Glc.
【００２３】例えばヒトトランスフェリン由来シアロ複
合型糖鎖のＤ−グルコース残基を有するＡｓｎ誘導体
（Ｆｍｏｃ−Ａｓｎ−Ｇｌｃ）への転移反応の例（化
２）を示す。ここで、ＮｅｕＡｃはシアル酸を、Ｇａｌ
はＤ−ガラクトースを、ＭａｎはＤ−マンノースを、ま
たＦｍｏｃはアミノ酸（Ａｓｎ）のＮ末端の保護基９−
フルオレニルメチルオキシカルボニル基を示す。点線で
囲った部分は、天然の元の形の糖鎖（上）及び形成され
たＧｌｃＮＡｃ−Ｇｌｃを含む新しい配列の糖鎖（下）
を示す。For example, an example of a transfer reaction of a sialo complex type sugar chain derived from human transferrin to an Asn derivative having a D-glucose residue (Fmoc-Asn-Glc) is shown below. Here, NeuAc replaces sialic acid with Gal
Represents D-galactose, Man represents D-mannose, and Fmoc represents the N-terminal protecting group 9- of amino acid (Asn).
Shows a fluorenylmethyloxycarbonyl group. The portions surrounded by the dotted line are the sugar chains in the natural original form (top) and the sugar chains of the new sequence containing the formed GlcNAc-Glc (bottom).
Is shown.
【化２】 Embedded image
【００２４】用いるエンドグリコシダーゼとしては、エ
ンド−β−Ｎ−アセチルグルコサミニダーゼ（ＥＣ３．
２．１．９６）であり、複合型糖鎖の転移活性を有する
ものが使われる。例えば、エンド−Ｍ等が用いられる。The endoglycosidase used is endo-β-N-acetylglucosaminidase (EC3.
2.1.96), which has a complex type sugar chain transfer activity. For example, End-M or the like is used.
【００２５】本発明の糖鎖転移反応は、基質の糖鎖供与
体である複合型糖質、糖鎖受容体および酵素のエンドグ
リコシダーゼを緩衝溶液中で混合することにより行われ
る。この時、基質である糖鎖供与体と糖鎖受容体の仕込
濃度を共に高濃度にすることにより反応が高収率で進行
する。また酵素の添加量を制限して反応を行うことによ
り高い収率が得られる。The transglycosylation reaction of the present invention is carried out by mixing a complex type carbohydrate as a sugar chain donor as a substrate, a sugar chain acceptor and an enzyme endoglycosidase in a buffer solution. At this time, the reaction proceeds at a high yield by increasing the charged concentrations of the sugar chain donor and the sugar chain acceptor that are substrates. In addition, a high yield can be obtained by performing the reaction while limiting the amount of the enzyme to be added.
【００２６】緩衝液としては、ｐＨ５〜８程度の適当な
緩衝液が用いられる。エンド−Ｍの場合、通常ｐＨ５．
５〜６．５の酢酸あるいはリン酸緩衝液中で反応が行わ
れる。反応温度は通常、室温〜５０℃程度、好ましくは
３０〜４０℃で行われ、反応時間は１〜２４時間程度で
ある。エンド−Ｍ酵素の場合、通常、温度３７℃で３〜
１８時間程度反応が行われる。As the buffer, an appropriate buffer having a pH of about 5 to 8 is used. In the case of Endo-M, usually pH5.
The reaction is performed in a 5-6.5 acetic acid or phosphate buffer. The reaction temperature is usually from room temperature to about 50 ° C, preferably from 30 to 40 ° C, and the reaction time is about 1 to 24 hours. In the case of the endo-M enzyme, the temperature is usually 3 to 37 ° C.
The reaction is performed for about 18 hours.
【００２７】生成した新規な複合型糖質は公知の手段に
従って反応終了液から容易に分離精製することが出来
る。例えば、ゲルろ過カラムクロマトグラフィー、イオ
ン交換樹脂カラムクロマトグラフィー、レクチンカラム
クロマトグラフィー、高速液体クロマトグラフィー（Ｈ
ＰＬＣ）等により反応終了液から反応生成物新規複合型
糖質を分離し、更に濃縮、脱塩、凍結乾燥等を行えばよ
い。The novel complex-type saccharide thus produced can be easily separated and purified from the reaction solution according to known means. For example, gel filtration column chromatography, ion exchange resin column chromatography, lectin column chromatography, high performance liquid chromatography (H
The reaction product novel complex type saccharide may be separated from the reaction completed solution by PLC) or the like, and then concentrated, desalted, freeze-dried, or the like.
【００２８】[0028]
【実施例】以下に実施例をあげて本発明を更に具体的に
説明するが、本発明はこれらに限定されるものではな
い。EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.
【００２９】[0029]
【実施例１】複合型糖鎖のパラ−ニトロフェニル（ＰＮ
Ｐ）グリコシドへの転移反応：糖鎖供与体として、ヒト
トランスフェリン（生化学工業）をプロナーゼ処理とセ
ファデックスＧ−２５ゲルろ過を繰り返して得たＡｓｎ
残基のみを有する２本鎖複合型糖鎖からなるシアロ糖ペ
プチド（以下ＴＦ−ＳＧＰと略する、分子量２３３８）
を調製した。ＴＦ−ＳＧＰをノイラミニダーゼ処理して
シアル酸のないアシアロ糖ペプチド（以下ＴＦ−ＡＳＧ
Ｐと略する）を調製した。糖鎖受容体は各種の糖の１位
のＰＮＰ誘導体（ＰＮＰ−糖）（生化学工業、シグマ
社）を用いた。ＴＦ−ＳＧＰ １μｍｏｌとＰＮＰ−糖
５００ｎｍｏｌを０．１Ｍリン酸緩衝液（ｐＨ６．２
５）２４μｌに溶解し、エンド−Ｍ １６０μＵを含む
酵素溶液１６μｌを加え、３７℃で３時間反応した。加
熱処理により反応を停止後反応液を蒸留水で１ｍｌに希
釈して、反応生成物をＨＰＬＣで分析した。各種ＰＮＰ
−糖へのシアロ複合型糖鎖の転移反応収率を、ＰＮＰ−
β−Ｄ−ＧｌｃＮＡｃを糖鎖受容体とした時の転移反応
収率（１２．２％）を１００（％）として、その相対値
で先の表１に示した。これら糖鎖転移反応生成物をＨＰ
ＬＣにより単離し、質量分析の結果、ＰＮＰ−糖へのシ
アロ複合型糖鎖の転移生成物であることを確認した。Ｔ
Ｆ−ＡＳＧＰを糖鎖供与体として同様に反応した時、Ｐ
ＮＰ−β−ＧｌｃＮＡｃ、ＰＮＰ−β−グルコースへの
相対反応収率は各８１％及び８４％であった。Example 1 Complex-type sugar chain para-nitrophenyl (PN
P) Transfer reaction to glycoside: Asn obtained by repeatedly treating human transferrin (Seikagaku Corporation) with pronase and Sephadex G-25 gel filtration as a sugar chain donor
Sialoglycopeptide comprising a double-stranded complex type sugar chain having only residues (hereinafter abbreviated as TF-SGP, molecular weight 2338)
Was prepared. A sialic acid-free asialoglycopeptide (hereinafter TF-ASG) is obtained by treating TF-SGP with neuraminidase.
P). As the sugar chain receptor, PNP derivatives at the 1-position of various sugars (PNP-sugars) (Seikagaku Corporation, Sigma) were used. 1 μmol of TF-SGP and 500 nmol of PNP-sugar were added to a 0.1 M phosphate buffer (pH 6.2).
5) Dissolved in 24 μl, added 16 μl of an enzyme solution containing 160 μU of Endo-M, and reacted at 37 ° C. for 3 hours. After terminating the reaction by heat treatment, the reaction solution was diluted to 1 ml with distilled water, and the reaction product was analyzed by HPLC. Various PNP
-The transfer reaction yield of the sialo complex type sugar chain to the sugar was determined by PNP-
Assuming that the transfer reaction yield (12.2%) when β-D-GlcNAc was used as the sugar chain receptor was 100 (%), the relative values are shown in Table 1 above. These sugar chain transfer products are converted to HP
It was isolated by LC, and as a result of mass spectrometry, it was confirmed that it was a transfer product of a sialo complex type sugar chain to a PNP-sugar. T
When F-ASGP was similarly reacted as a sugar chain donor,
The relative reaction yields for NP-β-GlcNAc and PNP-β-glucose were 81% and 84%, respectively.
【００３０】[0030]
【実施例２】ヒトトランスフェリン由来シアロ糖鎖のＦ
ｍｏｃ−Ａｓｎ−Ｇｌｃへの糖鎖転移反応： 糖鎖受容
体にはＮ末をＦｍｏｃ（９−フルオレニルメチルオキシ
カルボニル）で保護したＡｓｎにβ−Ｄ−グルコースを
結合させたＦｍｏｃ−Ａｓｎ−Ｇｌｃ（分子量５１７）
を用いた。この合成はＴ・イナヅらの先の文献記載のＦ
ｍｏｃ−Ａｓｎ−ＧｌｃＮＡｃの合成法に準じて行っ
た。ＴＦ−ＳＧＰ １μｍｏｌ（２．２４ｍｇ）とＦｍ
ｏｃ−Ａｓｎ−Ｇｌｃ ５００ｎｍｏｌ（０．２５８ｍ
ｇ）を０．１Ｍリン酸緩衝液（ｐＨ６．２５）２４μｌ
に溶解し、エンド−Ｍ １６０μＵを含む酵素溶液１６
μｌを加え、３７℃で６時間反応した。加熱処理により
反応を停止後反応液を蒸留水で１ｍｌに希釈して、反応
生成物をＨＰＬＣで分析した。転移反応生成物が１５．
８％（対仕込糖鎖受容体、モル比）の収率で得られた。
反応生成物をＨＰＬＣ分取により単離し、質量分析の結
果、ｍ／ｚ［Ｍ−Ｈ］２５１８にシグナルが観測され、
トランスフェリン由来のシアル酸が２個ついた（ジシア
ロ）２本鎖複合型糖鎖がＦｍｏｃ−Ａｓｎ−Ｇｌｃに転
移した化合物（分子量２５１９）であることが確認され
た。これをシアリダーゼで処理するとアシアロ体の化合
物（分子量１９３７）が得られシアル酸の存在が確認さ
れた。Example 2 F of sialo-glycan derived from human transferrin
Sugar chain transfer reaction to moc-Asn-Glc: The sugar chain receptor is Fmoc-Asn- in which β-D-glucose is bound to Asn in which the N-terminal is protected by Fmoc (9-fluorenylmethyloxycarbonyl). Glc (molecular weight: 517)
Was used. This synthesis was carried out by F. Inapura et al.
This was performed according to the method of synthesizing moc-Asn-GlcNAc. 1 μmol (2.24 mg) of TF-SGP and Fm
oc-Asn-Glc 500 nmol (0.258 m
g) in 0.1 μM phosphate buffer (pH 6.25) 24 μl
And an enzyme solution 16 containing 160 μU of Endo-M
μl was added and reacted at 37 ° C. for 6 hours. After terminating the reaction by heat treatment, the reaction solution was diluted to 1 ml with distilled water, and the reaction product was analyzed by HPLC. 15. The transfer reaction product is 15.
It was obtained in a yield of 8% (to the charged sugar chain receptor, molar ratio).
The reaction product was isolated by HPLC fractionation, and as a result of mass spectrometry, a signal was observed at m / z [MH] 2518,
It was confirmed that the compound was a compound (molecular weight: 2519) in which a (dicialo) double-stranded complex type sugar chain having two transferrin-derived sialic acids was transferred to Fmoc-Asn-Glc. When this was treated with sialidase, an asialo compound (molecular weight 1937) was obtained, and the presence of sialic acid was confirmed.
【００３１】[0031]
【実施例３】ヒトトランスフェリン由来シアロ糖鎖のＧ
ｌｃ残基を有するペプチドへの糖鎖転移反応：糖鎖受容
体にはＡｓｎとアラニン（Ａｌａ）からなるペプチドの
Ａｓｎ残基にβ−Ｄ−グルコースを結合させたＦｍｏｃ
−Ａｓｎ（Ｇｌｃ）−Ａｌａ（分子量５８８）を、Ｔ．
イナヅらの文献記載の方法に準じてＡｌａとＦｍｏｃ−
Ａｓｎ（Ｇｌｃ）とから合成した。ＴＦ−ＳＧＰ １μ
ｍｏｌ（２．２４ｍｇ）とＦｍｏｃ−Ａｓｎ（Ｇｌｃ）
−Ａｌａ ５００ｎｍｏｌ（０．２９４ｍｇ）を０．１
Ｍリン酸緩衝液（ｐＨ６．２５）２４μｌに溶解し、エ
ンド−Ｍ １６０μＵを含む酵素溶液１６μｌを加え、
３７℃で６時間反応した。加熱処理により反応を停止後
反応液を蒸留水で１ｍｌに希釈して、反応生成物をＨＰ
ＬＣで分析した。転移反応生成物が１６．３％（対仕込
糖鎖受容体、モル比）の収率で得られた。反応生成物を
ＨＰＬＣ分取により単離し、質量分析の結果、ｍ／ｚ
［Ｍ−Ｈ］２５９０にシグナルが観測され、トランスフ
ェリン由来のシアル酸が２個ついた（ジシアロ）２本鎖
複合型糖鎖がＦｍｏｃ−Ａｓｎ（Ｇｌｃ）−Ａｌａに転
移した化合物（分子量２５９０）であることが確認され
た。Example 3 G of sialo-glycan derived from human transferrin
Glycosyl transfer reaction to peptide having lc residue: Fmoc in which β-D-glucose is bound to Asn residue of peptide consisting of Asn and alanine (Ala) as a sugar chain receptor
-Asn (Glc) -Ala (molecular weight 588) was obtained from T.I.
Ala and Fmoc- according to the method described in the literature of Inapura et al.
It was synthesized from Asn (Glc). TF-SGP 1μ
mol (2.24 mg) and Fmoc-Asn (Glc)
-Ala 500 nmol (0.294 mg) to 0.1
M-phosphate buffer (pH 6.25) was dissolved in 24 μl, and 16 μl of an enzyme solution containing 160 μU of Endo-M was added.
The reaction was performed at 37 ° C. for 6 hours. After terminating the reaction by heat treatment, the reaction solution was diluted to 1 ml with distilled water, and the reaction product was HP
Analyzed by LC. The transfer reaction product was obtained in a yield of 16.3% (to the charged sugar chain acceptor, molar ratio). The reaction product was isolated by HPLC fractionation, and as a result of mass spectrometry, m / z
[MH] 2590, a signal was observed, and a compound (molecular weight 2590) in which a (dicialo) double-stranded complex type sugar chain with two sialic acids derived from transferrin was transferred to Fmoc-Asn (Glc) -Ala. It was confirmed that there was.
【００３２】[0032]
【発明の効果】本発明により、複合型糖鎖をβ−Ｇｌｃ
ＮＡｃ以外の糖残基を有する他の糖質、複合糖質あるい
はペプチド等（糖鎖受容体）に転移させて新規な配列の
複合型糖鎖を有する複合糖質を容易に合成することが可
能となった。例えば、糖鎖受容体としてβ−ＧｌｃＮＡ
ｃ以外の糖残基を持つ生理活性ペプチドを合成して糖鎖
を転移付加させることにより、天然には無かった新規な
糖鎖配列の複合糖ペプチドを合成出来る。このような糖
鎖は生体内の糖鎖分解酵素に対して安定化することが期
待され、本発明は、医薬への応用に有効である。また、
天然にはない複合型糖鎖の調製法としても有用である。According to the present invention, the complex type sugar chain is converted to β-Glc
It is possible to easily synthesize a complex carbohydrate having a complex type sugar chain having a novel sequence by transferring to another carbohydrate, glycoconjugate or peptide (sugar chain receptor) having a sugar residue other than NAc. It became. For example, β-GlcNA as a sugar chain receptor
By synthesizing a physiologically active peptide having a sugar residue other than c and transferring and adding a sugar chain, it is possible to synthesize a complex glycopeptide having a novel sugar chain sequence that has not existed in nature. Such sugar chains are expected to stabilize in vivo sugar chain-degrading enzymes, and the present invention is effective for application to pharmaceuticals. Also,
It is also useful as a method for preparing a complex type sugar chain that is not found in nature.
【００３３】[0033]
【化１】酵素エンドグリコシダーゼによる糖鎖転移の受
容体となる４位水酸基がエクアトリアルな立体配置をと
るヘキソピラノースあるいはペントピラノースの１位パ
ラ−ニトロフェニル（ＰＮＰ）誘導体の構造を示す。１
はＰＮＰ−β−Ｄ−ＧｌｃＮＡｃ、２はＰＮＰ−α−Ｄ
−ＧｌｃＮＡｃ、３はＰＮＰ−β−Ｄ−キトビオース、
４はＰＮＰ−β−Ｄ−グルコース、５はＰＮＰ−α−Ｄ
−グルコース、６はＰＮＰ−１ーＳ−β−Ｄ−グルコー
ス、７はＰＮＰ−β−Ｄ−マンノースまた８はＰＮＰ−
β−Ｄ−キシロピラノースを示す。## STR1 ## The structure of a 1-position para-nitrophenyl (PNP) derivative of hexopyranose or pentopyranose, in which the 4-position hydroxyl group serving as an acceptor of sugar chain transfer by the enzyme endoglycosidase has an equatorial configuration, is shown. 1
Is PNP-β-D-GlcNAc, 2 is PNP-α-D
-GlcNAc, 3 is PNP-β-D-chitobiose,
4 is PNP-β-D-glucose, 5 is PNP-α-D
Glucose, 6 is PNP-1-S-β-D-glucose, 7 is PNP-β-D-mannose and 8 is PNP-
1 shows β-D-xylopyranose.
【化２】Ｎ末をＦｍｏｃで保護したアスパラギン（Ａｓ
ｎ）残基に結合したＤ−グルコース（Ｇｌｃ）を有する
糖質（Ｆｍｏｃ−Ａｓｎ−Ｇｌｃ）への酵素エンド−Ｍ
によるシアロ複合型糖鎖の転移反応の反応式を示す。点
線で囲った部分が元の天然複合型糖鎖（上）および新た
に形成された非天然の複合型糖鎖（下）である。## STR2 ## Asparagine (As) with N-terminal protected by Fmoc
n) Enzyme endo-M to carbohydrate (Fmoc-Asn-Glc) with D-glucose (Glc) attached to the residue
1 shows a reaction formula of a transfer reaction of a sialo-complex type sugar chain by the following method. The portions surrounded by dotted lines are the original natural complex type sugar chain (top) and the newly formed non-natural complex type sugar chain (bottom).
【手続補正書】[Procedure amendment]
【提出日】平成８年９月１２日[Submission date] September 12, 1996
【手続補正１】[Procedure amendment 1]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】図面の簡単な説明[Correction target item name] Brief description of drawings
【補正方法】削除[Correction method] Deleted
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 // Ａ６１Ｋ 31/715 Ａ６１Ｋ 37/20 ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 6 Identification code Agency reference number FI Technical indication // A61K 31/715 A61K 37/20
Claims (5)
アセチル−Ｄ−グルコサミン（β−ＧｌｃＮＡｃ）以外
の糖残基を有する糖質あるいは複合糖質（糖鎖受容体）
に複合型糖鎖を転移させることにより複合糖質を製造す
る方法。(1) β-N- in the presence of endoglycosidase
Carbohydrate or glycoconjugate having a sugar residue other than acetyl-D-glucosamine (β-GlcNAc) (sugar chain receptor)
A method for producing a complex saccharide by transferring a complex type sugar chain to a saccharide.
酸基がエクアトリアルな立体配置をとるヘキソピラノー
スあるいはペントピラノースである請求項１に記載の方
法。2. The method according to claim 1, wherein the sugar residue of the sugar chain receptor according to claim 1 is hexopyranose or pentopyranose in which the 4-position hydroxyl group has an equatorial configuration.
アセチル−Ｄ−グルコサミン（α−ＧｌｃＮＡｃ）、Ｄ
−グルコース（Ｇｌｃ）、Ｄ−マンノース（Ｍａｎ）あ
るいはＤ−キシロピラノース（Ｘｙｌ）である請求項１
に記載の方法。3. The sugar chain of the sugar chain receptor according to claim 2, wherein the sugar residue is α-N-
Acetyl-D-glucosamine (α-GlcNAc), D
2. Glucose (Glc), D-mannose (Man) or D-xylopyranose (Xyl).
The method described in.
アセチルグルコサミニダーゼ（ＥＣ３．２．１．９６）
である請求項１に記載の方法。4. The method according to claim 1, wherein the endoglycosidase is endo-β-N-.
Acetylglucosaminidase (EC 3.2.1.96)
The method of claim 1, wherein
ｃＮＡｃ以外の糖残基を有する糖質あるいは複合糖質
（糖鎖受容体）に複合型糖鎖を転移させることにより製
造される新規複合糖質。5. The method according to claim 5, wherein β-Gl is present in the presence of endoglycosidase.
A novel complex saccharide produced by transferring a complex type sugar chain to a sugar having a sugar residue other than cNAc or a complex sugar (sugar chain receptor).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22310596A JP3811527B2 (en) | 1996-08-05 | 1996-08-05 | Process for producing new glycoconjugates |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22310596A JP3811527B2 (en) | 1996-08-05 | 1996-08-05 | Process for producing new glycoconjugates |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH1045788A true JPH1045788A (en) | 1998-02-17 |
| JP3811527B2 JP3811527B2 (en) | 2006-08-23 |
Family
ID=16792912
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP22310596A Expired - Fee Related JP3811527B2 (en) | 1996-08-05 | 1996-08-05 | Process for producing new glycoconjugates |
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| Country | Link |
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| JP (1) | JP3811527B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106905472A (en) * | 2017-04-18 | 2017-06-30 | 北京蛋白质组研究中心 | A kind of functionalization responsive to temperature type polymer and preparation method and application |
-
1996
- 1996-08-05 JP JP22310596A patent/JP3811527B2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106905472A (en) * | 2017-04-18 | 2017-06-30 | 北京蛋白质组研究中心 | A kind of functionalization responsive to temperature type polymer and preparation method and application |
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| Publication number | Publication date |
|---|---|
| JP3811527B2 (en) | 2006-08-23 |
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