JP3776952B2 - Production process of complex carbohydrates - Google Patents

Description

（式中、Ｒ¹はＨ、アミノ保護基、アミノ酸、ペプチドあるいはＮ末端アミノ酸のαアミノ基を保護したペプチドを示す。Ｒ²はＯＨ、カルボキシル保護基、アミノ酸、ペプチドあるいはＣ末端アミノ酸のカルボキシル基を保護したペプチドを示す。ｎは２である。）で示される化合物である。
【００１６】
（化１）に示すＧｌｃＮＡｃ残基を有する合成基質の合成は如何なる方法によってもよいが、例えばＴ．イナヅ（T. Inazu）ら［ペプチド ケミストリー １９９３（Peptide Chemistry 1993）、第１０１-１０４頁（１９９４）］の報告した方法に準じて合成される。
【００１７】
ＧｌｃＮＡｃがアミド基に結合したグルタミン（ＧｌｃＮＡｃ-Ｇｌｎ）をＧｌｎに代えて用いることによりＧｌｃＮＡｃがＧｌｎに結合したペプチド（ＧｌｃＮＡｃ-Ｇｌｎ-ペプチド）が合成される。（化１）のｎが２の化合物がこれに当たる。
【００１８】
（化１）のＲ¹にある末端アミノ酸のαアミノ基の保護基としては、例えば、９-フルオレニルメチルオキシカルボニル（Ｆｍｏｃ）基、第３ブチルオキシカルボニル（Ｂｏｃ）基、３-ニトロ-２-ピリジンスルフェニル（Ｎｐｙｓ）基、ベンジルオキシカルボニル（Ｚ）基あるいはダンシル（ＤＮＳ）基等が用いられる。
【００１９】
Ｎ末端アミノ酸の保護基は糖鎖転移反応後に常法により外すか、あるいは予め保護基を外した後に糖鎖転移反応に供してもよい。
【００２０】
（化１）のＲ²にあるカルボキシル基の保護基としては、第３ブチル（Ｂｕ^t）基、ベンジル（Ｂｚｌ）基あるいはメチル（Ｍｅ）基等であるが、水への溶解性を上げるために保護基を外し、遊離型で用いることが多い。
【００２１】
本発明の第２の構成は、糖鎖受容体である合成基質への糖鎖供与体からの糖鎖の転移反応による付加である。
【００２２】
エンドグリコシダーゼの存在下、下記式（式１）：
Ｘ-ＧｌｃＮＡｃ-Ｙ ＋ Ｚ → Ｘ-ＧｌｃＮＡｃ-Ｚ ＋ Ｙ （式１）
［式中、Ｘは複合糖鎖、Ｙは糖質あるいは複合糖質、Ｚは（化１）に示した合成基質］
で表される転移反応を行うことを特徴とする。
【００２３】
本発明に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、例えば、エンド-Ａやエンド-Ｍ等が用いられる。該酵素は下記式（式２）：
Ｒ-ＧｌｃＮＡｃ-ＧｌｃＮＡｃ-Ａｓｎ-（ペプチドまたはタンパク質）（式２）
（式中Ｒ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部分（ＧｌｃＮＡｃ-ＧｌｃＮＡｃ）の間を加水分解するが、この時に適当な糖鎖受容体があると、受容体に糖鎖（Ｒ-ＧｌｃＮＡｃ）部分が転移する。（式１）の反応はそれを利用したものである。
【００２４】
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となるのは、通常、Ａｓｎ-Ｘ-Ｓｅｒ（Ｔｈｒ）配列を含むペプチドのＡｓｎに結合したＧｌｃＮＡｃ残基である。
【００２５】
驚くべきことに、天然には存在しないグルタミン（Ｇｌｎ）に結合したＧｌｃＮＡｃ残基を含むペプチド（ＧｌｃＮＡｃ-Ｇｌｎ-ペプチド）のＧｌｃＮＡｃ残基にもＡｓｎの場合と同様に糖鎖の転移反応が起きることが分かった。
【００２６】
（化１）に示すＧｌｃＮＡｃ残基を有する合成基質はかかる知見に基づき調製されたものであり、この合成基質への酵素による糖鎖の転移反応を行う本発明を完成させた。
【００２７】
酵素の糖鎖供与体の基質特異性については、エンド-Ａは高マンノース型糖鎖のみに作用するが、エンド-Ｍは高マンノース型のみならず複合型糖鎖や混成型糖鎖にも作用する。
【００２８】
これらの酵素の本来の機能は加水分解であり、（式１）のＺの代わりに水が入り加水分解反応が転移反応とともに副反応として進行する。また、（式１）で生成した転移反応生成物（Ｘ-ＧｌｃＮＡｃ-Ｚ）は加水分解反応の基質となり再分解を受ける。
【００２９】
糖鎖転移反応を効率よく行わせるには加水分解反応を抑えて（式１）の反応を優先的に行わせることが必要である。与酵素量を減らし、基質である糖鎖供与体（Ｘ-ＧｌｃＮＡｃ-Ｙ）と糖鎖受容体（Ｚ）の仕込濃度を、そのモル比を１近くにしつつ高濃度にすることにより反応の場（酵素の活性中心）における水の影響を排して転移反応を効率よく進行させることができる。
【００３０】
反応系で重要な点は、反応を酵素律速条件下で行うことである。即ち糖鎖供与体から受容体への糖鎖転移反応の速度が与酵素量に依存し、その速度を最大にするに必要な最少量の酵素量になるように酵素の添加量を制限して反応する。例えばエンド-Ｍの場合、糖鎖供与体に対して５００ユニット（Ｕ）／モル（供与体）以下、望ましくは８０-４００Ｕ／モル（供与体）程度の酵素量を加える。なおここで、酵素の１ユニット（Ｕ）は、ヒトトランスフェリン由来のアシアロ複合型糖鎖のダンシル化（ＤＮＳ）誘導体を加水分解して、３７℃、１分間に１マイクロモル（μｍｏｌ）のＮ-アセチルグルコサミニル-アスパラギンのＤＮＳ誘導体（ＧｌｃＮＡｃ-Ａｓｎ-ＤＮＳ）を生成させるに必要な酵素量である。
【００３１】
糖鎖供与体と受容体の仕込濃度を著しく高めることも重要である。与酵素量を制限しつつ両基質を高濃度に仕込むことにより、糖鎖転移反応が促進され副反応が抑えられて反応収率は飛躍的に向上する。その濃度は、糖鎖供与体が１０ｍＭ以上、望ましくは１５-７５ｍＭがよい。また糖鎖受容体は２．５ｍＭ以上、望ましくは７．５-３５ｍＭ程度がよい。糖鎖受容体の濃度は高い方が望ましいが、合成基質の溶解度が低い場合には２．５ｍＭ程度の濃度でも用いられる。
【００３２】
本発明に用いる糖鎖供与体としては、高マンノース型、複合型、混成型いずれの糖鎖も用いられる。高マンノース型糖鎖は例えば卵白アルブミン等から、またシアル酸を含有する複合型糖鎖は例えばヒトトランスフェリンや牛フェツイン等から調製され、シアリダーゼ処理等によりシアル酸を外せばアシアロ複合型糖鎖が調製される。酵素的あるいは化学的に修飾された糖鎖、あるいは化学合成された糖鎖も用いることができる。
【００３３】
本発明の反応は、基質の糖鎖供与体、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。先述のごとく、糖鎖供与体の濃度を１０ｍＭ以上、望ましくは１５-７５ｍＭ、糖鎖受容体の濃度を２．５ｍＭ以上、望ましくは７．５-３５ｍＭになるように加える。酵素量は５００Ｕ／モル（供与体）以下、望ましくは８０-４００Ｕ／モル（供与体）程度に制限し、例えば、エンド-Ｍの場合、２-１０ｍＵ／ｍｌ程度の量で用いる。緩衝液としては、ｐＨ５-８程度、濃度２５-２００ｍＭ、望ましくは５０-１００ｍＭ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、濃度５０-１００ｍＭの酢酸あるいはリン酸緩衝液中で反応が行われる。基本的な反応液組成の一例は、糖鎖供与体２５ｍＭ、糖鎖受容体１０ｍＭ、エンド-Ｍ４ｍＵ／ｍｌおよび６０ｍＭリン酸緩衝液（ｐＨ６．２５）である。
【００３４】
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時間である。例えば、エンド-Ｍ酵素の場合、通常、３７℃で３-１８時間程度反応が行われる。
【００３５】
生成した複合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（ＨＰＬＣ）等により反応終了液から反応生成物の複合糖質を分離し、更に濃縮、脱塩、凍結乾燥等を行えばよい。
【００３６】
【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。
【実施例1】
高マンノース型糖鎖のＧｌｃＮＡｃ-Ｇｌｎ-Ｆｍｏｃへの糖鎖転移反応：
糖鎖受容体のＧｌｃＮＡｃ-Ｇｌｎ-Ｆｍｏｃ（分子量５７２）はＡｓｎの代わりにＧｌｎ（グルタミン）が入った化合物としてＧｌｃＮＡｃ-Ａｓｎ-Ｆｍｏｃの合成法に準じて合成した。酵素反応にはナトリウム塩にして用いた。糖鎖供与体として、卵白アルブミンをプロナーゼ処理、セファデックスＧ - ２５ゲルろ過更にＤｏｗｅｘ５０イオン交換クロマトにより分離精製して得た高マンノース型糖鎖（Ｍａｎ）₆-（ＧｌｃＮＡｃ）₂-Ａｓｎ（分子量１６５１）を１μｍｏｌ（終濃度２５ｍＭ）、糖鎖受容体としてＧｌｃＮＡｃ-Ｇｌｎ-Ｆｍｏｃ（分子量５７２）を２００ｎｍｏｌ（同５ｍＭ）、エンド-Ｍを１６０μＵ（同４ｍＵ／ｍｌ）加え、６０ｍＭリン酸緩衝液（ｐＨ６．２５）４０μｌ中で３７℃、３時間反応させた。反応停止後、反応液を蒸留水で１ｍｌに希釈し反応生成物をＨＰＬＣで分析した。転移反応生成物が７．５％の収率で得られた。転移反応生成物をＨＰＬＣ分取し、質量分析したところ、分子量１７４８に相当するイオンピーク（ｍ／ｚ）が認められ、（Ｍａｎ）₆-（ＧｌｃＮＡｃ）₂-ＡｓｎからＧｌｃＮＡｃ-Ｇｌｎ-Ｆｍｏｃへの転移反応生成物、即ち（Ｍａｎ）₆-（ＧｌｃＮＡｃ）₂-Ｇｌｎ-Ｆｍｏｃであることが確認された。
【００３７】
【実施例２】
ヒトトランスフェリン由来複合型糖鎖のＧｌｃＮＡｃ-Ｇｌｎ-Ｆｍｏｃ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、セファデックスＧ - ２５ゲルろ過を繰り返して得たＡｓｎ残基のみを有するシアロ糖ペプチド（ＴＦ - ＳＧＰ、分子量２３３８）、さらにシアリダーゼ処理してシアル酸を外したアシアロ糖ペプチド（ＴＦ - ＡＳＧＰ、分子量１７５６）を１μｍｏｌ（終濃度２５ｍＭ）とＧｌｃＮＡｃ-Ｇｌｎ-Ｆｍｏｃを２００ｎｍｏｌ（同５ｍＭ）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１６μｌを加え、３７℃で３時間反応した。反応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ＴＦ-ＳＧＰの場合５．０％、ＴＦ-ＡＳＧＰの場合１３．７％の収率で得られた。転移反応生成物をＨＰＬＣ分取により単離し、質量分析の結果、ＴＦ-ＳＧＰの転移反応生成物には分子量２５７４に相当するイオンピークが、またＴＦ-ＡＳＧＰの転移反応生成物には分子量１９９２に相当するイオンピーク（ｍ／ｚ［Ｍ-Ｈ］ １９９２）が観測され、各々ジシアロ２本鎖複合型糖鎖がＧｌｃＮＡｃ-Ｇｌｎ-Ｆｍｏｃに転移した化合物およびアシアロ２本鎖複合型糖鎖がＧｌｃＮＡｃ-Ｇｌｎ-Ｆｍｏｃに転移した化合物であることが確認された。
【００３８】
【実施例３】
ヒトトランスフェリン由来アシアロ糖鎖のＧｌｃＮＡｃ-Ｇｌｎ-ＤＮＳへの糖鎖転移反応：
糖鎖受容体としてＧｌｃＮＡｃ-Ｇｌｎのダンシル化（ＤＮＳ）誘導体（ＧｌｃＮＡｃ-Ｇｌｎ-ＤＮＳ）（分子量５８５）はＧｌｎのαアミノ基をＤＮＳ基で保護した化合物として合成した。糖鎖供与体としてアシアロ糖ペプチド（ＴＦ-ＡＳＧＰ）を用い、糖鎖供与体１μｍｏｌ（終濃度２５ｍＭ）と糖鎖受容体５００ｎｍｏｌ（同１２．５ｍＭ）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１６μｌを加え、３７℃で３時間反応させた。反応生成物をＨＰＬＣ分取し、質量分析したところ、分子量２００４に相当するイオンピーク（ｍ／ｚ）観察され、アシアロ２本鎖複合型糖鎖がＧｌｃＮＡｃ-Ｇｌｎ-ＤＮＳに転移した化合物であることが確認された。
【００３９】
【発明の効果】
本発明により、ＧｌｃＮＡｃ残基を有する合成基質に酵素的に糖鎖を付加して新規複合糖ペプチドを容易に合成することが可能となった。糖鎖受容体のペプチドはペプチド鎖のアミノ酸配列の中にグルタミン（Ｇｌｎ）があればＧｌｃＮＡｃ残基を介してＮ結合型糖鎖を付加することができ、天然には無い全く新しい複合糖ペプチドを合成できる。付加する糖鎖は高マンノース型、シアル酸を含む複合型糖鎖いずれでもよく、望み通りの複合糖ペプチドを合成できる。
本発明は、医薬への応用とともに複合糖質の糖鎖の果たしている生理的役割を解明するための研究手法を提供する。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a physiologically active complex glycopeptide that combines a peptide synthesis reaction and an enzymatic glycosylation reaction. The present invention is applied to the pharmaceutical field.
[0002]
[Prior art]
Saccharides and complex carbohydrates exist in living cells and body fluids and are deeply involved in cell substrate recognition and cell-cell recognition. Carbohydrates are related to the rate of metabolism such as absorption and decomposition of in vivo substances. Proteins having a sugar chain are known, for example, erythropoietin and tissue plasminogen activator, and these glycoproteins made by genetic engineering using animal cells are used as pharmaceuticals. Also, peptide hormones having sugar chains are known, such as human chorionic gonadotropin (hCG). In these glycoproteins or glycopeptides, the sugar chain is an N-linked sugar chain and is linked to Asn of the peptide chain via GlcNAc.
[0003]
It is expected to be useful for strengthening physiological functions or modifying physiological activities by attaching sugar chains to proteins or physiologically active peptides, or replacing existing sugar chains with other sugar chains. As a method for enzymatically modifying a sugar chain, 1) a method using a transferase or exoglycosidase, and 2) a method using endoglycosidase can be considered.
[0004]
As a method of 1), for example, D.I. H. There is a report of DH Joziasse et al [Eur. J. Biochem., Vol. 191, pp. 75-83 (1990)]. It is a sequential reaction. In addition, recently, M.M. M. Schuster et al. [J. Amer. Chem. Soc., 116, pp. 1135-1136 (1994)] is a glycan anchor that combines several glycosyltransferases. The phase synthesis method is reported.
[0005]
On the other hand, as the transglycosylation reaction using endoglycosidase of 2), R.I. B. Endoscope from Flavobacterium meningosepticum of RB Trimble et al. [J. Biol. Chem., 261, 12000-12005 (1986)]. for β-N-acetylglucosaminidase (endo-F), R.I. M.M. Endo-α-N-acetyl from Diprococcuspneumoniae in RM Bardales et al. [J. Biol. Chem., 264, 19893-19897 (1989)]. There is a report on galactosaminidase. The former reports that glycerol is a receptor, and the latter is glycerol, p-nitrophenol, serine, threonine and the like. Thereafter, Takekawa et al. [Japanese Patent Laid-Open No. 5-64594 (1993)] and K.K. Takegawa et al. [J. Biol. Chem., 270, 3094-3099 (1995)] is an endo-derived from Arthrobacter protophormiae. The transglycosylation reaction to carbohydrate by β-N-acetylglucosaminidase (endo-A) is also described in K. K. Yamamoto et al. [Biochem. Biophys. Res. Commun., Vol. 203, pp. 244-252 (1994)] is an endo-M derived from Mucor hiemalis. The transglycosylation reaction to carbohydrate was reported.
[0006]
[Problems to be solved by the invention]
The complex carbohydrate is composed of a sugar chain part and a protein, peptide, ceramide part or the like to which sugar chains are added. Natural glycoconjugates with the in vivo stability and biological activity of glycoconjugates by so-called sugar chain modification (remodeling), in which carbohydrates are newly added to sugar chains or replaced with other sugar chains It is useful when applied to pharmaceuticals if it is enhanced or added with a biological function that is not found in nature. 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 so far, but they provide important means for analyzing their roles. .
[0007]
As a method for newly adding or modifying a sugar chain to a carbohydrate or complex carbohydrate, an enzyme method in which sugar residues are sequentially added one by one using exoglycosidase or glycosyltransferase can be considered. In addition, endoglycosidase methods such as endo-A and endo-M provide a more efficient method of transferring a complex sugar chain to a carbohydrate or complex carbohydrate as a block.
[0008]
The sugar chain of a natural glycoprotein or glycopeptide is usually an N-linked sugar chain and has an amino acid sequence of Asn-X-Ser (Thr) [X is any amino acid, Ser (Thr) is serine or threonine]. It binds via GlcNAc bound to the amide group of Asn of the peptide chain. That is, a sugar chain having a GlcNAc residue at the end is added to Asn of this amino acid sequence and further modified to biosynthesize an N-linked complex sugar chain. Therefore, N-linked sugar chains other than the sugar chain bonded to Asn of this amino acid sequence have not been found in nature.
[0009]
The addition or modification of a sugar chain by the above-mentioned enzyme requires that a protein or peptide that is a sugar chain receptor has a GlcNAc residue, and the addition or modification of a sugar chain has conventionally been a natural glycoprotein or glycopeptide. The sugar chain was cut out with endoglycosidase or exoglycosidase leaving a GlcNAc residue, and then the sugar chain prepared from another glycoprotein was replaced.
[0010]
Many proteins and peptides have an Asn-X-Ser (Thr) sequence but do not have a sugar chain, such as calcitonin. Even if Asn is present, sugar chains cannot be added at the biosynthesis stage without this sequence. Of course, it is impossible to add a sugar chain to glutamine (Gln) biosynthetically even by genetic engineering techniques.
[0011]
If a glycopeptide in which GlcNAc is bound to an Asn residue (GlcNAc-Asn-peptide) can be chemically synthesized, a new complex glycopeptide in which a sugar chain is added to the GlcNAc residue bound to Asn can be synthesized by the enzymatic method described above. In this case, the Asn-X-Ser (Thr) sequence is not necessarily required on the peptide side, and only Asn is sufficient. In addition, if a glycopeptide in which a GlcNAc residue is added to glutamine (Gln), which is an amino acid related to Asn, is synthesized, synthesis of the same complex glycopeptide can be expected.
[0012]
Peptides are synthesized industrially by the solid phase synthesis method up to several tens of residues.
[0013]
The present invention, glutamine bound sugar (Gln) chemically synthesizing peptides i.e. synthetic substrate comprising at least one, new complex sugar if ask a sugar or a sugar chain to the sugar residues of the foothold enzymatically transferred It was developed based on the idea that peptides can be synthesized. End - beta - N - acetylglucosaminidase is based on the inventors' discovery of being able also to transfer additional sugar chains to a GlcNAc attached to one long Gln in the side chain methylene chain than Asn.
[0014]
[Means for Solving the Problems]
The present invention can be summarized as follows: 1) synthesis of a synthetic substrate having a GlcNAc residue to be a sugar chain receptor, and 2) enzymatic transfer of a sugar chain to a synthetic substrate having a GlcNAc residue. Consists of configuration.
[0015]
The synthetic substrate having a GlcNAc residue referred to in the present invention is the following formula (Formula 1)
[Chemical 2]

(In the formula, R ¹ represents H, an amino protecting group, an amino acid, a peptide or a peptide in which the α-amino group of the N-terminal amino acid is protected. R ² represents OH, a carboxyl protecting group, an amino acid, a peptide, or a carboxyl group of the C-terminal amino acid. Is a compound in which n is 2 .
[0016]
Synthesis of the synthetic substrate having the GlcNAc residue shown in (Chemical Formula 1) may be carried out by any method. Synthesized according to the method reported by T. Inazu et al. [Peptide Chemistry 1993, pp. 101-104 (1994)].
[0017]
By using glutamine (GlcNAc-Gln) in which GlcNAc is bound to an amide group instead of Gln, a peptide in which GlcNAc is bound to Gln (GlcNAc-Gln-peptide) is synthesized. This is the compound in which n is 2 in (Chemical Formula 1).
[0018]
Examples of the protecting group for the α-amino group of the terminal amino acid at R ¹ in (Chemical Formula 1) include 9-fluorenylmethyloxycarbonyl (Fmoc) group, tertiary butyloxycarbonyl (Boc) group, 3-nitro- A 2-pyridinesulfenyl (Npys) group, a benzyloxycarbonyl (Z) group, a dansyl (DNS) group, or the like is used.
[0019]
The protecting group of the N-terminal amino acid may be removed by a conventional method after the transglycosylation reaction, or may be subjected to the transglycosylation reaction after removing the protecting group in advance.
[0020]
The protecting group of the carboxyl group in R ² of the formula 1, tert-butyl (Bu ^t) group, benzyl (Bzl) is a group or a methyl (Me) group and the like, to increase the solubility in water In many cases, the protecting group is removed and used in a free form.
[0021]
The second configuration of the present invention is addition by a sugar chain transfer reaction from a sugar chain donor to a synthetic substrate which is a sugar chain acceptor.
[0022]
In the presence of endoglycosidase, the following formula (Formula 1):
X-GlcNAc-Y + Z → X-GlcNAc-Z + Y (Formula 1)
[Wherein X is a complex sugar chain, Y is a carbohydrate or complex carbohydrate, and Z is a synthetic substrate shown in (Chemical Formula 1)].
It carries out the transfer reaction represented by these.
[0023]
The endoglycosidase used in the present invention is endo-β-N-acetylglucosaminidase (EC 3.2.1.96), and examples thereof include endo-A and endo-M. The enzyme has the following formula (Formula 2):
R-GlcNAc-GlcNAc-Asn- (peptide or protein) (Formula 2)
(Wherein R represents a complex sugar chain)
Hydrolyzes between the chitobiose part (GlcNAc-GlcNAc) of the asparagine (Asn) -linked sugar chain of the asparagine. If there is an appropriate sugar chain receptor at this time, the sugar chain (R-GlcNAc) part is transferred to the receptor. To do. The reaction of (Formula 1) utilizes it.
[0024]
The sugar chain acceptor of the sugar chain transfer reaction by endoglycosidase is usually a GlcNAc residue bound to Asn of a peptide containing an Asn-X-Ser (Thr) sequence.
[0025]
Surprisingly, GlcNAc residues of peptides containing GlcNAc residues bound to non-naturally occurring glutamine (Gln) (GlcNAc-Gln-peptide) undergo a sugar chain transfer reaction as in the case of Asn. I understood.
[0026]
A synthetic substrate having a GlcNAc residue shown in (Chemical Formula 1) was prepared based on such findings, and the present invention for carrying out a sugar chain transfer reaction by an enzyme to this synthetic substrate was completed.
[0027]
As for the substrate specificity of the enzyme sugar chain donor, End-A acts only on the high mannose type sugar chain, while End-M acts on not only the high mannose type but also the complex type sugar chain and the hybrid sugar chain. To do.
[0028]
The original function of these enzymes is hydrolysis, and water enters instead of Z in (Formula 1) and the hydrolysis reaction proceeds as a side reaction along with the transfer reaction. Further, the transfer reaction product (X-GlcNAc-Z) generated in (Formula 1) becomes a substrate for the hydrolysis reaction and undergoes re-decomposition.
[0029]
In order to perform the transglycosylation reaction efficiently, it is necessary to suppress the hydrolysis reaction and to preferentially perform the reaction of (Formula 1). The amount of the enzyme is reduced, and the reaction concentration of the sugar chain donor (X-GlcNAc-Y) and sugar chain acceptor (Z), which are substrates, is set to a high concentration while maintaining the molar ratio close to 1. The transfer reaction can be efficiently advanced by eliminating the influence of water on the (active center of the enzyme).
[0030]
The important point in the reaction system is that the reaction is performed under enzyme-controlled conditions. In other words, the rate of the glycan transfer reaction from the glycan donor to the acceptor depends on the amount of the enzyme, and the amount of the enzyme added is limited so that the minimum amount of enzyme necessary to maximize the rate is reached. react. For example, in the case of endo-M, an enzyme amount of 500 units (U) / mol (donor) or less, preferably about 80-400 U / mol (donor) is added to the sugar chain donor. Here, one unit (U) of the enzyme hydrolyzes a dansylated (DNS) derivative of an asialo complex-type sugar chain derived from human transferrin, and 1 micromole (μmol) of N- This is the amount of enzyme required to produce a DNS derivative of acetylglucosaminyl-asparagine (GlcNAc-Asn-DNS).
[0031]
It is also important to significantly increase the concentration of sugar chain donor and acceptor. By charging both substrates at a high concentration while limiting the amount of enzyme, the transglycosylation reaction is promoted, side reactions are suppressed, and the reaction yield is dramatically improved. The concentration of the sugar chain donor is 10 mM or more, preferably 15-75 mM. The sugar chain receptor is 2.5 mM or more, preferably about 7.5-35 mM. A higher sugar chain receptor concentration is desirable, but when the solubility of the synthetic substrate is low, a concentration of about 2.5 mM is also used.
[0032]
As the sugar chain donor used in the present invention, any sugar chain of high mannose type, complex type or hybrid type can be used. High mannose type sugar chains are prepared from, for example, ovalbumin, etc., and complex type sugar chains containing sialic acid are prepared from, for example, human transferrin, bovine fetuin, etc., and asialo complex type sugar chains are prepared by removing sialic acid by sialidase treatment etc. Is done. Enzymatically or chemically modified sugar chains or chemically synthesized sugar chains can also be used.
[0033]
The reaction of the present invention is carried out by mixing a substrate sugar chain donor, a sugar chain acceptor and an enzyme endoglycosidase in a buffer solution. As described above, the sugar chain donor is added at a concentration of 10 mM or more, preferably 15-75 mM, and the sugar chain acceptor concentration is 2.5 mM or more, preferably 7.5-35 mM. The amount of the enzyme is limited to 500 U / mol (donor) or less, preferably about 80-400 U / mol (donor). For example, in the case of Endo-M, it is used in an amount of about 2-10 mU / ml. As the buffer solution, an appropriate buffer solution having a pH of about 5-8 and a concentration of 25-200 mM, preferably 50-100 mM is used. In the case of Endo-M, the reaction is usually carried out in an acetic acid or phosphate buffer solution having a pH of 5.5 to 6.5 and a concentration of 50 to 100 mM. An example of a basic reaction solution composition is a sugar chain donor 25 mM, a sugar chain acceptor 10 mM, endo-M4 mU / ml, and a 60 mM phosphate buffer (pH 6.25).
[0034]
The reaction temperature is usually about room temperature-50 ° C, preferably 30-40 ° C, and the reaction time is 1-24 hours. For example, in the case of endo-M enzyme, the reaction is usually carried out at 37 ° C. for about 3-18 hours.
[0035]
The produced complex carbohydrate can be easily separated and purified from the reaction-finished solution according to known means. For example, the complex carbohydrate of the reaction product is separated from the reaction end solution by gel filtration column chromatography, ion exchange resin column chromatography, lectin column chromatography, high performance liquid chromatography (HPLC), etc., and further concentrated, desalted, What is necessary is just to perform freeze-drying etc.
[0036]
【Example】
The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto.
[Example 1]
Glycosyltransferase reaction of high mannose type sugar chain to GlcNAc-Gln-Fmoc:
The sugar chain receptor GlcNAc-Gln-Fmoc (molecular weight 572) was synthesized as a compound containing Gln (glutamine) instead of Asn according to the synthesis method of GlcNAc-Asn-Fmoc. Sodium salt was used for the enzyme reaction. As a sugar chain donor , high mannose-type sugar chain (Man) _6- (GlcNAc) ₂ -Asn (molecular weight 1651) obtained by subjecting ovalbumin to pronase treatment, Sephadex G - 25 gel filtration and separation and purification by Dowex 50 ion exchange chromatography ) 1 μmol (final concentration 25 mM), GlcNAc-Gln-Fmoc (molecular weight 572) as a sugar chain receptor 200 nmol (5 mM), Endo-M 160 μU (4 mU / ml), and 60 mM phosphate buffer (pH 6) .25) The reaction was carried out in 40 μl at 37 ° C. for 3 hours. After stopping the reaction, the reaction solution was diluted to 1 ml with distilled water, and the reaction product was analyzed by HPLC. The transfer product was obtained in a yield of 7.5%. The transfer reaction product was subjected to HPLC fractionation and mass spectrometry. As a result, an ion peak (m / z) corresponding to a molecular weight of 1748 was observed. From (Man) _6- (GlcNAc) ₂ -Asn to GlcNAc-Gln-Fmoc The transfer reaction product, ie, (Man) _6- (GlcNAc) ₂ -Gln-Fmoc was confirmed.
[0037]
[Example 2]
Transfer reaction of human transferrin-derived complex sugar chain to GlcNAc-Gln-Fmoc:
As a sugar chain donor, human transferrin (Seikagaku Corporation) is treated with pronase, a sialoglycopeptide having only Asn residues obtained by repeating Sephadex G - 25 gel filtration (TF - SGP, molecular weight 2338), and further treated with sialidase 1 μmol (final concentration 25 mM) of asialoglycopeptide (TF - ASGP, molecular weight 1756) from which sialic acid was removed and 200 nmol (5 mM) of GlcNAc-Gln-Fmoc 0.1 M phosphate buffer (pH 6.25) Dissolved in 24 μl, added 16 μl of enzyme solution containing 160 μU of endo-M, and reacted at 37 ° C. for 3 hours. After stopping the reaction, the reaction solution was diluted to 1 ml with distilled water, and the reaction product was analyzed by HPLC. The transfer reaction product was obtained in a yield of 5.0% when TF-SGP and 13.7% when TF-ASGP. The transfer reaction product was isolated by HPLC preparative analysis. As a result of mass spectrometry, the transfer reaction product of TF-SGP had an ion peak corresponding to a molecular weight of 2574, and the transfer reaction product of TF-ASGP had a molecular weight of 1992. Corresponding ion peaks (m / z [M−H] 1992) were observed, and each compound in which the disialo double-chain complex sugar chain was transferred to GlcNAc-Gln-Fmoc and the asialo double-chain complex sugar chain were GlcNAc- It was confirmed that the compound was transferred to Gln-Fmoc.
[0038]
[Example 3]
Glycosyltransferase reaction of human transferrin-derived asialo sugar chain to GlcNAc-Gln-DNS:
GlcNAc-Gln dansylated (DNS) derivative (GlcNAc-Gln-DNS) (molecular weight 585) as a sugar chain receptor was synthesized as a compound in which the α amino group of Gln was protected with a DNS group. Asialoglycopeptide (TF-ASGP) was used as a sugar chain donor, and 1 μmol (final concentration 25 mM) of the sugar chain donor and 500 nmol (12.5 mM) of the sugar chain acceptor were added to a 0.1 M phosphate buffer (pH 6.25). ) Dissolve in 24 μl, Endo - M 16 μl of an enzyme solution containing 160 μU was added and reacted at 37 ° C. for 3 hours. The reaction product was subjected to HPLC fractionation and mass spectrometry. As a result, an ion peak (m / z) corresponding to a molecular weight of 2004 was observed, and the asialo double-chain complex sugar chain was transferred to GlcNAc-Gln-DNS. Was confirmed.
[0039]
【The invention's effect】
According to the present invention, a novel complex glycopeptide can be easily synthesized by enzymatically adding a sugar chain to a synthetic substrate having a GlcNAc residue. If the peptide of the sugar chain receptor has glutamine (Gln) in the amino acid sequence of the peptide chain, an N-linked sugar chain can be added via a GlcNAc residue. Can be synthesized. The sugar chain to be added may be either a high-mannose type or a complex type sugar chain containing sialic acid, and a desired complex glycopeptide can be synthesized.
The present invention provides a research technique for elucidating the physiological role played by sugar chains of glycoconjugates together with application to medicine.

Claims (3)

End - beta - N - presence of acetylglucosaminidase (EC3.2.1.96), indicated a sugar chain of complex carbohydrates are sugar donors by the following formula is a carbohydrate receptor (of 1) N -A method for producing a non-natural complex glycopeptide by transferring to a synthetic substrate having an acetylglucosamine (GlcNAc) residue.

(In the formula, R ¹ represents H, an amino protecting group, an amino acid, a peptide or a peptide in which the α-amino group of the N-terminal amino acid is protected. R ² represents OH, a carboxyl protecting group, an amino acid, a peptide, or a carboxyl group of the C-terminal amino acid. Represents a peptide in which n is 2. n is 2. The protecting group for the α-amino group of the N-terminal amino acid is a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a tertiary butyloxycarbonyl (Boc) group, a 3-nitro-2-pyridinesulfenyl (Npys) group, benzyloxy The method according to claim 1, which is a carbonyl (Z) group or a dansyl (DNS) group. Protecting group is tert-carboxyl group of the C-terminal amino acids (Bu ^t), benzyl (Bzl) or a method according to claim 1 or 2 is methyl (Me) group.