JP2012158729A - Acidic sugar chain sample preparation method - Google Patents
Acidic sugar chain sample preparation method Download PDFInfo
- Publication number
- JP2012158729A JP2012158729A JP2011021313A JP2011021313A JP2012158729A JP 2012158729 A JP2012158729 A JP 2012158729A JP 2011021313 A JP2011021313 A JP 2011021313A JP 2011021313 A JP2011021313 A JP 2011021313A JP 2012158729 A JP2012158729 A JP 2012158729A
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- Prior art keywords
- sugar chain
- acidic
- acidic sugar
- preparing
- compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
Description
本発明は、酸性糖鎖の試料調製方法に関する。 The present invention relates to a sample preparation method for acidic sugar chains.
生化学分野において、近年、核酸、タンパク質に続く第三の鎖として糖鎖分子が注目されている。特に細胞の分化や癌化、免疫反応や受精などのかかわりが研究され、新たな医薬や医療材料を創製しようとする試みが続けられている。
また、糖鎖は多くの毒素、ウィルス及びバクテリアなどの病原性外来因子受容体であり、また、癌のマーカーとしても注目されており、こちらの分野においても、同様に新たな医薬や医療材料を創製しようとする試みが続けられている。
生体内において糖タンパク質、糖ペプチドは、細胞膜表面に存在して細胞膜受容体として働いたり、血清などの体液中に分泌されたり、また細胞外マトリックスの構成物として存在している。近年、こうした糖鎖が、生体活動に重要な役割を果たしていることが明らかにされて来ている。
このような糖鎖と細胞の分化増殖、細胞認識、免疫反応及び細胞癌化との関係が明確にされれば、この糖鎖と、細胞工学あるいは臓器工学とを密接に関連させ、新たな糖鎖工学の展開を図ることが期待される。
In the field of biochemistry, in recent years, sugar chain molecules have attracted attention as the third chain following nucleic acids and proteins. In particular, research on cell differentiation, canceration, immune reaction, fertilization, etc. has been conducted, and attempts to create new medicines and medical materials are continuing.
In addition, sugar chains are pathogenous foreign factor receptors such as many toxins, viruses and bacteria, and are also attracting attention as markers for cancer. There are ongoing attempts to create.
In vivo, glycoproteins and glycopeptides exist on the surface of cell membranes and function as cell membrane receptors, are secreted into body fluids such as serum, and exist as components of extracellular matrix. In recent years, it has been revealed that these sugar chains play an important role in biological activities.
Once the relationship between such sugar chains and cell differentiation and proliferation, cell recognition, immune response, and cell carcinogenesis is clarified, this sugar chain is closely related to cell engineering or organ engineering, and a new sugar It is expected to develop chain engineering.
グリコサミノグリカン（ＧＡＧ）類は動物の結合組織を中心にあらゆる組織に普遍的に存在する。動物のみが産生し、植物からの単離の報告はない。
ＧＡＧ類は、ヘパリン、ヘパラン硫酸、ヒアルロン酸、コンドロイチン硫酸、デルマタン硫酸、ケラタン硫酸などが存在しており、主に動物の結合組織に多数存在し、また、ヘパリンは抗血液凝固剤として有名であり、医薬品に利用されている。
ＧＡＧ類は、生体内で種々のタンパク質と特異的な相互作用をしており、それにより生体機能を調製していることが知られている。
Glycosaminoglycans (GAGs) are ubiquitous in all tissues, mainly in the connective tissues of animals. Only animals produce and there are no reports of isolation from plants.
GAGs include heparin, heparan sulfate, hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, etc., mainly present in animal connective tissues, and heparin is famous as an anticoagulant. It is used for medicines.
GAGs are known to have specific interactions with various proteins in vivo, thereby preparing biological functions.
ＧＡＧ類は、硫酸基が付加した2糖の繰り返し構造からなる。うち１つはアミノ糖（ ガラクトサミン、ガラクトサミン、グルコサミン、グルコサミン）であり、もう１つはウロン酸（グルクロン酸、イズロン酸）または ガラクトースである。ＧＡＧ類は多数の硫酸基とカルボキシル基を持つ酸性糖鎖である。これら酸性糖鎖は様々な細胞増殖因子や細胞外マトリックス成分と相互作用し，細胞接着，移動，増殖，分化，形態形成といった細胞活動を制御したり，炎症や癌細胞の転移などの病的な現象にも関わったりしている。従ってこうした酸性糖鎖とタンパク質との相互作用を調べ、機能の調製を検証することは学術的な意味だけではなく、医学の発展に重要なテーマである。 GAGs have a repeating structure of disaccharides to which sulfate groups are added. One of them is an amino sugar (galactosamine, galactosamine, glucosamine, glucosamine), and the other is uronic acid (glucuronic acid, iduronic acid) or galactose. GAGs are acidic sugar chains having a large number of sulfate groups and carboxyl groups. These acidic sugar chains interact with various cell growth factors and extracellular matrix components to control cellular activities such as cell adhesion, migration, proliferation, differentiation and morphogenesis, and pathological factors such as inflammation and cancer cell metastasis. It is also related to the phenomenon. Therefore, examining the interaction between these acidic sugar chains and proteins and verifying the functional preparation is not only an academic meaning, but an important theme for the development of medicine.
しかし、生体試料中には酸性糖鎖以外の物質も多数含まれており、酸性糖鎖を分離精製する方法について非特許文献１に詳細に記されているが、有機溶媒を大量に使用したり大量の塩類を使用したりと煩雑な作業となる（特開２０１０−１２４８１１号）。 However, biological samples contain many substances other than acidic sugar chains, and a method for separating and purifying acidic sugar chains is described in detail in Non-Patent Document 1, but a large amount of organic solvent is used. Use of a large amount of salts is a complicated operation (Japanese Patent Laid-Open No. 2010-124811).
本発明は、簡便に酸性糖鎖を回収、精製しる酸性糖鎖の調製方法であり、該酸性糖鎖を提供することを目的とする。 The present invention is a method for preparing an acidic sugar chain that simply recovers and purifies the acidic sugar chain, and an object thereof is to provide the acidic sugar chain.
このような目的は、下記（１）〜（２１）に記載の本発明により達成される。
（１）酸性糖鎖を含有する溶液から酸性糖鎖を調製する方法であって、
酸性糖鎖を含有する溶液を、糖鎖固相担体と接触させて酸性糖鎖を捕捉する工程と、
前記酸性糖鎖を捕捉した糖鎖固相担体より前記酸性糖鎖を切り出し、該酸性糖鎖を回収する工程と、を有する酸性糖鎖の調製方法
（２）前記酸性糖鎖を回収する工程において、前記酸性糖鎖を切り出し、次いで該酸性糖鎖を標識化合物で標識し、標識化された酸性糖鎖を回収する（１）記載の酸性糖鎖の調製方法
（３）前記酸性糖鎖が、コンドロイチン硫酸、ヘパラン硫酸、ヒアルロン酸、コンドロイチン、およびヘパリン中から選ばれる１以上の酸性多糖である（１）または（２）記載の酸性糖鎖の調製方法。
（４）前記酸性多糖を構成する単糖、または前記酸性糖鎖を構成する二糖以上のオリゴ糖、または前記酸性多糖の断片糖鎖である（３）記載の酸性糖鎖の調製方法。
（５）前記糖鎖固相担体が、乾燥重量１ｍｇあたり１μｍｏｌ以上のヒドラジド基を有するポリマー粒子である（１）記載の酸性糖鎖の調製方法。
（６）前記糖鎖固相担体が下記の（式１）で表される構造を有するポリマー粒子である（１）または（４）記載の酸性糖鎖の調製方法。
（７）前記固相担体が下記の（式２）で表される構造を有するポリマー粒子である（１）ないし（６）いずれか１項に記載の酸性糖鎖の調製方法。
（ａ）酸性多糖から酵素処理により断片糖鎖を作製する工程と、
（ｂ）該酸性糖鎖と糖鎖固相担体をヒドラゾン結合により結合させる工程と、
（ｃ）糖鎖を固相担体から切り離した後、アミノ基を有する化合物を作用させて、還元的アミノ化反応により前記化合物に結合させる工程を含む酸性糖鎖の調製方法。
（９）前記アミノ基を有する化合物が紫外可視吸収特性又は蛍光特性を有するものである（８）記載の酸性糖鎖の調製方法。
（１０）前記アミノ基を有する化合物が、
8-Aminopyrene-1,3,6-trisulfonate, 8-Aminonaphthalene-1,3,6-trisulphonate, 7-Amino-1,3-naphtalenedisulfonic acid, 2-Amino9(10H)-acridone,5-Aminofluorescein,Dansylethylenediamie, 2-Aminopyridine, 7-Amino-4-methylcoumarine, 2-Aminobenzamide, 2-Aminobenzoic acid, 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3-(Aｃetｈylamino)-6-aminoacridine, 2-Amino-6-cyanoethylpyridine,Ethyl p-aminobenzoate, p-Aminobenzonitrile, 及び7-aminonaphothalene-1,3-disulfonic acid
から選ばれる少なくとも１つである（８）又は（９）記載の酸性糖鎖の調製方法。
（１１）前記（１）ないし（１０）いずれか１項に記載の酸性糖鎖の調製方法により調製した酸性糖鎖試料。
（１２）（１）ないし（７）いずれか１項に記載の酸性糖鎖の調製方法で
（ａ）酸性多糖から酵素処理により断片糖鎖を作製する工程と、
（ｂ）該酸性糖鎖と糖鎖固相担体をヒドラゾン結合により結合させる工程と、
（ｃ）ヒドラジド基を有する化合物、またはアミノオキシ基を有する化合物を作用させて、ヒドラゾン−オキシム交換反応により糖鎖を固相担体から切り離しつつ、前記化合物に結合させる工程を含む酸性糖鎖の調製方法。
（１３）（ａ）工程が、前記酸性多糖を糖鎖分解酵素で処理し前記酸性多糖を構成する単糖、二糖以上のオリゴ糖である断片糖鎖を作製する工程である（８）又は（１２）に記載の酸性糖鎖の調製方法。
（１４）前記糖鎖分解酵素が、コンドロイチナーゼ、ヒアルロニダーゼ、ヘパリナーゼ、ヘパリチナーゼ、ケラタナーゼからなる酵素の少なくとも１種である（８）、（１２）または（１３）いずれか１項に記載の酸性糖鎖の調製方法。
（１５）（ｂ）工程が、前記酸性糖鎖を含有する溶液と前記糖鎖固相担体と混合してインキュベートする（８）又は（１２）記載の酸性糖鎖の調製方法。
（１６）前記インキュベートが４℃以上９０℃以下の条件で、５分以上２４時間以下の時間行われる（１５）記載の酸性糖鎖の調製方法。
（１７）前記インキュベートが５０℃以上９０℃以下の条件で、１５分以上１２０分以下の時間行われる（１５）または（１７）記載の酸性糖鎖の調製方法。
（１８）（ｃ）工程のヒドラジド基を有する化合物が下記から選ばれた物質またはその塩である（１２）記載の酸性糖鎖の調製方法。
5-Dimethylaminonaphthalene-1-sulfonyl hydrazine (Dansylhydrazine)； 2-hydrazinopyridine；9-fluorenylmethyl carbazate (Fmoc hydrazine)；benzylhydrazine；4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionoc acid, hydrazide；2-(6,8-difluoro-7-hydroxy-4-methylcoumarin)acetohydrazide；7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH)；phenylhydrazine；1-Naphthaleneacethydrazide；2-hydrazinobenzoic acid；biotin hydrazide；phenylacetic hydrazide.
（１９）（ｃ）工程のアミノオキシ基を有する化合物が下記から選ばれた物質またはその塩である（１２）記載の酸性糖鎖の調製方法。
O-benzylhydroxylamine;O-phenylhydroxylamine; O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine; O-(4-nitrobenzyl)hydroxylamine; 2-aminooxypyridine; 2-aminooxymethylpyridine; 4-[(aminooxyacetyl)amino]benzoic acid methyl ester; 4-[(aminooxyacetyl)amino]benzoic acid ethyl ester; 4-[(aminooxyacetyl)amino]benzoic acid n-butyl ester；N-aminooxyacetyl-tryptophyl(arginine methyl ester)
（２０）前記アミノオキシ基を有する化合物がアルギニン残基、トリプトファン残基、フェニルアラニン残基、チロシン残基、システイン残基およびこれら誘導体の少なくとも一つからなる部分を含む（１９）記載の酸性糖鎖の調製方法。
（２１）前記アミノオキシ基を有する化合物が下記（式３）で表される構造を有する（１９）または（２０）記載の酸性糖鎖の調製方法。
Such an object is achieved by the present invention described in the following (1) to (21).
(1) A method for preparing an acidic sugar chain from a solution containing the acidic sugar chain,
A step of capturing the acidic sugar chain by contacting a solution containing the acidic sugar chain with a sugar chain solid phase carrier;
In the step of recovering the acidic sugar chain, a method for preparing the acidic sugar chain, comprising: cutting out the acidic sugar chain from the sugar chain solid phase carrier that has captured the acidic sugar chain, and collecting the acidic sugar chain. The method for preparing an acidic sugar chain according to (1), wherein the acidic sugar chain is excised, then labeled with a labeling compound, and the labeled acidic sugar chain is recovered (3) The method for preparing an acidic sugar chain according to (1) or (2), which is one or more acidic polysaccharides selected from chondroitin sulfate, heparan sulfate, hyaluronic acid, chondroitin, and heparin.
(4) The method for preparing an acidic sugar chain according to (3), which is a monosaccharide constituting the acidic polysaccharide, an oligosaccharide or more of a disaccharide constituting the acidic sugar chain, or a fragment sugar chain of the acidic polysaccharide.
(5) The method for preparing an acidic sugar chain according to (1), wherein the sugar chain solid phase carrier is polymer particles having a hydrazide group of 1 μmol or more per 1 mg of dry weight.
(6) The method for preparing an acidic sugar chain according to (1) or (4), wherein the sugar chain solid phase carrier is polymer particles having a structure represented by the following (formula 1).
(7) The method for preparing an acidic sugar chain according to any one of (1) to (6), wherein the solid phase carrier is polymer particles having a structure represented by the following (formula 2).
(B) a step of binding the acidic sugar chain and the sugar chain solid phase carrier by a hydrazone bond;
(C) A method for preparing an acidic sugar chain, which comprises a step of separating a sugar chain from a solid phase carrier, allowing a compound having an amino group to act, and binding to the compound by a reductive amination reaction.
(9) The method for preparing an acidic sugar chain according to (8), wherein the compound having an amino group has ultraviolet-visible absorption characteristics or fluorescence characteristics.
(10) The compound having an amino group is
8-Aminopyrene-1,3,6-trisulfonate, 8-Aminonaphthalene-1,3,6-trisulphonate, 7-Amino-1,3-naphtalenedisulfonic acid, 2-Amino9 (10H) -acridone, 5-Aminofluorescein, Dansylethylenediamie, 2-Aminopyridine, 7-Amino-4-methylcoumarine, 2-Aminobenzamide, 2-Aminobenzoic acid, 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3- (Acetylamino) -6-aminoacridine, 2-Amino-6- cyanoethylpyridine, Ethyl p-aminobenzoate, p-Aminobenzonitrile, and 7-aminonaphothalene-1,3-disulfonic acid
The method for preparing an acidic sugar chain according to (8) or (9), which is at least one selected from:
(11) An acidic sugar chain sample prepared by the method for preparing acidic sugar chains according to any one of (1) to (10).
(12) (a) a method for producing a fragment sugar chain from an acidic polysaccharide by enzymatic treatment in the method for preparing an acidic sugar chain according to any one of (1) to (7);
(B) a step of binding the acidic sugar chain and the sugar chain solid phase carrier by a hydrazone bond;
(C) Preparation of an acidic sugar chain comprising a step of allowing a compound having a hydrazide group or a compound having an aminooxy group to act and separating the sugar chain from a solid phase carrier by a hydrazone-oxime exchange reaction and binding to the compound Method.
(13) The step (a) is a step in which the acidic polysaccharide is treated with a glycolytic enzyme to produce a fragment sugar chain that is a monosaccharide or disaccharide or higher oligosaccharide constituting the acidic polysaccharide (8) or The method for preparing an acidic sugar chain according to (12).
(14) The acidic sugar according to any one of (8), (12) and (13), wherein the sugar chain degrading enzyme is at least one of enzymes consisting of chondroitinase, hyaluronidase, heparinase, heparitinase, and keratanase. Chain preparation method.
(15) The method for preparing an acidic sugar chain according to (8) or (12), wherein the step (b) comprises mixing and incubating the solution containing the acidic sugar chain and the sugar chain solid phase carrier.
(16) The method for preparing an acidic sugar chain according to (15), wherein the incubation is performed at a temperature of 4 ° C. or higher and 90 ° C. or lower for a period of 5 minutes to 24 hours.
(17) The method for preparing an acidic sugar chain according to (15) or (17), wherein the incubation is performed at a temperature of 50 ° C. to 90 ° C. for a period of 15 minutes to 120 minutes.
(18) The method for preparing an acidic sugar chain according to (12), wherein the compound having a hydrazide group in step (c) is a substance selected from the following or a salt thereof.
5-Dimethylaminonaphthalene-1-sulfonyl hydrazine (Dansylhydrazine); 2-hydrazinopyridine; 9-fluorenylmethyl carbazate (Fmoc hydrazine); benzylhydrazine; 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s -indacene-3-propionoc acid, hydrazide; 2- (6,8-difluoro-7-hydroxy-4-methylcoumarin) acetohydrazide; 7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH); phenylhydrazine; 1-Naphthaleneacethydrazide; 2 -hydrazinobenzoic acid; biotin hydrazide; phenylacetic hydrazide.
(19) The method for preparing an acidic sugar chain according to (12), wherein the compound having an aminooxy group in step (c) is a substance selected from the following or a salt thereof.
O-benzylhydroxylamine; O-phenylhydroxylamine; O- (2,3,4,5,6-pentafluorobenzyl) hydroxylamine; O- (4-nitrobenzyl) hydroxylamine; 2-aminooxypyridine; 2-aminooxymethylpyridine; 4-[(aminooxyacetyl) amino] 4-[(aminooxyacetyl) amino] benzoic acid ethyl ester; 4-[(aminooxyacetyl) amino] benzoic acid n-butyl ester; N-aminooxyacetyl-tryptophyl (arginine methyl ester)
(20) The acidic sugar chain according to (19), wherein the compound having an aminooxy group includes a moiety comprising at least one of an arginine residue, a tryptophan residue, a phenylalanine residue, a tyrosine residue, a cysteine residue, and a derivative thereof. Preparation method.
(21) The method for preparing an acidic sugar chain according to (19) or (20), wherein the compound having an aminooxy group has a structure represented by the following (formula 3).
本発明によれば、生体由来材料のような夾雑物混在状態から酸性糖鎖を簡便に精製した酸性糖鎖を調製することが可能となる。また、必要に応じて、酸性糖鎖を標識した試料を簡便に調製することができる。 ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to prepare the acidic sugar chain which refine | purified the acidic sugar chain simply from the contamination mixed state like biological material. Moreover, if necessary, a sample labeled with an acidic sugar chain can be easily prepared.
本発明に述べるところの酸性糖鎖含有溶液中から酸性糖鎖を精製させる方法は、図１に示すように、酸性糖鎖を含有する溶液を、糖鎖固相担体と接触させて酸性糖鎖を捕捉する工程と前記酸性糖鎖を捕捉した糖鎖固相担体より酸性糖鎖を切り出し、酸性糖鎖を回収する工程による酸性糖鎖試料調製方法である。
切り出しの際に、標識化合物で標識し、標識された酸性糖鎖として回収してもよい。
As shown in FIG. 1, the method for purifying an acidic sugar chain from an acidic sugar chain-containing solution described in the present invention comprises contacting a solution containing an acidic sugar chain with a sugar chain solid phase carrier, and And a method for preparing an acidic sugar chain sample by cutting out an acidic sugar chain from a sugar chain solid phase carrier that has captured the acidic sugar chain and recovering the acidic sugar chain.
At the time of excision, it may be labeled with a labeling compound and recovered as a labeled acidic sugar chain.
酸性糖鎖含有溶液とは、酸性糖鎖を含む溶液であり、その由来は、生体由来物質、有機合成物、または酵素合成物である場合が多い。生体由来物質の場合は、組織由来、動物細胞由来や血液などの体液由来物質であり、また、有機合成物の場合は、反応触媒や反応中間体を含み、酵素合成物の場合は、合成酵素を含んでいる。 An acidic sugar chain-containing solution is a solution containing an acidic sugar chain, and its origin is often a biological substance, an organic compound, or an enzyme compound. In the case of a biological substance, it is a substance derived from a tissue, animal cell, or body fluid such as blood. In the case of an organic compound, it contains a reaction catalyst or a reaction intermediate. In the case of an enzyme compound, a synthetic enzyme Is included.
本発明における酸性糖鎖とは、糖鎖を構成する糖中に硫酸基あるいは、カルボキシル基の酸性基を有する糖を含む糖鎖を示し、具体的には、コンドロイチン硫酸、ヘパラン硫酸、ヒアルロン酸、コンドロイチン、またはヘパリンである酸性多糖のうちの少なくとも１種、または、前記酸性多糖を構成する単糖、二糖以上のオリゴ糖、または前記酸性多糖を糖鎖分解酵素で処理した断片糖鎖である。オリゴ糖は、単糖類同士がグリコシド結合によって結合した化合物の中で、多糖類というほどは分子量が大きくない糖類のことであり、通常は、二糖以上の糖鎖構造物を示し、分子量としては３百−３千程度のものを指す。 The acidic sugar chain in the present invention refers to a sugar chain containing a sugar having a sulfate group or a carboxyl group acidic group in the sugar constituting the sugar chain. Specifically, chondroitin sulfate, heparan sulfate, hyaluronic acid, At least one of acidic polysaccharides that are chondroitin or heparin, or a monosaccharide, disaccharide or higher oligosaccharide constituting the acidic polysaccharide, or a fragment sugar chain obtained by treating the acidic polysaccharide with a glycolytic enzyme . An oligosaccharide is a saccharide whose molecular weight is not so large as a polysaccharide among compounds in which monosaccharides are linked by glycosidic bonds, and usually indicates a sugar chain structure of disaccharide or higher, and the molecular weight is The thing of about 300-3,000 points.
（糖鎖精製用担体）
当該糖鎖精製担体は、糖鎖を捕捉するための反応性の一級アミノ基をその表面に有する担体であり、該一級アミノとしてオキシルアミノ基またはヒドラジド基を有することが望ましい。これは、酵素やカップリング試薬などの非存在下においても糖鎖還元末端であるアルデヒド基と反応し結合可能であるから好適である。
(Carbohydrate purification carrier)
The sugar chain purification carrier is a carrier having a reactive primary amino group for capturing a sugar chain on its surface, and preferably has an oxylamino group or a hydrazide group as the primary amino. This is preferable because it can react with and bind to the aldehyde group which is the sugar chain reducing end even in the absence of an enzyme or a coupling reagent.
前記糖鎖精製用担体は、下記一般式〔１〕で表される構造が好ましい。
また、前記担体は、水溶液や有機溶媒に不溶性の担体であることがのぞましく、材質は特に限定するものではないが、ガラスや耐有機溶剤性に優れた樹脂、例えばシリコン、ポリスチレン、エチレン−無水マレイン酸共重合物、ポリメタクリル酸メチル等を選ぶことができる。 In addition, the carrier is preferably a carrier insoluble in an aqueous solution or an organic solvent, and the material is not particularly limited, but glass or a resin excellent in organic solvent resistance such as silicon, polystyrene, ethylene, and the like. -Maleic anhydride copolymer, polymethyl methacrylate and the like can be selected.
前記担体は、下記一般式〔２〕の構造を有する架橋ポリマー構造を有するポリマーマトリックスで構成される粒子であることが好ましい。
前記糖鎖精製用担体の形態は、特に限定するものではないが、粒子またはプレート状の形態であることが好ましい。糖鎖ライブラリーを作製するためには、同時に多数の試料を処理する可能性があり、その際には、カラムに粒子を充填したものを使用する事で連続的な処理が可能である。また、マルチウェルプレートであれば同時に多検体を処理することが可能である。マルチウェルプレートとしては、６、１２、２４，４８、９６，３８４ウェルなどのマルチウェルプレートを適宜使用することが出来る。 The form of the sugar chain purification carrier is not particularly limited, but is preferably in the form of particles or plates. In order to prepare a sugar chain library, a large number of samples may be processed at the same time, and in this case, continuous processing is possible by using a column packed with particles. Moreover, if it is a multiwell plate, it is possible to process many samples simultaneously. As the multiwell plate, multiwell plates such as 6, 12, 24, 48, 96, and 384 wells can be appropriately used.
式〔２〕の担体以外では、粒子として無機物質を用いることができる。該担体としては、粒子状のものを用いることができ、例えばシリカ粒子、アルミナ粒子、ガラス粒子、金属粒子などが挙げられる。また、有機高分子物質としては、アガロース、セファロースに代表される多糖類ゲル、ビニル化合物の重合体であるポリマーを粒子状にしたものを使用することが出来る。
また、粒子としたときの形状は球であることが好ましく、平均粒径０．１μｍ以上５００μｍ以下の粒子である。この場合の平均粒径は光学顕微鏡視野において観察される各粒子の直径を計測することにより求めたものである。このような範囲の粒径を有する担体の粒子は、遠心分離、 フィルターなどによる回収が容易であり、かつ、充分な表面積を有しているために糖鎖との反応効率も高いと考えられる。粒径が上記の範囲よりも大幅に大きい場合、表面積が小さくなるために糖鎖との反応効率が低くなることがある。また、粒径が上記の範囲よりも大幅に小さい場合、特にフィルターによる粒子の回収が難しくなることがある。さらに、粒子をカラムに充填して用いる場合、粒径が過小であると通液の際の圧力損失が大きくなってしまうことがある。
Other than the carrier of the formula [2], an inorganic substance can be used as the particles. The carrier can be in the form of particles, and examples thereof include silica particles, alumina particles, glass particles, and metal particles. In addition, as the organic polymer substance, a polysaccharide gel typified by agarose or sepharose, or a polymer obtained by polymerizing a polymer of a vinyl compound can be used.
The shape of the particles is preferably a sphere, and the particles have an average particle size of 0.1 μm to 500 μm. The average particle diameter in this case is obtained by measuring the diameter of each particle observed in the optical microscope field of view. It is considered that the carrier particles having a particle size in such a range can be easily collected by centrifugation, a filter, etc., and have a sufficient surface area, so that the reaction efficiency with sugar chains is high. When the particle size is significantly larger than the above range, the reaction efficiency with the sugar chain may be lowered due to the small surface area. In addition, when the particle size is significantly smaller than the above range, it may be difficult to collect the particles using a filter. Furthermore, when the particles are packed in a column and used, if the particle size is too small, the pressure loss at the time of liquid passage may increase.
（酸性多糖前処理）
前記酸性多糖から酸性糖鎖を得るには酵素処理により得ることができる。
ここで用いる酵素は、糖鎖分解酵素であり、具体的にはコンドロイチナーゼ、ヒアルロニダーゼ、ヘパリナーゼ、ヘパリチナーゼ、ケラタナーゼからなる酵素の少なくとも1種類であるが、特にこれらに限定されるものではない。
(Acid polysaccharide pretreatment)
An acidic sugar chain can be obtained from the acidic polysaccharide by enzymatic treatment.
The enzyme used here is a sugar chain degrading enzyme. Specifically, it is at least one of the enzymes consisting of chondroitinase, hyaluronidase, heparinase, heparitinase, and keratanase, but is not particularly limited thereto.
具体的な処理方法としては、例えば、グリコサミノグリカン約１μｇ含まれる試料を用いた場合、まず試料を凍結乾燥し緩衝液に再溶解する。酵素活性量として１〜５ｍＵ程度の酵素（コンドロイチナーゼ、ヘパリナーゼ、ヘパリチナーゼなど）を加え、３７℃で数時間反応させる。
なお、この酸性多糖前処理工程は、使用する酸性多糖の精製度などの状況により省略することも可能である。
As a specific treatment method, for example, when a sample containing about 1 μg of glycosaminoglycan is used, the sample is first lyophilized and redissolved in a buffer solution. Enzymes having an enzyme activity of about 1 to 5 mU (chondroitinase, heparinase, heparitinase, etc.) are added and reacted at 37 ° C. for several hours.
This acidic polysaccharide pretreatment step can be omitted depending on the conditions such as the degree of purification of the acidic polysaccharide used.
（酸性糖鎖捕捉）
前記糖鎖精製担体で末端還元糖鎖と一級アミノ基の結合反応の条件の一具体例は、ｐＨが２〜７、反応温度が５０〜１００℃、好ましくは６０〜９０℃、より好ましくは７０〜８５℃、反応時間が１５〜１２０分である。最も好ましい条件はｐＨ３〜６、反応温度が８０℃、反応時間が１時間である。
ｐＨが３未満、または７を越える場合は、中間体であるイミン体の生成が遅くなるため、捕捉効率が落ちる。反応温度は、５０℃未満の場合、反応効率が著しく悪化する場合があり、糖鎖を十分に捕捉することができない。反応は、開放系で行って溶媒を完全に蒸発させることが好ましい。これは、溶媒が蒸発するにつれで溶液濃度が無限濃縮されることにより十分な反応をおこさせることが目的である。
また、９０℃を超える場合は、酸性糖鎖自身に悪影響を及ぼすと共に、担体がプラスチックの場合は種類によって変形、溶融を発生することがある。
反応時間が３０分より短い場合は十分な結合反応が得られない場合があり、糖鎖を十分に捕捉することが出来ない。また９０分を超えた反応は、更なる糖鎖の捕捉は見られず時間をかけただけの効果がない。
(Acid sugar chain capture)
One specific example of the conditions for the coupling reaction between the terminal reduced sugar chain and the primary amino group on the sugar chain purification carrier is that the pH is 2 to 7, the reaction temperature is 50 to 100 ° C, preferably 60 to 90 ° C, more preferably 70. ˜85 ° C., reaction time is 15 to 120 minutes. The most preferred conditions are pH 3-6, reaction temperature 80 ° C., and reaction time 1 hour.
When the pH is less than 3 or more than 7, the production of an imine that is an intermediate is slowed, and the capture efficiency is lowered. When the reaction temperature is less than 50 ° C., the reaction efficiency may be remarkably deteriorated, and sugar chains cannot be sufficiently captured. The reaction is preferably carried out in an open system to completely evaporate the solvent. The purpose of this is to cause a sufficient reaction by infinitely concentrating the solution concentration as the solvent evaporates.
When the temperature exceeds 90 ° C., the acidic sugar chain itself is adversely affected, and when the carrier is a plastic, deformation and melting may occur depending on the type.
When the reaction time is shorter than 30 minutes, a sufficient binding reaction may not be obtained, and sugar chains cannot be captured sufficiently. In addition, the reaction exceeding 90 minutes does not show the effect of only taking time without further capturing of sugar chains.
（夾雑物の除去）
糖鎖を捕捉した状態の糖鎖精製担体は、夾雑物を取り除くために洗浄する必要がある。
ここで、洗浄液に用いられる溶液としては、メタノール、エタノールなどのアルコール類；水および水性緩衝液などが使用される。ここで、洗浄に水溶液が用いられる場合、この水溶液のｐＨは中性付近であることが好ましく、そのｐＨは４〜１０、より好ましくは６〜８である。
前期糖鎖捕捉した担体は、洗浄により、精製原料中の糖鎖以外の夾雑物を簡単に除去することが可能で、糖鎖のみを担体ごと回収することができる。
洗浄方法としては、粒子の場合は、洗浄液に浸漬し、洗浄液の交換を繰り返すことで洗浄することができる。
(Removal of impurities)
It is necessary to wash the sugar chain purification carrier in the state of capturing the sugar chain in order to remove impurities.
Here, as a solution used for the washing liquid, alcohols such as methanol and ethanol; water and an aqueous buffer are used. Here, when an aqueous solution is used for washing, the pH of the aqueous solution is preferably near neutral, and the pH is 4 to 10, more preferably 6 to 8.
The carrier obtained by capturing the sugar chain in the previous stage can easily remove impurities other than the sugar chain in the purified raw material by washing, and only the sugar chain can be recovered together with the carrier.
As a cleaning method, in the case of particles, the particles can be cleaned by immersing them in a cleaning solution and repeating the replacement of the cleaning solution.
例えば、遠心チューブ内に粒子を入れ、洗浄液を加え、粒子を自然沈降、または、遠心分離により強制的に沈降させた後、上清を除去する操作を繰り返すことで洗浄することができる。前記洗浄操作は３〜６回行うことが好ましい。
プレートの場合は、各ウェル内に洗浄液を分注、吸引除去を繰り返すことで簡便に洗浄することができる。また、必要に応じてプレートを遠心可能な遠心分離機を用いても良い。
For example, the particles can be washed by adding particles into a centrifuge tube, adding a washing solution, allowing the particles to settle naturally or by forcible sedimentation by centrifugation, and then removing the supernatant. The washing operation is preferably performed 3 to 6 times.
In the case of a plate, the washing liquid can be simply washed by dispensing and sucking and removing in each well. Moreover, you may use the centrifuge which can centrifuge a plate as needed.
また、チューブ状の容器であって、底面部に、液体透過可能で該粒子が不透過な孔径を有するフィルターを装着するフィルターチューブを用いることも可能である。該フィルターチューブに粒子を入れて使用することで、洗浄に要した洗浄液を、フィルターを介して除去することが可能となり、前記の遠心操作後の上清除去の工程が必要なくなり、作業性の向上を図ることができる。
また、６〜３８４穴のマルチウェルプレートの底部が前記フィルターを装着したものが各種市販されており、これらのプレートを用いることでハイスループット化することが可能である。特に９６穴マルチウェルプレートは、溶液分注機器、吸引除去システム、およびプレートの搬送システム等が開発されており、ハイスループット化に最適である。
Moreover, it is also possible to use a filter tube which is a tube-like container and is equipped with a filter having a pore size which allows liquid permeation and does not allow the particles to permeate on the bottom surface. By using particles in the filter tube, it is possible to remove the cleaning solution required for washing through the filter, eliminating the need for the step of removing the supernatant after the centrifugation, and improving workability. Can be achieved.
Various types of multi-well plates having 6 to 384 holes with the filter attached are commercially available, and high throughput can be achieved by using these plates. In particular, a 96-well multi-well plate has been developed as a solution dispensing device, a suction removal system, a plate transport system, and the like, and is optimal for high throughput.
なお、前記洗浄工程は、当初の生体試料の状態、例えば糖鎖以外の物質の混在の程度によっては行わなくても構わない。 The washing step may not be performed depending on the state of the initial biological sample, for example, the degree of mixing of substances other than sugar chains.
（酸性糖鎖分離、精製）
次のステップとして、担体ごと回収した酸性糖鎖を担体から遊離させ、酸性糖鎖試料として回収することについて以下に述べる。
このステップは、担体ごと回収した酸性糖鎖を担体から遊離させると同時に標識する方法と、酸性糖鎖を担体から遊離させた後に、標識する２つの方法がある。
(Acid sugar chain separation, purification)
As the next step, it will be described below that the acidic sugar chain recovered together with the carrier is released from the carrier and recovered as an acidic sugar chain sample.
In this step, there are two methods: a method in which the acidic sugar chain recovered together with the carrier is released from the carrier and labeling, and a method in which the acidic sugar chain is released from the carrier and then labeled.
遊離操作の基本は、担体ごと回収した糖鎖を酸性溶媒中、加熱反応を行うことで糖鎖を担体から切り出すことができる。
具体的な方法を以下に示す。
The basis of the releasing operation is that the sugar chain recovered together with the carrier can be excised from the carrier by performing a heating reaction in an acidic solvent.
A specific method is shown below.
反応を行う溶媒は、酸と水が反応に必要であることから、酸と水と有機溶媒の混合溶媒が好ましい。酸と水と有機溶媒の混合溶媒の場合、水の含有率は好ましくは０．１％〜９０％、より好ましくは０．１％〜８０％、さらに好ましくは０．１％〜５０％である。水の代わりに水性緩衝液を含有しても良い。混合前の緩衝液の濃度は好ましくは０．１ｍＭ〜１Ｍ、より好ましくは０．１ｍＭ〜５００ｍＭ、さらに好ましくは１ｍＭ〜１００ｍＭである。反応溶液のｐＨは好ましくは２〜９、より好ましくは２〜７であり、さらに好ましくは２〜６である。使用する酸は例えば、酢酸、ギ酸、トリフルオロ酢酸、塩酸、クエン酸、リン酸、硫酸が好ましく、より好ましくは酢酸、ギ酸、トリフルオロ酢酸、リン酸、さらに好ましくは酢酸、トリフルオロ酢酸である。 A solvent for the reaction is preferably a mixed solvent of an acid, water and an organic solvent because an acid and water are necessary for the reaction. In the case of a mixed solvent of acid, water and organic solvent, the water content is preferably 0.1% to 90%, more preferably 0.1% to 80%, and even more preferably 0.1% to 50%. . An aqueous buffer may be contained instead of water. The concentration of the buffer before mixing is preferably 0.1 mM to 1 M, more preferably 0.1 mM to 500 mM, and even more preferably 1 mM to 100 mM. The pH of the reaction solution is preferably 2-9, more preferably 2-7, and even more preferably 2-6. The acid used is preferably, for example, acetic acid, formic acid, trifluoroacetic acid, hydrochloric acid, citric acid, phosphoric acid, sulfuric acid, more preferably acetic acid, formic acid, trifluoroacetic acid, phosphoric acid, and more preferably acetic acid, trifluoroacetic acid. .
反応温度に関しては４〜９０℃が好ましく、好ましくは２５〜９０℃で、さらに好ましくは５０〜９０℃である。反応時間は、５分間〜２４時間、好ましくは１０分間〜８時間、より好ましくは１５分間〜１２０分間である。反応は、開放系で行って溶媒を完全に蒸発させることが好ましい。これは、溶媒が蒸発するにつれで溶液濃度が無限濃縮されることにより十分な反応をおこさせることが目的である。 The reaction temperature is preferably 4 to 90 ° C, preferably 25 to 90 ° C, more preferably 50 to 90 ° C. The reaction time is 5 minutes to 24 hours, preferably 10 minutes to 8 hours, more preferably 15 minutes to 120 minutes. The reaction is preferably carried out in an open system to completely evaporate the solvent. The purpose of this is to cause a sufficient reaction by infinitely concentrating the solution concentration as the solvent evaporates.
ｐＨ２の酸性から中性付近で、糖鎖切り出し反応を行うことができるため、従来の強酸性処理、たとえば１０％トリフルオロ酢酸処理による切出しのような強酸の存在下での切出し反応に比べて、シアル酸残基の脱離など糖鎖の加水分解などを引き起こすことを抑制することができるようになる。 Since the sugar chain cleaving reaction can be performed in the vicinity of pH 2 from acidic to neutral, compared to the conventional cleaving reaction in the presence of a strong acid such as cleaving by 10% trifluoroacetic acid treatment, It is possible to suppress the occurrence of sugar chain hydrolysis such as elimination of sialic acid residues.
また、担体ごと回収した糖鎖を遊離させ、さらに、高速液体クロマトグラフィーなどの方法で分画・分析をする場合には、糖鎖を蛍光標識する必要がある。酸性糖鎖の遊離ならびに蛍光標識について以下に記載する。また、ＭＡＬＤＩ−ＴＯＦ ＭＳやＥＳＩ−ＭＳによる検出感度を向上させるために、糖鎖を化合物で標識する必要がある。 In addition, when the sugar chain collected together with the carrier is released and further fractionated / analyzed by a method such as high performance liquid chromatography, it is necessary to fluorescently label the sugar chain. The release of acidic sugar chains and fluorescent labeling are described below. Moreover, in order to improve the detection sensitivity by MALDI-TOF MS or ESI-MS, it is necessary to label a sugar chain with a compound.
この化合物で標識する方法としては、担体ごと回収した酸性糖鎖を担体から遊離させた後に標識する方法と、酸性糖鎖を担体から遊離させると同時に標識する、２つの方法がある。 As a method of labeling with this compound, there are two methods: a method in which the acidic sugar chain recovered together with the carrier is released from the carrier and then a labeling method, and a method in which the acidic sugar chain is released from the carrier and labeled at the same time.
酸性糖鎖を糖鎖捕捉担体から切り出して後に標識する方法としては、アミノ基を有する標識化合物を作用させて、還元アミノ化反応により酸性糖鎖を前記標識化合物に結合させることができる。 As a method for excising the acidic sugar chain from the sugar chain-trapping carrier and labeling it later, the acidic sugar chain can be bound to the labeling compound by a reductive amination reaction by acting a labeling compound having an amino group.
この方法は、連続的に行うことができるため、酸性糖鎖の回収、精製から検出までの自動化が可能となる。 Since this method can be carried out continuously, it is possible to automate from recovery, purification to detection of acidic sugar chains.
まず、糖鎖捕捉担体から切り出す操作を行う。遊離操作の基本は、前記のように担体ごと回収した糖鎖を酸性溶媒中、加熱反応を行うことで糖鎖を担体から切り出すことである。用いる試薬や反応条件は前記の通りである。 First, an operation of cutting out from the sugar chain-trapping carrier is performed. The basis of the releasing operation is to cut out the sugar chain from the carrier by performing a heating reaction on the sugar chain collected together with the carrier as described above in an acidic solvent. The reagents and reaction conditions used are as described above.
続いて、回収した酸性糖鎖を、溶液中でアミノ基を含む還元アミノ化反応により標識化合部と結合させる。 Subsequently, the collected acidic sugar chain is bound to the labeled binding site by a reductive amination reaction containing an amino group in the solution.
前記のアミノ基を含む化合物は、具体的には蛍光物質またはＵＶ吸収基を有する化合物が、下記のアミノ基を含む物質からなる群から選ぶことが、好ましい。
8-Aminopyrene-1,3,6-trisulfonate, 8-Aminonaphthalene-1,3,6-trisulphonate, 7-Amino-1,3-naphtalenedisulfonic acid, 2-Amino9(10H)-acridone,5-Aminofluorescein,Dansylethylenediamie, 2-Aminopyridine, 7-Amino-4-methylcoumarine, 2-Aminobenzamide, 2-Aminobenzoic acid, 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3-(Acethylamino)-6-aminoacridine, 2-Amino-6-cyanoethylpyridine,Ethyl p-aminobenzoate, p-Aminobenzonitrile, 及び7-aminonaphothalene-1,3-disulfonic acid
Specifically, the compound containing an amino group is preferably a fluorescent substance or a compound having a UV absorbing group selected from the group consisting of the following substances containing an amino group.
8-Aminopyrene-1,3,6-trisulfonate, 8-Aminonaphthalene-1,3,6-trisulphonate, 7-Amino-1,3-naphtalenedisulfonic acid, 2-Amino9 (10H) -acridone, 5-Aminofluorescein, Dansylethylenediamie, 2-Aminopyridine, 7-Amino-4-methylcoumarine, 2-Aminobenzamide, 2-Aminobenzoic acid, 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3- (Acethylamino) -6-aminoacridine, 2-Amino-6- cyanoethylpyridine, Ethyl p-aminobenzoate, p-Aminobenzonitrile, and 7-aminonaphothalene-1,3-disulfonic acid
（還元アミノ化反応による標識方法）
具体的には、2-Aminobenzamideによる標識の場合、担体から酸性糖鎖を遊離後、反応容器に0.35 M 2-Aminobenzamid, 1 M sodium cyanoborohydride, ３０％ 酢酸水溶液を加え、６０℃で数時間反応する事で達成される。
(Labeling method by reductive amination reaction)
Specifically, in the case of labeling with 2-Aminobenzamide, 0.35 M 2-Aminobenzamid, 1 M sodium cyanoborohydride, 30% acetic acid aqueous solution is added to the reaction vessel after releasing the acidic sugar chain from the carrier and reacted at 60 ° C. for several hours. Achieved by things.
一方、酸性糖鎖を糖鎖捕捉担体から切り出すと同時に標識する方法としては、ヒドラジド基、またはアミノオキシ基を有する標識化合物を作用させて、ヒドラゾン−ヒドラゾンあるいはヒドラゾン−オキシム交換反応により酸性糖鎖を固相担体から切り離しすると同時に前記標識化合物に結合させることで可能である。 On the other hand, as a method of labeling the acidic sugar chain at the same time as cutting out from the sugar chain-capturing carrier, a labeling compound having a hydrazide group or an aminooxy group is allowed to act, and the acidic sugar chain is removed by hydrazone-hydrazone or hydrazone-oxime exchange reaction. This can be achieved by separating from the solid support and simultaneously binding to the labeled compound.
この方法は、連続的に行うことができるため、酸性糖鎖の回収、精製から検出までの自動化が可能となる。 Since this method can be carried out continuously, it is possible to automate from recovery, purification to detection of acidic sugar chains.
ヒドラジド基を含む標識化合物の作製方法としては、上記糖鎖を遊離する工程において、ヒドラジド基を含有しており、蛍光物質またはＵＶ吸収基を有する化合物を接触させ糖鎖を遊離させると同時に蛍光物質またはＵＶ吸収物質で糖鎖を標識する方法がある。反応の溶媒、反応温度、時間の詳細は前記の通りであるが、アルデヒド基を含む化合物、例えば糖鎖に対して１０等量以上の化合物を添加することで達成できる。 As a method for producing a labeling compound containing a hydrazide group, in the step of releasing the sugar chain, a fluorescent substance containing a hydrazide group is contacted with a fluorescent substance or a compound having a UV absorbing group to release the sugar chain simultaneously. Alternatively, there is a method of labeling sugar chains with UV absorbing substances. The details of the solvent, reaction temperature, and time for the reaction are as described above, but can be achieved by adding a compound containing an aldehyde group, for example, 10 equivalents or more of the compound to the sugar chain.
前記ヒドラジド基を含む物質、具体的には蛍光物質またはＵＶ吸収基を有する化合物が、下記のヒドラジド基を含む物質である群から選ぶことができる。
5-Dimethylaminonaphthalene-1-sulfonyl hydrazine (Dansylhydrazine)； 2-hydrazinopyridine；9-fluorenylmethyl carbazate (Fmoc hydrazine)；benzylhydrazine；4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionoc acid, hydrazide；2-(6,8-difluoro-7-hydroxy-4-methylcoumarin)acetohydrazide；7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH)；phenylhydrazine；1-Naphthaleneacethydrazide；2-hydrazinobenzoic acid；biotin hydrazide；phenylacetic hydrazide.
The substance containing a hydrazide group, specifically, a fluorescent substance or a compound having a UV absorbing group can be selected from the group consisting of the following substances containing a hydrazide group.
5-Dimethylaminonaphthalene-1-sulfonyl hydrazine (Dansylhydrazine); 2-hydrazinopyridine; 9-fluorenylmethyl carbazate (Fmoc hydrazine); benzylhydrazine; 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s -indacene-3-propionoc acid, hydrazide; 2- (6,8-difluoro-7-hydroxy-4-methylcoumarin) acetohydrazide; 7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH); phenylhydrazine; 1-Naphthaleneacethydrazide; 2 -hydrazinobenzoic acid; biotin hydrazide; phenylacetic hydrazide.
アミノオキシ基を含む標識化合物としては、アミノオキシ基含有の蛍光物質またはＵＶ吸収基を有する化合物を接触させ糖鎖を遊離させると同時に蛍光物質またはＵＶ吸収基を有する化合物で糖鎖を標識する方法である。反応の溶媒、反応温度、時間の詳細は前記の通りであるが、アミノ基を含む化合物、例えば糖鎖に対して１０等量以上の化合物を添加することで達成できる。 As a labeling compound containing an aminooxy group, a method of labeling a sugar chain with a compound having a fluorescent substance or a UV absorbing group at the same time as releasing a sugar chain by contacting a fluorescent substance containing an aminooxy group or a compound having a UV absorbing group It is. The details of the reaction solvent, reaction temperature, and time are as described above, but it can be achieved by adding a compound containing an amino group, for example, 10 equivalents or more of the compound to the sugar chain.
上記アミノオキシ化合物が下記の化合物から選ばれる事が望ましい。
O-benzylhydroxylamine;O-phenylhydroxylamine; O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine; O-(4-nitrobenzyl)hydroxylamine; 2-aminooxypyridine; 2-aminooxymethylpyridine; 4-[(aminooxyacetyl)amino]benzoic acid methyl ester; 4-[(aminooxyacetyl)amino]benzoic acid ethyl ester; 4-[(aminooxyacetyl)amino]benzoic acid n-butyl ester.
The aminooxy compound is preferably selected from the following compounds.
O-benzylhydroxylamine; O-phenylhydroxylamine; O- (2,3,4,5,6-pentafluorobenzyl) hydroxylamine; O- (4-nitrobenzyl) hydroxylamine; 2-aminooxypyridine; 2-aminooxymethylpyridine; 4-[(aminooxyacetyl) amino] benzoic acid methyl ester; 4-[(aminooxyacetyl) amino] benzoic acid ethyl ester; 4-[(aminooxyacetyl) amino] benzoic acid n-butyl ester.
前記アミノオキシ基を有する化合物がアルギニン残基、トリプトファン残基、フェニルアラニン残基、チロシン残基、システイン残基およびこれら誘導体の少なくとも一つからなる部分を含む化合物であることが好ましく、下記（式３）で表される構造を有した化合物であることが好ましい。
（酸性糖鎖試料保存方法）
前記の方法により得られた標識された酸性糖鎖は溶液状態で試料として回収される。この溶液状の酸性糖鎖試料は、目的に応じて、すぐに使用してもよいし、必要時まで保存しておいてよい。保存方法は、そのまま市販の蓋付きチューブなど、密閉容器に移し冷凍保存することが好ましい。さらに好ましくは凍結乾燥させてから冷凍保存する方法である。冷凍保存の温度は、−２０℃以下が好ましく、より好ましくは−８０℃以下である。
(Acid sugar chain sample storage method)
The labeled acidic sugar chain obtained by the above method is recovered as a sample in a solution state. This solution-like acidic sugar chain sample may be used immediately or may be stored until necessary according to the purpose. The storage method is preferably transferred to a closed container such as a commercially available tube with a lid as it is and stored frozen. More preferably, the method is freeze-dried and then stored frozen. The temperature for frozen storage is preferably −20 ° C. or lower, more preferably −80 ° C. or lower.
以下、本発明を実施例および比較例に基づいて詳細に説明するが、本発明はこれに限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a comparative example, this invention is not limited to this.
（実施例１）
（グリコサミノグリカンの分解）
１ｍｇ／ｍＬとなるよう１０ｍＭリン酸バッファーにヘパラン硫酸ナトリウム塩（ウシ腎臓由来、生化学工業株式会社）を溶解させた溶液４μＬとα-ＭＥＭ（インビトロジェン社製、）に１０％ウシ胎児血清（ＢＳＡ，インビトロジェン社製、）を加えた培地１００μＬと超純水（超純水製造装置MilliQにて製造、ミリポア社製）４００μＬを混合した溶液を限外濾過膜Ａｍｉｃｏｎ Ｕｌｔｒａ １０Ｋ、０．５ｍＬ（ミリポア社製、ＵＦＣ５０１０９６）を用いて２５℃、１４０００Ｇで３０分間遠心分離をかけた。再度水４８０μＬを加えて２５℃、１４０００Ｇで４０分間遠心分離をかけた後、回収した溶液２５μＬのうち１０μＬに消化用バッファー（１０ｍＭ酢酸ナトリウム三水和物、ナカライテスク株式会社、３１１１５−０５）、１０ｎＭ酢酸カルシウム一水和物（ナカライテスク株式会社、０６７１７−９２、ｐＨ = ７．０に調製）６μＬと０．１％ＢＳＡ（Ｅｑｕｉｔｅｃｈ−ｂｉｏ ｉｎｃ社製、ＢＡＣ６５−００１０）水溶液に溶解したヘパリナーゼ（生化学工業株式会社、１００７００）溶液５μＬ、０．１％ＢＳＡ水溶液に溶解したヘパリチナーゼ（生化学工業株式会社、１００７０３）溶液５μLを加え、全量が３０μLとなるよう超純水を加えて、３７℃で１時間インキュベートした。
Example 1
(Degradation of glycosaminoglycan)
10% fetal bovine serum (BSA) in 4 μL of a solution of heparan sulfate sodium salt (from bovine kidney, Seikagaku Corporation) dissolved in 10 mM phosphate buffer to 1 mg / mL and α-MEM (Invitrogen). , Manufactured by Invitrogen Corporation) and 400 μL of ultrapure water (manufactured by MilliQ manufactured by MilliQ, manufactured by Millipore) and ultrafiltration membrane Amicon Ultra 10K, 0.5 mL (Millipore) Manufactured by UFC501096) and centrifuged at 14,000 G for 30 minutes. After adding 480 μL of water again and centrifuging at 25 ° C. and 14000 G for 40 minutes, 10 μL of 25 μL of the collected solution was digested with a buffer for digestion (10 mM sodium acetate trihydrate, Nacalai Tesque, 31115-05), Heparinase dissolved in 10 nM calcium acetate monohydrate (Nacalai Tesque, Inc., 06717-92, prepared at pH = 7.0) and 0.1% BSA (Equitech-bio inc, BAC65-0010) aqueous solution ( Seikagaku Corporation, 100700) 5 μL of solution, 5 μL of heparitinase (Seikagaku Corporation, 100703) solution dissolved in 0.1% BSA aqueous solution was added, and ultrapure water was added so that the total amount was 30 μL, and 37 ° C. And incubated for 1 hour.
（グリコサミノグリカン分解物（二糖）の回収、標識）
糖鎖捕捉用の担体であるヒドラジド基を有する粒子５ｍｇ（ＢｌｏｔＧｌｙｃｏ（Ｒ））、住友ベークライト株式会社製、ＢＳ−４５６０１Ｓ、式２の構造を有し、モノマー仕込み比がｍ：ｎ＝２０：１のポリマー）が入ったディスポカラムに上記グリコサミノグリカン分解溶液２０μＬおよび１８０μＬの２％酢酸／アセトニトリル溶液を加え、８０℃で１時間反応させた。反応は開放系で行い、溶媒が完全に蒸発し粒子が乾固した状態であることを目視で確認した。グアニジン溶液、水、メタノール、トリエチルアミン溶液にて粒子を洗浄後、１０％無水酢酸／メタノールを添加し、室温で３０分間反応させ、未反応のヒドラジド基をキャッピングした。キャッピング後、メタノール、塩酸水溶液、水にて粒子を洗浄した。
(Recovery and labeling of glycosaminoglycan degradation products (disaccharides))
Particles having a hydrazide group, which is a carrier for capturing sugar chains (BlotGlyco®), manufactured by Sumitomo Bakelite Co., Ltd., BS-45601S, having the structure of Formula 2, and the monomer charge ratio is m: n = 20: 1 20 μL of the glycosaminoglycan decomposition solution and 180 μL of a 2% acetic acid / acetonitrile solution were added to the disposable column containing the polymer (2), and reacted at 80 ° C. for 1 hour. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the particles were dried. After washing the particles with guanidine solution, water, methanol and triethylamine solution, 10% acetic anhydride / methanol was added and reacted at room temperature for 30 minutes to cap unreacted hydrazide groups. After capping, the particles were washed with methanol, aqueous hydrochloric acid and water.
続いて、粒子の入ったディスポカラムに超純水２０μLおよび２％酢酸／アセトニトリル溶液１８０μＬを加え、８０℃で１．５時間反応させた。反応は開放系で行い、溶媒が完全に蒸発し粒子が乾固した状態であることを目視で確認した。
２−ａｍｉｎｏｂｅｎｚａｍｉｄｅ（２−ＡＢ、和光純薬、５７４−９２４４１）による標識を行った。粒子の入ったディスポカラムに、２−ＡＢおよびシアノ水素化ホウ素ナトリウムの終濃度がそれぞれ０．３５Ｍ、１Ｍになるように３０％酢酸／ジメチルスルホシキド（ＤＭＳＯ）混合溶媒に溶解させて調製した溶液５０μLを添加し、６０℃で２時間反応させた。
Subsequently, 20 μL of ultrapure water and 180 μL of a 2% acetic acid / acetonitrile solution were added to the disposable column containing the particles and reacted at 80 ° C. for 1.5 hours. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the particles were dried.
Labeling with 2-aminobenzamide (2-AB, Wako Pure Chemicals, 574-92441) was performed. It was prepared by dissolving in a 30% acetic acid / dimethylsulfoxide (DMSO) mixed solvent in a disposable column containing particles so that the final concentrations of 2-AB and sodium cyanoborohydride were 0.35M and 1M, respectively. 50 μL of the solution was added and reacted at 60 ° C. for 2 hours.
反応溶液５０μＬを回収し、アセトニトリルで１０倍に希釈した後、シリカカラム (ＢｌｏｔＧｌｙｃｏキット付属品)に添加してシリカゲルに標識糖鎖を吸着させた。アセトニトリルにてカラムを洗浄後、超純水５０μＬにて標識糖鎖を回収した。 50 μL of the reaction solution was recovered and diluted 10-fold with acetonitrile, and then added to a silica column (attached to the BlotGlyco kit) to adsorb the labeled sugar chain on the silica gel. After washing the column with acetonitrile, the labeled sugar chain was recovered with 50 μL of ultrapure water.
（標準二糖の作製）
不飽和ヘパラン／へパリン-二糖キット（生化学工業株式会社）の６種類の標準二糖溶液２０μＬ（１００μＭ）を用いて、２−ａｍｉｎｏｂｅｎｚａｍｉｄｅ（２−ＡＢ、和光, ５７４−９２４４１）による標識を行った。２−ＡＢおよびシアノ水素化ホウ素ナトリウムの終濃度がそれぞれ０．３５Ｍ、１Ｍになるように３０％酢酸／ＤＭＳＯ混合溶媒に溶解させて調製した溶液５０μLを添加し、６０℃で２時間反応させた。反応後は、アセトニトリルで１０倍に希釈した後、シリカカラム (ＢｌｏｔＧｌｙｃｏキット付属品)に添加してシリカゲルに標識糖鎖を吸着させた。アセトニトリル、アセトニトリル／水混合溶液（９５：５）にてカラムを洗浄後、超純水５０μＬにて標識糖鎖を回収した。６種類の糖鎖とは、以下の通りである。2-acetamido-2-deoxy-4-O-(4-deoxy-α-L-threo-hex-enopyranosyluronic acid)-D-glucose、2-deoxy-2-sulfamino-4-O-(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid)-D-glucose、2-acetamido-2-deoxy-4-O-(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose、2-deoxy-2-sulfamino-(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose、2-deoxy-2-sulfamino-(4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronc acid)-D-glucose、2-deoxy-2-sulfamino-(4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronc acid)-6-O-Sulfo-D-glucoseである。
(Production of standard disaccharide)
Labeling with 2-aminobenzamide (2-AB, Wako, 574-92441) was performed using 20 μL (100 μM) of six standard disaccharide solutions of an unsaturated heparan / heparin-disaccharide kit (Seikagaku Corporation). went. 50 μL of a solution prepared by dissolving in a 30% acetic acid / DMSO mixed solvent was added so that the final concentrations of 2-AB and sodium cyanoborohydride were 0.35 M and 1 M, respectively, and reacted at 60 ° C. for 2 hours. . After the reaction, the reaction mixture was diluted 10-fold with acetonitrile, and then added to a silica column (attached to the BlotGlyco kit) to adsorb the labeled sugar chain on the silica gel. After washing the column with acetonitrile / acetonitrile / water mixed solution (95: 5), the labeled sugar chain was recovered with 50 μL of ultrapure water. The six types of sugar chains are as follows. 2-acetamido-2-deoxy-4-O- (4-deoxy-α-L-threo-hex-enopyranosyluronic acid) -D-glucose, 2-deoxy-2-sulfamino-4-O- (4-deoxy- α-L-threo-hex-4-enopyranosyluronic acid) -D-glucose, 2-acetamido-2-deoxy-4-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6 -O-sulfo-D-glucose, 2-deoxy-2-sulfamino- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose, 2-deoxy- 2-sulfamino- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronc acid) -D-glucose, 2-deoxy-2-sulfamino- (4-deoxy-2-O- sulfo-α-L-threo-hex-4-enopyranosyluronc acid) -6-O-Sulfo-D-glucose.
（グリコサミノグリカン鎖由来２糖の検出）
回収した糖鎖水溶液５μLを、高速液体クロマトグラフ（ＬａＣｈｒｏｍ Ｅｌｉｔｅ、株式会社日立ハイテクノロジーズ製）にて解析した。カラムは、順相クロマトグラフィーカラム（ＴＳＫ−ＧＥＬ Ａｍｉｄｅ−８０、東ソー株式会社）を用い、移動相には５０ｍＭ ギ酸アンモニウム水溶液(ｐＨ４．４）とアセトニトリルを使用した。移動相は、５０ｍＭ ギ酸アンモニウム水溶液(ｐＨ４．４）が２０％から４１．７％に８０分かけて変化するように勾配をつけ、流速０．４ｍＬ／ｍｉｎ、カラム温度３０℃で流し、蛍光（励起波長３３０ ｎｍ、蛍光波長４２０ ｎｍ）にて検出した。得られたＨＰＬＣチャートを比較した結果、標準二糖のピークの溶出時間と一致する時間にグリコサミノグリカン分解物のピークも得られた（図２、３）。本手法により、生体試料中からグリコサミノグリカン糖鎖由来のピークが精製、標識できる事を確認した。
（実施例２）
（グリコサミノグリカンの分解）
実施例１と同様の方法にて処理を行った。
(Detection of disaccharide derived from glycosaminoglycan chain)
5 μL of the recovered sugar chain aqueous solution was analyzed by a high performance liquid chromatograph (LaChrom Elite, manufactured by Hitachi High-Technologies Corporation). The column used was a normal phase chromatography column (TSK-GEL Amide-80, Tosoh Corporation), and 50 mM ammonium formate aqueous solution (pH 4.4) and acetonitrile were used for the mobile phase. The mobile phase was graded so that the 50 mM ammonium formate aqueous solution (pH 4.4) changed from 20% to 41.7% over 80 minutes, and flowed at a flow rate of 0.4 mL / min and a column temperature of 30 ° C., and fluorescence ( Detection was performed at an excitation wavelength of 330 nm and a fluorescence wavelength of 420 nm. As a result of comparing the obtained HPLC charts, a glycosaminoglycan degradation product peak was also obtained at a time that coincided with the elution time of the standard disaccharide peak (FIGS. 2 and 3). By this method, it was confirmed that a peak derived from a glycosaminoglycan sugar chain could be purified and labeled from a biological sample.
(Example 2)
(Degradation of glycosaminoglycan)
The treatment was performed in the same manner as in Example 1.
（グリコサミノグリカン分解物（二糖）の回収、標識）
糖鎖捕捉用の担体であるヒドラジド基を有する粒子５ｍｇ（ＢｌｏｔＧｌｙｃｏ（Ｒ））、住友ベークライト株式会社製、ＢＳ−４５６０１Ｓ、式２の構造を有し、モノマー仕込み比がｍ：ｎ＝２０：１のポリマー）が入ったディスポカラムに上記グリコサミノグリカン分解溶液２０μＬおよび１８０μＬの２％酢酸／アセトニトリル溶液を加え、８０℃で１時間反応させた。反応は開放系で行い、溶媒が完全に蒸発し粒子が乾固した状態であることを目視で確認した。グアニジン溶液、水、メタノール、トリエチルアミン溶液にて粒子を洗浄後、１０％無水酢酸／メタノールを添加し、室温で３０分間反応させ、未反応のヒドラジド基をキャッピングした。キャッピング後、メタノール、塩酸水溶液、水にて粒子を洗浄した。
(Recovery and labeling of glycosaminoglycan degradation products (disaccharides))
Particles having a hydrazide group, which is a carrier for capturing sugar chains (BlotGlyco®), manufactured by Sumitomo Bakelite Co., Ltd., BS-45601S, having the structure of Formula 2, and the monomer charge ratio is m: n = 20: 1 20 μL of the glycosaminoglycan decomposition solution and 180 μL of a 2% acetic acid / acetonitrile solution were added to the disposable column containing the polymer (2), and reacted at 80 ° C. for 1 hour. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the particles were dried. After washing the particles with guanidine solution, water, methanol and triethylamine solution, 10% acetic anhydride / methanol was added and reacted at room temperature for 30 minutes to cap unreacted hydrazide groups. After capping, the particles were washed with methanol, aqueous hydrochloric acid and water.
続いて、粒子の入ったディスポカラムに２０ｍＭ ２−ａｍｉｎｏｂｅｎｚｈｙｄｒａｚｉｄｅ（２ＡＢｈ、Ａｌｄｒｉｃｈ、５６５４９０）水溶液２０μＬおよび２％酢酸／アセトニトリル溶液１８０μＬを加え、７０℃で１．５時間反応させた。反応は開放系で行い、溶媒が完全に蒸発し粒子が乾固した状態であることを目視で確認した。 Subsequently, 20 μL of 20 mM 2-aminobenzhydrazide (2ABh, Aldrich, 565490) aqueous solution and 180 μL of 2% acetic acid / acetonitrile solution were added to the disposable column containing the particles and reacted at 70 ° C. for 1.5 hours. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the particles were dried.
水５０μＬを添加し、２ＡＢｈで標識された糖鎖を回収し、アセトニトリルで１０倍に希釈した後、シリカカラム (ＢｌｏｔＧｌｙｃｏキット付属品)に添加してシリカゲルに標識糖鎖を吸着させた。アセトニトリルにてカラムを洗浄後、超純水５０μＬにて標識糖鎖を回収した。 50 μL of water was added, and the sugar chain labeled with 2ABh was recovered, diluted 10-fold with acetonitrile, and then added to a silica column (attached to the BlotGlyco kit) to adsorb the labeled sugar chain onto silica gel. After washing the column with acetonitrile, the labeled sugar chain was recovered with 50 μL of ultrapure water.
（グリコサミノグリカン鎖由来２糖の検出）
回収した糖鎖水溶液５μLを、高速液体クロマトグラフ（ＬａＣｈｒｏｍ Ｅｌｉｔｅ、株式会社日立ハイテクノロジーズ製）にて解析した。カラムは、順相クロマトグラフィーカラム（ＴＳＫ−ＧＥＬ Ａｍｉｄｅ−８０、東ソー株式会社）を用い、移動相には５０ｍＭ ギ酸アンモニウム水溶液(ｐＨ４．４）とアセトニトリルを使用した。移動相は、５０ｍＭ ギ酸アンモニウム水溶液(ｐＨ４．４）が２０％から４１．７％に８０分かけて変化するように勾配をつけ、流速０．４ｍＬ／ｍｉｎ、カラム温度３０℃で流し、蛍光（励起波長３３０ ｎｍ、蛍光波長４２０ ｎｍ）にて検出した。２ＡＢｈで標識された糖鎖由来のピークが検出された。
(Detection of disaccharide derived from glycosaminoglycan chain)
5 μL of the recovered sugar chain aqueous solution was analyzed by a high performance liquid chromatograph (LaChrom Elite, manufactured by Hitachi High-Technologies Corporation). The column used was a normal phase chromatography column (TSK-GEL Amide-80, Tosoh Corporation), and 50 mM ammonium formate aqueous solution (pH 4.4) and acetonitrile were used for the mobile phase. The mobile phase was graded so that the 50 mM ammonium formate aqueous solution (pH 4.4) changed from 20% to 41.7% over 80 minutes, and flowed at a flow rate of 0.4 mL / min and a column temperature of 30 ° C., and fluorescence ( Detection was performed at an excitation wavelength of 330 nm and a fluorescence wavelength of 420 nm. A peak derived from a sugar chain labeled with 2ABh was detected.
本発明により、簡便に酸性糖鎖を回収、精製し、必要に応じて標識化合物による標識を実施でき、該酸性糖鎖を提供することが可能となった。 According to the present invention, acidic sugar chains can be easily recovered and purified, and labeled with a labeling compound as necessary, and the acidic sugar chains can be provided.
Claims (21)
酸性糖鎖を含有する溶液を、糖鎖固相担体と接触させて酸性糖鎖を捕捉する工程と、
前記酸性糖鎖を捕捉した糖鎖固相担体より前記酸性糖鎖を切り出し、該酸性糖鎖を回収する工程と、を有する酸性糖鎖の調製方法 A method for preparing acidic sugar chains from a solution containing acidic sugar chains,
A step of capturing the acidic sugar chain by contacting a solution containing the acidic sugar chain with a sugar chain solid phase carrier;
A method of preparing an acidic sugar chain, comprising: cutting out the acidic sugar chain from a sugar chain solid phase carrier that has captured the acidic sugar chain, and recovering the acidic sugar chain
（ａ）酸性多糖から酵素処理により断片糖鎖を作製する工程と、
（ｂ）該酸性糖鎖と糖鎖固相担体をヒドラゾン結合により結合させる工程と、
（ｃ）糖鎖を固相担体から切り離した後、アミノ基を有する化合物を作用させて、還元的アミノ化反応により前記化合物に結合させる工程を含む酸性糖鎖の調製方法。 (A) a step of producing a fragment sugar chain from an acidic polysaccharide by enzymatic treatment in the method for preparing an acidic sugar chain according to any one of claims 1 to 7;
(B) a step of binding the acidic sugar chain and the sugar chain solid phase carrier by a hydrazone bond;
(C) A method for preparing an acidic sugar chain, which comprises a step of separating a sugar chain from a solid phase carrier, allowing a compound having an amino group to act, and binding to the compound by a reductive amination reaction.
8-Aminopyrene-1,3,6-trisulfonate, 8-Aminonaphthalene-1,3,6-trisulphonate, 7-Amino-1,3-naphtalenedisulfonic acid, 2-Amino9(10H)-acridone,5-Aminofluorescein,Dansylethylenediamie, 2-Aminopyridine, 7-Amino-4-methylcoumarine, 2-Aminobenzamide, 2-Aminobenzoic acid, 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3-(Aｃetｈylamino)-6-aminoacridine, 2-Amino-6-cyanoethylpyridine,Ethyl p-aminobenzoate, p-Aminobenzonitrile, 及び7-aminonaphothalene-1,3-disulfonic acid
から選ばれる少なくとも１つである請求項８又は９記載の酸性糖鎖の調製方法。 The compound having an amino group is
8-Aminopyrene-1,3,6-trisulfonate, 8-Aminonaphthalene-1,3,6-trisulphonate, 7-Amino-1,3-naphtalenedisulfonic acid, 2-Amino9 (10H) -acridone, 5-Aminofluorescein, Dansylethylenediamie, 2-Aminopyridine, 7-Amino-4-methylcoumarine, 2-Aminobenzamide, 2-Aminobenzoic acid, 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3- (Acetylamino) -6-aminoacridine, 2-Amino-6- cyanoethylpyridine, Ethyl p-aminobenzoate, p-Aminobenzonitrile, and 7-aminonaphothalene-1,3-disulfonic acid
The method for preparing an acidic sugar chain according to claim 8 or 9, which is at least one selected from the group consisting of:
（ａ）酸性多糖から酵素処理により断片糖鎖を作製する工程と、
（ｂ）該酸性糖鎖と糖鎖固相担体をヒドラゾン結合により結合させる工程と、
（ｃ）ヒドラジド基を有する化合物、またはアミノオキシ基を有する化合物を作用させて、ヒドラゾン−オキシム交換反応により糖鎖を固相担体から切り離しつつ、前記化合物に結合させる工程を含む酸性糖鎖の調製方法。 (A) a step of producing a fragment sugar chain from an acidic polysaccharide by enzymatic treatment in the method for preparing an acidic sugar chain according to any one of claims 1 to 7;
(B) a step of binding the acidic sugar chain and the sugar chain solid phase carrier by a hydrazone bond;
(C) Preparation of an acidic sugar chain comprising a step of allowing a compound having a hydrazide group or a compound having an aminooxy group to act and separating the sugar chain from a solid phase carrier by a hydrazone-oxime exchange reaction and binding to the compound Method.
5-Dimethylaminonaphthalene-1-sulfonyl hydrazine (Dansylhydrazine)； 2-hydrazinopyridine；9-fluorenylmethyl carbazate (Fmoc hydrazine)；benzylhydrazine；4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionoc acid, hydrazide；2-(6,8-difluoro-7-hydroxy-4-methylcoumarin)acetohydrazide；7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH)；phenylhydrazine；1-Naphthaleneacethydrazide；2-hydrazinobenzoic acid；biotin hydrazide；phenylacetic hydrazide. The method for preparing an acidic sugar chain according to claim 12, wherein the compound having a hydrazide group in step (c) is a substance selected from the following or a salt thereof.
5-Dimethylaminonaphthalene-1-sulfonyl hydrazine (Dansylhydrazine); 2-hydrazinopyridine; 9-fluorenylmethyl carbazate (Fmoc hydrazine); benzylhydrazine; 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s -indacene-3-propionoc acid, hydrazide; 2- (6,8-difluoro-7-hydroxy-4-methylcoumarin) acetohydrazide; 7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH); phenylhydrazine; 1-Naphthaleneacethydrazide; 2 -hydrazinobenzoic acid; biotin hydrazide; phenylacetic hydrazide.
O-benzylhydroxylamine;O-phenylhydroxylamine; O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine; O-(4-nitrobenzyl)hydroxylamine; 2-aminooxypyridine; 2-aminooxymethylpyridine; 4-[(aminooxyacetyl)amino]benzoic acid methyl ester; 4-[(aminooxyacetyl)amino]benzoic acid ethyl ester; 4-[(aminooxyacetyl)amino]benzoic acid n-butyl ester；N-aminooxyacetyl-tryptophyl(arginine methyl ester) The method for preparing an acidic sugar chain according to claim 12, wherein the compound having an aminooxy group in step (c) is a substance selected from the following or a salt thereof.
O-benzylhydroxylamine; O-phenylhydroxylamine; O- (2,3,4,5,6-pentafluorobenzyl) hydroxylamine; O- (4-nitrobenzyl) hydroxylamine; 2-aminooxypyridine; 2-aminooxymethylpyridine; 4-[(aminooxyacetyl) amino] 4-[(aminooxyacetyl) amino] benzoic acid ethyl ester; 4-[(aminooxyacetyl) amino] benzoic acid n-butyl ester; N-aminooxyacetyl-tryptophyl (arginine methyl ester)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103641935A (en) * | 2013-11-29 | 2014-03-19 | 济南天天香有限公司 | Extraction method of chondroitin sulfate |
| JP2017067677A (en) * | 2015-10-01 | 2017-04-06 | 住友ベークライト株式会社 | Method for preparing purified substance of label sugar |
| CN109652350A (en) * | 2019-03-01 | 2019-04-19 | 中国农业科学院农业资源与农业区划研究所 | One plant of tylosin degradation bacteria and its application |
| JP2020173228A (en) * | 2019-04-12 | 2020-10-22 | 株式会社島津製作所 | Glycosaminoglycan analysis method |
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| WO2008018170A1 (en) * | 2006-08-09 | 2008-02-14 | Sumitomo Bakelite Co., Ltd. | Sugar chain-capturing substance and use thereof |
| WO2009133696A1 (en) * | 2008-04-30 | 2009-11-05 | 住友ベークライト株式会社 | Method for labelling sugar chains |
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| JPS5968301A (en) * | 1982-10-12 | 1984-04-18 | Sumitomo Chem Co Ltd | Production of polysaccharide |
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| CN103641935A (en) * | 2013-11-29 | 2014-03-19 | 济南天天香有限公司 | Extraction method of chondroitin sulfate |
| JP2017067677A (en) * | 2015-10-01 | 2017-04-06 | 住友ベークライト株式会社 | Method for preparing purified substance of label sugar |
| CN109652350A (en) * | 2019-03-01 | 2019-04-19 | 中国农业科学院农业资源与农业区划研究所 | One plant of tylosin degradation bacteria and its application |
| CN109652350B (en) * | 2019-03-01 | 2022-01-28 | 中国农业科学院农业资源与农业区划研究所 | Tylosin degrading bacterium and application thereof |
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| JP7185232B2 (en) | 2019-04-12 | 2022-12-07 | 株式会社島津製作所 | Methods for analyzing glycosaminoglycans |
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