JP2015091953A - Method for labeling carbohydrate chain by 2-aminopyridine - Google Patents
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本発明は，還元末端が２−アミノピリジンで標識された糖鎖を得るための方法に関する。 The present invention relates to a method for obtaining a sugar chain whose reducing end is labeled with 2-aminopyridine.
糖蛋白質の糖鎖構造の分析方法として，糖蛋白質をトリプシン処理し，次いでグリコシダーゼで処理して，糖蛋白質から糖鎖を遊離させた後，この糖鎖を２−アミノピリジンで標識（２−アミノピリジン標識）し，これを順相カラムクロマトグラフィーに付して分離分析する方法が知られている（非特許文献１）。 As a method for analyzing the sugar chain structure of a glycoprotein, the glycoprotein is treated with trypsin and then treated with glycosidase to release the sugar chain from the glycoprotein, and then this sugar chain is labeled with 2-aminopyridine (2-aminopyridine). A method of separating and analyzing by subjecting to normal phase column chromatography is known (Non-patent Document 1).
糖鎖の２−アミノピリジン標識は，例えば，糖鎖を含む試料に，２−アミノピリジン溶液を添加して加熱反応させてイミンを形成させた後，ボラン−ジメチルアミン錯体溶液を添加して加熱し，イミンを還元することにより行われる（非特許文献１）。次いで，未反応の２−アミノピリジン等を除去するため，反応後の溶液を，トリエチルアミンとメタノールの１：１混合液，及びトルエンを添加してから乾燥させて乾燥物とし，この乾燥物を，メタノール及びトルエンに溶解させた後，再度乾燥させてから，水性溶媒に溶解させ，この溶液を，ゲルろ過カラムクロマトグラフィーに付して，２−アミノピリジン標識された糖鎖を分取する。このように，糖鎖を２−アミノピリジンで標識する方法は，トルエン等の人体に有害な有機溶媒を気化させて除去する工程を含む（特許文献１，非特許文献２）。従って，糖鎖を２−アミノピリジンで標識するために使用する特殊な装置（Ｔａｋａｒａ ＰＡＬＳＴＡＴＩＯＮｍｏｄｅｌ ４０００（タカラバイオ社））が市販されていた。 For example, a 2-aminopyridine label of a sugar chain is heated by adding a 2-aminopyridine solution to a sample containing a sugar chain and reacting it with heating to form an imine, and then adding a borane-dimethylamine complex solution. The imine is reduced (Non-patent Document 1). Next, in order to remove unreacted 2-aminopyridine and the like, the solution after the reaction is dried by adding a 1: 1 mixture of triethylamine and methanol and toluene, and dried to obtain a dried product. After dissolving in methanol and toluene, drying again, and then dissolving in an aqueous solvent, this solution is subjected to gel filtration column chromatography to fractionate 2-aminopyridine-labeled sugar chains. As described above, the method for labeling sugar chains with 2-aminopyridine includes a step of vaporizing and removing an organic solvent harmful to the human body such as toluene (Patent Document 1, Non-Patent Document 2). Therefore, a special apparatus (Takara PALSTATION model 4000 (Takara Bio Inc.)) used for labeling sugar chains with 2-aminopyridine was commercially available.
上記背景の下で，本発明の目的は，還元末端が２−アミノピリジンで標識された糖鎖を得るための新たな方法を提供することである。 In view of the above background, an object of the present invention is to provide a new method for obtaining a sugar chain whose reducing end is labeled with 2-aminopyridine.
上記目的に向けた研究において，本発明者は，鋭意検討を重ねた結果，糖鎖の還元末端を２−アミノピリジン標識させる反応において，反応過程において気化させて除去する有機溶媒の量を減じることができる新たな方法を見出し，本発明を完成した。すなわち，本発明は以下を提供する。
（１）還元末端が２−アミノピリジンで標識された糖鎖を得るための方法であって，
還元末端を有する糖鎖と２−アミノピリジンとを含む溶液を加熱して，該還元末端と該２−アミノピリジンとを反応させることによりイミンを形成させた後，該溶液にボラン−ジメチルアミン錯体を添加して加熱し，該イミンを還元させることにより，還元末端が２−アミノピリジンで標識された標識化糖鎖を含む反応液を得るステップと，
該反応液にトルエンを添加し，該標識化糖鎖を沈殿させるとともに，未反応の２−アミノピリジンをトルエンとともに除去するステップと，
該沈殿を水性溶媒に溶解させて得られた溶液を，ゲルろ過カラムクロマトグラフィーに付して，該標識化糖鎖を含有する画分を回収するステップとを，
この順で含んでなるものである方法。
（２）該ゲルろ過カラムクロマトグラフィーにおいて，カラムに充填される樹脂の排除限界が，１．５×１０３Ｄａを超えないものである上記（１）に記載の方法。
（３）該ゲルろ過カラムクロマトグラフィーにおいて，カラムに充填される樹脂の排除限界が，６．５×１０２〜７．５×１０２Ｄａである上記（１）に記載の方法。
（４）該糖鎖が，糖蛋白質から切り出して得られるものである，上記（１）〜（３）のいずれかに記載の方法。
（５）該糖蛋白質が，組換え糖蛋白質である，上記（４）に記載の方法。
（６）該糖蛋白質が，イズロン酸−２−スルファターゼ，α−ガラクトシダーゼＡ，酸性スフィンゴミエリナーゼ，α−Ｌ−イズロニダーゼ，Ｎ−アセチルガラクトサミン−４−スルファターゼ，グルコセレブロシダーゼ（グルコシルセラミダーゼ），ガルスルファーゼ，リソソーム酸性リパーゼ，酸性α−グルコシダーゼ等のリソソーム酵素，組織プラスミノーゲンアクチベーター（ｔ−ＰＡ），血液凝固第ＶＩＩ因子，血液凝固第ＶＩＩＩ因子，血液凝固第ＩＸ因子等の血液凝固因子，エリスロポエチン，インターフェロン，トロンボモジュリン，卵胞刺激ホルモン，甲状腺刺激ホルモン，ＧＭ−ＣＳＦ，Ｇ−ＣＳＦ，Ｍ−ＣＳＦ，及び抗体からなる群から選択されるものである，上記（４）又は（５）に記載の方法。As a result of extensive studies, the present inventor has reduced the amount of the organic solvent to be removed by vaporization in the reaction process in the reaction of labeling the reducing end of the sugar chain with 2-aminopyridine. The present inventors have found a new method that can do this and completed the present invention. That is, the present invention provides the following.
(1) A method for obtaining a sugar chain in which the reducing end is labeled with 2-aminopyridine,
A solution containing a sugar chain having a reducing end and 2-aminopyridine is heated to form an imine by reacting the reducing end with the 2-aminopyridine, and then the borane-dimethylamine complex is added to the solution. Adding and heating to reduce the imine, thereby obtaining a reaction solution containing a labeled sugar chain in which the reducing end is labeled with 2-aminopyridine;
Adding toluene to the reaction solution to precipitate the labeled sugar chain and removing unreacted 2-aminopyridine together with toluene;
Subjecting the solution obtained by dissolving the precipitate to an aqueous solvent to gel filtration column chromatography, and collecting the fraction containing the labeled sugar chain;
A method that comprises in this order.
(2) The method according to (1) above, wherein in the gel filtration column chromatography, the exclusion limit of the resin packed in the column does not exceed 1.5 × 10 3 Da.
(3) The method according to (1) above, wherein in the gel filtration column chromatography, the exclusion limit of the resin packed in the column is 6.5 × 10 2 to 7.5 × 10 2 Da.
(4) The method according to any one of (1) to (3) above, wherein the sugar chain is obtained by cutting out from a glycoprotein.
(5) The method according to (4) above, wherein the glycoprotein is a recombinant glycoprotein.
(6) The glycoprotein is iduronic acid-2-sulfatase, α-galactosidase A, acid sphingomyelinase, α-L-iduronidase, N-acetylgalactosamine-4-sulfatase, glucocerebrosidase (glucosylceramidase), galsulfase, Lysosomal enzymes such as lysosomal acid lipase and acid α-glucosidase, tissue plasminogen activator (t-PA), blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor such as blood coagulation factor IX, erythropoietin, The method according to (4) or (5) above, which is selected from the group consisting of interferon, thrombomodulin, follicle stimulating hormone, thyroid stimulating hormone, GM-CSF, G-CSF, M-CSF, and antibody.
本発明によれば，多量のトルエンを気化させることなく，還元末端が２−アミノピリジンで標識された糖鎖を得ることができる。 According to the present invention, a sugar chain having a reducing end labeled with 2-aminopyridine can be obtained without vaporizing a large amount of toluene.
糖又は糖鎖の還元末端においては，環状構造をとる糖が開環して鎖状構造となったときにアルデヒド基又はケトン基が生じ得る。本発明において，２−アミノピリジン標識（又はアミノピリジン標識）というときは，糖又は糖鎖の還元末端を２−アミノピリジンと反応させることによりイミンを形成させ，次いでのこのイミンをボラン−ジメチルアミン錯体等を用いて還元させることにより，糖又は糖鎖の還元末端に２−アミノピリジンを付加させることをいう。また，本発明において，２−アミノピリジン標識化糖鎖，アミノピリジン標識化糖鎖，又は標識化糖鎖というときは，２−アミノピリジン標識（又はアミノピリジン標識）された糖又は糖鎖のことをいう。 At the reducing end of a sugar or sugar chain, an aldehyde group or a ketone group can be generated when a sugar having a cyclic structure is opened to form a chain structure. In the present invention, when 2-aminopyridine labeling (or aminopyridine labeling) is used, an imine is formed by reacting the reducing end of a sugar or sugar chain with 2-aminopyridine, and this imine is then converted to borane-dimethylamine. This refers to addition of 2-aminopyridine to the reducing end of a sugar or sugar chain by reduction using a complex or the like. In the present invention, the term “2-aminopyridine labeled sugar chain, aminopyridine labeled sugar chain, or labeled sugar chain” refers to a sugar or sugar chain labeled with 2-aminopyridine (or aminopyridine labeled). Say.
アミノピリジン標識における，糖又は糖鎖の還元末端と２−アミノピリジンとを反応させてイミンを形成させる反応の一例を，下記の式（Ｉ）に示す。式（Ｉ）で示された反応は，糖鎖の還元末端に位置するＮ−アセチルグルコサミン（ＧｌｃＮＡｃ）と２−アミノピリジンとの反応である。 An example of a reaction for reacting a reducing end of a sugar or sugar chain with 2-aminopyridine to form an imine in aminopyridine labeling is shown in the following formula (I). The reaction represented by the formula (I) is a reaction between N-acetylglucosamine (GlcNAc) located at the reducing end of the sugar chain and 2-aminopyridine.
アミノピリジン標識における，イミンをボラン−ジメチルアミン錯体により還元させる反応の一例を下記の式（ＩＩ）に示す。式（ＩＩ）で示された反応は，式（Ｉ）の反応で生じたイミンの，ボラン−ジメチルアミン錯体による還元反応である。 An example of a reaction for reducing an imine with a borane-dimethylamine complex in an aminopyridine label is shown in the following formula (II). The reaction represented by the formula (II) is a reduction reaction of the imine produced by the reaction of the formula (I) with a borane-dimethylamine complex.
本発明において，糖鎖の還元末端と２−アミノピリジンとを反応させてイミンを形成させるときの温度及び時間は，かかる反応が促進される温度及び時間であれば特に限定はないが，好ましくは７５〜８５℃で４０分〜２時間であり，より好ましくは約８０℃で約１時間である。また，ボラン−ジメチルアミン錯体により該イミンを還元させるときの温度及び時間は，かかる反応が促進される温度及び時間であれば特に限定はないが，好ましくは７５〜８５℃で４０分〜２時間であり，より好ましくは約８０℃で約１時間である。 In the present invention, the temperature and time when the reducing end of the sugar chain is reacted with 2-aminopyridine to form an imine are not particularly limited as long as the temperature and time are such that the reaction is promoted. It is 40 minutes to 2 hours at 75 to 85 ° C, more preferably about 1 hour at about 80 ° C. The temperature and time when the imine is reduced by the borane-dimethylamine complex are not particularly limited as long as the reaction and the temperature are accelerated, but preferably at 75 to 85 ° C. for 40 minutes to 2 hours. And more preferably at about 80 ° C. for about 1 hour.
本発明において，ボラン−ジメチルアミン錯体により該イミンを還元することにより，糖鎖の還元末端をアミノピリジンで標識する反応は完了する。しかし，反応後の溶液中には未反応の２−アミノピリジンが残存するので，糖鎖の分析を行うためには，上記還元反応後の溶液から未反応の２−アミノピリジンを除去する必要がある。未反応の２−アミノピリジンは，還元反応後の溶液にトルエンを添加し，トルエンで抽出することにより除去することができる。 In the present invention, the reaction for labeling the reducing end of the sugar chain with aminopyridine is completed by reducing the imine with a borane-dimethylamine complex. However, since unreacted 2-aminopyridine remains in the solution after the reaction, it is necessary to remove the unreacted 2-aminopyridine from the solution after the reduction reaction in order to analyze the sugar chain. is there. Unreacted 2-aminopyridine can be removed by adding toluene to the solution after the reduction reaction and extracting with toluene.
未反応の２−アミノピリジンを除去するために還元反応後の溶液に添加するトルエンの量は，好ましくは当該溶液の２〜４倍容であり，より好ましくは約２．５倍容である。トルエンを添加して混和した後，遠心分離又は静置して，糖鎖を沈殿させるとともにトルエン層の上層を除去する。このトルエンにより未反応の２−アミノピリジンの除去する操作（トルエン抽出）は，好ましくは３〜５回，より好ましくは３回繰り返して行われる。最後のトルエン抽出の際には，沈殿を残すようにして，可能な限りの溶液を除去する。ここで除去したトルエンを含む液は，有機溶媒廃棄用の容器に回収されるので，この過程でトルエンが気化することはほとんどない。 The amount of toluene added to the solution after the reduction reaction in order to remove unreacted 2-aminopyridine is preferably 2 to 4 times the volume of the solution, more preferably about 2.5 times the volume. Toluene is added and mixed, and then centrifuged or allowed to stand to precipitate sugar chains and remove the upper layer of the toluene layer. The operation of removing unreacted 2-aminopyridine with toluene (toluene extraction) is preferably performed 3 to 5 times, more preferably 3 times. During the final toluene extraction, remove as much of the solution as possible, leaving a precipitate. Since the liquid containing toluene removed here is collected in a container for organic solvent disposal, toluene is hardly vaporized in this process.
トルエンにより未反応の２−アミノピリジンを除去した後，沈殿した糖鎖は乾燥される。このときの乾燥は，好ましくは減圧乾燥により行われる。このときトルエンが気化するが，その量は極わずかである。 After removing unreacted 2-aminopyridine with toluene, the precipitated sugar chain is dried. The drying at this time is preferably performed by drying under reduced pressure. At this time, toluene is vaporized, but the amount is very small.
減圧乾燥されたアミノピリジン標識化糖鎖は，水性溶媒に溶解されて，次いでゲルろ過カラムクロマトグラフィーに付される。ゲルろ過カラムクロマトグラフィーにより，トルエン抽出により除去されなかった未反応の２−アミノピリジンが更に除去されるとともに，バファー交換がされる。このとき，アミノピリジン標識化糖鎖を溶解させるための水性溶媒は，好ましくは純水である。また，ゲルろ過カラムクロマトグラフィーに用いるカラムに充填されるべき樹脂の球状蛋白質に対する排除限界は，好ましくは１．５×１０３Ｄａを超えないものであり，より好ましくは６×１０２Ｄａ〜１．５×１０３Ｄａであり，更に好ましくは６．５×１０２Ｄａ〜７．５×１０２Ｄａであり，更により好ましくは約７×１０２Ｄａである。市販のＳＥＰＨＡＤＥＸＴＭＧ−１０（排除限界：７×１０２Ｄａ），ＳＥＰＨＡＤＥＸＴＭＧ−１５（排除限界：１．５×１０３Ｄａ）等は，本発明において好適に使用できる。The aminopyridine-labeled sugar chain dried under reduced pressure is dissolved in an aqueous solvent and then subjected to gel filtration column chromatography. By gel filtration column chromatography, unreacted 2-aminopyridine that has not been removed by toluene extraction is further removed, and buffer exchange is performed. At this time, the aqueous solvent for dissolving the aminopyridine-labeled sugar chain is preferably pure water. Moreover, the exclusion limit with respect to the globular protein of the resin to be packed in the column used for gel filtration column chromatography is preferably not more than 1.5 × 10 3 Da, more preferably 6 × 10 2 Da to 1 .5 × a 10 3 Da, more preferably from 6.5 × 10 2 Da~7.5 × 10 2 Da, even more preferably from about 7 × 10 2 Da. Commercially available SEPHADEX ™ G-10 (exclusion limit: 7 × 10 2 Da), SEPHADEX ™ G-15 (exclusion limit: 1.5 × 10 3 Da) and the like can be suitably used in the present invention.
本発明の方法で得られたアミノピリジン標識化糖鎖は，これを順相カラムクロマトグラフィーに付し，カラム通過後の溶液に，蛍光検出器で励起光を連続的に照射して，流出液から放射される蛍光の強度を測定することにより分析できる。このとき用いられる順相カラムクロマトグラフィーでカラムに充填されるべき担体は，分析対象の糖鎖の分子量に応じて適宜選択されるべきものであるが，アミノプロピル基を官能基として保持する担体が，この目的で好適に使用できる。 The aminopyridine-labeled sugar chain obtained by the method of the present invention is subjected to normal phase column chromatography, and the solution after passing through the column is continuously irradiated with excitation light with a fluorescence detector to produce an effluent. Can be analyzed by measuring the intensity of the fluorescence emitted from. The carrier to be packed in the column by normal phase column chromatography used at this time should be appropriately selected according to the molecular weight of the sugar chain to be analyzed, but a carrier that holds an aminopropyl group as a functional group is used. , Can be suitably used for this purpose.
本発明において，糖蛋白質の糖鎖の還元末端をアミノピリジン標識する場合，糖蛋白質から糖鎖を予め切り出す必要がある。糖蛋白質からの糖鎖の切り出しは，酵素処理又は化学処理して行う。酵素処理は，Ｎ−グリコシダーゼ，グリコペプチダーゼＡ，Ｏ−グリコシダーゼ等を用いて行い，化学処理は，ヒドラジン分解等により行う。これらの処理により，糖蛋白質中のアスパラギン残基，セリン残基，スレオニン残基等に結合した糖鎖が切り出される。 In the present invention, when the reducing end of a sugar chain of a glycoprotein is labeled with aminopyridine, it is necessary to cut out the sugar chain from the glycoprotein in advance. Cutting out sugar chains from glycoproteins is performed by enzymatic treatment or chemical treatment. The enzyme treatment is performed using N-glycosidase, glycopeptidase A, O-glycosidase, etc., and the chemical treatment is performed by hydrazine degradation or the like. By these treatments, sugar chains bound to asparagine residues, serine residues, threonine residues and the like in glycoproteins are cut out.
本発明において，糖鎖が分析対象となる糖蛋白質は，特に限定はないが，好ましくはイズロン酸−２−スルファターゼ，α−ガラクトシダーゼＡ，酸性スフィンゴミエリナーゼ，α−Ｌ−イズロニダーゼ，Ｎ−アセチルガラクトサミン−４−スルファターゼ，グルコセレブロシダーゼ（グルコシルセラミダーゼ），ガルスルファーゼ，リソソーム酸性リパーゼ，酸性α−グルコシダーゼ等のリソソーム酵素，組織プラスミノーゲンアクチベーター（ｔ−ＰＡ），血液凝固第ＶＩＩ因子，血液凝固第ＶＩＩＩ因子，血液凝固第ＩＸ因子等の血液凝固因子，エリスロポエチン，インターフェロン，トロンボモジュリン，卵胞刺激ホルモン，甲状腺刺激ホルモン，ＧＭ−ＣＳＦ，Ｇ−ＣＳＦ，Ｍ−ＣＳＦ，及び抗体であり，特に，組換え体技術を用いて合成されたヒト等の哺乳動物の組換え糖蛋白質である。 In the present invention, the glycoprotein whose sugar chain is to be analyzed is not particularly limited, but preferably iduronic acid-2-sulfatase, α-galactosidase A, acid sphingomyelinase, α-L-iduronidase, N-acetylgalactosamine. -4-sulfatase, glucocerebrosidase (glucosylceramidase), galsulfase, lysosomal acid lipase, lysosomal enzymes such as acid α-glucosidase, tissue plasminogen activator (t-PA), blood coagulation factor VII, blood coagulation factor VIII Factors, blood coagulation factors such as blood coagulation factor IX, erythropoietin, interferon, thrombomodulin, follicle-stimulating hormone, thyroid-stimulating hormone, GM-CSF, G-CSF, M-CSF, and antibodies, especially recombinant technology Using It is a recombinant glycoprotein synthesized mammal such as human.
以下，実施例を参照して本発明を更に詳細に説明するが，本発明が実施例に限定されることは意図しない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, it is not intended that the present invention be limited to the examples.
〔ヒトイズロン酸−２−スルファターゼのグリコシダーゼ処理〕
組換え体ヒトイズロン酸−２−スルファターゼ（ｒｈｌ２Ｓ）の精製品を，公知の手法を用いて準備した（米国特許公報５７９８２３９）。ｒｈｌ２Ｓ溶液にＮ−グリコシダーゼＦ（ロシュ・ダイアグノスティックス製）を加えて振り混ぜ，２〜８℃で３時間静置した。次いで反応液を９５℃で５分間加熱してグリコシダーゼを失活させてから減圧乾固した。[Glycosidase treatment of human iduronic acid-2-sulfatase]
A purified product of recombinant human iduronate-2-sulfatase (rhl2S) was prepared using known techniques (US Pat. No. 5,798,239). N-glycosidase F (manufactured by Roche Diagnostics) was added to the rhl2S solution and shaken and allowed to stand at 2-8 ° C. for 3 hours. Next, the reaction solution was heated at 95 ° C. for 5 minutes to deactivate glycosidase, and then dried under reduced pressure.
〔糖鎖のアミノピリジン処理〕
上記の減圧乾固させた糖鎖に，２０μＬの２−アミノピリジン溶液（１５０ｍｇの２−アミノピリジンを５０μＬの酢酸に溶解したもの）を加えて振り混ぜ，８０℃で１時間反応させた。次いで，２０μＬのボラン−ジメチルアミン錯体溶液（２０ｍｇのボラン−ジメチルアミン錯体を１００μＬの酢酸に溶解したもの）を加えて振り混ぜ，８０℃で１時間反応させた。次いで，１００μＬのトルエンを加えて振り混ぜた後，遠心して，糖鎖を沈殿させるとともにトルエン層の上層を除去した。このトルエンによる抽出操作を，更に２回繰り返して行い，未反応の２−アミノピリジンを除去した。また，最後のトルエン抽出の際には，沈殿を残すようにして，可能な限りの溶液を除去した。ここで除去したトルエンを含む液は，有機溶媒廃棄用の容器に回収した。次いで，沈殿させた糖鎖を滅圧乾固させた後，５０μＬの水に溶解して糖鎖溶解液とし，これをゲルろ過カラムクロマトフィーに付して，アミノピリジン標識化糖鎖を含む画分を分取した。このときゲルろ過カラムクロマトフィーは，純水で平衡化させたＳＥＰＨＡＤＥＸＴＭＧ−１０（カラム径５ｍｍ，カラム長１５０ｍｍ，ＧＥヘルスケア）に糖鎖溶解液を付し，次いで，室温で８ｍＬ／分の流速で０．１％（ｖ／ｖ）酢酸水溶液を通して，蛍光検出器（励起波長３２０ｎｍ，蛍光波長４００ｎｍ）で蛍光強度をモニターしながら行った。このとき得られたクロマトグラムを図１に示す。図１において，ピークＡがアミノピリジン標識化糖鎖に由来するピークである。分取したアミノピリジン標識化糖鎖を含む画分を凍結乾燥した後，１ｍＬの水に溶かし，減圧乾固した。[Aminopyridine treatment of sugar chains]
To the sugar chain dried under reduced pressure, 20 μL of 2-aminopyridine solution (150 mg of 2-aminopyridine dissolved in 50 μL of acetic acid) was added and shaken and reacted at 80 ° C. for 1 hour. Next, 20 μL of borane-dimethylamine complex solution (20 mg of borane-dimethylamine complex dissolved in 100 μL of acetic acid) was added and shaken and reacted at 80 ° C. for 1 hour. Next, 100 μL of toluene was added and shaken, and then centrifuged to precipitate sugar chains and remove the upper layer of the toluene layer. This extraction operation with toluene was further repeated twice to remove unreacted 2-aminopyridine. In addition, as much as possible solution was removed so as to leave a precipitate during the final toluene extraction. The liquid containing toluene removed here was collected in a container for organic solvent disposal. Next, the precipitated sugar chain is vacuum-dried and then dissolved in 50 μL of water to obtain a sugar chain solution, which is subjected to gel filtration column chromatography to obtain a fraction containing an aminopyridine-labeled sugar chain. The fraction was collected. At this time, the gel filtration column chromatography was performed by attaching a sugar chain solution to SEPHADEX ™ G-10 (column diameter 5 mm, column length 150 mm, GE Healthcare) equilibrated with pure water, and then at room temperature, 8 mL / min. The fluorescence intensity was monitored with a fluorescence detector (excitation wavelength: 320 nm, fluorescence wavelength: 400 nm) through a 0.1% (v / v) acetic acid aqueous solution at a flow rate of 5 nm. The chromatogram obtained at this time is shown in FIG. In FIG. 1, peak A is a peak derived from an aminopyridine-labeled sugar chain. The fraction containing the aminopyridine-labeled sugar chain was lyophilized, dissolved in 1 mL of water, and dried under reduced pressure.
〔順相クロマトグラフィーによるアミノピリジン標識された糖の分析〕
（１）装置
島津ＨＰＬＣシステムＬＣ−２０Ａ（島津製作所）に順相クロマトグラフィー用カラム（ＡｓａｈｉｐａｋＴＭ ＮＨ２Ｐ−５０ ４Ｅ（４．６ｍｍ Ｉ．Ｄ．×２５０ｍｍ），官能基：アミノプロピル基，昭和電工）をセットした。カラムオーブンでカラムを５０℃に加熱するとともに，カラムの流出口の下流に蛍光検出器を設置し，カラム通過後の溶液に，励起光として波長３２０ｎｍの紫外線を照射し，波長４００ｎｍの蛍光を検出するようにした。
（２）移動相の調製
移動相として，０．１％（ｖ／ｖ）酢酸水溶液（溶液Ａ）と，０．５Ｍ ＮａＣｌを含有する０．１％（ｖ／ｖ）酢酸水溶液（溶液Ｂ）とを調製した。
（３）操作手順
上記の減圧乾固したアミノピリジン標識化糖鎖を溶液Ａに溶解し，試料溶液とした。還元糖分析システムのオートサンプラーに，溶液Ａと溶液Ｂをセットし，溶液Ａと溶液Ｂの体積比率が７０：３０である溶液でカラムを平衡化した後，試料溶液をカラムに負荷した。[Analysis of aminopyridine-labeled sugars by normal phase chromatography]
(1) Apparatus Shimadzu HPLC system LC-20A (Shimadzu Corporation) with normal phase chromatography column (Asahipak ™ NH2P-50 4E (4.6 mm ID × 250 mm), functional group: aminopropyl group, Showa Denko) Set. The column is heated to 50 ° C in a column oven, and a fluorescence detector is installed downstream of the column outlet. The solution after passing through the column is irradiated with ultraviolet light with a wavelength of 320 nm as excitation light, and fluorescence with a wavelength of 400 nm is detected. I tried to do it.
(2) Preparation of mobile phase As mobile phase, 0.1% (v / v) acetic acid aqueous solution (solution A) and 0.1% (v / v) acetic acid aqueous solution (solution B) containing 0.5 M NaCl And were prepared.
(3) Operating procedure The aminopyridine-labeled sugar chain was dried in vacuo and dissolved in solution A to obtain a sample solution. The solution A and the solution B were set in the autosampler of the reducing sugar analysis system, the column was equilibrated with a solution having a volume ratio of the solution A and the solution B of 70:30, and then the sample solution was loaded onto the column.
カラムに試料溶液を負荷した後，溶液Ａと溶液Ｂの体積比率が７０：３０の移動相を，０．６ｍＬ／分の流速で２分間カラムに流した後，同一流速で７８分かけて，移動相における溶液Ｂの体積比率を９５％にまで直線的に高め，次いで２５分間溶液Ｂの体積比率を９５％として同一流速で流し，更に，３５分間溶液Ａと溶液Ｂの体積比率を７０：３０にして同一流速で流した。カラム通過後の溶液に，励起光（波長３２０ｎｍ）を連続的に照射して該流出液から放射される蛍光（波長４００ｎｍ）の強度を連続的に測定した。 After loading the sample solution onto the column, a mobile phase with a volume ratio of solution A and solution B of 70:30 was passed through the column for 2 minutes at a flow rate of 0.6 mL / min, The volume ratio of the solution B in the mobile phase is increased linearly to 95%, then the volume ratio of the solution B is set to 95% for 25 minutes, and the same flow rate is applied for 35 minutes. 30 at the same flow rate. The solution after passing through the column was continuously irradiated with excitation light (wavelength 320 nm), and the intensity of fluorescence (wavelength 400 nm) emitted from the effluent was measured continuously.
〔分析結果の評価〕
順相クロマトグラフィーによる分析の結果，アミノピリジン標識化糖鎖がピークＡ〜Ｅとして検出された（図２）。ピークＥは，アミノピリジン標識されたマンノース６リン酸を含む糖鎖に由来するものであった。[Evaluation of analysis results]
As a result of analysis by normal phase chromatography, aminopyridine-labeled sugar chains were detected as peaks A to E (FIG. 2). Peak E was derived from a sugar chain containing aminopyridine-labeled mannose 6-phosphate.
本発明によれば，多量のトルエンを気化させることなく，遺伝子組換え技術を用いて製造した糖蛋白質の糖鎖の還元末端を２−アミノピリジンで標識することができる。 According to the present invention, the reducing end of a sugar chain of a glycoprotein produced using a gene recombination technique can be labeled with 2-aminopyridine without vaporizing a large amount of toluene.
Claims (6)
還元末端を有する糖鎖と２−アミノピリジンとを含む溶液を加熱して，該還元末端と該２−アミノピリジンとを反応させることによりイミンを形成させた後，該溶液にボラン−ジメチルアミン錯体を添加して加熱し，該イミンを還元させることにより，還元末端が２−アミノピリジンで標識された標識化糖鎖を含む反応液を得るステップと，
該反応液にトルエンを添加し，該標識化糖鎖を沈殿させるとともに，未反応の２−アミノピリジンをトルエンとともに除去するステップと，
該沈殿を水性溶媒に溶解させて得られた溶液を，ゲルろ過カラムクロマトグラフィーに付して，該標識化糖鎖を含有する画分を回収するステップとを，
この順で含んでなるものである方法。A method for obtaining a sugar chain whose reducing end is labeled with 2-aminopyridine,
A solution containing a sugar chain having a reducing end and 2-aminopyridine is heated to form an imine by reacting the reducing end with the 2-aminopyridine, and then the borane-dimethylamine complex is added to the solution. Adding and heating to reduce the imine, thereby obtaining a reaction solution containing a labeled sugar chain in which the reducing end is labeled with 2-aminopyridine;
Adding toluene to the reaction solution to precipitate the labeled sugar chain and removing unreacted 2-aminopyridine together with toluene;
Subjecting the solution obtained by dissolving the precipitate to an aqueous solvent to gel filtration column chromatography, and collecting the fraction containing the labeled sugar chain;
A method that comprises in this order.
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