JP2006291102A - Purified oligosaccharide fraction - Google Patents
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- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 97
- 150000002482 oligosaccharides Chemical class 0.000 title claims abstract description 95
- 235000000346 sugar Nutrition 0.000 claims description 24
- 239000000203 mixture Substances 0.000 description 23
- 238000000746 purification Methods 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 8
- 150000008163 sugars Chemical class 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- 229920002971 Heparan sulfate Polymers 0.000 description 6
- 238000001641 gel filtration chromatography Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 150000004044 tetrasaccharides Chemical class 0.000 description 6
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 5
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 5
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 229950006780 n-acetylglucosamine Drugs 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 238000005571 anion exchange chromatography Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 3
- 229920002683 Glycosaminoglycan Polymers 0.000 description 3
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000000593 degrading effect Effects 0.000 description 3
- 150000002016 disaccharides Chemical class 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 229940097043 glucuronic acid Drugs 0.000 description 3
- 229960002897 heparin Drugs 0.000 description 3
- 108010083213 heparitinsulfate lyase Proteins 0.000 description 3
- 238000005040 ion trap Methods 0.000 description 3
- 238000001698 laser desorption ionisation Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- WXTMDXOMEHJXQO-UHFFFAOYSA-N 2,5-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC(O)=CC=C1O WXTMDXOMEHJXQO-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000589565 Flavobacterium Species 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical group CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 2
- WRYNUJYAXVDTCB-UHFFFAOYSA-M acetyloxymercury Chemical compound CC(=O)O[Hg] WRYNUJYAXVDTCB-UHFFFAOYSA-M 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 229920002674 hyaluronan Polymers 0.000 description 2
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-MNSSHETKSA-N 0.000 description 2
- 229940099552 hyaluronan Drugs 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- MSWZFWKMSRAUBD-UHFFFAOYSA-N 2-Amino-2-Deoxy-Hexose Chemical compound NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- WFIYPADYPQQLNN-UHFFFAOYSA-N 2-[2-(4-bromopyrazol-1-yl)ethyl]isoindole-1,3-dione Chemical compound C1=C(Br)C=NN1CCN1C(=O)C2=CC=CC=C2C1=O WFIYPADYPQQLNN-UHFFFAOYSA-N 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 241000606860 Pasteurella Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 description 1
- 239000001639 calcium acetate Substances 0.000 description 1
- 229960005147 calcium acetate Drugs 0.000 description 1
- 235000011092 calcium acetate Nutrition 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 150000004043 trisaccharides Chemical class 0.000 description 1
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Abstract
【課題】 Ｎ−アセチルヘパロサン由来の純度の高いオリゴ糖画分を提供すること。
【解決手段】 Ｎ−アセチルヘパロサン構造を有するオリゴ糖を含む、精製オリゴ糖画分。
【選択図】 なしPROBLEM TO BE SOLVED: To provide a high-purity oligosaccharide fraction derived from N-acetylheparosan.
A purified oligosaccharide fraction comprising an oligosaccharide having an N-acetylheparosan structure.
[Selection figure] None
Description
本発明は、オリゴ糖画分に関し、特にＮ−アセチルヘパロサンから得られる高純度の精製オリゴ糖画分に関する。 The present invention relates to an oligosaccharide fraction, and more particularly to a high-purity purified oligosaccharide fraction obtained from N-acetylheparosan.
Ｎ−アセチルヘパロサンは、生体内においてヘパリン、ヘパラン硫酸の合成前駆体となるグリコサミノグリカン（GAG）であり、同様の構造体を大腸菌Ｋ５株の培養液から得ることができる（例えば、特許文献１参照。）。このＮ−アセチルヘパロサンはヒアルロナンと同様に、Ｎ−アセチルグルコサミンとグルクロン酸の交互繰り返し構造であり、硫酸基を持たない。このＮ−アセチルヘパロサンを低分子化し精製して得るオリゴ糖は、ヘパリン・ヘパラン硫酸の生合成の研究や、関連酵素の研究、またそれら酵素の阻害剤開発研究、さらには種々の複雑な構造の糖鎖の化学合成原料など、種々の局面において非常に有用な化合物となり得る。
非特許文献１においては、Ｎ−アセチルヘパロサンオリゴ糖を高速液体クロマトグラフィーとＥＳＩ−ＭＳにより分析しているが、それぞれのサイズのオリゴ糖の単離精製は行われていない。
In
本発明は、上記事情に鑑みなされたものであって、その目的はＮ−アセチルヘパロサン由来の純度の高い精製オリゴ糖画分を提供することである。 The present invention has been made in view of the above circumstances, and an object thereof is to provide a purified oligosaccharide fraction having high purity derived from N-acetylheparosan.
上記課題は、以下の手段により解決される。
１． Ｎ−アセチルヘパロサン構造を有するオリゴ糖を含む精製オリゴ糖画分。
２． Ｎ−アセチルヘパロサンを低分子化して得られたことを特徴とする上記１に記載の精製オリゴ糖画分。
３． オリゴ糖の純度が６０％以上であることを特徴とする上記１又は２に記載の精製オリゴ糖画分。
４． 前記オリゴ糖が３〜１００個の糖残基からなることを特徴とする上記１〜３のいずれかに記載の精製オリゴ画分。
５． 前記オリゴ糖が偶数糖であることを特徴とする上記１〜４のいずれかに記載の精製オリゴ画分。
６． 前記オリゴ糖が奇数糖であることを特徴とする上記１〜４のいずれかに記載の精製オリゴ画分。
The above problem is solved by the following means.
1. A purified oligosaccharide fraction containing an oligosaccharide having an N-acetylheparosan structure.
2. 2. The purified oligosaccharide fraction according to 1 above, obtained by reducing the molecular weight of N-acetylheparosan.
3. 3. The purified oligosaccharide fraction according to 1 or 2 above, wherein the purity of the oligosaccharide is 60% or more.
4). 4. The purified oligo fraction according to any one of 1 to 3 above, wherein the oligosaccharide comprises 3 to 100 sugar residues.
5. 5. The purified oligo fraction according to any one of 1 to 4 above, wherein the oligosaccharide is an even-numbered sugar.
6). 5. The purified oligo fraction according to any one of 1 to 4 above, wherein the oligosaccharide is an odd-numbered sugar.
本発明によれば、Ｎ−アセチルへパロサン由来の純度の高い新規精製オリゴ糖画分が得られる。この新規精製オリゴ糖画分は、ヘパリン・ヘパラン硫酸の生合成の研究や、関連酵素の研究、またそれら酵素の阻害剤開発研究、さらには種々の複雑な構造の糖鎖の化学合成原料など、種々の局面において非常に有用である。 According to the present invention, a novel purified oligosaccharide fraction having high purity derived from N-acetylheparosan can be obtained. This newly-purified oligosaccharide fraction includes research on biosynthesis of heparin and heparan sulfate, research on related enzymes, research on the development of inhibitors for these enzymes, and raw materials for chemical synthesis of sugar chains of various complex structures. It is very useful in various aspects.
以下、本発明を詳細に説明する。
本発明の精製オリゴ糖画分は、Ｎ−アセチルヘパロサン構造、即ちグルクロン酸がＮ−アセチルグルコサミンにβ１，４結合し、Ｎ−アセチルグルコサミンがグルクロン酸にα１，４結合した直鎖状の繰り返し構造を有するオリゴ糖を含むものである。精製オリゴ糖画分は不純物が少ないことが好ましいが、必ずしも完全に精製されている必要はなく、部分的に精製されたものもこれに含まれる。オリゴ糖画分中におけるオリゴ糖の純度は、６０％〜１００％であることが好ましく、純度７０％〜１００％であることがより好ましく、９０％〜１００％であることがさらに好ましい。
Hereinafter, the present invention will be described in detail.
The purified oligosaccharide fraction of the present invention has an N-acetylheparosan structure, that is, a linear repeating structure in which glucuronic acid is β1,4 linked to N-acetylglucosamine and N-acetylglucosamine is α1,4 linked to glucuronic acid. It includes an oligosaccharide having a structure. The purified oligosaccharide fraction preferably has few impurities, but does not necessarily need to be completely purified, and includes partially purified products. The oligosaccharide purity in the oligosaccharide fraction is preferably 60% to 100%, more preferably 70% to 100%, and even more preferably 90% to 100%.
また、精製オリゴ糖画分に含まれるオリゴ糖は、下記に示す、偶数の糖残基からなる偶数オリゴ糖でもよいし、奇数の糖残基からなる奇数オリゴ糖でもよい。偶数オリゴ糖としては、例えば、非還元末端に不飽和ウロン酸を有する偶数糖が挙げられ、奇数オリゴ糖としては、例えば、非還元末端にＮ−アセチルグルコサミンを有する奇数糖（奇数オリゴ糖）が挙げられる。 The oligosaccharide contained in the purified oligosaccharide fraction may be an even-numbered oligosaccharide composed of an even-numbered sugar residue or an odd-numbered oligosaccharide composed of an odd-numbered sugar residue as shown below. Examples of even oligosaccharides include even sugars having unsaturated uronic acid at the non-reducing end, and odd oligosaccharides include, for example, odd sugars (odd oligosaccharides) having N-acetylglucosamine at the non-reducing end. Can be mentioned.
オリゴ糖の糖鎖長としては特に制限はないが、３〜１００個の糖からなることが好ましく、３〜５０個の糖からなることがより好ましく、３〜２１個の糖からなることがさらに好ましい。
また、オリゴ糖の分子量としては、５００〜２００００の範囲が好ましく、５００〜４１００の範囲がより好ましい。
本発明のオリゴ糖画分は、生体において重要な働きを担うグリコサミノグリカンであるヒアルロナンを構成する糖を含んでいるため、薬剤、食品、化粧品等の原材料等として有望である。
The sugar chain length of the oligosaccharide is not particularly limited, but is preferably composed of 3 to 100 sugars, more preferably 3 to 50 sugars, and further preferably 3 to 21 sugars. preferable.
Moreover, as molecular weight of oligosaccharide, the range of 500-20000 is preferable and the range of 500-4100 is more preferable.
The oligosaccharide fraction of the present invention is promising as a raw material for pharmaceuticals, foods, cosmetics and the like because it contains a sugar constituting hyaluronan, which is a glycosaminoglycan that plays an important role in the living body.
本発明の精製オリゴ糖画分は、Ｎ−アセチルヘパロサンを低分子化した後、単離精製することにより得られる。
Ｎ−アセチルヘパロサンは、未だ動物の生体組織から採取された実績は無いが、Ｎ−アセチルヘパロサンを産生する細菌等から採取することが可能である。Ｎ−アセチルヘパロサンを産生する細菌としては、大腸菌、パスツレラ菌等が挙げられる。大腸菌からＮ−アセチルヘパロサンを採取・単離する方法としては、特開平５−２７１３０５号公報、特開２００４−１８８４０号公報等に記載の方法を用いることができる。
The purified oligosaccharide fraction of the present invention can be obtained by isolating and purifying N-acetylheparosan after reducing its molecular weight.
N-acetylheparosan has not yet been collected from animal tissues, but can be collected from bacteria that produce N-acetylheparosan. Examples of bacteria that produce N-acetylheparosan include Escherichia coli and Pasteurella. As a method for collecting and isolating N-acetylheparosan from E. coli, methods described in JP-A Nos. 5-271305 and 2004-18840 can be used.
Ｎ−アセチルヘパロサンは、Ｎ−アセチルヘパロサンの分解酵素で処理することにより低分子化することができる。Ｎ−アセチルヘパロサンの分解酵素としては、フラボバクテリウム由来ヘパリチナーゼI、K5リアーゼ等を用いることができる。
また、ヘパリチナーゼIを用いる場合の処理条件としては、２５〜６０℃（より好ましくは３６〜４５℃）で０．５〜１２０分間（より好ましくは２０〜６０分間）インキュベーションすることが好ましい。
N-acetylheparosan can be reduced in molecular weight by treating with N-acetylheparosan degrading enzyme. Flavobacterium-derived heparitinase I, K5 lyase, or the like can be used as a degrading enzyme for N-acetylheparosan.
Further, as treatment conditions when heparitinase I is used, it is preferable to incubate at 25 to 60 ° C. (more preferably 36 to 45 ° C.) for 0.5 to 120 minutes (more preferably 20 to 60 minutes).
上記のようにＮ−アセチルヘパロサンを分解酵素により処理すると、通常、種々のサイズの偶数オリゴ糖を含む混合物が得られる。この偶数オリゴ糖の混合物を精製することで、上記のような高い純度を有する偶数オリゴ糖を含む精製画分が得られる。オリゴ糖の混合物の精製は公知の精製方法を用いて行うことができ、例えば、ゲル濾過クロマトグラフィー、陰イオン交換クロマトグラフィー等によって精製することができる。 When N-acetylheparosan is treated with a degrading enzyme as described above, a mixture containing even-numbered oligosaccharides of various sizes is usually obtained. By purifying the mixture of even oligosaccharides, a purified fraction containing the even oligosaccharides having high purity as described above can be obtained. The mixture of oligosaccharides can be purified using a known purification method, for example, gel filtration chromatography, anion exchange chromatography, or the like.
また、Ｎ−アセチルヘパロサンを分解酵素により処理して得られた偶数オリゴ糖を酢酸水銀と反応させることで、偶数オリゴ糖の不飽和ウロン酸を除去し、奇数オリゴ糖を含む画分を得ることができる。この方法により奇数オリゴ糖を生成するには、Glycobiology vol.10 no.10 pp.1033-1039 (2000)に記載の方法に従って行うことができる。
なお、奇数オリゴ糖の生成は、偶数オリゴ糖の精製の後に行ってもよいし、種々のサイズを含む偶数オリゴ糖の混合物から上記方法により奇数オリゴ糖を生成した後に、これを精製して精製オリゴ糖画分を得てもよい。
Further, by reacting even-oligosaccharide obtained by treating N-acetylheparosan with a decomposing enzyme with mercury acetate, unsaturated uronic acid of the even-oligosaccharide is removed, and a fraction containing odd-oligosaccharide is obtained. be able to. Odd oligosaccharides can be produced by this method according to the method described in Glycobiology vol.10 no.10 pp.1033-1039 (2000).
The production of odd oligosaccharides may be carried out after purification of even oligosaccharides, or after production of odd oligosaccharides from a mixture of even oligosaccharides containing various sizes by the above method, purification is performed. An oligosaccharide fraction may be obtained.
以下、本発明を実施例に基づき更に詳細に説明する。
［実施例１］
（偶数オリゴ糖混合物の調製）
特開２００４−１８８４０号公報の実施例１に記載の方法と同様にして大腸菌Ｋ５菌株を培養し、Ｎ−アセチルヘパロサン画分を得て、さらに、同公報の実施例２に記載の方法と同様にしてこのＮ−アセチルヘパロサン画分を高純度に精製した。
精製したＮ−アセチルヘパロサン画分（100mg）を濃度が10mg/mlになるように、5mM酢酸カルシウムを含む50mM酢酸ナトリウム緩衝液（pH7.0）に溶解した。このN−アセチルヘパロサン溶解液に、フラボバクテリウム由来ヘパリチナーゼI（０．２ユニット）を添加し、３７℃で４５分間インキュベーションした。
その後、沸騰水浴中で反応を停止させ、沈殿物を遠心分離（８，０００ｒｐｍ、２０分）によって除去し、上清液を凍結乾燥した。以上のようにして、非還元末端に不飽和ウロン酸を有するN−アセチルヘパロサンのオリゴ糖混合物の凍結乾燥物を131.25mg得た（塩類を含む）。
Hereinafter, the present invention will be described in more detail based on examples.
[Example 1]
(Preparation of even oligosaccharide mixture)
E. coli K5 strain is cultured in the same manner as described in Example 1 of JP-A-2004-18840 to obtain an N-acetylheparosan fraction, and further, the method described in Example 2 of the same publication Similarly, this N-acetylheparosan fraction was purified to high purity.
The purified N-acetylheparosan fraction (100 mg) was dissolved in 50 mM sodium acetate buffer (pH 7.0) containing 5 mM calcium acetate so as to have a concentration of 10 mg / ml. Flavobacterium-derived heparitinase I (0.2 units) was added to this N-acetylheparosan lysate and incubated at 37 ° C. for 45 minutes.
Thereafter, the reaction was stopped in a boiling water bath, the precipitate was removed by centrifugation (8,000 rpm, 20 minutes), and the supernatant was lyophilized. As described above, 131.25 mg (including salts) of a lyophilized product of an N-acetylheparosan oligosaccharide mixture having an unsaturated uronic acid at the non-reducing end was obtained.
得られたオリゴ糖混合物を、マトリックス支援レーザー脱離イオン化飛行時間型質量分析装置（MALDI-TOF-MS）にて分析したところ、図１に示すように、主として４〜２６糖の偶数糖混合物であることが確認された。（なお、測定条件上、質量電荷比６００以下を検出していないが、実際には２糖も生成している。また、２８糖以上の偶数オリゴ糖も、僅少量生成していると考えられる。）
なお、質量分析装置は、ブルカー・ダルトニクス社製のUltraflexを使用し、測定用マトリックスとしてはDHB(2,5-dihydroxybenzoic acid)を用いた。
When the obtained oligosaccharide mixture was analyzed with a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF-MS), as shown in FIG. It was confirmed that there was. (Note that although the mass-to-charge ratio of 600 or less was not detected in the measurement conditions, disaccharides were actually produced. In addition, it is considered that even-numbered oligosaccharides of 28 or more sugars were produced in a small amount. .)
The mass spectrometer used was Ultraflex manufactured by Bruker Daltonics, and DHB (2,5-dihydroxybenzoic acid) was used as the measurement matrix.
（偶数オリゴ糖混合物の精製）
上記で得た偶数オリゴ糖混合物の凍結乾燥物（２mg）を、ゲル濾過クロマトグラフィーにて分取精製し凍結乾燥して、精製オリゴ糖画分を得た（図２参照。）。なお、ゲル濾過クロマトグラフィーには、アマシャム・バイオサイエンス社製のSuperdex Peptide 10/300GLを二本継ぎで使用した。
分取した各偶数精製オリゴ糖画分の純度を陰イオン交換HPLCにて確認した。なお、印イオン交換ＨＰＬＣの分析条件は以下の通りとした。この結果を図３に示す。
HPLCカラム： YMC-Pack Polyamine II （株）ワイエムシィ
移動相（溶媒）： 50mMから500mMまでのNaH2PO4による30分グラジェント
流速： 1.2ml/min.
検出： UV210nm
温度： 室温
(Purification of even oligosaccharide mixture)
The lyophilized product (2 mg) of the even oligosaccharide mixture obtained above was fractionated and purified by gel filtration chromatography and lyophilized to obtain a purified oligosaccharide fraction (see FIG. 2). For gel filtration chromatography,
The purity of each even-numbered purified oligosaccharide fraction was confirmed by anion exchange HPLC. The analysis conditions of the sign ion exchange HPLC were as follows. The result is shown in FIG.
HPLC column: YMC-Pack Polyamine II YMC Co., Ltd. Mobile phase (solvent): 30-minute gradient with NaH 2 PO 4 from 50 mM to 500 mM Flow rate: 1.2 ml / min.
Detection: UV210nm
Temperature: Room temperature
さらに、MALDI-TOF-MS（Ultraflex，ブルカー・ダルトニクス社製）およびESI-IT-MS（エレクトロスプレー−イオントラップ型質量分析装置；Ｅｓｑｕｉｒｅ ３０００ ｐｌｕｓ，ブルカー・ダルトニクス社製）にて、その分子量を確認した。この結果をそれぞれ図４及び５に示す。
Furthermore, the molecular weight was confirmed by MALDI-TOF-MS (Ultraflex, Bruker Daltonics) and ESI-IT-MS (Electrospray-ion trap mass spectrometer;
［実施例２］
（偶数オリゴ糖混合物の精製）
偶数オリゴ糖の精製は、陰イオン交換HPLCによっても実施した。
上記で得た偶数オリゴ糖混合物の凍結乾燥物（３mg）を、ＹＭＣ−ＰＡ−１２０−Ｓ５イオン交換カラム（ワイエムシィ（株））にアプライし、流速0.5mL/min.で４５分間に硫酸ナトリウム溶液を20mMから100mMまでの直線濃度勾配で溶出した。カラムから溶出した液をフラクション・コレクターにて１分毎に分取した。分取液はUV210ｎｍの吸収にてモニターし、N−アセチルヘパロサン２糖、４糖、６糖、８糖と考えられる画分を集めた。以上の操作を１５回繰り返して得た画分をセファデックスG-25カラムにより脱塩後、濃縮、凍結乾燥した。これらの操作により得た精製２糖、４糖、６糖、８糖の収量はそれぞれ、１０ｍｇ、８ｍｇ、６ｍｇ、４ｍｇであった。
[Example 2]
(Purification of even oligosaccharide mixture)
The purification of even oligosaccharides was also performed by anion exchange HPLC.
The lyophilized product (3 mg) of the even oligosaccharide mixture obtained above was applied to a YMC-PA-120-S5 ion exchange column (YMC Co., Ltd.) and a sodium sulfate solution at a flow rate of 0.5 mL / min for 45 minutes. Was eluted with a linear concentration gradient from 20 mM to 100 mM. The liquid eluted from the column was fractionated every minute with a fraction collector. The collected liquid was monitored by absorption at UV 210 nm, and fractions considered to be N-acetylheparosan disaccharide, tetrasaccharide, hexasaccharide, and octasaccharide were collected. Fractions obtained by repeating the above operation 15 times were desalted with a Sephadex G-25 column, concentrated and lyophilized. The yields of purified disaccharide, tetrasaccharide, hexasaccharide and octasaccharide obtained by these operations were 10 mg, 8 mg, 6 mg and 4 mg, respectively.
得られたオリゴ糖のうち、精製４糖、６糖、８糖について、1H-NMRスペクトルを測定した。測定装置はVARIAN社製UNITY INOVA500 (500MHZ)を使用し、各オリゴ糖試料をALDRICH社製重水に溶解し、７０℃で測定を行った。内部標準をtert-ブタノール（1.23ppm）とした場合の、各オリゴ糖の測定結果は以下のとおりであり、精製４糖、６糖、８糖についてのＮＭＲスペクトルをそれぞれ図１０〜１２に示す。これらの結果より、それぞれの鎖長のN-アセチルヘパロサンオリゴ糖であることが支持された。 Among the obtained oligosaccharides, 1 H-NMR spectra were measured for purified tetrasaccharide, hexasaccharide and octasaccharide. The measuring apparatus used was UNITY INOVA500 (500MHZ) manufactured by VARIAN, and each oligosaccharide sample was dissolved in heavy water manufactured by ALDRICH and measured at 70 ° C. The measurement results of each oligosaccharide when the internal standard is tert-butanol (1.23 ppm) are as follows, and the NMR spectra of purified tetrasaccharide, hexasaccharide and octasaccharide are shown in FIGS. These results supported N-acetylheparosan oligosaccharides of each chain length.
（精製４糖）
δ(ppm) = 5.782(br, 1H, H-4ｄ), 5.371(br, 1H, H-1c), 5.206(br, 0.6H, H-1aα), 5,075(m, 1H, H-1ｄ), 4.707(m, 0.4H, H-1aβ), 4.528(brd, 1H, H-1b), 3.929〜3.368(m), 2.997 (brt, 2H), 2.032(s, 3H, NH- COCH3), 2.026(s, 3H, NH-COCH3)
(Purified tetrasaccharide)
δ (ppm) = 5.782 (br, 1H, H-4d), 5.371 (br, 1H, H-1c), 5.206 (br, 0.6H, H-1aα), 5,075 (m, 1H, H-1d), 4.707 (m, 0.4H, H-1aβ), 4.528 (brd, 1H, H-1b), 3.929-3.368 (m), 2.997 (brt, 2H), 2.032 (s, 3H, NH- COCH 3 ), 2.026 (s, 3H, NH-COCH 3 )
（精製６糖）
δ(ppm) = 5.778(br, 1H, H-4f), 5.366(br, 2H, H-1c, H-1e), 5.199(brs, 0.6H, H-1aα), 5,067(m, 1H, H-1f), 4.707(m, 0.4H, H-1aβ), 4.527(brd, 1H, H-1b or H-1ｄ), 4.487(brd, 1H, H-1ｄ or H-1b), 3.947〜3.623(m), 3.367 (brt, 3H), 2.034(s, 3H, NH- COCH3), 2.029(s, 3H, NH- COCH3), 2.025(s, 3H, NH- COCH3)
(Purified hexasaccharide)
δ (ppm) = 5.778 (br, 1H, H-4f), 5.366 (br, 2H, H-1c, H-1e), 5.199 (brs, 0.6H, H-1aα), 5,067 (m, 1H, H -1f), 4.707 (m, 0.4H, H-1aβ), 4.527 (brd, 1H, H-1b or H-1d), 4.487 (brd, 1H, H-1d or H-1b), 3.947 to 3.623 ( m), 3.367 (brt, 3H), 2.034 (s, 3H, NH- COCH 3 ), 2.029 (s, 3H, NH- COCH 3 ), 2.025 (s, 3H, NH- COCH 3 )
（精製８糖）
δ(ppm) = 5.778(m, 1H, H-4h), 5.366(br, 3H, H-1c, H-1e, H-1g), 5.199(brs, 0.6H, H-1aα), 5,068(d, 1H, H-1h), 4.707(brs, 0.4H, H-1aβ), 4.526(d, 1H, H-1b or H-1ｄ or H-f), 4.481(d, 2H, H-1ｄ or H-1f or H-1b), 3.946〜3.621(m), 3.367 (t, 4H), 2.038(s, 3H, NH- COCH3), 2.035(s, 3H, NH- COCH3), 2.026(s, 6H, NH- COCH3)
(Purified octasaccharide)
δ (ppm) = 5.778 (m, 1H, H-4h), 5.366 (br, 3H, H-1c, H-1e, H-1g), 5.199 (brs, 0.6H, H-1aα), 5,068 (d , 1H, H-1h), 4.707 (brs, 0.4H, H-1aβ), 4.526 (d, 1H, H-1b or H-1d or Hf), 4.481 (d, 2H, H-1d or H-1f or H-1b), 3.946-3.621 (m), 3.367 (t, 4H), 2.038 (s, 3H, NH-COCH 3 ), 2.035 (s, 3H, NH- COCH 3 ), 2.026 (s, 6H, NH- COCH 3 )
［実施例３］
（奇数オリゴ糖混合物の調製）
上記実施例１の「偶数オリゴ糖混合物の調製」で得た偶数オリゴ糖混合物（100mg）を濃度が20mg/mlになるように蒸留水に溶解し、酢酸を用いてpHを5.0に調整した。同量の70mM酢酸水銀（pH5.0）を偶数オリゴ糖混合物の溶液に混合し、室温に１０分間放置した。
その後、Hg2+を除くために陽イオン交換体（ダイヤイオン PK220；30ml）カラムを用い、酸性画分を集め、中和した後、凍結乾燥した。この凍結乾燥物（342.3mg）を少量の蒸留水に再溶解し、セファデックスG-10カラム（2.2×115cm）にアプライし、蒸留水を用いて奇数オリゴ糖混合物を溶出した。
UV210/232nmおよび電気伝導度をモニタリングし、UV210(+)・UV232(-)画分を集め、凍結乾燥した。以上のようにして、非還元末端にヘキソサミンを有するN−アセチルヘパロサンのオリゴ糖混合物の凍結乾燥物を44.7mg得た（塩類を含む）。
得られたオリゴ糖混合物を実施例１と同様にしてMALDI-TOF-MSにて分析したところ、図１に示すように、主として５〜２５糖の偶数糖混合物であることが確認された。（測定条件上、質量電荷比６００以下を検出していないが、実際には単糖（N-アセチルグルコサミン）、３糖も生成している。また、２７糖以上の奇数オリゴ糖も、僅少量生成していると考えられる。）
[Example 3]
(Preparation of odd oligosaccharide mixture)
The even oligosaccharide mixture (100 mg) obtained in “Preparation of the even oligosaccharide mixture” in Example 1 was dissolved in distilled water to a concentration of 20 mg / ml, and the pH was adjusted to 5.0 with acetic acid. The same amount of 70 mM mercury acetate (pH 5.0) was mixed with the even oligosaccharide mixture solution and left at room temperature for 10 minutes.
Thereafter, an acidic fraction was collected using a cation exchanger (Diaion PK220; 30 ml) column to remove Hg 2+ , neutralized and then lyophilized. This lyophilized product (342.3 mg) was redissolved in a small amount of distilled water, applied to a Sephadex G-10 column (2.2 × 115 cm), and the odd oligosaccharide mixture was eluted using distilled water.
UV210 / 232nm and electrical conductivity were monitored and UV210 (+) and UV232 (-) fractions were collected and lyophilized. As described above, 44.7 mg (including salts) of a lyophilized product of the oligosaccharide mixture of N-acetylheparosan having hexosamine at the non-reducing end was obtained.
When the obtained oligosaccharide mixture was analyzed by MALDI-TOF-MS in the same manner as in Example 1, it was confirmed that the mixture was mainly an even sugar mixture of 5 to 25 sugars as shown in FIG. (In terms of measurement conditions, mass-to-charge ratio of 600 or less was not detected, but in fact, monosaccharide (N-acetylglucosamine) and trisaccharide were also produced. It is thought that it is generated.)
（奇数オリゴ糖混合物の精製）
上記で得た奇数オリゴ糖混合物の凍結乾燥物（２mg）をゲル濾過クロマトグラフィー（実施例１と同様の装置を使用）にて分取精製し凍結乾燥して、精製オリゴ糖画分を得た。（図６参照。）
分取した各精製奇数オリゴ糖画分の純度を、上記実施例１と同様にして陰イオン交換HPLCにて確認した。この結果を図７に示す。（なお、図３及び図７において、各糖鎖のピークが二分されて見える場合があるのは、オリゴ糖構造中、還元末端のα／βアノマーの共存によるピーク分離であり、これは水溶液中で平衡状態にあるものであるから問題とはならない。）
また、MALDI-TOF-MSおよびESI-IT-MSにて、その分子量を確認した。この結果を図８及び９に示す。
以上の実施例より、Ｎ−アセチルヘパロサンの偶数糖及び奇数糖の精製オリゴ糖画分を高い純度で得られることがわかった。
(Purification of odd oligosaccharide mixture)
The lyophilized product (2 mg) of the odd oligosaccharide mixture obtained above was fractionated and purified by gel filtration chromatography (using the same apparatus as in Example 1) and lyophilized to obtain a purified oligosaccharide fraction. . (See Figure 6.)
The purity of each purified odd oligosaccharide fraction was confirmed by anion exchange HPLC in the same manner as in Example 1. The result is shown in FIG. (Note that in FIGS. 3 and 7, the peaks of each sugar chain may appear to be divided into two due to the peak separation due to the coexistence of the reducing end α / β anomer in the oligosaccharide structure. (This is not a problem because it is in equilibrium.)
Moreover, the molecular weight was confirmed by MALDI-TOF-MS and ESI-IT-MS. The results are shown in FIGS.
From the above examples, it was found that purified oligosaccharide fractions of even-numbered and odd-numbered sugars of N-acetylheparosan can be obtained with high purity.
Claims (6)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012233204A (en) * | 2005-06-28 | 2012-11-29 | Seikagaku Kogyo Co Ltd | Method of measuring enzyme activity |
| JP2018172375A (en) * | 2017-03-31 | 2018-11-08 | ロート製薬株式会社 | Method for producing composition containing hyaluronic acid oligosaccharide |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01282201A (en) * | 1988-03-10 | 1989-11-14 | Akzo Nv | Sulfated k5 antigen and sulfated k% antigen segment |
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2005
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01282201A (en) * | 1988-03-10 | 1989-11-14 | Akzo Nv | Sulfated k5 antigen and sulfated k% antigen segment |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012233204A (en) * | 2005-06-28 | 2012-11-29 | Seikagaku Kogyo Co Ltd | Method of measuring enzyme activity |
| JP2018172375A (en) * | 2017-03-31 | 2018-11-08 | ロート製薬株式会社 | Method for producing composition containing hyaluronic acid oligosaccharide |
| JP7285048B2 (en) | 2017-03-31 | 2023-06-01 | ロート製薬株式会社 | Method for producing composition containing hyaluronic acid oligosaccharide |
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