JP2005232064A - Sulfated saccharide library - Google Patents
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本発明は、硫酸化剤による非選択的な硫酸化、および、6位選択的脱硫酸化剤による脱硫酸化を用い、硫酸化の全ての組合せを網羅した硫酸化ヘキソピラノシル誘導体を合成する硫酸糖誘導体の製造法に関するものである。 The present invention relates to a sulfated sugar derivative that synthesizes a sulfated hexopyranosyl derivative that covers all combinations of sulfation using non-selective sulfation with a sulfating agent and desulfation with a 6-position selective desulfating agent. It relates to the manufacturing method.
硫酸基を持つ糖鎖には、ヘパラン硫酸、デルマタン硫酸等の様に様々な生理作用を有するものが知られている。例えば、造血が行われている器官である骨髄中には種々の細胞外マトリックス成分と共にヘパリンなどの硫酸糖が多量に含まれている。これら硫酸糖は、生体内で極微量で種々の生理活性を示す多くのサイトカインや増殖因子と親和性が高く、これらの因子の局在化を生じ、造血作用などの種々の生理作用を促進していると言われている。
こうした硫酸糖の機能を代替する新たな生理活性物質を探索する試みが種々行われている。例えば、ヒト造血細胞の培養液にサイトカインを添加する際にヘパリンを共存させるとサイトカインの造血細胞増殖促進作用が大幅に増強されることが知られており、これはサイトカインとヘパリンとの間に親和性があるためとされている(非特許文献1参照)。そこで、遊離のヘパリンを用いるのではなく、動物細胞が接着する基質表面にヘパリンを固定化することにより、動物細胞近傍にサイトカインを局在化させ、より効果的に細胞培養を促進しようとする試みもなされてきた(非特許文献2参照)。また、細胞の接着基質であるポリエステル表面に酸素プラズマ放電で水酸基を導入し、これに続く数段の反応でヘパリンを共有結合により固定化する方法も開発された(非特許文献3参照)。
As sugar chains having a sulfate group, those having various physiological functions such as heparan sulfate and dermatan sulfate are known. For example, bone marrow, an organ undergoing hematopoiesis, contains a large amount of sulfate sugars such as heparin together with various extracellular matrix components. These sulfated sugars have a high affinity with many cytokines and growth factors that exhibit various physiological activities in a very small amount in the living body, cause localization of these factors, and promote various physiological functions such as hematopoiesis. It is said that
Various attempts have been made to search for new physiologically active substances that substitute for the function of sulfated sugar. For example, it is known that when heparin is coexisted in the culture medium of human hematopoietic cells, hematopoietic cell proliferation-promoting action of cytokine is greatly enhanced, and this has an affinity between cytokine and heparin. This is considered to be due to the nature (see Non-Patent Document 1). Therefore, instead of using free heparin, by immobilizing heparin on the substrate surface to which animal cells adhere, attempts to localize cytokines in the vicinity of animal cells and promote cell culture more effectively Has also been made (see Non-Patent Document 2). In addition, a method has also been developed in which hydroxyl groups are introduced into a polyester surface, which is a cell adhesion substrate, by oxygen plasma discharge, and heparin is immobilized by covalent bonding in several subsequent reactions (see Non-Patent Document 3).
我々はこれまでに、簡単な構造を持つ低分子である、各種の糖脂質アナログを合成し(特許文献1参照)、その脂質類似構造の疎水性を利用して、細胞培養素材に固定化することにより、単糖誘導体でも肝臓細胞の培養等に有効に利用できることを示してきた(特許文献2、非特許文献4、5参照)。また、参考例1に示す様に、ガラクトース−6−硫酸誘導体を合成して、参考例2に示す様に、造血細胞の培養に有効であることが示された(特願2003−47144)。つまり、ヘパリン等の天然由来の複雑な多糖の混合物を用いずに、簡単に合成できる単糖誘導体を用いて、従来よりも簡便な工程で動物細胞培養素材が得られることが明らかになった。さらに、参考例3に示す様に、硫酸化剤の当量を増すことにより、ガラクトース−2,6−二硫酸誘導体、ガラクトース−3,6−二硫酸誘導体、および、ガラクトース−4,6−二硫酸誘導体が得られることを明らかにした(特願2003−48499)。
しかし、参考例に示したランダム硫酸化は、短工程で多くの硫酸糖誘導体を得る優れた方法ではあるが、6位が硫酸化された誘導体の生成が優先するために、6位が遊離の水酸基である硫酸糖誘導体が得られないので、短工程で、硫酸化の全ての組合せを網羅した硫酸化ヘキソピラノシル誘導体が得られる硫酸糖誘導体の製造法が望まれていた。
However, the random sulfation shown in the Reference Example is an excellent method for obtaining many sulfated sugar derivatives in a short process, but since the formation of a derivative in which the 6-position is sulfated has priority, the 6-position is free. Since a sulfated sugar derivative that is a hydroxyl group cannot be obtained, a method for producing a sulfated sugar derivative capable of obtaining a sulfated hexopyranosyl derivative covering all combinations of sulfation in a short process has been desired.
本発明の目的は、2、3、4および6位の水酸基が無保護のヘキソピラノシル誘導体を原料として、短工程で、6位が遊離の水酸基である硫酸糖誘導体を含む、全ての硫酸化の場合を尽くした硫酸化ヘキソピラノシル誘導体を得ることを特徴とした硫酸糖誘導体の製造法を提供することにある。 The object of the present invention is the case of all sulfation using a hexopyranosyl derivative in which the hydroxyl groups at positions 2, 3, 4 and 6 are unprotected as raw materials and including a sulfated sugar derivative in which the 6-position is a free hydroxyl group. It is an object of the present invention to provide a method for producing a sulfated sugar derivative characterized by obtaining a sulfated hexopyranosyl derivative exhausted.
上記課題を鋭意検討した結果、本発明者らは、まず、当量と反応条件を変化させることで、6位の水酸基のみが硫酸化されたヘキソピラノシル誘導体、あるいは6位を含む水酸基が硫酸化された二、三または四硫酸化ヘキソピラノシル誘導体を合成し、次に、この6位を含む水酸基が硫酸化された二、三または四硫酸化ヘキソピラノシル誘導体を原料として、6位選択的脱硫酸化剤を反応させ、6位が遊離の水酸基である一、二または三硫酸化ヘキソピラノシル誘導体を合成することにより、硫酸化の全ての組合せを網羅した硫酸化ヘキソピラノシル誘導体が調製できることを見出し、本発明を完成するに至った。 As a result of diligent examination of the above problems, the present inventors first changed the equivalent and reaction conditions, so that only the 6-position hydroxyl group was sulfated, or the 6-position hydroxyl group was sulfated. A di-, tri- or tetrasulfated hexopyranosyl derivative was synthesized, and then a 6-position selective desulfating agent was reacted using the di-, tri- or tetrasulfated hexopyranosyl derivative in which the hydroxyl group containing 6-position was sulfated. The present inventors have found that a sulfated hexopyranosyl derivative covering all combinations of sulfation can be prepared by synthesizing a mono-, di- or trisulfated hexopyranosyl derivative in which the 6-position is a free hydroxyl group, and the present invention has been completed. It was.
すなわち、2、3、4および6位の水酸基が無保護のヘキソピラノシル誘導体を原料として、硫酸化剤を反応させ、6位の水酸基のみが硫酸化されたヘキソピラノシル誘導体、あるいは6位を含む水酸基が硫酸化された二、三または四硫酸化ヘキソピラノシル誘導体を合成し、次に、この6位を含む水酸基が硫酸化された二、三または四硫酸化ヘキソピラノシル誘導体を原料として、6位選択的脱硫酸化剤を反応させ、6位が遊離の水酸基である一、二または三硫酸化ヘキソピラノシル誘導体を合成することにより、硫酸化の全ての組合せを網羅した硫酸化ヘキソピラノシル誘導体を得ることを特徴とした硫酸糖誘導体の製造法、および、この製造法によって調製される全ての硫酸化ヘキソピラノシル誘導体の内、6位が遊離の水酸基である硫酸化ヘキソピラノシル誘導体を1つ以上含み、6位が硫酸化された硫酸化ヘキソピラノシル誘導体を含んでいてもよい、複数の硫酸化ヘキソピラノシル誘導体からなる硫酸糖ライブラリー、および、式(1)で示される硫酸化ガラクトース誘導体である。
以下に、本発明を詳細に説明する。なお、DMFはジメチルホルムアミド、MTSTFAはN−メチル−N−(トリメチルシリル)トリフルオロアセトアミド、BTSAはN,O−ビス(トリメチルシリル)アセトアミドの略号である。 The present invention is described in detail below. DMF is an abbreviation for dimethylformamide, MTSTFA is an abbreviation for N-methyl-N- (trimethylsilyl) trifluoroacetamide, and BTSA is an abbreviation for N, O-bis (trimethylsilyl) acetamide.
本発明の原料である2、3、4および6位が無保護のヘキソピラノシル誘導体の合成は如何なる方法によっても構わない。また、ヘキソピラノシル基とそれ以外の構造は、酸素を介する通常のグリコシド結合で結ばれていても、炭素を介するCグリコシド結合で結ばれていても、あるいは、それ以外のヘテロ化合物を介する結合で結ばれていても構わない。
2、3、4および6位の水酸基が無保護のヘキソピラノシル誘導体を原料として、硫酸化剤を、例えばDMF等の適当な溶媒中、適当な当量(1から100当量)、適当な反応温度(0から100℃)および反応時間(10分から2日間)の下に反応させ、6位の水酸基のみが硫酸化された6−硫酸化ヘキソピラノシル誘導体、あるいは6位を含む水酸基が硫酸化された2,6−二硫酸化ヘキソピラノシル誘導体、3,6−二硫酸化ヘキソピラノシル誘導体、4,6−二硫酸化ヘキソピラノシル誘導体、2,3,6−三硫酸化ヘキソピラノシル誘導体、2,4,6−三硫酸化ヘキソピラノシル誘導体、3,4,6−三硫酸化ヘキソピラノシル誘導体、および、2,3,4,6−四硫酸化ヘキソピラノシル誘導体を合成する。得られた化合物は、必要に応じてシリカゲル等を用いた高性能薄層クロマトグラフィー、あるいは、アミドカラム等を用いた高性能液体クロマトグラフィー等を用いて分離・精製する。
硫酸化剤としては、糖質化合物の硫酸化に用いられるものであれば何でもよいが、例えば、三酸化イオウ−ピリジン錯体、三酸化イオウ−トリメチルアミン錯体、クロロスルホン酸−ピリジン錯体、ジシクロヘキシルカルボジイミド−硫酸等を挙げることができ、好ましくは、三酸化イオウ−ピリジン錯体、三酸化イオウ−トリメチルアミン錯体が挙げられる。
The synthesis of the unprotected hexopyranosyl derivative at positions 2, 3, 4 and 6 as the raw material of the present invention may be carried out by any method. In addition, the hexopyranosyl group and the other structure may be linked by a normal glycoside bond via oxygen, a C glycoside bond via carbon, or a bond via other hetero compound. It does not matter.
Starting from a hexopyranosyl derivative in which the hydroxyl groups at positions 2, 3, 4, and 6 are unprotected, a sulfating agent is added in an appropriate amount (1 to 100 equivalents) in an appropriate solvent such as DMF, an appropriate reaction temperature (0 And 6-sulfated hexopyranosyl derivative in which only the hydroxyl group at 6-position is sulfated, or the hydroxyl group containing 6-position is sulfated 2,6 -Disulfated hexopyranosyl derivatives, 3,6-disulfated hexopyranosyl derivatives, 4,6-disulfated hexopyranosyl derivatives, 2,3,6-trisulfated hexopyranosyl derivatives, 2,4,6-trisulfated hexopyranosyl derivatives 3,3,4-trisulfated hexopyranosyl derivative and 2,3,4,6-tetrasulfated hexopyranosyl derivative are synthesized. The obtained compound is separated and purified using high performance thin layer chromatography using silica gel or the like, or high performance liquid chromatography using an amide column or the like, if necessary.
Any sulfating agent may be used as long as it is used for sulfation of carbohydrate compounds. For example, sulfur trioxide-pyridine complex, sulfur trioxide-trimethylamine complex, chlorosulfonic acid-pyridine complex, dicyclohexylcarbodiimide-sulfuric acid. Preferably, a sulfur trioxide-pyridine complex and a sulfur trioxide-trimethylamine complex are mentioned.
次に、上記の反応で得られた6位を含む水酸基が硫酸化された2,6−二硫酸化ヘキソピラノシル誘導体、3,6−二硫酸化ヘキソピラノシル誘導体、4,6−二硫酸化ヘキソピラノシル誘導体、2,3,6−三硫酸化ヘキソピラノシル誘導体、2,4,6−三硫酸化ヘキソピラノシル誘導体、3,4,6−三硫酸化ヘキソピラノシル誘導体、および、2,3,4,6−四硫酸化ヘキソピラノシル誘導体を原料として、6位選択的脱硫酸化剤を、例えばピリジン等の適当な溶媒中、適当な当量(4から100当量)、適当な反応温度(20から100℃)および反応時間(1分から1時間)の下に反応させ、水を含む溶液で後処理することにより、2,6−二硫酸化ヘキソピラノシル誘導体、3,6−二硫酸化ヘキソピラノシル誘導体、および、4,6−二硫酸化ヘキソピラノシル誘導体からは、それぞれ、2−硫酸化ヘキソピラノシル誘導体、3−硫酸化ヘキソピラノシル誘導体、および、4−硫酸化ヘキソピラノシル誘導体が、2,3,6−三硫酸化ヘキソピラノシル誘導体、2,4,6−三硫酸化ヘキソピラノシル誘導体、および、3,4,6−三硫酸化ヘキソピラノシル誘導体からは、それぞれ、2,3−二硫酸化ヘキソピラノシル誘導体、2,4−二硫酸化ヘキソピラノシル誘導体、および、3,4−二硫酸化ヘキソピラノシル誘導体が、2,3,4,6−四硫酸化ヘキソピラノシル誘導体からは2,3,4−三硫酸化ヘキソピラノシル誘導体が、単体または混合物として得られる。得られた化合物は、必要に応じてシリカゲル等を用いた高性能薄層クロマトグラフィー、あるいは、アミドカラム等を用いた高性能液体クロマトグラフィー等を用いて分離・精製する。
6位選択的脱硫酸化剤としては、硫酸糖の6位選択的脱硫酸化に用いられるものであれば何でもよいが、例えば、MTSTFA、BTSA、N−メチル−N−(トリメチルシリル)アセトアミド、ヘキサメチルジシラザン、ペプタメチルジシラザン、N,N−ジメチルアミノトリメチルシラン等を挙げることができ、好ましくは、MTSTFA、BTSAが挙げられる。
以下に、本発明を更に具体的に説明するが、本発明はこれに限定されるものではない。
Next, a 2,6-disulfated hexopyranosyl derivative, a 3,6-disulfated hexopyranosyl derivative, a 4,6-disulfated hexopyranosyl derivative, wherein a hydroxyl group containing 6-position obtained by the above reaction is sulfated, 2,3,4-trisulfated hexopyranosyl derivative, 2,4,6-trisulfated hexopyranosyl derivative, 3,4,6-trisulfated hexopyranosyl derivative, and 2,3,4,6-tetrasulfated hexopyranosyl Starting from the derivative, the 6-position selective desulfating agent is mixed with a suitable equivalent (4 to 100 equivalent), a suitable reaction temperature (20 to 100 ° C.) and a reaction time (1 to 1 minutes) in a suitable solvent such as pyridine. 2), and after-treatment with a solution containing water, 2,6-disulfated hexopyranosyl derivative, 3,6-disulfated hexopyranosyl derivative, and 4,6-disulfated hexopyranosyl derivatives include 2-sulfated hexopyranosyl derivatives, 3-sulfated hexopyranosyl derivatives, and 4-sulfated hexopyranosyl derivatives, respectively, and 2,3,6-trisulfated hexopyranosyl derivatives. 2,4,6-trisulfated hexopyranosyl derivative and 3,4,6-trisulfated hexopyranosyl derivative are respectively 2,3-disulfated hexopyranosyl derivative and 2,4-disulfated hexopyranosyl derivative. 3,4-disulfated hexopyranosyl derivatives, and 2,3,4,6-tetrasulfated hexopyranosyl derivatives, 2,3,4-trisulfated hexopyranosyl derivatives are obtained as a simple substance or as a mixture. The obtained compound is separated and purified using high performance thin layer chromatography using silica gel or the like, or high performance liquid chromatography using an amide column or the like, if necessary.
Any 6-position selective desulfating agent may be used as long as it is used for 6-position selective desulfation of sulfated sugar. For example, MTSTFA, BTSA, N-methyl-N- (trimethylsilyl) acetamide, hexamethyldisulfate Silazane, peptamethyldisilazane, N, N-dimethylaminotrimethylsilane and the like can be mentioned, and MTSTFA and BTSA are preferable.
Hereinafter, the present invention will be described more specifically, but the present invention is not limited thereto.
(N−(O―β―(2−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド、および、N−(O―β―(3−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド、および、N−(O―β―(4−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミドの合成)
参考例3に従って合成した、N−(O―β―(2,6−ジ−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド、および、N−(O―β―(3,6−ジ−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド、および、N−(O―β―(4,6−ジ−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミドの混合物(8.4mg, 8.8μmol) をピリジン(4ml)に溶解し、アルゴン気流下、室温にて5時間撹拌した後、溶媒を留去した。残渣をピリジン(2ml)に溶解し、MTSTFA(0.14ml, 0.71mmol)を加え、アルゴン気流下、60℃で2時間撹拌した。反応液に、水(8ml)と 5mM酢酸アンモニウム/メタノール溶液(2ml)を加え、アルゴン気流下、室温で0.5時間撹拌した。反応終了後溶媒を留去し、シリカゲル(イアトロビーズ)カラムクロマトグラフィー(クロロホルム:5mM酢酸アンモニウム/メタノール溶液:水=130:50:9)にて精製し、目的物(3.7mg, 48%)を混合物として得た。
1H−NMR(400MHz, DMSO-d6/D2O=98:2, 60℃)により、δ 4.22、4.14および4.08に、それぞれのガラクトース部分の1位のプロトンシグナルが観測され、N−(O―β―(2−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド、および、N−(O―β―(3−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド、および、N−(O―β―(4−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミドの生成を確認した。
(N- (O-β- (2-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide and N- (O-β- (3-O- Sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide and N- (O-β- (4-O-sulfogalactopyranosyl) -6-oxyhexyl)- Synthesis of 3,5-bis (dodecyloxy) benzamide)
N- (O-β- (2,6-di-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide synthesized according to Reference Example 3, and N- (O-β- (3,6-di-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide and N- (O-β- (4,6 -A mixture of di-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide (8.4 mg, 8.8 μmol) was dissolved in pyridine (4 ml) at room temperature under a stream of argon. Then, the solvent was distilled off. The residue was dissolved in pyridine (2 ml), MTSTFA (0.14 ml, 0.71 mmol) was added, and the mixture was stirred at 60 ° C. for 2 hours under an argon stream. Water (8 ml) and 5 mM ammonium acetate / methanol solution (2 ml) were added to the reaction solution, and the mixture was stirred at room temperature for 0.5 hours under an argon stream. After completion of the reaction, the solvent was distilled off, and the residue was purified by silica gel (Iatrobeads) column chromatography (chloroform: 5 mM ammonium acetate / methanol solution: water = 130: 50: 9) to obtain the desired product (3.7 mg, 48%) as a mixture. Got as.
By 1 H-NMR (400 MHz, DMSO-d 6 / D 2 O = 98: 2, 60 ° C.), proton signals at position 1 of each galactose moiety were observed at δ 4.22, 4.14 and 4.08, and N- ( O-β- (2-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide and N- (O-β- (3-O-sulfogalactopyrano) Syl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide and N- (O-β- (4-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5- Formation of bis (dodecyloxy) benzamide was confirmed.
[参考例1]
(N−(O−β−(6−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミドの合成)
N−(O−β−ガラクトピラノシル−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド(51.7mg, 0.0687mmol)をDMF2mlに溶解し、三酸化硫黄・トリメチルアミン錯体(336mg, 2.41mmol)を加え、アルゴン雰囲気下、2時間撹拌した。反応液をそのままゲルろ過(LH−20、クロロホルム−メタノール,1:1)し、目的物を含む画分を濃縮した。さらに、シリカゲルカラムクロマトフィー(クロロホルム−メタノール,2:1)により精製し、目的物(14.2mg, 0.0171mmol)を得た。
MALDI−TOF/MS(マトリックス、ジヒドロキシ安息香酸):m/z 831.53 (C43H77NO12Sとして計算:831.52,[M−H]-)
[Reference Example 1]
(Synthesis of N- (O-β- (6-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide)
N- (O-β-galactopyranosyl-6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide (51.7 mg, 0.0687 mmol) was dissolved in 2 ml of DMF, and sulfur trioxide / trimethylamine complex (336 mg, 2.41 mmol) and stirred for 2 hours under an argon atmosphere. The reaction solution was directly subjected to gel filtration (LH-20, chloroform-methanol, 1: 1), and the fraction containing the target product was concentrated. The product was further purified by silica gel column chromatography (chloroform-methanol, 2: 1) to obtain the desired product (14.2 mg, 0.0171 mmol).
MALDI-TOF / MS (matrix, dihydroxybenzoic acid): m / z 831.53 (calculated as C 43 H 77 NO 12 S: 831.52, [M−H] − )
[参考例2]
(硫酸糖脂質アナログが臍帯血前駆細胞増殖に及ぼす影響)
N−(O−β−(6−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド3.5mgを200 mlのエタノールに溶かし−20℃で保存した。使用時に、この保存溶液1.8mlにエタノール8.2mlを加えて希釈した後に、24穴プレートと同じ断面積で厚さ100μmに裁断したポリエステル不織布(旭化成製Y-15050、)を1枚入れた24穴プレートに1 mlずつ加え揮発させた。また、濃度による影響を検討するため、この溶液をエタノールでさらに10倍希釈して同様にポリエステル不織布にコーティングした。
動物細胞として、ヒト臍帯血からフィコール密度勾配遠心分離により得られた単核造血細胞を用いた。
予め、ヒト臍帯血単核造血細胞1×105cellsとヒト骨髄初代ストローマ細胞5×105cellsとを、無血清無サイトカイン培地X-VIVO10(BioWhittaker,Walkersville,USA)を用いて、24ウェルプレート中で37℃,5%CO2雰囲気下で1週間共培養した後、その培養上清を得た。
上記のN−(O−β−(6−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミドをコーティングした不織布、同じく10倍希釈した溶液でコーティングした不織布、または、コーティングしていない不織布、いずれかの不織布を入れた24穴プレートに、上記の培養上清を用いて、ヒト臍帯血単核造血細胞1×105 cellsを播種し、37℃、5%CO2雰囲気下で1週間培養した。
その後、培養後の不織布および培養液の両方から回収した造血細胞を集め、コロニーフォーミングユニットアッセイを行い、培養後の造血前駆細胞の濃度を計数した。
表1に示すように、培養後の造血前駆細胞濃度は、無処理の不織布に比べ、N−(O−β−(6−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミドを10倍希釈してコーティングしたものでもほぼ同等、N−(O−β−(6−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミドを1倍濃度でコーティングしたもので約1.6倍であり、明らかな増殖活性を示した。
(Effects of sulfated glycolipid analog on cord blood progenitor cell proliferation)
N- (O-β- (6-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide (3.5 mg) was dissolved in 200 ml of ethanol and stored at -20 ° C. 24 holes with 1 piece of polyester nonwoven fabric (Y-15050, manufactured by Asahi Kasei) cut into 100 μm thickness with the same cross-sectional area as the 24-hole plate after diluting by adding 8.2 ml of ethanol to 1.8 ml of the stock solution at the time of use 1 ml was added to the plate and evaporated. Further, in order to examine the influence of the concentration, this solution was further diluted 10 times with ethanol and coated on a polyester nonwoven fabric in the same manner.
Mononucleated hematopoietic cells obtained by ficoll density gradient centrifugation from human umbilical cord blood were used as animal cells.
In advance, human umbilical cord blood mononuclear hematopoietic cells 1 × 10 5 cells and human bone marrow primary stromal cells 5 × 10 5 cells were added to a 24-well plate using serum-free cytokine-free medium X-VIVO10 (BioWhittaker, Walkersville, USA). The culture supernatant was obtained after co-culture at 37 ° C. in a 5% CO 2 atmosphere for 1 week.
Nonwoven fabric coated with the above-mentioned N- (O-β- (6-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide, also coated with a 10-fold diluted solution Using the above culture supernatant, 1 × 10 5 human umbilical cord blood mononuclear hematopoietic cells are seeded on a 24-well plate containing either a non-woven fabric or a non-coated non-woven fabric, at 37 ° C. The cells were cultured for 1 week in a 5% CO 2 atmosphere.
Thereafter, hematopoietic cells collected from both the non-woven fabric and the culture solution after culture were collected, colony forming unit assay was performed, and the concentration of hematopoietic progenitor cells after culture was counted.
As shown in Table 1, the hematopoietic progenitor cell concentration after culture was N- (O-β- (6-sulfogalactopyranosyl) -6-oxyhexyl) -3,5- N- (O-β- (6-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide is almost equivalent even when coated with 10-fold diluted bis (dodecyloxy) benzamide Was coated at a 1-fold concentration, and was about 1.6 times, showing a clear proliferative activity.
[参考例3]
(N−(O―β―(2,6−ジ−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド、および、N−(O―β―(3,6−ジ−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド、および、N−(O―β―(4,6−ジ−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミドの合成)
N−(O―β―(ガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド(30.0mg, 0.040mmol)をDMF(2.2ml)に溶解させ、三酸化硫黄・ピリジン錯体(32.4mg, 0.20mmol, 5当量)を加え、アルゴン気流下、室温にて2.5時間撹拌した。反応終了後、メタノール(20ml)を加え、溶媒を留去した。残渣をゲル濾過カラムクロマトグラフィー(クロロホルム:メタノール:水=108:58:1、トリエチルアミン1%)にて分取し、さらに、シリカゲルカラムクロマトグラフィー(クロロホルム:メタノール=4:1、トリエチルアミン1%)にて精製し、目的物(27.8mg, 84%)を混合物として得た。
これらの化合物がエタノールに可溶であることを確認した。
1H-NMR (400MHz, DMSO-d6/D2O=98:2, 60℃)
[Reference Example 3]
(N- (O-β- (2,6-di-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide and N- (O-β- ( 3,6-di-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide and N- (O-β- (4,6-di-O-sulfo) Synthesis of galactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide
N- (O-β- (galactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide (30.0 mg, 0.040 mmol) was dissolved in DMF (2.2 ml), sulfur trioxide, Pyridine complex (32.4 mg, 0.20 mmol, 5 equivalents) was added, and the mixture was stirred at room temperature for 2.5 hours under a stream of argon. After completion of the reaction, methanol (20 ml) was added and the solvent was distilled off. The residue was separated by gel filtration column chromatography (chloroform: methanol: water = 108: 58: 1, triethylamine 1%) and further subjected to silica gel column chromatography (chloroform: methanol = 4: 1, triethylamine 1%). To obtain the desired product (27.8 mg, 84%) as a mixture.
It was confirmed that these compounds are soluble in ethanol.
1 H-NMR (400MHz, DMSO-d 6 / D 2 O = 98: 2, 60 ° C)
N−(O―β―(2,6−ジ−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド
δ 6.96 (t, J = 2.4 Hz), 6.55 (bs), 4.22 (d, J = 8.3 Hz), 4.09 (dd, J = 8.3, 9.3 Hz), 3.98 (t, J = 6.5 Hz), 3.86 (dd, J = 6.2, 10.3 Hz), 3.76 (dd, J = 6.2, 10.3 Hz), 3.70 (m), 3.64 (dd, J = 2.0, 8.6 Hz), 3.58 (t, J = 6.2 Hz), 3.50 (m), 3.47 (m), 3.20 (m), 1.69 (m), 1.53 (m), 1.41 (m), 1.29 (m), 0.85 (t, J = 6.9 Hz)
N- (O-β- (2,6-di-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide δ 6.96 (t, J = 2.4 Hz), 6.55 (bs), 4.22 (d, J = 8.3 Hz), 4.09 (dd, J = 8.3, 9.3 Hz), 3.98 (t, J = 6.5 Hz), 3.86 (dd, J = 6.2, 10.3 Hz), 3.76 (dd, J = 6.2, 10.3 Hz) , 3.70 (m), 3.64 (dd, J = 2.0, 8.6 Hz), 3.58 (t, J = 6.2 Hz), 3.50 (m), 3.47 (m), 3.20 (m), 1.69 (m), 1.53 ( m), 1.41 (m), 1.29 (m), 0.85 (t, J = 6.9 Hz)
N−(O―β―(3,6−ジ−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド
δ 6.96 (t, J = 2.4 Hz), 6.55 (bs), 4.15 (d, J = 7.6 Hz), 3.98 (t, J = 6.5 Hz), 3.95 (dd, J = 3.4, 9.6 Hz), 3.87 (bd, J = 3.4 Hz), 3.86 (dd, J = 6.2, 10.3 Hz), 3.76 (dd, J = 6.2, 10.3 Hz), 3.58 (t, J = 6.2 Hz), 3.47 (m), 3.43 (dd, J = 7.6, 9.6 Hz), 3.20 (m), 1.69 (m), 1.53 (m), 1.41 (m), 1.29 (m), 0.85 (t, J = 6.9 Hz)
N- (O-β- (3,6-di-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide δ 6.96 (t, J = 2.4 Hz), 6.55 (bs), 4.15 (d, J = 7.6 Hz), 3.98 (t, J = 6.5 Hz), 3.95 (dd, J = 3.4, 9.6 Hz), 3.87 (bd, J = 3.4 Hz), 3.86 (dd, J = 6.2, 10.3 Hz), 3.76 (dd, J = 6.2, 10.3 Hz), 3.58 (t, J = 6.2 Hz), 3.47 (m), 3.43 (dd, J = 7.6, 9.6 Hz), 3.20 (m ), 1.69 (m), 1.53 (m), 1.41 (m), 1.29 (m), 0.85 (t, J = 6.9 Hz)
N−(O―β―(4,6−ジ−O−スルホガラクトピラノシル)−6−オキシヘキシル)−3,5−ビス(ドデシロキシ)ベンズアミド
δ 6.96 (t, J = 2.4 Hz), 6.55 (bs), 4.34 (d, J = 3.4 Hz), 4.06 (d, J = 7.6 Hz), 3.98 (t, J = 6.5 Hz), 3.94 (m), 3.76 (dd, J = 6.2, 10.3 Hz), 3.70 (m), 3.47 (m), 3.34 (dd, J = 3.4, 9.6 Hz), 3.20 (m), 1.69 (m), 1.53 (m), 1.41 (m), 1.29 (m), 0.85 (t, J = 6.9 Hz)
N- (O-β- (4,6-di-O-sulfogalactopyranosyl) -6-oxyhexyl) -3,5-bis (dodecyloxy) benzamide δ 6.96 (t, J = 2.4 Hz), 6.55 (bs), 4.34 (d, J = 3.4 Hz), 4.06 (d, J = 7.6 Hz), 3.98 (t, J = 6.5 Hz), 3.94 (m), 3.76 (dd, J = 6.2, 10.3 Hz) , 3.70 (m), 3.47 (m), 3.34 (dd, J = 3.4, 9.6 Hz), 3.20 (m), 1.69 (m), 1.53 (m), 1.41 (m), 1.29 (m), 0.85 ( t, J = 6.9 Hz)
本発明は、2、3、4および6位の水酸基が無保護のヘキソピラノシル誘導体を原料として、硫酸化剤を反応させ、6位の水酸基のみが硫酸化されたヘキソピラノシル誘導体、あるいは6位を含む水酸基が硫酸化された二、三または四硫酸化ヘキソピラノシル誘導体を合成し、次に、この6位を含む水酸基が硫酸化された二、三または四硫酸化ヘキソピラノシル誘導体を原料として、6位選択的脱硫酸化剤を反応させ、6位が遊離の水酸基である一、二または三硫酸化ヘキソピラノシル誘導体を合成することにより、硫酸化の全ての組合せを網羅した硫酸化ヘキソピラノシル誘導体を得ることを特徴とした硫酸糖誘導体の製造法を提供するものであり、各種の細胞培養材料等の医療材料、血液抗凝固剤等の医薬品の製造に用いることができる。 The present invention relates to a hexopyranosyl derivative obtained by reacting a sulfating agent using a hexopyranosyl derivative in which the hydroxyl groups at positions 2, 3, 4 and 6 are unprotected as a raw material, or a hydroxyl group containing 6-position. Sulphated di-, tri- or tetra-sulphated hexopyranosyl derivative, and then 6-position selective desulfurization using the sulphated di-, tri- or tetrasulphated hexopyranosyl derivative in which the hydroxyl group containing 6-position is sulfated Sulfuric acid characterized by obtaining a sulfated hexopyranosyl derivative covering all combinations of sulfation by reacting an oxidant and synthesizing a mono-, di- or tri-sulfated hexopyranosyl derivative having a free hydroxyl group at the 6-position The present invention provides a method for producing a sugar derivative and can be used for the production of medical materials such as various cell culture materials and pharmaceuticals such as blood anticoagulants.
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05230090A (en) * | 1992-02-25 | 1993-09-07 | Seikagaku Kogyo Co Ltd | Method for selectively desulfating and oxidizing sulfated saccharide |
JP2001122889A (en) * | 1999-10-26 | 2001-05-08 | Noguchi Inst | Glycolipid analog compound |
JP2002030091A (en) * | 2000-07-13 | 2002-01-29 | Noguchi Inst | Double chain glycolipide analog |
JP2004256413A (en) * | 2003-02-25 | 2004-09-16 | Mutsumi Takagi | Animal cell culture raw material |
JP2004256433A (en) * | 2003-02-26 | 2004-09-16 | Noguchi Inst | Galactose disulfate derivative |
-
2004
- 2004-02-18 JP JP2004042128A patent/JP4675048B2/en not_active Expired - Fee Related
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05230090A (en) * | 1992-02-25 | 1993-09-07 | Seikagaku Kogyo Co Ltd | Method for selectively desulfating and oxidizing sulfated saccharide |
JP2001122889A (en) * | 1999-10-26 | 2001-05-08 | Noguchi Inst | Glycolipid analog compound |
JP2002030091A (en) * | 2000-07-13 | 2002-01-29 | Noguchi Inst | Double chain glycolipide analog |
JP2004256413A (en) * | 2003-02-25 | 2004-09-16 | Mutsumi Takagi | Animal cell culture raw material |
JP2004256433A (en) * | 2003-02-26 | 2004-09-16 | Noguchi Inst | Galactose disulfate derivative |
Non-Patent Citations (3)
Title |
---|
JPN6010027379, Biosci. Biotech. Biochem., 1992, 56, 1577−1580 * |
JPN6010027380, Biosci. Biotech. Biochem., 1992, 56, 1413−1416 * |
JPN6010027381, J. Carbohydrate Chem., 1995, 14, 885−888 * |
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KR20180120186A (en) | 2016-03-09 | 2018-11-05 | 가부시키가이샤 도우사 고가쿠 겐큐쇼 | A method for producing a sugar having a sulfate group and / or a phosphate group |
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