JP4942014B2 - O-linked sugar amino acids - Google Patents
O-linked sugar amino acids Download PDFInfo
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- JP4942014B2 JP4942014B2 JP2005264181A JP2005264181A JP4942014B2 JP 4942014 B2 JP4942014 B2 JP 4942014B2 JP 2005264181 A JP2005264181 A JP 2005264181A JP 2005264181 A JP2005264181 A JP 2005264181A JP 4942014 B2 JP4942014 B2 JP 4942014B2
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- 235000000346 sugar Nutrition 0.000 title claims description 81
- 150000001413 amino acids Chemical class 0.000 title description 35
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- 238000004519 manufacturing process Methods 0.000 claims description 7
- 239000007858 starting material Substances 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 4
- 230000009471 action Effects 0.000 claims description 2
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- 229910052739 hydrogen Inorganic materials 0.000 description 13
- 239000001257 hydrogen Substances 0.000 description 13
- -1 9-fluorenyloxycarbonyl group Chemical group 0.000 description 12
- XCSQXCKDEVRTHN-UHFFFAOYSA-N cyclohexa-1,4-diene-1-carboxylic acid Chemical compound OC(=O)C1=CCC=CC1 XCSQXCKDEVRTHN-UHFFFAOYSA-N 0.000 description 12
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Landscapes
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Description
本発明は種々のＯ−結合型糖アミノ酸の合成のための出発化合物として有用な糖アミノ酸、及びそれを用いるＯ−結合型糖アミノ酸の製造方法に関する。 The present invention relates to a sugar amino acid useful as a starting compound for the synthesis of various O-linked sugar amino acids, and a method for producing an O-linked sugar amino acid using the same.
近年のゲノム研究の発展により、核酸とタンパク質に関する研究は急速に発展した。タンパク質が実際に機能するためには正しくフォールディングし、特定の部位へ輸送される必要があるが、これに糖鎖が深く関わっていることが近年報告され、その詳細な機構の解明が期待されている。さらに、特定の部位に配置されたタンパク質が様々な相互作用を利用して機能する場面においても、多くの場合、糖鎖との相互作用は避けて通ることのできない重要な情報伝達経路である。 Recent advances in genome research have led to rapid development of nucleic acid and protein research. In order for proteins to actually function, they need to be correctly folded and transported to specific sites, but it has recently been reported that sugar chains are deeply involved in this, and the detailed mechanism is expected to be elucidated. Yes. Furthermore, even in a situation where a protein arranged at a specific site functions using various interactions, in many cases, an interaction with a sugar chain is an important information transmission pathway that cannot be avoided.
このようにポストゲノム分野において糖鎖はきわめて重要な役割を果たしているにもかかわらず、その詳細な機能の解明はあまり進んでいない。その主な理由として糖鎖はタンパク質などとの複合体として機能していることが多く、さらに特定のタンパク質に特定の糖鎖が修飾されるわけではなく、加齢、病変などに伴いその糖鎖構造は多様に変化することがあげられる。また、特定のタンパク質上の糖鎖構造の変化を測定する技術に関してもまだ有力なものは開発されていない。 Thus, although the sugar chain plays a very important role in the post-genomic field, elucidation of its detailed function has not progressed much. The main reason is that sugar chains often function as a complex with proteins, etc., and specific sugar chains are not modified to specific proteins. The structure can be variously changed. In addition, a powerful technique for measuring the change in sugar chain structure on a specific protein has not been developed yet.
糖鎖の機能をより詳細に解明するには、まず、構造が明確で純粋な（均一な）複合糖質サンプルを容易にしかも迅速に合成できなければならない。 In order to elucidate the function of glycans in more detail, it is first necessary to be able to synthesize easily and rapidly a pure (homogeneous) complex carbohydrate sample with a well-defined structure.
しかしながら、現在のところ標準糖鎖として入手可能な糖タンパク質関連糖鎖はほぼＮ−結合型糖アミノ酸のものに限られており（例えば、特開２００１−１１５６１６号公報、特開平１１−２５５８０７号公報）、Ｏ−結合型糖アミノ酸の標準サンプルは入手は非常に困難である。一部のＯ−結合型糖アミノ酸について報告されているが、その簡便な合成法についは知られていない。 However, currently, glycoprotein-related sugar chains available as standard sugar chains are almost limited to those of N-linked sugar amino acids (for example, JP-A Nos. 2001-115616 and 11-255807). ), Standard samples of O-linked sugar amino acids are very difficult to obtain. Although some O-linked sugar amino acids have been reported, no simple synthesis method is known.
多様な糖鎖を有する種々のＯ−結合型糖アミノ酸の簡便な製造方法及びそのための原料化合物、並びに新規なＯ−結合型糖アミノ酸を提供することを目的とする。 An object is to provide a simple method for producing various O-linked sugar amino acids having various sugar chains, a raw material compound therefor, and a novel O-linked sugar amino acid.
本発明者らは、上記課題を解決するために鋭意検討した結果、特定の糖アミノ酸を出発化合物として用い、これに糖転移酵素を作用させることにより、前記課題を解決できることを見出し、本発明を完成させるに至った。 As a result of intensive investigations to solve the above problems, the present inventors have found that the above problems can be solved by using a specific sugar amino acid as a starting compound and allowing a glycosyltransferase to act on the compound. It came to complete.
即ち、本発明は以下の発明を包含する。
（１）下記式（Ｉ）：
Ｒは、
That is, the present invention includes the following inventions.
(1) The following formula (I):
R is
Ｘ、Ｙは、それぞれ独立して、水素、単糖残基又はオリゴ糖残基であり；
Z１は、水素、アミノ基の保護基又は標識基であり；
Z２は、ヒドロキシル基、カルボキシル基の保護基、活性化基又は標識基である。]
で表される化合物。
但し、Ｘ及びＹが同時に水素となることはなく、また下記化合物を除く：
X and Y are each independently hydrogen, a monosaccharide residue or an oligosaccharide residue;
Z 1 is hydrogen, an amino-protecting group or a labeling group;
Z 2 is a hydroxyl group, a protecting group for a carboxyl group, an activating group or a labeling group. ]
A compound represented by
However, X and Y do not simultaneously become hydrogen and exclude the following compounds:
（２）Ｒは、
Ｘ及びＹは、それぞれ独立して、水素、
(2) R is
X and Y are each independently hydrogen,
Ｚは、ヒドロキシル基、
Z is a hydroxyl group,
Ｒ３、Ｒ６及びＲ６’は、互いに独立して、水素、又は
R 3 , R 6 and R 6 ′ are independently of each other hydrogen, or
Ｒ３’及びＲ３”は、互いに独立して、水素又は、
R 3 ′ and R 3 ″ are independently of each other hydrogen or
ｎは１〜２０までの整数である；
の前記（１）記載の化合物。
n is an integer from 1 to 20;
The compound according to (1) above.
（３）下記式：
（４）下記式：
（５）前記（１）記載の式（Ｉ）の化合物を出発原料として用いるＯ−結合型糖アミノ酸の製造方法。
（６）糖供与体の存在下で、前記（１）記載の式（Ｉ）の化合物に糖転移酵素を作用させて、式（Ｉ）の化合物の糖鎖を伸長することを特徴とするＯ−結合型糖アミノ酸の製造方法。
(5) A method for producing an O-linked sugar amino acid using the compound of formula (I) described in (1) as a starting material.
(6) O-ring characterized by extending a sugar chain of the compound of the formula (I) by allowing a glycosyltransferase to act on the compound of the formula (I) described in the above (1) in the presence of a sugar donor. -Production method of conjugated sugar amino acid.
本発明の方法によれば、特定のコア構造を有する糖アミノ酸を出発化合物として、これに糖転移酵素を作用させることにより、糖鎖が均一なＯ−結合型糖アミノ酸を容易に得ることができる。また、本発明の新規なＯ−結合型糖アミノ酸は、糖鎖分析用の標準サンプルとして、又はタンパク質との相互作用解析用サンプル等として有用である。さらには、本発明のＯ−結合型糖アミノ酸は、これ自体を原料とし、糖転移酵素を作用させることにより糖鎖を伸長させることもできる。 According to the method of the present invention, a sugar amino acid having a specific core structure is used as a starting compound, and a glycosyltransferase is allowed to act thereon, whereby an O-linked sugar amino acid having a uniform sugar chain can be easily obtained. . In addition, the novel O-linked sugar amino acid of the present invention is useful as a standard sample for sugar chain analysis, a sample for analysis of interaction with proteins, or the like. Furthermore, the O-linked sugar amino acid of the present invention can be used as a raw material, and the sugar chain can be extended by the action of glycosyltransferase.
以下に本発明を詳細に説明する。
本発明の化合物は、下記式（Ｉ）：
Ｒは、
The present invention is described in detail below.
The compound of the present invention has the following formula (I):
R is
上記Ｘ及びＹとしては、互いに独立して、水素、糖残基又は２〜３０糖の糖鎖が挙げられる。前記糖残基又は２〜３０糖の糖鎖としては、単糖及びオリゴ糖（２〜３０糖、好ましくは２〜１５糖、さらに好ましくは２〜１０糖）のいずれでもよく、また、各構成糖はペントース、ヘキソース及びヘプトース等のいずれであってもよい。３糖以上のオリゴ糖鎖である場合には直鎖状糖鎖及び分岐状糖鎖のいずれでもよい。前記糖残基又は糖鎖は、その水酸基の１つ以上が硫酸基、リン酸基、メチル基及び／又はアセチル基により置換されていてもよい。前記糖残基又は糖鎖の糖鎖構造は天然型および非天然型のいずれでもよい。 As said X and Y, hydrogen, a sugar residue, or a sugar chain of 2-30 sugars is mentioned mutually independently. The sugar residue or sugar chain of 2 to 30 sugars may be any of monosaccharides and oligosaccharides (2 to 30 sugars, preferably 2 to 15 sugars, more preferably 2 to 10 sugars). The sugar may be any of pentose, hexose, heptose and the like. In the case of an oligosaccharide chain having 3 or more sugars, either a linear sugar chain or a branched sugar chain may be used. In the sugar residue or sugar chain, one or more of the hydroxyl groups may be substituted with a sulfate group, a phosphate group, a methyl group and / or an acetyl group. The sugar residue or sugar chain structure of the sugar chain may be either a natural type or a non-natural type.
前記糖残基又は糖鎖の具体例としては、例えば、Ｎ−アセチルグルコサミン、Ｎ−アセチルガラクトサミン、キシロース、マンノース、ガラクトース、グルコース、フコース、Ｎ−アセチルノイラミン酸、Ｎ−グリコイルノイラミン酸、Ｎ−アセチルラクトサミン、リボース、ラクトース、マルトース等の単糖類、並びにこれらの構成糖単位を含み直鎖状又は分岐状に結合したオリゴ糖が挙げられる。 Specific examples of the sugar residue or sugar chain include, for example, N-acetylglucosamine, N-acetylgalactosamine, xylose, mannose, galactose, glucose, fucose, N-acetylneuraminic acid, N-glycolneuraminic acid, Examples thereof include monosaccharides such as N-acetyllactosamine, ribose, lactose and maltose, and oligosaccharides containing these constituent sugar units and bound in a linear or branched manner.
Z１は水素、アミノ基の保護基、又は標識基である。アミノ基の保護基としては例えば、９−フルオレニルオキシカルボニル基（Ｆｍｏｃ基）、ベンジルオキシカルボニル基、第３ブチルオキシカルボニル基等が挙げられ、特にＦｍｏｃ基が好ましい。また、標識基としては発色団、蛍光基又は放射性同位体が挙げられる。 Z 1 is hydrogen, an amino-protecting group, or a labeling group. Examples of the amino-protecting group include 9-fluorenyloxycarbonyl group (Fmoc group), benzyloxycarbonyl group, tertiary butyloxycarbonyl group and the like, and Fmoc group is particularly preferable. Examples of the labeling group include a chromophore, a fluorescent group, and a radioisotope.
Z２はヒドロキシル基、カルボキシル基の保護基、標識基、又はカルボキシル基の活性化基である。通常は遊離酸（即ち、Z２はヒドロキシル基）の状態で使用されるが、必要に応じて発色団、蛍光基、放射性同位体等の標識基を導入してもよく、ペプチド合成を行う場合には、保護基により保護したり、活性化してもよい。カルボキシル基の活性化基とは、カルボキシル基のカルボニル炭素の反応性を高める目的で導入される官能基である。カルボキシル基の保護基及び活性化基Ｒ２としては、例えば、−ＯＲ（エステル：−ＣＯＯＲとして）、Ｐｆｐ（ペンタフルオロフェニル基）、フッ素基、Ｓ−アルキル基及びＳ−アリール基（チオエステル：−ＣＯＳＲとして）等が挙げられる。ここで、前記Ｒは低級アルキル又はアリール基である。 Z 2 is a hydroxyl group, a protecting group for a carboxyl group, a labeling group, or an activating group for a carboxyl group. Usually used as a free acid (ie, Z 2 is a hydroxyl group), but if necessary, labeling groups such as chromophores, fluorescent groups, and radioisotopes may be introduced. May be protected by a protecting group or activated. The activating group of the carboxyl group is a functional group introduced for the purpose of increasing the reactivity of the carbonyl carbon of the carboxyl group. The protecting group of the carboxyl group and activating group R 2, for example, -OR (ester as -COOR), Pfp (pentafluorophenyl group), a fluorine group, S- alkyl and S- aryl groups (thioester: - As COSR). Here, R is a lower alkyl or aryl group.
本明細書で言う発色団とはＵＶ−ＶＩＳ（可視）領域の電磁波（２００〜８００ｎｍが好ましい）を吸収する官能基であり、蛍光基とはＵＶ−ＶＩＳ領域の電磁波を吸収することによりＶＩＳ領域の電磁波（300〜900ｎｍが好ましい）を発する官能基を指す。そのような発色団及び蛍光基の具体例としては、例えば、フルオレセイン基、ダンシル基、ＡＭＣＡ基、ＤＩＤＳ基、ピリジルアミノ基、ローダミン基、テトラメチルローダミン基、クマリン基、７−メトキシクマリン基、ピレン基、ＮＢＤ基等が挙げられる。 The chromophore referred to in this specification is a functional group that absorbs electromagnetic waves in the UV-VIS (visible) region (200 to 800 nm is preferable), and the fluorescent group is a VIS region that absorbs electromagnetic waves in the UV-VIS region. The functional group which emits electromagnetic waves (300 to 900 nm is preferable). Specific examples of such chromophores and fluorescent groups include, for example, fluorescein group, dansyl group, AMCA group, DIDS group, pyridylamino group, rhodamine group, tetramethylrhodamine group, coumarin group, 7-methoxycoumarin group, and pyrene group. , NBD group and the like.
但し、本発明の化合物は、式（Ｉ）においてＸ及びＹが同時に水素となることはなく、また下記化合物は除外される： However, in the compounds of the present invention, X and Y in formula (I) are not simultaneously hydrogen, and the following compounds are excluded:
好ましくは、Ｒは
好ましくは、Ｘ、Ｙは、それぞれ独立して、水素、
好ましくは、前記Ｚは、水素、
好ましくは、前記Ｒ３、Ｒ６及びＲ６’は、互いに独立して、水素又は、
好ましくは、前記Ｒ３'及びＲ３”は、互いに独立して、水素又は、
好ましくは、前記ｎは１〜２０までの整数である。
本発明のＯ−結合型糖アミノ酸は糖転移酵素を用いて製造することができる。出発化合物としては、特に限定されるものではないが、以下のコア構造を有する糖アミノ酸：
Preferably, n is an integer from 1 to 20.
The O-linked sugar amino acid of the present invention can be produced using a glycosyltransferase. The starting compound is not particularly limited, but is a sugar amino acid having the following core structure:
Ｃｏｒｅ２と呼ばれる分岐型３糖を有する上記糖アミノ酸化合物１から出発して、糖転移酵素を用いて、例えば、以下のようなスキームにより多様なＯ−結合型糖アミノ酸を製造することができる。 Starting from the above sugar amino acid compound 1 having a branched trisaccharide called Core2, various O-linked sugar amino acids can be produced by using a glycosyltransferase according to the following scheme, for example.
以下に、上記化合物１を出発化合物とする本発明のＯ−結合型糖アミノ酸の製造方法について例を示して説明するが、本発明はこれに限定されるものではない。なお、生成したＯ−結合型糖アミノ酸の分離精製及び分析感度の点から、そのアミノ基部位は非極性の置換基であるＦｍｏｃ基が望ましい。 Hereinafter, the method for producing an O-linked sugar amino acid of the present invention using Compound 1 as a starting compound will be described with reference to examples, but the present invention is not limited thereto. In view of separation and purification of the produced O-linked sugar amino acid and analytical sensitivity, the amino group site is preferably a Fmoc group which is a nonpolar substituent.
糖転移酵素としては、例えば、グルコース転移酵素（ＧｌｃＴ）、ガラクトース転移酵素（ＧａｌＴ）、N-アセチルグルコサミン転移酵素（ＧｌｃＮＡｃＴまたはＧｎＴ）、N-アセチルガラクトサミン転移酵素（ＧａｌＮＡｃＴ）、グルクロン酸転移酵素（ＧｌｃＡＴ）、マンノース転移酵素（ＭａｎＴ）、フコース転移酵素（ＦｕｃＴ）、シアル酸転移酵素（ＳｉａＴ）、キシロース転移酵素（ＸｙｌＴ）、ガラクツロン酸転移酵素（ＧａｌＡＴ）等が挙げられ、より具体的には、β−１，４−ＧｌｃＴ、α−１，２−ＧｌｃＴ、α−１，３−ＧｌｃＴ、α−１，４−ＧｌｃＴ、β−１，２−ＧｌｃＮＡｃＴ、β−１，３−ＧｌｃＮＡｃＴ、β−１，４−ＧｌｃＮＡｃＴ、β−１，６−ＧｌｃＮＡｃＴ、β−１，３−ＧａｌＮＡｃＴ、β−１，４−ＧａｌＮＡｃＴ、β−１，６−ＧａｌＮＡｃＴ、α−１，２−ＧａｌＮＡｃＴ、α−１，３−ＧａｌＮＡｃＴ、α−１，４−ＧａｌＮＡｃＴ、α−１，６−ＧａｌＮＡｃＴ、β−１，４−ＧａｌＴ、β−１，３−ＧａｌＴ、α−１，４−ＧａｌＴ、α−１，３−ＧａｌＴ、α−１，２−ＭａｎＴ、α−１，３−ＭａｎＴ、α−１，６−ＭａｎＴ、α−２，３（Ｏ）−ＳｉａＴ、α−２，３（Ｎ）−ＳｉａＴ、α−２，３−ＳｉａＴ、α−２，６（Ｎ）−ＳｉａＴ、α−２，６−ＳｉａＴ、α−２，３／８−ＳｉａＴ、α−２，８−ＳｉａＴ、α−２，８／９−ＳｉａＴ、α−１，２−ＦｕｃＴＩ、α−１，２−ＦｕｃＴII、α−１，３／４−ＦｕｃＴIII、α−１，３／４−ＦｕｃＴIII、α−１，３−ＦｕｃＴIV、α−１，３−ＦｕｃＴＶ、α−１，３−ＦｕｃＴVI、α−１，３−ＦｕｃＴVII、α−１，６−ＦｕｃＴVIII、α−１，３−ＦｕｃＴIX、α−１，４−ＧａｌＡＴ、α−１，３−ＸｙｌＴ、β−１，２−ＸｙｌＴ等が挙げられる。 Examples of the glycosyltransferase include glucose transferase (GlcT), galactose transferase (GalT), N-acetylglucosamine transferase (GlcNAcT or GnT), N-acetylgalactosamine transferase (GalNAcT), glucuronic acid transferase (GlcAT). ), Mannose transferase (ManT), fucose transferase (FucT), sialic acid transferase (SiaT), xylose transferase (XylT), galacturonic acid transferase (GalAT) and the like, more specifically, β -1,4-GlcT, α-1,2-GlcT, α-1,3-GlcT, α-1,4-GlcT, β-1,2-GlcNAcT, β-1,3-GlcNAcT, β-1 , 4-GlcNAcT, β-1,6-GlcNAcT, β-1,3-GalNAcT, β-1,4- alNAcT, β-1,6-GalNAcT, α-1,2-GalNAcT, α-1,3-GalNAcT, α-1,4-GalNAcT, α-1,6-GalNAcT, β-1,4-GalT, β-1,3-GalT, α-1,4-GalT, α-1,3-GalT, α-1,2-ManT, α-1,3-ManT, α-1,6-ManT, α- 2,3 (O) -SiaT, α-2,3 (N) -SiaT, α-2,3-SiaT, α-2,6 (N) -SiaT, α-2,6-SiaT, α-2 , 3 / 8-SiaT, α-2,8-SiaT, α-2,8 / 9-SiaT, α-1,2-FucTI, α-1,2-FucTII, α-1,3 / 4-FucTIII , Α-1,3 / 4-FucTIII, α-1,3-FucTIV, α-1,3-FucTV, α-1,3-FucTVI α-1,3-FucTVII, α-1,6-FucTVIII, α-1,3-FucTIX, α-1,4-GalAT, α-1,3-XylT, β-1,2-XylT, etc. It is done.
本発明に用いる糖鎖供与体としては、糖転移酵素の基質となり得るものであれば特に限定されるものではなく、例えば、ＵＤＰ−Ｇｌｃ、ＵＤＰ−Ｇａｌ、ＵＤＰ−ＧｌｃＮＡｃ、ＵＤＰ−ＧａｌＮＡｃ、ＵＤＰ−ＧｌｃＡ、ＵＤＰ−ＧａｌＡ、ＵＤＰ−Ｘｙｌ、ＧＤＰ−Ｇｌｃ、ＧＤＰ−Ｍａｎ、ＧＤＰ−Ｆｕｃ、ＣＭＰ−ＮｅｕＡｃ、及びこれらのナトリウム塩等が挙げられる。また、酵素的あるいは化学的に修飾された糖鎖、あるいは化学合成された糖鎖も用いることができる。 The sugar chain donor used in the present invention is not particularly limited as long as it can serve as a glycosyltransferase substrate. For example, UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, UDP- Examples include GlcA, UDP-GalA, UDP-Xyl, GDP-Glc, GDP-Man, GDP-Fuc, CMP-NeuAc, and sodium salts thereof. In addition, enzymatically or chemically modified sugar chains or chemically synthesized sugar chains can also be used.
本発明の反応は、基質の糖供与体、糖受容体である糖アミノ酸及び糖転移酵素を緩衝溶液中で混合することにより行われる。通常、糖供与体の濃度を１ｍＭ以上、好ましくは１０〜１００ｍＭ、糖アミノ酸の濃度を０．１ｍｇ／ｍｌ以上、好ましくは１．０〜２０ｍｇ／ｍｌになるように加える。酵素量は、好ましくは０．１〜５００ｍＵ、さらに好ましくは５〜２００ｍＵ程度の量で用いる。緩衝液としては、ｐＨ５〜１０程度、濃度１０〜２００ｍＭ、好ましくは２０〜１００ｍＭの適当な緩衝液（例えば、ＨＥＰＥＳ、ＭＥＳ緩衝液）が用いられる。また、必要に応じて金属塩を追加してもよい。添加できる金属塩としてはＭｇ、Ｍｎ、Ｃａ、Ｃｏ、Ｚｎ、Ｃｕ等があり、通常塩化物等の形で添加することができる。 The reaction of the present invention is carried out by mixing a sugar donor as a substrate, a sugar amino acid as a sugar acceptor, and a glycosyltransferase in a buffer solution. Usually, the sugar donor concentration is 1 mM or more, preferably 10 to 100 mM, and the sugar amino acid concentration is 0.1 mg / ml or more, preferably 1.0 to 20 mg / ml. The amount of the enzyme is preferably 0.1 to 500 mU, more preferably about 5 to 200 mU. As the buffer solution, a suitable buffer solution (for example, HEPES, MES buffer solution) having a pH of about 5 to 10 and a concentration of 10 to 200 mM, preferably 20 to 100 mM is used. Moreover, you may add a metal salt as needed. Examples of metal salts that can be added include Mg, Mn, Ca, Co, Zn, Cu, and the like, which can be usually added in the form of chlorides.
反応温度は通常、酵素の至適温度、例えば、１０〜５０℃程度、好ましくは２０〜４０℃で行われ、反応時間は１〜４８時間、好ましくは１〜２４時間程度である。 The reaction temperature is usually an optimum enzyme temperature, for example, about 10 to 50 ° C., preferably 20 to 40 ° C., and the reaction time is 1 to 48 hours, preferably about 1 to 24 hours.
生成したＯ−結合型糖アミノ酸は公知の手段に従って反応終了液から容易に分離精製することができる。例えば、ゲルろ過カラムクロマトグラフィー、イオン交換樹脂カラムクロマトグラフィー、レクチンカラムクロマトグラフィー、高速液体クロマトグラフィー（ＨＰＬＣ）等により反応終了液から反応生成物のＯ−結合型糖アミノ酸を分離し、更に必要に応じて濃縮、脱塩、凍結乾燥等を行えばよい。
さらに糖鎖を伸長する場合には、上記の操作を繰り返し行えばよい。
The produced O-linked sugar amino acid can be easily separated and purified from the reaction completion solution according to known means. For example, the O-linked sugar amino acid of the reaction product is separated from the reaction completion solution by gel filtration column chromatography, ion exchange resin column chromatography, lectin column chromatography, high performance liquid chromatography (HPLC), etc. Accordingly, concentration, desalting, lyophilization and the like may be performed.
Further, when the sugar chain is extended, the above operation may be repeated.
このように作用させる糖転移酵素の種類、順番を変えることにより、さまざまな構造のＯ−結合型糖アミノ酸を調製することができる。 O-linked sugar amino acids having various structures can be prepared by changing the type and order of glycosyltransferases to act in this way.
上記の方法により、例えば、以下のようなＯ−結合型糖アミノ酸を製造できる：
このような化合物の具体例としては、以下の化合物：
By the above method, for example, the following O-linked sugar amino acids can be produced:
Specific examples of such compounds include the following compounds:
また、本発明の化合物のアミノ酸残基（即ち、トレオニン又はセリン）に他のアミノ酸を結合してペプチドを形成してもよい。 Moreover, you may form a peptide by couple | bonding another amino acid with the amino acid residue (namely, threonine or serine) of the compound of this invention.
ペプチド鎖を形成するには公知のペプチド合成反応を利用して行うことができる。例えば、公知の縮合剤（例えば、ジフェニルホスホリルアジド(ＤＰＰＡ)、ジエチルホスホリルシアニデート、アジドトリス(ジメチルアミノ)ホスホニウムヘキサフルオロリン酸塩等の有機リン化合物、Ｎ−エトキシカルボニル−２−エトキシ−１,２−ジハイドロキノリン(ＥＥＤＱ)、１−イソブチル−２−イソブチル−１,２−ジヒドロキシキノリン等のキノリン系ペプチド縮合剤、２-(１Ｈ-ベンゾトリアゾール-1-イル)-1,1,3,3-テトラメチルウロニウムヘキサフルオロホスフェート(ＨＢＴＵ）、ＤＣＣ等のカルボジイミド類等)を用いて、本発明の化合物のトレオニン又はセリン残基のアミノ基又はカルボキシル基に、順次アミノ酸を結合していくことによりペプチド鎖を伸長することができる。また、マイクロ波を照射しながら行ってもよい。本明細書で言う「マイクロ波」とは波長約０．３〜３０ｃｍ程度の電磁波（１〜１００ＧＨｚに相当する）のことをいう。マイクロ波の波長としては、好ましくは２３００〜２６００ＭＨｚであるが、さらに好ましくは２３５０〜２５５０ＭＨｚ、特に好ましくは２４００〜２５００ＭＨｚである。 A peptide chain can be formed using a known peptide synthesis reaction. For example, known condensing agents (for example, organic phosphorus compounds such as diphenylphosphoryl azide (DPPA), diethylphosphoryl cyanidate, azidotris (dimethylamino) phosphonium hexafluorophosphate, N-ethoxycarbonyl-2-ethoxy-1, Quinoline peptide condensing agents such as 2-dihydroquinoline (EEDQ) and 1-isobutyl-2-isobutyl-1,2-dihydroxyquinoline, 2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate (HBTU), carbodiimides such as DCC, etc.), and sequentially binding amino acids to the amino group or carboxyl group of the threonine or serine residue of the compound of the present invention. Can elongate the peptide chain. Moreover, you may carry out, irradiating a microwave. As used herein, “microwave” refers to an electromagnetic wave (corresponding to 1 to 100 GHz) having a wavelength of about 0.3 to 30 cm. The wavelength of the microwave is preferably 2300 to 2600 MHz, more preferably 2350 to 2550 MHz, and particularly preferably 2400 to 2500 MHz.
このペプチド合成は固相反応により行ってもよく、例えば、固相担体に本発明の化合物のＮ末端アミノ基又はＣ末端カルボキシル基を結合して（例えば、Ｃ末端のカルボキシル基をクロロトリチル樹脂、クロルメチル樹脂、オキシメチル樹脂、ｐ−アルコキシベンジルアルコール樹脂等の担体に結合する）、通常の固相合成反応と同様にして行うことができる。 This peptide synthesis may be carried out by solid phase reaction, for example, by binding the N-terminal amino group or C-terminal carboxyl group of the compound of the present invention to a solid phase carrier (for example, the C-terminal carboxyl group is chlorotrityl resin, It binds to a carrier such as chloromethyl resin, oxymethyl resin, or p-alkoxybenzyl alcohol resin), and can be carried out in the same manner as in a normal solid phase synthesis reaction.
以下に本発明を実施例によりさらに具体的に説明するが、本発明は、これらに限定されるものではない。 The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.
ＨＰＬＣによる生成物の分析
以下に示される条件で分析を行った。また、生成物の分取も同条件で行った。
カラム：Ｉｎｅｒｔｓｉｌ ＯＤＳ−３（ＧＬサイエンス製、内径４．６ｍｍ、長さ２５０ｍｍ）
移動相：Ａ：０．１％トリフロロ酢酸、Ｂ：アセトニトリル（０．１％トリフロロ酢酸）
０→５ｍｉｎ Ａ：Ｂ＝８５：１５
５→３５ｍｉｎ Ａ：Ｂ＝８５：１５→４５：５５
３５→３５．１ｍｉｎ Ａ：Ｂ＝４５：５５→１０：９０
３５．１→４５ｍｉｎ Ａ：Ｂ＝１０：９０
流速：１ｍｌ／ｍｉｎ
カラム温度：４０℃
検出：ＵＶ２５４ｎｍ
参考例 コア(Core)２トレオニン誘導体の調製
下記の合成スキームにしたがってコア２トレオニン誘導体(化合物８)を調製した。
Analysis of the product by HPLC The analysis was performed under the following conditions. The product was also collected under the same conditions.
Column: Inertsil ODS-3 (GL Science, inner diameter 4.6 mm, length 250 mm)
Mobile phase: A: 0.1% trifluoroacetic acid, B: acetonitrile (0.1% trifluoroacetic acid)
0 → 5min A: B = 85: 15
5 → 35 min A: B = 85: 15 → 45: 55
35 → 35.1 min A: B = 45: 55 → 10: 90
35.1 → 45 min A: B = 10: 90
Flow rate: 1 ml / min
Column temperature: 40 ° C
Detection: UV254nm
Reference Example Preparation of Core 2 Threonine Derivative A core 2 threonine derivative (Compound 8) was prepared according to the following synthesis scheme.
化合物（１）の合成
３，４，６−トリ−Ｏ−アセチル−Ｄ−ガラクタール（６８．９ｇ）を乾燥アセトニトリル（１０００ｍｌ）に溶かし、−２０℃に冷却した。−２０℃まで冷えたらセリウムアンモニウムナイトレート（２１６ｇ）及びアジ化ナトリウム（１３．０ｇ）を加え、−２０℃で激しく攪拌した。１時間後、さらにセリウムアンモニウムナイトレート（２００ｇ）及びアジ化ナトリウム（１１．７ｇ）を加え、−２０℃で攪拌した。６時間後、反応液にエーテルと水とを加え、分液操作により有機層を集め、水層からエーテルで２回抽出操作を行った。有機層を集め、硫酸マグネシウム乾燥した後、減圧濃縮した。残渣をシリカゲル（２００ｇ）を充填したショートカラムに加え、ヘキサン−酢酸エチル（１：１）で溶出した。得られた粗生成物画分を集め、濃縮し、その残渣をアセトン−水（１：１）混合液（１０００ｍｌ）に溶かし、これに炭酸カルシウム（２５ｇ）を加え、室温で３日間攪拌した。反応懸濁液からカルシウム塩を濾別したのち、濾液を半量になるまで濃縮し、酢酸エチルを用いて３回抽出した。有機層を乾燥、濃縮し、残渣をシリカゲルカラムクロマトグラフィー［トルエン−酢酸エチル（２：１）］により精製し、化合物（１）（４３ｇ、５２％）を得た。化合物（１）：Ｒｆ０．３［ヘキサン−酢酸エチル（１：１）］。
Synthesis of Compound (1) 3,4,6-Tri-O-acetyl-D-galactal (68.9 g) was dissolved in dry acetonitrile (1000 ml) and cooled to −20 ° C. When cooled to -20 ° C, cerium ammonium nitrate (216 g) and sodium azide (13.0 g) were added, and the mixture was vigorously stirred at -20 ° C. After 1 hour, cerium ammonium nitrate (200 g) and sodium azide (11.7 g) were further added, and the mixture was stirred at -20 ° C. After 6 hours, ether and water were added to the reaction solution, the organic layer was collected by liquid separation operation, and extraction operation was performed twice from the aqueous layer with ether. The organic layer was collected, dried over magnesium sulfate, and concentrated under reduced pressure. The residue was added to a short column packed with silica gel (200 g) and eluted with hexane-ethyl acetate (1: 1). The obtained crude product fractions were collected and concentrated, and the residue was dissolved in an acetone-water (1: 1) mixture (1000 ml), calcium carbonate (25 g) was added thereto, and the mixture was stirred at room temperature for 3 days. After the calcium salt was filtered off from the reaction suspension, the filtrate was concentrated to half volume and extracted three times with ethyl acetate. The organic layer was dried and concentrated, and the residue was purified by silica gel column chromatography [toluene-ethyl acetate (2: 1)] to obtain compound (1) (43 g, 52%). Compound (1): Rf 0.3 [hexane-ethyl acetate (1: 1)].
化合物（２）の合成
化合物（１）（４．０５ｇ）とトリクロロアセトニトリル（２．４５ｍｌ）とのジクロロメタン（４０ｍｌ）溶液に無水炭酸カリウム（１．６９ｇ）を加え、室温で７時間激しく攪拌した。炭酸カリウムをセライトろ過により濾別した後、濾液を濃縮し、残渣をシリカゲルカラムクロマトグラフィー［トルエン−酢酸エチル−トリエチルアミン（１５０：５０：１）］により精製し、化合物（２）（４．６ｇ、７９％）を得た。化合物（２）：Ｒｆ０．４［トルエン−酢酸エチル−トリエチルアミン（１５０：５０：１）］。
Synthesis of Compound (2) To a solution of compound (1) (4.05 g) and trichloroacetonitrile (2.45 ml) in dichloromethane (40 ml) was added anhydrous potassium carbonate (1.69 g), and the mixture was vigorously stirred at room temperature for 7 hours. After potassium carbonate was filtered off by Celite filtration, the filtrate was concentrated, and the residue was purified by silica gel column chromatography [toluene-ethyl acetate-triethylamine (150: 50: 1)] to obtain compound (2) (4.6 g, 79%). Compound (2): Rf 0.4 [toluene-ethyl acetate-triethylamine (150: 50: 1)].
化合物（３）の合成
化合物（２）（２５．０ｇ）とN−α−Fmoc−L−トレオニン−ｔ−ブチルエステル（１１．４ｇ）をジクロロメタン−エーテル（１：１）の乾燥混合液（２００ｍｌ）に溶かし、−３０℃に冷却した後、ＴＭＳＯＴｆ（５１６μｌ）を滴下した。反応液を−３０℃で３０分間攪拌した後、トリエチルアミン（４００μｌ）で中和し、クロロホルムで希釈した後、０．１Ｍ 塩酸で洗浄、硫酸マグネシウム乾燥、濃縮を順次行った。残渣を減圧乾燥した後、乾燥メタノールに溶かし、これに、ｐＨ８〜９の範囲に保つように注意しながら０．５Ｍ ナトリウムメトキシドメタノール溶液を滴下した。ＴＬＣで目的物（Ｒｆ０．５；酢酸エチル）の蓄積を確認した後、反応液に酢酸（２．０ｍｌ）を加え、減圧濃縮した。次に、残渣をクロロホルムに溶かし、飽和炭酸水素ナトリウム水、水で順次洗浄した後、有機層を硫酸マグネシウム乾燥、濃縮し、減圧乾燥した。続いて、残渣を乾燥アセトニトリルに溶かし、ベンズアルデヒドジメチルアセタール（８．５ｍｌ）とＣＳＡ（２００ｍｇ）を加え、室温で３時間攪拌した。反応液に飽和炭酸水素ナトリウム水を加え、この懸濁液からクロロホルムで３回抽出した。抽出液を硫酸マグネシウム乾燥、濃縮し、残渣をシリカゲルカラムクロマトグラフィー［ヘキサン−酢酸エチル（３：２）］により精製し、化合物（３）（７．２ｇ、３８％）を得た。化合物（３）：Ｒｆ０．６［ヘキサン−酢酸エチル（１：１）］。
Synthesis of Compound (3) Compound (2) (25.0 g) and N-α-Fmoc-L-threonine-t-butyl ester (11.4 g) were mixed in a dichloromethane-ether (1: 1) dry mixture (200 ml) ) And cooled to −30 ° C., TMSOTf (516 μl) was added dropwise. The reaction solution was stirred at −30 ° C. for 30 minutes, neutralized with triethylamine (400 μl), diluted with chloroform, washed with 0.1 M hydrochloric acid, dried over magnesium sulfate, and concentrated. The residue was dried under reduced pressure, dissolved in dry methanol, and 0.5M sodium methoxide methanol solution was added dropwise thereto while taking care to keep the pH in the range of 8-9. After confirming the accumulation of the desired product (Rf0.5; ethyl acetate) by TLC, acetic acid (2.0 ml) was added to the reaction solution, followed by concentration under reduced pressure. Next, the residue was dissolved in chloroform and washed successively with saturated aqueous sodium hydrogen carbonate and water, and then the organic layer was dried over magnesium sulfate, concentrated and dried under reduced pressure. Subsequently, the residue was dissolved in dry acetonitrile, benzaldehyde dimethyl acetal (8.5 ml) and CSA (200 mg) were added, and the mixture was stirred at room temperature for 3 hours. Saturated aqueous sodium hydrogen carbonate was added to the reaction mixture, and the suspension was extracted 3 times with chloroform. The extract was dried over magnesium sulfate and concentrated, and the residue was purified by silica gel column chromatography [hexane-ethyl acetate (3: 2)] to obtain compound (3) (7.2 g, 38%). Compound (3): Rf 0.6 [hexane-ethyl acetate (1: 1)].
化合物（４）の合成
化合物（３）（２００ｍｇ）、２,３,４,６−テトラ−Ｏ−アセチル−α−Ｄ−ガラクトーストリクロロアセトイミデート（２００ｍｇ）のジクロロメタン（４ｍｌ）溶液を０℃に冷却し、ＴＭＳＯＴｆ（１１μｌ）を滴下した。０℃で時間攪拌した後、反応液にトリエチルアミン（８μｌ）を加え、反応液に０．１Ｍ塩酸を加えた。有機層の分液及び水層のクロロホルム抽出を行った後、有機層を合わせ、硫酸マグネシウム乾燥し、濃縮した。残渣をシリカゲルカラムクロマトグラフィー［ヘキサン−酢酸エチル（１：１）］により精製し、化合物（４）（２４９ｍｇ、８４％）を得た。化合物（４）：Ｒｆ０．４［ヘキサン−酢酸エチル（１：１）］。
Synthesis of Compound (4) Compound (3) (200 mg), 2,3,4,6-tetra-O-acetyl-α-D-galactose trichloroacetimidate (200 mg) in dichloromethane (4 ml) was brought to 0 ° C. After cooling, TMSOTf (11 μl) was added dropwise. After stirring at 0 ° C. for a time, triethylamine (8 μl) was added to the reaction solution, and 0.1 M hydrochloric acid was added to the reaction solution. After separation of the organic layer and chloroform extraction of the aqueous layer, the organic layers were combined, dried over magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography [hexane-ethyl acetate (1: 1)] to obtain Compound (4) (249 mg, 84%). Compound (4): Rf 0.4 [hexane-ethyl acetate (1: 1)].
化合物（５）の合成
化合物（４）（２２４ｍｇ）を８０％酢酸水に溶かし、８０℃で２時間攪拌した。反応液の濃縮、トルエン共沸を行い、残渣をシリカゲルカラムクロマトグラフィー［ヘキサン−酢酸エチル（１：３）］により精製し、化合物（５）（１７６ｍｇ、８６％）を得た。化合物（５）：Ｒｆ０．２［ヘキサン−酢酸エチル（１：３）］。
Synthesis of Compound (5) Compound (4) (224 mg) was dissolved in 80% aqueous acetic acid and stirred at 80 ° C. for 2 hours. The reaction solution was concentrated and azeotroped with toluene, and the residue was purified by silica gel column chromatography [hexane-ethyl acetate (1: 3)] to obtain compound (5) (176 mg, 86%). Compound (5): Rf0.2 [hexane-ethyl acetate (1: 3)].
化合物（６）の合成
化合物（５）（１．７１ｇ）と３,４,６−トリ−Ｏ−アセチル−Ｎ−（２,２,２−トリクロロエチルオキシカルボニル）−Ｄ−グルコサミントリクロロイミデート（２．２８ｇ）の乾燥ジクロロメタン（５０ｍｌ）溶液を−４０℃に冷却し、ＴＭＳＯＴｆ（４８μｌ）を滴下した。１０分後、反応液にトリエチルアミン（３８μｌ）を加え、反応液に０．１Ｍ塩酸を加えた。有機層の分液及び水層のクロロホルム抽出を行った後、有機層を合わせ、硫酸マグネシウム乾燥し、濃縮した。残渣をシリカゲルカラムクロマトグラフィー［ヘキサン−酢酸エチル（１：２）］により精製し、化合物（６）（３．１５ｇ、９２％）を得た。化合物（６）：Ｒｆ０．６［ヘキサン−酢酸エチル（１：３）］。
Synthesis of Compound (6) Compound (5) (1.71 g) and 3,4,6-tri-O-acetyl-N- (2,2,2-trichloroethyloxycarbonyl) -D-glucosamine trichloroimidate ( 2.28 g) of dry dichloromethane (50 ml) was cooled to −40 ° C. and TMSOTf (48 μl) was added dropwise. After 10 minutes, triethylamine (38 μl) was added to the reaction solution, and 0.1 M hydrochloric acid was added to the reaction solution. After separation of the organic layer and chloroform extraction of the aqueous layer, the organic layers were combined, dried over magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography [hexane-ethyl acetate (1: 2)] to obtain Compound (6) (3.15 g, 92%). Compound (6): Rf 0.6 [hexane-ethyl acetate (1: 3)].
化合物（７）の合成
化合物（６）（３．５ｇ）及び酢酸（８．０ｍｌ）の酢酸エチル溶液（６０ｍｌ）に亜鉛粉末（１９ｇ）を加え、激しく攪拌した。３０分後、反応液に無水酢酸（８．０ｍｌ）を加え、さらに３０分間攪拌した。未反応の亜鉛末をセライトろ過により濾別した後、濾液を０．１Ｍ塩酸及び水で順次洗浄し、硫酸マグネシウム乾燥、濃縮した。残渣にシリカゲルカラムクロマトグラフィー［アセトン−ヘキサン（２：１）］を行い（７）の粗生成物（３．０ｇ、９４％）を得た。さらにクロロホルム−ヘキサンより３回再結晶することにより化合物（７）の精製物（１．９ｇ、６０％）を得た。化合物（７）：Ｒｆ０．３［アセトン−ヘキサン（２：１）］。
Synthesis of Compound (7) Zinc powder (19 g) was added to an ethyl acetate solution (60 ml) of compound (6) (3.5 g) and acetic acid (8.0 ml), and vigorously stirred. After 30 minutes, acetic anhydride (8.0 ml) was added to the reaction solution, and the mixture was further stirred for 30 minutes. Unreacted zinc powder was filtered off through Celite filtration, and the filtrate was washed successively with 0.1M hydrochloric acid and water, dried over magnesium sulfate, and concentrated. The residue was subjected to silica gel column chromatography [acetone-hexane (2: 1)] to obtain a crude product of (7) (3.0 g, 94%). Further, recrystallization from chloroform-hexane three times gave a purified product (1.9 g, 60%) of compound (7). Compound (7): Rf0.3 [acetone-hexane (2: 1)].
化合物（８）の合成
化合物（７）（１．８ｇ）を９５％ＴＦＡに溶かし、室温で１時間攪拌した。反応液をトルエンで希釈した後濃縮し、さらにトルエン共沸を２回行い、化合物（８）（１．７ｇ、１００％）を得た。化合物（８）：Ｒｆ０．５［クロロホルム−メタノール（４：１）］。化合物８の水酸基を保護するアセチル基を定法により脱保護して、以下の実施例で使用する化合物１を得た。
Synthesis of Compound (8) Compound (7) (1.8 g) was dissolved in 95% TFA and stirred at room temperature for 1 hour. The reaction mixture was diluted with toluene and concentrated, and toluene azeotropy was further performed twice to obtain compound (8) (1.7 g, 100%). Compound (8): Rf0.5 [chloroform-methanol (4: 1)]. The acetyl group protecting the hydroxyl group of Compound 8 was deprotected by a conventional method to obtain Compound 1 used in the following Examples.
実施例１ 化合物２の合成Example 1 Synthesis of Compound 2
β１，４−ＧａｌＴ（東洋紡製）を１００ｍＵ／ｍｌ、ＵＤＰ−Ｇａｌを２５ｍＭ、ＭｎＣｌ２を１０ｍＭ、ＢＳＡを０．１％含む５０ｍＭＨＥＰＥＳ緩衝液（ｐＨ７．０）１ｍｌに化合物１を４．６ｍｇ加え、２５℃で２４時間反応させた。反応後、９５℃で３分間加熱し、反応を停止した。反応液１μｌを蒸留水で５０倍希釈し、ＨＰＬＣで分析したところ、原料である化合物１に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物４．３ｍｇを得た。
得られた生成物を、２，５−ジヒドロ安息香酸をマトリックスとする、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析したところ、１０９４．３［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１０９４．４）にピークが検出され、化合物２であることが確認された。
4.6 mg of Compound 1 was added to 1 ml of 50 mM HEPES buffer (pH 7.0) containing 100 mM U / ml of β1,4-GalT (Toyobo), 25 mM of UDP-Gal, 10 mM of MnCl 2 and 0.1% of BSA, The reaction was performed at 25 ° C. for 24 hours. After the reaction, the reaction was stopped by heating at 95 ° C. for 3 minutes. When 1 μl of the reaction solution was diluted 50-fold with distilled water and analyzed by HPLC, the peak corresponding to the raw material compound 1 disappeared, and a new peak derived from the product was confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 4.3 mg of product.
The obtained product was analyzed by MALDI-TOF-MS using 2,5-dihydrobenzoic acid as a matrix, and peaked at 1094.3 [M + Na] + (theoretical value [M + Na] + = 1094.4). Was detected and confirmed to be compound 2.
実施例２ 化合物３及び６の合成Example 2 Synthesis of Compounds 3 and 6
Glycobiology, 9, 1061-1071(1999)に従って調製したNeisseria menigitidis MC58株（ATCC BAA-335D）由来のβ1,3-GlcNAcTを３０ｍU／ｍｌ、ＵＤＰ−ＧｌｃＮＡｃを５ｍＭ、ＭｇＣｌ２を１０ｍＭ、ＭｎＣｌ２を１０ｍＭ、ＢＳＡを０．０１％含む１００ｍＭグリシン緩衝液（ｐＨ１０．０）２ｍｌに実施例１で得られた化合物２を２．２ｍｇ加え、20℃で２４時間反応させた。反応後、９５℃で３分間加熱し、反応を停止した。反応液５μｌを蒸留水で１０倍希釈し、ＨＰＬＣで分析したところ、原料である化合物２に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物２．０ｍｇを得た。
得られた生成物を、２，５−ジヒドロ安息香酸をマトリックスとする、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析したところ、１２９６．３［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１０９７．５）にピークが検出され、化合物３であることが確認された。
なお、同様に48時間反応した後、熱処理したサンプルをHPLCで分析したところ、化合物３に対応するピークの他に新たなピークの出現が確認された。HPLCを用いて当該ピークを分取し、凍結乾燥後、得られた生成物を ２，５−ジヒドロ安息香酸をマトリックスとする、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析したところ、化合物６（理論値［Ｍ＋Ｎａ］＋＝１５００．５）に対応するにピーク １４９９．５［Ｍ＋Ｎａ］＋を確認した。
Β1,3-GlcNAcT derived from Neisseria menigitidis MC58 strain (ATCC BAA-335D) prepared according to Glycobiology, 9, 1061-1071 (1999), 30 mU / ml, UDP-GlcNAc 5 mM, MgCl 2 10 mM, MnCl 2 10 mM Then, 2.2 mg of the compound 2 obtained in Example 1 was added to 2 ml of 100 mM glycine buffer (pH 10.0) containing 0.01% BSA, and reacted at 20 ° C. for 24 hours. After the reaction, the reaction was stopped by heating at 95 ° C. for 3 minutes. When 5 μl of the reaction solution was diluted 10 times with distilled water and analyzed by HPLC, the peak corresponding to the raw material compound 2 disappeared, and a new peak derived from the product was confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 2.0 mg of product.
The obtained product was analyzed by MALDI-TOF-MS using 2,5-dihydrobenzoic acid as a matrix, and peaked at 1296.3 [M + Na] + (theoretical value [M + Na] + = 1097.5). Was detected and confirmed to be compound 3.
In addition, after reacting similarly for 48 hours, when the heat-processed sample was analyzed by HPLC, appearance of a new peak other than the peak corresponding to the compound 3 was confirmed. The peak was fractionated using HPLC, and after freeze-drying, the obtained product was analyzed by MALDI-TOF-MS using 2,5-dihydrobenzoic acid as a matrix. Compound 6 (theoretical value [M + Na ] + = 1500.5) corresponding to peak 1499.5 [M + Na] + was confirmed.
実施例３ 化合物４の合成Example 3 Synthesis of Compound 4
Neisseria meningitidis ＭＣ５８株由来β１，３−ＧｌｃＮＡｃＴを３０ｍＵ／ｍｌ、ＵＤＰ−ＧｌｃＮＡｃを５ｍＭ、ＭｇＣｌ２を１０ｍＭ、ＭｎＣｌ２を１０ｍＭ、ＢＳＡを０．１ｍｇ／ｍｌ含む５０ｍＭグリシン緩衝液（ｐＨ１０．０）１ｍｌに化合物１を１．０ｍｇ加え、２０℃で２４時間反応させた。反応後、９５℃で３分間加熱し、反応を停止した。反応液１μｌを蒸留水で１０倍希釈し、ＨＰＬＣで分析したところ、原料である化合物１に相当するピークの他に、生成物に由来する新たなピークの出現を確認した。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥し、これを２，５−ジヒドロ安息香酸をマトリックスとする、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析したところ、化合物４（理論値［Ｍ＋Ｎａ］＋＝１１３５．４）に対応するにピーク １１３５．４［Ｍ＋Ｎａ］＋を確認した。 Neisseria meningitidis MC58 strain-derived β1,3-GlcNAcT at 30 mU / ml, UDP-GlcNAc at 5 mM, MgCl 2 at 10 mM, MnCl 2 at 10 mM and BSA at 0.1 mg / ml in 1 ml of 50 mM glycine buffer (pH 10.0) 1.0 mg of compound 1 was added and reacted at 20 ° C. for 24 hours. After the reaction, the reaction was stopped by heating at 95 ° C. for 3 minutes. When 1 μl of the reaction solution was diluted 10-fold with distilled water and analyzed by HPLC, the appearance of a new peak derived from the product was confirmed in addition to the peak corresponding to compound 1 as the raw material. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was freeze-dried and analyzed by MALDI-TOF-MS using 2,5-dihydrobenzoic acid as a matrix, which corresponds to compound 4 (theoretical value [M + Na] + = 1135.4). The peak 1135.4 [M + Na] + was confirmed in FIG.
実施例４ 化合物５の合成Example 4 Synthesis of Compound 5
α２，３（Ｏ）−ＳｉａＴ（カルビオケム社製）を１６９ｍＵ／ｍｌ、ＣＭＰ−ＮｅｕＡｃを２５ｍＭ、ＭｇＣｌ２を１０ｍＭ、トリトンＣＦ−５４を０．１％、ＢＳＡを０．１％含む５０ｍＭＨＥＰＥＳ緩衝液（ｐＨ７．０）１ｍｌに化合物１を４．６ｍｇ加え、２５℃で２４時間反応させた。反応後、実施例１と同様の方法で反応の停止及びＨＰＬＣ分析を行った。その結果、原料である化合物１に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物４．８ｍｇを得た。
得られた生成物を、実施例１と同様の方法でＭＡＬＤＩ−ＴＯＦ−ＭＳ分析したところ、１２２３．２［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１２２３．４）にピークが検出され、化合物５であることが確認された。
50 mM HEPES buffer containing 169 mU / ml α2,3 (O) -SiaT (Calbiochem), 25 mM CMP-NeuAc, 10 mM MgCl 2 , 0.1% Triton CF-54 and 0.1% BSA ( 4.6 mg of Compound 1 was added to 1 ml of pH 7.0) and reacted at 25 ° C. for 24 hours. After the reaction, the reaction was stopped and HPLC analysis was performed in the same manner as in Example 1. As a result, the peak corresponding to the raw material compound 1 disappeared, and a new peak derived from the product was confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 4.8 mg of product.
The obtained product was analyzed by MALDI-TOF-MS in the same manner as in Example 1. As a result, a peak was detected at 1223.2 [M + Na] + (theoretical value [M + Na] + = 1223.4). 5 was confirmed.
実施例５ 化合物７の合成Example 5 Synthesis of Compound 7
α２，３（Ｎ）−ＳｉａＴ（カルビオケム社製）を２０ｍＵ／ｍｌ、ＣＭＰ−ＮｅｕＡｃを２５ｍＭ、ＭｇＣｌ２を１０ｍＭ、トリトンＣＦ−５４を０．１％、ＢＳＡを０．１％含む５０ｍＭＭＥＳ緩衝液（ｐＨ６．５）１ｍｌに実施例１で得た化合物２を５．４ｍｇ加え、２５℃で２時間反応させた。反応後、実施例１と同様の方法で反応の停止及びＨＰＬＣ分析を行った。その結果、原料である化合物２に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物５．４ｍｇを得た。
得られた生成物を、実施例１と同様の方法でＭＡＬＤＩ−ＴＯＦ−ＭＳ分析したところ、１３８６．３［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１３８５．５）にピークが検出され、化合物７であることが確認された。
50 mM MES buffer containing 20 mU / ml α2,3 (N) -SiaT (Calbiochem), 25 mM CMP-NeuAc, 10 mM MgCl 2 , 0.1% Triton CF-54, 0.1% BSA ( pH 6.5) 5.4 mg of the compound 2 obtained in Example 1 was added to 1 ml and reacted at 25 ° C. for 2 hours. After the reaction, the reaction was stopped and HPLC analysis was performed in the same manner as in Example 1. As a result, the peak corresponding to the raw material compound 2 disappeared, and a new peak derived from the product was confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 5.4 mg of product.
The obtained product was analyzed by MALDI-TOF-MS in the same manner as in Example 1. As a result, a peak was detected at 1386.3 [M + Na] + (theoretical value [M + Na] + = 1385.5). 7 was confirmed.
実施例６ 化合物８の合成Example 6 Synthesis of Compound 8
化合物１に代えて実施例１で得た化合物２を５．４ｍｇ用いる以外は実施例４と同様の方法で反応を行った。反応後、実施例１と同様の方法で反応の停止及びＨＰＬＣ分析を行った。その結果、原料である化合物２に相当するピークは消失しており、生成物に由来する新たなピーク（但し、実施例５の生成物とは異なる保持時間）が確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物５．５ｍｇを得た。
得られた生成物を、実施例１と同様の方法でＭＡＬＤＩ−ＴＯＦ−ＭＳ分析したところ、１３８５．４［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１３８５．５）にピークが検出され、化合物８であることが確認された。
The reaction was conducted in the same manner as in Example 4 except that 5.4 mg of compound 2 obtained in Example 1 was used instead of compound 1. After the reaction, the reaction was stopped and HPLC analysis was performed in the same manner as in Example 1. As a result, the peak corresponding to the compound 2 as the raw material disappeared, and a new peak derived from the product (however, a different retention time from the product of Example 5) was confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 5.5 mg of product.
The obtained product was analyzed by MALDI-TOF-MS in the same manner as in Example 1. As a result, a peak was detected at 1385.4 [M + Na] + (theoretical value [M + Na] + = 1385.5). 8 was confirmed.
実施例７ 化合物９の合成Example 7 Synthesis of Compound 9
化合物２に代えて実施例６で得た化合物８を６．８ｍｇ用いる以外は実施例５と同様の方法で反応を行った。反応後、実施例１と同様の方法で反応の停止及びＨＰＬＣ分析を行った。その結果、原料である化合物８に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物６．６ｍｇを得た。
得られた生成物を、実施例１と同様の方法でＭＡＬＤＩ−ＴＯＦ−ＭＳ分析したところ、１６７７．２［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１６７６．６）にピークが検出され、化合物９であることが確認された。
The reaction was conducted in the same manner as in Example 5 except that 6.8 mg of compound 8 obtained in Example 6 was used instead of compound 2. After the reaction, the reaction was stopped and HPLC analysis was performed in the same manner as in Example 1. As a result, the peak corresponding to the raw material compound 8 disappeared, and a new peak derived from the product was confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 6.6 mg of product.
The obtained product was analyzed by MALDI-TOF-MS in the same manner as in Example 1. As a result, a peak was detected at 1677.2 [M + Na] + (theoretical value [M + Na] + = 1676.6). 9 was confirmed.
実施例８ 化合物９の合成Example 8 Synthesis of Compound 9
化合物２に代えて実施例５で得た化合物７を６．８ｍｇ用いる以外は実施例６と同様の方法で反応を行った。反応後、実施例１と同様の方法で反応の停止及びＨＰＬＣ分析を行った。その結果、原料である化合物７に相当するピークは消失しており、生成物に由来する新たなピーク（実施例７の生成物と同じ保持時間）が確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物６．５ｍｇを得た。
得られた生成物を、実施例１と同様の方法でＭＡＬＤＩ−ＴＯＦ−ＭＳ分析したところ、１６７６．９［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１６７６．６）にピークが検出され、化合物９であることが確認された。
The reaction was conducted in the same manner as in Example 6 except that 6.8 mg of compound 7 obtained in Example 5 was used instead of compound 2. After the reaction, the reaction was stopped and HPLC analysis was performed in the same manner as in Example 1. As a result, the peak corresponding to compound 7 as the raw material disappeared, and a new peak derived from the product (the same retention time as the product of Example 7) was confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 6.5 mg of product.
The obtained product was analyzed by MALDI-TOF-MS in the same manner as in Example 1. As a result, a peak was detected at 1676.9 [M + Na] + (theoretical value [M + Na] + = 1676.6). 9 was confirmed.
実施例９ 化合物１０の合成Example 9 Synthesis of Compound 10
α１，３−ＦｕｃＴＶ（カルビオケム社製）を５０ｍＵ／ｍｌ、ＧＤＰ−Ｆｕｃを２５ｍＭ、ＭｎＣｌ２を２０ｍＭ、トリトンＣＦ−５４を０．１％、ＢＳＡを０．１％含む５０ｍＭＨＥＰＥＳ緩衝液（ｐＨ７．５）１ｍｌに実施例７で得た化合物９を８．３ｍｇ加え、２５℃で２４時間反応させた。反応後、実施例１と同様の方法で反応の停止及びＨＰＬＣ分析を行った。その結果、原料である化合物９に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物７．２ｍｇを得た。
得られた生成物を、実施例１と同様の方法でＭＡＬＤＩ−ＴＯＦ−ＭＳ分析したところ、１８２４．０［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１８２４．６）にピークが検出され、化合物１０であることが確認された。
50 mM HEPES buffer (pH 7.5) containing 50 mU / ml α1,3-FucTV (Calbiochem), 25 mM GDP-Fuc, 20 mM MnCl 2 , 0.1% Triton CF-54, 0.1% BSA ) 8.3 mg of compound 9 obtained in Example 7 was added to 1 ml and reacted at 25 ° C. for 24 hours. After the reaction, the reaction was stopped and HPLC analysis was performed in the same manner as in Example 1. As a result, the peak corresponding to compound 9 as a raw material disappeared, and a new peak derived from the product was confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 7.2 mg of product.
The obtained product was analyzed by MALDI-TOF-MS in the same manner as in Example 1. As a result, a peak was detected at 1824.0 [M + Na] + (theoretical value [M + Na] + = 1824.6). 10 was confirmed.
実施例１０ 化合物１１の合成Example 10 Synthesis of Compound 11
化合物９に代えて実施例５で得た化合物７を６．８ｍｇ用いる以外は実施例７と同様の方法で反応を行った。反応後、実施例１と同様の方法で反応の停止及びＨＰＬＣ分析を行った。その結果、原料である化合物７に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物６．０ｍｇを得た。
得られた生成物を、実施例１と同様の方法でＭＡＬＤＩ−ＴＯＦ−ＭＳ分析したところ、１５３３．１［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１５３３．５）にピークが検出され、化合物１１であることが確認された。
The reaction was conducted in the same manner as in Example 7 except that 6.8 mg of compound 7 obtained in Example 5 was used instead of compound 9. After the reaction, the reaction was stopped and HPLC analysis was performed in the same manner as in Example 1. As a result, the peak corresponding to the raw material compound 7 disappeared, and a new peak derived from the product was confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 6.0 mg of product.
The obtained product was analyzed by MALDI-TOF-MS in the same manner as in Example 1. As a result, a peak was detected at 1533.1 [M + Na] + (theoretical value [M + Na] + = 1533.5). 11 was confirmed.
実施例１１ 化合物１２の合成Example 11 Synthesis of Compound 12
化合物９に代えて実施例６で得た化合物８を６．８ｍｇ用いる以外は実施例９と同様の方法で反応を行った。反応後、実施例１と同様の方法で反応の停止及びＨＰＬＣ分析を行った。その結果、原料である化合物８に相当するピークは消失しており、生成物に由来する新たなピーク（但し、実施例１０の生成物とは異なる保持時間）が確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物５．９ｍｇを得た。
得られた生成物を、実施例１と同様の方法でＭＡＬＤＩ−ＴＯＦ−ＭＳ分析したところ、１５３２．８［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１５３３．５）にピークが検出され、化合物１２であることが確認された。
The reaction was conducted in the same manner as in Example 9 except that 6.8 mg of compound 8 obtained in Example 6 was used instead of compound 9. After the reaction, the reaction was stopped and HPLC analysis was performed in the same manner as in Example 1. As a result, the peak corresponding to the raw material compound 8 disappeared, and a new peak derived from the product (however, a different retention time from the product of Example 10) was confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 5.9 mg of product.
The obtained product was analyzed by MALDI-TOF-MS in the same manner as in Example 1. As a result, a peak was detected at 1532.8 [M + Na] + (theoretical value [M + Na] + = 1533.5). 12 was confirmed.
実施例１２ 化合物１３の合成Example 12 Synthesis of Compound 13
β１，４−ＧａｌＴ（東洋紡製）を１００ｍＵ／ｍｌ、ＵＤＰ−Ｇａｌを５ｍＭ、ＭｎＣｌ２を１０ｍＭ、ＢＳＡを０．１ｍｇ／ｍｌ含む５０ｍＭＨＥＰＰＥＳ緩衝液（ｐＨ７．０）１．５ｍｌに化合物３を１．９ｍｇ加え、２５℃、２４時間反応させた。反応後、９５℃で３分間加熱し、反応液５μｌを１０倍希釈し、ＨＰＬＣで分析したところ、原料である化合物３に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物１．８ｍｇを得た。
得られた生成物を、２，５−ジヒドロ安息香酸をマトリックスとする、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析したところ、１４４６．５［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１４４５．５）にピークが検出され、化合物１３であることが確認された。
1. Compound 3 in 1.5 ml of 50 mM HEPES buffer (pH 7.0) containing 100 mU / ml of β1,4-GalT (manufactured by Toyobo), 5 mM of UDP-Gal, 10 mM of MnCl 2 and 0.1 mg / ml of BSA. 9 mg was added and reacted at 25 ° C. for 24 hours. After the reaction, it was heated at 95 ° C. for 3 minutes, 5 μl of the reaction solution was diluted 10 times, and analyzed by HPLC. As a result, the peak corresponding to the compound 3 as a raw material disappeared, and a new peak derived from the product was found. confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 1.8 mg of product.
The obtained product was analyzed by MALDI-TOF-MS using 2,5-dihydrobenzoic acid as a matrix, and peaked at 1446.5 [M + Na] + (theoretical value [M + Na] + = 1445.5). Was detected and confirmed to be Compound 13.
実施例１３ 化合物１４の合成Example 13 Synthesis of Compound 14
Neisseria meningitidis ＭＣ５８株由来β１，３−ＧｌｃＮＡｃＴを３０ｍＵ／ｍｌ、ＵＤＰ−ＧｌｃＮＡｃを５ｍＭ、ＭｇＣｌ２を１０ｍＭ、ＭｎＣｌ２を１０ｍＭ、ＢＳＡを０．１ｍｇ／ｍｌ含む５０ｍＭグリシン緩衝液（ｐＨ１０．０）１ｍｌに化合物１３を１．６ｍｇ加え、２０℃で２４時間反応させた。反応後、９５℃で３分間加熱し、反応を停止した。反応液２μｌを蒸留水で１０倍希釈し、ＨＰＬＣで分析したところ、原料である化合物１３に相当するピークはほぼ消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物１．５ｍｇを得た。
得られた生成物を、２，５−ジヒドロ安息香酸をマトリックスとする、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析したところ、１６４８．２［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１６４８．６）にピークが検出され、化合物１４であることが確認された。
Neisseria meningitidis MC58 strain-derived β1,3-GlcNAcT at 30 mU / ml, UDP-GlcNAc at 5 mM, MgCl 2 at 10 mM, MnCl 2 at 10 mM and BSA at 0.1 mg / ml in 1 ml of 50 mM glycine buffer (pH 10.0) 1.6 mg of compound 13 was added and reacted at 20 ° C. for 24 hours. After the reaction, the reaction was stopped by heating at 95 ° C. for 3 minutes. When 2 μl of the reaction solution was diluted 10-fold with distilled water and analyzed by HPLC, the peak corresponding to the compound 13 as the raw material almost disappeared, and a new peak derived from the product was confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 1.5 mg of product.
The obtained product was analyzed by MALDI-TOF-MS using 2,5-dihydrobenzoic acid as a matrix, and peaked at 1648.2 [M + Na] + (theoretical value [M + Na] + = 1648.6). Was detected and confirmed to be Compound 14.
実施例１４ 化合物１５の合成Example 14 Synthesis of Compound 15
β１，４−ＧａｌＴ（東洋紡製）を１００ｍＵ／ｍｌ、ＵＤＰ−Ｇａｌを５ｍＭ、ＭｎＣｌ２を１０ｍＭ、ＢＳＡを０．１ｍｇ／ｍｌ含む５０ｍＭＨＥＰＰＥＳ緩衝液（ｐＨ７．０）０．８ｍｌに化合物１４を１．３ｍｇ加え、２５℃、２４時間反応させた。反応後、９５℃で３分間加熱し、反応液２μｌを１０倍希釈し、ＨＰＬＣで分析したところ、原料である化合物１４に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物１．３ｍｇを得た。
得られた生成物を、２，５−ジヒドロ安息香酸をマトリックスとする、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析したところ、１８１０．６［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１８１０．６）にピークが検出され、化合物１５であることが確認された。
Compound 14 was added to 0.8 ml of 50 mM HEPES buffer (pH 7.0) containing 100 mU / ml of β1,4-GalT (manufactured by Toyobo), 5 mM of UDP-Gal, 10 mM of MnCl 2 and 0.1 mg / ml of BSA. 3 mg was added and reacted at 25 ° C. for 24 hours. After the reaction, the mixture was heated at 95 ° C. for 3 minutes, and 2 μl of the reaction solution was diluted 10-fold and analyzed by HPLC. As a result, the peak corresponding to the raw material compound 14 disappeared, and a new peak derived from the product was found. confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 1.3 mg of product.
The obtained product was analyzed by MALDI-TOF-MS using 2,5-dihydrobenzoic acid as a matrix, and peaked at 1810.6 [M + Na] + (theoretical value [M + Na] + = 1810.6). Was detected and confirmed to be Compound 15.
実施例１５ 化合物１６の合成Example 15 Synthesis of Compound 16
Neisseria meningitidis ＭＣ５８株由来β１，３−ＧｌｃＮＡｃＴを３０ｍＵ／ｍｌ、ＵＤＰ−ＧｌｃＮＡｃを５ｍＭ、ＭｇＣｌ２を１０ｍＭ、ＭｎＣｌ２を１０ｍＭ、ＢＳＡを０．１ｍｇ／ｍｌ含む５０ｍＭグリシン緩衝液（ｐＨ１０．０）０．８ｍｌに化合物１５を１．１ｍｇ加え、２０℃で２４時間反応させた。反応後、９５℃で３分間加熱し、反応を停止した。反応液２μｌを蒸留水で１０倍希釈し、ＨＰＬＣで分析したところ、原料である化合物１５に相当するピークと共に、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物０．４ｍｇを得た。
得られた生成物を、２，５−ジヒドロ安息香酸をマトリックスとする、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析したところ、２０１３．９［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝２０１３．７）にピークが検出され、化合物１６であることが確認された。
50 mM glycine buffer (pH 10.0) containing β1,3-GlcNAcT derived from Neisseria meningitidis strain MC58 at 30 mU / ml, UDP-GlcNAc at 5 mM, MgCl 2 at 10 mM, MnCl 2 at 10 mM, and BSA at 0.1 mg / ml. To 8 ml, 1.1 mg of compound 15 was added and reacted at 20 ° C. for 24 hours. After the reaction, the reaction was stopped by heating at 95 ° C. for 3 minutes. When 2 μl of the reaction solution was diluted 10-fold with distilled water and analyzed by HPLC, a new peak derived from the product was confirmed along with a peak corresponding to the compound 15 as a raw material. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 0.4 mg of product.
The obtained product was analyzed by MALDI-TOF-MS using 2,5-dihydrobenzoic acid as a matrix, and peaked at 20133.9 [M + Na] + (theoretical value [M + Na] + = 20133.7). Was detected and confirmed to be Compound 16.
実施例１６ 化合物１７の合成Example 16 Synthesis of Compound 17
β１，４−ＧａｌＴ（東洋紡製）を１００ｍＵ／ｍｌ、ＵＤＰ−Ｇａｌを５ｍＭ、ＭｎＣｌ２を１０ｍＭ、ＢＳＡを０．１ｍｇ／ｍｌ含む５０ｍＭＨＥＰＰＥＳ緩衝液（ｐＨ７．０）０．３ｍｌに化合物１６を０．３ｍｇ加え、２５℃、２４時間反応させた。反応後、９５℃で３分間加熱し、反応液２μｌを１０倍希釈し、ＨＰＬＣで分析したところ、原料である化合物１６に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物０．３ｍｇを得た。
得られた生成物を、２，５−ジヒドロ安息香酸をマトリックスとする、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析したところ、２１７５．８［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝２１７５．８）にピークが検出され、化合物１７であることが確認された。
Compound 16 was added to 0.3 ml of 50 mM HEPES buffer (pH 7.0) containing 100 mU / ml of β1,4-GalT (manufactured by Toyobo), 5 mM of UDP-Gal, 10 mM of MnCl 2 and 0.1 mg / ml of BSA. 3 mg was added and reacted at 25 ° C. for 24 hours. After the reaction, the mixture was heated at 95 ° C. for 3 minutes, and 2 μl of the reaction solution was diluted 10-fold and analyzed by HPLC. As a result, the peak corresponding to the compound 16 as a raw material disappeared, and a new peak derived from the product was found. confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 0.3 mg of product.
The obtained product was analyzed by MALDI-TOF-MS using 2,5-dihydrobenzoic acid as a matrix, and peaked at 2175.8 [M + Na] + (theoretical value [M + Na] + = 2175.8). Was detected and confirmed to be Compound 17.
実施例１７ 化合物１８の合成Example 17 Synthesis of Compound 18
β１，４−ＧａｌＴ（東洋紡製）を１００ｍＵ／ｍｌ、ＵＤＰ−Ｇａｌを５ｍＭ、ＭｎＣｌ２を１０ｍＭ、ＢＳＡを０．１ｍｇ／ｍｌ含む５０ｍＭＨＥＰＰＥＳ緩衝液（ｐＨ７．０）１．０ｍｌに化合物６を１．３ｍｇ加え、２５℃、２４時間反応させた。反応後、９５℃で３分間加熱し、反応液２μｌを１０倍希釈し、ＨＰＬＣで分析したところ、原料である化合物６に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物１．５ｍｇを得た。
得られた生成物を、２，５−ジヒドロ安息香酸をマトリックスとする、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析したところ、１８１０．５［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝１８１０．６）にピークが検出され、化合物１８であることが確認された。
Compound 1 was added to 1.0 ml of 50 mM HEPES buffer (pH 7.0) containing 100 mU / ml of β1,4-GalT (manufactured by Toyobo), 5 mM of UDP-Gal, 10 mM of MnCl 2 and 0.1 mg / ml of BSA. 3 mg was added and reacted at 25 ° C. for 24 hours. After the reaction, the mixture was heated at 95 ° C. for 3 minutes, and 2 μl of the reaction solution was diluted 10-fold and analyzed by HPLC. As a result, the peak corresponding to compound 6 as a raw material disappeared, and a new peak derived from the product was found. confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 1.5 mg of product.
The obtained product was analyzed by MALDI-TOF-MS using 2,5-dihydrobenzoic acid as a matrix, and peaked at 1810.5 [M + Na] + (theoretical value [M + Na] + = 1810.6). Was detected and confirmed to be Compound 18.
実施例１８ 化合物１９の合成Example 18 Synthesis of Compound 19
Neisseria meningitidis ＭＣ５８株由来β１，３−ＧｌｃＮＡｃＴを３０ｍＵ／ｍｌ、ＵＤＰ−ＧｌｃＮＡｃを５ｍＭ、ＭｇＣｌ２を１０ｍＭ、ＭｎＣｌ２を１０ｍＭ、ＢＳＡを０．１ｍｇ／ｍｌ含む５０ｍＭグリシン緩衝液（ｐＨ１０．０）０．８ｍｌに化合物１８を１．３ｍｇ加え、２０℃で２４時間反応させた。反応後、９５℃で３分間加熱し、反応を停止した。反応液２μｌを蒸留水で１０倍希釈し、ＨＰＬＣで分析したところ、原料である化合物１８に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物１．４ｍｇを得た。
得られた生成物を、２，５−ジヒドロ安息香酸をマトリックスとする、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析したところ、２２１６．９［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝２２１６．８）にピークが検出され、化合物１９であることが確認された。
50 mM glycine buffer (pH 10.0) containing β1,3-GlcNAcT derived from Neisseria meningitidis strain MC58 at 30 mU / ml, UDP-GlcNAc at 5 mM, MgCl 2 at 10 mM, MnCl 2 at 10 mM, and BSA at 0.1 mg / ml. To 8 ml, 1.3 mg of compound 18 was added and reacted at 20 ° C. for 24 hours. After the reaction, the reaction was stopped by heating at 95 ° C. for 3 minutes. When 2 μl of the reaction solution was diluted 10 times with distilled water and analyzed by HPLC, the peak corresponding to the compound 18 as a raw material disappeared, and a new peak derived from the product was confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 1.4 mg of product.
The obtained product was analyzed by MALDI-TOF-MS using 2,5-dihydrobenzoic acid as a matrix, and peaked at 2216.9 [M + Na] + (theoretical value [M + Na] + = 2216.8). Was detected and confirmed to be Compound 19.
実施例１９ 化合物２０の合成Example 19 Synthesis of Compound 20
β１，４−ＧａｌＴ（東洋紡製）を１００ｍＵ／ｍｌ、ＵＤＰ−Ｇａｌを５ｍＭ、ＭｎＣｌ２を１０ｍＭ、ＢＳＡを０．１ｍｇ／ｍｌ含む５０ｍＭＨＥＰＰＥＳ緩衝液（ｐＨ７．０）０．８ｍｌに化合物１９を１．０ｍｇ加え、２５℃、２４時間反応させた。反応後、９５℃で３分間加熱し、反応液２μｌを１０倍希釈し、ＨＰＬＣで分析したところ、原料である化合物１９に相当するピークは消失しており、生成物に由来する新たなピークが確認された。反応液より、ＨＰＬＣを用いて生成物に由来するピークを分取することにより、生成物を分離精製した。生成物画分を凍結乾燥することにより生成物１．１ｍｇを得た。
得られた生成物を、２，５−ジヒドロ安息香酸をマトリックスとする、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析したところ、２５４０．９［Ｍ＋Ｎａ］＋（理論値［Ｍ＋Ｎａ］＋＝２５４０．９）にピークが検出され、化合物２０であることが確認された。
1. Compound 19 in 100 ml of β1,4-GalT (Toyobo), 5 mM of UDP-Gal, 10 mM of MnCl 2 and 0.1 mg / ml of BSA in 0.8 ml of 50 mM HEPPES buffer (pH 7.0) 0 mg was added and reacted at 25 ° C. for 24 hours. After the reaction, the mixture was heated at 95 ° C. for 3 minutes, and 2 μl of the reaction solution was diluted 10 times and analyzed by HPLC. As a result, the peak corresponding to the compound 19 as a raw material disappeared, and a new peak derived from the product was found. confirmed. The product was separated and purified from the reaction solution by separating a peak derived from the product using HPLC. The product fraction was lyophilized to give 1.1 mg of product.
The obtained product was analyzed by MALDI-TOF-MS using 2,5-dihydrobenzoic acid as a matrix, and peaked at 2540.9 [M + Na] + (theoretical value [M + Na] + = 2540.9). Was detected and confirmed to be Compound 20.
本発明のＯ−結合型糖アミノ酸は糖アミノ酸の糖部分の水酸基が遊離であり、糖ペプチド合成用原料に限らず、それ自体で天然糖鎖の標準サンプルとしても有用である。また、タンパク質との相互作用解析用サンプルとしても使用することができる。 The O-linked sugar amino acid of the present invention has a free hydroxyl group in the sugar moiety of the sugar amino acid, and is useful not only as a raw material for synthesizing glycopeptides but also as a standard sample of natural sugar chains. It can also be used as a sample for analyzing interaction with proteins.
さらに本発明の糖アミノ酸から出発して多様なＯ−結合型糖アミノ酸を製造することができ、Ｏ−結合型糖アミノ酸ライブラリー合成を可能となり、糖タンパク質の機能解析や構造解析が飛躍的に進行するものと期待される。 Furthermore, starting from the sugar amino acids of the present invention, various O-linked sugar amino acids can be produced, O-linked sugar amino acid libraries can be synthesized, and functional analysis and structural analysis of glycoproteins can be dramatically improved. Expected to progress.
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