JP4844920B2 - Glycolipid-containing compound, biosensor on which it is immobilized, glycolipid-containing microparticle, detection reagent containing the microparticle, and method for detecting a sugar-binding compound - Google Patents

Description

The present invention relates to a glycolipid-containing compound having a sugar chain moiety, a side chain moiety, and a linker moiety.
The present invention also relates to a biosensor containing the glycolipid-containing compound, a glycolipid-containing fine particle in which the glycolipid-containing compound is immobilized on the surface of a metal fine particle, and a sugar-binding compound detection reagent containing the fine particle.
Furthermore, the present invention relates to a method for detecting a sugar binding compound using the biosensor or the sugar binding compound detection reagent.

In fields such as biotechnology, biochemical research, clinical testing, and food testing, a highly sensitive detection and quantification method for a sugar-binding compound such as a target sugar-binding protein is required.
At present, many sensing methods using antigen-antibody reactions are employed in sensing methods used in clinical tests and the like. However, since the conventional method requires complicated work and labeling substances, surface plasmon resonance (SPR) or quartz crystal resonators that can detect changes in ligands with high sensitivity without using labeling substances Biosensors using Quartz Crystal Microbalance (QCM) are also used. This SPR phenomenon utilizes an optical phenomenon that occurs when surface plasmons are excited at the metal / liquid interface. That is, since a minute mass change occurring on the surface of the sensor chip is detected as an SPR signal, for example, a change in binding and dissociation between two molecules between a substance immobilized on a gold surface and a specimen is quantitatively measured. be able to. On the other hand, the QCM method is a method for determining the amount of adsorption of a substance by converting the frequency into a mass, and binding between two molecules of a substance (ligand molecule) immobilized on a gold surface and a specimen (analyte). Information on change can be considered quantitatively.

  By the way, as a general biosensor using a sugar compound, a sensor in which a sugar compound is immobilized on the surface of a substrate such as gold has been proposed (Patent Document 1). This biosensor has a structure in which a group having a sugar compound is covalently bonded directly to a gold surface. In this sensor, a low molecular weight compound such as a hydrocarbon is bound to a sugar compound, and then a thiol group is bound to the hydrocarbon, and further, a direct covalent bond or adsorption between the gold surface and the thiol group (-SH) is performed. A method of immobilizing the sugar compound on the substrate is employed.

  Bonding via a thiol group is stable, but sugar compounds have many hydroxyl groups and complex steric structures unlike antigens, antibodies, enzymes, and the like. For this reason, in order to introduce | transduce the said thiol group into a sugar compound, many processes and labor are required, and there exists a problem that a thiol group cannot be introduce | transduced into a sugar compound simply.

  On the other hand, it is theoretically possible to introduce a group containing a thiol group to the gold surface and then bind a separately synthesized sugar compound, but this method is poor in efficiency of introducing the sugar compound onto the gold surface and is practically used. Can not.

  Therefore, in order to immobilize the natural sugar compound on the surface of the sensor chip for SPR, the chip surface is treated in advance with hydrophobic treatment, and the previous sugar chain is immobilized on the chip surface using hydrophobic interaction. Later, attempts have been made to detect cholera toxin (Non-Patent Document 1). Furthermore, in order to highly control the sugar chains and at the same time immobilize the sugar chains at a high density, the Langmuir-Blodgett film-forming method is used to form a simple glycolipid air-water interface on the quartz crystal sensor chip. A method of applying a verotoxin after detection of a molecular film is also known (Patent Document 2, Non-Patent Document 2).

  That is, the latter method (Patent Document 2 and Non-Patent Document 2) is to apply a hydrophobic treatment by previously forming a monomolecular film of a long-chain alkyl chain on the chip surface, and develop a LB film of glycolipid on the surface. Thus, the glycolipid can be immobilized on the sensor chip by hydrophobic interaction. However, immobilization by hydrophobic interaction has relatively weak immobilization ability, and it is possible that sugar chains are sometimes detached from the substrate when immersed in water or buffer for a long time or when the sensor chip is used repeatedly. There was a problem of lack of stability and durability.

  In the former method (Non-Patent Document 1), since only the hydrophobic interaction is performed, high orientation and high density immobilization cannot be performed. Had a problem of lack of practicality. Furthermore, in the LB film forming method, after preparing a monomolecular film, it is necessary to transfer (accumulate) it to an appropriate substrate. There is a problem that it is difficult to process. In other words, the work of accumulating the monomolecular film on the hydrophobically treated gold surface has a problem in that it is limited to be performed quickly and in large quantities and is not practical.

  In order to simplify the immobilization work, a linker compound capable of introducing and immobilizing oligosaccharide chains in a single stage on a support for protein analysis such as a sensor chip such as SPR or a carrier for affinity chromatography, and the linker A ligand using a compound and an oligosaccharide chain immobilization method using the ligand have also been tried (Patent Document 3). However, even with the above-described method, high-density fixation with high orientation cannot be performed, and further improvement in performance is required as a method for producing a highly sensitive sensor chip.

  On the other hand, as a sensing method that can be performed quickly and easily, a detection method using a gold colloid aggregation method in which a sugar compound is bound to the surface of a gold fine particle has been proposed. The colloidal gold method is a method in which a specific binding substance is fixed chemically and / or physicochemically on the surface of a gold microparticle, and the gold colloid is aggregated by causing specific binding between the microparticle and the measurement target. This is a method for detecting a change in color tone of a colloidal gold solution. Changes in color can be detected visually or with a spectrometer.

  As a method for immobilizing a specific binding substance on gold fine particles, substance immobilization by an immune reaction such as an antigen-antibody reaction is generally known (Non-patent Document 3). Further, regarding a method of colloidal gold agglutination by the reaction of a colloidal gold-labeled antibody and an antigen and changing the color tone, the method is known in the case of colloidal gold-labeled rabbit anti-mannan antibody and yeast mannan (non-patented). Reference 4). Furthermore, it is also known about aggregation between human hemoglobin and colloidal gold labeled anti-human hemoglobin antibody (Patent Document 4).

  Of these, biosensing in which a sugar compound is immobilized on the surface of gold fine particles has been actively studied in recent years. For example, surface modification of gold fine particles by a compound having an alkyl chain or a polyoxyethylene chain at both ends of a disulfide group capable of gold immobilization and having sugar chains bonded at both ends is known (Non-patent Document 5). . In addition, a specific adsorption method for a sugar recognition protein is known in which a polyethylene glycol chain having a thiol group is first introduced on the gold surface, and a separately synthesized sugar compound is bound using an aldehyde group present at the terminal. (Non-patent document 6).

It is described that a mixture of glycosphingolipid and glycerolipid can be used for binding to the gold surface (Non-patent Documents 7 and 8).
U.S. Pat. No. 3,321,733 JP 2002-022745 A JP 2003-83969 A Japanese Patent Laid-Open No. 2-141665 Biochemistry, Vol.35, P.6375, 1996 Biomacromolecules, vol. 3, p411, 2002 J. Histochem. Cytochem., Vol. 25, P. 1187, 1977 J. Histochem. Cytochem., Vol. 25, P.295, 1977 Angew. Chem. Int. Ed., Vol.40, P.2257, 2001 J. Am. Chem. Soc., Vol. 123, P.8226, 2001 Tetrahedron Letters 40 (1999) 2011-2014 Tetrahedron 56 (2000) 9975-9984

  Accordingly, there has been a demand for a sensor, a detection reagent, and a detection method thereof that can easily fix a sugar compound with high orientation and high density, and that can quantitate and evaluate a sugar-binding compound easily and quickly with high sensitivity.

Non-Patent Documents 7 and 8 describe that a mixture of glycosphingolipid and glycerolipid can be used for binding to the gold surface. These documents state that glycerolipids are mixed to mimic natural cell membranes (biological membranes) and may suppress non-specific adsorption.
However, a sensor using glycosphingolipid and glycerolipid described in the document is not specifically disclosed. Furthermore, as shown in the examples of the present specification, the lipids of this document have a dispersion stability of colloidal particles when applied to a detection method by a colloidal gold aggregation method in which a sugar compound is bound to the surface of gold fine particles. It's bad and can't be used as a sensor.

The present invention is capable of easily fixing a sugar compound to a substrate surface or fine particle surface with a high degree of orientation and an appropriate density, and to quantitatively evaluate a sugar-binding compound with high sensitivity and ease. It is an object of the present invention to provide a glycolipid-containing compound and a glycolipid-containing fine particle.
Furthermore, an object of the present invention is to provide a biosensor, a detection reagent, or a method for detecting the sugar-binding compound that can quantitatively evaluate the sugar-binding compound with high sensitivity, simply and quickly.

  As a result of intensive studies in view of the above problems, the present inventors have immobilized a glycolipid-containing compound having a sugar chain site, a specific linker site, and a specific side chain site on the surface of a metal substrate or metal fine particle. The immobilization can be carried out with high orientation and high density, and when a sensor containing the glycolipid-containing compound or a detection reagent containing a glycolipid-containing microparticle is used, even a very small amount of a sugar-binding compound is highly sensitive. Thus, the present invention was completed.

〔式中、G_NはＮ個（Ｎは１〜１０の整数）の糖が直鎖状または分岐状に共有結合した糖鎖部位を示し；
Ｘは、−Ｏ−または−ＮＨ−を示し；
Ｒ¹は、−Ｃ（＝Ｏ）−Ｃ_nＨ_2n−Ｙまたは−ＣＨ₂−Ｃ_nＨ_2n−Ｙ（式中、ｎは２〜１０の整数を示し、Ｙは１個又は２個以上のチオール基または１個又は２個以上ジスルフィド基を有する基を示す。）で表されるリンカー部位を示し；
Ｒ²は、置換基を有していてもよいＣ_２〜２０アルキル基、置換基を有していてもよいＣ_２〜２０アルケニル基、置換基を有していてもよいＣ_1〜21アルキルカルボニルオキシメチル基、置換基を有していてもよいＣ_2〜22アルキルオキシメチル基、置換基を有していてもよいＣ_2〜21アルケニルカルボニルオキシメチル基、または置換基を有していてもよいＣ_2〜22アルケニルオキシメチル基で示される側鎖部位を示す。〕で表されることを特徴とする糖脂質含有化合物。
〔２〕前記Ｒ²が、置換基を有していてもよいＣ_10〜２０アルキル基、置換基を有していてもよいＣ_10〜２０アルケニル基、置換基を有していてもよいＣ_10〜21アルキルカルボニルオキシメチル基、置換基を有していてもよいＣ_11〜22アルキルオキシメチル基、置換基を有していてもよいＣ_10〜21アルケニルカルボニルオキシメチル基、または置換基を有していてもよいＣ_11〜22アルケニルオキシメチル基である、〔１〕に記載の糖脂質含有化合物。
〔３〕 前記Ｒ¹の主鎖に含まれる原子の合計数（Ｎ１）（Ｙ中の硫黄原子とＸとを最長で結合する複数の原子を主鎖を構成する原子とし、Ｙがジスルフィド基の場合はそのうちのＸに近い硫黄原子側を主鎖上の原子とし、合計数Ｎ１には硫黄原子を含まない。）と、前記Ｒ²の主鎖に含まれる原子の合計数（Ｎ２）（Ｒ²末端とＸとを最長で結合する複数の原子を主鎖を構成する原子とする。）とが、Ｎ１：Ｎ２＝１：１．５〜１：５の範囲にある、〔１〕または〔２〕に記載の糖脂質含有化合物。
〔４〕 前記Ｒ²が、下記式（２）

（式中、ｐは１〜１７の整数を示す。）で表される基、
下記式（３）

（式中、ｑは１〜２０の整数を示す。）で表される基、または、
下記式（４）

（式中、ｑは１〜２０の整数を示す。）で表される基であることを特徴とする〔１〕に記載の糖脂質含有化合物。
〔５〕 前記式（１）で表される化合物が、下記式（５）

〔式中、G_NはＮ個（Ｎは１〜１０の整数）の糖が直鎖状または分岐状に共有結合した糖鎖部位を示し；
リンカー部位中のｎは２〜１０の整数を示し、Ｙは１個又は２個以上のチオール基または１個又は２個以上ジスルフィド基を有する基を示し；
側鎖部位中のｐは９〜１７の整数を示す。〕であることを特徴とする〔１〕に記載の糖脂質含有化合物。
〔６〕 前記式（１）で表される化合物が、下記式（６）

〔式中、G_NはＮ個（Ｎは１〜１０の整数）の糖が直鎖状または分岐状に共有結合した糖鎖部位を示し；
Ｒ¹は、−Ｃ（＝Ｏ）−Ｃ_nＨ_2n−Ｙまたは−ＣＨ₂−Ｃ_nＨ_2n−Ｙ（式中、ｎは２〜１０の整数を示し、Ｙは１個又は２個以上のチオール基または１個又は２個以上ジスルフィド基を有する基を示す。）で表されるリンカー部位を示し；
Ｒ³は、−Ｃ（＝Ｏ）−Ｃ_ｑＨ_2ｑ−ＣＨ₃または−ＣＨ₂−Ｃ_ｑＨ_2ｑ−ＣＨ₃（式中、ｑは９〜２０の整数を示す。）で表される基を示す。〕であることを特徴とする〔１〕に記載の糖脂質含有化合物。
〔７〕前記式（６）で表される化合物が、下記式（７）

〔式中、G_NはＮ個（Ｎは１〜１０の整数）の糖が直鎖状または分岐状に共有結合した糖鎖部位を示し；
リンカー部位中のｎは２〜１０の整数を示し、Ｙは１個又は２個以上のチオール基または１個又は２個以上ジスルフィド基を有する基を示し；
側鎖部位中のｑは９〜２０の整数を示す。〕であることを特徴とする〔６〕に記載の糖脂質含有化合物。
〔８〕 前記式（６）で表される化合物が、下記式（８）

〔式中、G_NはＮ個（Ｎは１〜１０の整数）の糖が直鎖状または分岐状に共有結合した糖鎖部位を示し；
リンカー部位中のｎは２〜１０の整数を示し、Ｙは１個又は２個以上のチオール基または１個又は２個以上ジスルフィド基を有する基を示し；
側鎖部位中のｑは９〜２０の整数を示す。〕であることを特徴とする〔６〕に記載の糖脂質含有化合物。
〔９〕 前記式（６）で表される化合物が、下記式（９）

〔式中、G_NはＮ個（Ｎは１〜１０の整数）の糖が直鎖状または分岐状に共有結合した糖鎖部位を示し；
リンカー部位中のｎは２〜１０の整数を示し、Ｙは１個又は２個以上のチオール基または１個又は２個以上ジスルフィド基を有する基を示し；
側鎖部位中のｑは９〜２０の整数を示す。〕であることを特徴とする〔６〕に記載の糖脂質含有化合物。
〔１０〕 前記式（６）で表される化合物が、下記式（１０）

〔式中、G_NはＮ個（Ｎは１〜１０の整数）の糖が直鎖状または分岐状に共有結合した糖鎖部位を示し；
リンカー部位中のｎは２〜１０の整数を示し、Ｙは１個又は２個以上のチオール基または１個又は２個以上ジスルフィド基を有する基を示し；
側鎖部位中のｑは９〜２０の整数を示す。〕であることを特徴とする〔６〕に記載の糖脂質含有化合物。
〔１１〕 前記Ｇ_N−が、下記式(i)〜(xv)

（式(xii)中、Ｗ¹、Ｗ²は、水素原子又は−ＳＯ₃Ｍ（Ｍは水素原子又はその生理的に許容される塩を形成する基を示す）を示し、Ｗ¹、Ｗ²のうちの少なくとも一方は−ＳＯ₃Ｍである；
式(xiii)および(xiv)中、Ｒ₁は−ＮＨＡｃまたは−ＯＨであり、−ＣＯＯＭは−ＣＯＯＨまたはその生理的に許容される塩を示す。）
で表されるいずれかの基である、〔１〕〜〔１０〕のいずれかに記載の糖脂質含有化合物。
〔１２〕〔１〕〜〔１１〕のいずれかに記載の糖脂質含有化合物が、リンカー部位を介して金属基板表面に固定されていることを特徴とするバイオセンサー。
〔１３〕 前記糖脂質含有化合物が金属基板表面に固定されたときの密度が、0.1〜10個／nm²である、〔１２〕に記載のバイオセンサー。
〔１４〕 前記金属基板が、金基板であることを特徴とする〔１２〕または〔１３〕に記載のバイオセンサー。
〔１５〕 前記バイオセンサーが、リシン毒素、大腸菌Ｏ−１５７、ベロ毒素、ボツリヌス毒素、ＨＩＶまたはインフルエンザウイルスの検出用バイオセンサーである、〔１２〕〜〔１４〕のいずれかに記載のバイオセンサー。
〔１６〕 下記の工程からなる、糖結合性化合物の検出方法：
（１）試験化合物を、〔１２〕〜〔１４〕のいずれかに記載のバイオセンサーの基板表面に接触させる工程、
（２）基板に結合した糖結合性化合物を検出する工程。
〔１７〕 前記Ｇ_N−が、下記式(i)〜(v)

のいずれかで表される基である、〔１６〕に記載の方法。
〔１８〕 前記Ｇ_N−が、下記式(vi)または(vii)

で表される、〔１６〕に記載の方法。
〔１９〕 前記Ｇ_N−が、下記式(i)、(viii)〜(xi)

で表されるいずれかの基である、〔１６〕に記載の方法。
〔２０〕 前記Ｇ_N−が、下記式(i)、(xii)

（式(xii)中、Ｗ¹、Ｗ²は、水素原子又は−ＳＯ₃Ｍ（Ｍは水素原子又はその生理的に許容される塩を形成する基を示す）を示し、Ｗ¹、Ｗ²のうちの少なくとも一方は−ＳＯ₃Ｍである。）で表されるいずれかの基である、〔１６〕に記載の方法。
〔２１〕 前記Ｇ_N−が、下記式(xiii)または(xiv)

（式中、Ｒ₁は−ＮＨＡｃまたは−ＯＨであり、−ＣＯＯＭは−ＣＯＯＨまたはその生理的に許容される塩を示す。）で表されるいずれかの基である、〔１６〕に記載の方法。
〔２２〕 前記糖結合性化合物が、リシン毒素である、〔１７〕に記載の方法。
〔２３〕 前記糖結合性化合物が、大腸菌Ｏ−１５７またはベロ毒素である、〔１８〕に記載の方法。
〔２４〕 前記糖結合性化合物が、ボツリヌス毒素である、〔１９〕に記載の方法。
〔２５〕 前記糖結合性化合物が、ＨＩＶである、〔２０〕に記載の方法。
〔２６〕 前記糖結合性化合物が、インフルエンザウイルスである、〔２１〕に記載の方法。
〔２７〕 〔１〕〜〔１１〕のいずれかに記載の糖脂質含有化合物が、リンカー部位を介して金属微粒子表面に固定されていることを特徴とする糖脂質含有微粒子。
〔２８〕 前記金属微粒子の平均粒子径が、１〜１００ｎｍの範囲にあることを特徴とする〔２７〕に記載の糖脂質含有微粒子。
〔２９〕 前記糖脂質含有化合物が金属微粒子表面に固定されたときの密度が、0.1〜10個／nm²である、〔２７〕に記載の糖脂質含有微粒子。
〔３０〕 前記金属微粒子が金微粒子であることを特徴とする〔２７〕〜〔２９〕のいずれかに記載の糖脂質含有微粒子。
〔３１〕 〔２７〕〜〔３０〕のいずれかに記載の糖脂質含有微粒子を含有することを特徴とする糖結合性化合物検出試薬。
〔３２〕 前記糖脂質含有微粒子が、溶液中にコロイドで存在していることを特徴とする〔３１〕に記載の糖結合性化合物検出試薬。
〔３３〕 前記糖結合性化合物検出試薬が、リシン毒素、大腸菌Ｏ−１５７、ベロ毒素、ボツリヌス毒素、ＨＩＶまたはインフルエンザウイルスの検出試薬である、〔３１〕または〔３２〕に記載の糖結合性化合物検出試薬。
〔３４〕 下記の工程からなる、糖結合性化合物の検出方法：
（１）試験化合物を、〔３１〕または〔３２〕に記載の糖結合性化合物検出試薬に添加して、糖脂質含有微粒子に接触させる工程、
（２）糖脂質含有微粒子に結合した糖結合性化合物を検出する工程。
〔３５〕 前記Ｇ_N−が、下記式(i)〜(v)

のいずれかで表される基である、〔３４〕に記載の方法。
〔３６〕 前記Ｇ_N−が、下記式(vi)または(vii)

で表される、〔３４〕に記載の方法。
〔３７〕 前記Ｇ_N−が、下記式(i)、(viii)〜(xi)

で表されるいずれかの基である、〔３４〕に記載の方法。
〔３８〕 前記Ｇ_N−が、下記式(i)、(xii)

（式(xii)中、Ｗ¹、Ｗ²は、水素原子又は−ＳＯ₃Ｍ（Ｍは水素原子又はその生理的に許容される塩を形成する基を示す）を示し、Ｗ¹、Ｗ²のうちの少なくとも一方は−ＳＯ₃Ｍである。）で表されるいずれかの基である、〔３４〕に記載の方法。
〔３９〕 前記Ｇ_N−が、下記式(xiii)または(xiv)

（式中、Ｒ₁は−ＮＨＡｃまたは−ＯＨであり、−ＣＯＯＭは−ＣＯＯＨまたはその生理的に許容される塩を示す。）で表されるいずれかの基である、〔３４〕に記載の方法。
〔４０〕 前記糖結合性化合物が、リシン毒素である、〔３５〕に記載の方法。
〔４１〕 前記糖結合性化合物が、大腸菌Ｏ−１５７またはベロ毒素である、〔３６〕に記載の方法。
〔４２〕 前記糖結合性化合物が、ボツリヌス毒素である、〔３７〕に記載の方法。
〔４３〕 前記糖結合性化合物が、ＨＩＶである、〔３８〕に記載の方法。
〔４４〕 前記糖結合性化合物が、インフルエンザウイルスである、〔３９〕に記載の方法。That is, this invention includes the following.
[1] The following general formula (1)

Wherein, G _N is N (N is an integer of from 1 to 10) shows the sugar chain site of sugar is covalently bonded to a linear or branched;
X represents —O— or —NH—;
R ¹ represents —C (═O) —C _n H _2n —Y or —CH ₂ —C _n H _2n —Y (wherein n represents an integer of 2 to 10, Y represents 1 or 2 or more) A thiol group or a group having one or more disulfide groups.)
R ² is a C _2-20 alkyl group which may have a substituent, a C _2-20 alkenyl group which may have a substituent, a C _1-21 alkyl which may have a substituent. carbonyl oxymethyl group, have an optionally substituted C _{2 to 22} alkyloxymethyl group, which may have a substituent C _{2 to 21} alkenylcarbonyloxy methyl group or a substituent, The side chain site _| part shown by a _C2-22 alkenyloxymethyl group which may be good is shown. A glycolipid-containing compound represented by the formula:
[2] The R ² may have a C _10-20 alkyl group which may have a substituent, a C _10-20 alkenyl group which may have a substituent, or a C which may have a substituent. _10-21 alkylcarbonyloxymethyl group, which may have a substituent C _{11 to 22} alkyl oxymethyl group which may have a substituent C _10-21 alkenylcarbonyl oxymethyl group, or a substituent The glycolipid-containing compound according to [1], which is a C _11-22 alkenyloxymethyl group which may have.
[3] Total number of atoms (N1) contained in the main chain of R ¹ (A plurality of atoms that combine the longest sulfur atom and X in Y are atoms constituting the main chain, and Y is a disulfide group) In this case, the sulfur atom side close to X is an atom on the main chain, and the total number N1 does not include a sulfur atom.) And the total number of atoms (N2) (R2) in the main chain of R ² A plurality of atoms connecting the ^two ends and X at the longest are atoms constituting the main chain.) Are in the range of N1: N2 = 1: 1.5 to 1: 5 [1] or [ [2] The glycolipid-containing compound of [2].
[4] R ² represents the following formula (2)

(Wherein p represents an integer of 1 to 17),
Following formula (3)

(Wherein q represents an integer of 1 to 20), or
Following formula (4)

(Wherein q represents an integer of 1 to 20). The glycolipid-containing compound according to [1], which is a group represented by
[5] The compound represented by the formula (1) is represented by the following formula (5):

Wherein, G _N is N (N is an integer of from 1 to 10) shows the sugar chain site of sugar is covalently bonded to a linear or branched;
N in the linker moiety represents an integer of 2 to 10, and Y represents a group having one or more thiol groups or one or more disulfide groups;
P in a side chain part shows the integer of 9-17. The glycolipid-containing compound according to [1], wherein
[6] The compound represented by the formula (1) is represented by the following formula (6):

Wherein, G _N is N (N is an integer of from 1 to 10) shows the sugar chain site of sugar is covalently bonded to a linear or branched;
R ¹ represents —C (═O) —C _n H _2n —Y or —CH ₂ —C _n H _2n —Y (wherein n represents an integer of 2 to 10, Y represents 1 or 2 or more) A thiol group or a group having one or more disulfide groups.)
R ³ is a group represented by —C (═O) —C _q H _2q —CH ₃ or —CH ₂ —C _q H _2q —CH ₃ (wherein q represents an integer of 9 to 20). Indicates. The glycolipid-containing compound according to [1], wherein
[7] The compound represented by the formula (6) is represented by the following formula (7):

Wherein, G _N is N (N is an integer of from 1 to 10) shows the sugar chain site of sugar is covalently bonded to a linear or branched;
N in the linker moiety represents an integer of 2 to 10, and Y represents a group having one or more thiol groups or one or more disulfide groups;
Q in a side chain site | part shows the integer of 9-20. [6] The glycolipid-containing compound according to [6].
[8] The compound represented by the formula (6) is represented by the following formula (8):

Wherein, G _N is N (N is an integer of from 1 to 10) shows the sugar chain site of sugar is covalently bonded to a linear or branched;
N in the linker moiety represents an integer of 2 to 10, and Y represents a group having one or more thiol groups or one or more disulfide groups;
Q in a side chain site | part shows the integer of 9-20. [6] The glycolipid-containing compound according to [6].
[9] The compound represented by the formula (6) is represented by the following formula (9).

Wherein, G _N is N (N is an integer of from 1 to 10) shows the sugar chain site of sugar is covalently bonded to a linear or branched;
N in the linker moiety represents an integer of 2 to 10, and Y represents a group having one or more thiol groups or one or more disulfide groups;
Q in a side chain site | part shows the integer of 9-20. [6] The glycolipid-containing compound according to [6].
[10] The compound represented by the formula (6) is represented by the following formula (10):

Wherein, G _N is N (N is an integer of from 1 to 10) shows the sugar chain site of sugar is covalently bonded to a linear or branched;
N in the linker moiety represents an integer of 2 to 10, and Y represents a group having one or more thiol groups or one or more disulfide groups;
Q in a side chain site | part shows the integer of 9-20. [6] The glycolipid-containing compound according to [6].
[11] The G _N - is represented by the following formula (i) ~ (xv)

(In formula (xii), W ¹ and W ² represent a hydrogen atom or —SO ₃ M (M represents a hydrogen atom or a group that forms a physiologically acceptable salt thereof), and W ¹ , W ² at least one of the is -SO ₃ M;
In the formulas (xiii) and (xiv), R ₁ is —NHAc or —OH, and —COOM represents —COOH or a physiologically acceptable salt thereof. )
The glycolipid-containing compound according to any one of [1] to [10], which is any group represented by:
[12] A biosensor, wherein the glycolipid-containing compound according to any one of [1] to [11] is immobilized on the surface of a metal substrate via a linker site.
[13] density when the glycolipid-containing compound is fixed to the metal substrate surface is 0.1 to 10 pieces / nm ^2, biosensor according to [12].
[14] The biosensor according to [12] or [13], wherein the metal substrate is a gold substrate.
[15] The biosensor according to any one of [12] to [14], wherein the biosensor is a biosensor for detecting ricin toxin, E. coli O-157, verotoxin, botulinum toxin, HIV or influenza virus.
[16] A method for detecting a sugar-binding compound comprising the following steps:
(1) A step of bringing a test compound into contact with the substrate surface of the biosensor according to any one of [12] to [14],
(2) A step of detecting a sugar-binding compound bound to the substrate.
[17] The _GN − represents the following formulas (i) to (v):

The method according to [16], which is a group represented by any one of:
[18] The above G _N − is represented by the following formula (vi) or (vii)

The method according to [16], represented by:
[19] The above G _N − represents the following formulas (i), (viii) to (xi)

The method according to [16], which is any group represented by:
[20] The above _GN − represents the following formulas (i) and (xii)

(In formula (xii), W ¹ and W ² represent a hydrogen atom or —SO ₃ M (M represents a hydrogen atom or a group that forms a physiologically acceptable salt thereof), and W ¹ , W ² At least one of them is —SO ₃ M.), the method according to [16].
[21] The above G _N − is represented by the following formula (xiii) or (xiv)

(Wherein R ₁ is —NHAc or —OH, —COOM represents —COOH or a physiologically acceptable salt thereof), Method.
[22] The method according to [17], wherein the sugar-binding compound is a ricin toxin.
[23] The method according to [18], wherein the sugar-binding compound is E. coli O-157 or verotoxin.
[24] The method according to [19], wherein the sugar-binding compound is botulinum toxin.
[25] The method according to [20], wherein the sugar-binding compound is HIV.
[26] The method according to [21], wherein the sugar-binding compound is an influenza virus.
[27] A glycolipid-containing fine particle, wherein the glycolipid-containing compound according to any one of [1] to [11] is immobilized on the surface of the metal fine particle via a linker site.
[28] The glycolipid-containing fine particles according to [27], wherein the metal fine particles have an average particle diameter in the range of 1 to 100 nm.
[29] density when the glycolipid-containing compound is fixed to the metal fine particle surface is 0.1 to 10 pieces / nm is ^2, glycolipid-containing particle according to [27].
[30] The glycolipid-containing fine particles according to any one of [27] to [29], wherein the metal fine particles are gold fine particles.
[31] A sugar-binding compound detection reagent comprising the glycolipid-containing fine particles according to any one of [27] to [30].
[32] The sugar-binding compound detection reagent according to [31], wherein the glycolipid-containing fine particles are present in a colloid in a solution.
[33] The sugar-binding compound according to [31] or [32], wherein the sugar-binding compound detection reagent is a detection reagent for ricin toxin, E. coli O-157, verotoxin, botulinum toxin, HIV or influenza virus. Detection reagent.
[34] A method for detecting a sugar-binding compound comprising the following steps:
(1) A step of adding a test compound to the sugar-binding compound detection reagent according to [31] or [32] and bringing the test compound into contact with the glycolipid-containing microparticles;
(2) A step of detecting a sugar-binding compound bound to the glycolipid-containing fine particles.
[35] The _GN − represents the following formulas (i) to (v):

The method according to [34], which is a group represented by any one of:
[36] The above-mentioned G _N − represents the following formula (vi) or (vii)

The method according to [34], represented by:
[37] The above _GN − represents the following formulas (i), (viii) to (xi)

The method of [34], which is any group represented by:
[38] The above _GN − is represented by the following formulas (i), (xii)

(In formula (xii), W ¹ and W ² represent a hydrogen atom or —SO ₃ M (M represents a hydrogen atom or a group that forms a physiologically acceptable salt thereof), and W ¹ , W ² At least one of them is —SO ₃ M.), the method according to [34].
[39] The above G _N − is represented by the following formula (xiii) or (xiv)

(Wherein R ₁ represents —NHAc or —OH, and —COOM represents —COOH or a physiologically acceptable salt thereof), according to [34], Method.
[40] The method according to [35], wherein the sugar-binding compound is a ricin toxin.
[41] The method according to [36], wherein the sugar-binding compound is Escherichia coli O-157 or Verotoxin.
[42] The method according to [37], wherein the sugar-binding compound is botulinum toxin.
[43] The method according to [38], wherein the sugar-binding compound is HIV.
[44] The method according to [39], wherein the sugar-binding compound is an influenza virus.

FIG. 1 is a ¹ H-NMR spectrum (5.0 ppm to 3.0 ppm) of the compound (1d). FIG. 2 is a ¹ H-NMR spectrum (5.0 ppm to 3.0 ppm) of the compound (2d). FIG. 3 is an SPR sensorgram of the interaction between the surface of the sensor chip on which the compound (2b), compound (3b) or compound (4b) having galactose at the sugar chain site is immobilized and ricin 120. The lysine 120 solution was injected at a flow rate of 10 μL / min with 100 μL of a 1000 ng / mL (8.3 nM) solution. FIG. 4 is an SPR sensorgram of the interaction between the surface of the sensor chip on which compound (2c) or compound (3c) having lactose at the sugar chain site is immobilized, and ricin 120. The lysine 120 solution was injected at a flow rate of 10 μL / min with 100 μL of a 1000 ng / mL (8.3 nM) solution. FIG. 5 is a sensorgram of the interaction between the surface of the sensor chip on which the compound (2b) is immobilized and each concentration of lysine 120. The injection amount of the lysine 120 solution is 10000, 1000, 100, and 100 μL of a 10 ng / mL solution injected at a flow rate of 10 μL / min. FIG. 6 is a sensorgram of the interaction between the surface of the sensor chip on which the compound (2c) is immobilized and each concentration of lysine 120. The injection amount of the lysine 120 solution is 10000, 1000, 100, and 100 μL of a 10 ng / mL solution injected at a flow rate of 10 μL / min. FIG. 7 is a sensorgram of the interaction between the surface of the sensor chip on which the compound (2d) is immobilized and botulinum toxin C at various concentrations. The injection amount of the botulinum toxin C solution is 1000, 500, 100, and 100 μL of a 10 ng / mL solution at a flow rate of 10 μL / min.

Hereinafter, the glycolipid-containing compound, biosensor, and sugar-binding compound detection reagent will be specifically described.
<Glycolipid-containing compound>
The glycolipid-containing compound according to the present invention is represented by the following general formula (1).

Wherein, G _N is N (N is an integer of from 1 to 10) indicating the sugar chain portion sugar covalently attached to a linear or branched.
_GN is determined and not limited by the relationship with the sugar-binding compound, and may be any sugar chain.
For example, as for the sugar chain part to which the sugar was covalently bonded in a straight chain, N is preferably 1 to 8, more preferably 1 to 5. In the case of a branched shape, N is preferably a sugar chain moiety of 5 to 10.

Specific examples of the linear sugar chain moiety (G _N ) include the following sugar chain moieties (i) to (vii).

Of these, for example, a galactose unit represented by (i), an N-acetylgalactosamine unit represented by (ii), a disaccharide lactose unit represented by (iii), and (iv) The disaccharide lactosamine unit represented by (v) and the disaccharide unit (Galβ1-3GalNAc) in which galactose and N-acetylgalactosamine represented by (v) are β-linked are effective for detecting ricin toxin.
In addition, two galactoses represented by (vi) above are α-linked disaccharide units (Galα1-4Galβ1-) and two galactoses represented by (vii) are α-linked, and glucose is further added. A linked trisaccharide unit (Galα1-4Galβ1-4Glcβ1-) is effective for detection of verotoxin produced by E. coli O-157 or E. coli O-157.
The sugar chain site represented by (i) is also effective in detecting botulinum toxin B produced by Clostridium botulinum.

式(xii)中、Ｗ¹、Ｗ²は、水素原子又は−ＳＯ₃Ｍ（Ｍは水素原子又はその生理的に許容される塩を形成する基を示す）を示し、Ｗ¹、Ｗ²のうちの少なくとも一方は−ＳＯ₃Ｍである。
具体的には、下記(xv)〜(xvii)で表される基が挙げられる。

前記(i)、(xii)で表される基は、AIDS（後天性免疫不全症候群）の原因となるHIV (human immunodeficiency virus；ヒト免疫不全ウイルス) （別冊日系サイエンス１１１、８９〜１０１ページ）の検出に有効である。Furthermore, as a specific example in the case of a linear sugar chain moiety (G _N ), a sugar chain moiety such as the following (xii) can also be mentioned.

In formula (xii), W ¹ and W ² represent a hydrogen atom or —SO ₃ M (M represents a hydrogen atom or a group that forms a physiologically acceptable salt thereof), and W ¹ and W ² At least one of them is —SO ₃ M.
Specific examples include groups represented by the following (xv) to (xvii).

The groups represented by (i) and (xii) are those of HIV (human immunodeficiency virus) that causes AIDS (acquired immune deficiency syndrome) Effective for detection.

（式中、Ｒ₁は−ＮＨＡｃまたは−ＯＨであり、−ＣＯＯＭは−ＣＯＯＨまたはその生理的に許容される塩を示す。）で表されるいずれかの基であってもよい。
前記(xiii)、(xiv)で表される基は、インフルエンザウイルス（別冊日系サイエンス１１１、８９〜１０１ページ）の検出に有効である。Furthermore, the G _N - is represented by the following formula (xiii) or (xiv)

(Wherein R ₁ represents —NHAc or —OH, and —COOM represents —COOH or a physiologically acceptable salt thereof).
The groups represented by the above (xiii) and (xiv) are effective for detection of influenza virus (separate volume Nikkei Science 111, pages 89 to 101).

The -SO ₃ M, when -CO ₂ M are physiologically acceptable salts, in the form of its salt is not particularly limited, for example, inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, Examples thereof include acidic or basic amino acid salts.

  Preferable examples of inorganic acid salts include hydrochloride, hydrobromide, sulfate, nitrate, phosphate and the like, and preferable examples of organic acid salts include, for example, acetate, succinate and fumarate. Acid salts, maleates, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, p-toluenesulfonate, and the like.

  Preferable examples of the inorganic base salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt and ammonium salt. Preferred examples of organic base salts Examples thereof include diethylamine salt, triethylamine salt, diethanolamine salt, meglumine salt, N, N′-dibenzylethylenediamine salt and the like.

  Preferable examples of the acidic amino acid salt include aspartate and glutamate, and preferable examples of the basic amino acid salt include arginine salt, lysine salt and ornithine salt.

More specific examples in the case of a branched sugar chain moiety include sugar chain moieties such as the following (viii) to (xi).

The glycolipid-containing compound of the present invention is also effective for detecting botulinum toxin. Examples of botulinum toxin include botulinum toxin A, B, C, D, E, and F.
The sugar chain site of the hexasaccharide represented by (viii) is effective in detecting botulinum toxins A and B produced by Clostridium botulinum.
The sugar chain site of the heptasaccharide represented by (ix) is effective for detecting botulinum toxins A, B, C, and F produced by Clostridium botulinum.
The sugar chain site of the octasaccharide represented by (x) is effective in detecting botulinum toxins A and E produced by Clostridium botulinum.
The sugar chain site of hexasaccharide represented by (xi) is effective in detecting botulinum toxin B produced by Clostridium botulinum.

  For example, the sugar chain part of the octasaccharide represented by the formula (x) may be represented by the following formula.

NeuAc (α2-8) NeuAc (α2-3) Gal (β1-3) GalNAc (β1-4) (NeuAc (α2-8) NeuAc (α2-3)) Gal (β1-4) Glc (β)-

NeuAc represents N-acetylneuraminic acid (sialic acid), Gal represents galactose, GalNAc represents N-acetylgalactosamine, and Glc represents glucose. Numbers and Greek letters indicate the bonding mode between sugars. For example, Gal (β1-4) Glc means that the 1-position of galactose is linked to the 4-position of glucose by a β bond. Ac represents an acetyl group (—C (═O) CH ₃ ).

  X is a divalent group represented by —O— or —NH—.

R ¹ represents a linker moiety represented by —C (═O) —C _n H _2n —Y or —CH ₂ —C _n H _2n —Y. In formula, n shows the integer of 2-10, Preferably it is 3-8, More preferably, it is 3-5.
When n is in the above range, the sensitivity of the sensor when the glycolipid-containing compound is bound to the substrate surface or the fine particle surface is improved, and the substrate surface or the surface has a density and orientation suitable for binding to the sugar-binding compound. It can be effectively bonded to the surface of the fine particles.

  In said formula (1), Y shows group which has 1 or 2 or more thiol groups or 1 or 2 or more disulfide groups. In the present specification, the “thiol group” is a group represented by —SH, and the “disulfide group” is a group having a structure represented by —S—S—.

  Of these, disulfide groups are preferred. In the case of the disulfide group, the glycolipid-containing compound can be firmly and stably immobilized on the substrate surface or fine particle surface. In particular, in the present invention, steric hindrance of the sugar chain part, the linker site, and the side chain is predicted, and a decrease in bond density, a decrease in sensitivity, etc. can be considered. However, as in the present invention, a specific linker, side chain, and disulfide group are considered. By combining these, it becomes possible to achieve a strong and stable immobilization, and it is possible to obtain a highly sensitive sensor without the influence of steric hindrance.

  The disulfide group is preferably a cyclic disulfide group, of which a 5-membered dithiolanyl group, such as a 1,2-dithiolan-3-yl group or a 1,2-dithiolan-4-yl group, is a 6-membered group. Examples of the ring dithianyl group include a 1,2-dithian-3-yl group and a 1,2-dithian-4-yl group. Of these, a 5-membered dithiolanyl group is preferable, and a 1,2-dithiolan-3-yl group is more preferable. A group derived from thioctic acid (lipoic acid) can also be used.

R ² is a C _2-20 alkyl group which may have a substituent, a C _2-20 alkenyl group which may have a substituent, a C _1-21 alkyl which may have a substituent. carbonyl oxymethyl group, have an optionally substituted C _{2 to 22} alkyloxymethyl group, which may have a substituent C _{2 to 21} alkenylcarbonyloxy methyl group or a substituent, The side chain site _| part shown by a _C2-22 alkenyloxymethyl group which may be good is shown.

Further, R ² may preferably have a C _10-20 alkyl group which may have a substituent, a C _10-20 alkenyl group which may have a substituent, or a substituent. good C _{10 to 21} alkylcarbonyloxymethyl group, which may have a substituent C _{11 to 22} alkyl oxymethyl group which may have a substituent C _{10 to 21} alkenylcarbonyloxy methyl group or substituted, C _11-22 alkenyloxymethyl group which may have a group.

Further, the total number of atoms (N1) contained in the main chain of R ¹ and the total number of atoms (N2) contained in the main chain of R ² are preferably N1: N2 = 1: 1.5. ˜1: 5, more preferably in the range of 1: 2 to 1: 3.
In N1, a plurality of atoms that bond the longest sulfur atom in Y and X are the atoms constituting the main chain, and when Y is a disulfide group, the sulfur atom side close to X is the main chain. The total number N1 does not include a sulfur atom. In N2, a plurality of atoms that bond the R ² terminal and X at the longest are atoms constituting the main chain.

When such a side chain site is included, the detection sensitivity can be significantly improved as compared with the case where no side chain site is present. It is speculated that this may be based on a moderate hydrophobic interaction between the side chain site and a sugar-binding compound such as a protein to be analyzed.
Furthermore, when the side chain site is composed of such carbon number or number of atoms, and the side chain site has a length longer than the linker site, the glycolipid-containing compound is suitable for binding to the sugar-binding compound. It can be immobilized on a substrate or fine particles with a higher degree of orientation, and when applied to a biosensor containing the glycolipid-containing compound or a detection reagent containing a glycolipid-containing fine particle, even a very small amount of a sugar-binding compound can be used. Sensitivity can be detected and quantified.
This is because the side chain site has a folded structure depending on the length of the linker site, and the folded side chain site allows the glycolipid-containing compounds to have high orientation suitable for binding to a sugar-binding compound. It is speculated that it may be effectively bonded to the surface of the fine particles.
Whereas the above-mentioned Non-Patent Documents 7 and 8 are imitations of cell membranes (biological membranes), the feature of the present application is that it is possible to provide substrates and microparticles in which glycolipid compounds are immobilized with a more appropriate orientation than cell membranes, The detection sensitivity is greatly improved.

In the present invention, R ² preferably contains a hydrophobic group represented by —C _m H _2m —CH ₃ . In formula, m shows the integer of 1-21, Preferably m is 10-20, More preferably, it is 13-18.
By having such a hydrophobic side chain site, it is possible to fix the glycolipid-containing compound to the substrate and the fine particles with high orientation and high density, compared to the case where the side chain site does not exist, By using a biosensor containing the glycolipid-containing compound or a detection reagent containing glycolipid-containing microparticles, even a very small amount of a sugar-binding compound can be detected and quantified with high sensitivity.

The “C _2-20 alkyl group” is an alkyl group having 2 to 20 carbon atoms, which may be a linear or branched alkyl group, and is preferably a linear alkyl group.

The “C _2-20 alkenyl group” is an alkenyl group having 2 to 20 carbon atoms, which may be linear or branched, but is preferably a linear alkenyl group. One or more unsaturated bonds may be contained. In the case of containing a plurality of unsaturated bonds, an arrangement such as —CH═CH—CH ₂ —CH═CH— may be included.

The “C _1-20 alkylcarbonyloxymethyl group” is a group represented by a C _1-20 alkyl group —CO—O—CH ₂ —, wherein the C _1-20 alkyl group is a linear or branched alkyl group. Any of the groups may be used, but a linear alkyl group is preferable.

The “C _2-21 alkyloxymethyl group” is a group represented by a C _2-21 alkyl group —O—CH ₂ —, and the C _2-21 alkyl group is either a linear or branched alkyl group. Preferably, it is a linear alkyl group.

The “C _2-20 alkenylcarbonyloxymethyl group” is a group represented by a C _2-20 alkenyl group —CO—O—CH ₂ —, wherein the C _2-20 alkenyl group is linear or branched. Either is acceptable. One or more unsaturated bonds may be contained. In the case of containing a plurality of unsaturated bonds, an arrangement such as —CH═CH—CH ₂ —CH═CH— may be included.

The “C _2-21 alkenyloxymethyl group” is a group represented by a C _2-21 alkenyl group —O—CH ₂ —, and the C _2-21 alkenyl group may be linear or branched. . One or more unsaturated bonds may be contained. In the case of containing a plurality of unsaturated bonds, an arrangement such as —CH═CH—CH ₂ —CH═CH— may be included.

  The phrase “which may have a substituent” means that one or more substituents may be arbitrarily combined at substitutable sites. Specific examples of the substituent include groups selected from the following substituent group A.

<Substituent group A>
A halogen atom, a hydroxyl group, a C _1-6 alkoxy group, a trifluoromethyl group, a trifluoromethoxy group, an amino group, a C _1-6 alkylamino group;

Of these substituents, a hydroxyl group or a C _1-6 alkoxy group is preferred.

The “optionally substituted alkyl group having 2 to 20 carbon atoms” means an alkyl group having 2 to 20 carbon atoms (—C _t H _{2t + 2} (t is 2 to 20)). To do.

式中、ｐは１〜１７の整数を示す。前記ｐは好ましくは７〜１７、さらに好ましくは１０〜１５である。Of the “alkylene group having 2 to 20 carbon atoms which may have a substituent”, a group represented by the following formula (2) is preferable.

In formula, p shows the integer of 1-17. The p is preferably 7 to 17, more preferably 10 to 15.

式中、Ｇ_Nは式（１）中のそれと同意義である。リンカー部位中のｎは２〜１０の整数を示し、好ましくは３〜８、さらに好ましくは３〜５である。Ｙは１個又は２個以上のチオール基または１個又は２個以上ジスルフィド基を有する基を示し、好ましくは１個のジスルフィド基である。側鎖部位中のｐは好ましくは１〜１７の整数を示し、さらに好ましくは７〜１７、特に好ましくは１０〜１５である。Of the compounds (1) having a group represented by the above (2), a compound represented by the following formula (5) is preferable.

In the formula, _GN is the same as that in formula (1). N in a linker part shows the integer of 2-10, Preferably it is 3-8, More preferably, it is 3-5. Y represents a group having one or more thiol groups or one or more disulfide groups, preferably one disulfide group. P in the side chain site preferably represents an integer of 1 to 17, more preferably 7 to 17, and particularly preferably 10 to 15.

Furthermore, the total number of atoms (N1) contained in the main chain of the —COC _n H _2n Y and the total number of atoms contained in the main chain of the —C (OH) C═CC _p H _2p CH ₃ (N2 ) Is preferably in the range of N1: N2 = 1: 1.5 to 1: 5, more preferably 1: 2 to 1: 3.
In N1, a plurality of atoms that bond the longest sulfur atom in Y and X are the atoms constituting the main chain, and when Y is a disulfide group, the sulfur atom side close to X is the main chain. The total number N1 does not include a sulfur atom. In N2, a plurality of atoms that bond the R ² terminal and X at the longest are atoms constituting the main chain.

As described above, when such a side chain moiety is included, the detection sensitivity can be significantly improved as compared to the case where no side chain moiety is present.
Further, when the side chain site is composed of such carbon number or number of atoms and the side chain site has a length longer than the linker site, the glycolipid-containing compound is densely substrated with a higher degree of orientation. When a biosensor containing the glycolipid-containing compound or a detection reagent containing the glycolipid-containing microparticle is used, even a very small amount of a sugar-binding compound can be detected and quantified with high sensitivity.
In Formula (5), —NH and —OH adjacent thereto are preferably D-erythro.

  Such a compound represented by (5) can be derived from a glycosphingolipid (a compound in which a sugar is bound to a ceramide in which a sphingoid and a fatty acid are bound), which is present on the front side of the cell bilayer membrane, It is also preferable from the viewpoint of easy availability of raw materials.

式中、ｑは１〜２０の整数を示し、好ましくは１０〜２０、さらに好ましくは１３〜１８である。Of the “C _1-20 alkylcarbonyloxymethyl group”, a group represented by the following formula (3) is preferable.

In formula, q shows the integer of 1-20, Preferably it is 10-20, More preferably, it is 13-18.

式中、ｑは１〜２０の整数を示し、好ましくは１０〜２０、さらに好ましくは１３〜１８である。Of the “C _2-21 alkyloxymethyl group”, a group represented by the following formula (4) is preferable.

In formula, q shows the integer of 1-20, Preferably it is 10-20, More preferably, it is 13-18.

式中、G_NはＮ個（Ｎは１〜１０の整数）の糖が直鎖状または分岐状に共有結合した糖鎖部位を示し；
Ｒ¹は、−Ｃ（＝Ｏ）−Ｃ_nＨ_2n−Ｙまたは−ＣＨ₂−Ｃ_nＨ_2n−Ｙで表されるリンカー部位を示す。式中、ｎは２〜１０の整数を示し、好ましくは３〜８、さらに好ましくは３〜５である。Ｙは１個又は２個以上のチオール基または１個又は２個以上ジスルフィド基を有する基を示し、好ましくは１個のジスルフィド基である。
Ｒ³は、−Ｃ（＝Ｏ）−Ｃ_ｑＨ_2ｑ−ＣＨ₃または−ＣＨ₂−Ｃ_ｑＨ_2ｑ−ＣＨ₃で表される基を示す。式中、ｑは１〜２０の整数を示し、好ましくは１０〜２０、さらに好ましくは１３〜１８である。Of the compounds (1) having a group represented by the above (3) or (4), a compound represented by the following formula (6) is preferable.

Wherein, G _N is N (N is an integer of from 1 to 10) shows the sugar chain site of sugar is covalently bonded to a linear or branched;
R ¹ represents a linker moiety represented by —C (═O) —C _n H _2n —Y or —CH ₂ —C _n H _2n —Y. In formula, n shows the integer of 2-10, Preferably it is 3-8, More preferably, it is 3-5. Y represents a group having one or more thiol groups or one or more disulfide groups, preferably one disulfide group.
R ³ represents a group represented by —C (═O) —C _q H _2q —CH ₃ or —CH ₂ —C _q H _2q —CH ₃ . In formula, q shows the integer of 1-20, Preferably it is 10-20, More preferably, it is 13-18.

Specific examples of the compound represented by the formula (6) include compounds represented by the following formulas (7) to (10).

In the formula, _GN is as defined above. Y represents a group having one or two or more thiol groups or one or two or more disulfide groups as described above, and preferably one disulfide group. n shows the integer of 2-10 as above-mentioned, Preferably it is 3-8, More preferably, it is 3-5. q shows the integer of 1-20, Preferably it is 10-20, More preferably, it is 13-18.

Furthermore, the total number (N1) of atoms contained in the main chain of the —COC _n H _2n Y and —CH ₂ C _n H _2n Y, the —CH ₂ O (CO) C _q H _2q CH ₃ and —CH The total number of atoms (N2) contained in the main chain of ₂ OCH ₂ C _q H _2q CH ₃ is preferably N1: N2 = 1: 1.5 to 1: 5, more preferably 1: 2 to 1: It is in the range of 3.
In N1, a plurality of atoms that bond the longest sulfur atom in Y and X are the atoms constituting the main chain, and when Y is a disulfide group, the sulfur atom side close to X is the main chain. The total number N1 does not include a sulfur atom. In N2, a plurality of atoms that bond the R ² terminal and X at the longest are atoms constituting the main chain.

As described above, when such a side chain moiety is included, the detection sensitivity can be significantly improved as compared to the case where no side chain moiety is present.
Further, when the side chain moiety is composed of such carbon number or number of atoms, and the side chain moiety has a length larger than the length of the linker moiety, the glycolipid-containing compound can be added to the substrate or fine particles with a higher degree of orientation. When a biosensor containing the glycolipid-containing compound or a detection reagent containing a glycolipid-containing microparticle is used, even a very small amount of a sugar-binding compound can be detected and quantified with high sensitivity.

  Such a compound represented by the formula (6) or the formulas (7) to (10) is a glyceroglycolipid (sugar as a hydrophilic group, a glyceride moiety as a fat-soluble group, or a glyceroglycolipid present in a large amount on the front side of a cell bilayer membrane. It can be derived from a glycolipid having a diacylglycerol moiety, etc., and is also preferred from the viewpoint of easy availability of raw materials.

  Having such a linker site and side chain site, the glycolipid-containing compound can be immobilized on a substrate or fine particles with high orientation and high density, and a biosensor or sugar containing the glycolipid-containing compound can be used. When a detection reagent containing lipid-containing fine particles is used, even a very small amount of a sugar-binding compound can be detected and quantified with high sensitivity.

Method for producing glycolipid-containing compound
Although there is no limitation in the manufacturing method of the glycolipid containing compound (1) based on this invention, For example, the method of using naturally derived sphingoglycolipid as a starting material as shown in the following process 1 is mentioned.

スフィンゴ糖脂質（１ａ）（式中、Ｇ_Nは式（１）中のＧ_Nと同様であり、Ｒ⁴は−Ｃ_10〜18Ｈ_20〜30−ＣＨ₃であり、Ｒ^'は−Ｃ_10〜20Ｈ_20〜40−ＣＨ₃である。）は、１個以上の糖が直鎖あるいは分岐鎖で共有結合している糖鎖部位と、スフィンゴシン構造またはスフィンゴシン部位に脂肪酸がN-アシル結合したセラミドと呼ばれる脂質を骨格としている。該スフィンゴ糖脂質は、脊椎動物、無脊椎動物、植物などに広く存在する化合物である。 Process 1

During glycosphingolipids (1a) (wherein, G _N is the same as G _N in the formula (1), R ⁴ is _{-C 10~18 H 20~30 -CH 3, R} ' is -C _{10 ˜20} H _20-40 —CH ₃ ) is a fatty acid N-acyl-bonded to a sugar chain site where one or more sugars are covalently bonded in a straight chain or branched chain, and a sphingosine structure or sphingosine site. The skeleton is a lipid called ceramide. The glycosphingolipid is a compound widely present in vertebrates, invertebrates, plants and the like.

  For example, by subjecting the glycosphingolipid (1a) to an enzyme treatment using a known method (J. Biol. Chem., Vol. 250, P. 24370, 1995), the amide bond at the ceramide site is selectively selected. To obtain a compound (2a) (lyso form) which is a single-chain glycolipid in which a sphingosine structure having an amino group or a sphingosine site is a hydrophobic part. The enzyme reaction uses an enzyme capable of specifically acting on glycosphingolipids and sphingomyelin, Sphingolipid ceramide N-deacylase (SCDase, E.C. 3.5.1.69). The enzyme acts on all glycosphingolipids and sphingomyelin.

  The SCDase can efficiently catalyze a condensation reaction and a fatty acid exchange reaction in addition to hydrolysis depending on the pH of the reaction solution and the surfactant concentration (J. Lipid Res., Vol. 38, P. 1923, 1997). Year).

  In the hydrolysis reaction by the enzyme, SCDase is preferably added in an amount of 1 to 5 U with respect to 10 μmol of glycosphingolipid, and preferably in a buffer solution of pH 5.5 to 6.5, for example, sodium taurodeoxycholate 0.5% as a surfactant. It is preferable to carry out at 30 to 40 ° C. in the presence of% to 2.0%. Furthermore, the compound (2a) can be obtained in a high yield by adding a hydrocarbon solvent having 10 to 18 carbon atoms corresponding to 10 times the amount of the reaction solution to the upper layer of the reaction solution.

Next, the compound (3a), which is a linker site capable of being chemically and / or physicochemically fixed to the surface of the metal substrate or metal fine particle, is bonded to the amino group of the obtained compound (2a) by chemical bonding. Compound (4a) can be obtained. Examples of such a compound include thioctic acid represented by HOOC-C _n H _2n Y (3a) (wherein n and Y are the same as n and Y in the formula (1)). , Fatty acids having a thiol group at the end, and fatty acids having a disulfide group.

Further, as the compound (3a) for inducing a linker site, a compound in which a carboxyl group is previously activated with N-hydroxysuccinimide (NHS) or the like may be used.
The reaction of the amino group of the lyso form (2a) and the carboxyl group of the compound (3a) is carried out in a solvent, if necessary, in the presence of a dehydration condensing agent. It is preferable that the usage-amount of a compound (3a) exists in the range of 1-3 equivalent with respect to a lyso body (2a).
Examples of the solvent include N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), organic solvents such as acetonitrile and ethanol, water, and the like. A solvent can be used individually by 1 type or in mixture of 2 or more types.

When a dehydrating condensing agent is used, for example, 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholine salt (DMT-MM), dicyclohexylcarbodiimide (DCC), 1- Examples include ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC or WSC). In addition, an activator such as N-methylmorpholine (NMP) or NHS can be used in combination with the dehydrating condensing agent.
The reaction is preferably carried out at a temperature in the range of 0 ° C. to 40 ° C., preferably for about 1 to 48 hours.

Process 2
As shown in Step 2, the glycolipid-containing compound may further convert the carbon number of the side chain portion by introducing an epoxy group or a hydroxyl group into the double bond of the side chain portion or converting it to an aldehyde. it can.

In Step 2, R ⁵ is —C _n H _2n —Y. R ⁴ is a group represented by -C _p H _2p -CH _3. R ^{4 ′} is a group represented by —C _p H _2p —CH ₃ different from R ⁴ .
G _N , n, and p are as defined above.

In order to introduce an epoxy group into the olefin part, alkaline hydrogen peroxide (Prilezhaev epoxidation reaction), m-chloroperbenzoic acid (MCPBA oxidation), Ti (Oi-Pr) ₄ and diethyl methanol tartrate In the presence of tert-butyl hydroperoxide (Kazuki-Sharpless asymmetric oxidation) and the like.

  To introduce a hydroxyl group into an olefin, an acid such as camphorsulfonic acid (CSA) or p-toluenesulfonic acid (pTsOH), or a base such as sodium hydroxide (NaOH) or potassium hydroxide (KOH) Below, the epoxy group can be cleaved to introduce a hydroxyl group (diol).

Alternatively, it may be reductively cleaved with sodium cyanoborohydride (NaBH ₃ CN). Alternatively, BH ₃ can be allowed to act on the olefin and then treated with hydrogen peroxide to convert it to monool (monoalcohol) (hydroboration).

  Furthermore, when an olefin is ozone-oxidized under an ozone stream, an aldehyde is obtained. A hydroxyl group (diol) obtained by cleaving an epoxy group can be oxidized with periodate to obtain a similar derivative (however, the sugar hydroxyl group is protected with an acyl protecting group such as an acetyl group, for example). There is a need).

  The resulting aldehyde can then be reacted with phosphorus ylide having an arbitrary length of hydrocarbon by Wittig reaction to arbitrarily convert the length of the side chain in formula (5).

Process 3
Furthermore, as shown in Step 3, there is a method using a glyceroglycolipid derived from nature as a starting material.

Wherein, G _N has the same meaning as G _N in the formula ^{(1), R 6, R} 7 are, -C _r H _2r -CH ₃ (wherein independently of one another, r is 1 to 20 of an integer It is shown.)

The compound 6a can be obtained by treating the acyl group part of the compound 5a with ordinary sodium hydroxide, potassium hydroxide, sodium methylate, lithium hydroxide or the like and performing deacylation.
In addition, when a lipase that selectively acts at the 2-position of the acyl moiety is used, it can be converted to compound 6b. Similarly, when a 1,3-specific lipase is allowed to act, it can be converted to compound 6c. These reactions are preferably performed in a buffer solution.

Next, using HOOC-C _n H _2n Y (7a), or this vinyl ester, trichloroacetyl ester (active ester) form, organic solvent (ether, chloroform, toluene, methylene chloride, acetonitrile, THF, DMF, etc.) In particular, when lipase is allowed to act, a transesterification reaction occurs, and the corresponding compounds 8a to 8c can be obtained.

  In addition, the above reaction does not need to protect especially the hydroxyl group in sugar. If necessary, the 6-position of the sugar can be replaced with a silyl-protecting group (t-butyldimethylsilyl group, t-butyldiphenylsilyl group), an ether-based protecting group such as a trityl group, and 4- and 6-positions with isopropylidene. It may be selectively protected with an acetal type protective group such as a group or a benzylidene group, or a levuloyl group.

  The silyl protecting group is tetrabutylammonium fluoride in the presence of acetic acid, and the trityl group can be removed by CSA, p-TsOH, or catalytic hydrogenation. Acetal-based protecting groups can also be removed by acid treatment. When the side chain contains a saturated hydrocarbon group, the benzylidene group can also be deprotected by catalytic hydrogenation. The levuloyl group can be selectively removed with hydrazine-acetic acid. Any of these deprotection conditions can be removed without affecting the acyl group of the glycerol moiety.

Further, when R ^{6 in the} side chain portion has a double bond or the like, another substituent can be introduced in the same manner as described above.

In the glycolipid-containing compound obtained as described above, the sugar chain site can be further converted by an enzymatic reaction as shown in Step 4 below.
Process 4

As the enzyme, glycosyltransferase and sugar hydrolase can be used.
Examples of the glycosyltransferase include sialyltransferase, galactosyltransferase, and the like, respectively, sialic acid at the 3rd or 6th position on the non-reducing end side of galactose or lactose or lactosamine, and galactose at the 4th position of glucose. Can be transferred.

  Examples of the sugar hydrolase include galactosidase, N-acetylhexosaminidase, sialidase, etc., respectively, galactose at the 4-position of glucose, N-acetylglucosamine at glucose and galactose, and sialic acid at galactose, etc. Can be transferred to.

In addition, in the reaction with a transferase, a buffer solution is used, and in the reaction with a hydrolase, in addition to a substrate concentration close to supersaturation, acetonitrile, methanol, THF, and DMF may be used as a co-solvent as necessary.
Under these enzyme reaction conditions, another type of sugar can be introduced without affecting the original sugar chain, depending on the original sugar chain structure and the substrate specificity of the enzyme.

  More specifically, the sugar chain sites (i) to (xvii) exemplified above can be obtained by the following method, for example.

  The ceramide type glycolipids whose sugar chain sites are represented by the formulas (i), (iii), (vii) to (xi) are all commercially available products (Cosmo Bio, Sigma, Seikagaku, Takara Bio, etc.) Can be derived from. These ceramide derivatives can be converted to lyso form by selective hydrolysis with sphingolipid ceramide N-deacylase (EC 3.5.1.69), and after reacting a free amino group with a thiocarboxylic acid such as lipoic acid, Can be fixed to the sensor chip.

The derivative having the formula (ii) at the sugar chain site uses N-acetylgalactosamine as a starting material, and any of its complete acetyl, 1-bromo, 1-trichloroacetimidate, 1-fluoro, etc. After derivatization, natural ceramide can be synthesized into the aglycone part of the sugar using a catalyst such as trimethylsilyl trifluoromethanesulfonate, boron trifluoride etherate, silver perchlorate, or trimethylsilyl silver salt. it can.
Next, the protecting group is deprotected (removed), and the amide bond in the side chain is selectively hydrolyzed by sphingolipid ceramide N-deacylase (EC 3.5.1.69) to convert it into a lyso form in which the amino group is liberated. Alternatively, single-stranded sphingosine may be introduced into any of complete acetyl, 1-bromo, 1-trichloroacetimidate, 1-fluoro and the like by a method similar to the above. In this case, since sphingosine is used as a starting material, a free amino group is present, and hydrolysis with sphingolipid ceramide N-deacylase is unnecessary. In either case, the liberated amino group can be reacted with a thiocarboxylic acid such as lipoic acid and immobilized on the sensor chip.

  N-acetylglucosaminyl ceramide is synthesized by using N-acetylglucosamine as a starting material instead of N-acetylgalactosamine and coupling it with ceramide by a method similar to the derivative having formula (ii). it can. Alternatively, N-acetylglucosaminyl sphingosine can be synthesized by coupling N-acetylglucosamine and sphingosine.

  Next, if galactose is selectively transferred to these 4-positions using galactose transferase, it can be converted into a disaccharide derivative having a sugar chain site (iv). Subsequent conversion is the same as described above. In addition, ceramide and sphingosine may be introduced into disaccharides, which are commercially available N-acetyllactosamine, from the beginning. This method is in accordance with the chemical conversion method described above. In addition, after coupling N-acetyllactosamine disaccharide and ceramide, the amide bond of ceramide is hydrolyzed with the previous sphingolipid ceramide N-deacylase to release the amino group and bind to lipoic acid. And you can make a sensor chip. When sphingosine is used, hydrolysis by this enzyme is unnecessary, and it can be used for a sensor chip after lipoic acid is directly introduced into sphingosine.

  Derivatives having (v) at the sugar moiety can also be prepared by a method similar to the above. That is, after the glycolipid corresponding to (ii) is prepared, galactose is selectively introduced into the 3-position of this (it is preferable to repeat chemical protection and deprotection), or Gal · 1- After chemically synthesizing the disaccharide structure of 3GalNAc, ceramide or sphingosine may be introduced by the glycosylation reaction shown above. As described above, when ceramide is introduced, a hydrolysis process using sphingolipid ceramide N-deacylase is required.

  Derivatives having (vi) at the sugar chain site can be prepared in the same manner as described in the literature, Biomacromolecules, 3, 411-414 (2002).

  The formula (xii) means three of the formulas (xv), (xvi), (xvii), and these are two documents, Chem. Commun., 1998, 2311-2312; ChemBioChem, 4, It can be prepared by the method described in 640-647 (2003).

  Derivatives having the formula (xiii) and formula (xiv) at the sugar chain site can be obtained by selectively using commercially available pNP N-acetylglucosamine and galactose at the 4-position of N-acetylglucosamine using a hydrolase such as galactosidase. These sugar chain sites can be induced by transferring them to disaccharides and then allowing α2,3-sialyltransferase or α2,6-sialyltransferase to act.

<Biosensor>
The biosensor according to the present invention is characterized in that the glycolipid-containing compound is immobilized on the surface of a metal substrate via a linker site. The glycolipid-containing compounds can be used alone or in combination of two or more.

  Examples of the material for the metal substrate include gold, silver, platinum, copper, palladium, and the like. Among these, gold or platinum, and particularly preferably gold is more preferable. The shape of the substrate is not limited, and examples thereof include a plate shape, a tubular shape, and a spherical shape. Of these, a substrate having a plate shape can be preferably used.

  Self-assembled monolayer (SAM: Self-Assembled Monolayer) has a hydrophobic effect on the side chain when immobilizing the glycolipid-containing compound on a metal substrate such as gold, and has high orientation and high density. It is speculated that it will be possible to achieve proper immobilization. This effect is improved when the side chain site has a certain length.

Specifically, when R ² in the side chain moiety is a C _2-20 alkyl group (—C _m H _2m —CH ₃ ), m is 2-20, more preferably 3-19, more preferably 9-19. Particularly preferably, the effect is improved as it becomes 12-17.

Also, if the group R ² is represented by the formula (2), the -C _p H _2p -CH ₃ in the formula (2), p is preferably 1 to 17, more preferably 7 to 17, further Preferably, the effect is improved as it becomes 10-15.

Further, when R ² is a group represented by the formula (3) or (4), in —C _q H _2q —CH ₃ in the formula (3) or (4), q is preferably 1 to 20, More preferably, the effect is improved as it becomes 10 to 20, more preferably 13 to 18.

  The self-organization refers to a desirable structure in which molecules and atoms necessary for forming a specific structure (aggregate) are assembled and arranged by the interaction between those molecules and atoms. Means to form naturally.

  As described above, the side chain moiety and the linker moiety preferably have a certain range of length or relationship, and a sensor with higher sensitivity can be obtained when the length is within the above range.

  Immobilization to the metal substrate is performed by direct covalent bonding or adsorption between the metal surface and a thiol group (-SH) or disulfide group (-SS-) of a linker site having a certain length. It is presumed that a high degree of orientation and high-density fixation are possible due to a synergistic effect by the self-assembly of the side chain sites.

  Furthermore, this self-organization desirably has naturally occurring side chains such as sphingosine structures or sphingosine sites.

  The SAM method is a method in which a compound having a thiol group or a disulfide group is specifically adsorbed with gold or the like and forms SAM by van der Waals force between alkyl chains. Construction of a monomolecular film on the surface of a metal substrate using a glycolipid-containing compound does not require a special apparatus, and can be easily carried out simply by immersing the substrate in a solution of these derivatives. The structure and orientation can be controlled by the immersion conditions (solvent, concentration, temperature, time). The solvent used here is not particularly limited, but is preferably an organic solvent in which the derivative to be immobilized is dissolved.

The binding rate of the glycolipid-containing compound on the surface of the metal substrate is preferably in the range of 0.1 to 10 / nm ² , more preferably 1 to 10 / nm ² . With such a binding ratio, excellent detection sensitivity can be exhibited.
In particular, in the glycolipid-containing compound of the present invention, a side chain site and a linker site are configured with a specific number of atoms. In this case, the side chain site is longer than the linker site, the side chain site takes a folded structure according to the length of the linker site, and the glycolipid-containing compounds are linked to each other by the folded side chain site. It is presumed that it may be effectively bonded to the substrate surface or fine particle surface with an orientation suitable for bonding with the substrate. Thereby, it is presumed that the preferable bond ratio as described above was achieved.

Method for detecting sugar-binding compound (1)
The method for detecting a sugar-binding compound according to the present invention comprises the following steps.
(1) A step of bringing a test compound into contact with the substrate surface of the biosensor,
(2) A step of detecting a sugar-binding compound bound to the substrate.

  Examples of such sugar-binding compounds include sugar-binding proteins, lectins, glycoproteins, toxins, antibodies, surface proteins possessed by bacteria and viruses.

The contact between the biosensor in which the glycolipid-containing compound (1) according to the present invention is immobilized on a metal substrate and the sugar-binding compound is usually preferably performed in the presence of a solvent. In the detection of sugar-binding compounds using the biosensor, it is necessary to select a solvent that does not denature these sugar-binding compounds, and for example, water or a buffer solution is used.
The pH of the solvent is preferably in the range of 6-8, and the temperature is preferably in the range of 0-60 ° C, more preferably in the range of 25-40 ° C.

  The buffer varies depending on the type of the sugar-binding compound. For example, phosphate buffer, acetate buffer, HEPES buffer, or Tris buffer, more preferably HEPES buffer (10 mM, pH: 7.5, NaCl: 0.15). N).

  Hereinafter, among the biosensors according to the present invention, an example of a method for detecting a sugar-binding protein by surface plasmon resonance (SPR) is shown. In the detection method by SPR, a change in the analyte (analyte compound) can be detected without using a labeling substance. This SPR phenomenon uses an optical phenomenon that occurs when surface plasmon is excited at the metal / liquid interface, and a small amount of mass change that occurs on the surface of the sensor chip is detected as an SPR signal. Changes in the binding and dissociation between the molecule and the immobilized substance can be quantitatively obtained.

  For detection of a sugar-binding protein, the temperature is preferably kept constant in a range of 0 to 60 ° C., more preferably 25 to 40 ° C., preferably a phosphate buffer, an acetate buffer, a HEPES buffer, or a Tris buffer. Solution, more preferably HEPES buffer.

  A sugar-binding protein is slowly injected into this. When determining the affinity constant, it is desirable to measure 2 to 8 times by changing the concentration of the sugar-binding protein. The reaction time is preferably about 3 to 30 minutes for both the binding and dissociation reactions, but the measurement time may be changed depending on the intended interaction.

Further, the flow rate of the buffer is preferably 10 to 30 μL / min, and the amount of the sample is preferably about 100 to 300 μL per measurement of nM to mM. Affinity constants takes measured concentration of 2-8 points, the sensorgram at each concentration to calculate the affinity constant (k _a and k _d) to create a Scatchard plot the equilibrium state.

Such a surface plasmon resonance biosensor using the biosensor of the present invention can detect even a very small amount (<0.01 nM) of a sugar-binding compound with high sensitivity without using pretreatment or a labeling substance. Biosensors and binding rate constant of sugar-binding compound (k _a), binding constant (K _A) and dissociation constants (K _D), a very high value when compared to having no side chain site. For example, the binding rate constant and dissociation constant of those having sphingosine bases are about 1000 times the values of those having no sphingosine structure or sphingosine site, and have a very strong binding force.

  Such effects are achieved by highly oriented and high-density immobilization by the sphingosine structure or the self-organizing function of the side chain site such as the sphingosine site, and multiple adjacent sugar chain sites are efficiently sugar-binding compounds. It is presumed to be strongly coupled.

<Glycolipid-containing fine particles, sugar-binding compound detection reagent>
Glycolipid-containing microparticles The glycolipid-containing microparticles according to the present invention are microparticles in which the glycolipid-containing compound according to the present invention is fixed to the surface of the metal microparticles via a linker site. The glycolipid-containing compounds can be used alone or in combination of two or more.

  The material for the metal fine particles is preferably gold, silver, platinum, copper, palladium, etc. Among these, gold or platinum is more preferable, and gold is particularly preferable.

  Such metal fine particles have an average particle diameter of preferably 1 to 100 nm, more preferably 1 to 50 nm. The metal fine particles preferably have a monodispersed particle size. By using metal fine particles in this range, a visually detectable signal color (orange to red) can be obtained, dispersion stability in the solvent can be improved, and detection time can be shortened with high sensitivity. it can.

  The metal fine particles used in the present invention can be produced by a known method. For example, gold fine particles can be easily produced in various sizes by using the method of Frens (Nature Phys. Sci., Vol. 241, P. 20, 1973).

Specifically, for example, a method for producing gold fine particles can be performed by heating and boiling a gold chloride solution and then mixing with a sodium citrate solution to reduce gold chloride. As soon as the two solutions are mixed, the boiling solution turns pale blue and nucleation begins. Then, immediately, the color changes from blue to red to produce monodispersed fine particles, and the reduction of gold chloride is completed by further boiling.
The particle diameter of the obtained fine particles can be adjusted by changing the concentration of the sodium citrate solution. It is preferable that such gold fine particles can be dispersed in a solution, and the state is not particularly limited, but it is preferable that the gold fine particles exist in a stable dispersed state. For example, a colloidal state (dispersed colloidal state) is more preferable.

  As a method for immobilizing the glycolipid-containing compound of the present invention on metal fine particles, a method for immobilizing a substance related to an immune reaction typified by an antigen-antibody reaction (for example, J. Histochem. Cytochem., Vol. 30, P. .691, 1982), it is possible to apply a method in which a glycolipid-containing compound is immobilized on a sensor chip according to the present invention. That is, the solution in which the metal fine particles are dispersed and the glycolipid-containing compound solution are mixed for 5 minutes or more. At this time, if a sufficient amount of the glycolipid-containing compound is fixed to the metal fine particles, the metal colloid is stably dispersed, and glycolipid-containing fine particles can be obtained. Here, the binding ratio of the glycolipid-containing compound on the surface of the metal fine particles can be controlled by the concentration of the glycolipid-containing compound in the solution and other conditions (for example, solvent, temperature, time, etc.).

Usually, the binding rate of the glycolipid-containing compound on the surface of the metal fine particles is preferably in the range of 0.1 to 10 particles / nm ² , more preferably 1 to 10 particles / nm ² . With such a binding ratio, excellent detection sensitivity can be exhibited.
In particular, in the glycolipid-containing compound of the present invention, a side chain site and a linker site are configured with a specific number of atoms. In this case, the side chain site is longer than the linker site, the side chain site takes a folded structure according to the length of the linker site, and the glycolipid-containing compounds are linked to each other by the folded side chain site. It is presumed that it may be effectively bonded to the substrate surface or fine particle surface with an orientation suitable for bonding with the substrate. Thereby, it is presumed that the preferable bond ratio as described above was achieved.

Further, such glycolipid-containing fine particles are preferably stored in a solution form from the viewpoint of storage stability of the glycolipid-containing fine particles.
In this case, the fine particles glycolipid-containing compounds of the present invention is bonded, in ^{R 1, -C (= O)} -C n H 2n -Y or linker moiety represented by -CH ₂ -C _n H _2n -Y However, as n is preferably an integer of 2 to 10, more preferably 3 to 8, particularly preferably 3 to 5, the length of the linker site is within a specific range, so that the dispersion stability in the solution is improved. Very good. Therefore, the reagent containing the fine particles to which the glycolipid-containing compound of the present invention is bound is particularly excellent in use in a solution.

  The immobilization of the glycolipid-containing compound on the metal fine particles is aimed at high orientation and high-density immobilization of the glycolipid-containing compound, so that the necessary and sufficient amount of the glycolipid-containing compound is mixed and immobilized. However, when an excessive amount of the glycolipid-containing compound is mixed, it is necessary to remove the glycolipid-containing compound from the mixed solution.

For example, in the case of gold fine particles, the optimal mixing amount of the glycolipid-containing compound is determined based on the dispersion stability of the dispersion solution of the gold fine particles to which the glycolipid-containing compound is immobilized, and the relationship between the addition amount of the sugar-binding compound and the color change. Can be determined.
That is, when the amount of the added compound is less than the optimum amount, it does not reach a sufficient amount for self-organization of the glycolipid-containing compound, so that the hydrophobicity of the gold fine particle surface increases, the dispersion stability decreases, and the color tone Changes and aggregation-precipitation occurs.
In addition, when the added compound is more than the optimum amount, the aggregation reaction is inhibited by the compound added excessively after the sugar-binding compound is added, and the color tone change becomes small. Therefore, optimal immobilization can be performed by controlling the color tone within a range in which color tone change and aggregation do not occur and when a sugar-binding compound is added.

Glycolipid-containing compound detection reagent Glycolipid-containing microparticles can be obtained by immobilizing the glycolipid-containing compound to the metal microparticles, but the solution containing the glycolipid-containing microparticles is used as it is to detect the sugar-binding compound of the present invention. It can be used as a reagent.
For this reason, it is necessary for the solvent used for the immobilization that the sugar-binding compound to be detected is not denatured, and for example, water or a buffer solution is used.
The pH of the solvent is preferably in the range of 6-8, and the temperature is preferably in the range of 0-60 ° C, more preferably in the range of 25-40 ° C.

  The buffer varies depending on the type of the sugar-binding compound. For example, phosphate buffer, acetate buffer, HEPES buffer, or Tris buffer, more preferably HEPES buffer (10 mM, pH: 7.5, NaCl: 0.15). N).

  In this way, a reagent containing the glycolipid-containing fine particles in the solvent is obtained with the synthesis of the glycolipid-containing fine particles. In such a sugar-binding compound detection reagent, the glycolipid-containing fine particles are preferably present in the form of a colloid in the solution, and more preferably the colloid is present in a dispersed state.

For example, in the case of gold colloid, an equal amount of 10% sodium chloride aqueous solution is added to the mixed solution, and if there is no change in the color of the gold colloid, the confirmation is made as to whether the metal colloid of the glycolipid-containing fine particles has been stabilized. Can be certified.
The concentration of the glycolipid-containing fine particles in the sugar-binding compound detection reagent is preferably 1.0 × 10 ^{5 to} 1.0 × 10 ¹⁵ particles / L, more preferably 1.0 × 10 ^{7 to} 1.0 × 10. The range is ¹³ / L.
Within such a concentration range, the colloid of the glycolipid-containing fine particles is dispersed and stably present, so that the storage stability can be improved and the detection sensitivity of the sugar-binding compound can be improved.

Method for detecting sugar-binding compound (2)
The method for detecting a sugar-binding compound according to the present invention comprises the following steps.
(1) adding a test compound to the sugar-binding compound detection reagent and bringing it into contact with the glycolipid-containing microparticles;
(2) A step of detecting a sugar-binding compound bound to the glycolipid-containing fine particles.

As a means for detecting a sugar-binding compound bound to a glycolipid-containing microparticle, for example, in a colloidal solution of the glycolipid-containing microparticle, the sugar-binding compound is detected from a change in color when the glycolipid-containing microparticle binds to the sugar-binding compound. The method of performing detection of this is mentioned.
Specifically, for example, in the case of gold fine particles, the solution shows a red color in a state where the colloidal particles of the glycolipid-containing fine particles are dispersed in a state where the sugar-binding compound is not bonded to the glycolipid-containing fine particles. When the active compound is bonded, the glycolipid-containing fine particles are aggregated and the red color fades or changes to colorless and transparent.

  In the case of ricin toxin, the test compound may be pretreated and used as necessary. Whether the test compound (containing ricin toxin) is provided as a powder (white powder) or supplied as a solution cannot be uniquely determined, but in either case, the test compound is directly The test compound may be reacted with the described glycolipid-containing microparticles, or the test compound may be treated with a reducing agent such as mercaptoethanol, dithiothreitol, dithioerythritol and used as a sample.

  The amount of these reducing agents to be used may be equivalent (1 molar equivalent) or excessive (2 molar equivalents to 100 molar equivalents) with respect to the test compound, but as little as possible in consideration of the stability of the glycolipid-containing fine particles. It is desirable to use a reducing agent of, preferably 1 to 10 molar equivalents. The reaction time is preferably 1 minute to 24 hours, more preferably 2 minutes to 12 hours. The reaction temperature is preferably 0 ° C to 60 ° C, more preferably 4 ° C to 40 ° C.

  As a method for measuring a color change, it is possible to easily semi-quantify the presence of a measurement target substance based on the result of visual observation.

It is also possible to detect a change in color tone with high sensitivity using a spectrophotometer. In this case, the wavelength for measuring the color tone change is not particularly limited as long as the color tone change can be measured. For example, in the case of gold fine particles, the measurement is performed in the vicinity of the maximum absorption wavelength of 500 to 550 nm of the gold fine particles. This is desirable because the sensitivity can be increased. It goes without saying that highly accurate detection is possible by measuring absorbance at multiple wavelengths.
Examples of apparatuses that can measure color change include a colorimeter, a spectrophotometer, a microplate reader, and a biochemical automatic analyzer.

  By measuring such a change in color tone, the target compound can be detected with high sensitivity. For example, as shown in Examples, optimized glycolipid-containing fine particles (in the formula (1), the sugar chain portion is the formula (i), n is 4 and X is a 5-membered disulfide group, m No. 12 and gold fine particles (average particle diameter 20 nm), and using a lysine 120 as a sugar-binding compound visually from the color tone change of the gold fine particle dispersion, its detection limit concentration was determined. The detection limit was 1 to 5 μg / mL, and the lethal dose of lysine was 150 μg / person, so that it was predicted to be extremely sensitive. In addition, when the inhibition test was also evaluated in the same manner, the color change due to these external effects did not occur in distilled water, tap water, and rainwater, and sugar-binding compounds present in the environment could be detected well. .

〔化合物（１ｂ）の合成〕
Galactosyl-ceramide 10.0mg（13.7μmol）（SIGMA社製）を1.0%タウロデオキシコール酸ナトリウムの50mM酢酸緩衝液（pH:6.0）10mLに溶かし500mL梨型フラスコに入れた。次にスフィンゴ脂質セラミドN-デアシラーゼ（SCDase）1.5U（タカラバイオ社製）を加え、ｎ-デカン100mLを反応液と混ざらないように静かに加えて37℃で24時間反応させた後、さらにSCDase0.5Uを加えて37℃で24時間反応させた。上層部であるｎ-デカンを取り除いた後、反応液を沸騰した水浴中で3分間熱した。さらに、反応液はフィルターで濾過した後、濃縮した。
ODSカラムクロマトグラフィーで精製した。その結果、化合物（１ｂ）を収率64%（4.0mg）で得た。
¹H-NMR(400MHz, DMF-d7=8.028ppm):δ5.645(ddd, -CH(OH)-CH=CH-CH₂-, J=6.4Hz, 6.8Hz, 15.6Hz), 5.544(dd, -CH(OH)-CH=CH-CH₂-, J=7.2Hz, 15.6Hz), 4.426(brd, H-4, J=4.0Hz), 4.385(t, H-2, J=7.2Hz), 4.171(d, H-1, J=7.2Hz), 3.833(q, Gal-O-CH ₂-CH(NH₂)-), 1.284(brs, -(CH ₂)-), 0.878(t, -CH₂-CH ₃).
ESI(+)-MS m/z=462.4 (M+H)⁺
ESI(-)-MS m/z=460.3(M-H)^-, 506.3(M+HCOO)^- EXAMPLES Examples and comparative examples of the present invention will be described below in detail, but the present invention is not limited by these.
Synthesis of glycolipid-containing compound [Example 1]

[Synthesis of Compound (1b)]
Galactosyl-ceramide 10.0 mg (13.7 μmol) (manufactured by SIGMA) was dissolved in 10 mL of 50% acetic acid buffer (pH: 6.0) of 1.0% sodium taurodeoxycholate and placed in a 500 mL pear-shaped flask. Next, sphingolipid ceramide N-deacylase (SCDase) 1.5U (manufactured by Takara Bio Inc.) is added, and 100 mL of n-decane is gently added so as not to mix with the reaction solution, followed by reaction at 37 ° C. for 24 hours, and further SCDase 0 .5U was added and reacted at 37 ° C. for 24 hours. After removing n-decane as the upper layer, the reaction solution was heated in a boiling water bath for 3 minutes. Further, the reaction solution was filtered and then concentrated.
Purified by ODS column chromatography. As a result, compound (1b) was obtained with a yield of 64% (4.0 mg).
¹ H-NMR (400 MHz, DMF-d7 = 8.028 ppm): δ 5.645 (ddd, -CH (OH) -CH = C H -CH _2- , J = 6.4 Hz, 6.8 Hz, 15.6 Hz), 5.544 ( dd, -CH (OH) -C H = CH-CH _2- , J = 7.2Hz, 15.6Hz), 4.426 (brd, H-4, J = 4.0Hz), 4.385 (t, H-2, J = 7.2Hz), 4.171 (d, H-1, J = 7.2Hz), 3.833 (q, Gal-OC H ₂ -CH (NH ₂ )-), 1.284 (brs,-(C H ₂ )-), 0.878 (t, -CH ₂ -C H ₃ ).
ESI (+)-MS m / z = 462.4 (M + H) ⁺
ESI (-)-MS m / z = 460.3 (MH) ^- , 506.3 (M + HCOO) ^-

[Synthesis of Compound (2b)]
Compound (1b) 3.7 mg (8.0 μmol) and thioctic acid 2.0 mg (9.7 μmol) were dissolved in 3 mL of ethanol and stirred with 4- (4,6-dimethoxy-1,3,5-triazin-2-yl)- 2 mL of an ethanol solution containing 2.7 mg (9.8 μmol) of 4-methylmorpholine salt (DMT-MM) was added and reacted at room temperature for 12 hours. The reaction solution was concentrated and then purified by ODS column chromatography. As a result, compound (2b) was obtained in a yield of 76% (3.9 mg).
¹ H-NMR (400 MHz, DMF-d7 = 8.028 ppm): δ 7.466 (d, -N H -CO), 5.631 (ddd, -CH (OH) -CH = C H -CH _2- , J = 6.8 Hz, 7.2Hz, 15.2Hz), 5.500 (dd, -CH (OH) -C H = CH-CH _2- , J = 6.8Hz, 15.2Hz), 4.187 (d, H-1, J = 7.2Hz) , 3.28-3.12 (m, N-thioctyl), 2.33, 2.01, 1.84, 1.75, 1.55, 1.43 (6xm, five of six are N-thioctyl), 1.113 (brs,-(C H ₂ )-), 0.707 ( t, -CH ₂ -C H ₃ ).
ESI (+)-MS m / z = 650.1 (M + H) ⁺
ESI (-)-MS m / z = 648.1 (MH) ^- , 694.2 (M + HCOO) ^-

〔化合物（２ｃ）の合成〕
実施例１の化合物（１ｂ）の合成と同様な手法で合成した化合物（１ｃ）10.0mg（16.0μmol）をDMF5mLに溶かし、撹拌しながらあらかじめN-ヒドロキシこはく酸イミド、ジシクロヘキシルカルボジイミドで活性化させたチオクト酸10.0mg（33.0μmol）を含むDMF溶液2mLを加え室温で24時間反応させた。反応液は濃縮した後、ODSカラムクロマトグラフィーで精製した。その結果、化合物（２ｃ）を収率71％（9.2mg）で得た。
^１H-NMR(600MHz, MeOH-d4):δ5.642(dt, -CH(OH)-CH=CH-CH₂-, J=6.6Hz, 15.4Hz), 5.444(dd, -CH(OH)-CH=CH-CH₂-, J=7.7Hz, 15.4Hz), 4.354(d, Gal H-1, J=7.7Hz), 4.302(d, Glc H-1, J=7.7Hz), 4.16-4.14(m, -O-CH ₂-CH(NH-)-, 4.073(t, -CH(NH-)-CH(OH)-, J=7.7Hz), 3.98-3.97(m, -CH(NH-)-CH(OH)-), 3.898(brd, Glc H-6, J=10.6Hz), 3.833(dd, Glc H-6’, J=4.4Hz, 12.1Hz), 3.806(d, Gal H-4, J=3.3Hz), 3.770(dd, Gal H-6, J=7.5Hz, 11.6Hz), 3.692(dd, Gal H-6’, J=4.6Hz, 11.6Hz), 3.60-3.51(m, Glc H-3, H-4, Gal H-2, H-5, -Glc-O-CH ₂-CH(NH-)-, CH₂-CH(S-)- ), 3.471(dd, Gal H-3, J=3.3Hz, 9.5Hz), 3.43-3.40(m, Glc H-5), 3.28(overlap, Glc H-2), 3.19-3.15(m, -CH₂-CH ₂-S-), 3.12-3.07(m, -CH₂-CH ₂-S-), 2.49-2.43(m, -CH ₂-CH₂-S-), 2.21-2.16(m, -C(O)-CH ₂-CH₂-), 2.04-2.00(br, -CH=CH-CH ₂-CH₂-), 1.91-1.85(m, -CH ₂-CH₂-S-), 1.75-1.57, 1.50-1.41(2xm, N-thioctyl), 1.39-1.25 (m, -(CH ₂)-), 0.891(t, -CH₂-CH ₃, 7.0Hz).
ESI(+)-MS m/z=812.6(M+H)⁺
ESI(-)-MS m/z=810.7(M-H)^-, 856.6(M+HCOO)^- [Example 2]

[Synthesis of Compound (2c)]
10.0 mg (16.0 μmol) of the compound (1c) synthesized by the same method as the synthesis of the compound (1b) of Example 1 was dissolved in 5 mL of DMF, and activated with N-hydroxysuccinimide and dicyclohexylcarbodiimide in advance while stirring. 2 mL of DMF solution containing 10.0 mg (33.0 μmol) of thioctic acid was added and reacted at room temperature for 24 hours. The reaction solution was concentrated and then purified by ODS column chromatography. As a result, compound (2c) was obtained with a yield of 71% (9.2 mg).
¹ H-NMR (600 MHz, MeOH-d4): δ 5.642 (dt, -CH (OH) -CH = C H -CH _2- , J = 6.6 Hz, 15.4 Hz), 5.444 (dd, -CH (OH ) -C H = CH-CH _2- , J = 7.7Hz, 15.4Hz), 4.354 (d, Gal H-1, J = 7.7Hz), 4.302 (d, Glc H-1, J = 7.7Hz), 4.16-4.14 (m, -OC H ₂ -CH (NH-)-, 4.073 (t, -CH (NH-)-C H (OH)-, J = 7.7Hz), 3.98-3.97 (m, -C H (NH-)-CH (OH)-), 3.898 (brd, Glc H-6, J = 10.6Hz), 3.833 (dd, Glc H-6 ', J = 4.4Hz, 12.1Hz), 3.806 (d , Gal H-4, J = 3.3Hz), 3.770 (dd, Gal H-6, J = 7.5Hz, 11.6Hz), 3.692 (dd, Gal H-6 ', J = 4.6Hz, 11.6Hz), 3.60 -3.51 (m, Glc H-3, H-4, Gal H-2, H-5, -Glc-OC H ₂ -CH (NH-)-, CH ₂ -C H (S-)-), 3.471 (dd, Gal H-3, J = 3.3Hz, 9.5Hz), 3.43-3.40 (m, Glc H-5), 3.28 (overlap, Glc H-2), 3.19-3.15 (m, -CH ₂ -C H ₂ -S-), 3.12-3.07 (m, -CH ₂ -C H ₂ -S-), 2.49-2.43 (m, -C H ₂ -CH ₂ -S-), 2.21-2.16 (m,- C (O) -C H ₂ -CH _2- ), 2.04-2.00 (br, -CH = CH-C H ₂ -CH _2- ), 1.91-1.85 (m, -C H ₂ -CH ₂ -S- ), 1.75-1.57, 1.50-1.41 (2xm, N-thioctyl), 1.39-1.25 (m,-(C H ₂ )-), 0.891 (t, -CH ₂ -C H ₃ , 7.0Hz).
ESI (+)-MS m / z = 812.6 (M + H) ⁺
ESI (-)-MS m / z = 810.7 (MH) ^- , 856.6 (M + HCOO) ^-

〔化合物（１ｄ）の合成〕
Ganglioside GT1b 5.0mg（2.3μmol）を1.0％タウロデオキシコール酸ナトリウムの50mM酢酸緩衝液（pH：6.0）10mLに溶かし500mL 梨型フラスコに入れた。次にSCDase 1Uを加え、ｎ-デカン100mLを反応液と混ざらないように静かに加えて37℃で24時間反応させた。さらにSCDase 0.5Uを加えて37℃で24時間反応させた。上層部のｎ-デカンを取り除いた後、反応液を沸騰した水浴中で3分間熱した。反応液はフィルターで濾過したのち、ODSカラムクロマトグラフィーで精製した結果、化合物（１ｄ）を収率70％（3.1 mg）で得た。
^１H-NMR(600MHz, MeOH-d4):δ5.642(dt, -CH(OH)-CH=CH-CH₂-, J=6.6Hz, 15.4Hz), 5.444(dd, -CH(OH)-CH=CH-CH₂-, J=7.7Hz, 15.4Hz), 2.80-2.77(m, 3He of NeuAc), 2.04-2.00(overlap, -CH=CH-CH ₂-CH₂-, NHAc), 1.94-1.85(m, 3Ha of NeuAc), 1.36-1.25 (m, -(CH ₂)-), 0.892(t, -CH₂-CH ₃, 7.0Hz).
また得られた化合物（１ｄ）の¹Ｈ-ＮＭＲスペクトル（5.0ppm〜3.0ppm）を図１に示す。
ESI(-)-MS m/z=944.5(M-2H)^2- Example 3

[Synthesis of Compound (1d)]
Ganglioside GT1b 5.0 mg (2.3 μmol) was dissolved in 10 mL of 50% acetic acid buffer (pH: 6.0) of 1.0% sodium taurodeoxycholate and placed in a 500 mL pear-shaped flask. Next, SCDase 1U was added, 100 mL of n-decane was gently added so as not to mix with the reaction solution, and the mixture was reacted at 37 ° C. for 24 hours. Further, SCDase 0.5U was added and reacted at 37 ° C. for 24 hours. After removing the upper n-decane, the reaction was heated in a boiling water bath for 3 minutes. The reaction solution was filtered through a filter and purified by ODS column chromatography. As a result, compound (1d) was obtained in a yield of 70% (3.1 mg).
¹ H-NMR (600 MHz, MeOH-d4): δ 5.642 (dt, -CH (OH) -CH = C H -CH _2- , J = 6.6 Hz, 15.4 Hz), 5.444 (dd, -CH (OH ) -C H = CH-CH _2- , J = 7.7Hz, 15.4Hz), 2.80-2.77 (m, 3He of NeuAc), 2.04-2.00 (overlap, -CH = CH-C H ₂ -CH _2- , NHAc), 1.94-1.85 (m, 3Ha of NeuAc), 1.36-1.25 (m,-(C H ₂ )-), 0.892 (t, -CH ₂ -C H ₃ , 7.0Hz).
The ¹ H-NMR spectrum (5.0 ppm to 3.0 ppm) of the obtained compound (1d) is shown in FIG.
ESI (-)-MS m / z = 944.5 (M-2H) ^2-

[Synthesis of Compound (2d)]
N, N-dimethylformamide solution containing 2.0 mg (6.6 μmol) of thioctic acid, in which 3.0 mg (1.6 μmol) of compound (1d) is dissolved in 3 mL of DMF and pre-activated with N-hydroxysuccinimide and dicyclohexylcarbodiimide with stirring 1 mL was added and reacted at room temperature for 24 hours. The reaction solution was concentrated and then purified by ODS column chromatography. As a result, compound (2d) was obtained with a yield of 54% (1.8 mg).
¹ H-NMR (600 MHz, MeOH-d4): δ 5.645 (dt, -CH (OH) -CH = C H -CH _2- , J = 6.6 Hz, 15.4 Hz), 5.444 (dd, -CH (OH ) -C H = CH-CH _2- , J = 7.7Hz, 15.4Hz), 3.19-3.15 (m, -CH ₂ -C H ₂ -S-), 3.11-3.07 (m, -CH ₂ -C H ₂ -S-), 2.85-2.80 (m, 3He of NeuAc), 2.48-2.43 (m, -C H ₂ -CH ₂ -S-), 2.22-2.17 (m, -C (O) -C H ₂ -CH _2- ), 2.05-1.97 (overlap, -CH = CH-C H ₂ -CH _2- , NHAc), 1.94-1.85 (overlap, -C H ₂ -CH ₂ -S-, 3Ha of NeuAc), 1.75-1.58, 1.50-1.41 (2xm, N-thioctyl), 1.35-1.25 (m,-(C H ₂ )-), 0.892 (t, -CH ₂ -C H ₃ , 7.0Hz).
In addition, FIG. 2 shows the ¹ H-NMR spectrum (5.0 ppm to 3.0 ppm) of the obtained compound (2d).
ESI (-)-MS m / z = 692.1 (M-3H) ^3- , 1039.0 (M-2H) ^2-
[Comparative Example 1]

〔化合物（３ｂ）の合成〕
p-Aminophenylβ-D-galactopyranoside 10mg（36.9μmol）およびチオクト酸9.1mg（44.1μmol）をメタノール5mLに溶かし、撹拌しながらDMT-MM 12.2mg（44.1μmol）を含むメタノール溶液5mLを加え室温で12時間反応させた。反応液は濃縮した後、ODSカラムクロマトグラフィーで精製した。その結果、化合物（３ｂ）を収率82％（13.9mg）で得た。
〔比較例２〕

[Synthesis of Compound (3b)]
Dissolve 10 mg (36.9 μmol) of p-Aminophenylβ-D-galactopyranoside and 9.1 mg (44.1 μmol) of thioctic acid in 5 mL of methanol, add 5 mL of methanol solution containing 12.2 mg (44.1 μmol) of DMT-MM with stirring, and then at room temperature for 12 hours. Reacted. The reaction solution was concentrated and then purified by ODS column chromatography. As a result, Compound (3b) was obtained with a yield of 82% (13.9 mg).
[Comparative Example 2]

〔化合物（３ｃ）の合成〕
p-Aminophenyl D-lactoside 10mg（23.1μmol）をDMF3mLに溶かし、撹拌しながらあらかじめN-ヒドロキシこはく酸イミド、ジシクロヘキシルカルボジイミドで活性化させたチオクト酸10.0mg（33.0μmol）を含むDMF溶液2mLを加え室温で24時間反応させた。反応液は濃縮した後、ODSカラムクロマトグラフィーで精製した。その結果、化合物（３ｃ）を収率86％（12.3mg）で得た。
〔比較例３〕

[Synthesis of Compound (3c)]
Dissolve 10 mg (23.1 μmol) of p-Aminophenyl D-lactoside in 3 mL of DMF, add 2 mL of DMF solution containing 10.0 mg (33.0 μmol) of thioctic acid previously activated with N-hydroxysuccinimide and dicyclohexylcarbodiimide while stirring. For 24 hours. The reaction solution was concentrated and then purified by ODS column chromatography. As a result, compound (3c) was obtained with a yield of 86% (12.3 mg).
[Comparative Example 3]

〔化合物（４ｂ）の合成〕
化合物（１ｂ）10.0mg（21.7μmol）およびメタノールから再結晶により精製した16-Mercaptohexadecanoic acid（Aldrich社製）6.3mg（21.7μmol）をメタノール3mLに溶かし、撹拌しながら4-(4,6-ジメトキシ-1,3,5-トリアジン-2-イル)-4-メチルモルホリン塩（DMT-MM）7.5mg（27.0μmol）を含むメタノール溶液2mLを加え室温で24時間反応させた。反応液は濃縮した後、ODSカラムクロマトグラフィーで精製した。その結果、化合物（４ｂ）を収率42%（6.8mg）で得た。

[Synthesis of Compound (4b)]
Compound (1b) 10.0 mg (21.7 μmol) and 6.3 mg (21.7 μmol) of 16-Mercaptohexadecanoic acid (Aldrich) purified by recrystallization from methanol are dissolved in 3 mL of methanol, and 4- (4,6-dimethoxy is stirred. 2 mL of a methanol solution containing 7.5 mg (27.0 μmol) of -1,3,5-triazin-2-yl) -4-methylmorpholine salt (DMT-MM) was added and reacted at room temperature for 24 hours. The reaction solution was concentrated and then purified by ODS column chromatography. As a result, compound (4b) was obtained with a yield of 42% (6.8 mg).

Detection of lysine 120 by SPR sensor (creation of sensor chip)
A gold substrate of SIA kit Au commercially available from Biacore was washed with an ozone cleaner and immersed in 2 mL of a methanol solution in which 100 nmol of the compound (2b) synthesized in Example 1 was dissolved at room temperature for 24 hours. Then, it wash | cleaned 3 times in order of methanol and water, and created the sensor chip (1). Further, using the compound (2c) of Example 2, the compound (3b) of Comparative Example 1, the compound (3c) of Comparative Example 2, and the compound (4b) of Comparative Example 3 in the same manner as above, respectively, (2) A sensor chip (3), a sensor chip (4), and a sensor chip (5) were prepared.

(SPR measurement)
In order to measure the specific adsorption property of the sugar-binding protein to the obtained sensor chip (1), the sensor chip (1) on which the compound (2b) of Example 1 was immobilized was replaced with a commercially available surface plasmon resonance biosensor ( (Biacore 2000, manufactured by Biacore) and a HEPES (10 mM, pH: 7.5, NaCl: 0.15 M) buffer solution was allowed to flow at a set temperature of 25 ° C. and a flow rate of 10 μL / min. It was confirmed that the sensorgram was stable before measurement.
As the sugar-binding protein, ricin 120 (Ricinus communis agglutinin 120, manufactured by Wako Pure Chemical Industries, Ltd.) was used, and it was prepared at 1000 ng / mL (8.3 nM) with HEPES (10 mM, pH: 7.5, NaCl: 0.15 M) buffer. The ricin 120 solution was injected at 100 μL at a flow rate of 10 μL / min.
Similarly, a sensor chip (2) in which the compound (2c) of Example 2, the compound (3b) of Comparative Example 1, the compound (3c) of Comparative Example 2, and the compound (4b) of Comparative Example 3 were immobilized, respectively. Sensor chip (3), sensor chip (4), and sensor chip (5) were also measured. As a result, as shown in FIGS. 3 and 4, the detection intensity of lysine 120 was 1214 RU in Example 1, 420 RU in Comparative Example 1, 1640 RU in Example 2, 630 RU in Comparative Example 2, and 957 RU in Comparative Example 3. there were.
Note that “RU” is an abbreviation of “Resonance unit” and means that when a substance is adsorbed, adhered, or immobilized on the substrate surface, the value increases in accordance with the amount. 1RU = about 1 pg / mm ² (Biacore).

  From these results, the sensor chip (1-5) of Example 1, Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3 can detect lysine 120, but Example 1 having a sphingosine structure or sphingosine site. In Example 2, it was confirmed that an extremely strong signal was obtained as compared with the results of Comparative Examples 1 and 2. This is presumed that the sugar-binding compound was detected with high sensitivity by the sphingosine structure or sphingosine site which is the side chain site of the compounds (2b) and (2c). In addition, when comparing the sensor chip (1) of Example 1 and the sensor chip (5) of Comparative Example 3, Example 1 having a lipoic acid having a disulfide group and an alkyl chain length of n = 5 in Example 1 has a thiol group. It was found that the sensitivity was much higher than that of Comparative Example 3 having a long-chain alkyl group n = 15.

(SPR measurement at various concentrations of lysine 120)
The sensor chip (1) on which the compound (2b) of Example 1 was immobilized was mounted on a surface plasmon resonance biosensor (Biacore 2000, manufactured by Biacore) in the same manner as described above, and the setting temperature was 25 ° C. and the flow rate was 10 μL / min. HEPES (10 mM, pH: 7.5, NaCl: 0.15 M) buffer was run. It was confirmed that the sensorgram was stable before measurement.
Measurement is performed using ricin 120 (Ricinus communis agglutinin 120, manufactured by Wako Pure Chemical Industries, Ltd.) and 10000 ng / mL (83 nM) and 1000 ng / mL (8.3 nM) in HEPES (10 mM, pH: 7.5, NaCl: 0.15 M) buffer, respectively. , 100 ng / mL (0.83 nM) and 10 ng / mL (0.083 nM). The ricin 120 solution was injected at 100 μL at a flow rate of 10 μL / min. Similarly, the sensor chip (2) on which the compound (2c) of Example 2 was immobilized was also measured.

  As a result, as shown in FIG. 5 and FIG. 6, a good sensorgram was obtained at any concentration. The detection intensity of lysine 120 at each concentration was 2536RU, 1214RU, 423RU and 267RU in Example 1, and Examples 2 was 2352RU, 1640RU, 494RU and 228RU. From this result, it was confirmed that even a very small amount (<0.01 nM) of lysine 120 can be detected with high sensitivity without using pretreatment or a labeling substance.

(Calculation of coupling constant)
From the SPR measurement results, the optimum conditions for obtaining the coupling constant were estimated. That is, the sensor chip (1) on which the compound (2b) of Example 1 was immobilized was attached to a surface plasmon resonance biosensor, and HEPES (10 mM, pH: 7.5, NaCl: at a set temperature of 25 ° C. and a flow rate of 30 μL / min. 0.15M) buffer was run. It was confirmed that the sensorgram was stable before measurement. The binding constant was ricin 120 (Ricinus communis agglutinin 120, manufactured by Wako Pure Chemical Industries, Ltd.) and 1000 ng / mL (8.3 nM) and 750 ng / mL (6.3 nM) in HEPES (10 mM, pH: 7.5, NaCl: 0.15 M) buffer, respectively. ), 500 ng / mL (4.2 nM) and 100 ng / mL (0.83 nM). 100 μL of lysine 120 solution was injected at 30 μL / min.

  Further, the sensor chip (2), the sensor chip in which the compound (2c) of Example 2, the compound (3b) of Comparative Example 1, the compound (3c) of Comparative Example 2, and the compound (4b) of Comparative Example 3 were immobilized, respectively. (3) The sensor chip (4) and the sensor chip (5) were also processed in the same manner, but the sensor chip (3) and the sensor chip (4) have very small SPR signals, so The concentration was measured at 1000 times the amount.

As a result of obtaining the 1: 1 reaction binding constant from the obtained sensorgram, as shown in Table 1, in Example 1, Example 2 and Comparative Example 3 having a sphingosine structure or a sphingosine site, Comparative Example 1 and Comparative Example compared with the results of example 2, association rate constant _(k a), binding constant _{(K a)} and dissociation constant _{(K D)} shows a value of about 10 to 1000 times, to have a very strong bonding force It could be confirmed. This is an influence of the side chain part contained in the structure of the immobilized glycolipid-containing compound. Also, the impact of the structure of the linker site, comparing Comparative Example 3 having a long chain alkyl group having Example 1 and the thiol group with lipoic acid having a disulfide group, association rate constant (k _a) is significantly improved I was able to confirm. This involves both the effect of the length of the side chain site and the linker site and the effect of the difference between the disulfide group and the thiol group contained in the linker site, and the orientation of the glycolipid-containing compound on the sensor chip surface. It is speculated that it had some influence.

Detection of botulinum toxin using SPR sensor (creation of sensor chip)
A gold substrate of SIA kit Au commercially available from Biacore was washed with an ozone cleaner and immersed in 2 mL of a methanol solution in which 100 nmol of the compound (2d) synthesized in Example 3 was dissolved at room temperature for 24 hours. Thereafter, the sensor chip was prepared by washing three times in order of methanol and water.

(SPR measurement)
In order to measure the specific adsorption property of the sugar-binding protein to the obtained sensor chip, a commercially available surface plasmon resonance biosensor (Biacore 2000, manufactured by Biacore) was used as a sensor chip on which the compound (2d) of Example 3 was immobilized. The HEPES (10 mM, pH: 7.5, NaCl: 0.15 M) buffer was flowed at a set temperature of 25 ° C. and a flow rate of 10 μL / min. It was confirmed that the sensorgram was stable before measurement. Botulinus toxin C (Botulinus Toxin C, manufactured by Wako Pure Chemical Industries, Ltd.) is used as the sugar-binding protein, and 1000 ng / mL (2860 pM) and 500 ng / mL (1430 pM) in HEPES (10 mM, pH: 7.5, NaCl: 0.15 M) buffer, respectively. ), 100 ng / mL (286 pM) and 10 ng / mL (28.6 pM). Botulinum toxin C solution was injected at 100 μL at a flow rate of 10 μL / min. As a result, as shown in FIG. 7, a good sensorgram was obtained at any concentration, and the detection intensities of botulinum toxin C at each concentration were 579 RU, 258 RU, 92 RU, and 10 RU. From this result, it was confirmed that even botulinum toxins, which were conventionally difficult to measure, could be detected with high sensitivity without using pretreatment or labeling substances.

(Calculation of coupling constant)
From the SPR measurement results, the optimum conditions for obtaining the coupling constant were estimated. That is, a sensor chip on which the compound (2d) of Example 3 was immobilized was attached to a surface plasmon resonance biosensor, and HEPES (10 mM, pH: 7.5, NaCl: 0.15 M) at a set temperature of 25 ° C. and a flow rate of 20 μL / min. Buffer was run. It was confirmed that the sensorgram was stable before measurement. The binding constant was Botulinus Toxin C (Botulinus Toxin C, manufactured by Wako Pure Chemical Industries, Ltd.), 1000 ng / mL (2860 pM), 750 ng / mL (2160 pM) with HEPES (10 mM, pH: 7.5, NaCl: 0.15 M) buffer, respectively. Prepared to 500 ng / mL (1430 pM) and 100 ng / mL (286 pM). Botulinum toxin C solution was injected at 100 μL at 20 μL / min. From the resulting sensorgram 1: 1 result of determining binding constants of the reaction, as shown in Table 2, the compound (2d) the immobilized sensor chip and binding rate constants of the botulinum toxin C (k _a), binding constant From the values of (K _A ) and dissociation constant (K _D ), it was confirmed that they had a very strong binding force.

Detection of sugar-binding compounds using gold microparticle detection reagent (Preparation of glycolipid-containing microparticles and detection reagents with optimal immobilization amount)
Gold particle dispersion solution having a particle diameter of 20 nm (particle concentration: 5.0 × 10 ¹¹ cells / mL) while adding stirring 100μL ethanol solution dissolving the compounds of 0~10μg the (2b) in 500 [mu] L, 1 hour at room temperature, fixed Reaction.
During this time, the color tone of the gold fine particle dispersion changed to red immediately after the addition of the compound (2b), and the change varied depending on the addition concentration.

  Prepare lysine 120 (Ricinus communis agglutinin 120, manufactured by Wako Pure Chemical Industries, Ltd.) in HEPES (10 mM, pH: 7.5, NaCl: 0.15 N) buffer solution in 200 μL of gold fine particle dispersion solution fixed by changing the concentration of compound (2b). 50 μL of the 1.0 mg / mL solution was added and allowed to stand at room temperature for 1 hour.

  The results of the dispersion stability and the color tone change after addition of lysine 120 were evaluated visually from the color tone change of the gold fine particle dispersion solution 1. Here, the dispersion stability result is the result after 1 hour of fixing the compound (2b), and the color tone change result after the addition of lysine 120 is the result after 1 hour of addition.

  Further, using the compound (3b) synthesized in Comparative Example 1 or the compound (4b) synthesized in Comparative Example 3, gold fine particles in which the compound (3b) or the compound (4b) was immobilized by the same method as above. And a gold fine particle dispersion was prepared.

As is apparent from the results in Table 3, when the amount of the added compound (2b) is 1.5 μg or less, the amount of the compound (2b) sufficient for self-assembly is not reached, so that the hydrophobicity of the gold fine particle surface is reduced. It was confirmed that the dispersion stability decreased. Further, when the amount of the added compound (2b) was 5.0 μg or more, the change in color tone after addition of lysine 120 was small, so that the excessively added compound (2b) inhibited the aggregation reaction.
Therefore, the optimal immobilization amount of compound (2b) per 500 μL of gold fine particles with a particle size of 20 nm (fine particle concentration: 5.0 × 10 ¹¹ particles / mL) is 2.0 μg to 2.5 μg. I was able to set it. Further, in this case, about 5.5 pieces were immobilized per 1 nm ^{2 of} the gold surface, and it was confirmed that the bonds were extremely dense and highly oriented.

On the other hand, the added compound (3b) had poor dispersion stability as shown in Table 3, and lysine 120 could not be detected. In addition, the optimal immobilization amount of compound (3b) per 500 μL of gold fine particles (particle concentration: 5.0 × 10 ¹¹ particles / mL) having a particle size of 20 nm is 0.3 μg to 0.4 μg, which is compared with compound (2b). It was inferior. Further, in the case of this immobilization amount, the optimal immobilization amount of the compound (3b) on the surface of the metal fine particles is 0.3 μg to 0.4 μg, and about 1.3 are immobilized per 1 nm ² of the gold surface. Both density and orientation are inferior compared to the compound (2b).
Furthermore, in the compound (4b), the tendency that the dispersion stability of the gold fine particles is remarkably lowered was recognized. Although the dispersion stability of the gold fine particles was improved by adding an excessive amount, precipitation occurred after 24 hours. In addition, the dispersion 1 hour after addition of 10.0 μg of compound (4b) had a small change in color tone after addition of lysine 120, and was unusable as a detection reagent.
The above results indicate that when the compound (2b) is used, the glycolipid-containing compound is immobilized on the surface of the gold fine particles with high orientation and high density.

(Detection of sugar-binding compounds)
200 μL ethanol solution in which 5.0 μg of compound (2b), which is within the range of the optimum immobilization amount investigated above, was dissolved in 1000 μL of a gold particle dispersion solution (particle concentration: 5.0 × 10 ¹¹ particles / mL) having a particle diameter of 20 nm. Was added with stirring.

  The immobilization reaction was performed at room temperature for 1 hour. A HEPES (10 mM, pH: 7.5, NaCl: 0.15N) buffer solution containing lysine 120 (Ricinus communis agglutinin 120, manufactured by Wako Pure Chemical Industries, Ltd.) in a dispersion solution of glycolipid-containing fine particles to which the compound (2b) is immobilized (100 μL) 100 μL of 1.0 to 100 μg / mL) was added and allowed to stand at room temperature for a certain period of time.

  The change in color tone of the gold fine particle dispersion solution at each time was visually evaluated. As a comparative control, a 10 μg / mL solution of ricin 120 and 100 times the amount of D-galactose were added simultaneously.

As is clear from the results of Table 4, it was confirmed that when the compound (2b) was used, it was sufficiently detectable even when the concentration of lysine 120 was low. For example, even if the lysine 120 concentration was 1.0 μg / mL, it could be confirmed in about 1 hour.
On the other hand, D-galactose added simultaneously inhibited the aggregation reaction between compound (2b) and lysine 120, so it was confirmed that almost no non-specific adsorption occurred.

  Thus, in the case of this experiment, it was confirmed that lysine 120 could be sufficiently detected even at an extremely low concentration of 1.0 μg / mL by using a gold fine particle dispersion solution on which compound (2b) was immobilized.

On the other hand, for compound (3b), a 200 μL ethanol solution in which 0.8 μg of compound (3b), which is within the range of the optimal immobilization amount, was added with stirring, was the same as in the case of using compound (2b). By the method, a gold fine particle dispersion solution in which the compound (3b) was immobilized was prepared, and a detection test corresponding to the concentration of lysine 120 was performed.
As is clear from the results in Table 4, in the gold fine particle dispersion solution in which the compound (3b) was immobilized, a slight color change was observed at a lysine 120 concentration of 100 μg / mL, but at a lysine 120 concentration of 50 μg / mL, , Lysine 120 could not be detected.

(Inhibition experiment)
The recognizability of lysine 120 in the presence of various aqueous solutions was examined using a gold fine particle dispersion solution on which the compound (2b) prepared by the above method was immobilized.

  That is, 50 μL of each solution of distilled water, tap water, and rainwater was added to 100 μL of the gold fine particle dispersion solution on which the compound (2b) was immobilized, and the mixture was allowed to stand at room temperature for 1 hour. Thereafter, 50 μL of 50 μg / mL solution of ricin 120 (Ricinus communis agglutinin 120, manufactured by Wako Pure Chemical Industries, Ltd.) prepared with HEPES (10 mM, pH: 7.5, NaCl: 0.15N) buffer was added and allowed to stand at room temperature for 1 hour. .

  Table 5 shows the results of visual evaluation of the color tone change of each gold fine particle dispersion. In either case, it was confirmed that the color tone did not change with the addition of the solution, and the environmental resistance was small, indicating that sugar-binding compounds present in the environment could be detected well.

When the glycolipid-containing compound according to the present invention is immobilized on the surface of a substrate or fine particles, the immobilization can be performed with high orientation and high density, and a biosensor or a detection reagent containing the glycolipid-containing compound is: Even extremely small amounts of sugar-binding compounds can be detected with high sensitivity and speed.
When such a glycolipid-containing compound is used, it can be applied to uses such as quartz crystal microbalance (QCM) and immunochromatography with high sensitivity, or labeling of sugar chain recognition sites.

Claims (22)

Following formula (5)

[Wherein, n in the linker moiety represented by —C (═O) C _n H _2n Y is 4, and Y represents a 1,2-dithiolan-3-yl group;
_{P in the} side chain moiety represented by —CH (OH) CH═CHC pH _2p CH ₃ represents an integer of 12 to 14,
G _N − represents the following formulas (i) to (x)

The sugar chain part represented by these is shown. ] The glycolipid containing compound characterized by the above-mentioned. G _N − represents the following formulas (i) to (v), (viii) to (x)

The glycolipid-containing compound according to claim 1, which is any sugar chain moiety represented by the formula: The glycolipid-containing compound according to claim 1, represented by the following formula:

. The glycolipid-containing compound according to claim 1, represented by the following formula:

. The glycolipid-containing compound according to claim 1, represented by the following formula:

. A biosensor for detecting ricin toxin, verotoxin, or botulinum toxin, wherein the glycolipid-containing compound according to claim 1 is immobilized on the surface of a metal substrate through a linker site . Density when the glycolipid-containing compound is fixed to the metal substrate surface is 0.1 to 10 pieces / nm ^2, biosensor of claim 6.   The biosensor according to claim 6 or 7, wherein the metal substrate is a gold substrate. A method for detecting a sugar-binding compound selected from the group consisting of ricin toxin, verotoxin, and botulinum toxin, comprising the following steps:
(1) A step of bringing a test compound into contact with the substrate surface of the biosensor according to any one of claims 6 to 8,
(2) detecting the sugar-binding compound bound to the substrate. The G _N − represents the following formulas (i) to (v)

Ri Oh a group represented by either of, the sugar binding compound is a ricin toxin A method according to claim 9. G _N − represents the following formula (vi) or (vii)

10. The method of claim 9 , wherein the sugar binding compound is verotoxin . The _GN − represents the following formulas (i), (viii) to (x)

In Ri or a group Der represented, the sugar binding compound is a botulinum toxin, the method according to claim 9.   A glycolipid-containing fine particle, wherein the glycolipid-containing compound according to claim 1 is immobilized on the surface of the metal fine particle via a linker site. 14. The glycolipid-containing fine particles according to claim 13 , wherein the metal fine particles have an average particle diameter in the range of 1 to 100 nm. Density when the glycolipid-containing compound is fixed to the metal fine particle surface is 0.1 to 10 pieces / nm is ^2, glycolipid-containing microparticles of claim 13. The glycolipid-containing microparticle according to claim 13 , wherein the metal microparticle is a gold microparticle. A reagent for detecting a sugar-binding compound selected from the group consisting of ricin toxin, verotoxin, and botulinum toxin, comprising the glycolipid-containing microparticles according to any one of claims 13 to 16 . Detection reagents sugar-binding compound of claim 17, wherein the glycolipid-containing particles, characterized in that it exists in colloid solution. A method for detecting a sugar-binding compound selected from the group consisting of ricin toxin, verotoxin, and botulinum toxin, comprising the following steps:
(1) Test compounds are added to the detection reagent of the sugar binding compound according to claim 17 or 18, contacting the glycolipid-containing particles,
(2) detecting the sugar-binding compound bound to the glycolipid-containing microparticles. The G _N − represents the following formulas (i) to (v)

Ri Oh a group represented by either of, the sugar binding compound is a ricin toxin A method according to claim 19. G _N − represents the following formula (vi) or (vii)

21. The method of claim 19 , wherein the sugar binding compound is verotoxin . The _GN − represents the following formulas (i), (viii) to (x)

In Ri or a group Der represented, the sugar binding compound is a botulinum toxin, the method according to claim 19.

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