JP4742339B2 - Detection of sialic acid-containing trisaccharide compounds and thirsvirus or thirst spike proteins using the same - Google Patents

Description

  The present invention relates to a sialic acid-containing trisaccharide compound, a sensor chip for detecting a thirsvirus or a third spike protein using the same, a fine particle or adsorbent for detection, a decontamination agent, and a method for detecting a third virus or third spike protein.

  A new type of pneumonia (SARS: Severe Acute Respiratory Syndrome) was prevalent in 2003 in China, Taiwan and Canada. This infection is caused by SARS coronavirus (SARS-CoV), and the mortality rate reaches 10% or more. A large number of proteins called spike proteins are present on the surface of SARS-CoV, and it is considered that infection occurs when these spike proteins bind to specific receptors on the host cells. This virus has a new history after discovery, and there are many unclear points regarding human SARS virus receptors at this stage.

  Until now, gene diagnosis methods (RT-PCR method: Non-Patent Document 1), antibody methods (Non-Patent Document 2), and the like are known for the detection of Thurs virus. However, the genetic diagnosis method has a risk of contamination due to other than viruses, and is merely an estimate of the presence of viruses. The antibody method is a complicated method because it is necessary to store the antibody at a low temperature of 2 to 8 ° C. due to the problem of the stability of the antibody. For this reason, a highly accurate and simple method for detecting a Thurs virus has been desired.

On the other hand, in the fields of biotechnology, biochemical research, clinical testing, food testing, and the like, a method for detecting and quantifying sugar-binding proteins present in viruses or the like has been proposed (Patent Document 1).
However, so far, the structure of the sugar chain in which the Thirs virus serves as a receptor has not been elucidated, and there is no report on the detection and adsorption method of Thurs virus using the sugar chain.

U.S. Pat. No. 3,321,733 Journal of Virological Methods, vol. 122, pp. 29-36 (2004). Clinical Immunology, vol. 113, pp. 145-150 (2004).

  An object of the present invention is to provide a novel sugar compound capable of detecting a Thurs virus or Thurs spike protein.

  Furthermore, the present invention provides a biosensor chip, a detection reagent, a detection fine particle, an adsorbent, a slow dyeing agent, and a detection method for a third virus or a third spike protein that can detect a third virus or a third spike protein with high sensitivity, simply and quickly. The issue is to provide.

  In view of the above problems, the inventors of the present application have studied sugar chains that can be bound to Thurs virus with respect to the sugar chains synthesized by the present inventors. Specifically, we have eagerly studied sugar chains that can bind to proteins called spike proteins present on the surface of Thurs virus, and for the first time found that a specific sugar chain structure binds to Thurs virus spike proteins very effectively. It was. It has been found that by using a compound having this sugar chain structure, rapid detection of a thirsvirus or a third spike protein, an adsorbent, etc. can be provided, and the present invention has been completed.

〔式中、Ｒ_１は、−ＯＨ、または、−ＮＨＣＯＣＨ_３を示し；
Ｒ_２およびＲ_３のいずれか一方は下記式（２）

で示されるシアル酸残基（式中、−ＣＯＯＭは、−ＣＯＯＨまたはその生理的に許容される塩）であり、Ｒ_２およびＲ_３の残りの一方はＨを示し；
Ｘ_１およびＸ_２は、それぞれ独立に、直鎖もしくは分岐した−Ｃ_nＨ_2n−（ｎが２〜２０）で表されるアルキル基、または置換基を有していてもよい炭素原子数６〜１０の２価の芳香族基を示し；
Ｙは、−ＳＨまたはジスルフィド基を有する基を示す。〕で表される、シアル酸含有３糖化合物。
〔２〕 前記Ｒ_２が前記式（２）で表されるシアル酸残基であり、前記Ｒ_３がＨである、〔１〕に記載のシアル酸含有３糖化合物。
〔３〕 前記Ｒ_２がＨであり、前記Ｒ_３が前記式（２）で表されるシアル酸残基である、〔１〕に記載のシアル酸含有３糖化合物。
〔４〕 前記Ｘ_１が芳香族置換基であり、Ｘ_２が直鎖もしくは分岐した炭素原子数２〜２０のアルキル基である、〔１〕〜〔３〕のいずれかに記載のシアル酸含有３糖化合物。
〔５〕 前記Ｘ_１が、下記式（３）

で表される２価の基である、〔４〕に記載のシアル酸含有３糖化合物。
〔６〕 前記Ｘ_２が、-C_nH_2n- (n = 2-6)である、〔４〕または〔５〕に記載のシアル酸含有３糖化合物。
〔７〕 Ｙが、ジスルフィド基である、〔１〕〜〔６〕のいずれかに記載のシアル酸含有３糖化合物。
〔８〕 下記式（４）

〔式中、Ｒ_１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、〔１〕に記載のシアル酸含有３糖化合物。
〔９〕 下記式（５）

〔式中、Ｒ_１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、〔１〕に記載のシアル酸含有３糖化合物。
〔１０〕 下記一般式（６）

〔式中、Ｒ_１は、−ＯＨ、または、−ＮＨＣＯＣＨ_３を示し；
Ｒ_２およびＲ_３のいずれか一方は下記式（２）

で示されるシアル酸残基（式中、−ＣＯＯＭは、−ＣＯＯＨまたはその生理的に許容される塩）であり、Ｒ_２およびＲ_３の残りの一方はＨを示し；
Ｘ_３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_２〜10アルケニル基、置換基を有していてもよいＣ_２〜20アルキニル基、置換基を有していてもよいＣ_６〜10アリール基、置換基を有していてもよいＣ_1〜20アルキルカルボニルオキシプロピル基、置換基を有していてもよいＣ_2〜21アルキルオキシプロピル基、置換基を有していてもよいＣ_2〜20アルケニルカルボニルオキシプロピル基、または置換基を有していてもよいＣ_2〜21アルケニルオキシプロピル基を意味し、前記置換基は下記Ａ群から選ばれるいずれかの基である：
Ａ群：
ハロゲン原子、水酸基、Ｃ_１〜６アルコキシ基、トリフルオロメチル基、トリフルオロメトキシ基、アミノ基、Ｃ_１〜６アルキルアミノ基；
−ＮＨ（Ｃ＝Ｏ）Ｘ₂Ｙ（式中、Ｘ₂は、直鎖もしくは分岐した−Ｃ_nＨ_2n−で表されるアルキレン基、または下記Ｂ群から選ばれる置換基を有していてもよい炭素原子数６〜１０の２価の芳香族基を示す。）；
−ＮＨ−ＣＨ₂−Ｃ_nＨ_2n−Ｙ；
−Ｏ−Ｃ（＝Ｏ）−Ｃ_nＨ_2n−Ｙ；
−Ｏ−ＣＨ₂−Ｃ_nＨ_2n−Ｙ；
（Ａ群中、ｎは２〜２０の整数を示し、Ｙはチオール基またはジスルフィド基を有する基を示す。）
Ｂ群：ハロゲン原子、水酸基、Ｃ_１〜６アルコキシ基、トリフルオロメチル基、トリフルオロメトキシ基、アミノ基、Ｃ_１〜６アルキルアミノ基、ニトロ基〕で表されるシアル酸含有３糖化合物が、金属基板表面に固定されている、サーズウイルスまたはサーズスパイク蛋白質検出用センサーチップ。
〔１１〕 下記一般式（６）

〔式中、Ｒ_１は、−ＯＨ、または、−ＮＨＣＯＣＨ_３を示し；
Ｒ_２およびＲ_３のいずれか一方は下記式（２）

で示されるシアル酸残基（式中、−ＣＯＯＭは、−ＣＯＯＨまたはその生理的に許容される塩）であり、Ｒ_２およびＲ_３の残りの一方はＨを示し；
Ｘ_３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_２〜10アルケニル基、置換基を有していてもよいＣ_２〜20アルキニル基、置換基を有していてもよいＣ_６〜10アリール基、置換基を有していてもよいＣ_1〜20アルキルカルボニルオキシプロピル基、置換基を有していてもよいＣ_2〜21アルキルオキシプロピル基、置換基を有していてもよいＣ_2〜20アルケニルカルボニルオキシプロピル基、または置換基を有していてもよいＣ_2〜21アルケニルオキシプロピル基を意味し、前記置換基は下記Ａ群から選ばれるいずれかの基である：
Ａ群：
ハロゲン原子、水酸基、Ｃ_１〜６アルコキシ基、トリフルオロメチル基、トリフルオロメトキシ基、アミノ基、Ｃ_１〜６アルキルアミノ基；
−ＮＨ（Ｃ＝Ｏ）Ｘ₂Ｙ（式中、Ｘ₂は、直鎖もしくは分岐した−Ｃ_nＨ_2n−で表されるアルキレン基、または下記Ｂ群から選ばれる置換基を有していてもよい炭素原子数６〜１０の２価の芳香族基を示す。）；
−ＮＨ−ＣＨ₂−Ｃ_nＨ_2n−Ｙ；
−Ｏ−Ｃ（＝Ｏ）−Ｃ_nＨ_2n−Ｙ；
−Ｏ−ＣＨ₂−Ｃ_nＨ_2n−Ｙ；
（Ａ群中、ｎは２〜２０の整数を示し、Ｙはチオール基またはジスルフィド基を有する基を示す。）
Ｂ群：ハロゲン原子、水酸基、Ｃ_１〜６アルコキシ基、トリフルオロメチル基、トリフルオロメトキシ基、アミノ基、Ｃ_１〜６アルキルアミノ基、ニトロ基〕で表されるシアル酸含有３糖化合物が、金属微粒子表面に固定されている、サーズウイルスまたはサーズスパイク蛋白質検出用微粒子。
〔１２〕 〔１１〕に記載の前記微粒子が、溶液中にコロイドで存在している、サーズウイルスまたはサーズスパイク蛋白質検出用試薬。
〔１３〕 下記の工程からなる、サーズウイルスまたはサーズスパイク蛋白質検出方法：
（１）試験体を、〔１０〕に記載のセンサーチップの基板表面に接触させる工程、
（２）基板に結合したサーズウイルスのスパイク蛋白質またはサーズウイルスを検出する工程。
〔１４〕 下記の工程からなる、サーズウイルスまたはサーズスパイク蛋白質検出方法：
（１）試験体を、〔１２〕に記載の試薬に添加して、〔１１〕に記載の前記微粒子と接触させる工程、
（２）前記微粒子に結合したサーズウイルスのスパイク蛋白質またはサーズウイルスを検出する工程。
〔１５〕 下記一般式（６）

〔式中、Ｒ_１は、−ＯＨ、または、−ＮＨＣＯＣＨ_３を示し；
Ｒ_２およびＲ_３のいずれか一方は下記式（２）

で示されるシアル酸残基（式中、−ＣＯＯＭは、−ＣＯＯＨまたはその生理的に許容される塩）であり、Ｒ_２およびＲ_３の残りの一方はＨを示し；
Ｘ_３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_２〜10アルケニル基、置換基を有していてもよいＣ_２〜20アルキニル基、置換基を有していてもよいＣ_６〜10アリール基、置換基を有していてもよいＣ_1〜20アルキルカルボニルオキシプロピル基、置換基を有していてもよいＣ_2〜21アルキルオキシプロピル基、置換基を有していてもよいＣ_2〜20アルケニルカルボニルオキシプロピル基、または置換基を有していてもよいＣ_2〜21アルケニルオキシプロピル基を意味し、前記置換基は下記Ａ群から選ばれるいずれかの基である：
Ａ群：
ハロゲン原子、水酸基、Ｃ_１〜６アルコキシ基、トリフルオロメチル基、トリフルオロメトキシ基、アミノ基、Ｃ_１〜６アルキルアミノ基；
−ＮＨ（Ｃ＝Ｏ）Ｘ₂Ｙ（式中、Ｘ₂は、直鎖もしくは分岐した−Ｃ_nＨ_2n−で表されるアルキレン基、または下記Ｂ群から選ばれる置換基を有していてもよい炭素原子数６〜１０の２価の芳香族基を示す。）；
−ＮＨ−ＣＨ₂−Ｃ_nＨ_2n−Ｙ；
−Ｏ−Ｃ（＝Ｏ）−Ｃ_nＨ_2n−Ｙ；
−Ｏ−ＣＨ₂−Ｃ_nＨ_2n−Ｙ；
（Ａ群中、ｎは２〜２０の整数を示し、Ｙはチオール基またはジスルフィド基を有する基を示す。）
Ｂ群：ハロゲン原子、水酸基、Ｃ_１〜６アルコキシ基、トリフルオロメチル基、トリフルオロメトキシ基、アミノ基、Ｃ_１〜６アルキルアミノ基、ニトロ基〕で表されるシアル酸含有３糖化合物を含有する、サーズウイルスまたはサーズスパイク蛋白質吸着材。 That is, this invention includes the following.
[1] The following general formula (1):

[Wherein R ₁ represents —OH or —NHCOCH ₃ ;
One of R ₂ and R ₃ is represented by the following formula (2)

A sialic acid residue (wherein —COOM is —COOH or a physiologically acceptable salt thereof), and the other of R ₂ and R ₃ represents H;
X ₁ and X ₂ are each independently a linear or branched alkyl group represented by —C _n H _2n — (n is 2 to 20), or an optionally substituted carbon atom having 6 carbon atoms. 10 to 10 divalent aromatic groups;
Y represents —SH or a group having a disulfide group. A sialic acid-containing trisaccharide compound represented by the formula:
[2] The sialic acid-containing trisaccharide compound according to [1], wherein R ₂ is a sialic acid residue represented by the formula (2), and R ₃ is H.
[3] The sialic acid-containing trisaccharide compound according to [1], wherein R ₂ is H and R ₃ is a sialic acid residue represented by the formula (2).
[4] The sialic acid-containing product according to any one of [1] to [3], wherein X ₁ is an aromatic substituent, and X ₂ is a linear or branched alkyl group having 2 to 20 carbon atoms. Trisaccharide compound.
[5] X ₁ represents the following formula (3)

The sialic acid-containing trisaccharide compound according to [4], which is a divalent group represented by:
[6] The sialic acid-containing trisaccharide compound according to [4] or [5], wherein X ₂ is —C _n H _2n − (n = 2-6).
[7] The sialic acid-containing trisaccharide compound according to any one of [1] to [6], wherein Y is a disulfide group.
[8] The following formula (4)

[In the formula, R ₁ is —NHAc or —OH, and —COOM represents —COOH or a physiologically acceptable salt thereof] The sialic acid-containing trisaccharide according to [1] Compound.
[9] The following formula (5)

[In the formula, R ₁ is —NHAc or —OH, and —COOM represents —COOH or a physiologically acceptable salt thereof] The sialic acid-containing trisaccharide according to [1] Compound.
[10] The following general formula (6)

[Wherein R ₁ represents —OH or —NHCOCH ₃ ;
One of R ₂ and R ₃ is represented by the following formula (2)

A sialic acid residue (wherein —COOM is —COOH or a physiologically acceptable salt thereof), and the other of R ₂ and R ₃ represents H;
X ₃ is a C _2-20 alkyl group which may have a substituent, a C _2-10 alkenyl group which may have a substituent, or a C _2-20 alkynyl which may have a substituent. Group, C _6-10 aryl group _optionally having substituent (s), C _1-20 alkylcarbonyloxypropyl group _optionally having substituent (s), C _{2-21 optionally} having substituent (s) An alkyloxypropyl group, an optionally substituted C _2-20 alkenylcarbonyloxypropyl group, or an optionally substituted C _2-21 alkenyloxypropyl group, wherein the substituent is Any group selected from the following group A:
Group A:
Halogen atom, hydroxyl group, C _1-6 alkoxy group, trifluoromethyl group, trifluoromethoxy group, amino group, C _1-6 alkylamino group;
—NH (C═O) X ₂ Y (wherein X ₂ has a linear or branched alkylene group represented by —C _n H _2n — or a substituent selected from the following group B: Or a divalent aromatic group having 6 to 10 carbon atoms.);
_{-NH-CH 2 -C n H 2n} -Y;
-O-C (= O) -C n H 2n -Y;
_{-O-CH 2 -C n H 2n} -Y;
(In group A, n represents an integer of 2 to 20, and Y represents a group having a thiol group or a disulfide group.)
Group B: sialic acid-containing trisaccharide compound represented by 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, a nitro group] A sensor chip for detecting a thirsvirus or thirst spike protein, which is fixed to the surface of a metal substrate.
[11] The following general formula (6)

[Wherein R ₁ represents —OH or —NHCOCH ₃ ;
One of R ₂ and R ₃ is represented by the following formula (2)

A sialic acid residue (wherein —COOM is —COOH or a physiologically acceptable salt thereof), and the other of R ₂ and R ₃ represents H;
X ₃ is a C _2-20 alkyl group which may have a substituent, a C _2-10 alkenyl group which may have a substituent, or a C _2-20 alkynyl which may have a substituent. Group, C _6-10 aryl group _optionally having substituent (s), C _1-20 alkylcarbonyloxypropyl group _optionally having substituent (s), C _{2-21 optionally} having substituent (s) An alkyloxypropyl group, an optionally substituted C _2-20 alkenylcarbonyloxypropyl group, or an optionally substituted C _2-21 alkenyloxypropyl group, wherein the substituent is Any group selected from the following group A:
Group A:
Halogen atom, hydroxyl group, C _1-6 alkoxy group, trifluoromethyl group, trifluoromethoxy group, amino group, C _1-6 alkylamino group;
—NH (C═O) X ₂ Y (wherein X ₂ has a linear or branched alkylene group represented by —C _n H _2n — or a substituent selected from the following group B: Or a divalent aromatic group having 6 to 10 carbon atoms.);
_{-NH-CH 2 -C n H 2n} -Y;
-O-C (= O) -C n H 2n -Y;
_{-O-CH 2 -C n H 2n} -Y;
(In group A, n represents an integer of 2 to 20, and Y represents a group having a thiol group or a disulfide group.)
Group B: sialic acid-containing trisaccharide compound represented by 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, a nitro group] Fine particles for detection of thirsvirus or thirst spike protein fixed on the surface of metal fine particles.
[12] A reagent for detecting a third virus or a third spike protein, wherein the microparticle according to [11] is present in a colloid in a solution.
[13] A method for detecting a thirsvirus or thirst spike protein comprising the following steps:
(1) A step of bringing the specimen into contact with the substrate surface of the sensor chip according to [10],
(2) A step of detecting a thirsvirus spike protein or thirsvirus bound to the substrate.
[14] A method for detecting a thirsvirus or thirst spike protein comprising the following steps:
(1) A step of adding a test specimen to the reagent according to [12] and bringing the specimen into contact with the fine particles according to [11],
(2) A step of detecting a thirsvirus spike protein or a thirsvirus bound to the microparticles.
[15] The following general formula (6)

[Wherein R ₁ represents —OH or —NHCOCH ₃ ;
One of R ₂ and R ₃ is represented by the following formula (2)

A sialic acid residue (wherein —COOM is —COOH or a physiologically acceptable salt thereof), and the other of R ₂ and R ₃ represents H;
X ₃ is a C _2-20 alkyl group which may have a substituent, a C _2-10 alkenyl group which may have a substituent, or a C _2-20 alkynyl which may have a substituent. Group, C _6-10 aryl group _optionally having substituent (s), C _1-20 alkylcarbonyloxypropyl group _optionally having substituent (s), C _{2-21 optionally} having substituent (s) An alkyloxypropyl group, an optionally substituted C _2-20 alkenylcarbonyloxypropyl group, or an optionally substituted C _2-21 alkenyloxypropyl group, wherein the substituent is Any group selected from the following group A:
Group A:
Halogen atom, hydroxyl group, C _1-6 alkoxy group, trifluoromethyl group, trifluoromethoxy group, amino group, C _1-6 alkylamino group;
—NH (C═O) X ₂ Y (wherein X ₂ has a linear or branched alkylene group represented by —C _n H _2n — or a substituent selected from the following group B: Or a divalent aromatic group having 6 to 10 carbon atoms.);
_{-NH-CH 2 -C n H 2n} -Y;
-O-C (= O) -C n H 2n -Y;
_{-O-CH 2 -C n H 2n} -Y;
(In group A, n represents an integer of 2 to 20, and Y represents a group having a thiol group or a disulfide group.)
B group: sialic acid-containing trisaccharide compound represented by halogen atom, hydroxyl group, C _1-6 alkoxy group, trifluoromethyl group, trifluoromethoxy group, amino group, C _1-6 alkylamino group, nitro group] A thirsvirus or thirst spike protein adsorbent containing.

  Hereinafter, a sialic acid-containing trisaccharide compound, a sensor chip for detecting a thirsvirus or a third spike protein, a thirsvirus or a third spike protein detection reagent, a third virus or a third spike protein detection method, and an adsorbent will be specifically described.

<Sialic acid-containing trisaccharide compound>
A preferred sialic acid-containing trisaccharide compound that can be used for the detection and adsorption of the Thurs virus or Thurs spike protein according to the present invention is represented by the following general formula (6).

In the formula, R ₁ represents —OH or —NHCOCH ₃ . Of these, —NHCOCH ₃ is preferred. In the case of —NHCOCH ₃ , binding to Thurs virus, more specifically binding to spike protein existing on the surface of Thurs virus is stronger than —OH.

  In the present specification, the “spike protein” is a protein characteristic of coronavirus, and this spike protein is a protruding protein present on the surface of a membrane of a thirsvirus (Thirdsvirus belongs to coronavirus). This is composed of S1 subunit and S2 subunit (Clinical Immunology, 113, 145-150 (2004)). In addition, it is a protein necessary for infecting (binding to) host cells (details of infection mechanism are described in Trends in Microbiology, 12, 466-472 (2004)). Until now, there is no fact that it binds to sugar chains on host cells.

で示されるシアル酸残基（式中、−ＣＯＯＭは、−ＣＯＯＨまたはその生理的に許容される塩）であり、Ｒ_２およびＲ_３の残りの一方はＨを示す。 One of R ₂ and R ₃ is represented by the following formula (2)

(Wherein —COOM is —COOH or a physiologically acceptable salt thereof), the other of R ₂ and R ₃ represents H.

  In the above formula (6), the trisaccharide moiety into which the sialic acid residue is introduced specifically binds to the Thurs virus spike protein.

When the —CO ₂ M is a physiologically acceptable salt, the form of the salt is not particularly limited, and examples thereof include inorganic acid salts, organic acid salts, inorganic basic salts, organic basic salts, acidic or basic amino acids. Examples include salt.

  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, trimethylamine salt, pyridinium salt, and among these, trimethylamine salt and pyridinium salt are more preferable. .

  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.

In the formula, X ₃ is an _optionally substituted C _2-20 alkyl group, an _optionally substituted C _2-10 alkenyl group, and an _optionally substituted C _{2. -20} alkynyl group, C _6-10 aryl group _optionally having substituent, C _1-20 alkylcarbonyloxypropyl group _optionally having substituent, C _optionally having substituent _It means a _2-21 alkyloxypropyl group, an optionally substituted _C2-20 alkenylcarbonyloxypropyl group, or an optionally substituted _C2-21 alkenyloxypropyl group.
The substituent can be selected from the following group A.
Group A:
Halogen atom, hydroxyl group, C _1-6 alkoxy group, trifluoromethyl group, trifluoromethoxy group, amino group, C _1-6 alkylamino group;
—NH (C═O) X ₂ Y (wherein X ₂ has a linear or branched alkylene group represented by —C _n H _2n — or a substituent selected from the following group B: Or a divalent aromatic group having 6 to 10 carbon atoms.);
_{-NH-CH 2 -C n H 2n} -Y;
-O-C (= O) -C n H 2n -Y;
_{-O-CH 2 -C n H 2n} -Y;
(In group A, n represents an integer of 2 to 20, and Y represents a group having a thiol group or a disulfide group.)
Group B: halogen atom, hydroxyl group, C _1-6 alkoxy group, trifluoromethyl group, trifluoromethoxy group, amino group, C _1-6 alkylamino group, nitro group.

The “C _2-20 alkyl group” is an alkyl group having 2 to 20 carbon atoms, which may be either a linear or branched alkyl group, but is preferably a linear alkyl group. The number of carbon atoms is preferably 4 to 20, more preferably 10 to 20, and particularly preferably 13 to 18.

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. The number of carbon atoms is preferably 4 to 20, more preferably 10 to 20, and particularly preferably 13 to 18. 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 _2-20 alkynyl group” is an alkynyl group having 2 to 20 carbon atoms, which may be linear or branched, but is preferably a linear alkynyl group. The number of carbon atoms is preferably 4 to 20, more preferably 10 to 20, and particularly preferably 13 to 18. One or more unsaturated bonds may be contained.

The “C _6-10 aryl group” refers to an aromatic hydrocarbon cyclic group having 6 to 10 carbon atoms, and specifically includes, for example, a phenyl group, a 1-naphthyl group, a 2-naphthyl group, and the like. It is done.

The “C _1-20 alkylcarbonyloxypropyl group” is a group represented by a C _1-20 alkyl group —CO—O—C ₃ H ₆ —, and the C _1-20 alkyl group is linear or branched. Any alkyl group may be used, but a linear alkyl group is preferred.

The “C _2-21 alkyloxypropyl group” is a group represented by a C _2-21 alkyl group —O—C ₃ H ₆ —, and the C _2-21 alkyl group is a linear or branched alkyl group. However, it is preferably a linear alkyl group.

The “C _2-20 alkenylcarbonyloxypropyl group” is a group represented by a C _2-20 alkenyl group —CO—O—C ₃ H ₆ —, and the C _2-20 alkenyl group is linear or branched. Any of these may be used. 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 alkenyloxypropyl group” is a group represented by a C _2-21 alkenyl group —O—C ₃ H ₆ —, and the C _2-21 alkenyl group is linear or branched. But you can. 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.

Among these X _3, a C _2-20 alkyl group which may have a substituent, a C _6-10 aryl group which may have a substituent, and a substituent may be preferable. C _{1 to 20} alkyl carbonyloxy propyl group which may have a substituent C _{2 to 21} alkyloxy propyl group which may have a substituent C _{2 to 20} alkenylcarbonyloxy propyl group or a substituted group, C _2-21 alkenyloxypropyl group which may have

In these, the substituent is preferably —NH (C═O) X ₂ Y, —O—C (═O) —C _n H _2n —Y, or —O—CH ₂ —C _n H _2n —. Y.

Among these, when a C _2-20 alkyl group which may have a substituent or a C _6-10 aryl group which may have a substituent has a substituent, the substituent may be the above-mentioned —NH ( A group represented by C═O) X ₂ Y is preferred.

Further, the C _1-20 alkylcarbonyloxypropyl group which may have a substituent, the C _2-21 alkyloxypropyl group which may have a substituent, and the C ₂ which may have a substituent. as the substituent when having ₂₀ alkenylcarbonyloxy propyl or optionally substituted C _{2 to 21} alkenyloxy propyl group substituents,, -NH (C = O) X 2 Y, - O—C (═O) —C _n H _2n —Y or —O—CH ₂ —C _n H _2n —Y is preferred.

More specifically, as X ₃ in the formula (6), for example, groups represented by the following formulas (7) to (11) are more preferable.

  In the formulas (7) to (11), n preferably represents an integer of 2 to 20, more preferably 2 to 10, still more preferably 3 to 8, particularly preferably 3 to 5. The p is preferably an integer of 1 to 17, more preferably 7 to 17, and still more preferably 10 to 15. q preferably represents an integer of 1 to 20, more preferably 10 to 20, and still more preferably 13 to 18.

As represented by the formulas (7) to (11), the linker site (—COC _n H _2n Y or —CH ₂ C _n H _2n Y) and the side chain site (—CH (OH) C═CC _p H _2p CH _3, as having a _{-CH 2 O (CO) C q} H 2q CH 3 or _{-CH 2 OCH 2 C q H 2q} CH 3), orientation suitable sialic acid-containing trisaccharide compound to the sensor It can be fixed to a substrate and fine particles at high density with a biosensor containing a sialic acid-containing trisaccharide compound or a detection reagent containing a glycolipid-containing fine particle, and even a very small amount of thirsvirus can be detected with high sensitivity. -Can be quantified.

  In particular, in the formulas (7) to (11), when n is in the above range, the sensitivity of the sensor when the sialic acid-containing trisaccharide compound is bound to the substrate surface or the fine particle surface is improved, and It can be effectively bonded to the substrate surface or fine particle surface with a density and orientation suitable for bonding.

  In the above formula, Y represents a group having one or more thiol groups or one 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—.

  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.

  Of these, disulfide groups are preferred. In the case of a disulfide group, the sialic acid-containing trisaccharide compound can be firmly and stably immobilized on the substrate surface or fine particle surface. In particular, in the groups represented by the formulas (7) to (11), steric hindrance of the sialic acid-containing trisaccharide moiety, the linker moiety, and the side chain moiety is predicted, and a decrease in bond density, a decrease in sensitivity, and the like are considered. However, the combination of a specific linker, a side chain, and a disulfide group as in the present invention enables strong and stable immobilization, and can be a highly sensitive sensor without the influence of steric hindrance.

Furthermore, in the formula (7), the total number (N1) of atoms contained in the main chain of the —COC _n H _2n Y and the main chain of the —CH (OH) C═CC _p H _2p CH ₃ The total number of atoms (N2) is preferably in the range of N1: N2 = 1: 1.5 to 1: 5, more preferably 1: 2 to 1: 3.
In the formulas (8) to (11), the total number (N1) of atoms contained in the main chain of the —COC _n H _2n Y or —CH ₂ C _n H _2n Y and the —CH ₂ O (CO ) C _q H _2q CH ₃ or _{-CH 2 OCH 2 C q H 2q} CH total number of atoms contained in the main chain of ₃ (N2) is preferably N1: N2 = 1: 1.5~1: 5 More preferably, it is in the range of 1: 2 to 1: 3.

In N1, a plurality of atoms that bond the longest sulfur atom in Y to —CO in —COC _n H _2n Y as the atoms constituting the main chain, and when Y is a disulfide group, The sulfur atom side close to —CO is defined as an atom on the main chain, and the total number N1 does not include a sulfur atom. In the N2, the _{-CH (OH) C = CC p} H 2p CH 3, -CH 2 O (CO) C q H 2q CH 3 or -CH ₂ OCH ₂ C _q H _2q in CH ₃ terminus of - A plurality of atoms that bond CH ₃ and —CH (OH) or —CH ₂ O at the longest are atoms constituting the main chain.

  When such a side chain site | part of Formula (7)-(11) is contained, compared with the case where a side chain site | part does not exist, a detection sensitivity can be improved significantly. Furthermore, 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 sialic acid-containing trisaccharide compound can be formed into a substrate with a higher degree of orientation. It can be immobilized on microparticles, and with a biosensor containing the sialic acid-containing trisaccharide compound or a detection reagent containing glycolipid-containing microparticles, even smaller amounts of thirsvirus or third spike protein can be detected with high sensitivity. Can be quantified.

  Such a compound represented by the formula (6) or the formulas (7) to (11) is a glyceroglycolipid (sugar as a hydrophilic group, glyceride moiety as a fat-soluble group or 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.

X ₃ may be bonded to any polymer.

式中、Ｒ_１、Ｒ_２およびＲ_３は前述と同じである。糖鎖末端の、−Ｘ_１−ＮＨ−（Ｃ＝Ｏ）−Ｘ_２−Ｙは、センサーチップを基板表面あるいは金属微粒子表面に固定化する部分である。 X ₃ is also preferably a group (12) represented by —X ₁ NH— (CO) —X ₂ Y. Specifically, such a compound (6) can be represented by the following formula (1).

In the formula, R ₁ , R ₂ and R ₃ are the same as described above. The sugar chain terminal —X ₁ —NH— (C═O) —X ₂ —Y is a portion for immobilizing the sensor chip on the surface of the substrate or the surface of the metal fine particles.

X ₁ and X ₂ each independently have a linear or branched alkyl group represented by —C _n H _2n — (n is 2 to 20, preferably 2 to 6), or a substituent. And a divalent aromatic group having 6 to 10 carbon atoms, preferably 6 to 8 carbon atoms.

で表される基がさらに好ましい。 Among these, X ₁ is preferably an aromatic group, and the aromatic group is represented by the following formula (3):

Is more preferable.

X ₂ is preferably a linear or branched alkyl group represented by —C _n H _2n — (n is 2 to 20, preferably 2 to 6), and among these, more preferably is a linear group, The case where n = 4 is particularly preferable.

  Y represents —SH or a disulfide group, and a disulfide group is preferable. The “disulfide group” is the same as described above.

More specifically, examples of such a compound include compounds represented by the following formulas (4) and (5).

  Such a compound according to the present invention binds to a thirsvirus, particularly a thirsvirus spike protein, very effectively.

Production method of sialic acid-containing trisaccharide compound The production method of the sialic acid-containing trisaccharide compound according to the present invention is not limited, and examples thereof include the methods shown below.
<Synthesis of sialic acid-containing trisaccharide compound>
As an example, the glycoside part on the reduction end side is shown for a trisaccharide having a p-nitrophenyl (pNP) group, but the glycoside part is not limited thereto.

Scheme 1

As shown in Synthesis Scheme 1 of 1a , commercially available pNP N-acetylglucosamine is used as an acceptor, lactose, pNP lactose and the like are allowed to coexist as donors, and a hydrolase such as galactosidase is used to form 4 of N-acetylglucosamine. Compound 1a can be obtained by selectively rearranging galactose at the position.

  In the enzyme reaction, a rearrangement enzyme such as galactosyltransferase may be used. For example, UDP-galactose can be used as a donor in this case. This disaccharide linkage mode is β1-4 linkage. Depending on the origin of the hydrolase, it is also possible to synthesize β1-3 linked disaccharides, but these are also sugar chains that can bind to the Thurs virus and are effective in the development of detection sensors and adsorbents. .

  The enzyme reaction can be performed, for example, in a HEPES buffer, a phosphate buffer, or an acetate buffer, preferably at 20-80 ° C, more preferably at 30-45 ° C. The pH of the buffer is preferably pH 1-10, more preferably pH 3-8. The reaction time is preferably 30 minutes to 30 days, more preferably 3 hours to 7 days. An organic solvent such as acetonitrile or methanol may be added to the buffer solution. The ratio of adding the organic solvent is preferably 1-80%, more preferably 10-50%. The ratio of the acceptor (pNP N-acetylglucosamine) and the donor (lactose, pNP lactose, etc.) is preferably about 1:99 to 99: 1, more preferably about 1:10. The commercially available hydrolase contains contaminating N-acetylhexosaminidase, which may hydrolyze the acceptor. If necessary, the enzyme is roughly purified with a column such as phenyl sepharose. It is preferable to purify. The number of units of the enzyme used is preferably 0.1 to 100 units, more preferably 0.2 to 10 units.

Synthesis of 2a and 3a Next, using this disaccharide compound 1a as an acceptor, the donor CMP-NeuAc, serum albumin, manganese ion (usually MnCl ₂ ), and alkaline phosphatase coexist, α2,3- If sialyltransferase is added, trisaccharide compound 2a having a sialic acid residue rearranged at the 3-position of compound 1a can be obtained.
On the other hand, if α2,6-sialyltransferase is used instead of α2,3-sialyltransferase, a trisaccharide compound 3a having a sialic acid residue rearranged at the 6-position of 1a can be obtained.

The enzyme reaction conditions are the same as described above. As the buffer solution, a morpholine ethanesulfonic acid (MES) buffer solution may be used instead of the HEPES buffer solution. The pH is preferably pH 4-9, more preferably pH 5-7, and particularly preferably pH 6-7. The alkaline phosphatase to be added is preferably 1-1000 equivalents, more preferably 5-20 equivalents, for each sialyltransferase.
The ratio of acceptor 1a to donor (CMP-NeuAc) is the same as described above, but is preferably 1: 1 to 1.5.
The reaction is preferably carried out while monitoring by HPLC. As the column, for example, an ODS-C18 column can be used, and the eluent is analyzed by adding 0.1-1% TFA (trifluoroacetic acid) with, for example, a 15% acetonitrile aqueous solution. You may use the column for molecular sieves, such as biogel P-2.

<Conversion method of glycoside moiety: synthesis of compound of formula (4)>
Scheme 2

Trisaccharide 4a can be obtained by reducing compound 2a in which the glycoside part is a p-nitrophenyl group to a p-aminophenyl group.
This reaction is carried out in the presence of a catalyst such as palladium, palladium-activated carbon, or palladium hydroxide under a hydrogen stream. Instead of palladium, ammonium formate or the like may be used.
As the solvent, water, methanol, ethanol, or a mixed solution thereof can be used. The reaction temperature is preferably 10-80 ° C, more preferably 20-40 ° C. The pressure is preferably 1 atm to 100 atm, more preferably 1 atm. The reaction time is preferably 30 minutes to 3 days, more preferably 1 hour to 24 hours.

Next, the trisaccharide compound 4a is reacted with lipoic acid in the presence of 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM), It can be converted to the trisaccharide compound 5a.
As the solvent, ethanol, methanol, water, isopropyl alcohol, tetrahydrofuran, DMF, DMSO, or a mixed solution thereof can be used, and among these, ethanol, water, or methanol is preferable. The reaction temperature is preferably 10 ° C-80 ° C, more preferably 20-40 ° C. Lipoic acid is preferably 0.9-10 equivalents, more preferably 0.9-2 equivalents relative to the trisaccharide compound 4a. Further, DMT-MM is preferably 0.9-10 equivalent, more preferably 0.9-2 equivalent.

<Conversion method of glycoside moiety: synthesis of compound of formula (5)>
Scheme 3

In the same manner as in Compound 5a, Compound 7a can be obtained from Compound 3a having a sialic acid residue at the 6-position of galactose.

<Method of converting glycoside moiety: Synthesis of compound having glycoside moiety of formula (7)>
A sialic acid-containing trisaccharide compound having a glycoside moiety of formula (7) has various glycoside moieties, for example, by a method using a naturally occurring sphingoglycolipid as a starting material, as shown in Scheme 4 below. A compound can be obtained.

Scheme 4

Glycosphingolipid (1b) (wherein, G _N represents a sugar chain portion, e.g., a glucose unit, R ^{3 'is} _{-C 10~18 H 20~30 -CH 3, R} ' is -C _{10 to 20} H _{20 to 40} —CH ₃ ) is a sugar chain site in which one or more sugars are covalently bonded in a linear or branched chain, and a ceramide in which a fatty acid is N-acyl bonded to a sphingosine base. It has a skeleton called lipid. The glycosphingolipid is a compound widely present in vertebrates, invertebrates, plants and the like. Commercially available products such as Takara Bio and Sigma can be used.

  For example, by subjecting the glycosphingolipid (1b) 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 used. To obtain compound (2b) (lyso form) which is a single-chain glycolipid in which a sphingosine base having an amino group becomes 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 30-40 degreeC in presence of% -2.0%. Furthermore, the compound (2b) 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 (3b), which is a linker site capable of being chemically fixed and / or fixed by physical adsorption, is bonded to the amino group of the obtained compound (2b) by a chemical bond. To give compound (4b). Such a compound is represented by, for example, HOOC-C _n H _2n Y (3b) (wherein n and Y are the same as n and Y in the above formulas (7) to (11)). Thiooctic acid, fatty acid having a thiol group at its terminal, and fatty acid having a disulfide group.

In addition, as the compound (3b) 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 (2b) and the carboxyl group of the compound (3b) is carried out in a solvent, if necessary, in the presence of a dehydrating condensing agent. It is preferable that the usage-amount of a compound (3b) exists in the range of 1-3 equivalent with respect to a lyso body (2b).
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.

Scheme 5
As shown in Scheme 5, the number of carbons in the side chain portion can be converted by introducing an epoxy group or a hydroxyl group into the double bond in the side chain portion, or converting it into an aldehyde.

In the above scheme 5, 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 (7).

  The compound 4b or the like having a side chain of the formula (7) thus obtained has, for example, a glucose unit. For example, the glucose unit contains a sialic acid by the same method as shown in Scheme 1. Can be converted to trisaccharide units.

<Conversion method of glycoside moiety: Synthesis of compounds having glycoside moieties of formulas (8) to (11)>
Scheme 6
Furthermore, a sialic acid-containing trisaccharide compound having a glycoside moiety of formulas (8) to (11) can be synthesized by a method using a glyceroglycolipid derived from nature as a starting material as shown in Scheme 6. .

In the formula, _GN is a sugar chain site, for example, a glucose unit. R ⁶ and R ⁷ are each independently —C _r H _2r —CH ₃ (wherein r represents an integer of 1 to 20). Commercial products such as Sigma can be used for these.

The compound 6b can be obtained by treating the acyl group part of the compound 5b with ordinary sodium hydroxide, potassium hydroxide, sodium methylate, lithium hydroxide or the like and deacylating it.
In addition, when a lipase that selectively acts at the 2-position of the acyl moiety is used, it can be converted to compound 6c. Similarly, when 1,3-specific lipase is allowed to act, it can be converted to compound 6d. 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, an 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 8b to 8d 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 ^{4 in the} side chain portion has a double bond or the like, another substituent can be introduced in the same manner as described above.

  The compound thus obtained is converted from a GN unit into a sialic acid-containing trisaccharide unit, for example, by the same method as shown in Scheme 1.

<Sensor chip for detection of thirsvirus or thirs spike protein>
In the sensor chip for detecting a thirsvirus or a third spike protein according to the present invention, the sialic acid-containing trisaccharide compound is immobilized on the surface of a metal substrate.

  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.

The immobilization on the metal substrate can be preferably performed by direct covalent bond or adsorption between the metal surface and the thiol group (—SH) or disulfide group (—SS—).
Therefore, in this case, among sialic acid-containing trisaccharide compounds, compounds having —SH or a disulfide group at the terminal at the terminal of the glycoside are preferable. That is, it is preferable to use a compound in which X ₃ is represented by the formulas (7) to (11) in the formula (1) or the formula (6).

In the formula (6), when X ₃ is a group represented by the formulas (7) to (11), a higher degree of orientation and high density fixation can be achieved by a synergistic effect due to the self-assembly of the side chain sites. It becomes possible. Furthermore, it is desirable for this self-assembly to have a naturally occurring side chain such as a sphingosine base. This is presumably because the surface of the substrate such as the sensor chip is in a state that approximates the surface state of the cell membrane.

Bonding to the substrate can be performed using a self-assembled monolayer (SAM) method or the like. That is, this is a method in which a compound having a thiol group or a disulfide group is specifically adsorbed with gold or the like, and SAM is formed by van der Waals force between alkyl chains.
When the sialic acid-containing trisaccharide compound has —SH or a disulfide group at the terminal, the construction of the monomolecular film on the surface of the metal substrate by the sialic acid-containing trisaccharide compound does not require a special apparatus, and these derivatives are in solution. This can be easily carried out simply by immersing the substrate in the substrate. 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.

Usually, the binding rate of the sialic acid-containing trisaccharide 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 formulas (7) to (11), when 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 folded side chain site It is presumed that the acid-containing trisaccharide compounds may be effectively bonded to the substrate surface or the fine particle surface with an orientation suitable for binding with the Thurs virus. Thereby, it is presumed that the preferable bond ratio as described above was achieved.

<Method of detecting a thirsvirus or a thirst spike protein (1)>
The method for detecting a thirsvirus or a third spike protein 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 sensor chip,
(2) A step of detecting a thirsvirus spike protein or thirsvirus bound to the substrate.

The contact between the sensor chip in which the sialic acid-containing trisaccharide compound is immobilized on a metal substrate and the thirsvirus or the thirsvirus spike protein is usually preferably performed in the presence of a solvent. In the detection of Thurs virus or its spike protein using the sensor chip, it is necessary to select a solvent in which these do not denature, for example, water or buffer.
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.

  Examples of the buffer include phosphate buffer, acetate buffer, HEPES buffer, or Tris buffer, more preferably HEPES buffer (10 mM, pH: 7.5, NaCl: 0.15N).

  Below, an example of the detection method of a virus by surface plasmon resonance (Surface Plasmon Resonance: SPR) among the sensor chips based on this invention is shown. In the detection method by SPR, a change in a test compound (also referred to as an analyte or an analysis target compound) can be detected without using a labeling substance. This SPR phenomenon utilizes an optical phenomenon that occurs when surface plasmons are excited at the metal / liquid interface. For example, a small plasmon resonance angle (SPR signal) generated on the surface of the sensor chip is detected. Changes in the binding and dissociation between the two molecules between the substance immobilized on the sample and the specimen can be quantitatively obtained.

  The detection of the Thurs virus or its spike protein is preferably performed by keeping the temperature constant in the range of 0 to 60 ° C., more preferably 25 to 40 ° C., preferably phosphate buffer, acetate buffer, HEPES buffer, or Tris buffer, more preferably HEPES buffer.

  This is slowly infused with a solution containing Thurs virus or its spike protein. When determining the affinity constant, it is desirable to measure 2 to 8 times by changing the concentration of Thurs virus or its spike 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.

The flow rate of the buffer is preferably 3 to 50 μl / min, and the sample volume is preferably about 5 to 1000 μl for each measurement of nM to mM. The affinity constant takes 2 to 8 measured concentrations, and in each sensorgram, a Scatchard plot is created from the equilibrium state to calculate affinity constants (K _d and K _a ).

Such a surface plasmon resonance biosensor using the sensor chip of the present invention can detect a trace amount (<0.01 nM) of a thirsvirus or its spike protein with high sensitivity without using pretreatment or a labeling substance. it can.
That is, the binding rate constant (K _a ) and dissociation constant (K _D ) between the sensor chip and Thurs virus or its spike protein are high and small in the sialic acid-containing trisaccharide compound represented by the above formula (1), respectively. Indicates the value.

In Formula (6), when X ₃ is a group represented by Formulas (7) to (11), Ka indicates a higher value. This effect is due to the high degree of orientation and high-density immobilization due to the self-assembly function of the side chain sites such as sphingosine bases, and multiple adjacent sugar chain sites efficiently and strongly bind the virus. It is.

<Microparticles for detecting Thurs virus or Third Spike protein, Reagent for detecting Thurs virus or Third Spike protein>
Microparticles for detecting thirsvirus or thirst spike protein The microparticles for detecting a thirsvirus according to the present invention are microparticles in which the sialic acid-containing trisaccharide compound is immobilized on the surface of the metal microparticles.

  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, 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 a sialic acid-containing trisaccharide compound on a metal fine particle, 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), a method in which a sialic acid-containing trisaccharide compound is immobilized on the sensor chip can be applied. That is, a solution in which fine metal particles are dispersed and a solution of the sialic acid-containing trisaccharide compound are mixed for 5 minutes or more. At this time, if a sufficient amount of the sialic acid-containing trisaccharide compound is fixed to the metal microparticles, the metal colloid is stably dispersed, and microparticles for detection of the virus can be obtained. Here, the binding ratio of the sialic acid-containing trisaccharide compound on the surface of the metal fine particles can be controlled by the concentration of the sialic acid-containing trisaccharide compound in the solution and other conditions (for example, solvent, temperature, time, etc.).

Usually, the binding rate of the sialic acid-containing trisaccharide compound on the surface of the metal fine particles 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 sialic acid-containing trisaccharide compounds of the formulas (7) to (11), a side chain site and a linker site are configured with a specific number of atoms. In this case, in particular, when 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 sialic acid-containing trisaccharide compounds are bound to each other by the folded side chain site. It is presumed that it may be effectively bonded to the substrate surface or the fine particle surface with an orientation suitable for binding with the Thurs virus. Thereby, it is presumed that the preferable bond ratio as described above was achieved.

In addition, it is preferable that such microparticles for detecting a thirsvirus or a third spike protein are stored in a solution form from the viewpoint of the storage stability of the microparticles.
In this case, the fine particles the formula (7) to sialic acid-containing trisaccharide compound represented by (11) is attached _{is, -C (= O) -C n} H 2n -Y or -CH ₂ -C _n H _2n In the linker moiety represented by -Y, since n is preferably an integer of 2 to 10, more preferably 3 to 8, particularly preferably 3 to 5, the length of the linker moiety is within a specific range, Excellent dispersion stability in solution. Therefore, the reagent containing fine particles to which the sialic acid-containing trisaccharide compound of the present invention is bound is particularly excellent in use in a solution.

  The immobilization of the sialic acid-containing trisaccharide compound on the metal fine particles is aimed at high orientation and high-density fixation of the sialic acid-containing trisaccharide compound. It is preferable to fix the mixture by mixing the compounds. However, when an excessive amount of the sialic acid-containing trisaccharide compound is mixed, a removal operation from the mixed solution is required.

For example, in the case of gold fine particles, a sialic acid-containing trisaccharide compound is obtained from the dispersion stability of a dispersion solution of gold fine particles in which a sialic acid-containing trisaccharide compound is immobilized, and the relationship between the amount of addition of a thirsvirus or its spike protein and the color change. The optimum mixing amount can be determined.
That is, when the added compound is less than the optimum amount, it does not reach a sufficient amount for self-organizing the sialic acid-containing trisaccharide compound, so that the hydrophobicity of the gold fine particle surface increases and the dispersion stability decreases. Color change and agglomeration-precipitation occur.
In addition, when the added compound is more than the optimum amount, the agglutination reaction is inhibited by the compound added excessively after adding the virus or spike protein thereof, and the color tone change becomes small. Therefore, optimal immobilization can be carried out by controlling the color tone to be within a large range when no change in color tone or aggregation occurs, and when Thurs virus or spike protein thereof is added.

A reagent for detecting a thirsvirus or a third spike protein By immobilizing a sialic acid-containing trisaccharide compound to the metal fine particles, a fine particle for detecting a thirsvirus or a third spike protein can be obtained. It can be used as a reagent for detecting a thirsvirus of the invention.
For this reason, it is necessary that the solvent used for the immobilization does not denature the virus to be detected or its spike protein. 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.

  Examples of the buffer include phosphate buffer, acetate buffer, HEPES buffer, or Tris buffer, more preferably HEPES buffer (10 mM, pH: 7.5, NaCl: 0.15N).

  In this way, with the synthesis of the microparticles for detecting a thursvirus or a third spike protein, a thirsvirus detection reagent containing the thirsvirus or the third spike protein detecting microparticle in a solvent is obtained. In such a third virus or third spike protein detection reagent, fine particles are preferably present in the form of a colloid in the solution, and the colloid is more preferably dispersed.

For example, in the case of gold colloid, confirm that the colloidal gold has been stabilized if there is no change in the color of the gold colloid by adding an equal volume of 10% sodium chloride aqueous solution to the mixed solution. can do.
The concentration of the third virus for detecting a third virus in the third virus 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 microparticles for detecting the virus or the spike protein can be dispersed and exist stably, improving the storage stability and improving the detection sensitivity of the virus or spike protein. Can be improved.

<Method of detection of Thurs virus or Thurs spike protein (2)>
The method of detecting a Thurs virus or Thurs spike protein according to the present invention comprises the following steps.
(1) A step of adding a test compound to the above-mentioned reagent for detecting a thirsvirus or thirst spike protein and contacting the thirs virus or thirst spike protein detecting microparticles;
(2) A step of detecting a thirsvirus spike protein or a thirsvirus bound to a thirsvirus or thirst spike protein detection microparticle.

As a means for detecting a thirsvirus spike protein or thirsvirus bound to a thirsvirus or thirst spike protein detection microparticle, for example, in a colloidal solution of a thirsvirus detection microparticle, A method of detecting the spike protein of the Thurs virus or Thurs virus from the color change upon binding is mentioned.
Specifically, for example, in the case of gold microparticles, the solution shows a red color in a state where the colloidal particles of the microparticles are dispersed in a state where the spike protein of Thurs virus or Thurs virus is not bound to the microparticles for detecting Thurs virus. When the spiked protein of Thurs virus or Thurs virus is bound, the fine particles are aggregated and the red color is faded or becomes colorless and transparent, or aggregates (precipitates) are formed.

  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.

<Third virus or Third spike protein adsorbent>
The sialic acid-containing trisaccharide compound can be used as an adsorbent for a thurs virus or a thirst spike protein because the trisaccharide moiety into which a sialic acid residue has been introduced specifically binds to the thirsvirus spike protein.
Examples of the adsorbent include sheet-like materials such as non-woven fabrics and woven fabrics, and spray-like materials sprayed in a solution.

  For example, if a solution containing the sialic acid-containing trisaccharide compound in an aqueous solution such as physiological saline is used as a mouthwash or aerosol spray, the infection prevention effect is high, and it should be used as a nosocomial infection prevention reagent. You can also.

Moreover, the adsorption sheet which removes a thirs virus can also be obtained by swelling the solution containing a sialic acid containing trisaccharide compound in a nonwoven fabric or a woven fabric, and drying a water | moisture content.
For example, non-woven masks such as the N95 mask (Sumitomo 3M, USA) approved by the National Institute of Occupational Safety and Health are known to prevent infection with Thurs virus, but are difficult to breathe. Thus, by adsorbing the sialic acid-containing trisaccharide compound to a normal mask or the like, it is possible to trap the Thurs virus without feeling breathlessness. In addition, it is possible to remove the thirs virus by adsorbing a sialic acid-containing trisaccharide compound to an air conditioner filter, an air cleaner filter, or the like.

Examples of the present invention will be specifically described below, but the present invention is not limited to these examples.
In the following examples, the third spike protein was purchased from Protein Sciences Corporation (Connecticut, Meriden). BIAcore 3000 (Pharmacia Biosensor AB, Uppsala, Sweden) was used as the surface plasmon resonance (SPR) device and sensor chip. ¹ H-NMR was measured in heavy water using a JEOL LA-600 spectrometer. The enzyme used was Sigma or Glycoscience.
[Example 1]
Process 1

(1) Synthesis of Compound 1a As a hydrolase, β-galactosidase (Sigma) derived from Bacillus circulans was used. Usually, the hydrolase β-galactosidase contains β-N-acetylhexosaminidase (hereinafter referred to as NAHase) as a contaminant enzyme, and this contaminant enzyme is p-nitrophenyl β-DN— Since acetylglucosamine (hereinafter referred to as GlcNAcβ-pNP) may be hydrolyzed, it is necessary to purify it in advance and remove contaminating enzymes. NAHase activity was removed using Phenyl Sepharose 6 Fast Flow (Amersham Biosciences).

Next, GlcNAcβ-pNP (1.0 g) and lactose (4.0 g) were dissolved in 35 mL of sodium phosphate buffer (20 mM, pH 6.8) containing 50% acetonitrile, and the purified β-galactosidase (3.2 U) was added. The mixture was reacted at 30 ° C. and monitored by HPLC using Amide-80 column (manufactured by Tosoh Corporation).
The reaction was stopped 24 hours after the maximum amount of Compound 1a, and purification was performed with ODS-C18 (Merck), Toyopearl HW-40S (Tosoh) and BioGel P2 (BioRad) to obtain 265 mg of Compound 1a.
By this simple and simple method, it was possible to synthesize compound 1a in large quantities using β-galactosidase.

Compound 1a: ¹ H NMR (600 MHz, D ₂ O) δ 8.205 (d, J = 9.0 Hz, 2H, o-Ph), 7.149 (d, J = 9.0 Hz, 2H, m-Ph), 5.303 (d , J = 7.8 Hz, 1H, H-1), 4.476 (d, J = 7.8 Hz, 1H, H'-1), and 1.987 (s, 3H, NHAc).

(2) Synthesis of Compound 2a The sialylation of Compound 1a was performed using recombinant rat-derived α2,3- (N) -sialyltransferase to synthesize cyaryllactosamine trisaccharide compound 2a.
40 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.3) containing Compound 1a (10 mg, 15.8 μmol), CMP-Neu5Ac (10.5 mg, 16.0 μmol), BSA (1.2 mg / mL) and 20 mM MnCl ₂ , 0.8 mL), 25 U alkaline phosphatase and α2,3-sialyltransferase (20 mU) (derived from recombinant rats) were incubated at 30 ° C.

The reaction was monitored by HPLC. After 48 hours of incubation, HPLC analysis confirmed that most of compound 1a was sialylated.
The reaction mixture was purified by chromatography on ODS (15% acetonitrile (containing 0.1% TFA as eluent) and BioGel P2 column, and the cyaryllactosamine trisaccharide compound 2a was 91% based on donor. (14.5 mg).

Compound 2a: ¹ H NMR (600 MHz, D ₂ O) δ 8.183 (d, J = 9.0 Hz, 2H, o-Ph), 7.141 (d, J = 9.0 Hz, 2H, m-Ph), 5.310 (d , J = 7.8 Hz, 1H, H-1), 4.568 (d, J = 7.8 Hz, 1H, H'-1), 2.730 (dd, J = 12.0, 4.8 Hz, 1H, H-3 _e ``), 2.010, 1.990 (2s, 6H, 2 NHAc), and 1.782 (t, J = 12.0 Hz, 1H, H-3 _a "); ¹³ C NMR δ 77.120 (C-3); [α] _D =-46.7 ( 1.20, water).

Process 2

(3) Synthesis of compound 5a:
Compound 2a was reduced in the presence of hydrogen and converted to p-aminophenyl derivative 4a.
Next, 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methlmorpholinium chloride (DMT-MM, 2.9 mg) was added to lipoic acid (1.9 mg) and reduced form 4a ( 8 mg) in ethanol solution. The mixture was stirred at room temperature for 24 hours, and ethanol was removed by concentration under reduced pressure. The residue was purified with an ODS column to obtain 7.4 mg of compound 5a.

Compound 4a: ¹ H NMR δ 6.925 (d, J = 8.4 Hz, 2H, o-Ph), 6.825 (d, J = 8.4 Hz, 2H, m-Ph), 4.991 (d, J = 8.4 Hz, 1H, H-1), 4.539 (d, J = 7.6 Hz, 1H, H'-1), 2.719 (dd, J = 12.0, 4.8 Hz, 1H, H-3 _e ``), 2.010, 2.001 (2s, 6H, 2 NHAc), and 1.771 (t, J = 12.0 Hz, 1H, H-3 <sub>a</sub> ``).
Compound 5a: ¹ H NMR (600 MHz, D ₂ O) δ 7.317 (d, J = 9.0 Hz, 2H, o-Ph), 7.038 (d, J = 9.0 Hz, 2H, m-Ph), 5.116 (d , J = 8.4 Hz, 1H, H-1), 4.586 (d, J = 7.8 Hz, 1H, H'-1), 2.723 (dd, J = 12.0, 4.8 Hz, 1H, H-3 _e "), 1.999, 1.983 (2s, 6H, NHAc), and 1.772 (t, J = 12.0 Hz, 1H, H-3 _a ``).

[Example 2]
Process 3

(1) Preparation of compound 3a:
Using rat liver α2,6-sialyltransferase as an enzyme, compound 3a was obtained from compound 1a (10 mg) in high yield (15.0 mg, calculated based on 93% donor) in the same manner as the synthesis of compound 2a. .
In both syntheses of compounds 2a and 3a, although the donor CMP-Neu5Ac was used in an equimolar ratio to the acceptor, compound 1a was efficiently sialylated. Although donor CMP-Neu5Ac is expensive, in the present invention, a high yield is obtained by using an equimolar amount of donor / acceptor. Therefore, this method is an attractive and practical method.

Compound 3a: ¹ H NMR δ 8.232 (d, J = 9.6 Hz, 2H, o-Ph), 7.176 (d, J = 9.6 Hz, 2H, m-Ph), 5.351 (d, J = 8.4 Hz, 1H, H-1), 4.465 (d, J = 7.2 Hz, 1H, H'-1), 2.668 (dd, J = 12.0, 4.8 Hz, 1H, H-3 _e ``), 2.010, 1.998 (2s, 6H, 2 NHAc), and 1.723 (t, J = 12.0 Hz, 1H, H-3 _a "); ¹³ C NMRδ65.086 (C-6); [α] _D =-132.7 (0.43, water)

(1) Synthesis of Siaryl Conjugate 7a Compound 3a was reduced in the presence of hydrogen and converted to p-aminophenyl derivative 6a. Next, DMT-MM (2.3 mg) in 1.0 mL of ethanol was added to an ethanol solution of lipoic acid (1.5 mg) and compound 6a (6 mg) at room temperature. After stirring at room temperature for 24 hours, ethanol was removed by concentration under reduced pressure. The residue was purified by ODS column to give compound 7a (5.2 mg).

Compound 6a: ¹ H NMR δ6.979 (d, J = 8.4 Hz, 2H, o-Ph), 6.908 (d, J = 8.4 Hz, 2H, m-Ph), 5.064 (d, J = 8.8 Hz, 1H, H-1), 4.437 (d, J = 8.0 Hz, 1H, H'-1), 2.639 (dd, J = 12.0, 4.8 Hz, 1H, H-3 _e ``), 2.025, 1.992 (2s, 6H, 2 NHAc), and 1.698 (t, J = 12.0 Hz, 1H, H-3 <sub>a</sub> ``).
Compound 7a: ¹ H NMR (600 MHz, D ₂ O) δ 7.320 (d, J = 9.0 Hz, 2H, o-Ph), 7.047 (d, J = 9.0 Hz, 2H, m-Ph), 5.155 ( d, J = 8.4 Hz, 1H, H-1), 4.444 (d, J = 7.8 Hz, 1H, H'-1), 2.644 (dd, J = 12.0, 4.8 Hz, 1H, H-3 _e ") , 2.019, 1.991 (2s, 6H, NHAc), and 1.747 (t, J = 12.0 Hz, 1H, H-3 _a ``).

Example 3
Preparation of sensor chip 1 bonded with sialic acid-containing trisaccharide compound (1) Preparation of sensor chip 1 bonded with compound 5a on the surface As a gold surface for immobilization of sialyl conjugate, SIA kit Au (manufactured by BIAcore) Using. The SIA kit Au plate washed with an ozone cleaner was immersed in a methanol solution (2 mL, 60 μM) containing compound 5a. After leaving at room temperature for 24 hours, the gold plate was taken out of the reaction solution, washed with methanol and water, and dried under a nitrogen stream to obtain a sensor chip 1.

(2) Preparation of sensor chip having compound 7a bonded to the surface In the same manner as the sensor chip having compound 5a bonded to the surface, sensor chip 2 having compound 7a bonded to the surface was obtained.

Example 4
Analysis of interaction between the surge spike protein and the sensor chips 1 and 2 each having the compound 5a and the compound 7a bound to the surface
The SARS spike protein solution having a concentration of 1 μg / mL was injected into an SPR device equipped with the sensor chip 1 or 2 having the compound 5a or compound 7a bound to the surface. As a result, it was found that SARS spike protein was bound on the surface of sensor chip 1 to which compound 5a was bound and sensor chip 2 to which compound 7a was bound.
The test result of the sensor chip 1 is shown in FIG. 1, and the test result of the sensor chip 2 is shown in FIG.

[Comparative Example 1]

  Commercially available p-nitrophenyl α-D-glucopyranoside (compound 9) (250 mg) was dissolved in water, palladium black (10 mg) was added, and reduction was performed in the presence of hydrogen at 1 atm and room temperature. The mixture was filtered through Celite to remove the catalyst, and concentrated under reduced pressure to obtain 225 mg (99%) of Compound 10. Subsequently, Compound 10 (225 mg, 0.83 mmol) and succinimidyl 1,2-dithiolane-3-pentanoate (300 mg, 0.99 mmol) were dissolved in 5 ml of dry DMF and reacted at 25 ° C. for 48 hours. Compound 11 was obtained (200 mg, 59%).

¹ H NMR (400 MHz, CD ₃ OD) of Compound 11: δ 7.45 (d, aromatics, J = 9.0 Hz), 7.119 (d, aromatics, J = 9.0 Hz), 5.416 (d, H-1, J = 3.6 Hz), 3.829 (t, H-3, J = 9.4 Hz), 3.542 (dd, H-2, J = 3.6 and 9.6 Hz), 3.404 (t, H-4, J = 9.2 Hz), 3.21 -3.05 (m, dithiolane of lipoic acid, 2H), 2.50-2.41 (m, dithiolane of lipoic acid, 1H), 2.353 (t, -CH ₂ -CONH-, J = 7.4Hz), 1.94-1.84 (m, dithiolane of lipoic acid, 1H), 1.80-1.62 (m, -CH _2- , 4H) and 1.56-1.45 (m, -CH _2- , 2H).

[Comparative Example 2]
Preparation of sensor chip 3 to which compound 11 was bound In the same manner as in Example 3, sensor chip 3 having the surface bound to compound 11 produced in Comparative Example 1 was obtained.

[Comparative Example 3]
FIG. 3 shows the SPR when the SARS spike protein is injected into the sensor chip 3 of Comparative Example 2. FIGS. 1 and 2 show the SPR results of SARS spike protein for sensor chip 1 and sensor chip 2 to which sialic acid-containing trisaccharide compounds 5a and 7a are bound, respectively. As shown in FIG. In sensor chip 3 to which 11 was bound, SARS spike protein did not bind at all. This indicates that the SARS spike protein is specifically bound to the sensor chip 1 and the sensor chip 2 to which the compound 5a or 7a is bound, respectively.

  The sensor chip or reagent containing metal microparticles, in which the sialic acid-containing trisaccharide compound according to the present invention is immobilized on the substrate or the surface of the microparticles, can detect a very small amount of thirsvirus or its spike protein with high sensitivity and speed.

  By applying the sensor chip to a surface plasmon resonance (SPR) or a quartz crystal (Quartz Crystal Microbalance: QCM) that can detect a change in a ligand with high sensitivity without using a labeling substance, It is possible to detect a trace amount of Thurs virus or Thurs spike protein.

  The colloid detection method using metal fine particles, for example, the gold colloid aggregation method using gold fine particles, is a method for sensing a thirsvirus or a thirst spike protein that can be performed quickly and easily.

  The adsorbent containing the sialic acid-containing trisaccharide compound can be used as an adsorbent sheet for masks, filters, and the like, and is effective for preventing infection with Thurs virus by using a solution containing the adsorbent as a spray.

FIG. 1 is an SPR sensorgram of the interaction between the surface of the sensor chip 1 on which the compound 5a is immobilized and the spike protein of Thurs virus. FIG. 2 is an SPR sensorgram of the interaction between the surface of the sensor chip 2 on which the compound 7a is immobilized and the spike protein of Thurs virus. FIG. 3 is an SPR sensorgram of the interaction between the surface of the sensor chip 3 on which the compound 11 is immobilized and the spike protein of Thurs virus.

Claims (9)

The following general formula (1) :

[Wherein R ₁ represents —OH or —NHCOCH ₃ ;
One of R ₂ and R ₃ is represented by the following formula (2)

A sialic acid residue (wherein —COOM is —COOH or a physiologically acceptable salt thereof), and the other of R ₂ and R ₃ represents H;
X ₁ is represented by the following formula (3)

A divalent group represented by:
X ₂ is —C _n H _2n − (n = 2-6);
Y is selected from the group consisting of a 1,2-dithiolan-3-yl group, a 1,2-dithiolan-4-yl group, a 1,2-dithian-3-yl group, and a 1,2-dithian-4-yl group. The disulfide group selected is shown. ] A sensor chip for detecting a thirsvirus or a thirst spike protein, wherein a sialic acid-containing trisaccharide compound represented by the formula: The sensor chip according to claim 1, wherein n is 4 and Y is a 1,2-dithiolan-3-yl group. The following general formula (1) :

[Wherein R ₁ represents —OH or —NHCOCH ₃ ;
One of R ₂ and R ₃ is represented by the following formula (2)

A sialic acid residue (wherein —COOM is —COOH or a physiologically acceptable salt thereof), and the other of R ₂ and R ₃ represents H;
X ₁ is represented by the following formula (3)

A divalent group represented by:
X ₂ is —C _n H _2n − (n = 2-6);
Y is selected from the group consisting of a 1,2-dithiolan-3-yl group, a 1,2-dithiolan-4-yl group, a 1,2-dithian-3-yl group, and a 1,2-dithian-4-yl group. The disulfide group selected is shown. ] A microparticle for detecting a thirsvirus or a thirst spike protein, wherein the sialic acid-containing trisaccharide compound represented by the formula is immobilized on the surface of the metal microparticle.   The fine particles according to claim 3, wherein n is 4 and Y is a 1,2-dithiolan-3-yl group. A reagent for detecting a thirsvirus or a third spike protein, wherein the microparticles according to claim 3 or 4 are present in a colloid in a solution. A method for detecting a Thurs virus or Thurs spike protein comprising the following steps:
(1) A step of bringing a test body into contact with the substrate surface of the sensor chip according to claim 1 or 2 ,
(2) A step of detecting a thirsvirus spike protein or thirsvirus bound to the substrate. A method for detecting a Thurs virus or Thurs spike protein comprising the following steps:
(1) adding a test specimen to the reagent according to claim 5 and bringing the specimen into contact with the fine particles according to claim 3 or 4 ;
(2) A step of detecting a thirsvirus spike protein or a thirsvirus bound to the microparticles. The following general formula (1) :

[Wherein R ₁ represents —OH or —NHCOCH ₃ ;
One of R ₂ and R ₃ is represented by the following formula (2)

A sialic acid residue (wherein —COOM is —COOH or a physiologically acceptable salt thereof), and the other of R ₂ and R ₃ represents H;
X ₁ is represented by the following formula (3)

A divalent group represented by:
X ₂ is —C _n H _2n − (n = 2-6);
Y is selected from the group consisting of a 1,2-dithiolan-3-yl group, a 1,2-dithiolan-4-yl group, a 1,2-dithian-3-yl group, and a 1,2-dithian-4-yl group. The disulfide group selected is shown. Or a third spike protein adsorbent containing a sialic acid-containing trisaccharide compound represented by the formula: The adsorbent according to claim 8, wherein n is 4 and Y is a 1,2-dithiolan-3-yl group.

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