JP2007298334A - Saccharide immobilized body and use thereof - Google Patents

Abstract

The present invention provides a saccharide-immobilized product that realizes suppression of non-specific adsorption and firm immobilization on a surface in immobilization of saccharide on a carrier surface.
A saccharide-immobilized product in which a saccharide compound comprising a saccharide and a polyether is immobilized on the surface of a carrier via the polyether. More specifically, the sugar compound is immobilized on the surface of the carrier through covalent bonding or interaction between biologically relevant substances via the polyether.
[Selection figure] None

Description

  The present invention relates to an immobilized saccharide and use thereof. Specifically, a saccharide-immobilized product obtained by immobilizing a saccharide compound composed of a saccharide and a polyether on the surface of a carrier via the polyether, a sensor using the same, a method for analyzing a sample, and purification of a substance to be detected The present invention relates to a method, a specimen detection apparatus, and a functional molecule.

  In recent years, saccharides have attracted attention because of their role in the living body and their importance in relation to diseases and infectious diseases. It is very useful to clarify the relationship between biomolecules and saccharides, and to apply the information obtained thereby to medicine and diagnosis. As an example, it can be applied to a method for examining the concentration and dynamics of a specific biomolecule in a living body, and a method for detecting bacteria, viruses, toxins and the like in a specimen.

  Immobilized functional organic molecules that have the ability to bind to the substance to be detected on the surface of a solid carrier for the purpose of measuring the concentration of the substance to be detected in a specimen or for applications such as biosensors. ) Has been reported. As these functional organic molecules, which are also called “ligands”, many examples using proteins have been reported. As a use of the immobilized body in which the protein is immobilized, detection of a target substance using an antigen-antibody reaction or a sugar-lectin interaction has been reported.

An example using saccharides as a ligand has also been reported. As applications of the immobilized form in which saccharides are immobilized, application to detection of viruses, bacteria, toxins, and the like have been reported.
When immobilizing saccharides on a carrier and analyzing the interaction with biomolecules, what is important is the suppression of non-specific adsorption on the surface on which saccharides are immobilized and the stability of the immobilized saccharides.
In general, when a saccharide is used as a functional molecule, a portion connecting the saccharide and a carrier is necessary for immobilization. Compared with proteins, immobilization of small molecules such as saccharides results in a relatively large proportion of the connecting portions, and a greater proportion of the portions other than the saccharide exposed on the surface of the immobilized body. This exposed part is considered to be related to non-specific adsorption. Suppression of non-specific adsorption is important for reducing misrecognition of other coexisting substances when detecting a substance to be detected.

  In addition, proteins have a relatively strong hydrophobicity because they have a hydrophobic part, and they are also immobilized by hydrophobic interaction. However, because saccharides themselves are hydrophilic, such a method cannot be taken. Furthermore, proteins have many functional groups such as amino groups, hydroxycarbonyl groups, disulfide groups, etc., and are easy to immobilize using them. In many cases, saccharides have only hydroxyl groups and are immobilized. For this purpose, it is necessary to introduce a functional group, which is difficult in that it is necessary to perform an appropriate derivatization.

In order to solve these problems, for example, it has been known that polyether suppresses nonspecific adsorption in the field of surface modification. Have been reported (see Non-Patent Documents 1 and 2). The immobilized body described in Non-Patent Document 1 is obtained by binding a part of a saccharide to a carrier by a hydrophobic bond using a polyether. On the other hand, in the immobilized body described in Non-Patent Document 2, a ligand in which a part of saccharide is a polyether is immobilized in the same manner, but a polyether having a plurality of functional groups previously added is physically adsorbed by a drying process. It is produced by immobilizing the saccharide on the carrier and binding the saccharide to the immobilized polyether via the functional group.
Pohl et al., J. Am. Chem. Soc., 2005, 127, 13162-13163 Kataoka, K., Langmuir, 2004, 20 (26), 11285-11287

As described above, when saccharide is immobilized on a carrier and analysis of interaction with a biomolecule is performed, suppression of non-specific adsorption and stability of the immobilized saccharide are important.
Conventionally, hydrophobic bonds have been frequently used for immobilization of sugar compounds. The reason is that the synthesis step can be simplified and the immobilization is easy because the step of introducing a specific functional group or the like into the sugar compound to be immobilized is easy. However, the hydrophobic bond cannot be said to be a strong bond, and if the bond to the carrier is weak, the sugar compound may be detached during washing or contact with the specimen, which may be inappropriate in practice. In the detection of biomolecules, washing operations for separating signals derived from bound and free analytes are widely used, and it is also common to use a surfactant during washing and detection, A compound immobilized with a weak bond such as a hydrophobic bond may be released.

On the other hand, it has been known that bonds such as covalent bonds and interactions between biological substances are strong and useful. However, synthesis of sugar compounds is generally difficult, and it is necessary to identify these strong bonds. The introduction of functional groups and the like further complicates the process.
In addition, it was also known to introduce a polyether into a part of the molecule for the purpose of suppressing nonspecific adsorption, but there are few reports of a compound in which a saccharide and a polyether are directly bonded, and it is more suitable for this. There were no examples of synthesizing a sugar compound suitable for an immobilized body prepared by binding a functional group or the like and using it for a covalent bond or the like. In order to be used for a sensor or the like, synthesis and immobilization must be performed while maintaining the function of the saccharide as much as possible so that the reaction in the living body can be reproduced, and further advanced techniques are required.

  For example, the immobilized body described in Non-Patent Document 1 is intended to suppress non-specific adsorption by using a part of saccharide as a polyether, and this is immobilized on a carrier using a hydrophobic bond. Therefore, it cannot be said that the bonding force is sufficient, and there is a problem that it is lost from the surface by the cleaning operation. The immobilization product described in Non-Patent Document 2 is also not strong because the immobilization of the polyether to the carrier surface is a physical adsorption method by drying, and the production method is complicated and affects the function of sugars. There was a risk of affecting.

Thus, there are few reports of saccharide-immobilized bodies that can suppress non-specific adsorption and are firmly immobilized, and the molecular structure of a preferred sugar compound to be used for the immobilization has not been clarified. It was. There has been a demand for the preparation of a highly stable sugar compound or sugar-immobilized body that can be used suitably for applications such as sensors, which solves such problems.
The present invention has been made in view of the above problems, and the object thereof is to use a saccharide-immobilized body capable of suppressing non-specific adsorption and firmly immobilizing on a carrier surface, and using the same. It is intended to provide a sensor, a specimen detection method and a detection apparatus, and a functional molecule used therein.

  In view of the above problems, the present inventors have intensively studied a novel saccharide-immobilized product. As a result, a saccharide compound composed of a saccharide and a polyether is immobilized on a carrier surface by covalent bond or interaction between biologically relevant substances. It has been found that by using the obtained saccharide-immobilized product, it is possible to produce an immobilized product that satisfies both the suppression of non-specific adsorption and the strong immobilization on the surface of the carrier. The obtained immobilized body had sufficient sensitivity even when this was used as a sensor.

That is, according to the present invention,
(1) A saccharide-immobilized product, wherein a saccharide compound comprising a saccharide and a polyether is immobilized on the surface of a carrier via the polyether,
(2) The immobilized body according to (1) above, wherein the immobilization is performed by a covalent bond,
(3) The immobilized body according to (1) above, wherein the immobilization is performed using an interaction between biologically relevant substances having a dissociation constant of 1 × 10 ⁻⁸ or less,
(4) The immobilized body according to (3) above, wherein the interaction between the biological substances is a biotin-avidin interaction,
(5) The immobilized body according to any one of (1) to (4) above, wherein the sugar compound is further immobilized on the surface of the carrier via a spacer,
(6) The spacer is a divalent hydrocarbon group, -S (sulfur atom)-, -O (oxygen atom)-, carbonyl group, amino group, -Si (silicon atom)-, maleimide group, protein and these The immobilized body according to (5) above, which is selected from the group consisting of any one of the combinations,
(7) The bond between the spacer and the carrier is a metal-S bond, C-Si bond, O-Si bond, C (= O) -NH bond, C (= O) -O bond, NH-C (= O ) Bond, N═C bond, NH—CH bond, O—C (═O) bond, and triazole, the fixing according to (5) or (6) above, Chemical,
(8) The fixing according to any one of (5) to (7) above, wherein the spacer is formed by bonding a bifunctional thiol or a bifunctional trialkoxysilane to the support surface. Chemical,
(9) The polyether is characterized in that the hydrocarbon group constituting it is selected from the group consisting of a divalent alkylene group, a divalent arylene group, and a combination thereof. (1) to the immobilized body according to any one of (8),
(10) The immobilized body according to any one of (1) to (9) above, wherein the polyether is polyethylene glycol having 2 or more repeating units,
(11) The saccharide-polyether bond is selected from the group consisting of an ether bond, glycoside bond, sulfur glycoside bond, N (nitrogen atom) glycoside bond, C = N double bond, and reductive amination reaction product. The immobilized body according to any one of (1) to (10) above,
(12) The immobilized body according to any one of (1) to (11) above, wherein the saccharide is a monosaccharide or a polysaccharide.
(13) Any of (1) to (12) above, wherein the saccharide is a monosaccharide selected from the group consisting of sialic acid, galactose, glucose, fucose and mannose, or a derivative thereof, or a polysaccharide containing them. The immobilization body according to claim 1,
(14) The immobilization according to any one of (1) to (13) above, wherein the saccharide has a sialic acid derivative which may have a substituent as a non-reducing terminal sugar. The body is provided.

Furthermore, according to the present invention,
(15) The sensor, wherein the immobilized body according to any one of (1) to (14) above forms a sensor surface,
(16) A detection principle of at least one selected from the group consisting of a quartz crystal microbalance, a cantilever, a field effect transistor, color development, fluorescence emission, optical interference, chemiluminescence, surface plasmon resonance, optical waveguide, and surface acoustic wave The sensor as described in (15) above is provided, wherein the sensor is used for detection.

According to another aspect of the present invention,
(17) The sensor according to the above (15) or (16) is brought into contact with a specimen containing a detected substance that can specifically interact with the sugar compound, and the interaction between the sensor and the detected substance is measured. A method of analyzing a specimen, characterized by detecting,
(18) The immobilized body according to any one of (1) to (14) is brought into contact with a specimen containing a target substance that can specifically interact with the sugar compound, and the immobilized body. An analyte analysis method comprising: detecting a target substance specifically bound to the sugar compound after separating the analyte from the analyte;
(19) The immobilized body according to any one of the above (1) to (14) is brought into contact with a specimen containing a target substance that can specifically interact with the sugar compound, and the immobilized body. A substance to be detected that is specifically bound to the sugar compound, after separating the sample from the sample,
(20) The method according to (18) or (19) above, wherein a magnetic material is used for the carrier,
(21) The substance to be detected is at least one selected from the group consisting of RNA, DNA, sugars, proteins, toxins, glycolipids, oligopeptides, viruses, bacteria, and derivatives thereof, The method according to any one of (17) to (20) above,
(22) The sensor according to (15) or (16) above, a sample supply unit that supplies the sample to bring the sample into contact with the sensor, and detection for detecting an interaction between the sensor and the sample And a specimen detecting device.

According to yet another aspect,
(23) A functional molecule comprising at least a saccharide, a polyether, and a spacer, wherein the polyether is bonded to the saccharide at one end and the spacer at the other end, and the spacer is a carrier. A molecule characterized by being capable of binding to a biological bond or an interaction between biological materials,
(24) The number of monosaccharides constituting the saccharide is 1 or more and 20 or less, and the hydrocarbon group constituting the polyether is a divalent alkylene group, a divalent arylene group, or a combination thereof The functional group of the spacer is selected from the group consisting of amino group, mercapto group, biotin, hydroxycarbonyl group, N-hydroxysuccinimide ester, maleimide group, formyl group, azide group, alkoxysilyl group. And a molecule as described in (23) above, which is at least one functional group selected from the group consisting of metal chelate polyamines.

  ADVANTAGE OF THE INVENTION According to this invention, it is a saccharide fixed body which fix | immobilized the saccharide compound on the surface, Comprising: Nonspecific adsorption | suction is suppressed and the fixed body firmly fixed to the support | carrier surface is provided.

Hereinafter, the present invention will be described in detail, but the present invention is not limited to the following description, and various modifications can be made within the scope of the gist of the present invention.
[I. Basic concept of the present invention]
In the immobilized body of the present invention, a sugar compound is immobilized by a strong bond such as a covalent bond or an interaction between biologically related substances, and it is used under normal storage conditions or by a sensor using the immobilized body. Alteration under sample measurement conditions can be prevented. In addition, when performing these bonds, unlike the case of hydrophobic bonds or the like, a sugar compound and a specific functional group for bonding to the carrier surface are required, but a specific place on the carrier surface is treated in advance. By doing so, it becomes possible to bind the sugar compound only to a specific region, and there is also an advantage that the position where the sugar compound is immobilized can be specified in the preparation of the immobilization carrier.
In addition, the use of a saccharide compound composed of a saccharide and a polyether is one of the major features of the immobilized body of the present invention. Since the polyether is appropriately used, the immobilized body has an effect of suppressing nonspecific adsorption.

[II. Saccharide immobilized body]
The saccharide-immobilized product according to the present invention (hereinafter sometimes abbreviated as “immobilized product of the present invention” or simply “immobilized product”) is a saccharide compound having a saccharide-polyether structure. It is immobilized on the surface of the carrier via ether.

[II-1. Sugar compounds and spacers]
The sugar compound used in the immobilized body of the present invention is not particularly limited as long as it has a saccharide-polyether structure in the molecule.
Moreover, it is preferable that a saccharide | sugar compound further has a spacer containing the functional group (bonding group) etc. for fixation to a support | carrier. Hereinafter, each of these components and preferred combinations thereof will be described in detail.

<II-1-1. Sugar>
In the present invention, “saccharide” refers to a monosaccharide and derivatives thereof (hereinafter collectively referred to as “monosaccharide”), and a compound in which two or more monosaccharides are linked by a glycosidic bond (hereinafter collectively referred to as “monosaccharide”). "Polysaccharide"). In addition, polysaccharides are sometimes referred to as “sugar chains”. The saccharide may be a naturally occurring molecule or a synthesized molecule. In addition, it may be a monosaccharide having a molecular structure that exists in nature or a polysaccharide composed thereof, and may be modified by addition or substitution of a functional group, but it may be a simple sugar having a molecular structure that exists in nature. Saccharides or polysaccharides composed thereof are preferred.

-Monosaccharide The kind of monosaccharide is not particularly limited, and any monosaccharide can be used. Specifically, either aldose or ketose may be used. The number of carbon atoms in the main skeleton is not particularly limited, but is usually 3 or more, preferably 4 or more, more preferably 5 or more, and usually 9 or less, preferably 8 or less, more preferably 6 or less. That is, pentose (carbon number 5) and hexose (carbon number 6) are particularly preferable, and hexose (carbon number 6) is most preferable. Specific examples of pentose include xylose, ribose, ribulose, xylulose and the like. Specific examples of hexose include glucose, mannose, fructose, galactose and the like. As other naturally-derived sugars, sialic acid (9 carbon atoms) is preferable.

  Examples of monosaccharides having a naturally occurring molecular structure include amino sugars (glycosamine) such as glucosamine, galactosamine, neuraminic acid; uronic acids such as glucuronic acid and iduronic acid; aldonic acids such as gluconic acid; Sorbitol), ribitol, xylitol and the like; deoxy sugars such as oxyribose, fucose and the like. In addition, a monosaccharide having a naturally occurring molecular structure having a substituent such as an acetyl group on a nitrogen atom or an oxygen atom of the monosaccharide described above can also be used. As examples of such derivatives of monosaccharides having an acetyl group on the nitrogen atom, N-acetylglucosamine, N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid, N -Sialic acid such as glycylneuraminic acid.

  Each of the above-mentioned monosaccharides and derivatives thereof has two types of isomers, D-type and L-type (hereinafter, D-type isomer is “D-form” and L-type isomer is “L-form”). Abbreviated). Any of these may be used and may be appropriately selected according to the purpose, but the D-form having a large natural abundance ratio can often favor the interaction with the specimen.

-Polysaccharides Polysaccharides (sugar chains) are those in which two or more monosaccharides (monosaccharides and derivatives thereof) are directly connected by glycosidic bonds as described above. The number of monosaccharides constituting the polysaccharide is not particularly limited, and may be selected from a practical viewpoint such as ease of preparation and its function. In the present invention, the polysaccharide is formed by directly binding 20 or less monosaccharides. (This may be referred to as “oligosaccharide”) is particularly preferably used. The number of monosaccharides constituting the oligosaccharide is preferably 10 or less, more preferably 5 or less, particularly 4 or less, and especially 3 or less.
The kind of monosaccharides constituting the polysaccharide is arbitrary, and can be arbitrarily selected from the above-mentioned monosaccharides (monosaccharides or derivatives thereof) according to the purpose. The polysaccharide may be composed of one kind of monosaccharide (homopolysaccharide, simple polysaccharide), or may be composed of two or more kinds of monosaccharide (heteropolysaccharide, complex polysaccharide). .
Also, the type of glycosidic bond between the monosaccharides is not particularly limited, and may be either α bond or β bond.

  Among the monosaccharides constituting the polysaccharide, it is preferable to use a specific monosaccharide, particularly as the non-reducing terminal sugar. Specific examples include sialic acid, galactose, galactosamine, glucose, fucose, mannose and the like. Of these, sialic acid and galactosamine are preferable, and sialic acid is particularly preferable. Sialic acid has a substituent on the amino group or hydroxyl group of neuraminic acid, among which N-acetylneuraminic acid, N-glycolneuraminic acid, 9-O-acetyl-5-N -Acetylneuraminic acid.

Polysaccharides (oligosaccharides) whose non-reducing terminal sugar is sialic acid are collectively referred to as “sialyl sugar chains”. Examples of sialyl sugar chains include sialyl lactose sugar chains, sialyl ganglio sugar chains, sialyl lactose sugar chains, and the like.
Examples of sialyllacto-type sugar chains include type I and type II, and examples of type I include Neu5Acα2-6 (3) Galβ1-3GlcNAcβ1- and Neu5Acα2-6 (3) Galβ1-3GlcNAcβ1-3Galβ1. Examples include Neu5Acα2-6 (3) Galβ1-4GlcNAcβ1- and Neu5Acα2-6 (3) Galβ1-4GlcNAcβ1-3Galβ1. In the above formula, “2-6 (3)” indicates that the linking position of the sugar may be a 2-6 bond or a 2-3 bond, and may be properly used depending on the purpose. The following description is the same.

Examples of sialylganglio sugar chains include Neu5Acα2-6 (3) Galβ1-4GalNAcβ1-, Neu5Acα2-6 (3) Galβ1-4GalNAcβ1-4Galβ1-, Neu5Acα2-6 (3) Galβ1-3GalNAcβ1-, and Neu5Ac2- 3) Galβ1-3GalNAcβ1-4Galβ1- is mentioned.
Examples of sialyllactose sugar chains include Neu5Acα2-6 (3) Galβ1-4Glcβ1- and Neu5Acα2-6 (3) Galβ1-3Glcβ1-. Examples of other sialyllacto-based sugar chains include 9-O-NeuAcα2-6 (3) Galβ1-, Neu5Acα2-3Galβ1-4Glcβ1-, and 5-N-glycol Neuα2-3Galβ1-4Glcβ1-.

Examples of the acidic oligosaccharide include those having HO3S-3Gal1- at the non-reducing end, and include sugar chains contained in sulfatides.
Other oligosaccharides include those having Galβ1-3GalNAcβ1-4Glcβ1- and GalNAcβ1-4Glcβ1-.
Examples of proteoglycan and glycosaminoglycan include hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratan sulfate.
Among the above saccharides, those having the above-mentioned monosaccharides or polysaccharides formed by directly binding 20 or less monosaccharides are preferred, and the monosaccharides selected from the group consisting of sialic acid, galactose, glucose, fucose and mannose Alternatively, those having derivatives thereof or polysaccharides containing them are particularly preferred.

<II-1-2. Polyether>
The saccharide compound used in the immobilized body of the present invention comprises the above saccharide (monosaccharide or polysaccharide) and polyether, and is characterized in that they are directly bonded. As the saccharide compound to be immobilized, it is preferable to use a non-specific adsorption at a portion other than the saccharide and a highly hydrophilic one. From this viewpoint, a polyether was selected. Further, by directly bonding a polyether to a saccharide, a nonspecific adsorption factor can be eliminated without affecting the function of the saccharide, and the compound can be immobilized.

  The polyether used in the present invention is formed by alternately bonding a plurality of divalent hydrocarbon groups via oxygen atoms (hereinafter, these may be referred to as “polyether groups”). The divalent hydrocarbon group constituting this is preferably a polyether group containing at least one group selected from an alkylene group or an arylene group, and may be a combination thereof. In particular, an alkylene group is preferred.

  The number of carbon atoms of the divalent hydrocarbon group that is the structural unit of the polyether group is usually preferably 2 or more, and usually 12 or less, especially 6 or less, more preferably 4 or less, and particularly preferably 3 or less. Most preferred. Such divalent hydrocarbon groups include hexadecylene group (-n-C16H32-), pentadecylene group (-n-C15H30-), tetradecylene group (-n-C14H28-), tridecylene group (-n-C13H26-). ), Dodecylene group (-n-C13H26-), undecylene group (-n-C11H22-), decylene group (-n-C10H20-), nonylene group (-n-C9H18-), octylene group (-n-C8H16-) ), Heptylene group (-n-C7H14-), hexylene group (-n-C6H12-), pentylene group (-n-C5H10-), butylene group (-n-C4H8-), propylene group (-CH3CH6-), An ethylene group (—CH 2 CH 4 —) is preferable, among which a butylene group, a propylene group and an ethylene group are preferable, a propylene group and an ethylene group are particularly preferable, and an ethylene group is particularly preferable. This is because the ethylene group has the highest hydrophilicity and is unlikely to cause nonspecific adsorption.

  Moreover, the hydrocarbon group which becomes a structural unit of a polyether group may have a substituent. Examples of the substituent include a hydrocarbon group, an amino group, a halogen atom, a hydroxyl group, an alkoxy group, an aryloxy group, an aminocarbonyl group, a hydroxycarbonyl group, an alkoxycarbonyl group, and an alkylcarbonyl group. The hydrocarbon group may have one or two or more substituents, and when having two or more substituents, these substituents may be the same as or different from each other. It may be. In addition, it is preferable that these substituents are selected so as to avoid or minimize undesirable side reactions at the time of immobilization of the sugar compound, and if selected in relation to immobilization conditions and functional groups. Good. Economically, those having no substituent are often preferred.

  When having a substituent, the plurality of hydrocarbon groups constituting the polyether group may be the same as or different from each other including the substituent. From the viewpoint of ease of production, it is preferable that all the structural units are the same. However, the objective functions such as strength of interaction with the specimen, hydrophilic / hydrophobic properties, non-specific adsorption, resistance, etc. You should choose it with a balance. Economically, the same thing is often preferable.

  The number of hydrocarbon groups constituting the polyether group (the number of repeating units of the polyether group) is not particularly limited, and may be selected according to the type of hydrocarbon group and the entire length of the polyether. Above, preferably 3 or more, particularly preferably 4 or more, and particularly preferably 5 or more. Further, it is usually 50 or less, preferably 20 or less, more preferably 8 or less, and particularly preferably 6 or less.

  As for the length of the polyether group, if the polyether group is too long, the functional sugar moiety is covered with the polyether group, and depending on the reaction conditions, the interaction with the sample may be hindered. . On the other hand, if the polyether group is too short, it may not be long enough to interact with the receptor. The length of the polyether, in terms of the number of atoms in the main chain, is usually 8 or more, more preferably 11 or more, particularly 14 or more, usually 60 or less, especially 45 or less, more preferably 36 or less, and particularly preferably 24 or less. . When the sugar is a monosaccharide, the size thereof is smaller than that of a polysaccharide, and thus a long polyether is preferable. The number of atoms in the main chain is usually 11 or more, more preferably 14 or more, particularly 17 or more, usually 60. In the following, it is preferable that the range be 45 or less, particularly 24 or less. However, the length of the polyether group in this embodiment is the number of atoms on the shortest path when the covalent bond is sequentially traced from the atom of the functional group bonded to the carrier to the atom of the polyether group bonded to the saccharide. Define.

  Among the above, polyethylene glycol having 2 or more repeating units is preferably used for the saccharide-immobilized product of the present invention, among which polyethylene glycol having 3 or more repeating units is preferably used, and among them, 4 or more repeating units are used. Polyethylene glycol is particularly preferably used, and polyethylene glycol having 5 or more repeating units is most preferably used. The upper limit of the repeating unit of polyethylene glycol is not limited, but polyethylene glycol having a repeating unit of 200 or less is preferably used. Among them, polyethylene glycol having a repeating unit of 100 or less is preferably used, and among them, polyethylene having a repeating unit of 50 or less. Glycol is particularly preferably used, and polyethylene glycol having a repeating unit of 14 or less is most preferably used.

The polyether is bonded to a saccharide at one end, and is bonded to a carrier or a spacer described later at the other end.
The bond between the saccharide and the polyether is formed by, for example, an ether bond, a glycoside bond, a sulfur glycoside bond, an N (nitrogen atom) glycoside bond, a C = N double bond, or the like. Moreover, a reductive amination reaction product may be sufficient. A C = N double bond and a reductive amination reaction product are obtained by reacting a reducing end of a sugar with a polyether having an amino group at the end.

  The polyether may be bonded at any position of the saccharide. The position where the saccharide-polyether bond is present in the saccharide compound is more suitable for the purpose as it is farther from the site deeply involved in the function expression, but may be selected in view of the ease of preparation. As an example, there may be a carbon atom called anomer position, or other positions, but it is preferably bonded to a polyether (glycoside bond) at the anomeric position. In the case of a polysaccharide, it is a glycoside bond or a reductive amino acid. It is preferable that the reducing end sugar is bonded in such a manner that it is ring-opened by the reaction.

  When the saccharide and the polyether are glycosidically bonded, the atom bonded to the anomeric carbon of the saccharide is shared as the terminal atom of the polyether. Examples of this atom (hereinafter referred to as “bonding atom”) include an oxygen atom, a sulfur atom, a nitrogen atom, and a carbon atom. Among these, an oxygen atom, a sulfur atom and a nitrogen atom are preferable, an oxygen atom and a sulfur atom are particularly preferable, and an oxygen atom is most preferable. In this case, the structure of the sugar compound is “sugar- [bonding atom] -polyether- [functional group]”.

<II-1-3. Spacer>
In the saccharide-immobilized product of the present invention, a saccharide compound comprising a saccharide and a polyether is immobilized on a carrier through the polyether, but the saccharide compound is further immobilized through a spacer. Is preferred. That is, in this case, the structure of the immobilized body is “saccharide-polyether-spacer-carrier”.

In the present specification, the spacer includes all those having the ability to bind a polyether and a carrier in the above structure. The term “bond” as used herein is preferably a bond that utilizes a covalent bond or an interaction between biological substances.
A covalent bond is a bond that is generated when atoms share a closed shell structure and stabilize when electrons share electrons. Therefore, the bond between the atoms is very strong. The interaction between biological substances is a strong affinity generated between an organic compound and a biological substance existing in a natural living body or a material synthesized from the biological substance. The dissociation constant is 1 × 10 ⁻⁸ or less. There are many types of combinations of substances exhibiting the interaction between the biological substances, but those having 1 × 10 ⁻¹⁴ or less are particularly preferable, and examples thereof include biotin-avidin bond and the like.

  Examples of the spacer include a divalent hydrocarbon group, -S (sulfur atom)-, -O (oxygen atom)-, carbonyl group, amino group, -Si (silicon atom)-, maleimide group, protein and these The thing etc. which consist of a combination are mentioned. Examples of the bond between the spacer formed using such a spacer and the carrier include, for example, a metal-S bond, a C-Si bond, an O-Si bond, a C (= O) -NH bond, and C (= O). Examples include —O bond, NH—C (═O) bond, N═C bond, NH—CH bond, O—C (═O) bond, and triazole. In addition, a spacer having a structure formed by bonding a bifunctional thiol or a bifunctional trialkoxysilane to the support surface is also used.

  More specifically, examples of the spacer used for the covalent immobilization include a case where only a functional group is used, and a case where a protein, a dienofile and the like are used. For example, the functional group includes amino group, azide group, mercapto group, disulfide group, hydroxycarbonyl group, maleimide group, formyl group, alkoxysilyl group, hydroxyl group, vinyl group, acetylene group, butadienyl group, oxyethylene group, etc. Among these, an amino group, an azide group, a mercapto group, a vinyl group, and the like are preferable, an amino group or an azide group is preferable, and an amino group is particularly preferable. Examples of the protein include avidin, albumin and the like. In the case of avidin, the type is not limited, but streptavidin is preferable. As albumin, bovine serum albumin (hereinafter sometimes abbreviated as “BSA”) and porcine serum albumin (hereinafter sometimes abbreviated as “PSA”) are preferable, and BSA is particularly preferable. . Moreover, as a dienophile, an acetylene group etc. are illustrated, for example.

  For example, when the above-mentioned divalent hydrocarbon group is selected as the spacer and covalently bonded to the carrier, the structure of the sugar compound is [saccharide]-[polyether group]-[hydrocarbon group]-[ The structure is a functional group for covalent bonding]. In such a sugar compound, if a divalent hydrocarbon group having a large number of carbon atoms is used as the hydrocarbon group, the combination of the functional group and the material of the carrier can be selected appropriately to bring the sugar compound into contact with the carrier. The hydrocarbon group part forms a monomolecular film. Therefore, the immobilization process can be simplified compared to the process of immobilizing the sugar compound after forming a monomolecular film on the surface of the carrier as described later. Such a divalent hydrocarbon group preferably has 4 or more carbon atoms, more preferably 6 or more, and particularly preferably 10 or more. In addition, when the sugar compound is immobilized on the carrier, the structure is [saccharide]-[polyether group]-[hydrocarbon group]-[carrier], and the hydrocarbon group is covered with the polyether group. It will be in the same state as Therefore, when the obtained immobilized body of the present invention is used for a sensor or the like, nonspecific adsorption can be further reduced. Further, since such a sugar compound forms a liposome in water, it may be bound to the carrier as it is, or may be bound to the carrier by forming a monomolecular film by contacting with the carrier. .

  In this case, the carbon number of the divalent hydrocarbon group to be bonded to the polyether is usually 2 or more, and usually 20 or less, particularly 16 or less, more preferably 12 or less, and particularly preferably 6 or less. Examples of such a divalent hydrocarbon group include a hexadecylene group, a pentadecylene group, a tetradecylene group, a dodecylene group, an undecylene group, a decylene group, a nonylene group, an octylene group, a heptylene group, a hexylene group, a pentylene group, a butylene group, and a propylene group. , Ethylene group, phenylene group, biphenylene group, naphthylene group and the like. Of these, a decylene group, a butylene group, a propylene group, an ethylene group, a phenylene group, a biphenylene group, a naphthylene group, and the like are preferable, and among them, a decylene group, a butylene group, a propylene group, and an ethylene group are particularly preferable.

A mercapto group or disulfide group is suitably used as a functional group when a metal such as gold, silver or platinum is present on the surface of the support. For example, when a spacer having a mercapto group or a disulfide group as a functional group comes into contact with gold on the surface of the carrier, a gold atom-sulfur atom bond is generated, thereby immobilizing the sugar compound on the surface of the carrier.
In addition, a vinyl group, an acetylene group, a butadienyl group, and an oxyethylene group are suitably used as a functional group when the carrier surface is formed of silicon or the like. When a spacer having these groups as functional groups comes into contact with silicon on the support surface, a Si-alkyl group is formed by reaction with the Si—H group on the silicon surface, thereby immobilizing the sugar compound on the support surface.

Examples of the spacer used for immobilization by the interaction between biological substances include small molecules or proteins. Examples of small molecules include molecules that bind strongly to proteins with dissociation constants as described above, such as biotin, metal chelate polyamine groups, polyhistidine, flag tags, and glutathione. Among these, biotin is preferred. . Examples of the protein include avidin, GST protein, etc. Among them, avidin is preferable, and streptavidin is more preferable among them. Polyhistidine, flag tag, and glutathione are oligopeptides. Among them, polyhistidine and glutathione are preferably used.
Examples of these spacers will be described in more detail in the following specific examples and description of the immobilization method.

<II-1-4. Specific examples of sugar compounds and spacers>
Among the saccharide compounds and spacers used in the saccharide immobilization product of the present invention, particularly preferred specific examples are shown below. These are immobilized on the surface of the carrier by a functional group contained in the spacer. However, the present invention is not limited by the following examples unless it exceeds the gist. In the following formulae, “Me” represents a methyl group, “Et” represents an ethyl group, and “Ac” represents an acetyl group.

  In addition, specific examples of the structure in a state where the sugar compound is immobilized on a carrier are shown below. Note that atoms on the support surface are displayed at the right end of the structural formula (“Au” or “Si—O” corresponds to this).

  Avidin is immobilized on a self-organized membrane formed on a gold surface by an amide bond, and a sugar compound is immobilized by biotin-avidin interaction. Three representative examples of 16-mercaptopalmitic acid-derived portions constituting the self-assembled membrane are exemplified.

[II-2. Carrier
There are no particular limitations on the material of the carrier used in the immobilized body of the present invention, and any material can be used as long as it can immobilize the above-described sugar compound. Examples include glass, quartz, silicon, metal, metal oxide, plastic and the like. These materials may be properly used depending on the use of the immobilized body, the type of sugar compound, the binding method used, and the like.

For example, when the immobilized body of the present invention is used for a QCM (Quartz Crystal Microbalance) sensor, quartz or gold is preferably used as the carrier material.
In addition, when the immobilized body of the present invention is used for an FET (field-effect transistor) sensor, gold, silicon, functionalized carbon nanotubes, or the like is preferably used as the carrier material. The

Further, when the immobilized body of the present invention is used for an SPR (Surface Plasmon Resonance) sensor, gold or the like is preferably used as the carrier material.
Further, when the immobilized body of the present invention is used for a sensor based on MEMS (Micro-Electro-Mechanical Systems) technology, silicon or the like is preferably used as the carrier material.
In the case of using the sugar compound of the present invention immobilized on magnetic particles, a magnetic material such as a metal oxide is preferably used as the carrier material.

In addition, when the immobilized body of the present invention is used for ELISA (enzyme-linked immunosorbent assay), plastic or glass is preferably used as the carrier material.
The carrier materials listed above may be immobilized on a support made of another material, and the target sugar compound may be immobilized using this as a carrier. For example, for SPR sensor applications, a gold thin film can be formed on a support made of glass or plastic and used as a carrier. For QCM sensor applications, a gold thin film can be formed on a support made of quartz and used as a carrier.

When plastic is used as a carrier material, it is possible to immobilize a sugar compound by a method described later by immobilizing a functional group such as an amino group on the surface of the plastic (functionalization). is there. Such an example is illustrated in M. Schwarz et al., Glycobiology, 2003, Vol. 13, No. 11, pp. 749-754.
There is no restriction | limiting in particular about the shape of a support | carrier, What is necessary is just to select suitably according to the function of a fixed body, a use, etc. Examples include a flat plate shape and a particle shape. Further, the size of the carrier is not particularly limited, and may be selected in consideration of functionality and economy. For example, when gold is formed on a support and used as a carrier, the thickness of the gold thin film is usually 1 nm or more, preferably 5 nm or more, and usually 10 μm or less, preferably 1 μm or less.

  When the carrier is in the form of particles, it may be useful for separation and purification of a substance to be detected that specifically binds to a sugar compound or for detection using the same. Examples of the material of such particles include polymers, metals, metal oxides, and composites thereof. Examples of the polymer include latex, polyester, polyamide and the like. Examples of the metal include those containing gold, silver, platinum, iron, tin, and a rare earth metal. As the material having ferromagnetism, one containing iron or a rare earth metal is preferable, and among them, an alloy containing iron is particularly preferable. Gold, silver, and platinum are preferred as those that do not have ferromagnetism, and those containing gold are particularly preferred.

Commercially available insoluble carriers widely used for detection of antigen-antibody reaction and the like can also be used. For example, magnetic particles, polymers such as polystyrene, or latex thereof, gelatin, liposomes and the like are preferably used. Among them, magnetic particles are particularly preferable from the viewpoint of realizing quick and simple solid-liquid separation using magnetic force, and specifically, for example, triiron tetroxide (Fe ₃ O ₄ ), diiron trioxide (Fe ₂ O). ₃ ) Magnetic particles such as fine particles made of various ferrites, metals such as iron, manganese, nickel, cobalt and chromium, and alloys such as cobalt, nickel and manganese are preferably used. In addition, it is possible to preferably use those magnetic particles prepared in a form contained in a polymer latex such as polystyrene, gelatin or liposome, or immobilized on the surface.

[II-3. (Method of immobilizing sugar compounds)
Examples of the method for immobilizing the sugar compound of the present invention on the carrier described above include the following methods (i) to (v).
(I) A method of immobilizing a sugar compound directly or via the spacer on the carrier surface.
(Ii) A method of immobilizing a sugar compound directly or via the spacer on the self-assembled film formed on the surface of the carrier.
(Iii) A method of immobilizing a sugar compound directly or via the spacer using a polymer or the like immobilized on the surface of a self-assembled film formed on the surface of the carrier.
(Iv) A method of immobilizing a protein or the like on the surface of a carrier and immobilizing a sugar compound on the protein or the like directly or via the spacer.
(V) Other.

Hereinafter, each method (i) to (v) will be described.
(I) Method for Immobilizing a Sugar Compound on the Support Surface Directly or via the Spacer When immobilizing the sugar compound of the present invention directly on the support surface, usable bonds and reactions include metal-sulfur atom bonding, O Examples include -Si bond, ester bond, amide bond, biotin-avidin bond, Schiff basification reaction, reductive amination reaction, hydrosilylation reaction, Diels-Alder reaction, reaction of azide group and acetylene group. Among these, a metal-sulfur atom bond, an amide bond, a biotin-avidin bond, and a Schiff basification reaction are preferable, among which a metal-sulfur atom bond and an amide bond are particularly preferable, and among them, a metal-sulfur atom is particularly preferable. Bonding is preferred.

  Further, as a technique for immobilizing the sugar compound of the present invention on the support surface, a sugar compound having a mercapto group as a functional group contained in the spacer is used, and a metal atom is formed on the support surface having gold, silver, or platinum atoms. -Immobilization by sulfur atom bonding, or by using a sugar compound having an alkoxysilyl group as a functional group and reacting with a Si-OH group on the surface of a carrier made of glass, quartz, etc. to form an O-Si bond Immobilization by immobilizing and reacting with the carrier surface made of silicon using a sugar compound having a vinyl group, acetylene group, butadienyl group, oxyethylene group, etc. as a functional group to form a Si-C bond For example. As a method of immobilizing by a metal atom-sulfur atom bond, a method of immobilizing by a gold atom-sulfur atom bond is preferable. In a sugar compound having an alkoxysilyl group as a functional group, a triethoxyxysilyl group is preferred as the functional group.

  In addition, when a divalent hydrocarbon group is selected as the spacer, the structure of the saccharide compound is [saccharide]-[polyether group]-[hydrocarbon group]-[functional group for covalent bond] as described above. Thus, for a specific number of hydrocarbons, a divalent hydrocarbon group may form a self-assembled film. By selecting such a hydrocarbon group, the step of forming a self-assembled film in advance as described later can be omitted.

Further, when utilizing the reaction between an azide group and an acetylene group, a sugar compound having an azide group as a functional group is used, and 1, 1 with respect to an acetylene group present on the surface of a carrier such as gold, glass, silicon, or plastic. The sugar compound can be immobilized by causing a 3-cycloaddition reaction to form the corresponding triazole. Such an example is exemplified in CH. Wong et al., Journal of the American Chemical Society, 2002, Vol. 124, No. 48, pp. 14397-14402.
The thickness of the immobilization layer of the sugar compound immobilized on the carrier surface from the carrier surface is not limited, but is usually 1 nm or more, usually 100 μm or less, especially 10 μm or less, more preferably 1000 nm or less, particularly 100 nm or less. Is preferred.

(Ii) A method of immobilizing a sugar compound directly or via a spacer on a self-assembled film formed on the surface of the carrier. Self-assembled monolayer (hereinafter referred to as “SAM”) on the surface of the carrier. As a method for forming the above, for example, a method in which a compound having thiol or disulfide is reacted with a support surface having gold, silver, or platinum atoms, or a method in which alkoxy is applied to the support surface made of glass or metal oxide. Examples thereof include a method of reacting a compound having a silyl group or a chlorosilyl group. The compounds used for the production of these self-assembled films may have a terminal functional group, and examples of such a terminal functional group include a hydroxycarbonyl group, a succinimide carbonyl group, an amino group, and a hydroxyl group. As a method of immobilizing by a metal atom-sulfur atom bond, a method of immobilizing by a gold atom-sulfur atom bond is preferable. In a sugar compound having an alkoxysilyl group as a functional group, a triethoxysilyl group is preferred as the functional group.

  As a method for binding a sugar compound to the self-assembled film formed on the support surface, for example, ω-hydroxycarbonyl α-thiol or bis (ω-hydroxycarbonylalkyl) disulfide is self-attached to the support surface having a gold atom. Examples thereof include a method of immobilizing a sugar compound by using an amide bond, an ester bond, a reaction with an azide group, or the like with respect to the hydroxycarbonyl group of the self-assembled film obtained. Examples of ω-hydroxycarbonyl α-thiol include 16-mercaptopalmitic acid, 14-mercaptomyristic acid, 12-mercaptolauric acid, 11-mercaptoundecanoic acid, 10-mercaptocapric acid, 9-mercaptopelargonic acid, 8- Examples include mercaptocaprylic acid, 7-mercaptoenanthic acid, 6-mercaptocaproic acid, 5-mercaptovaleric acid, 4-mercaptobutyric acid, and 3-mercaptopropionic acid. Examples of bis (ω-hydroxycarbonyl) disulfide include 4,4′-dithiodibutyric acid and the like.

  Various condensing agents can be used for immobilizing the sugar compound on the self-assembled membrane. Among them, the hydroxycarbonyl group of the self-assembled membrane is once converted to N-hydroxysuccinimide (N-hydroxysuccinimide). : The following is abbreviated as “NHS” as appropriate.) A method of amidation by reacting with an amino group of a sugar compound is preferred. In the NHS conversion, in addition to NHS, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (1-Ethyl-3- (3-dimethylaminopropyl) -carbodiimidehydrochloride: Can be used in combination. However, if a thiol or disulfide having a carbonyl group whose terminal is NHS is used in the production of the self-assembled film, this NHS step is not necessary.

  In addition, when ω-amino α-thiol or bis (ω-aminoalkyl) disulfide is self-assembled on the surface of a carrier having a gold atom, the surface of the carrier becomes an amino group and is fixed by an amide bond of the hydroxycarbonyl group of the sugar compound. And can be immobilized by a Schiff base bond of a formyl group of a sugar compound or a reductive amination reaction. Various reactants can be used in this amidation, but a method in which the hydroxycarbonyl group of the sugar compound is first converted to NHS is also possible. In the case of binding with a Schiff base, a dehydrating agent may be used. In the reductive amination reaction, the generated Schiff base is reduced to form a single bond between the carbon atom and the nitrogen atom. Sodium cyanoborohydride, diborane, picoline borane and the like are preferable as the reducing reagent. In the reductive amination reaction, the reducing reagent may coexist from the beginning or after the Schiff base is formed.

  The self-assembled film on the carrier may be subjected to functional group conversion before binding to the sugar compound. Examples of such methods include conversion of amino group to formyl group, conversion of hydroxycarbonyl group to NHS carbonyl group and avidin, conversion of hydroxyl group to formyl group, conversion to NHS carbonyl group and avidin, and the like. . These transformations may be performed using a reagent or may be performed by reacting with a bifunctional compound. As an example of this, there is a technique in which glutaraldehyde is reacted with a self-assembled film whose carrier surface is an amino group. In this case, the portion reacted with the amino group becomes a Schiff base or an aminomethylene group, and the unreacted functional group remains as a formyl group, so that it can be used for immobilizing a sugar compound having an amino group.

In a method of reacting a compound having an alkoxysilyl group or a chlorosilyl group with a support surface made of glass, metal oxide or the like, Si (glass) -O-Si or metal atom-O-Si is formed on the support surface. Can be fixed. Examples of such a reactive agent include aminopropyltriethoxysilane.
When a diene is used as the functional group at the terminal of the sugar compound, the sugar compound can be immobilized by reacting with Dienophile contained in the self-assembled film and Diels-Alder reaction. The diene includes a cyclopentadienyl group, and the dienophile includes a quinone. Such an example is illustrated in M. Mrksih et al., Chemistry & Biology, 2002, Vol. 9, pp. 443-454.

  When a maleimide group is used as the terminal functional group of the sugar compound, the ω-mercapto group contained in the self-assembled film is reacted with the carbon-carbon double bond (C = C) of the maleimide group of the sugar compound. Thus, the sugar compound can be immobilized. Such an example is illustrated in I. Shin et al., Angewandte Chemie [International Edition], 2002, Vol. 41, No. 17, pp. 3180-3182.

  When a diene is used as the functional group at the terminal of the sugar compound, the sugar compound can be immobilized by reacting with Dienophile contained in the self-assembled film and Diels-Alder reaction. The diene includes a cyclopentadienyl group, and the dienophile includes a quinone. Such an example is illustrated in M. Mrksih et al., Chemistry & Biology, 2002, Vol. 9, pp. 443-454.

  When an azide group is used as the terminal functional group of a sugar compound, triazole can be formed by reacting with the acetylene group contained in the self-assembled film, and the sugar compound can be immobilized. Conversely, it is also possible to use an acetylene group as the terminal functional group of the sugar compound, and to make the functional group of the self-assembled film an azide group.

(Iii) A method of immobilizing a sugar compound directly or via the spacer using a polymer or the like immobilized on the surface of a self-assembled film formed on the surface of the carrier Self-assembled on a solid surface When a polymer is immobilized on a membrane and a sugar compound is immobilized on the polymer, the type of polymer that can be used is not particularly limited, but examples include polysaccharides and organic polymers. Examples of polysaccharides include agarose, dextran, carrageenan, alginic acid, cellulose and the like. Examples of the organic polymer include polyvinyl alcohol, polyacrylic acid, polyacrylamide and the like. These functional groups may be subjected to derivatization treatment such as methylation.

  A general bond can be used as a bond for immobilizing the sugar compound to the polymer. Examples include ester bond, amide bond, Schiff basification reaction, reductive amination reaction, Diels-Alder reaction, azide group-acetylene group reaction, and the like. As an example of such a technique, a technique for immobilizing a sugar compound on a nitrocellulose membrane is exemplified in T. Feizi et al., Nature Biotechnology, 2002, Vol. 20, pp. 1011-1017.

(Iv) A method of immobilizing a protein or the like on the surface of a carrier and immobilizing a sugar compound on the protein or the like directly or via the spacer The protein is immobilized and immobilized by covalent bonding using a functional group on the surface of the carrier. It is also possible to immobilize a sugar compound on the protein. Examples of such a carrier material include plastic, metal, and glass, and plastic or metal is preferable. Of the plastics, polystyrene is particularly preferable. Among metals, gold is particularly preferable.

  Examples of a method for immobilizing a protein by a covalent bond include a method of forming an amide bond with an amino group in a protein by making the carrier surface a hydroxycarbonyl group and then NHS by the above-described method. The surface of the carrier may be functional group-converted by a self-assembled film by the method described above. As the protein, a protein that hardly causes nonspecific adsorption is preferable. Specifically, albumin or avidin is preferable. Examples of a method for immobilizing a sugar compound on an immobilized protein include a method in which a hydroxycarbonyl group on a protein is converted to an NHS group and then reacted with an amino group of the sugar compound to form an amide bond. Further, when the protein is avidin, it can be immobilized by a biotin-avidin bond by using a sugar compound having biotin. As albumin, bovine serum albumin (hereinafter abbreviated as “BSA” where appropriate) and porcine serum albumin (hereinafter abbreviated as “PSA” where appropriate) are preferable, and BSA is particularly preferable. Although there is no restriction | limiting in the kind of avidin, Streptavidin is mentioned. Such an example is exemplified in H. Seeberger, Angewandte Chemie [International Edition], 2002, Vol. 41, No. 19, pp. 3583-3586. The protein may be immobilized by contacting a protein dissolved in a buffer solution or pure water with a functionalized carrier at an appropriate temperature for an appropriate time, but the use of a buffer solution is preferred. Such a method is exemplified in W. G. T. Willats et al., Proteomics, 2002, Vol. 2, pp. 1666-1671. Avidin may be immobilized by biotin-avidin interaction after biotinylation of the carrier surface.

(V) Other:
Other techniques include, for example, a technique of using a monomer to which a saccharide compound is bonded and immobilizing a polymer obtained by polymerizing the monomer on the surface of the carrier.

[II-4. Conditions for immobilization of sugar compounds)
Conventionally used conditions can be used as the reaction conditions when the sugar compound of the present invention is immobilized on the sensor surface. Specifically, the reaction temperature is usually 0 ° C. or higher, especially 4 ° C. or higher, more preferably 10 ° C. or higher, particularly 20 ° C. or higher, usually 100 ° C. or lower, especially 60 ° C. or lower, more preferably 40 ° C. or lower, especially 30 ° C. or lower. Is preferred. The reaction pressure is preferably atmospheric pressure. As a solvent used for immobilization, an aqueous solvent or an organic solvent can be used. As the aqueous solvent, water, buffer solution, dilute DMSO aqueous solution and the like can be used. As the organic solvent, methanol, ethanol, acetone and the like are preferable, and methanol or ethanol is preferable among them.

[III. sensor〕
The above-described immobilized body of the present invention can be used for various applications, and particularly preferable applications include application to biosensors (hereinafter sometimes simply referred to as “sensors”). . The biosensor having the immobilized body of the present invention (hereinafter referred to as “the sensor of the present invention”) by immobilizing the sugar compound of the present invention by the above-described procedure using the sensor surface of the biosensor as the carrier surface. Is obtained).

Details of the sensor of the present invention will be described below.
[III-1. Sensor type)
Examples of biosensors to which the present invention can be applied include sensors using QCM, cantilevers, FETs, chemiluminescence, SPR, magnetic materials, optical waveguides, surface acoustic waves, and the like.
When the sensor of the present invention is configured as a quartz crystal microbalance, a sugar compound is immobilized on the surface of the quartz crystal serving as the sensor surface, or a gold thin film is formed on the surface of the quartz crystal, and then the gold thin film is formed on Immobilize the sugar compound. Alternatively, a polymer film or the like may be formed on the surface of the crystal oscillator or the gold thin film, and the sugar compound may be immobilized thereon. Thereby, the interaction between the sugar compound and the specimen can be observed as a change in frequency.

  When the sensor of the present invention is configured as a cantilever sensor, the surface of the cantilever serving as the sensor surface is formed of a material such as silicon, gold, or silicon oxide, and a sugar compound is immobilized on the surface. Thereby, the interaction between the sugar compound and the specimen can be observed as a shape change such as warpage of the cantilever. As a method for observing the change in the shape of the cantilever, there are a method in which a change in reflected light is detected by applying a laser beam, a method in which the cantilever is configured as a piezo element, and an electric observation is performed. In addition, when the cantilever is vibrated, the interaction between the sugar compound and the specimen can be detected as a change in frequency or a change in amplitude. It is also possible to detect a sample immobilized on a solid surface like an atomic force microscope (hereinafter abbreviated as “AFM”) using a cantilever on which a sugar compound is immobilized. It is.

When the sensor of the present invention is configured as an FET sensor, the interaction between the sugar compound and the specimen can be detected as a change in potential by immobilizing the sugar compound on or near the gate electrode serving as the sensor surface. it can.
When the sensor of the present invention is configured as an SPR sensor, the interaction between the saccharide compound and the sample is determined by surface plasmon resonance by immobilizing the saccharide compound on the gold surface serving as the sensor surface directly or via a polymer. It can be detected as a change in reflection angle due to or a change in reflection intensity at a specific reflection angle.

When the sugar compound of the present invention is immobilized on a magnetic substance, the magnetic substance is made into a magnetic particle or magnetic nanoparticle, and the sugar compound is immobilized on the surface thereof, so that the interaction between the sugar compound and the sample is superconducting quantum. It can be detected by a device using a superconducting quantum interference device (hereinafter abbreviated as “SQUID”) or a Hall element.
When the sensor of the present invention is configured as an optical waveguide sensor, the optical path serving as the sensor surface is divided into two and a sugar compound is immobilized on one surface. When an interaction between the sugar compound and the specimen occurs, a change in the speed or phase of light occurs, and this can be detected as interference with the other light.

  When the sensor of the present invention is applied to a surface acoustic wave detection device, by immobilizing a sugar compound on the sensor surface, the interaction between the sugar compound and the specimen can be detected as a change in stationary acoustic waves.

[III-2. (Sample)
Examples of substances (analytes, substances to be detected) that can be detected using the sensor of the present invention include RNA, DNA, sugar chains, proteins, lectins, protozoa, viruses, bacteria, toxins, glycolipids, etc., or fragments thereof, These mixtures are mentioned. A sugar compound may be selected according to the target substance to be detected.

・ Virus:
Examples of viruses that can be samples of the sensor of the present invention include hepatitis virus, HIV, influenza virus, Sendai virus, Newcastle disease virus, reovirus, rotavirus, coxsackie virus, echovirus, enterovirus, Japanese encephalitis virus, poliovirus, rhinovirus. Examples include viruses and herpes virus viruses. Examples of the hepatitis virus include hepatitis B virus. Influenza viruses include type A, type B, type C, or their coexistence. Reovirus includes type 1 and type 2. Examples of HIV include HIV-1 type, HIV-2 type, SIV and the like. Examples of the herpes virus include herpes type 1 virus. Only one of these viruses may be the detection target, but two or more types of viruses may be the detection target.

・ Lectin:
Examples of lectins that can serve as the sample of the sensor of the present invention include Japanese elder collectin, concavalin A and the like.
・ Bacteria:
Examples of bacteria that can be a sample of the sensor of the present invention include Escherichia coli and Helicobacter pylori.

·toxin:
A toxin is a general term for anything that affects the normal metabolism of an organism and stops its function. Examples of toxins that can be samples of the sensor of the present invention include, for example, verotoxin, botulinum toxin, and the like.
-Glycolipids Examples of glycolipids that can serve as a sample of the sensor of the present invention include glycosphingolipids in which a sugar chain and ceramide are bound, and glyceroglycolipids in which a sugar chain is bound with diciacylglycerol or alkylacylglycerol.

  When analysis is performed using the sensor having the immobilized body of the present invention (sensor of the present invention), the analysis is usually performed using various devices (specimen detection devices). The configuration of such an apparatus is not particularly limited, and may be appropriately selected according to the analysis method to be used and the type of specimen. As typical apparatus configurations, the sensor of the present invention described above and the sensor And a sample supply unit that supplies the sample to contact the sample that can specifically interact with the saccharide, and a detection unit that detects the interaction between the sensor and the sample. In this case, the sensor may be configured to be detachable.

[IV. (Sample analysis method)
By using the sensor of the present invention described above, a specimen containing a substance to be detected that can specifically interact with the sugar compound is brought into contact, and the interaction between the sensor and the substance to be detected is detected. Analysis can be performed.
The sensor and specimen used are as described above. The detection method can be performed by arbitrarily selecting or combining known methods known per se.
In the sensor of the present invention, the immobilized saccharide of the present invention forms the sensor surface, and is stably immobilized in a state where the function of the saccharide is maintained high. Therefore, the interaction between the sensor and the substance to be detected can be detected by contacting a specimen containing the substance to be detected that can specifically interact with the sugar compound. Non-specific adsorption is suppressed in the immobilized body of the present invention, and it can be preferably used for such a reaction. In particular, when a biological sample or the like is used as a specimen, it is often a problem that a contaminant present in the specimen causes a nonspecific reaction. However, in the immobilized body of the present invention, the sugar compound is stably present. Since it is immobilized, it is unlikely that the sugar compound is released even if operations such as washing and separation are performed, and detection with higher sensitivity and specificity can be performed.

[V. (Purification method of detected substance)
The immobilized body of the present invention can also be used in a method for purifying a substance to be detected.
Contacting the immobilized body with a specimen containing a substance to be detected that can specifically interact with the sugar compound, separating the immobilized body and the specimen, and then washing the sugar compound, Obtain the specifically bound substance to be detected. Since the saccharide compound is stably immobilized in the immobilized body of the present invention, it is unlikely that the saccharide compound will be released even if washing, separation, etc., and purification with high sensitivity and specificity is performed. Can do.

The purification method can be carried out by arbitrarily selecting a commonly used method known per se.
In such a case, for example, it is preferable to use the particulate carrier as described above as the carrier used for preparing the immobilized body. More specifically, particles made of a resin such as latex, and magnetic particles containing a metal in the particles are preferably used. As an example, the substance to be detected can also be dissociated by bringing the particles into contact with a liquid containing a separating agent and changing conditions such as temperature and pH. If the particles are removed, a solution containing only the substance to be detected can be prepared, so that the substance to be detected can be purified. When magnetic particles are used as a carrier, there is an advantage that they are easily transported by a magnetic field, and washing and separation processes are simplified. The purification operation using the magnetic particles and the like can also be appropriately performed by applying a known technique.

In addition, by analyzing the immobilized body on which the substance to be detected is immobilized by spectroscopic techniques, mass spectrometry, etc., it is possible to identify the substance to be detected and obtain information on the concentration. Become. Moreover, the liquid which liberated the to-be-detected to-be-detected substance can be used for detections, such as a diagnosis.
The substance to be detected may be any substance that binds to sugar, and examples thereof include proteins, viruses, bacteria, and toxins. Proteins are preferred.

[VI. (Sample detection device)
The specimen detection apparatus of the present invention includes a sensor according to the present invention, a specimen supply section that supplies the specimen to bring the specimen into contact with the sensor, and a detection section that detects an interaction between the sensor and the specimen. It is characterized by having.
The configuration and function of each part can be arbitrarily selected from known techniques.
By adopting such an apparatus configuration, automatic analysis using the saccharide immobilization product of the present invention becomes possible.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to a following example, unless the summary is exceeded.
In the following description, when referring to a compound represented in the reaction formula, the number attached to the compound is appended with parentheses, and is described as “compound (1)”. In the following formulae, “Me” represents a methyl group, “Et” represents an ethyl group, “Ac” represents an acetyl group, and “Bn” represents a benzyl group. Moreover, as a reagent and a solvent used for reaction, all the commercial items were used.

[Example 1: Synthesis of sugar compound]
In order to investigate the immobilization state and the amount of immobilization of sugar compounds over time, two monosaccharide sugar compounds having amino groups at the ends and fluorine atoms introduced were synthesized, their immobilization reaction and surface analysis. Went. Furthermore, a sugar compound containing a polysaccharide having an amino group at the end and a sugar compound containing a monosaccharide or a polysaccharide having a terminal biotin were synthesized.

[Synthesis of sugar compounds 1]
First, as steps 1 to 13 below, compounds (9) and (16) were synthesized as monosaccharide sugar compounds having a terminal amino group and a fluorine atom introduced.
-Step 1 (fluorination reaction):

First, a starting compound (compound (1)) was synthesized by the method described in Ito et al., Tetrahedron, 1990, Vol. 46, pp. 89-102. 1.02 g (1.5 mmol) of the obtained compound (1) was dissolved in 13.5 ml of dehydrated DMF (N, N-dimethylformamide) under a nitrogen atmosphere, and Selectfluor (registered trademark: 1-chloromethyl-4- 1) was dissolved. 2.17 g (6.1 mmol) of fluoro-1,4-diazoniabicyclo- [2.2.2] -octane / bis- (tetrafluoroborate)) and 4.5 ml of water were added, and the mixture was stirred at 60 ° C. for 1.5 hours. After cooling to room temperature, the mixture was diluted with ethyl acetate, washed with water and then with saturated brine, and dried by adding sodium sulfate. After the solvent was distilled off, the desired fluorinated product (compound (2)) was isolated by silica gel column chromatography (hexane / ethyl acetate = 3/2). The yield was 739 mg, and the yield was 69%. The obtained compound was identified by NMR. The results are shown below.
1H NMR (400MHZ, CDCl3, J in Hz); 4.88 (dd, 1H, J = 2.1 & 50.0), 4.85 (d, 1H, J = 11.8), 4.64 (d, 1H, J = 11.8), 4.64 (s , 2H), 4.59 (s, 2H), 4.55 (s, 1H, J = 11.8), 4.51 (d, 1H, J = 10.9), 4.42 (d, 1H, J = 11.8), 3.99 (dd, 1H, J = 2.2 & 10.8), 3.99 (m, 1H), 3.81 (t, 1H, J = 8.3), 3.80 (dd, 1H, J = 1.4 & 6.5), 3.77 (s, 3H), 3.72 (dd, 1H , J = 4.5 & 10.7), 1.60 (s, 3H).
13C NMR (100MHz, CDCl3, J in Hz); 94.22 (d, J = 24.7), 86.07 (d, J = 184.1), 78.47, 73.47 (d, J = 17.5), 70.09, 60.48, 53.26, 48.66, 23.60 .
Step 2 (TMS reaction):

Under a nitrogen atmosphere, 550 mg (0.8 mmol) of fluorohydrin (compound (2)) obtained in the previous step was dissolved in 10 ml of methylene chloride and cooled to -20 ° C. Trimethylsilyldiethylamine (0.30 ml, 8.0 mmol) was added and stirred for 1 hour. The reaction solution was poured into hydrochloric acid adjusted to 0.01 N with ice water and extracted with methylene chloride. Dried over sodium sulfate. After the solvent was distilled off, the desired methylated product (compound (3)) was isolated by silica gel column chromatography (hexane / ethyl acetate = 1/1). The yield was 470 mg and the yield was 78%. The obtained compound was identified by NMR. The results are shown below.
1H NMR (400MHz, CDCl3, J in Hz); 5.02 (dd, 1H, J = 1.5 & 51.2), 4.72 (d, 1H, J = 10.9), 4.70 (d, 1H, J = 11.1), 4.67 (s , 2H), 4.05 (m, 1H), 3.95 (m, 1H), 3.79 (dd, 1H, J = 2.1 & 10.7), 3.72 (d, 1H, J = 8.7), 3.67 (dd, 1H, J = 3.4 & 10.7), 3.58 (s, 3H), 1.77 (s, 3H), 0.20 (s, 9H).
13C NMR (100MHz, CDCl3, J in Hz); 96.67 (d, J = 17.3), 86.95 (d, J = 187.5), 77.68, 74.46, 74.42 (d, J = 18.9), 73.65, 73.30, 72.20, 71.08 , 72.28, 68.04, 52.44, 47.40, 21.04.
Step 3 (azidation reaction):

Under a nitrogen atmosphere, 5.0118 g (9.97 mmol) of tetraethylene glycol ditosylate (compound (4)) was added to 10.0 ml of dehydrated DMF in a 50 ml two-necked flask equipped with a reflux tube and dissolved. I let you. It was cooled to 0 ° C. with an ice water bath from the outside. While stirring, 0.3299 g (5.07 mmol) of sodium azide was added under a nitrogen atmosphere, and the temperature was raised to 50 ° C. after 50 minutes. The reaction solution was appropriately sampled, and the reaction was followed by thin layer chromatography (hereinafter abbreviated as “TLC”) (hexane / ethyl acetate = 1/1). After reacting at 50 ° C. for 1.5 hours, the reaction solution was cooled to room temperature, and the reaction solution was added to a mixed solvent of water 100 ml / ethyl acetate 200 ml. After oil-water separation, the aqueous phase was extracted twice with 100 ml of ethyl acetate, and all the ethyl acetate extracted phases were combined and washed with 100 ml of saturated aqueous sodium hydroxide. The extract was dried over sodium sulfate overnight, and then the solvent was distilled off. The target monoazide (compound (5)) was isolated by silica gel column chromatography (hexane / ethyl acetate = 1/1). The yield was 1.0919 g (2.92 mmol), and the yield was 29.3%. The obtained compound was identified by NMR. The results are shown below.
1H NMR (400MHz, CDCl3, J in Hz); 7.80 (dd, 2H, J = 8.20), 7.34 (dd, 2H, J = 8.20), 4.16 (t, 2H, J = 4.8), 3.69 (t, 6H , J = 5.0), 3.67 (t, 6H, J = 5.0), 3.60 (S, 4H), 3.38 (t, 2H, J = 5.1), 2.45 (S, 3H).
13C NMR (100MHz, CDCl3, J in Hz); 144.78, 132.99, 129.79, 127.96, 70.74, 70.65, 70.58, 70.02, 69.21, 68.66, 50.65, 21.62.
Step 4 (iodination reaction):

4. Dehydrated acetone containing 0.5069 g (1.36 mmol) of the monoazide (monotosylate, compound (5)) obtained in the previous step in a 50 ml two-necked flask equipped with a reflux tube under a nitrogen atmosphere. Added to 0 ml. At room temperature, 0.2987 g (1.99 mmol) of sodium iodide was added and stirred in a nitrogen atmosphere. Once dissolved, a white precipitate formed after 10 minutes. The mixture was heated in an oil bath 15 minutes after the addition of sodium iodide, and refluxing began 22 minutes later. The amount of precipitation gradually increased, but the reaction was stopped after 1 hour by cooling. After running off the solvent, the desired iodo compound (compound (6)) was isolated by silica gel column chromatography (hexane / ethyl acetate = 1/1). The yield was 0.3512 g (1.07 mmol), and the yield was 79.7%. The obtained compound was identified by NMR. The results are shown below.
1H NMR (400MHz, CDCl3, J in Hz); 3.76 (t, 2H, J = 7.0), 3.70-3.68 (m, 10H), 3.40 (t, 2H, J = 5.1), 3.27 (t, 2H, J = 6.8).
13C NMR (100MHz, CDCl3, J in Hz); 71.95, 70.70, 70.66, 70.21, 70.04, 50.67, 2.90.
Step 5 (coupling reaction):

Under nitrogen atmosphere, 0.3038 g (0.39 mmol) of the sialic acid derivative (OTMS form, compound (3)) obtained in Step 2, and 0.2545 g of the monoiodide form (Compound (6)) obtained in Step 4 (0.77 mmol) and 1.3 ml of dehydrated DMF were dissolved in a 10 ml flask. After dissolution, the mixture was cooled to −10 ° C., and 0.1054 g (0.69 mmol) of cesium fluoride (CsF) was added. After 15 hours of reaction, the reaction mixture was opened in 100 ml of hydrochloric acid adjusted to 0.01 N with ice water and extracted with 100 ml of ethyl acetate. The organic phase was separated and extracted again with 50 ml of ethyl acetate. The organic phases were combined, washed with saturated brine, dried over sodium sulfate, and the solvent was washed away. The target coupling product (compound (7)) was isolated by silica gel column chromatography (hexane / ethyl acetate = 9/1 to 4/6, the target product was 5/5 to 4/6). The yield was 0.1707 g (0.1890 mmol), and the yield was 48.1%. The obtained compound was identified by NMR. The results are shown below.
1H NMR (400MHz, CDCl3, J in Hz); 7.4-7.2 (m, 20H), 5.12 (dd, 1H, J = 38.6 & 1.6), 4.74 (s, 2H), 4.70 (s, 1H), 4.64 ( s, 2H), 4.58 (d, 2H, J = 3.9), 4.44 (m, 1H), 4.35 (s, 1H), 4.33 (s, 1H), 4.24 (dd, 1H, J = 8.1 & 1.3), 4.00 (m, 1H), 3.91 (m, 2H), 3.80 (m, 1H), 3.66-3.52 (m, 15H), 3.62 (s, 3H), 3. 36 (t, 2H, J = 3.8), 1.56 (s, 3H).
13C NMR (100MHz, CDCl3, J in Hz); 170.48, 166.52 (d, J = 3.3), 138.80, 138.40, 138.28, 137.64, 129.57, 128.52, 128.40, 128.17, 128.01, 127.97, 127.97, 127.84, 127.73, 127.61 , 127.34, 127.29, 97.93 (d, J = 19.1), 89.00 (d, J = 170.0), 79.63,74.13, 73.31 (d, J = 17.1), 73.38, 72.73, 71.89, 71.63, 70.65, 70.50, 70.48, 70.29, 69.97, 69.50, 69.41, 52.59, 50.66, 48.62, 46.62, 23.57.
Step 6 (hydrolysis reaction):

  0.1601 g (0.177 mmol) of the coupling product (methyl ester, compound (7)) obtained in the previous step was placed in a 25 ml flask, dissolved by adding 12.2 ml of methanol, and cooled to 0 ° C. A separately prepared homogeneous solution of lithium hydroxide monohydrate 0.0380 g (0.91 mmol) / water 3.1 ml was added. The ice water bath was removed and the temperature was gradually increased to room temperature. At one and a half hours later, no progress of the reaction was confirmed by TLC (hexane / ethyl acetate = 2/8). After 3 days, the completion of the reaction was confirmed, and an ion exchange resin (Diaion (registered trademark) SK1BH, H + type, manufactured by Mitsubishi Chemical Corporation) was added to adjust the pH to 4. After stirring at room temperature for 30 minutes, the mixture was filtered and the solvent was distilled off. The desired demethylated product (compound (8)) was isolated by silica gel column chromatography (ethyl acetate / methanol = 1/1). The yield was 118.6 mg (0.13 mmol), and the yield was 75.2%.

The obtained compound was identified by NMR. The results are shown below.
1H NMR (400MHz, CDCl3, J in Hz); 7.4-7.2 (m, 20H), 5.18 (dd, 1H, J = 38.7 & 1.2), 4.69 (s, 2H), 4.67 (s, 1H), 4.64 ( s, 2H), 4.55 (d, 2H, J = 3.6), 4.54-4.38 (m, 4H), 4.31 (dd, 1H, J = 21.1 & 8.1), 4.03 (dt, 1H, J = 2.1 & 3.9) , 3.93 (m, 2H), 3.73 (m, 1H), 3.67-3.51 (m, 15H), 3.35 (m, 2H), 1.53 (s, 3H)
13C NMR (100MHz, CDCl3, J in Hz); 171.17, 167.19 (d, J = 3.6), 138.67, 138.32, 138.16, 137.76, 129.32, 128.40, 128.34, 128.32, 128.24, 128.06, 127.97, 127.85, 127.81, 127.78 , 127.57, 127.46, 97.72 (d, J = 16.0), 87.04 (d, J = 187.7), 79.01, 74.34, 73.98 (d, 17.5), 73.35, 73.01, 72.50, 71.7, 71.49, 70.53, 70.50, 70.27, 70.09, 69.95, 69.79, 69.71, 64.30, 50.45, 48.45, 23.28.
Step 7 (reductive deprotection reaction):

0.0824 g (0.093 mmol) of the hydrolyzate (Bn-protected azide, compound (8)) obtained in the previous step was placed in a 10 ml flask, and 6.0 ml of methanol was added and dissolved. 1.8 ml of 1N hydrochloric acid prepared separately was added, 0.0158 g of 10% palladium carbon (Pd / C) catalyst was added, and the inside was replaced with hydrogen. The reaction was monitored by TLC (developed with methanol). The completion of the reaction was confirmed after 28 hours, and the solvent was distilled off after filtration. Purified by gel filtration (Amersham LH-20, methanol). After the solvent was distilled off, the residue was dissolved in methanol, an equal amount of 20 mM sodium acetate / methanol solution (4.8 ml) was added, and the mixture was stirred for 30 minutes at room temperature. Purification by gel filtration (Amersham LH-20, methanol) gave the target compound (compound (9)). The yield was 48.7 g (0.091 mmol), and the yield was 97.7%. The obtained compound was identified by NMR and mass spectrometry. The results are shown below. 1H NMR (400MHz, CDCl3, J in Hz); 5.06 (dd, 1H, J = 51.0 & 2.0), 5.23 (s, 1H), 5.10 (s, 1H), 4.18 (t, 1H, J = 10.7), 4.08 (t, 1H, J = 10.3), 3.98 (m, 2H), 3.39-3.67 (m, 16H), 3.54 (m, 2H), 2.02 (s, 3H).
13C NMR (100MHz, CDCl3, J in Hz); 175.89, 163.80 (d, J = 5.3), 99.88 (d, J = 46.4), 93.66 (d, J = 181.9), 75.04, 73.41, 71.81, 71.76, 71.46 , 71.17 (d, J = 24.3), 70.82, 70.34, 68.21, 66.28, 64.92, 49.22, 41.00, 23.11.
Mass spectrometry: ESI-MS method (Micromass type-ZMD)
Mass scanning conditions: 50-1500 m / e, 4.0 sec / scan
Applied voltage: 2.0 kV, Cone voltage: 30 V
M / z 503 (M + H) was observed by the ESI (+) method. Thereafter, m / z 525 (M + Na) and m / z 541 (M + K) were observed. In addition, m / z 501 (M−H) was observed by the ESI (−) method. The mass number of the target substance was MW502, which coincided with the mass number corresponding to the synthetic target product.
Step 8 (tosylation reaction):

A four-neck round bottom flask was charged with 24 ml of an aqueous 5.2 M sodium hydroxide (NaOH) solution and placed in an ice bath. 24 ml of a tetrahydrofuran (hereinafter abbreviated as “THF”) solution of 10.00 g (35.4 mmol) of hexaethylene glycol (Hexaethyleneglycol, compound (10)) was slowly added and stirred. 36 ml of a THF solution of 14.86 g (78.0 mmol) of tosyl chloride (TsCl) was added dropwise over 1 hour and stirred for 23 hours. A 10% hydrochloric acid (HCl) aqueous solution was added to adjust the pH to 4, followed by extraction with toluene (100 ml × 3). The organic phase was collected and washed with water (100 ml × 2), 0.5N sodium bicarbonate (NaHCO ₃ ) aqueous solution (100 ml) and water (100 ml) in this order. The organic phase was dehydrated with magnesium sulfate (MgSO ₄ ), filtered, and evaporated. Ditosyl compound (compound (11), yield 8.84 g, yield 83%) was isolated by silica gel column chromatography (spherical silica 100 g, n-hexane: ethyl acetate = 4: 1). The obtained compound was identified by NMR. The results are shown below.
1H NMR (400MHz, CDCl3, J in Hz); 7.80 (d, J = 8.3, 4H), 7.34 (d, J = 7.8, 4H), 4.15 (t, J = 3.9, 4H), 3.68 (t, J = 4.8, 4H), 3.62 (s, 8H), 3.58 (s, 8H), 2.45 (s, 6H).
13C NMR (100MHz, CDCl3, J in Hz); 144.77, 133.04, 129.81, 127.97, 70.74, 70.60, 70.54, 70.50, 69.22, 68.67, 21.62.
Step 9 (azidation reaction):

8.00 g (13.6 mmol) of ditosylate (compound (11)) obtained in the previous step was placed in a four-necked round bottom flask, and the atmosphere in the flask was replaced with nitrogen. Then, 27 ml of dehydrated DMF was added. Sodium nitride (NaN ₃ ) 0.882 g (13.6 mmol) was slowly added, heated to 50 ° C. and stirred for 3 hours. After cooling to room temperature, 200 ml of toluene and 100 ml of water were added to extract the organic phase. Further, the aqueous phase was extracted with toluene (100 ml × 2). The organic phase was collected, washed with water (100 ml × 3), dried over sodium sulfate (Na ₂ SO ₄ ), filtered, and then the solvent was distilled off. Monoazide compound (compound (12), 1.93 g, yield 31%) was isolated by performing silica gel column chromatography (spherical silica 200 g, n-hexane: ethyl acetate = 7: 13) twice. The obtained compound was identified by NMR. The results are shown below.
1H NMR (400MHz, CDCl3, J in Hz); 7.80 (d, J = 8.3, 2H), 7.34 (d, J = 7.8, 2H), 4.16 (t, J = 6.1, 2H), 3.69 (t, J = 5.9Hz, 2H), 3.66 (s, 6H), 3.65 (s, 4H), 3.63 (m, 4H), 3.59 (s, 4H), 3.38 (t, J = 5.1Hz, 2H), 2.45 (s , 3H).
13C NMR (100MHz, CDCl3, J in Hz); 144.77, 133.04, 129.81, 127.98, 70.76, 70.70, 70.67, 70.63, 70.58, 70.56, 70.52, 70.02, 69.22, 68.69, 50.70, 21.62.
Step 10 (iodination reaction):

  1.93 g (4.19 mmol) of monotosylate (monoazide compound, compound (12)) obtained in the previous step was placed in a four-necked round bottom flask, purged with nitrogen, and then 15 ml of dehydrated acetone was added. Sodium iodide (NaI) 0.753g (5.02mmol) was added, and it heated at 50 degreeC, and stirred for 2 hours. The reaction solution was evaporated. The iodinated product (compound (13), yield 1.38 g, yield 79%) was isolated by silica gel column chromatography (n-hexane: ethyl acetate = 1: 1).

The obtained compound was identified by NMR. The results are shown below.
1H NMR (400MHz, CDCl3, J in Hz); 3.76 (t, J = 6.9, 2H), 3.68 (t, J = 5.8, 2H), 3.67-3.66 (m, 16H), 3.39 (t, J = 5.1 , 2H), 3.26 (t, J = 6.9, 2H).
13C NMR (100MHz, CDCl3, J in Hz); 72.00, 70.69, 70.64, 70.61, 70.24, 70.03, 50.71, 2.87.
Step 11 (coupling reaction):

Cesium fluoride (CsF) 208 mg (1.37 mmol) was placed in a four-necked round bottom flask and cooled in a −10 ° C. bath. 1 ml of a dehydrated DMF solution of 1.35 g (3.23 mmol) of the monoiodide obtained in the previous step (compound (13)) and 502 mg (0) of the sialic acid derivative (compound (3)) obtained in step 2 .65 mmol) dehydrated DMF solution (1.2 ml) was added to the above cesium fluoride while being cooled to −10 ° C., stirred at the same temperature for 5 hours, and overnight in a refrigerator (−20 ° C.). I left it alone. From TLC, it is considered that the progress of the reaction was completed after 5 hours. 22 hours after the start of the reaction, ice-cooled ethyl acetate was added to dilute the reaction solution, and ice-cooled 0.01 M hydrochloric acid (HCl) 100 ml was added. The mixture was extracted with ethyl acetate (150 ml, 100 ml × 2), dehydrated with sodium sulfate (Na ₂ SO ₄ ), filtered, and then the solvent was distilled off. By silica gel column chromatography (n-hexane: ethyl acetate = 5: 95), 360 mg of the coupled product (compound (14)) was obtained in a yield of 56%. The obtained compound was identified by NMR. The results are shown below.
1H NMR (400 MHz, CDCl3, J in Hz); 7.39-7.26 (m, 20H), 5.07 (dd, J = 51.5 & 1.8, 1H), 4.74 (s, 2H), 4.70 (s, 1H), 4.63-4.57 (m, 4H), 4.38 (d, J = 10.6, 1H), 4.09 (m, 1H), 3.91 (m, 2H), 3.77-3.72 (m, 2H), 3.69-3.52 (m, 25H ), 3.36 (t, J = 5.3, 2H), 1.56 (s, 3H).
13C NMR (100 MHz, CDCl3, J in Hz); 170.41, 166.46 (d, J = 3.6), 138.75, 138.23, 138.17, 137.58, 129.45, 128.44, 128.33, 128.29, 128.21, 128.10, 127.92, 127.89, 127.78, 127.65, 127.54, 127.38, 127.22, 97.78 (d, J = 16.0), 86.94 (d, J = 189.1), 78.61, 73.91, 73.32, 72.83, 72.66, 72.34, 71.59, 71.51, 70.59, 70.56, 70.53, 70.48, 70.44, 69. 92, 69.81, 69.44, 64.34, 52.52, 50.60, 48.50 (d, J = 3.6), 23.49.
Step 12 (hydrolysis reaction):

349 mg (0.352 mmol) of the methyl ester product (coupling product, compound (14)) obtained in the previous step was placed in a four-necked round bottom flask, purged with nitrogen, and then 23 ml of methanol (MeOH) was added and stirred. After placing the reaction vessel in an ice bath, 6 ml of a 60 mM lithium hydroxide (LiOH) aqueous solution was slowly added. The reaction vessel was returned to room temperature and stirred for 1.5 hours. The mixture was allowed to stand at room temperature for 14 hours and then stirred again at that temperature for 4.5 hours. The reaction vessel was placed in an ice bath, an ion exchange resin (Amberlite 120B, Organo 120B, H + type) was added to adjust pH = 4, the mixture was stirred at room temperature for 30 minutes, filtered and the solvent was distilled off. The hydrolyzate (compound (15)) was isolated by silica gel column chromatography (ethyl acetate: methanol = 85: 15). The yield was 315 mg, and the yield was 92%. The obtained compound was identified by NMR. The results are shown below.
1H NMR (400 MHz, CDCl3, J in Hz); 7.45-7.19 (m, 20H), 5.43 (d, 53.3, 1H), 4.82 (t, J = 9.6), 4.69 (d, J = 10.4), 4.58 (d, J = 10.9), 4.53 (s, 4H), 4.00 (m, 1H), 3.94 (m, 2H), 3.91 (s, 1H), 3.88 (m, 1H), 3.75 (m, 2H), 3.56-3.45 (m, 21H), 3.30 (t, J = 4.8, 2H), 1.86 (s, 3H).
13C NMR (100 MHz, CDCl3, J in H); 170.47, 138.98, 138.71, 138.48, 138.41, 128. 65, 128.31, 128.13, 128.08, 128.03, 127.99, 127.56, 127.51, 127.26, 127.17, 99.51, 88.87 (d , J = 173.5), 78.95, 74.45, 73.14, 73.05, 71.16, 70.47, 69.68, 69.38, 69.01, 68.76, 68.53, 63.19, 50.22, 46.07, 23.46.
Step 13 (reductive deprotection reaction):

310.4 mg (0.320 mmol) of the hydrolyzate (Bn-protected azide, compound (15)) obtained in the previous step was placed in a four-necked round bottom flask, and 64 ml of methanol was added. Under an ice bath, 40.8 mg (0.291 mmol) of 10% palladium carbon (Pd / C) catalyst and 19 ml of 1N hydrochloric acid were added. The reaction vessel was returned to room temperature and purged with hydrogen, followed by stirring for 7 hours. It was left at room temperature overnight and purged with nitrogen. After filtration, the solvent was distilled off. Purified by gel filtration (Amersham LH-20, methanol). After the solvent was distilled off, the residue was dissolved in 10 ml of methanol, and 3.2 ml of 0.1 M lithium hydroxide (LiOH) aqueous solution was slowly added. In an ice bath, an ion exchange resin (Amberlite 120B manufactured by Organo Corporation, H + type) was added to adjust the pH to 6, followed by stirring at room temperature for 30 minutes, followed by filtration and evaporation of the solvent. Purification by gel filtration (Amersham LH-20, methanol) gave a deprotected reductant (compound (16)). The yield was 163.8 mg, and the yield was 87%. The obtained compound was identified by NMR. The results are shown below.
1H NMR (400 MHz, CD3OD, J in Hz); 5.20 (dd, J = 52.3 & 2.3, 1H), 4.11 (t, J = 10.6, 1H), 3.99 (dt, J = 12.4 & 3.8, 1H), 3.88-3.79 (m, 5H), 3.77-3.60 (m, 22H), 3.51 (dd, J = 9.1 & 1.5, 1H), 3.25 (dt, J = 6.5 & 2.3, 2H), 2.01 (s, 3H) .
13C NMR (100 MHz, CD3OD, J in Hz); 175.59, 174.25 (d, J = 2.9), 100.98 (d, J = 13.8), 93.03 (d, J = 183.3), 74.60, 73.02, 71.36, 71.32, 71.26, 71.17, 71.08, 70.96, 70.76, 70.68, 70.57, 70.52, 70.02, 68.12, 65.41, 64.51, 49.84, 22.68.

[Synthesis of sugar compounds 2]
Next, compound (20) was synthesized as a saccharide compound containing a polysaccharide having an amino group at the end as in steps 14 to 19 below.
Step 14 (diazide formation)

25.37 g (50.5 mmol) of a ditosylated compound (compound (4)) synthesized from tetraethylene glycol was placed in a four-necked round bottom flask, and the atmosphere in the flask was replaced with nitrogen. Sodium nitride (NaN ₃ ) 9.72 g (150 mmol) was slowly added, heated to 50 ° C., and stirred for 2.5 hours. After cooling to room temperature, 250 ml of ethyl acetate and 350 ml of water were added to extract the organic phase. Further, the aqueous phase was extracted with 100 ml of ethyl acetate. The organic phase was collected and washed with water (400 ml × 3), dehydrated with sodium sulfate (Na ₂ SO ₄ ), filtered, and then the solvent was distilled off. The diazide compound (compound (17), yield 9.76 g, yield 79%) was isolated by silica gel column chromatography (spherical silica 100 g, ethyl acetate: n-hexane = 1: 1). The obtained compound was identified by NMR. The results are shown below.
¹ H NMR (400 MHz, CDCl ₃ , J in Hz) δ 3.68 (m, 12H), 3.39 (t, J = 5.1 Hz, 4H).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 70.62, 69.94, 50.61.
Step 15 (reduction of azide)

25.37 g (50.5 mmol) of the diazide obtained in the previous step (compound (17)) was placed in a four-necked round bottom flask and purged with nitrogen, and then 131 ml of methanol was added. Under an ice bath, 232.4 mg (0.165 mmol) of 10% palladium carbon (Pd / C) and 39 ml of 1N hydrochloric acid were added. The reaction vessel was returned to room temperature and replaced with hydrogen, followed by stirring for 7 hours. After purging with nitrogen, 100.2 mg (0.0714 mmol) of palladium carbon (Pd / C) was further added, followed by purging with hydrogen and stirring for 17 hours. After purging with nitrogen, the mixture was filtered and the solvent was distilled off until the filtrate volume reached about 30 ml. After slowly adding 40 ml of 1N-lithium hydroxide (LiOH) aqueous solution and stirring at room temperature for 30 minutes, the solvent was distilled off. Gel filtration (Amersham LH-20, methanol) was performed twice to obtain diamine (compound (18), yield 2.51 g, yield 100%). The obtained compound was identified by NMR. The results are shown below.
¹ H NMR (400 MHz, CDCl ₃ , J in Hz) δ 3.66 (brs, 8H), 3.58 (t, J = 5.1 Hz, 4H), 3.35 (s, 4H), 2.88 (T, J = 5.1 Hz, 4H).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 71.67, 69, 44, 69.08, 40.56.
Step 16 (reductive amination)

After 0.531 g (2.75 mmol) of the diamine (compound (18)) obtained in the previous step was placed in a one-necked eggplant flask and purged with nitrogen, 50 mg (0.074 mmol) of trisaccharide (compound (19)), ethanol And 1.0 ml of a mixed solvent of acetic acid (10: 1) was added. The milky white suspension was stirred at room temperature for 17 hours. Then, 37 mg (0.349 mmol) of 2-picoline borane was added and stirred at 50 ° C. for 8 hours. The reaction solution was subjected to gel filtration (Amersham LH-20, methanol) four times to obtain a mixture of a sugar compound (compound (20)) and a diamine (compound (18)). Identification was performed by NMR and ESI-MS.
Mass spectrometry: ESI-MS method (Micromass type-ZMD)
Mass scanning conditions: 50 to 2000 m / e, 4.0 sec / scan
Applied voltage: 3.8 kV, Cone voltage: 30, 80 V
Solvent: m / z 849 (M−H) was observed by methanol ESI (−) method. The mass number of the target substance was MW850, which coincided with the mass number corresponding to the synthesis target product.
Step 17 (reduction)

1.50 g (6.17 mmol) of the diazide obtained in the previous step (6.17 mmol) was placed in a 100 ml four-necked round bottom flask and purged with nitrogen, and then 14 ml of 0.65 M aqueous phosphoric acid solution was added and stirred. . 11 ml of a diethyl ether (Et ₂ O) solution of 1.39 g (5.30 mmol) of triphenylphosphine (PPh ₃ ) was added dropwise over 80 minutes, followed by stirring at room temperature for 18 hours. H ₂ O (15 ml) was added and the aqueous phase was washed with Et ₂ O (50 ml × 3). After adding 0.867 g (21.7 mmol) of sodium hydroxide (NaOH) to the aqueous phase to make it basic, residual Et ₂ O contained in the aqueous phase was distilled off. The aqueous phase was cooled in an ice bath, and the precipitated white crystals were filtered and washed with H ₂ O (30 ml × 3). 17.5 g (438 mmol) of sodium hydroxide (NaOH) was added to the filtrate to form a 4M sodium hydroxide (NaOH) aqueous solution, followed by extraction with methylene chloride (100 ml × 12). The methylene chloride phase was dried over sodium sulfate (Na ₂ SO ₄ ) and after filtration, the solvent was distilled off. Monoamine (compound (21), yield 601 mg, yield 44%) was isolated by silica gel column chromatography (spherical silica 10 g, ethyl acetate: methanol: NEt ₃ = 1: 0: 0 to 45: 50: 5). . The obtained compound was identified by NMR. The results are shown below.
¹ H NMR (400 MHz, CDCl ₃ , J in Hz) δ 3.68 (m, 8H), 3.63 (m, 2H), 3.51 (t, J = 5.3 Hz, 2H), 3.39 (T, J = 5.1 Hz, 2H), 2.86 (t, J = 5.3 Hz, 2H), 1.39 (s, 2H)
¹³ C NMR (100 MHz, CDCl ₃ ) δ 73.41, 70.61, 70.56, 70.53, 70.18, 69.92, 50.57, 41.71
Step 18 (reductive amination)

Trisaccharide (compound (19)) 86.8 mg (0.129 mmol) and monoamine (compound (21)) 25 mg (0.115 mmol) were placed in a 10 ml eggplant flask and purged with nitrogen, and then a mixed solvent of methanol and acetic acid (10 1) 1 ml was added and stirred at room temperature for 45 hours. 393 mg (1.80 mmol) of monoamine (compound (21)) was added, and the mixture was heated at 40 ° C. for 2.5 hours and at 50 ° C. for 2.5 hours. 2-Picoline borane (45.9 mg, 0.429 mmol) was added and the mixture was heated at 50 ° C. for 6 hours, and then the solvent was distilled off. Gel filtration (Amersham LH-20, methanol) was performed. After adding 29.3 g (31.6 mmol) of benzaldehyde polystyrene and purging with nitrogen, 28.0 ml of methylene chloride was added. The mixture was stirred at 40 ° C. for 15 hours and filtered. The resin was washed with methylene chloride (30 ml × 5) and methanol (30 ml × 5), and the filtrate was evaporated to isolate a sugar compound (compound (22), yield 40 mg, yield 68%). . The obtained compound was identified by NMR and ESI-MS. The results are shown below.
¹ H NMR (400 MHz, CDCl ₃ , J in Hz) δ 4.45 (m, 1H), 4.17 to 4.09 (m, 2H), 3.87 to 3.77 (m, 8H), 3 .74 to 3.60 (m, 24H), 3.53 to 3.46 (m, 3H), 3.39 (t, J = 4.9 Hz, 2H), 3.16 (q, J = 7. 3 Hz, 1 H), 2.82 (brd, J = 10.4 Hz, 1 H), 2.08 (s, 3 H), 2.04 (s, 3 H), 1.63 (t, J = 10.5 Hz, 1H)
_{^{13 C NMR (100MHz, CDCl 3}} ) δ 175.39,172.69,165.72,105.24,98.50,82.60,74.49,73.14,71.62,71.54, 71.48, 71.37, 71.07, 67.45, 64.73, 51.7747.66, 22.66
Mass spectrometry: ESI-MS method (Micromass type-ZMD)
Mass scanning conditions: 50-1500 m / e, 4.0 sec / scan
Applied voltage: 3.8 kV, Cone voltage: +/− 50, −100V
Solvent: m / z 877 (M + H) was observed by methanol ESI (+) method. Thereafter, m / z 899 (M + Na) and m / z 921 (M−H + 2Na) were observed. Further, m / z 875 (M−H) was observed by the ESI (−) method. The mass number of the target substance was MW876, which coincided with the mass number corresponding to the synthesis target product.
Step 19 (reduction)

In a 100 ml four-necked round bottom flask, 21.4 mg (0.0244 mmol) of a sugar compound (compound (22)) was added, and 5 ml of methanol was added. 6.4 mg (0.005 mmol) of 10% palladium carbon (Pd / C) was added, and 1.3 ml of 1N hydrochloric acid was added in an ice bath. The reaction vessel was returned to room temperature and purged with hydrogen, followed by stirring for 12 hours. After purging with nitrogen, the mixture was filtered and the filtrate was evaporated. Gel filtration (gel filtration (Amersham LH-20, methanol) was performed and the solvent was distilled off. Dissolve in 4.3 ml of methanol and slowly add 4.33 ml of 0.01M sodium acetate in methanol in an ice bath. After the solvent was distilled off, the reduced product (compound (20), yield 19.9 mg, yield 96%) was obtained by gel filtration (Amersham LH-20, methanol). The obtained compound was identified by NMR and ESI-MS, and the results are shown below.
¹ H NMR (400 MHz, CDCl ₃ , J in Hz) δ 4.53 to 4.46 (m, 1H), 4.17 to 4.09 (m, 2H), 3.87 to 3.77 (m, 8H), 3.74 to 3.60 (m, 24H), 3.53 to 3.46 (m, 3H), 3.22 to 3.16 (m, 3H), 2.82 (brd, J = 10.1 Hz, 1 H), 2.02 (s, 6 H), 1.60 (m, 1 H), 1.14 (brs, 2 H)
Mass spectrometry: ESI-MS method (Micromass type-ZMD)
Mass scanning conditions: 50 to 2000 m / e, 4.0 sec / scan
Applied voltage: 3.8 kV, Cone voltage: 30, 80 V
Solvent: m / z 849 (M−H) was observed by methanol ESI (−) method. The mass number of the target substance was MW850, which coincided with the mass number corresponding to the synthesis target product.

[Synthesis of sugar compounds 3]
Finally, as in steps 20 and 21 below, compounds (24) and (25) were synthesized as sugar compounds containing a biotin-terminated monosaccharide or polysaccharide. The compound (24) is a biotinylated form of the compound (16), and the compound (25) is a biotinylated form of the compound (20).
Step 20 (biotinylation)

Into a 1 ml eggplant flask, 1.6 mg (2.7 μmol) of the sialic acid derivative (compound (16)) obtained in the previous step and 1.5 mg (27 μmol) of a commercially available biotinylating agent (compound (23)) are placed. Under a nitrogen atmosphere, dimethylformamide (DMF, 0.25 ml) and triethylamine (0.020 ml) were added. After stirring at room temperature for 6 hours, the solvent was distilled off. Purification by gel filtration (Amersham LH-20, methanol) was performed to isolate a biotinylated compound (compound (24), yield 2.5 mg, yield 101%).
¹ H NMR (400 MHz, CDCl ₃ , J in Hz) δ 5.17 (dd, J = 52.3, 2.0 Hz, 1H), 4.50 (dd, J = 7.8, 5.0 Hz, 1H) ), 4.31 (dd, J = 7.8, 4.3 Hz, 1H), 4.10 (t, J = 10.5 Hz, 1H), 3.98 (m, 1H), 3.90-3. .81 (m, 4H), 3.77 to 3.62 (m, 30H), 3.56 to 3.50 (m, 3H), 2.92 (dd, J = 12.9, 5.0 Hz, 1H), 2.70 (d, J = 12.9 Hz, 1H), 2.21 (t, J = 6.9 Hz, 2H), 2.19 (t, J = 7.1 Hz, 2H), 2. 00 (s, 3H), 1.69 to 1.59 (m, 4H), 1.52 (qn, J = 7.3 Hz, 2H), 1.46 (q, J = 7.6 Hz, 2H), 1.3 ~1.32 (m, 2H) 1.27 (t, J = 7.3Hz, 2H)
Step 21 (biotinylation)

  In a 5 ml eggplant flask, 2.0 mg (2.3 μmol) of the saccharide compound (compound (20)) obtained in the previous step and 1.3 mg (2.3 μmol) of a commercially available biotinylating agent (compound (23)) are placed. Under a nitrogen atmosphere, DMF (0.25 ml) and triethylamine (0.020 ml) were added. After stirring at room temperature for 4.5 hours, the solvent was distilled off. Purification by gel filtration (Amersham LH-20, methanol) was performed to obtain a biotinylated compound (compound (25), yield 0.9 mg, yield 32%).

The obtained compound was identified by ESI-MS. The results are shown below.
Mass spectrometry: ESI-MS method (Micromass type-ZMD)
Mass scanning conditions: 200 to 1800 m / e, 4.0 sec / scan
Applied voltage: +/- 3.8kV, Cone voltage: +/- 30, +/- 80V
Solvent: m / z 1188 (M−H) was observed by methanol ESI (−) method. The mass number of the target substance was MW1189, which coincided with the mass number corresponding to the synthesis target product.

[Example 2: Production of immobilized saccharide]
An alloy composed of Ni (80 wt%) / Cr (20 wt%) was deposited on a polyolefin plate to a thickness of 5 nm, and then gold was deposited at a thickness of 90 nm. The obtained carrier was cleaned with an ozone cleaner (Laser Techno Co., Ltd.) for 5 minutes and then immediately immersed in a 16-mercaptohexadecanoic acid / ethanol solution (5.0 mM) at room temperature for 70 hours. (SAM conversion step). The carrier after immersion was thoroughly washed with ethanol and dried overnight. The dried carrier was prepared so that N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) were 5.0 mM and 20.0 mM, respectively. It was immersed in the prepared aqueous solution for 1 hour (NHS process). The carrier after immersion is thoroughly washed with water and dried, and then is added to a 10 mM methanol solution of the compound (9) obtained in [Example 1] for a predetermined time (in the leftmost row of Table 1-1 described later). It was immersed for the stated time) (sugar compound immobilization step). Then, it fully washed with methanol and dried to obtain an immobilized body in which compound (9), which is a sugar compound, was immobilized on a carrier.
The contents of each treatment described above are schematically shown in the following reaction formula.

  For the compound (16) obtained in [Example 1], methanol solutions having concentrations of 10 mM and 30 mM were prepared and immobilized in the same procedure as described above. The immersion time is as described in the leftmost row of Table 1-2 described later.

[Example 3: Evaluation of immobilized saccharide by surface analysis]
・ Quantification with ToF-SIMS:
The immobilized body produced in [Example 2] was subjected to surface analysis by time-of-flight secondary ion mass spectrometry (ToF-SIMS). As a measuring device, TOF-SIMS IV manufactured by ION-TOF was used, the primary ions were Au +, the acceleration energy was 25 keV, and the irradiation current was 0.5 pA. Secondary ions were collected as positive and negative ions, the analysis area was 200 μm square, and the integration time was 150 seconds. Immersion time in the above-described sugar compound immobilization step (Example 2) (time in which the carrier was immersed in a methanol solution of compound (9) or compound (16)) and fluorine ions (F−) measured by ToF-SIMS Table 1-1 and Table 1-2 below, and the graph of FIG. In the graph of FIG. 1, the horizontal axis represents the carrier immersion time, the vertical axis represents the fluorine ion intensity measured by ToF-SIMS, and A represents a 10 mM methanol solution of compound (9). B represents a graph when a 30 mM methanol solution of the compound (16) was used, and C represents a graph when a 10 mM methanol solution of the compound (16) was used. As is clear from Table 1 and FIG. 1, the change with time in the amount of compound (9) or compound (16) immobilized could be clearly obtained. Under this experimental condition, it was considered that the compound (9) was immobilized at a higher density than the compound (16).

  The immersion time of 0 refers to the carrier before the immersion treatment in the sugar compound immobilization step (the carrier after the SAM formation step and the NHS conversion step).

・ Quantification with XPS:
XPS analysis was performed on the immobilized form of the compound (9) produced in [Example 2]. As a measuring device, Quantum 2000 manufactured by PHI was used. X-ray source is monochromatic Al-Kα, output is 16 kV-30 W (X-ray generation area 150 μmφ), spectral path energy is 187.85 eV (wide spectrum) and 58.7 eV (narrow spectrum), measurement area is 500 μm square The take-out angle was 45 ° (from the surface). The analysis results in each step of Example 2 (SAM conversion step, NHS conversion step, sugar compound immobilization step) are shown in Table 2.

[Example 4: Production of QCM sensor chip using immobilized saccharide]
・ Production of sensor chip:
The measuring device used was Affinix-Q manufactured by Inicium as a quartz crystal microbalance. The resonance frequency is 27 MHz. The sensor portion of the sensor chip has a surface made of gold, and the above compound (9) was immobilized on the gold surface by the following steps.

  The sensor part of the sensor chip was cleaned by contacting with sulfuric acid / hydrogen peroxide solution (= 3/1) for 5 minutes. After thoroughly washing with water, moisture was blown off by a nitrogen stream and dried. Thereafter, the sensor portion was immersed in a 16-mercaptohexadecanoic acid / ethanol solution (5.0 mM) for 70 hours (SAM conversion step). The carrier after immersion was thoroughly washed with ethanol and dried for 3 hours. After drying, an aqueous solution prepared so that N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) were 5.0 mM and 20 mM, respectively, was used as a sensor part. And left at room temperature for 15 minutes under high humidity conditions. It was thoroughly washed with water, dried with a nitrogen stream, and dried. The sensor part was immersed in a 9.0 mM methanol solution of the compound (9) obtained in [Example 1] for 23 hours (sugar compound immobilization step). Thereafter, it was thoroughly washed with methanol and dried. Thereafter, an ethanolamine aqueous solution (1M) was dropped onto the sensor part (ethanolamine treatment) and washed thoroughly with water. Subsequently, the sensor chip was set on the measuring device. 7.2 ml of a virus culture medium (containing 0.3% BSA) was placed in a well for measurement, and the temperature of the liquid was controlled at 25 ° C. and immersed for 1 hour while stirring at 600 rpm (BSA treatment). The BSA treatment is intended to suppress nonspecific adsorption.

Moreover, the sensor chip which fix | immobilized the compound (16) and the sensor chip which fix | immobilized the compound (20) were also produced by the same method. In addition, it carried out on the same conditions except having made the methanol solution of the compound (16) into 13.5 m, making immersion time into 48 hours, setting it into 13.6 mM of compound (20), and making immersion time into 21 hours.
The contents of the above ethanolamine treatment are schematically shown in the following reaction formula.

[Example 5: Detection of virus by immobilized saccharide using QCM]
Using a sensor chip obtained by immobilizing compound (9), compound (16), or compound (20) as a ligand obtained by the above procedure, a virus detection experiment was subsequently performed according to the following procedure.
・ Preparation of virus specimen solution:
The virus was cultured using influenza A (H3N2) / Fukuoka / C29 / 85 according to the following procedure. First, a canine kidney-derived cell line (MDCK cell) was cultured in a flask at 37 ° C. for 3 days, and then the cell growth medium was removed. Next, after inoculating the virus into MDCK cells and adsorbing at room temperature for 1 hour, the virus was removed, and an influenza medium containing bovine serum albumin (BSA) at a concentration of 0.3% was added at 37 ° C. Cultured for 3 days. Infected cells and culture supernatants that showed cytopathic effect (CPE) 3-4 + were collected and centrifuged at 4 ° C. and 3000 rpm for 10 minutes. The supernatant was dispensed in 0.5 ml portions as a virus solution. The virus titer was measured and found to be 1.3 × 10 ⁷ [TCID / ml].

・ Measurement of interaction between sugar compound and virus:
After setting the QCM sensor chip produced in Example 4 above in the apparatus, it was immersed in a medium for culture of virus (7.2 ml), and frequency measurement was started. When there was no change in frequency and stabilization, a virus culture solution (0.5 ml) was added dropwise. Since the virus culture solution is stirred and mixed with the medium, and the virus concentration is diluted to 6.5%, the titer can be calculated as 8.4 × 10 ⁵ [TCID / ml]. The temperature was 25 ° C. throughout and the stirring speed was 600 rpm. A frequency decrease was observed after the addition of virus. For comparison, a sensor chip treated in the same manner as above except that no sugar compound was immobilized on the QCM sensor (that is, a sensor chip in which only ethanolamine was immobilized after NHS was formed on the sensor surface) was the same as above. The virus detection experiment was performed according to the procedure.

  The measurement results are shown in FIG. In the graph of FIG. 2, A is a comparative sensor chip with ethanolamine immobilized, B is a sensor chip with compound (9) immobilized, and C is compound (16). When the immobilized sensor chip is used, D indicates the case where the sensor chip to which the compound (20) is immobilized is used. From FIG. 2, the amount of influenza virus adsorbed changes over time due to the interaction between the influenza virus in the sample liquid and the compounds (9), (16) and (20) which are sugar compounds. I understand that. No change was observed in the frequency when only the comparative ethanolamine was immobilized.

  In Example 3 above, it was considered that the compound (9) was immobilized at a higher density than the compound (16). However, as a result of this measurement, the compound (9) was immobilized rather than the chip on which the compound (9) was immobilized. The chip to which 16) was immobilized had a larger amount of virus adsorption. The amount of adsorbed virus was not necessarily proportional to the amount of immobilized sugar compound, and it was assumed that there was an optimum amount of immobilized sugar compound depending on the sugar, sugar compound or substance to be detected. Moreover, it was clearly shown that the length of the polyether contained in the saccharide compound and the difference in saccharide affect the reactivity. That is, it was found that a sensor suited to the purpose can be produced by appropriately adjusting the structure and the amount of immobilization of the sugar compound according to the combination of saccharide and the substance to be detected, reaction conditions, and the like.

[Example 6: Detection of lectin by immobilized saccharide using QCM]
-Preparation of lectin sample solution:
Japanese elder collectin (manufactured by J Oil Mills, SSA, freeze-dried powder) was dissolved in PBS buffer to prepare a solution having a concentration of 2 mg / mL.

・ Measurement of interaction between sugar compounds and lectins:
The QCM sensor chip on which the compound (20) prepared in Example 4 was immobilized was used. After setting the sensor chip in the apparatus, the sensor chip was immersed in a PBS buffer solution (7.95 ml), and frequency measurement was started. After stabilizing the frequency, the liquid was replaced with a 0.3% BSA buffer solution and stirred for 60 minutes (blocking). The probe was washed with PBS buffer solution and then stabilized again in PBS for 60 minutes. Thereafter, 100 μL of Mouse IgG was added, and the behavior of the frequency was observed for 30 minutes, but no significant signal fluctuation due to the addition of Mouse IgG was confirmed. Thereafter, when 100 μL of the above-mentioned Japanese elder collectin solution was added, a change in frequency was observed.
The measurement results are shown in FIG. It was found that the amount of adsorbed Japanese elder collectin changed with time due to the interaction between Japanese elder collectin (SSA) and compound (20) in the sample.

[Example 7: Production of saccharide-immobilized body by immobilization of saccharide compound to hydrogel]
Next, an immobilized body was prepared using a method of immobilizing a sugar compound on a hydrogel immobilized on the surface of a self-assembled film formed on the surface of the carrier. Here, the “protein / polymer composite hydrogel” refers to a hydrogel containing a protein in its structure and made from a protein and a hydrophilic polymer that can contain a large amount of water.

-Preparation of polymer As monomers, N-acryloylmorpholine (manufactured by KOHJIN, hereinafter abbreviated as NAM) 0.2834 g and N-acryloyloxysuccinimide (manufactured by ACROS ORGANIC, sometimes abbreviated as NAS) 0. 8457 g and dehydrated dioxane (manufactured by Wako Pure Chemical Industries, Ltd.) 20.8160 g were mixed well, poured into a 50 mL four-necked flask, and deaerated with nitrogen at room temperature for 30 minutes to prepare a monomer solution. The monomer solution was heated to 60 ° C. in an oil bath, and a solution obtained by dissolving 0.01231 g of azobisisobutyronitrile (AIBN, manufactured by Kishida Chemical Co., Ltd.) as a polymerization initiator in 0.5 g of dehydrated dioxane was added. The polymerization was started. The polymerization was performed for 8 hours under a nitrogen atmosphere.

The reaction solution after polymerization was added dropwise to 0.5 L of diethyl ether (manufactured by Kokusan Chemical Co., Ltd.) to reprecipitate the target polymer, and the solvent was removed to form a powder.
The obtained polymer was subjected to SEC measurement calibrated with standard polystyrene. As a result, the polymerization average molecular weight (Mw) was estimated to be about 86,000. The molar ratio of NAS to NAM (NAS / NAM) contained in the obtained polymer was estimated to be NAS / NAM = 30/70 from NMR measurement.

-Preparation and immobilization of protein / polymer composite hydrogel and immobilization of compound (25) Streptavidin (manufactured by WAKO) was dissolved in carbonate buffer (pH = 9.0, 100 mM), 3.0, 1.0 , 0.5, 0.25 and 0.13 wt% aqueous solutions were prepared respectively. The polymer was dissolved in water to prepare aqueous solutions having concentrations of 3.0, 1.0, 0.5, 0.25, and 0.13 wt%, respectively. A polymer aqueous solution and a streptavidin aqueous solution having the same concentration were mixed at a ratio of 1:10 to obtain five types of mixed solutions.

  A resin gold vapor deposition substrate (2.5 cm × 2.5 cm) was immersed in an ethanol solution (5.0 mM) of 16-mercaptohexadecanoic acid for 16 hours. Thereafter, it was washed with ethanol, washed with water, and dried with nitrogen. Subsequently, a succinimide group was introduced with an EDC / NHS aqueous solution in the same manner as in Example 4. 3 μL of the mixed solution of each concentration was spotted on this. The mixture was allowed to stand at room temperature for 30 minutes and dried to form a protein / polymer composite hydrogel. Then, the board | substrate was immersed in ethanolamine aqueous solution (pH = 8.5, 1M), and the unreacted succinimide group was converted into the ethanolamino group. Subsequently, the gold substrate was immersed in an aqueous PBS solution (1M, pH = 7.4) of BSA (manufactured by SIGMA) for 30 minutes, washed with water, and dried with nitrogen.

  A buffer solution of compound (25) (1 mM, 3 μL, pH = 9.0) was spotted on a protein / polymer composite hydrogel spot consisting of a polymer and streptavidin and allowed to stand at room temperature for 30 minutes. . Thereafter, it was washed with water and dried with nitrogen. As a result, an immobilized body in which the compound (25) was bound to the protein / polymer composite hydrogel immobilized on the substrate by a biotin-avidin bond was produced.

[Example 8: Detection of lectin by immobilized saccharide prepared using hydrogel]
Using the immobilized body prepared in Example 7 above, lectin was detected by fluorescence.
Measurement The gold substrate prepared above was placed in a buffer solution (pH = 7.4, 10 μg / mL) of Japanese elder collectin (manufactured by J Oil Mills, FITC-SSA) labeled with FITC (fluorescein isothiocyanate) for 30 minutes. Then, it was immersed at room temperature to carry out a sugar-lectin reaction. After completion of the reaction, it was washed with water and dried with nitrogen. Finally, using a fluorescence measuring apparatus FX PRO PLUS (manufactured by BioRAD), the amount of fluorescence derived from the bound Japanese elder collectin was measured. The excitation light wavelength at this time was 547 nm, and the detection wavelength was 563 nm.

The result is shown in FIG. The solid content concentration on the horizontal axis is the combined weight concentration of streptavidin and polymer contained in the mixed solution used in preparing the protein / polymer composite hydrogel. It has been found that the fluorescence intensity increases with increasing solids concentration. That is, it was found that as the amount of streptavidin increased, the amount of compound (25) immobilized increased, and the amount of adsorbed Japanese chick collectin interacting with compound (25) increased.
Thus, it was revealed that the saccharide-immobilized body of the present invention can be produced using a combination of various immobilization methods and surface treatment techniques.

  The immobilized body of the present invention in which a saccharide compound composed of a saccharide and a polyether is immobilized is excellent in terms of the stability of the immobilized saccharide and the suppression of non-specific adsorption. Etc. can be suitably used. The sensor of the present invention can be suitably used for detection, analysis, purification and the like of a substance to be detected in a specimen. Therefore, the present invention can be used in any industrial field, but can be used particularly preferably in fields such as diagnosis, medical care, pharmaceutical development, food analysis, and environmental measurement.

4 is a graph showing the relationship between the immersion time in each sugar compound solution in which a carrier is immersed in Example 2 and the fluorine ion intensity measured by ToF-SIMS in Example 3. FIG. It is a graph showing the time change of the frequency by the adhesion of the virus on the QCM sensor based on the interaction of the virus and the sugar compound measured in Example 5. It is a graph showing the time change of the frequency by adhesion of the lectin on the QCM sensor based on interaction of a lectin and a sugar compound measured in Example 6. It is a graph showing the fluorescence emission intensity change by adhesion | attachment of labeled lectin based on interaction of a lectin and a sugar compound measured in Example 8. FIG.

Claims (24)

A saccharide-immobilized product, wherein a saccharide compound comprising a saccharide and a polyether is immobilized on the surface of a carrier via the polyether. The immobilization body according to claim 1, wherein the immobilization is performed by a covalent bond. The immobilized body according to claim 1, wherein the immobilization is performed using an interaction between biologically relevant substances having a dissociation constant of 1 × 10 ⁻⁸ or less. The immobilized body according to claim 3, wherein the interaction between the biological substances is a biotin-avidin interaction. The immobilized body according to any one of claims 1 to 4, wherein the sugar compound is further immobilized on the surface of the carrier via a spacer. The spacer is any of a divalent hydrocarbon group, —S (sulfur atom) —, —O (oxygen atom) —, carbonyl group, amino group, —Si (silicon atom) —, maleimide group, protein, and combinations thereof. The immobilization body according to claim 5, wherein the immobilization body is selected from the group consisting of: The bond between the spacer and the carrier is a metal-S bond, C-Si bond, O-Si bond, C (= O) -NH bond, C (= O) -O bond, NH-C (= O) bond, The immobilized body according to claim 5 or 6, wherein the immobilized body is selected from the group consisting of any of N = C bond, NH-CH bond, OC (= O) bond and triazole. The immobilized body according to any one of claims 5 to 7, wherein the spacer is formed by bonding a bifunctional thiol or a bifunctional trialkoxysilane to the support surface. The polyether is characterized in that the hydrocarbon group constituting it is selected from the group consisting of a divalent alkylene group, a divalent arylene group, and a combination thereof. The immobilized body according to any one of 8. The immobilized body according to any one of claims 1 to 9, wherein the polyether is polyethylene glycol having 2 or more repeating units. The saccharide-polyether bond is selected from the group consisting of an ether bond, a glycoside bond, a sulfur glycoside bond, an N (nitrogen atom) glycoside bond, a C = N double bond, and a reductive amination reaction product. The immobilization body according to any one of claims 1 to 10, which is characterized. The immobilized body according to any one of claims 1 to 11, wherein the saccharide is a monosaccharide or a polysaccharide. The saccharide is a monosaccharide selected from the group consisting of sialic acid, galactose, glucose, fucose, and mannose, or a derivative thereof, or a polysaccharide containing them, according to any one of claims 1 to 12. Immobilized body. The immobilized body according to any one of claims 1 to 13, wherein the saccharide has a sialic acid derivative optionally having a substituent as a non-reducing terminal sugar. A sensor, wherein the immobilized body according to any one of claims 1 to 14 forms a sensor surface. Utilizing at least one detection principle selected from the group consisting of a quartz crystal microbalance, cantilever, field effect transistor, color development, fluorescence emission, optical interference, chemiluminescence, surface plasmon resonance, optical waveguide and surface acoustic wave The sensor according to claim 15, which performs detection. The sensor according to claim 15 or 16, wherein a specimen containing a substance to be detected that can specifically interact with the sugar compound is brought into contact, and the interaction between the sensor and the substance to be detected is detected. Analyte analysis method. The immobilized body according to any one of claims 1 to 14 is brought into contact with a specimen containing a substance to be detected that can specifically interact with the sugar compound, and the immobilized body and the specimen are separated. And then detecting a substance to be detected specifically bound to the sugar compound. The immobilized body according to any one of claims 1 to 14 is brought into contact with a specimen containing a substance to be detected that can specifically interact with the sugar compound, and the immobilized body and the specimen are separated. After that, a method for purifying a substance to be detected, comprising obtaining a substance to be detected specifically bound to the sugar compound. The method according to claim 18 or 19, wherein a magnetic material is used for the carrier. The substance to be detected is at least one selected from the group consisting of RNA, DNA, saccharides, proteins, toxins, glycolipids, oligopeptides, viruses, bacteria, and derivatives thereof. The method according to any one of -20. 17. A sensor according to claim 15 or 16, a sample supply unit for supplying the sample to bring the sample into contact with the sensor, and a detection unit for detecting an interaction between the sensor and the sample. Characteristic specimen detection device. A functional molecule comprising at least a saccharide, a polyether, and a spacer, wherein the polyether is bonded to the saccharide at one end and the spacer at the other end, and the spacer is covalently bonded to the carrier. Alternatively, a molecule characterized by being able to bind by interaction between biological substances. The number of monosaccharides constituting the saccharide is 1 or more and 20 or less, and the polyether is composed of any one of a divalent alkylene group, a divalent arylene group, and a combination thereof. The functional group of the spacer selected from the group is amino group, mercapto group, biotin, hydroxycarbonyl group, N-hydroxysuccinimide ester, maleimide group, formyl group, azide group, alkoxysilyl group, and metal chelate The molecule according to claim 23, wherein the molecule is at least one functional group selected from the group consisting of polyamines.

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