JP2008231013A - Immobilized sugar chain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immobilized sugar chain constituted of a sugar chain immobilized on a solid phase support, a method for preparing the same, and its applications. <P>SOLUTION: The immobilized sugar chain is prepared by immobilizing an intended sugar chain onto a solid phase support via a spacer comprising a group obtained by the reaction of an azido group and an alkyne group. An adsorbent comprising the immobilized sugar chain is used as a means for analyzing a sugar chain, or removing a virus, a toxin and a microorganism. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an immobilized sugar chain, a method for producing the same, and an adsorbent such as a toxin using the same. More specifically, the present invention relates to an immobilized glycan bound to a solid phase carrier by a spacer having a group obtained by a reaction between an azide group and an alkyne group, a method for producing the same, and an adsorbent such as a toxin using the same.

  Sugar chains are complex in structure, and development for industrial use is delayed compared to DNA and proteins, which are the same biomolecules. However, in recent years, research on sugar chains has progressed rapidly, and the search for industrial use has been promoted. In particular, new facts about the involvement of sugar chains in cell differentiation, canceration, immune reaction, etc. are being revealed. For example, sugar chains on the cell surface are involved in important processes such as cell differentiation, canceration, and immune reaction in vivo by interacting with proteins and lipids. In addition, it has become clear that sugar chains play an important role in cell recognition, adhesion and signal transduction in the cell surface layer.

  On the other hand, as a blood purification method, conventionally, a patient's blood or plasma is brought into contact with an adsorbent to remove harmful substances using a physicochemical phenomenon or an immune reaction. In this method, coconut shell adsorbent, activated carbon made from petroleum pitch, peat, charcoal, etc. as raw materials, styrene / divinylbenzene copolymer has amino groups, quaternary ammonium groups, etc. as active sites. Adsorbents such as anionic ion exchange resins and fibers obtained by immobilizing antibiotics on polystyrene derivative fibers are used. For example, anionic ion exchange resins having amino groups, quaternary ammonium groups, etc. as active sites in styrene / divinylbenzene copolymers are clinically used as adsorbents for bilirubin produced by the degradation of hemoglobin when aged erythrocytes break down. in use.

  If this technology can be applied to adsorbents with a sugar chain polymer as an active site in the future, medical materials can be obtained by immobilizing functional sugar chains that specifically bind to pathogens and toxins on solid carriers. It is possible to apply to. For example, it is known that a sugar chain polymer having globotrisaccharide specifically adsorbs verotoxin, and such an adsorbent for a harmful substance to which a functional sugar chain polymer is immobilized can be used for medical treatment. It is considered possible.

  In addition, since sugar chains are involved in various important processes in vivo by interacting with proteins and lipids, sugar chain arrays are used to analyze the mechanism at the molecular level. For example, Takara Bio Co., Ltd. sells a glycolipid array in which 24 types of glycolipids are spotted on a glass slide coated with butylsilane and immobilized using the hydrophobic interaction between the lipid portion of the glycolipid and the glass surface. ing.

  In such a technique, as a method of immobilizing a sugar chain, a method of immobilizing a sugar chain to a solid phase carrier by noncovalent bonding is known, but it cannot be stably immobilized, and a washing step Can cause sugar chains to be washed away. In addition, as a method for immobilizing a sugar chain to a solid phase carrier by covalent bond, a method for immobilizing by a Diels-Alder reaction and a method for immobilizing by a Michael addition reaction are also known. However, there are problems such as large non-specific adsorption with proteins and reduction of reaction points due to oxidation of thiols on the substrate.

  Patent Document 1 discloses a method for producing a sugar chain array in which sugar chains are bonded to a solid phase carrier by thioalkylation via a spacer group. However, it is necessary to thioalkylate a sugar chain via a spacer group, and to immobilize various sugar chains, it is necessary to obtain various sugar chains as raw materials and further thioalkylate.

JP 2006-78418

  As described above, the immobilized glycans currently used have problems that non-specific adsorption may occur and the process of modifying the glycan itself at the stage of immobilizing the glycan is complicated. Therefore, development of a specific and easy-to-create immobilized sugar chain is strongly desired.

  Therefore, an object of the present invention is to provide an immobilized sugar chain that can specifically interact with a detection target and that can be easily produced, a method for producing the same, and a method for applying the same.

  As a result of intensive studies, the present inventor has found that the above-described object can be achieved by the immobilized sugar chain, the preparation method thereof, and the application thereof, and has completed the present invention.

  That is, the present invention relates to an immobilized sugar chain in which a sugar chain containing at least two monosaccharide residues or derivatives thereof is bound to a solid phase carrier or a polymer, and the sugar chain includes a triazole ring. To provide an immobilized glycan bound to a solid phase carrier.

The immobilized sugar Kusaritai the formula _{(I) :( G 1 ··· G} n) -L-X- [ wherein, G _1, · · G _n is a monosaccharide residue each independently a cyclic structure Or a derivative thereof, which are glycosidically linked together to form a sugar chain; L is —O— (CH ₂ ) _m —, —S— (CH ₂ ) _m —, and —NH— (CH ₂ ) one or more linking groups selected from the group consisting of _m- ; n is an integer from 2 to 100; m is an integer from 8 to 20; and X is any group containing a triazole ring One or more sugar chain compounds represented by “meaning a spacer” may be contained.

  The sugar chain can be obtained by adding a sugar chain primer to an animal cell and culturing, and can be a compound obtained by binding a sugar chain primer or an alkyl chain having an azide group to an oligosaccharide chain.

  The sugar chain may include a plurality of the same or different types of sugar chains.

  The solid phase carrier may be one selected from the group consisting of a carrier containing a polymer compound, a carrier containing a metal, and a carrier made of paper.

  The solid phase carrier can be in the form of a plate or bead.

The present invention is also a method for producing an immobilized sugar chain in which a sugar chain is bound to a solid phase carrier,
There is provided a method comprising the step of reacting and binding a compound containing a sugar chain containing at least two or more monosaccharide residues or derivatives thereof, an alkyl group, and an azide group and an alkyne group.

  The alkyne group may be bound to a solid phase carrier.

The above compound has the formula (I): (G ₁ ... G _n ) -LN ₃ [wherein G ₁ ,... G _n are each independently a monosaccharide residue having a cyclic structure or a derivative thereof. And is a glycosidic bond so as to form a sugar chain with each other; L represents —O— (CH ₂ ) _m —, —S— (CH ₂ ) _m —, and —NH— (CH ₂ ) _m —. 1 or more linking groups selected from the group consisting of; n is an integer of 2 to 100, and m is an integer of 8 to 20.

  The sugar chain can be obtained by adding a sugar chain primer to animal cells and culturing, and the sugar chain primer can be a compound in which an alkyl chain having an azide group is bound to an oligosaccharide chain.

  The carrier may be one selected from the group consisting of a carrier containing a polymer compound, a carrier containing a metal, and a carrier made of paper.

  The present invention also provides a method of bringing the immobilized sugar chain into contact with a substance that binds to or interacts with the sugar chain moiety in the immobilized sugar chain.

  The substance may be one selected from the group consisting of viruses, microorganisms, protozoa, plant cells, and animal cells.

  The present invention also provides an adsorbent for viruses, bacteria, and / or toxins comprising the above-described immobilized sugar chain; neutralizing agent for viruses, bacteria, and / or toxins; biological defense system stimulation, anti-inflammatory, antibacterial, and Agents useful for anti-tumor metastasis; diagnostic agents; cell culture substrates or specific cell separation substrates; detection bases for viruses or bacterial toxins; or in vivo drug carriers.

  As described above, by applying the present invention, a quick, simple and specific immobilized sugar chain and a method for producing the same can be obtained. The novel immobilized sugar chain according to the present invention is preferably obtained by easily immobilizing a sugar chain compound produced by animal cells on a solid phase carrier using a click chemistry ligation reaction. Therefore, it is possible to easily and strongly immobilize sugar chains, and to provide various immobilized sugar chains. In the immobilized sugar chain of the present invention, the sugar chain and the solid phase carrier are firmly fixed by a covalent bond, exist stably in water or in a buffer solution, and the function of the immobilized sugar chain decreases for a long time. do not do. For this reason, the immobilized sugar chain of the present invention can be used for various applications.

  Embodiments of the present invention will be described below.

In the immobilized sugar chain of the present invention, a sugar chain containing at least two monosaccharide residues or derivatives thereof is bound to a solid phase carrier or a polymer, and bound to the solid phase carrier by a spacer containing a triazole ring. is doing. Here, but not limited to, immobilized sugar Kusaritai of the invention is a compound of Formula _{(I) :( G 1 ··· G} n) -L-X- [ wherein, G _1, · · G _n is Each is independently a monosaccharide residue having a cyclic structure or a derivative thereof, which are linked to form a sugar chain with each other; L is —O— (CH ₂ ) _m —, —S— (CH ₂ ) _m- and one or more linking groups selected from the group consisting of -NH- (CH ₂ ) _m- ; n is an integer from 2 to 100; m is an integer from 8 to 20; And X represents an arbitrary spacer including a triazole ring], and can also include one or more sugar chain compounds.

  The solid phase carrier used in the immobilized sugar chain of the present invention is not particularly limited as long as it allows the sugar chain to be bonded through the reaction of an azide group and an alkyne group for forming a triazole ring. Can be used. Examples of such solid phase carriers include solid phase carriers made of paper such as filter paper, commercially available membrane filters, glass solid phase carriers, silicon solid phase carriers, solid phase carriers containing other polymer compounds, gold, Examples include solid phase carriers containing metals such as silver, platinum, and iron. The solid phase carrier has a functional group for introducing a functional group such as a hydroxyl group, a carboxyl group, and an amino group in order to facilitate the preparation of the immobilized sugar chain of the present invention, or It is preferable to use a glass solid phase carrier into which a functional group such as an amino group has been introduced, a gold solid phase carrier in which a functional group can be introduced by an Au-S bond, or the like. For example, the filter paper has a hydroxyl group, and the solid phase carrier can be modified to have an alkyne moiety using the hydroxyl group. In addition, the term “immobilized sugar chain” in the present invention may refer to a polymerized sugar chain in which a sugar chain and a polymer compound are bonded.

  The sugar chain used in the present invention is not particularly limited. In the present specification, the term “sugar chain” refers to a chain formed by glycosidic bonding of a monosaccharide residue having a cyclic structure or a derivative thereof, and the type thereof is not particularly limited. Preferably, it is obtained by culturing animal cells under conditions such that sugar chains can be obtained. Animal cells are not particularly limited, and various animal cells can be used depending on the type of the desired sugar chain compound. Examples of animal cells include mammalian cells such as human origin, mouse origin, monkey origin, rat origin and the like, in particular B16 mouse melanoma cells, rat PC12 cells, mouse Neuro2a cells, rat RBL-2H3 cells, human MOLT- 4 cells, human HL-60 cells, monkey kidney-derived Vero cells and the like can be suitably used. Culturing is performed according to normal animal cell culture conditions.

  The conditions under which the sugar chain compound can be obtained include, for example, that the above animal cells are previously cultured in a culture medium such as DMEM / F12 at a temperature between room temperature and 45 ° C. for a period of 1 hour to 70 hours. After culturing, the medium is replaced with a medium supplemented with a saccharide primer, and further culturing is performed to carry out a glycan elongation reaction.

Here, the sugar chain primer can be arbitrarily selected depending on the cell type used and the desired sugar chain structure. Examples of preferred carbohydrate primers, but are not limited to, compounds of the formula (I) :( G1) x ( G2) y (G3) z-L-N 3 may be mentioned. Here, in the formula, G 1, G 2 and G 3 are each independently a monosaccharide residue having a cyclic structure or a derivative thereof, and L is —O— (CH ₂ ) m —, —S— (CH ₂ ). m-, and -NH- (CH ₂₎ a linking group selected from m-; x, y, and z are each independently 0 integer, preferably 0 to 5, more preferably Is 0 to 3, and x, y and z are not all 0 at the same time. M is an integer of 8 to 20, preferably 8 to 16, and particularly preferably 12. The monosaccharide is not limited, but pentose and hexose are preferably used. Either aldose or ketose can be used.

  When such a sugar chain primer is added to the culture medium of various cells and the cells are cultured, the cells take up the primer, add sugar to the sugar chain part to glycosylate, and introduce the sugar addition product into the cell. Secretes extracellularly without accumulating. The added sugar varies depending on the cell.

  The resulting cell culture or the supernatant obtained by removing cells from the cell culture contains a sugar chain compound that is a product obtained by extending from a sugar chain primer. For the preparation of the immobilized sugar chain of the present invention, the cell culture or the supernatant of the cell culture is used as it is or after it is subjected to a concentration and / or purification step by contacting the supernatant with a solid adsorbent or the like. Can be used.

The method for producing the immobilized sugar chain of the present invention is not limited, but the azide group added to the alkyl chain of the sugar chain and the alkyne portion of the solid support are bonded to the solid support by a click chemistry ligation reaction. Can be made. In the click chemistry ligation reaction, a method using Cu (PPh ₃ ) ₃ Br as a catalyst, a method using electron beam irradiation, and the like are known.

  Or, by a click chemistry ligation reaction, for example, a sugar chain obtained by culturing animal cells is modified to have a triazole ring obtained by previously combining an azide group and an alkyne group and another reactive group at the end. You can also keep it. For example, by reacting a sugar chain having an azide group end with a commercially available 2-propynylamine, a sugar chain having an amino group at the end is generated. When propargyl alcohol is used, a hydroxyl group is used, and when propargyl benzenesulfonate is used, a sulfonate group is formed. When propargyl bromide or propargyl chloride is used, a sugar chain having a halogen group at the terminal is generated. Even when not commercially available, the target functional group can be introduced by a condensation reaction or the like between the compound having a propargyl group and the compound having the target functional group to be introduced.

In the click chemistry ligation reaction, an azide group and an alkyne group are removed in a sufficient time (for example, 30 to 40 hours) to give a product in a refluxing solvent (toluene or the like) so that a triazole ring is formed. React. By adding a catalyst (such as copper) to the reaction mixture, the reaction can proceed in an aqueous solvent at room temperature to obtain a triazole ring component. Catalyzed by adding a catalytic amount of Cu (II) in the presence of a reducing agent to reduce Cu (II) to Cu (I). Preferred reducing agents include ascorbate, metallic copper, quinone, hydroquinone, vitamin K1, glutathione, cysteine, Fe ²⁺ , Co ²⁺ , and applied potential. Further preferred reducing agents include metals selected from the group consisting of Al, Be, Co, Cr, Fe, Mg, Mn, Ni and Zn.

  The click chemistry ligation reaction between an azide group and an alkyne group can also be catalyzed by conducting the reaction in a solution in contact with metallic copper. Metallic copper contributes directly or indirectly to catalysis for its click chemistry ligation reaction. In a preferred manner, the solution is an aqueous solution. In this case, the sugar chain having an azide group and the reaction compound having an alkyne group can be present in equimolar amounts.

Alternatively, the click chemistry ligation reaction between an azide group and an alkyne group is performed using a metal ion selected from the group consisting of Au, Ag, Hg, Cd, Zr, Ru, Fe, Co, Pt, Pd, Ni, Rh and W. Catalyzed by adding a catalytic amount of a metal salt. Such a click chemistry ligation reaction is performed in the presence of a reducing agent for reducing the metal ion to a catalytically active form. Preferred reducing agents include ascorbate, quinone, hydroquinone, vitamin K1, glutathione, cysteine, Fe ²⁺ , Co ²⁺ , applied potential, and Al, Be, Co, Cr, Fe, Mg, Mn, Ni and Zn A metal selected from the group consisting of:

Immobilized sugar chain of the present invention, when containing a formula _{(I) :( G 1 ··· G} n) a sugar chain compound represented by -L-X-, wherein, G _1, · · G _n Are each independently a monosaccharide residue having a cyclic structure or a derivative thereof, and are glycosidically bonded to each other so as to form a sugar chain. The monosaccharide is not limited, but pentose and hexose are preferably used. Either aldose or ketose can be used. N representing the number of monosaccharides is an integer of 2 to 100. L _{is, -O- (CH 2) m -} , - S- (CH 2) m -, and -NH- (CH _{2) m} - is one or more linking groups selected from the group consisting of, m Is an integer of 8-20. X means an arbitrary spacer containing a triazole ring, and it is desirable that the arbitrary spacer is linear and has a simple structure with little or few side chains.

  More specifically, it demonstrates, demonstrating the manufacturing method of the fixed sugar_chain | carbohydrate of this invention.

(1) A method of binding an azide group added to an alkyl chain of a sugar chain and an alkyne part of a solid phase carrier to a solid phase carrier by a click chemistry ligation reaction

  Sugar chains can be obtained by culturing animal cells and sugar chain primers together. This sugar chain is used for conjugation to a solid phase carrier after being subjected to a concentration and / or purification step as it is or in contact with a solid adsorbent or the like.

  On the other hand, for example, filter paper having a hydroxyl group can be used as the solid phase carrier. The hydroxyl group portion of this filter paper can be reacted to form, for example, a solid phase carrier having a carboxyl group end. The method for introducing the carboxyl group terminal is not particularly limited, but can be achieved by reacting the hydroxyl group of the filter paper with a halogenated acetic acid.

  Next, the resulting carboxymethylated filter is reacted with propanylamine to obtain a solid phase carrier having an amide-bonded propargyl group.

  The alkyne part of the solid phase carrier thus obtained is reacted with the azide group of the sugar chain, and the sugar chain is bound to the solid phase carrier by a click chemistry ligation reaction.

  Alternatively, by using rayon having a carboxyl group (trade name: Paramos, manufactured by Daiwabo Rayon) as a solid phase carrier, propynylamine can be reacted directly with the carboxyl group, and a solid phase carrier having an amide-bonded propargyl group is obtained. can get. Furthermore, when a compound obtained by bonding 2-mercaptopropionic acid and 2-propynylamine by a condensation reaction is used, a propargyl group can be introduced onto the gold surface by an Au—S bond.

  The alkyne part of the solid phase carrier thus obtained is reacted with the azide group of the sugar chain, and the sugar chain is bound to the solid phase carrier by a click chemistry ligation reaction.

(2) A method in which a sugar chain is modified to have a triazole ring and another reactive group at the end by a click chemistry ligation reaction, and then bound to a solid phase carrier

  Sugar chains can be obtained by culturing animal cells and sugar chain primers together. The sugar chain is modified after being subjected to a concentration and / or purification step by contacting this sugar chain as it is or with a solid adsorbent or the like. For example, when a sugar chain having an azide group terminal is reacted with a commercially available 2-propynylamine by a click chemistry ligation reaction, a sugar chain having an amino group at the terminal is generated.

  On the other hand, for example, filter paper having a hydroxyl group can be used as the solid phase carrier. The hydroxyl group portion of this filter paper can be reacted to form, for example, a solid phase carrier having a carboxyl group end. The method for introducing the carboxyl group terminal is not particularly limited, but can be achieved by reacting the hydroxyl group of the filter paper with a halogenated acetic acid.

  The carboxyl group of the solid phase carrier thus obtained is reacted with the amino group of the sugar chain, and the sugar chain is bound to the solid phase carrier by an amide bond.

The immobilized sugar chain of the present invention may be obtained by a method of purifying and immobilizing a single type or a plurality of types of sugar chains, but in a state where a plurality of types of sugar chains are contained, It is also possible to immobilize with purification. When purifying a single type of sugar chain, the immobilized sugar chain can be used as an adsorbent or neutralizing agent for toxins, viruses, bacteria, and the like. It is also possible to produce a sugar chain array or the like by immobilizing a plurality of types of sugar chains. In addition, by using an unpurified sugar chain, for example, a functional sugar chain can be screened from a cell-derived sugar chain compound. Various substances can be brought into contact with the immobilized sugar chain of the present invention to analyze and / or adsorb sugar chains or substances that interact with sugar chains. Substances that interact with sugar chains include, but are not limited to, viruses, microorganisms, protozoa, plant cells, and animal cells. The virus herein includes various DNA viruses, RNA viruses, and retroviruses in addition to pathogenic influenza virus, HIV virus, hepatitis virus, SARS virus, and the like. Microorganisms include bacteria, fungi, actinomycetes, and the like. In addition, the toxin is not limited, but it is an organic compound derived from a living body such as aconitine, ibotenic acid, muscimol, excitotoxin, erabtoxin, clare, colchicine, saxitoxin, ciguatoxin, tetanus toxin, tetrodotoxin, domoic acid, Synthetic organic compounds such as palytoxin, botulinum toxin, ricin, cycloxin, trigonelline, gramicidin, nicotine, phosgene, sarin, soman, tabun, VX, dioxin, toluene, biphenyl, monofluoroacetic acid, methylmercury chloride, tetra Some are derived from venom such as ethyl lead, and spines and fangs of toxic organisms. Other simple substance (fluorine (F2), ozone (oxygen allotrope, O ₃ ), phosphorus (white phosphorus / yellow phosphorus, P4) selenium (Se), arsenic (As) cadmium (Cd), mercury (Hg), lead (Pb ), Thallium (Tl), etc., plutonium (Pu), polonium (Po), etc., inorganic compounds (hydrogen fluoride (HF), hydrogen cyanide (HCN), hydrogen sulfide (H ₂ S), carbon monoxide (CO), etc. Potassium cyanide (potassium cyanide, KCN), sodium cyanide (NaCN), sodium hydroxide (caustic soda, NaOH), sodium azide (NaN ₃ ), etc. Mercury (II) chloride (raised, HgCl ₂ ), potassium dichromate (K ₂ Cr ₂ O ₇ ), chloroauric acid (HAuCl ₄ ), silver nitrate (AgNO ₃ ), etc.

  Thus, the immobilized glycan of the present invention comprises an adsorbent for viruses, bacteria, and / or toxins; virus, bacteria, and / or toxin neutralizers; biological defense system stimulation, anti-inflammatory, antibacterial, and It can also be used as an agent useful for anti-tumor metastasis; a diagnostic agent; a cell culture substrate or a specific cell separation substrate; a detection base for viruses or bacterial toxins; or an in vivo drug carrier.

  Specific examples of the method for purifying and concentrating the sugar chain compound of the present invention are shown below in the embodiments, but the present invention is not limited thereto.

  Using 12-azidodecyl β-lactoside, the immobilization on the filter paper in the present invention was performed according to Scheme 1 (FIG. 1). At this time, 12-azidodecyl β-lactoside to be immobilized was fluorescently labeled with DBD-COCl (manufactured by Tokyo Chemical Industry Co., Ltd.) to confirm that the final substance was immobilized.

Specifically, 12-azidodecyl β-lactoside was immobilized on filter paper as follows.
(1) A filter paper (manufactured by Whatman, No. 41, φ = 55 mm) (1 in FIG. 1) was immersed in a mixed solution of 1M NaOH aqueous solution and saturated saline (mixing ratio 1: 1) and stirred for 1 hour. Thereafter, 100 mg of bromoacetic acid per filter paper was added and stirred overnight. After completion of the reaction, the product was thoroughly washed with water until neutral and dried naturally to obtain a paper filter having a carboxyl group at the terminal (2 in FIG. 1). The introduced carboxyl group was quantified by neutralization titration based on a calibration curve using a commercially available acetic acid standard solution. As a result, the amount of carboxyl group introduced per filter paper was 16.0 μmol.

(2) Next, 20 filter papers obtained in (1) were immersed in 15 ml of water, 80 mg of 2-propynylamine and 200 mg of DMT-MM were added, and the mixture was stirred overnight at room temperature. After completion of the reaction, it was sufficiently washed with water and naturally dried to obtain a paper filter (3 in FIG. 1) having a propargyl group at the end. As a result of quantifying the carboxyl group by the neutralization titration method, it was confirmed that 12.3 μmol of propargyl group was introduced per filter paper from the amount of decrease of the carboxyl group before the reaction.

(3) After the glycosylation using boron trifluoride etherate (BF ₃ · OEt ₂ ), which is a Lewis acid as a catalyst, and alcohol or an alcohol having an azido group introduced, deacetylation reaction is performed. As a result, three types of sugar chain primers (12-azidodecyl β-lactoside, 2-azidodecyl β-lactoside, and dodecyl β-lactoside) were obtained (Murozuka, Y., Kasuya, MC, Kobayashi, M., Watanabe , Y., Sato, T. and Hatanaka, K. 2005, Chemistry and Biodiversity, 2, 1063, Kasuya, MC, Wang, L., Lee, YC, Mitsuki, M., Nakajima, N., Sato, T. , Hatanaka, K., Yamagata, S. and Yamagata, T. 2000, Carbohydr. Res., 329, 755.)

  Separately, 8.3 mg of DBD-COCl was dissolved in 1 ml of N, N-dimethylformamide to prepare a DBD-COCl solution. 500 nmol of three types of sugar chain primers (12-azidodecyl β-lactoside, 2-azidodecyl β-lactoside, and dodecyl β-lactoside) are dissolved in 1 ml of N, N-dimethylformamide containing 0.5 mM quinuclidine. A chain solution was prepared. These DBD-COCl solution and sugar chain solution were mixed, reacted at 60 ° C. for 3 hours, purified by Sep-Pac (manufactured by Waters), and the resulting methanol extract was evaporated to dryness. This dried product was dissolved in 0.5 ml of methanol, 5 times the amount of water was added, and unreacted DBD-COCl was removed by dialysis. After dialysis, fluorescently labeled sugar chains were obtained by lyophilization.

(4) The filter paper introduced with the propargyl group obtained in (2) is cut into a size of 1 cm ² , the 1 cm ² filter paper section, the fluorescently labeled sugar chain obtained in (3), Add 0.05 mg of N, N-diisopropylethylamine, 5 mg of Cu (PPh ₃ ) ₃ Br to a glass container containing 1 ml of tetrahydrofuran, and stir at room temperature for 48 hours (immobilization step by click chemistry ligation reaction) Then, an immobilized sugar chain (4 in FIG. 1) was obtained.

  After completion of the reaction, it was thoroughly washed with tetrahydrofuran and naturally dried, and then irradiated with ultraviolet rays of 365 nm and 450 nm, and it was confirmed whether or not sugar chains were immobilized by fluorescent color development. The result is shown in FIG. For 12-azidodecyl β-lactoside having an azido group at the end, relatively strong fluorescence was observed, but for dodecyl β-lactoside having no azide group, almost no fluorescence was observed. For 2-azidodecyl β-lactoside having an azide group at the base of the alkyl chain, fluorescent coloration was observed although it was very weak.

In each of 150 100 mm dishes, 2 × 10 ⁶ cells of mouse melanoma B16 cells were suspended and seeded in 7 ml of DMEM / F12 medium (Invitrogen) containing 10% FBS, and 37 times in a CO ₂ incubator. Pre-culture was performed for 48 hours at ° C.
After preculture, the medium was replaced with DMEM / F12 medium supplemented with 12-azidododecyl-β-lactoside and 1% ITSX (GIBCO) to a final concentration of 50 μM, and further 37 ° C. in a CO ₂ incubator. Incubated for 48 hours to carry out a sugar chain elongation reaction. After the sugar chain elongation reaction, the medium was collected, and the dish surface was washed with PBS to collect the whole medium supernatant.

  50 g of a styrene synthetic adsorbent Diaion HP20 was added to the obtained medium supernatant, and the mixture was shaken for 16 hours to adsorb the sugar chain compound to the adsorbent. Next, after washing with 100 ml of water five times, 50% concentration methanol, 70% concentration methanol, and 100% concentration methanol were sequentially passed through each 100 ml four times.

  The eluate with 50% methanol and 70% methanol is concentrated by a rotary evaporator and then re-adsorbed on the adsorbent in a column filled with 50 ml styrene synthetic adsorbent (Diaion HP20). After passing an appropriate amount of 40-50% methanol, GM3 type sugar chain compound as the target sugar chain compound was eluted with 200 ml of 70% methanol.

  On the other hand, the eluate with 100% methanol was concentrated by a rotary evaporator and then dried. After 3 ml of DMSO was added to the dried product and dissolved, 100 ml of water was added, followed by re-adsorption to the adsorbent in a column filled with 50 ml of a styrene synthetic adsorbent (Separbeads SP207). After washing with 200 ml of water, the GM3-type sugar chain compound as the target sugar chain compound was eluted with 200 ml of 70% strength methanol. After all the eluate was concentrated under reduced pressure and dried, GM3 type sugar chain compound was obtained as 18.5 mg of white powder.

Next, two filter papers introduced with propargyl group obtained in Example 1, 18.5 mg of GM3 type sugar chain compound obtained as described above, 2.1 mg of N, N-diisopropylethylamine, 0 .5 mg of Cu (PPh ₃ ) ₃ Br was added to a glass container containing 30 ml of tetrahydrofuran and stirred at room temperature for 48 hours (an immobilization step by a click chemistry ligation reaction). After completion of the reaction, the product was sufficiently washed with tetrahydrofuran and water and dried naturally.

  As in Example 1, 12-azidododecyl-β-lactoside without sialic acid added was immobilized on filter paper by a click chemistry ligation reaction.

  Next, the influenza virus (strain A / PR / 8/34) obtained by the above-described steps into the filter paper on which 12-azidododecyl-β-lactoside is immobilized and the filter paper on which the GM3 type sugar chain is immobilized. ) Was compared.

  Blocking was performed by adding 5 × Denhardt's solution containing 200 ul of 10 mg / ml salmon sperm DNA to the container in which each filter paper was placed, and incubating at room temperature for 30 minutes. After blocking, the blocking solution was removed.

Influenza virus solutions 3, 9, and 30 ul having a concentration of about 10 ⁹ pfu / ml were mixed with 1 ml of PBS (−) (phosphate buffer), respectively (× 3, × 9, and × 30, respectively). 100 ul of each prepared influenza virus solution was added to the blocked filter paper and incubated at room temperature for 10 minutes. Unadsorbed influenza virus was removed from the cellulose filter and washed three times with 200 ul of PBS (−).
Thereafter, 15 ul of SDS sample buffer was added, heat-treated at 100 ° C. for 5 minutes, and adsorbed virus was eluted by centrifugation.

  The obtained viral protein solution was subjected to SDS-PAGE, and the protein was blotted on a PVDF (polyvinylidene difluoride) membrane from the gel after electrophoresis. The blotted filter was immersed in PBS (−) containing 5% milk for 30 minutes and then immersed in an anti-PR / 8/34 antibody solution for 1 hour. Thereafter, the plate was washed 3 times with TBS-T (Tris buffer solution-Tween 20) for 5 minutes, and immersed in a horseradish peroxidase secondary antibody solution for 30 minutes. After the reaction, the plate was washed 3 times with TBS-T for 5 minutes, and developed with an ECL Western Blotting Detection System (GE Healthcare Biosciences).

  The results are shown in FIG. 3, but the influenza paper adsorption ability could be confirmed with the filter paper with the Gb3-type sugar chain immobilized, but the influenza virus adsorption ability could hardly be confirmed with the filter paper with lactose immobilized. .

  12-azidododecyl β-lactoside was used as a sugar chain compound as a target for introducing a functional group, and a thiol group was introduced by a click chemistry ligation reaction. The scheme shown in FIG. 4 was followed.

  106.14 mg (1 mmol) of 3-mercaptopropionic acid (Compound 1 of FIG. 4), 55.08 mg (1 mmol) of 2-propynylamine (Compound 2 of FIG. 4), and 300 mg (1.08 mmol) of DMT-MM Was dissolved in 10 ml of water and reacted overnight at room temperature. The produced precipitate was collected by centrifugation and then thoroughly washed with water. Then, it lyophilized | freeze-dried and obtained 20.2 mg powder (compound 3 of FIG. 4). Thin layer chromatography confirmed the disappearance of the raw material and one new spot.

20 mg (0.036 mmol) of 12-azidododecyl β-lactoside (compound 4 in FIG. 4), 5 mg (0.035 mmol) of compound 3 obtained above, 2.6 mg (0.02 mmol) of N, N— Diisopropynylethylamine and 0.6 mg (1.2 μmol) of Cu (PPh ₃ ) ₃ Br were dissolved in 200 μl of tetrahydrofuran and reacted at room temperature for 48 hours. After the reaction, the solvent was removed by blowing air, dissolved in 100 μl of dimethyl sulfoxide, 5 ml of water was added for reprecipitation, and the precipitate was collected by centrifugation. Further, the obtained precipitate was sufficiently washed with water and then lyophilized to obtain 22.5 mg of a powder (mixture of compound 5 and unreacted compound 4 in FIG. 4).

  With respect to the mixture of Compound 5 and unreacted Compound 4 obtained by the above operation, the introduction rate of thiol groups was measured by quantification of thiol groups by the DTNB method, and found to be 44.3%.

  As a result of analyzing the obtained compound by NMR, in addition to the peak derived from 12-azidododecyl β-lactoside as a raw material, a new peak that could be generated by the click chemistry ligation reaction was detected. It was confirmed that the desired sugar chain compound having a thiol group at the terminal could be obtained.

  12-azidododecyl β-lactoside was used as a sugar chain compound as a target for introducing a functional group, and 2-propynylamine was linked by a click chemistry ligation reaction to introduce an amino group. Further, the polymer was polymerized by condensation reaction with polyacrylic acid.

  Specifically, as described below, an amino group was introduced into 12-azidododecyl β-lactoside and further polymerized by a condensation reaction with polyacrylic acid. The scheme is shown in FIG.

Introduction of amino group 100 mg (0.181 mmol) 12-azidododecyl β-lactoside (compound 4 in FIG. 5), 10 mg (0.182 mmol) 2-propynylamine (compound 2 in FIG. 5), 13 mg (0.1 mmol) ) N, N-diisopropylethylamine and 3 mg (0.006 mmol) Cu (PPh ₃ ) ₃ Br were dissolved in 1 ml tetrahydrofuran and reacted at room temperature for 48 hours. Thereafter, the solvent was removed by blowing gas. The remaining dried product was dissolved in 500 ul of dimethyl sulfoxide, and 12 ml of water was added to reprecipitate the sugar chain compound. The precipitate was collected by centrifugation, then sufficiently washed with water and lyophilized to obtain 34.5 mg of a powder (mixture of compound 6 and unreacted compound 4 in FIG. 5). It was confirmed by thin layer chromatography that the disappearance of 2-propynylamine as a raw material and a new spot were colored by the ninhydrin reaction.

  As a result of analyzing the obtained compound by NMR, it was possible to detect a new peak that could be generated by the click chemistry ligation reaction in addition to the peak derived from the raw material 12-azidododecyl β-lactoside. It was confirmed that the sugar chain compound was produced.

Polymerization by Immobilization to Polyacrylic Acid 9.1 mg of powder containing 12-azidododecyl β-lactoside (compound 6 in FIG. 5) introduced with the amide group obtained by the above operation and polyacrylic acid (compound 7) ) 540 mg was dissolved in 600 μl of N, N-dimethylformamide, and 15.2 mg (40 μmol) of HATU was added and allowed to react overnight. After the reaction, the solvent was removed by blowing air, dissolved in 10 ml of water, and centrifuged to collect the supernatant. Next, the collected supernatant was lyophilized to obtain 18.7 mg of powder. When the obtained powder was analyzed by NMR, in addition to the peak derived from water-soluble polyacrylic acid, a peak derived from water-insoluble 12-azidododecyl β-lactoside could be detected. It was confirmed that a polymer (compound 8 in FIG. 5) was obtained by immobilization.

An example of a typical scheme showing the production process of the immobilized sugar chain of the present invention is shown. The image which detected the sugar chain fluorescently labeled on the filter paper is shown. The result of having assayed the virus adsorption ability to the filter paper which fix | immobilized GM3 is shown. A scheme for introducing a thiol group into a sugar chain is shown. A scheme for introducing an amino group into a sugar chain and an example of polymerizing the sugar chain are shown.

Claims (20)

An immobilized sugar chain in which a sugar chain containing at least two monosaccharide residues or derivatives thereof is bound to a solid phase carrier or a polymer,
An immobilized sugar chain in which the sugar chain is bound to a solid phase carrier by a spacer containing a triazole ring. Formula (I): (G ₁ ... G _n ) -L-X- [wherein G ₁ ,... G _n are each independently a monosaccharide residue having a cyclic structure or a derivative thereof, G is a glycosidic bond so as to form a sugar chain; L is a group consisting of —O— (CH ₂ ) _m —, —S— (CH ₂ ) _m —, and —NH— (CH ₂ ) _m —. One or more linking groups selected; n is an integer from 2 to 100; m is an integer from 8 to 20; and X represents any spacer containing a triazole ring] The immobilized sugar chain according to claim 1, comprising at least one sugar chain compound. The sugar chain is obtained by adding a sugar chain primer to an animal cell and culturing, and the sugar chain primer is a compound obtained by binding an alkyl chain having an azide group to an oligosaccharide chain. 2. The immobilized sugar chain according to 2. The immobilized sugar chain according to any one of claims 1 to 3, wherein the sugar chain comprises a plurality of sugar chains of the same or different types. The immobilized saccharide according to any one of claims 1 to 4, wherein the solid phase carrier is one selected from the group consisting of a carrier containing a polymer compound, a carrier containing a metal, and a carrier made of paper. Chain. The immobilized sugar chain according to any one of claims 1 to 5, wherein the solid phase carrier has a plate-like form or a bead-like form. A method for preparing an immobilized sugar chain in which a sugar chain is bound to a solid phase carrier,
A method comprising a step of reacting and binding a compound containing a sugar chain containing at least two or more monosaccharide residues or derivatives thereof, an alkyl group, and an azide group and an alkyne group. 8. The method for producing an immobilized sugar chain according to claim 7, wherein the alkyne group is bound to a solid phase carrier. The compound is represented by the formula (I): (G ₁ ... G _n ) -LN ₃ [wherein G ₁ ,... G _n are each independently a monosaccharide residue having a cyclic structure or a derivative thereof. And is a glycosidic bond so as to form a sugar chain with each other; L represents —O— (CH ₂ ) _m —, —S— (CH ₂ ) _m —, and —NH— (CH ₂ ) _m —. 9 or 1 is a linking group selected from the group consisting of: n is an integer of 2 to 100, and m is an integer of 8 to 20. To create an immobilized sugar chain of The immobilized sugar chain according to any one of claims 7 to 9, further comprising a step of adding a compound obtained by binding an alkyl chain having an azide group to an oligosaccharide chain to animal cells and culturing the compound to obtain a sugar chain. How to create a body. The immobilized sugar according to any one of claims 7 to 10, wherein the solid phase carrier is one selected from the group consisting of a carrier containing a polymer compound, a carrier containing a metal, and a carrier made of paper. How to create a chain. A method of bringing the immobilized sugar chain according to any one of claims 1 to 6 into contact with a substance that binds or interacts with a sugar chain part in the immobilized sugar chain. The method according to claim 12, wherein the substance is one selected from the group consisting of viruses, microorganisms, protozoa, plant cells, and animal cells. A virus, bacteria, and / or toxin adsorbent comprising the immobilized sugar chain according to any one of claims 1 to 6. A virus, bacteria, and / or toxin neutralizing agent comprising the immobilized sugar chain according to any one of claims 1 to 6. A drug containing the immobilized sugar chain according to any one of claims 1 to 6, which is useful for stimulating a host defense system, anti-inflammatory, antibacterial, and / or anti-tumor metastasis. A diagnostic agent comprising the immobilized sugar chain according to any one of claims 1 to 6. A cell culture substrate or a specific cell separation substrate comprising the immobilized sugar chain according to any one of claims 1 to 6. A detection base for a virus or bacterial toxin comprising the immobilized sugar chain according to any one of claims 1 to 6. An in vivo drug transporter comprising the immobilized sugar chain according to any one of claims 1 to 6.

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