JP2010068910A - Sugar chain fixed polymer substrate for hiv adsorption, and method of manufacturing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sugar chain-fixed polymer substrate for HIV absorption having high stability and blood compatibility for fixing a glucide to a polymer substrate without using an alkaline treatment and a compound having strong toxicity such as bromoacetate, 1,4-butanediol diglycidyl ether and the like, and a method of manufacturing the same. <P>SOLUTION: There are provided a sugar chain-fixed polymer substrate for HIV absorption having a sugar chain obtained by bringing a polymer substrate having a methylene group in a main chain in contact with a polymerizing compound having an ethylene unsaturated bond and a sugar chain or a polymerizing composition containing the polymerizing compound and then by irradiating it with an ionizing radiation ray, or polymer substrate irradiating it with the ionizing radiation ray and then bringing it in contacted with the polymerizing compound or the polymerizing composition containing the polymerizing compound, and a method of manufacturing the same. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a carbohydrate-immobilized polymer substrate for removing HIV (human immunodeficiency virus) and a method for producing a carbohydrate-immobilized polymer substrate using ionizing radiation graft polymerization.
The present invention is based on "patent application subject to Article 19 of the New Energy and Industrial Technology Development Organization Commissioned Research, Industrial Technology Strengthening Act Article 19" (formerly "2006 New Energy and Industrial Technology Development Organization Patent application subject to Article 30 of the Contract Research and Industrial Revitalization Special Measures Law ").

  Certain carbohydrates interact specifically with toxins, viruses and other pathogens, and carbohydrate macromolecules formed by binding such carbohydrates to macromolecules bind to pathogens such as viruses and proteins by the cluster effect. By doing so, it is known that toxicity and infectivity are inhibited. For example, influenza virus binds sialyl lactose to a carbohydrate polymer linked to a polymer, verotoxin binds to a galactosyl lactate linked to a polymer, and HIV is a sulfated polysaccharide. To join.

  Thus, by immobilizing a carbohydrate that specifically interacts with a pathogen, it becomes a polymer material applicable to various uses. For example, by fixing a carbohydrate polymer to a hollow fiber, non-woven fabric, or bead to form a column for extracorporeal circulation, it can be applied to uses such as removal of viruses and toxins, and separation and purification of toxins and viruses.

  As an example of the removal of such viruses and toxins, pathogenic fine particle-adsorbing hollow fibers formed by chemically bonding a carbohydrate polymer formed by binding a sugar chain to a polymer to the inner surface of the hollow fiber are known (patents). Reference 1 and Non-Patent Document 1). For example, in Patent Document 1, a sodium hydroxide aqueous solution is added to a polymer base material such as regenerated cellulose, and then a water-dimethyl sulfoxide mixed solution of 1,4-butanediol glycidyl ether is added. A method is described in which a sugar chain is introduced into a substrate by activating the substrate and then bringing it into contact with a sugar polymer solution having acrylamide as the main chain synthesized earlier. Non-Patent Document 1 discloses a carbohydrate polymer whose main chain is an acrylamide synthesized previously by activating the cellulose surface with sodium hydroxide, reacting with bromoacetic acid to introduce a carboxy group on the surface. And a method in which a carboxy group on the surface is condensed using WSC (Water Soluble Carbodiimide) and a sugar chain is introduced into a substrate.

  However, these methods require several steps to immobilize the sugar chain, and it is necessary to confirm by using surface analysis or the like that the target surface treatment is reliably performed in each step. It was complicated.

  Furthermore, these methods require alkali treatment and must use highly toxic compounds (allergic dermatitis, dermatitis, mucosal inflammation, etc.) such as bromoacetic acid and 1,4-butanediol diglycidyl ether. Therefore, a thorough cleaning process was necessary to ensure safety.

  In addition, when used for adsorption removal by extracorporeal circulation, it is necessary to continuously return the blood to the body after passing through the hollow fiber module placed outside the body, so that the blood does not coagulate during the adsorption removal. However, when a hydroxyl group or functional group is introduced to the substrate surface, the hydroxyl group or functional group that did not bind to the carbohydrate polymer remains unreacted on the substrate surface, resulting in blood coagulation factors. Is activated by these hydroxyl groups and functional groups (see Non-Patent Document 2), it has also been a factor of lowering blood compatibility.

As an example of immobilizing a sugar chain on a substrate, a sugar chain represented by Galα1 → 4Galβ1 → 4Glc (Gb ₃ ) or Galα1 → 4Galβ1 (Gb ₂ ) is used as a commercially available carboxymethyl such as “BIACORE CM5”. Surface treatment with dextran chain-immobilized chip surface via carboxyl group, gold surface with 1-thio-alkane-ω-amine, 1-thio-alkane-ω-carboxylic acid, lipoic acid, cysteamine, etc. And what is fix | immobilized on the chip | tip surface via those amino groups and carboxy groups is known (patent document 2). However, these require a step of heating and depositing gold under a high vacuum, so that raw material costs and manufacturing costs are required, and the steps are complicated. Moreover, since the obtained sugar chain immobilization base material is also used for sensors, the sugar chain density is very low, and as a result, the adsorption removal effect is very small. In addition, BIACORE CM5 is difficult to permeate into the dextran chain even if it is brought into contact with a toxin or virus due to steric hindrance of the dextran chain itself, and interacts only on the surface of the sensor chip. Even if it existed, the adsorption removal effect was not able to be exhibited.

JP 2003-135596 A JP 2003-226697 A A. Miyagawa et al Biomaterials, 27, 3304 (2006) Published by Hiroo Iwata, edited by the Society of Polymer Science, “Biomaterials”, Kyoritsu Publishing, 2005

  Therefore, the problem to be solved by the present invention is to fix a saccharide to a polymer substrate without using a toxic compound such as alkali treatment, bromoacetic acid, bromoacetic acid or 1,4-butanediol diglycidyl ether, An object of the present invention is to provide a sugar chain-immobilized polymer substrate for HIV adsorption having high safety and high blood compatibility, and a method for producing the same.

  As a result of diligent research to solve the above-mentioned problems, the present inventors polymerize and immobilize a polymerizable compound having a sugar chain on a polymer substrate by graft polymerization using a radical species generated by radiation irradiation as an initiator. Thus, it was found that sugar chains can be immobilized on a polymer substrate without using a highly toxic compound such as alkali treatment or bromoacetic acid, and the present invention has been completed.

  That is, the present invention provides a polymer base material having a methylene group in the main chain with the general formula (1)

(1)

Wherein R ¹ represents a hydrogen atom or a methyl group, W and X each independently represent a divalent linking group, and G represents a sugar chain, or the polymerizable compound A step (1) of contacting a polymerizable composition containing a compound, and a step (2) of irradiating ionizing radiation in a state where the polymerizable compound represented by the general formula (1) is brought into contact with the polymer substrate. And in this order, or
The step (3) of irradiating the polymer substrate with ionizing radiation, and the step of contacting the polymerizable compound or a polymerizable composition containing the polymerizable compound with the polymer substrate irradiated with ionizing radiation (4) ) In this order, a method for producing a sugar chain-immobilized polymer substrate for HIV adsorption is provided.

  The present invention also provides a polymer base material having a methylene group in the main chain, the repeating unit (M) represented by the general formula (3).

(3)

(Wherein R ¹ represents a hydrogen atom or a methyl group, W represents —CONH—, —CO—O—, or —R ² —CONH— (wherein R ² represents an aromatic group), and X represents alkylene. A group, or —R ³ —Z—R ⁴ — (wherein R ³ and R ⁴ represent an alkylene group, Z represents —O—, —NH—, —NR ⁰ — (where R ⁰ represents a hydrogen atom or a carbon atom number); 1 represents an alkyl group of 1 to 4) or represents -S-), G represents a sugar chain), or a repeating group represented by the general formula (3) The repeating unit (L) represented by the unit (M) and the general formula (4)

(4)

(Wherein R ⁵ represents a hydrogen atom or a methyl group, and V represents —COOH, —COOR ⁶ (where R ⁶ represents an alkyl group) or —CONH ₂ ). A sugar chain-immobilized polymer base material is provided.

  In the method for producing a sugar chain-immobilized polymer substrate of the present invention, a carbohydrate is immobilized on a polymer substrate without using an alkali treatment or a toxic compound such as bromoacetic acid or 1,4-butanediol diglycidyl ether. be able to. For this reason, a highly safe sugar chain-immobilized polymer substrate can be provided. In addition, the method for producing a sugar chain-immobilized polymer substrate of the present invention eliminates the cause of activating complement, which is a blood coagulation factor, because it is not necessary to introduce a hydroxyl group or a functional group to the substrate surface. As a result, the blood compatibility inherent in the substrate itself can be maintained, so that a sugar chain-immobilized polymer substrate having high blood compatibility can be provided.

-Method for producing sugar chain-immobilized polymer substrate The method for producing a sugar chain-immobilized polymer substrate according to the present invention comprises a polymer substrate having a methylene group in the main chain, represented by the general formula (1)

(1)

Wherein R ¹ represents a hydrogen atom or a methyl group, W and X each independently represent a divalent linking group, and G represents a sugar chain, or the polymerizable compound A step (1) of contacting a polymerizable composition containing a compound, and a step (2) of irradiating ionizing radiation in a state where the polymerizable compound represented by the general formula (1) is brought into contact with the polymer substrate. And in this order, or
A step (3) of irradiating the polymer substrate with ionizing radiation, and a step (4) of bringing the polymerizable compound or a polymerizable composition containing the polymerizable compound into contact with the polymer substrate irradiated with ionizing radiation. ) In this order.

-Polymer substrate The polymer substrate used in the present invention may be a polymer compound having a methylene group in the main chain because radicals are generated by irradiation with ionizing radiation. As such a polymer compound, various compounds having high blood compatibility can be used. For example, olefin resin, styrene resin, sulfone resin, acrylic resin, urethane resin, ester Resin, ether resin or cellulose acetate, and more specifically, polyethylene terephthalate, ethylene vinyl alcohol copolymer, polymethyl methacrylate, polysulfone, polyether sulfone, polyacrylonitrile, polyethylene, polypropylene or poly-4-methyl. An example is pentene.

The shape of the polymer substrate is not particularly limited, and can be used in various forms such as hollow fiber membranes, beads, and nonwoven fabrics. When applied to extracorporeal circulation, it can be used as beads or non-woven fabrics with a structure in which blood stays. It is desirable to use a hollow fiber membrane. Further, if the hollow fiber membrane is not used as a filtration membrane, it is not necessary to be porous, and therefore the form of the hollow fiber may be selected according to the application.

Polymerizable compound The polymerizable compound used in the present invention is represented by the general formula (1).

(1)

It is represented by In the formula, R ¹ represents a hydrogen atom or a methyl group.
In the above formula, W represents a divalent linking group, but different in general the type of sugar chains pathogens and later, -CONH -, - CO-O- , or an ^-R 2 -CONH-. R ² represents an aromatic group, for example, a divalent aromatic group such as a phenylene group or a naphthylene group.

In addition, in the formula, X represents a divalent linking group, and generally varies depending on the pathogen and the type of sugar chain described below, but an alkylene group, an element at any position other than a single or plural carbon (oxygen atom, nitrogen) An alkylene group substituted with an atom or a sulfur atom). As specifically the X alkylene group or ^-R 3 ^{-Z-R 4,} 1 to 30 carbon atoms - for example, a group represented by. Where R ³ and R ⁴ represent an alkylene group, specifically an alkylene group having 1 to 30 carbon atoms, Z represents —O—, —NH—, —NR ⁰ — (where R ⁰ represents a hydrogen atom or carbon Represents an alkyl group having 1 to 4 atoms) or -S-.

  Further, in the formula, G represents a sugar chain. The sugar chain used in the present invention is composed of various sugars and combinations thereof, and a sugar having excellent adsorption activity may be appropriately selected according to the virus to be adsorbed. The case is represented by the following general formula (2)

(2)

Galactosyl lactose (Gb3, ^Pk saccharide) represented by the formula (wherein n represents an integer of 0 to ¹ ), galactosylgalactose (Gb2) and the like are particularly preferable.

-Polymerizable composition Moreover, the polymeric composition which used 2 or more types of polymeric compounds from which sugar chain differs among the polymeric compounds mentioned above can also be used. Furthermore, in order to adjust to an optimal sugar chain density or to increase the amount of immobilization to the polymer substrate, other polymerizable compounds can be used in combination with the above-described polymerizable compound. As such other polymerizable compounds, those having high blood compatibility are preferred, and examples thereof include polymerizable compounds having an ethylenically unsaturated bond, such as acrylic acid, acrylic acid ester, methacrylic acid, methacrylic acid ester, Examples include acrylamide and methacrylamide.

-Contact method The contact method between the polymer substrate and the polymerizable compound or the polymerizable composition is a method in which the polymerizable compound or the polymerizable composition is dissolved in an aqueous solvent to form an aqueous solution, What is necessary is just to make it contact with a molecular base material. In the present invention, since the graft polymerization on the surface of the polymer substrate proceeds preferentially, the concentration of the polymerizable compound or the polymerizable composition in the aqueous solution is preferably lower. At a very high concentration, for example, 20% by mass or more, when a polymerizable compound that is easily polymerized, for example, a low molecular weight polymerizable compound such as acrylic acid or acrylamide is used, radicals diffuse into the solution and the polymerization reaction occurs simultaneously in the solution. May progress. Specifically, the upper limit is limited by the solubility of each polymerizable compound to be used, and is 0.1% by mass or more, preferably 0.1 to 20% by mass, more preferably 0.1 to 15% by mass. .

-Irradiation of ionizing radiation In this invention, the ethylenically unsaturated bond part which the said polymeric compound has is graft-polymerized to a polymer base material by the radical which generate | occur | produced by irradiating the said polymer base material with ionizing radiation. Examples of the ionizing radiation source used in the graft polymerization include α rays, β rays, γ rays, accelerated electron rays, X rays, and the like. Practically, γ rays and accelerated electron rays are preferable.

  The graft polymerization method is a simultaneous irradiation graft polymerization method in which the polymer base material and the polymerizable compound are brought into contact with each other and irradiated with ionizing radiation, and a pre-irradiation graft polymerization method in which the polymer base material is pre-irradiated and then contacted with the polymerizable compound. Either of these is possible and can be selected according to the purpose.

  In the graft polymerization method using ionizing radiation used in the present invention, the irradiation dose and acceleration voltage differ depending on the polymer base material, so the range cannot be determined unconditionally. It is necessary to consider and adjust accordingly. For example, when the irradiation amount is large, dielectric breakdown due to charging occurs, and when the irradiation amount is small, the polymerization reaction does not proceed. For this reason, in consideration of the material and form of the polymer base material, the amount of irradiation with which the dielectric breakdown due to charging does not occur and the polymerization reaction sufficiently proceeds may be appropriately adjusted. Further, the acceleration voltage is related to the permeability and varies depending on the thickness of the polymer substrate. In the case of a thin form such as a film, the acceleration voltage is generally small and may be selected depending on the form of the polymer substrate.

  For example, in the case of a hollow fiber having a thickness of 0.1 ([mu] m) to 10 ([mu] m) made of 4-methyl-1-pentene as the polymer base material, it is 10 (kGy) or more and 300 (kGy) or less. More desirably, it is 90 (kGy) or less. The acceleration voltage is 0.1 (kV) or more and 10 [kV] or less, preferably 5 (kV) or less.

  There is no particular limitation in the order of the contact step and the ionizing radiation irradiation step. For example, after contacting the polymerizable compound represented by the general formula (1) or the polymerizable composition containing the polymerizable compound with the polymer base material (step 1), irradiation with ionizing radiation is performed in this state. The step (2) may be performed in this order, or conversely, the polymer substrate is first irradiated with ionizing radiation (step (3)), and then the polymer substrate is subjected to the general formula (1). And the step (4) of contacting the polymerizable compound represented by (1) or the polymerizable composition containing the polymerizable compound may be performed in this order. In the present invention, the sugar chain can be immobilized on the polymer substrate by either method.

In the irradiation graft polymerization, radicals in the substrate after irradiation are quickly inactivated by temperature increase and contact with oxygen. Therefore, after irradiation, it is preferable to store it at a low temperature in a state where oxygen is sufficiently removed, and to quickly fix it. For the above reasons, the immobilization is preferably carried out under deoxygenation or in an inert gas.
What is necessary is just to remove the unreacted polymeric compound by various methods, such as water washing, to the sugar_chain | carbohydrate fixed polymer base material after superposition | polymerization. The unreacted polymerizable compound is originally water-soluble, and is used with alkali treatment, bromoacetic acid or 1,4-butanediol diglycidyl ether in order to bond the polymer having a higher molecular weight to the substrate. Compared with the conventional chemical bonding method, a simpler washing process may be used.

When the polymer base material is a hollow fiber membrane and the polymerizable compound or the polymerizable composition is fixed to the hollow fiber membrane, either the inner surface or the outer surface of the yarn is used depending on the embodiment. Alternatively, for example, when blood is refluxed inside the hollow fiber, the sugar chain is immobilized by contacting the polymerizable compound or polymerizable composition inside the hollow fiber. Conversely, at the time of external reflux, the polymerizable compound or polymerizable composition may be brought into contact with the outside of the hollow fiber to immobilize the sugar chain. Furthermore, when a hollow fiber membrane is used as a filtration membrane, the sugar chain may be immobilized by contacting both surfaces.

-Sugar chain-immobilized polymer substrate Next, the sugar chain-immobilized polymer substrate of the present invention will be described. The sugar chain-immobilized polymer substrate of the present invention has a repeating unit (M) represented by the general formula (3) on a polymer substrate having a methylene group in the main chain.

(3)

(Wherein R ¹ represents a hydrogen atom or a methyl group, W represents —CONH—, —CO—O—, or —R ² —CONH— (wherein R ² represents an aromatic group), and X represents alkylene. A group, or —R ³ —Z—R ⁴ — (wherein R ³ and R ⁴ represent an alkylene group, Z represents —O—, —NH—, —NR ⁰ — (where R ⁰ represents a hydrogen atom or a carbon atom number); Represents an alkyl group of 1 to 4) or represents -S-), and G represents a sugar chain).
Alternatively, the repeating unit (M) represented by the general formula (3) and the repeating unit (L) represented by the general formula (4)

(4)

(Wherein R ⁵ represents a hydrogen atom or a methyl group, V represents —COOH, —COOR ⁶ (wherein R ⁶ represents an alkyl group) or —CONH ₂ ), and To do.

However, R ² represents the same aromatic group as described for the polymerizable compound represented by the general formula (1), and R ³ and R ⁴ are polymerizable compounds represented by the general formula (1). Represents the same alkylene group as described. R ⁶ represents an alkyl group, preferably an alkyl group having 1 to 30 carbon atoms. G also represents the same sugar chain as described for the polymerizable compound represented by the general formula (1).

Furthermore, the repeating unit (M) in the said General formula (3) is 1 or more, Preferably it is 1-10000, More preferably, it is the range of 100-1000. Moreover, the repeating unit (L) in the said General formula (4) is 1 or more, Preferably it is 1-10000, More preferably, it is the range of 100-1000.
Further, the ratio of the repeating unit (M) in the general formula (3) to the repeating unit (L) represented by the general formula (4) is not particularly limited, and can be adjusted to an optimum sugar chain density. Or, it may be appropriately adjusted according to the case where the amount immobilized on the polymer substrate is increased.

  In addition, examples of the polymer substrate used for the sugar chain-immobilized polymer substrate in the present invention include the same as those in the general formula (1).

The sugar chain-immobilized polymer substrate of the present invention can be easily obtained by the production method. In this case, one end of the repeating units (M) and (L) on the side opposite to the substrate is the polymerizable compound residue described above.

-Use Since the method for producing a sugar chain-immobilized polymer base material of the present invention can be applied in a wide range with respect to the material and form of the polymer material, a sugar-immobilized polymer material according to the purpose and application can be obtained, In addition to the above uses, it can be used for purification and separation of pathogens.

  In addition, the sugar chain-immobilized polymer base material of the present invention has HIV adsorptivity and can adsorb and remove the target HIV according to the saccharide, and can be applied to an adsorption removal column for extracorporeal circulation of HIV. In particular, the sugar chain represented by W is represented by the following general formula (2)

(2)

(Wherein n represents an integer of 0 to 1), galactosyl lactose (Gb3, ^Pk saccharide) and galactosylgalactose (Gb2) It can be particularly preferably used.

・ ・ Medical device and method of use Any method may be used as long as the method of using the sugar chain-immobilized polymer substrate can be brought into contact with a body fluid such as blood, plasma, serum, etc., and can absorb and remove HIV in the body fluid, For example, the following method can be mentioned.
(1) A method in which an adsorbent is supported inside a hollow fiber and a body fluid is caused to flow through the adsorbent.
(2) A method in which an adsorbent is filled in a container equipped with a filter having an inlet and an outlet for bodily fluid, through which the bodily fluid passes but no adsorbent passes, and the bodily fluid flows through the container.
(3) A method in which body fluid is taken out and stored in a bag or the like, mixed with an adsorbent to remove HIV, and then adsorbent is filtered to obtain a body fluid from which HIV has been removed.

Either method can be used, but the methods (1) and (2) are preferable because they are easy to operate. By incorporating them into an external circulation circuit, HIV can be efficiently removed from a patient's body fluid online. Is possible. Among these, the method according to (1) is the most preferable method. In the methods (2) and (3), it is necessary to separate blood into blood cells and plasma and treat only the plasma in order to prevent blood coagulation, but the method (1) requires such separation. This is because the operation is the simplest and the burden on the patient is small. In addition, the hollow fiber surface has irregularities and has a high surface area compared to other substrates with a smooth surface, so the contact area with blood is increased and HIV in blood can be adsorbed efficiently. Can do.

  An example of a medical device using the sugar chain-immobilized polymer substrate of the present invention will be described based on the schematic cross-sectional view.

  The container shown in FIG. 6 includes a bundle of hollow fibers 4 made of a polymer base material having a liquid inlet / outlet 1, a liquid outlet / inlet 2, and a sugar chain 3 of the present invention immobilized on the inner wall. It is stored inside. Moreover, the end surface part of the hollow fiber is sealed with a partition wall 6 made of a urethane sealant or the like. The shape and material of the container are not particularly limited, but when applied to extracorporeal circulation, the container is preferably a cylindrical container having an internal liquid amount of 10 to 400 mL and an outer diameter of about 2 to 10 cm. Most preferably, it is a cylindrical container having an inner liquid amount of 20 to 200 mL and an outer diameter of about 2.5 to 4 cm.

Hereinafter, the immobilization to the hollow fiber and the adsorption of HIV will be described in more detail with an example using Gb3 as a carbohydrate, a poly-4-methyl-1-pentene hollow fiber membrane as a polymer base material, and HIV as a pathogen. To do.

(Reference Example 1)

  6- [2- (N-Acryloylamino) ethylthio] hexyl 4-O- [4-O- (α-D-galactopylanosyl) -β-D-galactopylanosyl] -β-D-glucopyroxide (hereinafter referred to as the following chemical formula) Gb3 monomer) has been published in the literature (VP Kamath et al, Carbohydrate Research, 339, 1141 (2004), K. Matsuoka et al, Tetrahedron Lett., 40, 7839 (1999), P. B. van Sevent. , Carbohydrate Research, 300, 369 (1997), Japanese Patent Application Laid-Open No. 2005-289907) (FIG. 1). . Details will be described below.

Synthesis Example 1 <6- [2- (N-Acryloylamino) ethylthio] hexyl 4-O- [4-O- (2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl)- Synthesis of 2,3,6-tri-O-actyl-β-D-galactopylanoyl] -2,3,6-tri-O-acetyl-β-D-glucopyroxide (2)>

n-hexenyl 4-O- [4-O- (2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl) -2,3,6-tri-O-acetyl-β-D- galactopyranosyl] -2,3,6-tri-O-acetyl-β-D-glucopyranoside (1) 50 mg (0.05 mmol) and aminoethanethiol hydrochloride 28 mg (0.25 mmol) were dissolved and suspended in 2 mL of THF, and AIBN 2 mg After substituting with nitrogen under reduced pressure, the mixture was heated and stirred at an oil bath temperature of 70 ° C. The progress of the reaction was confirmed by TLC (hexane / ethyl acetate = 1/2). After disappearance of the raw materials, ethyl acetate and an aqueous sodium hydrogen carbonate solution were added for liquid separation, and anhydrous ethyl sulfate was added to the ethyl acetate layer, dried, filtered, and used as it was in the next step.

  To the ethyl acetate solution, 12 mg (0.12 mmol) of triethylamine was added, and 5.4 mg (0.06 mmol) of acrylic acid chloride was added dropwise under ice cooling. After 10 minutes, the disappearance of the raw materials was confirmed by TLC (ethyl acetate / methanol = 1/2). Water was added to perform liquid separation and post-treatment, and purification was performed by silica gel column chromatography (toluene / ethyl acetate = 1/3). Yield 51.4 mg, yield 90%.

¹ H-NMR (300 MHz, CDCl ₃ ) δ = 6.29 (dd, 1H), 6.13 (br, 1H), 6.12 (dd, 1H), 5.65 (dd, 1H), 5. 58 (d, 1H), 5.39 (dd, 1H), 5.23-5.16 (m, 2H), 5.11 (dd, 1H), 4.99 (d, 1H), 4.88 (Dd, 1H), 4.74 (dd, 1H), 4.53-4.40 (m, 5H), 4.20-4.08 (m, 4H), 4.01 (d, 1H), 3.84-3.45 (m, 7H), 2.69 (t, 2H), 2.52 (t, 2H), 2.13-1.86 (m, 30H), 1.58-1. 35 (m, 8H)

¹³ C-NMR (75 MHz, CDCl ₃ ) δ = 170.58, 170.37, 170.00, 169.64, 169.47, 168.82, 165.45, 130.73, 126.46, 101. 03, 100.50, 99.55, 76.58, 76.44, 73.09, 72.76, 72.51, 71.79, 69.87, 69.00, 68.81, 67.90, 67.11, 67.06, 62.19, 61.33, 60.26, 38.38, 31.71, 31.55, 29.39, 29.19, 28.31, 25.32, 20. 84, 20.80, 20.64, 20.55, 20.51, 20.44

(Synthesis Example 2) <6- [2- (N-Acryloylamino) ethylthio] hexyl 4-O- [4-O- (α-D-galactopyranoylyl) -β-D-galactopyranoylyl] -β-D-glucopyroxide (Gb3 Monomer)>

51.4 mg (0.0452 mmol) of the compound obtained in Synthesis Example 1 was dissolved in 1 mL of methanol, 2.4 mg (0.0452 mmol) of MeONa was added, and the mixture was stirred at room temperature. The progress of the reaction was confirmed by LC (MeOH / H ₂ O = 20/80). The product was neutralized with an ion exchange resin and concentrated to dryness. Yield 30.5mg, Yield 90%

¹ H-NMR (300 MHz, D ₂ O) δ = 6.21-6.05 (m, 2H), 5.65 (d, 1H), 4.84 (d, 1H), 4.40 (d, 1H), 4.39 (d, 1H), 4.24 (t, 1H), 3.92-3.18 (m, 21H), 2.63 (t, 2H), 2.49 (t, 2H) ), 1.51-1.40 (m, 4H), 1.31-1.22 (m, 4H)

¹³ C-NMR (75 MHz, D ₂ O) δ = 169.45, 130.81, 128.26, 104.14, 102.87, 101.20, 79.63, 78.26, 76.29, 75 .66, 75.40, 73.81, 73.08, 71.83, 71.74, 71.43, 70.02, 69.84, 69.44, 61.42, 61.25, 60.99 , 39.71, 31.90, 31.19, 29.48, 28.46, 25.45.

In addition, it confirmed that the structure of the Gb3 monomer prepared in Reference Example 1 was consistent with the data described in the literature from ¹ H-NMR and ¹³ C-NMR.

Example 1
Gb3 monomer 0.2g was melt | dissolved in the ion-exchange water 1.8g, and it was set as 10 mass% aqueous solution, and dissolved oxygen in the aqueous solution was removed by reducing pressure while stirring.

Next, at 23 ° C., a hollow fiber bundle made of poly-4-methylpentene (hollow fiber surface area: 30 cm ² , manufactured by DIC Corporation) is put in a glass test tube and sealed with a rubber stopper. Was replaced with nitrogen, and then an electron beam of 90 (kGy) was irradiated with an acceleration energy of 4.8 (MeV) with an electron beam irradiation apparatus “DYNAMICRON 5 MeV-150 kW” manufactured by RDI.

  Next, at 23 ° C., the inside of the test tube containing the hollow fiber bundle irradiated with the electron beam was evacuated, and then the deoxygenated Gb3 monomer aqueous solution was added. After standing at 23 ° C. for 4 hours, the hollow fiber bundle was taken out and washed several times with water to remove unreacted monomers to obtain a hollow fiber having Gb3 immobilized thereon. The amount of sugar chain immobilized on the hollow fiber was confirmed by XPS (X-ray photoelectron Spectroscopy) “ESCA-800” (hereinafter abbreviated as XPS) manufactured by Shimadzu Corporation. Changes in surface shape and immobilized sugar chains were confirmed by “S-800” (hereinafter abbreviated as SEM). The results are shown in Table 1 and FIG.

  Compared with the untreated hollow fiber by XPS, oxygen derived from the Gb3 monomer increased, and nitrogen and sulfur elements were detected. Thus, it was confirmed that the hollow fiber surface was immobilized. Moreover, the graft polymerization chain extended | stretched from the surface has been confirmed from SEM.

(Example 2)
<Preparation of virus>
AD8 and NL43: After transfecting pro virus to HeLa cells with FugeneHD (Roche), the supernatant was collected and used 48 hours later.
-HXB2: After transfecting pro virus to MT2 cells by Electroporation, the supernatant was collected over time, and the supernatant with the highest amount of virus (high reverser enzyme activity) was used.
<Measurement of virus count>
The PCR method (Roche, AMPLICOR HIV MONITOR Test version 1.5 or COBAS TaqMan) was used. 1 mL of PBS solution (virus number 3 × 10 ³ copies / mL, AD8 only 3 × 10 ⁴ ) of each laboratory strain (JRCSF, AD8, HXB2, NL43) was added to 5 cm ² of the Gb3-immobilized hollow fiber prepared in Example 1. Rotate, sample at 2 and 16 hours, and count the number of viruses.

(Comparative Example 1)
As a comparative example, the same experiment was performed on 5 cm ² of lactose-immobilized hollow fiber. Further, as a control, the same experiment was performed by rotating only the virus solution without adding the hollow fiber.
The result of an Example and a comparative example is shown in FIGS.

It is a chart which shows the synthetic pathway of Gb3 monomer. The left figure is an SEM photograph of the outer surface of the hollow fiber after immobilization, and the right figure is an untreated hollow fiber outer surface. It is a figure which shows adsorption | suction in AD8 stock | strain. It is a figure which shows adsorption | suction in HXB2 stock | strain. It is a figure which shows adsorption | suction in NL43 stock | strain. It is a schematic sectional drawing which shows the adsorption | suction apparatus using the sugar_chain | carbohydrate fixed polymer base material of this invention.

Explanation of symbols

1 Outlet 2 Inlet 3 Sugar chain 4 Hollow fiber 5 Container 6 Bulkhead

Claims (12)

A polymer substrate having a methylene group in the main chain is represented by the general formula (1)
(1)
Wherein R ¹ represents a hydrogen atom or a methyl group, W and X each independently represent a divalent linking group, and G represents a sugar chain, or the polymerizable compound A step (1) of contacting a polymerizable composition containing a compound, and a step (2) of irradiating ionizing radiation in a state where the polymerizable compound represented by the general formula (1) is brought into contact with the polymer substrate. And in this order, or
A step (3) of irradiating the polymer substrate with ionizing radiation, and a step (4) of bringing the polymerizable compound or a polymerizable composition containing the polymerizable compound into contact with the polymer substrate irradiated with ionizing radiation. ) In this order. A method for producing a sugar chain-immobilized polymer substrate for HIV adsorption. The sugar chain represented by G is represented by the general formula (2)
(2)
The manufacturing method of the sugar_chain | carbohydrate fixed polymer base material for HIV adsorption | suction of Claim 1 represented by (In formula, n represents the integer of 0-1). The divalent linking group represented by W is, -CONH -, - CO-O- or ^-R 2 -CONH- (provided that ^{R 2} represents an aromatic group) for HIV adsorption according to claim 1, wherein the A method for producing a sugar chain-immobilized polymer substrate. The divalent linking group represented by X is an alkylene group or —R ³ —ZR ⁴ — (wherein R ³ and R ⁴ each independently represents an alkylene group, Z is —O—, —NH The sugar chain-immobilized polymer group for HIV adsorption according to claim 1, which is-, -NR ^0- (wherein R ⁰ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms) or -S-). A method of manufacturing the material. 2. The sugar chain fixing for HIV adsorption according to claim 1, wherein the polymer substrate is an olefin resin, styrene resin, sulfone resin, acrylic resin, urethane resin, ester resin, ether resin or cellulose acetate. Method for producing a polymer base material. The method for producing a sugar chain-immobilized polymer substrate for HIV adsorption according to claim 5, wherein the olefin resin is a polymer of 4-methyl-1-pentene. The method for producing a sugar chain-immobilized polymer substrate for HIV adsorption according to claim 1, wherein the polymer substrate is a hollow fiber membrane, and the polymerizable compound is brought into contact with the inside of the hollow fiber membrane. The method for producing a sugar chain-immobilized polymer substrate for HIV adsorption according to claim 1, wherein the irradiation intensity of ionizing radiation is in the range of a voltage of 0.1 to 5 [kV] and an absorbed dose of 10 to 300 [kGy]. Repeating unit (M) represented by general formula (3) on a polymer substrate having a methylene group in the main chain
(3)
(Wherein R ¹ represents a hydrogen atom or a methyl group, W represents —CONH—, —CO—O—, or —R ² —CONH— (wherein R ² represents an aromatic group), and X represents alkylene. A group, or —R ³ —Z—R ⁴ — (wherein R ³ and R ⁴ represent an alkylene group, Z represents —O—, —NH—, —NR ⁰ — (where R ⁰ represents a hydrogen atom or a carbon atom number); Represents an alkyl group of 1 to 4) or represents -S-), and G represents a sugar chain).
Alternatively, the repeating unit (M) represented by the general formula (3) and the repeating unit (L) represented by the general formula (4)
(4)
(Wherein R ⁵ represents a hydrogen atom or a methyl group, V represents —COOH, —COOR ⁶ (wherein R ⁶ represents an alkyl group) or —CONH ₂ ), and A sugar chain-immobilized polymer substrate for HIV adsorption. The sugar chain fixing for HIV adsorption according to claim 9, wherein the polymer substrate is an olefin resin, a styrene resin, a sulfone resin, an acrylic resin, a urethane resin, an ester resin, an ether resin, or cellulose acetate. Polymer base material. The sugar chain-immobilized polymer substrate for HIV adsorption according to claim 10, wherein the olefin resin is a polymer of 4-methyl-1-pentene. The sugar chain-immobilized polymer substrate for HIV adsorption according to any one of claims 9 to 11, wherein the polymer substrate is a hollow fiber membrane.