JP2008012460A - Lectin adsorbent using sugar-side-chain-type polymer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a lectin adsorbent which has a high adsorption capacity, enables the control of the adsorption capacity, adsorbing power and fixabilities, and allows a convenient manufacture. <P>SOLUTION: The lectin adsorbent uses one or more selected from sugar-side-chain-type urea polymers of formula (2) (wherein, R<SB>1</SB>is a hydrogen atom or a methyl group; n is an integer of equal to or more than 2; G is a sugar residue; and the urea polymers may have different sugar residues G), products of the urea polymers formed by copolymerization with a cross-linking material and products of the urea polymers formed by binding to a polymer material or inorganic fine particles. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a lectin adsorbent which has a molecular element for recognizing lectins and can efficiently separate and purify lectins.

A lectin, also called a blood agglutinin, is a protein or glycoprotein that interacts with sugar and has unique properties such as agglutinating cells such as red blood cells and sedimenting glycoconjugates. It is a physiologically active substance that exists in almost all living species such as plants and microorganisms.
At present, sugar-binding cell agglutinins having a valence of 2 or more, which are not immunological products such as antibodies, from plant to animal, are widely called lectins. In 1951-1952, W.C. T.A. J. et al. Morgan and W.W. M.M. Watkins discovered that the ABO blood group-specific lectin inhibits aggregation activity with different monosaccharides corresponding to the difference in blood group specificity. This finding provided the first clue to estimate the structure of the ABO blood group determinant. For example, Ulex europeus I (Harienishida), Lotus tetragonolobus (Miyakogusa), Ele serum (eel serum) are used to detect O-type determinants for binding to L-Fuc. Cytisus sesilifolios and Ulex europeus II are also used to detect O-type determinants because they bind to D-GlcNAcb1 → 4D-GlcNA. Phaseolus limensis, Dorichos biflorus, and Vicia cracca are used to detect A-type determinants in order to bind to D-GalcNAc. Griffonia simplicifolia I is used for detection of type B determinants in order to bind to D-Gal.

  In addition, various lectins are used for cell surface sugar search, and it is now possible to estimate cell surface glycoprotein sugar chains recognized by various lectins and sugar sequences in sugar chains.

In addition, glucose and mannose-binding lectins such as concanavalin A are known to act mainly on the complement system in immune function. That is, the activation pathway of the complement system has so far been the so-called classical pathway in which immunoglobulin is used as a foreign molecule recognition molecule and the first component of complement is activated, and the third component of complement is directly applied to foreign substances such as bacteria. In recent years, mannose or glucose-binding lectin specifically recognizes sugar chains on the surface of microorganisms such as glucose, mannose, N-acetylglucosamine, and the like. The presence of a third complement activation pathway, called the lectin pathway, that causes activation has been found. This lectin pathway is one of the natural immunity that exists universally, including invertebrates that do not have so-called acquired immunity to recognize and memorize antigens, and is a biological defense function that occupies an important position among them (J. Biol. Chem. 1987 262 7451-7454; J. Clin. Invest. 1989 84 1821-1829).
The starting point is that mannose-binding lectin recognizes and binds to mannose and N-glucosamine on the surface sugar chains of viruses and bacteria, and is a complex with MASP (mannose-binding lectin-associated serine protease). It activates the complement pathway during formation, causing killing of viruses and bacteria.

Similarly, it is known that mannose-binding lectins start from binding to viruses, bacteria, etc., and that phagocytic cells prey on them, that is, mannose-binding lectins have an opsonizing action. . A report that the susceptibility of MBL-deficient patients whose phagocytic cells do not cause phagocytosis to bacteria, etc., and that is easily infectious, was supplemented with mannose-binding lectin. (Scan. J. Immunol. 1998 48 116-123).
Furthermore, reports that mannose-binding lectins suppress influenza infection (Immunology 1999 97 385-392), reports that HIV infection is suppressed (J. Exp. Med. 1989 169 185-196), Reports of suppression (Proc. Natl. Acad. Sci. USA 1999 96 371-375) have been made so far.

  In addition, lectins have been used mainly for microscopic studies of cell surfaces, but in recent years, lectins have been found to be deeply involved in the control and differentiation mechanisms of various biological cells as bioactive substances. It has been revealed that certain lectins have a function to promote the synthesis of nerve growth factors that have been shown to be effective for the prevention and treatment of Alzheimer-type dementia. It is becoming.

  Along with this knowledge, research on the distribution of lectins in biological species and their properties has been actively conducted.

  Among these lectins, glucopyranosyl structure, maltose structure, mannopyranosyl structure, galactopyranosyl structure, 6-deoxygalactopyranosyl structure, N-acetylgalactosaminyl structure, N, N′-acetylchitobiose structure, There is a known group that has the property of binding to functional groups that contain lectins. May need to be purified in many cases.

In order to separate lectins from living organisms, extraction with an appropriate buffer is used. However, when further purifying the lectins, the fraction obtained by ammonium sulfate or the like and the property that lectins bind to sugar are usually used. Affinity chromatography or the like is employed as the main technique.
For example, a lectin having a property of binding to a functional group containing a glucopyranosyl structure, a mannopyranosyl structure, or a maltose structure is adsorbed to column chromatography, and then D-glucose, mannose, methyl-β- is used as an eluent from the adsorbent. Elute with D-mannopyranoside.
As the column chromatography to be adsorbed, a substrate having a mannose residue exposed on the surface is advantageously employed because the adsorption rate of the lectin is high. For example, an adsorbent in which mannose or mannan is immobilized on a commercially available agarose gel (Sepharose 4B, Pharmacia Biotech) is used, but the recovery rate of lectin is as low as 43% (for example, Non-Patent Document 1).
This is probably because mannose is bonded to the agarose surface in a single layer and the adsorption site is small. In addition, the molecules are only immobilized randomly on the agarose surface. In the case of mannan-immobilized agarose, there is no description of the amount of mannan and the like, and mannan having a controlled molecular weight is not immobilized, so that the immobilization force or the adsorption capacity cannot be controlled.
Similarly, a method in which a lectin having a property of binding to a functional group containing a galactopyranosyl structure is adsorbed by affinity chromatography and then eluted from the adsorbent is also employed.
The column chromatography to be adsorbed is advantageously employed because the substrate having a galactose residue exposed on the surface has a high adsorption rate of the lectin. For example, there are commercially available agarose gels and those obtained by adding a galactopyranosyl structure to the surface of various equipment. As in the case of mannose-binding lectin, lactose or galactose is bound to the agarose surface in a single layer, the adsorption site is small, and the molecules are immobilized randomly on the agarose surface. .

  In addition, a method of purifying glucose-binding lectin and mannose-binding lectin at an industrial level using a residue obtained by extracting immunoglobulin from the II + III fraction obtained by the low-temperature ethanol fractionation method of corn as a raw material has been reported (Patent Literature). 1). However, in this technique, as a pretreatment of an affinity chromatography treatment step using agarose (Sepharose CL4B, Pharmacia Biotech Co., Ltd.) to which mannan or the like is not immobilized, a plurality of normal filtration steps and a dehydration step for removing insoluble matters are performed. A filtration step using a lipid filter is required, which is complicated as a purification method and also requires equipment for that purpose.

Further, an adsorbent using amino sugar and covalently binding it to a support has been reported. For example, Patent Document 2 discloses a solid support derivatized with an amino sugar having a primary amino group. Specifically, a support obtained by coupling D-mannosamine or D-glucosamine to NHS-activated Sepharose FF is disclosed in Patent Document 3 as poly- (β-1,4-D-glucosamine). A polymer material in which a saccharide is bonded to a derivative via a glycopyranosylamide bond has been reported.
However, since the former method uses monosaccharides, the adsorption site is small, while the latter method is complicated in operation, such as deacetylation after introduction of sugar into the polymer using acetylated sugar. , There was a drawback.
J. et al. Biochem. 1983 94 937-947 Special Table 2002-517513 JP 2005-511537 A JP 2005-36163 A

  It is an object of the present invention to provide a substrate that can be easily manufactured with a large adsorption capacity and controlled immobilization force as compared with various sugar-binding gels that have a small adsorption capacity in the conventional method. .

As a result of research to solve such problems, the present inventors have found that a polymer obtained by polymerizing a urea-type vinyl monomer having a sugar in the side chain can be a molecular element, and have reached the present invention.
That is, the present invention
(1) a lectin adsorbent comprising a sugar side chain type urea polymer,
(2) The lectin adsorbent according to (1) above, wherein the sugar side chain urea polymer is a copolymer of two or more sugar side chain urea monomers,
(3) The lectin adsorbent according to (1) above, wherein the sugar side chain type urea polymer is a blend polymer of two or more sugar side chain type urea polymers,
(4) The lectin adsorbent according to (1) or (2) above, wherein the sugar side chain type urea polymer is copolymerized with a crosslinking material,
(5) A lectin adsorbent obtained by binding the sugar side chain type urea polymer according to any one of (1) to (3) to a polymer material or inorganic fine particles,
Is to provide.

The sugar side chain type urea polymer of the present invention can be a molecular element having a large molecular weight and capable of providing a huge binding site (Transactions of the Materials Research Society of Japan 2005 30 1139-1142). It can be controlled freely.
Therefore, the adsorption capacity is large, and the adsorption capacity, the adsorption force, and the immobilization force can be controlled.
In addition, since the sugar side chains are bonded by urea bonds, the association form is not spherical as in the case of benzamide type sugar side chain type polymers and the cohesive force is weak, or the sugar chain is linear as in the case of amide type polymers. There is no disadvantage that the isomers are mixed and the physical properties are not stable.

Hereinafter, the present invention will be described in detail.
The sugar side chain type urea polymer referred to in the present invention is a polymer represented by the formula (2) obtained by polymerizing the sugar side chain type urea monomer represented by the formula (1).
In the formula, R ₁ represents a hydrogen atom or a methyl group, n represents an integer of 2 or more, G represents a saccharide residue, and is a saccharide residue capable of converting the anomeric position to an amino group. Specific examples of such saccharides include glucose having a glucopyranosyl residue, cellobiose having a binding mode of β-1,4, cellotriose, cellotetraose, cellooligosaccharide obtained by decomposing cellulose, and mixtures thereof, α-1, Maltose, maltotriose, maltotetraose, malto-oligosaccharides having degraded amylose and mixtures thereof, galactose, lactose, galacto-oligosaccharides having galactopyranosyl residues, and β-1,6 bonds Examples thereof include reducing sugars such as raffinose, melibiose, and gentibiose.
The compound represented by formula (1) or formula (2) can be produced, for example, according to the method described in JP-A No. 2004-203870 developed by the present inventors.

  The lectin adsorbent of the present invention comprises the polymer of the above formula (2), and the saccharide residue of the polymer can be appropriately selected according to the target lectin.

Further, in the polymer represented by the formula (2), those having different saccharide residues (copolymerized compounds represented by the formula (1) having different saccharide residues) or different saccharide residues A blend of homopolymers having groups can also be used.
These include functional groups including glucopyranosyl structure, maltose structure, mannopyranosyl structure, galactopyranosyl structure, 6-deoxygalactopyranosyl structure, N-acetylgalactosaminyl structure, N, N′-acetylchitobiose structure It can be used as an adsorbent for two or more lectins as a multi-molecular element having specific affinity for two or more lectins having the property of binding to each other.

In the present invention, the polymer (homopolymer, copolymer) represented by the above formula (2) is converted into polysaccharide particles such as cellulose, glucomannan, pullulan and agarose, polyvinyl alcohol, polyhydroxyethyl acrylate, polyhydroxyethyl acrylamide, polystyrene and the like. And an adsorbent grafted on the surface of fine particles such as inorganic fine particles such as silica gel, alumina, titania and zirconia.
As the grafting method, a method known per se can be applied. For example, radicals may be generated in the fine particles and polymerized therefrom, or polymer may be obtained by polymerizing first and modified into fine particles.

  In the present invention, the compound represented by the above formula (1) is further converted into a crosslinking agent having a polymerizable group, such as methylene bisacrylamide, ethylene glycol dimethacrylate, ethylene glycol diacrylate, butylene bisacrylamide, trimethylol. It can also be made into a lump or fine particle adsorbent by copolymerizing with a polyfunctional monomer such as propane trimethacrylate, trimethylolpropane triacrylate, triallyl cyanurate, or divinylbenzene.

The method of using the adsorbent of the present invention can be specified as a base material for conventional column chromatography or by adding the adsorbent of the present invention to a solution containing a specific lectin and precipitating from the solution, followed by solid-liquid separation. Lectins can be recovered.
The elution of lectin can be performed by using a corresponding sugar or sugar alcohol according to a known method.

Hereinafter, the present invention will be described in more detail with reference to examples.
Production Example 1 Synthesis of 2- (methacryloyloxy) ethylureidomaltose polymer (Formula (3), Polymer 1) (1) Synthesis of maltosylamine 1.0 g (2.93 mmol) of maltose was dissolved in 50 ml of water in a 300 ml beaker, 18.5 g of ammonium hydrogen carbonate was added three times every 24 hours (total amount 55.5 g), and the mixture was stirred at 37 ° C. for 72 hours while being open. After completion of the reaction, 100 ml of distilled water was added and water was distilled off to 20 ml, and then 100 ml of water was added and concentrated to 10 ml. This operation was repeated until the ammonia odor disappeared and freeze-dried to obtain maltosylamine as a white solid. As a result of HPLC analysis of the white solid, the purity was 78.4%.
A ¹ H-NMR spectrum of the obtained maltosylamine (white solid) is shown in FIG. A peak derived from the β-type structure was observed at around 4.1 ppm, and a peak derived from the α-type structure was confirmed at around 4.3 ppm (when the amino group was bonded to the carbon atom at position 1 and shifted to the high magnetic field side. In addition, a peak common to the α-type structure and the β-type structure was observed in the vicinity of 4.5 ppm, and a peak presumed to be derived from the α-type structure of maltose was confirmed in the vicinity of 5.2 ppm. The structure of the product was confirmed from the result of these ¹ H-NMR spectra.

(2) Synthesis of 2- (methacryloyloxy) ethylureidomaltose 0.8 g (purity 78.4%, 1.84 mmol) obtained in (1) was dissolved in 20 ml of 1 × 10 ⁻³ M-KOH aqueous solution. It was. To this aqueous solution, 0.713 g (4.6 mmol) of 2-isocyanatoethyl methacrylate (2-IEM) was added and stirred vigorously for 12 hours while maintaining at 3 ° C. After completion of the reaction, the white solid deposited in the flask was filtered off, and the filtrate was washed 4 times with 30 ml of diethyl ether to remove unreacted 2-IEM, and then lyophilized. After completion of lyophilization, the obtained white solid was dissolved in a mixed solution of 2 ml of water and 3 ml of methanol, and dropped into a mixed solution of 100 ml of diethyl ether and 150 ml of acetone and cooled. The precipitated white solid was collected by filtration and dried under reduced pressure to obtain 0.55 g of 2- (methacryloyloxy) ethylureidomaltose as a white product.
The obtained white product was subjected to HPLC analysis. The chromatogram is shown in FIG. The maltosylamine peak disappeared and a new compound having absorption in the UV-visible region was observed. This is considered to be absorption derived from the vinyl group of the target product 2- (methacryloyloxy) ethylureidomaltose. When the purity was calculated from the peak area ratio, the purity was 65.0%.
The FT-IR spectrum of the obtained white product is shown in FIG. NH-CO-NH mutually stretching the 1570cm ^-1, C = O stretching vibration 1650cm ^-1 (urea), a peak derived from C = O stretching vibration (Ester), to have been confirmed in 1705 cm ^-1.
Further, the 1H-NMR spectrum is shown in FIG. A peak derived from a methyl group at 1.9 ppm, a methylene group near 4.2 ppm, a vinyl group at 5.7 ppm and 6.1 ppm was confirmed, and a peak derived from a sugar skeleton near 3.3-4.0 ppm And the structure of the target product was confirmed.

(3) Polymerization of 2- (methacryloyloxy) ethylureidomaltose 4.46 g (5.85 mmol) of 2- (methacryloyloxy) ethylureidomaltose obtained in (2) was dissolved in 20 ml of degassed water and cooled with ice. Nitrogen was bubbled for 30 minutes. Then, 100.8 mg (0.585 mmol) of N, N, N ′, N′-tetraethylethylenediamine (TEEDAm) was added, and 13.35 mg (0.0585 mmol) of ammonium persulfate (APS) was added as an initiator, The mixture was stirred for 3 hours under a nitrogen atmosphere under ice cooling. After completion of the reaction, the reaction solution was diluted 2 times and purified by dialysis for 3 days, followed by lyophilization to obtain 1.2 g of a white product (yield 60%).
FIG. 5 shows the FT-IR spectrum of the white product obtained. The spectrum is broader than that of the monomer shown in FIG. This is considered to be caused by a decrease in the degree of freedom of the polymer side chain accompanying polymerization.
The ¹ H-NMR spectrum is shown in FIG. In the spectrum, the broadening of the peak due to polymerization can be confirmed, the peaks derived from the vinyl groups of 5.7 ppm and 6.1 ppm disappear, and a new peak derived from the methylene group is observed around 1.5 ppm. The white product was confirmed to be 2- (methacryloyloxy) ethylureido maltose polymer.

Production Example 2 Synthesis of 2- (methacryloyloxy) ethylureido lactose polymer (Polymer 2) (1) Synthesis of lactosylamine Lactose 1.0 (2.93 mmol) was dissolved in 50 ml of water in a 300 ml beaker, and ammonium bicarbonate was dissolved. 18.5 g each was added 3 times every 24 hours (total amount 55.5 g), and the mixture was stirred at 37 ° C. for 72 hours while being open. After completion of the reaction, 100 ml of distilled water was added and water was distilled off to 20 ml, and then 100 ml of water was added and concentrated to 10 ml. This operation was repeated until the ammonia odor disappeared and freeze-dried to obtain lactosylamine as a white solid. The structure of the product was confirmed by ¹ H-NMR spectrum.

(2) Synthesis of 2- (methacryloyloxy) ethylureido lactose 0.8 g (purity 78.4%, 1.84 mmol) of lactosylamine obtained in (1) was dissolved in 20 ml of 1 × 10 ⁻³ M-KOH aqueous solution. I let you. To this aqueous solution, 0.713 g (4.6 mmol) of 2-isocyanatoethyl methacrylate (2-IEM) was added and stirred vigorously for 12 hours while keeping at 3 ° C. After completion of the reaction, the white solid deposited in the flask was filtered off, and the filtrate was washed 4 times with 30 ml of diethyl ether to remove unreacted 2-IEM, and then lyophilized. After completion of lyophilization, the obtained white solid was dissolved in a mixed solution of 2 ml of water and 3 ml of methanol, and dropped into a mixed solution of 100 ml of diethyl ether and 150 ml of acetone and cooled. The precipitated white solid was collected by filtration and dried under reduced pressure to obtain 0.55 g of 2- (methacryloyloxy) ethylureidolactose as a white product.
The structure of the product was confirmed by FT-IR spectrum and ¹ H-NMR spectrum.

(3) Polymerization of 2- (methacryloyloxy) ethylureidolactose 4.46 g (5.85 mmol) of 2- (methacryloyloxy) ethylureidolactose obtained in (2) was dissolved in 20 ml of degassed water under ice-cooling. Nitrogen was bubbled for 30 minutes. Then, 100.8 mg (0.585 mmol) of N, N, N ′, N′-tetraethylethylenediamine (TEEDAm) was added, and 13.35 mg (0.0585 mmol) of ammonium persulfate (APS) was added as an initiator, The mixture was stirred for 3 hours under a nitrogen atmosphere under ice cooling. After completion of the reaction, the reaction solution was diluted 2 times and purified by dialysis for 3 days, and then lyophilized to obtain 1.2 g of a white product (yield 83.6%).
The structure of the product was confirmed by FT-IR spectrum and ¹ H-NMR spectrum.

Production Example 3 Synthesis of 2- (methacryloyloxy) ethylureido cellobiose polymer (Polymer 4) According to the method described in Example 1 of JP-A-2004-203870, 2- (methacryloyloxy) ethylureido cellobiose polymer was obtained.

Example 1 Adsorption Experiment Using Urea Polymer Having Maltose Residue 27.75 mg of calcium chloride was dissolved in 250 ml of Tris-HCl buffer to prepare 1 mM calcium chloride / Tris-HCl buffer (I). Concanavalin A (Con A) (50 mg) was dissolved in (I) (100 ml) to prepare a 0.5 mg / ml Con A solution (II).
In addition, 5 mg of 2- (methacryloyloxy) ethylureidomaltose polymer obtained in Production Example 1 was dissolved in 10 ml of (I) (1 × 10 ⁻⁶ unit mol / ml, (III)), and 50 μl (5) from (III) × 10 ⁻⁸ unit mol), 100 μl (1 × 10 ⁻⁷ unit mol), and 500 μl (5 × 10 ⁻⁷ unit mol) were collected, and 1.5 ml of (II) was added to each and added at 25 ° C. for 15 minutes. The transmittance was measured at 500 nm every 2 hours.
The measurement results are shown in FIG. As is apparent from FIG. 7, all of these concentrations became cloudy by 15 minutes.

Example 2
Maltose, amylose, 2- (methacryloyloxy) ethylureidomaltose polymer (Polymer 1), 2- (methacryloyloxy) ethylureidomaltotriose polymer (Polymer 4), 2- (methacryloyloxy) ethylureido cellobiose polymer (Polymer 3) And polyvinyl benzamide maltose (PV-MA, manufactured by Seikagaku Corporation) according to Example 1 (mixed solution of the above (I), (II) and sugar solution), and the solution was allowed to stand at 25 ° C. for 1 hour. The transmittance at 500 nm was measured.
The results are shown in FIG.
As is clear from FIG. 8, a decrease in transmittance was confirmed in polymer 1, polymer 2, and PV-MA. This is thought to be because Con A, which selectively binds to α-glucose residues, binds to α-glucose residues of maltose and maltotriose in the polymer side chain to form a crosslinked structure. Furthermore, in the polymer 1 and the polymer 4, the increase in the transmittance due to the aggregation of the precipitate was confirmed when the concentration was higher than a certain level. On the other hand, PV-MA, which is a similar sugar side chain polymer, was white and turbid, but did not aggregate and precipitate. From this, it was suggested that the lectin adsorbent of the present invention shows stronger binding ability.
In addition, although maltose and amylose have an α-glucose residue, a decrease in permeability was not confirmed. Even when binding to Con A occurs, the α-glucose residue is only at one end. Since it was not present and a crosslinked structure could not be formed, in Polymer 3, since it was a β-glucose residue, binding to Con A did not occur, and a decrease in transmittance was not observed.

Production Example 4 Preparation of sugar side chain type urea polymer grafted inorganic fine particles (silica gel) (1) Preparation of mercaptotrimethoxy silica gel (Sil-MPS) 5.0 g of silica gel was added to 90 ml of dehydrated toluene, and external ultrasonic irradiation was performed for 30 minutes. And dispersed. Thereafter, 7.24 g (36.88 mmol) of 3-mercaptopropyl trimethoxysilane was added, and the mixture was stirred at 200 rpm and 50 ° C. for 24 hours. After completion of the reaction, the mixture was allowed to cool and the precipitate was collected by filtration, washed with toluene and diethyl ether, and dried under reduced pressure to obtain a white solid (Sil-MPS).
Elemental analysis value (measured value) H: 1.37%, C: 3.87%, S: 2.51%
From the results of elemental analysis, increases in carbon content and sulfur content were confirmed, and the amount of immobilized MPS was calculated to be 8.093%.

8.43 mg of 2,2-dithioviridine (2PDS) was dissolved in 250 ml of methanol, 10 ml was added to 3.5 mg of collected Sil-MPS, and external ultrasonic irradiation was performed for 30 minutes. Then, after stirring for 8 hours, UV spectrum measurement was performed. Absorbance at 360 nm was 0.266 for Sil-MPS. The result was substituted into a calibration curve (y = 0.5968x + 0.0202), and the immobilized amount of Sil-MPS was calculated as 5.87 × 10 ⁻⁴ mol / g.

(2) Grafting reaction In 9 ml of sufficiently degassed distilled water, 0.05 g (0.0392 mmol) of Sil-MPS and 2- (methacryloyloxy) ethylureidomaltose monomer 0.109 g, 0.218 g, 0.436 g,. 654 g (purity 89.4%, 0.196 mmol, 0.393 mmol, 0.789 mmol, 1.176 mmol respectively; 5 times (Sil-Mal 5), 10 times (Sil-Mal 10), respectively, relative to the MPS residue, 20 times (Sil-Mal 20) and 30 times (Sil-Mal 30) equivalent) were added, external ultrasonic irradiation was performed for 15 minutes to disperse Sil-MPS, and nitrogen aeration was performed at 5 ° C. for 30 minutes. Thereafter, 0.084 ml of TEEDAm was added, and 8.95 mg (0.0392 mmol) of APS was added as an initiator, followed by stirring at 5 ° C. under a nitrogen atmosphere for 3 hours. After completion of the reaction, the precipitate was collected by filtration, washed with distilled water, ethanol and diethyl ether in this order, and then dried under reduced pressure to obtain a white solid.
Qualitative analysis was performed by FT-IR, C = O ( urea) stretching vibration 1650 cm ^-1, a peak derived from C = O (ester) stretching vibration was observed at 1705 cm ^-1.
Elemental analysis value (measured value)
Sil-Mal5 H: 1.50%, C: 6.50%, N: 0.64%
Sil-Mal10 H: 1.47%, C: 6.61%, N: 0.65%
Sil-Mal20 H: 1.90%, C: 9.65%, N: 1.18%
Sil-Mal30 H: 2.34%, C: 12.3%, N: 1.41%

Example 3
3 mg each of Sil-Mal 5, Sil-Mal 10, Sil-Mal 20, Sil-Mal 30 and silica gel obtained in Production Example 4 was collected in a sample tube, and 900 μl of the buffer (I) used in Example 1 and Rhodamine-Con A solution were used. (5 mg / ml) 100 μl was added, and the mixture was allowed to stand in the dark for 1 hour, and then filtered through a disk filter. The filtrate was measured by fluorescence spectrum.
The results are shown in FIG.
FIG. 9 indicates that the fluorescence intensity is lowest when silica gel is added, and Con A is adsorbed on the silica gel. Regarding the decrease in the amount of adsorption in Sil-Mal5, the polymer chain was adsorbed on the silica gel of the carrier, the degree of freedom of maltose in the side chain was lost, and Con A could not be bound, and the adsorbed polymer chain This is probably because the adsorption of Con A did not occur on the silica gel itself. Moreover, since free maltose increases as the degree of polymerization increases, the adsorption amount of Con A increases. In Sil-Mai30, the adsorption amount is considered to increase at a stretch due to the polymer effect.

Production Example 5 Preparation of sugar side chain type urea polymer grafted polymer fine particles (cellulose) (1) Preparation of mercapto trimethoxy cellulose fine particles (Cell-MPS) After washing 23.81 g of cellulose beads with methanol, wash with toluene A white solid was obtained after drying under reduced pressure. Of these, 4 g was collected, 90 ml of dehydrated toluene was added, and external ultrasonic irradiation was performed for 30 minutes to disperse the cellulose fine particles. Then, 7.24 g (36.88 mmol) of 3-mercaptotrimethoxysilane was added and stirred at 200 rpm and 50 ° C. for 24 hours. After completion of the reaction, the mixture was allowed to cool, and the precipitate was collected by filtration, washed with toluene and diethyl ether, and dried under reduced pressure to obtain a white solid (Cell-MPS).
Elemental analysis value (measured value) H: 6.66%, C: 41.2%, S: 2.18%

8.43 mg of 2PDS was dissolved in 250 ml of methanol, 10 ml was added to 3.5 mg of the collected Cell-MPS, and external ultrasonic irradiation was performed for 30 minutes. Then, after stirring for 8 hours, UV spectrum measurement was performed. Absorbance at 360 nm was 0.266 for Cell-MPS. The result was substituted into a calibration curve (y = 0.5968x + 0.0202), and the immobilized amount of Cell-MPS was calculated to be 4.59 × 10 ⁻⁴ mol / l.

(2) Grafting reaction 0.13 g (0.0551 mmol) of Cell-MPS and 0.168 g, 0.457 g, 0.672 g of 2- (methacryloyloxy) ethylureidomaltose were added to 13.5 ml of sufficiently degassed distilled water. 1.008 g (purity 89.4%, 0.275 mmol, 0.0550 mmol, 1.10 mmol, 1.650 mmol, respectively) 5 times (Cell-Mal5), 10 times (Cell-Mal10) with respect to MPS residues ), 20 times (Cell-Mal 20), 30 times (Cell-Mal 30) equivalent), and external ultrasonic irradiation was performed for 15 minutes to disperse Sil-MPS, and nitrogen aeration was performed at 5 ° C. for 30 minutes. Thereafter, 0.119 ml of TEEDAm was added, 12.6 mg (0.0551 mmol) of APS was added as an initiator, and the mixture was stirred for 3 hours in a nitrogen atmosphere at 5 ° C. After completion of the reaction, the precipitate was collected by filtration, washed with distilled water, ethanol and diethyl ether in this order, and then dried under reduced pressure to obtain a white solid.
Qualitative analysis was performed by FT-IR, C = O ( urea) stretching vibration 1650 cm ^-1, a peak derived from C = O (ester) stretching vibration was observed at 1705 cm ^-1.
Elemental analysis value (measured value)
Cell-Mal5 H: 6.37%, C: 39.9%, N: 0.19%
Cell-Mal10 H: 6.42%, C: 40.5%, N: 0.25%
Cell-Mal20 H: 6.39%, C: 39.9%, N: 0.35%
Cell-Mal30 h: 6.34%, C: 40.4%, N: 0.42%

Example 4
3 mg of Cell-Mal5, Cell-Mal10, Cell-Mal20, Cell-Mal30 and cellulose fine particles obtained in Production Example 5 were collected in a sample tube, and 900 μl of Buffer (I) used in Example 1 and Rhodamine-Con A 100 μl of the solution (5 mg / ml) was added, and the mixture was allowed to stand in the dark for 1 hour and filtered through a disk filter. The filtrate was measured by fluorescence spectrum.
The results are shown in FIG.
From FIG. 10, the cellulose fine particles themselves did not adsorb, and it was confirmed that the adsorption amount increased with the increase in the degree of polymerization of the polymer chain.

Production Example 6 Lump by Copolymerization with Crosslinking Material 2- (methacryloyloxy) ethylureidomaltose monomer and methylenebisacrylamide (MBAAm) were added at a predetermined ratio as shown in Table 1 to 2.2 ml of sufficiently degassed distilled water. Mixing and nitrogen bubbling at 5 ° C. for 30 minutes. Thereafter, 0.0217 ml of TEEDAm was added, 2.3 mg (0.0101 mmol) of APS was added as an initiator, and the mixture was stirred in a nitrogen atmosphere at 5 ° C. for 3 hours. After completion of the reaction, the white solid was obtained by lyophilization after thoroughly washing with distilled water.
Qualitative identification was performed by FT-IR.

Example 5
3 mg each of [2- (methacryloyloxy) ethylureidomaltose monomer] / [MBAAm] = 0/10, 5/5, 6/4, 7/3, 8/2 was collected in a sample tube, and Example 1 900 μl of the buffer solution (I) used in 1) and 100 μl of Rhodamine-Con A solution (5 mg / ml) were added, left standing in the dark for 1 hour, filtered through a filter, and the filtrate was measured by fluorescence spectrum.
The results are shown in FIG.
From FIG. 11, it was confirmed that the amount of adsorption increased with an increase in the composition ratio of maltose.

  As described above, according to the present invention, there is provided a lectin adsorbent that can be easily manufactured and has a large adsorption capacity and can control the adsorption capacity, adsorption force, and immobilization force.

1 is a ¹ H-NMR spectrum of maltosylamine. It is a HPLC chromatogram of 2- (methacryloyloxy) ethylureidomaltose and maltosyamine. It is an FT-IR spectrum of 2- (methacryloyloxy) ethylureidomaltose. ^{1 is} a ¹ H-NMR spectrum of 2- (methacryloyloxy) ethylureidomaltose. It is a FT-IR spectrum of 2- (methacryloyloxy) ethylureido maltose polymer. ^{1 is} a ¹ H-NMR spectrum of 2- (methacryloyloxy) ethylureidomaltose polymer. It is the figure which showed the time-dependent change of the transmittance | permeability of 500 nm of the solution which mixed polymer 1 aqueous solution and Con A. FIG. It is the figure which showed the relationship between the polymer addition amount and the transmittance | permeability of 500 nm of the solution which mixed various polymer aqueous solution and Con A. It is the figure which showed the relationship between the polymerization degree of the polymer grafted on the silica, and the adsorption amount of Con A. It is the figure which showed the relationship between the polymerization degree of the polymer grafted on the cellulose, and the adsorption amount of Con A. It is the figure which showed the relationship between the raw material ratio of 2- (methacryloyloxy) ethylureido maltose and a methylenebisacrylamide, and the adsorption amount of Con A.

Claims (5)

  A lectin adsorbent comprising a sugar side chain type urea polymer.   The lectin adsorbent according to claim 1, wherein the sugar side chain type urea polymer is a copolymer of two or more types of sugar side chain type urea monomers.   The lectin adsorbent according to claim 1, wherein the sugar side chain type urea polymer is a blend polymer of two or more types of sugar side chain type urea polymers.   The lectin adsorbent according to claim 1 or 2, wherein the sugar side chain type urea polymer is copolymerized with a cross-linking material.   A lectin adsorbent obtained by binding the sugar side chain type urea polymer according to any one of claims 1 to 3 to a polymer material or inorganic fine particles.

Images