JP2006291225A - Method of surface modification of polymer gel filler - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly selective filler in the template polymerization of a saccharide. <P>SOLUTION: The polymer gel filler for chromatography is obtained by subjecting a monomer such as methacrylic acid or 4-vinylpyridine, a crosslinking agent such as ethylene dimethacrylate or methylene bisacrylamide, and a solvent such as toluene, to the 2-stage swelling polymerization allowing the resulting polymer to adsorb a saccharide such as galactose, glucose, or mannose, and then treating the product with heat. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for modifying a surface of a polymer gel filler.

  Researches on polymer hosts that specifically identify saccharides, such as lectins, which are proteins, are being actively conducted. As one of the preparation methods, a method using a molecular imprint method has been proposed.

  The molecular imprint method has attracted attention as a method for preparing a polymer having specific molecular recognition ability. As a general preparation method, a so-called bulk polymerization method in which a monomer serving as a host and a print molecule serving as a guest are placed in a test tube or the like and polymerized is generally used. Specifically, first, a print molecule and a monomer are added. At this time, a non-covalent interaction is formed between the print molecule and the monomer. Next, a crosslinking agent is added and polymerization is initiated. After the polymerization is completed, the polymer is washed to remove the print molecules. In the obtained polymer, a site storing the size of the print molecule and the position of the functional group is prepared. Such a molecular imprint method is schematically shown in FIG.

  This bulk polymerization method has an advantage that water does not exist in the system, and weak interactions such as hydrogen bonds formed between the host monomer and the print molecule are not inhibited. However, in this polymerization, there is a limitation that generally only oil-soluble print molecules can be applied, and it becomes a problem particularly when a synthetic polymer that specifically recognizes a water-soluble substance is prepared as an alternative to a biopolymer. . For example, a problem with the molecular imprint method using sugar as a template is that the template must be dissolved in a polar solvent such as water, and the interaction between the host monomer and the template is disadvantageous. .

  In recent years, a molecular imprint method in an aqueous medium using a monosaccharide as a template has been reported (for example, see Non-Patent Documents 1 and 2). In addition, it has been reported that the ability to discriminate the template increases by heating to 120 ° C. while the template is present in the host (see, for example, Non-Patent Document 3).

However, a sufficient discriminating ability has not yet been developed for the sugar used as a template.
Furthermore, although the polymer mass obtained by the bulk polymerization method can be used as a filler for HPLC (High Performance Liquid Chromatography) by pulverization, operations such as classification are complicated, and the particle size is small. There was a disadvantage that the performance of the column obtained from non-uniformity was not high.
Hosoya et al., The 45th Annual Meeting of the Polymer Society of Japan, 45 (1996) 1764 K. Tsukagoshi et al., Chem. Lett., 1994, 681) B. Sellergen et al., J. Chromatogr., 635 (1993) 31

  An object of the present invention is to overcome the problems of the conventional preparation methods (bulk polymerization method) and to provide a surface modification method for a polymer gel filler that specifically recognizes sugar.

  As a result of earnest research, the present inventors have conducted polymerization using methacrylic acid and a cross-linking agent, and when the heat treatment is performed in the presence of sugar as a template molecule, the selective discrimination with respect to saccharide is more greatly expressed. I found out. Furthermore, it was found that the selective discrimination extends not only to the template but also to sugars having a structure similar to the template. The present invention has been completed based on these findings.

  That is, the present invention provides a surface of a polymer gel filler characterized by adsorbing a sugar to a polymer obtained by polymerization using a monomer, which is methacrylic acid or 4-vinylpyridine, a cross-linking agent, and a solvent as raw materials, and performing a heat treatment. The present invention relates to a reforming method.

  The chromatographic filler comprising the polymer gel of the present invention prepared using saccharide as a template exhibits a greater selectivity for saccharide. Moreover, the selectivity did not identify only the template, but aldpentose / aldohexose and N-acetylhexosamine were each identified as two groups. Therefore, the present invention makes it possible to provide a highly selective filler in saccharide template polymerization, and is considered to greatly contribute to separation of complex carbohydrates as well as analysis of carbohydrates.

  Examples of the sugar (print molecule) used in the present invention include monosaccharides such as galactose (Gal), glucose (Glc), and mannose (Man). Examples of the monomer (host monomer) used in the present invention include methacrylic acid (MA) and vinylpyridine (VP). Moreover, as a crosslinking agent used in this invention, ethylene dimethacrylate (EDMA) or methylenebisacrylamide is mentioned. Furthermore, toluene is mentioned as a solvent used in this invention.

  In the present invention, polymerization conditions in the polymerization performed using a monomer, a crosslinking agent and a solvent are as follows. That is, the temperature is from room temperature to about 50 ° C., and the polymerization time is from 4 hours to 8 hours. In addition, photopolymerization can also be used as a polymerization method.

  After completion of the polymerization, the solvent is removed to obtain the target polymer gel. Furthermore, sugar is adsorbed to the polymer obtained by removing the solvent after completion of the polymerization, and heat treatment is performed. In this case, the heating temperature is generally preferably in the range of 100 ° C to 300 ° C, particularly preferably around 250 ° C.

  The polymer gel of the present invention thus produced has various chromatographic properties such as high performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) for the purpose of analyzing carbohydrates and separating complex carbohydrates in general. Can be used as a filler. The particle diameter of the polymer particles of the polymer gel when the polymer gel is used as a chromatographic filler can be appropriately set according to the type of chromatography used, the type of substance to be separated, and the like. A desirable particle diameter is usually 2 μm to 300 μm, preferably 2 μm to 20 μm, but is not limited to this range.

  The following examples further illustrate the present invention in detail but are not to be construed to limit the scope of the invention.

  2 ml (11 mmol) of ethylene dimethacrylate as a cross-linking agent and 4 ml of toluene as a solvent are subjected to a polymerization reaction at 50 ° C. by a two-stage swelling polymerization method. Further, 1 ml (12 mmol) of methacrylic acid as a host monomer on the surface of the obtained polymer gel As a cross-linking agent, 0.5 g (3 mmol) of methylenebisacrylamide was introduced by polymerization. After completion of the polymerization reaction, the polymer gel was washed, glucose was adsorbed on the surface of the polymer gel, and heat treatment was performed at 250 ° C. for 30 minutes. D-xylose (Xyl), L-arabinose (Ara), D-mannose (Man), N-acetyl D-glucosamine under the following conditions using high performance liquid chromatography using the obtained polymer particles as a filler The saccharide identification was examined using a sugar mixture of (GlcNAc), N-acetyl-D-galactosamine (GalNAc), D-glucose (Glc) and D-galactose (Gal).

Chromatographic conditions:
Mobile phase: 95% acetonitrile aqueous solution and 90% acetonitrile aqueous solution Flow rate: 1 ml / min Column size: 4.6 mm (id) x 150 mm
Column temperature: 30 ° C
Detection: RI
The results are shown in FIG. The following can be seen from FIG.
(1) When heat treatment is carried out without a mold, the selectivity for saccharides is not significantly changed from that before the heat treatment.
(2) Comparison of the effects of adding a template showed that the selectivity for saccharides having an amide group was greatly changed by heat treatment.
(3) The selectivity does not identify only the template, in particular acetylhexosamine and aldopentose / aldohexose as two groups.
(4) It is presumed that the heat treatment under the condition where the template exists causes some change in the state of the carboxyl group on the surface of the filler, and the interaction with the amide group changes.

  In FIG. 2, the symbols on the vertical axis and the horizontal axis represent the following meanings.

  D-galactose (Gal), L-arabinose (Ara), using high performance liquid chromatography using vinyl pyridine as a host monomer and the polymer particles described in Example 1 as a filler, under the following conditions: Sugar identification was examined using a sugar mixture of D-glucose (Glc), D-xylose (Xyl), D-mannose (Man) and D-lyxose (Lyx).

Chromatographic conditions:
Mobile phase: 85% aqueous acetonitrile flow rate: 1 ml / min Column size: 4.6 mm (id) x 150 mm
Column temperature: 50 ° C
Detection: RI
Injection amount: 140 nmol
The results are shown in Table 2. As shown in Table 2 below, when vinyl pyridine was used as the host monomer, saccharides were identified by group as in the case where methacrylic acid was used as the host monomer.

  When a change in ion exchange capacity with heating time was measured for a polymer using methacrylic acid as a host monomer, as shown in Table 3 below, the ion exchange capacity decreased as the heating time increased. The ion exchange capacity measurement conditions are as follows.

  The polymer was packed in a column with an inner diameter of 4.6 mm and a length of 150 mm. Next, 0.1N aqueous potassium chloride solution was passed through the column, and the elution solution was titrated with 0.1N aqueous potassium hydroxide solution to determine the ion exchange capacity.

  About the polymer which used methacrylic acid as a host monomer, it was confirmed by the FT-IR spectrum that the polymer composition change by heat processing was not seen.

The UV spectra of both were measured using an aqueous acetic acid solution having a concentration of 1.7 μmol / L and an aqueous solution containing 1.7 μmol / L acetic acid and 0.17 μmol / L D-glucose. Moreover, both UV spectra were measured in the same manner using a polymethacrylic acid aqueous solution having a concentration of 47 mg / L and an aqueous solution containing 47 mg / L polymethacrylic acid and 58 μmol / L D-glucose. The results are shown in FIG. 3 and FIG. From both figures, the following can be understood.
(1) In the absorption band due to the transition of n → π ° of polymethacrylic acid, due to the presence of glucose in water, a shallow color shift and a dark color effect are observed, and such a marked difference is not observed with monomeric acetic acid. It was.
(2) Polymethacrylic acid and glucose may interact with each other even in water through hydrogen bonding.

  As shown in Table 4 below, when the influence of the heating time on the discrimination ability of the saccharide and the functional group was examined, the heat treatment using the saccharide as a template tends to be relatively advantageous with respect to retention of monosaccharides and amines. showed that. Although the ion exchange groups disappear due to the heat treatment, it is presumed that the presence of the template suppressed the decrease, or the ion exchange groups remained concentrated at a certain site.

In addition, the measurement conditions of each substance in the said Table 4 are as follows.
(1) In the case of D-xylose, D-glucose, N-acetyl-D-glucosamine Mobile phase: 98% acetonitrile aqueous solution Flow rate: 1 mL / min Column size: 4.6 mm (id) x 150 mm
Column temperature: 30 ° C
Detection: RI
Injection amount: 140 nmol
(2) In the case of triethylamine and diethylenetriamine Mobile phase: pH 4.8 citrate buffer Flow rate: 1 mL / min Column size: 4.6 mm (id) x 150 mm
Column temperature: 30 ° C
Detection: RI
Injection amount: 140 nmol

It is a figure explaining the process of the molecular imprint method. It is a graph which shows the discrimination ability of the saccharide | sugar by the polymer gel filler prepared by heat-processing using methacrylic acid as a host monomer. It is a UV spectrum showing the discrimination of the sugar by acetic acid. It is UV spectrum showing the identification of the saccharide | sugar by polymethacrylic acid.

Claims (4)

  A method of modifying a surface of a polymer gel filler, comprising subjecting a polymer obtained by polymerization using a monomer, a cross-linking agent, and a solvent that are methacrylic acid or 4-vinylpyridine as a raw material, and subjecting the polymer to heat treatment.   The surface modification method according to claim 1, wherein the sugar is selected from the group consisting of galactose, glucose and mannose.   The surface modification method according to claim 1 or 2, wherein the crosslinking agent is ethylene dimethacrylate or methylene bisacrylamide.   The surface modification method according to any one of claims 1 to 3, wherein the solvent is toluene.

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