Sugar chain-immobilized polymer particles and method for producing the same

Abstract

The present invention provides: sugar chain immobilized polymer particles in which a hydrophobized sugar chain polypeptide is firmly immobilized to the outermost surface of the polymer particles and the sugar chain density on the surface is raised, thereby making it possible to vastly improve the specific adsorption performance of proteins and viruses that adsorb via sugar chains; and a method for producing the sugar chain immobilized polymer particles. The sugar chain immobilized polymer particles 1: include polymer particles 5, and hydrophobized sugar chain polypeptides 4 that have a plurality of sugar chains and a plurality of hydrophobic groups per molecule and that are immobilized non-covalently to the polymer particles 5 via a plurality of hydrophobic groups 3 embedded at a plurality of different locations on the polymer particles 5 so that each of the hydrophobic groups 3 individually faces from the surface toward the interior of the polymer particles 5; have an average grain size ranging from 50 nm to 1 μm; and are produced by performing soap-free emulsion polymerization in the presence of the hydrophobized sugar chain polypeptides 4 during the course of a polymerization reaction of hydrophobic polymerizable monomers.

Description

[Technical Field]

[0001] The present invention relates to sugar chain-immobilized polymer particles and a method for producing the same, and in particular to sugar chain-immobilized polymer particles and a method for producing the same, which can significantly improve the specific adsorption of proteins and viruses via sugar chains by immobilizing hydrophobic glycopolypeptides more firmly and densely to the polymer particles. [Background technology]

[0002] It is already known that sugar chains interact specifically with proteins and viruses, and they are used as biosensors for proteins, viruses, etc., or as a versatile means for separating and purifying them by utilizing their affinity. However, a single sugar chain molecule has very weak binding affinity with proteins or viruses, and therefore does not exhibit sufficient specific interaction with proteins or viruses, making it impossible to exhibit the desired specific adsorption properties. For this reason, microparticles are used as carriers, and many sugar chain molecules are immobilized on the surface of the carrier.

[0003] Here, polymer particles (which may contain magnetic particles) or inorganic particles such as glass or metal oxide composites such as titanium dioxide are used as microparticles. However, glycan-immobilized polymer particles are being considered because of their ease of production, including the method for immobilizing glycans, and the ability to impart a wide range of functions such as specific adsorption properties.

[0004] Examples of sugar chain-immobilized polymer particles include polymer particles that are polymers of vinyl monomers and have two or more oligosaccharides immobilized thereon (Patent Document 1), sugar chain polymers that have a polypeptide main chain as the polymer (Patent Document 2), and porous polymer particles that have a coating layer and are used as protein separation materials (Patent Document 3).

[0005] The present inventors have also proposed nano-level sugar chain-immobilized polymer particles with an average particle size of 1 μm or less and a narrow particle size distribution, in which sugar chains having hydrophobic groups are non-covalently immobilized via the hydrophobic groups to polymethyl methacrylate microparticles produced by a soap-free emulsion polymerization method (Non-Patent Document 1).

[0006] On the other hand, Patent Document 4 proposes nano-level polymer particles in which, instead of sugar chains, maleimide compounds having hydrophobic segments are non-covalently immobilized, although they are not sugar chain-immobilized polymer particles. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2002-145896 [Patent Document 2] Japanese Patent Application Publication No. 9-227600 [Patent Document 3] International Application No. 2017 / 155105 [Patent Document 4] Special Publication No. 2009-508936 [Non-patent literature]

[0008] [Non-Patent Document 1] N. Yamauchi, et. al., “One-pot formation of sugar-immobilized monodisperse polymethylemethacrylate particles by soap-free emulsion polymerization”, Colloids and surfaces, A 580, 123754, 2019, pp. 1-7 Summary of the Invention [Problem to be solved by the invention]

[0009] The particle size of sugar chain-immobilized polymer particles that specifically adsorb proteins or viruses is desirably an average particle size of 1 μm or less, preferably in the range of several tens to several hundreds of nm, in consideration of the size of the proteins or viruses to be adsorbed. In addition, polymer particles with a narrow particle size distribution and a nearly uniform particle size are desirable because they can uniformly exhibit nearly identical properties among all particles and reduce variation in specific adsorption properties.

[0010] Furthermore, when focusing on a single sugar chain-immobilized polymer particle, in order to obtain high and stable specific adsorption, it is necessary that the sugar chains on the surface of the polymer particle are firmly attached to the polymer particle and that the sugar chains are immobilized densely on the surface at a high concentration.

[0011] Furthermore, if the manufacturing process of sugar chain-immobilized polymer particles, including the sugar chain immobilization step, can be made more efficient and simplified, the cost can be reduced and the use of the particles can be expanded.

[0012] However, it was difficult for the polymer particles disclosed in the above Patent Documents 1 to 4 and Non-Patent Document 1 to simultaneously satisfy all of the above technical problems. That is, although the inventions described in Patent Documents 1 and 2 firmly immobilize the polymer particles and sugar chains via covalent bonds, the sugar chain-immobilized polymer particles themselves are composed of copolymers or polypeptides having sugar chain components on their side chains, and the sugar chain components are embedded even inside the polymer particles, making it difficult to arrange sugar chain components at high concentrations on the surface of the polymer particles.

[0013] Furthermore, the polymer particles disclosed in Patent Documents 1 to 3 require highly specialized techniques in the synthesis reaction carried out to form covalent bonds between the polymer particles and sugar chains, making it difficult to control the production process. In particular, the method for producing a separation material described in Patent Document 3 employs a method for forming a coating layer having first and second graft chains on the surface of porous polymer particles, which requires precise control of the synthesis reaction, making the polymer particle production process more complicated than the methods described in Patent Documents 1 and 2.

[0014] On the other hand, Patent Document 4 describes a method for forming spherical polymer nanometer (100-500 nm) particles by dispersing an oil layer containing a maleimide compound and a polymer in a water tank. However, the spherical polymer particles described in Patent Document 4 have a maleimide compound having a hydrophobic segment adsorbed to the surface of the polymer particles through hydrophobic interactions, and the hydrophobic segment is not embedded inside the polymer particles. Furthermore, the formation method described in Patent Document 4 tends to result in a somewhat broad particle size distribution of the polymer particles. Therefore, the polymer particles and their production method described in Patent Document 4 cannot be directly applied to the object of the present invention.

[0015] In light of the above, as a method for producing polymer particles more simply and for immobilizing sugar chain components more firmly than those disclosed in Patent Documents 1 to 4, nano-level polymer particles in which sugar chain components having hydrophobic groups are non-covalently immobilized to polymer particles by soap-free emulsion polymerization and a method for producing the same have been proposed, as disclosed in Non-Patent Document 1. Here, non-covalent immobilization to polymer particles is achieved in a manner in which the hydrophobic groups are embedded inside the polymer particles.

[0016] However, the sugar chain component having a hydrophobic group described in Non-Patent Document 1 is a low-molecular-weight compound containing only one hydrophobic segment of the hydrophobic group and one hydrophilic segment of the sugar chain per molecule. Therefore, the immobilization strength of the sugar chain component to the polymer particle is not necessarily sufficient, and further improvement of the immobilization strength was necessary. Furthermore, because it is difficult to surface-adsorb all of the low-molecular-weight compound added in soap-free emulsion polymerization, there are technical limitations to increasing the density of the sugar chain component on the polymer particle surface. Therefore, if the sugar chain component could be immobilized more firmly and at a high density on the polymer particle surface instead of the method described in Non-Patent Document 1, it is expected that the specific adsorption ability for proteins and viruses could be significantly improved. Therefore, polymer particles with such properties and characteristics and a method for producing them have been strongly desired.

[0017] The present invention has been made in consideration of the above-mentioned conventional problems, and provides sugar chain-immobilized polymer particles and a method for producing the same, which can significantly improve the specific adsorption of proteins and viruses adsorbed via sugar chains by firmly immobilizing a glycopolypeptide having a hydrophobic segment on the outermost surface of the polymer particle and densely introducing sugar chains onto the outermost surface of the polymer particle. [Means for solving the problem]

[0018] The present inventors discovered that the above problems can be solved by using a polypeptide having multiple sugar chains and multiple hydrophobic groups in its side chains in one molecule, instead of the conventional hydrophobicized glycopolypeptide having one hydrophobic group and one sugar chain as hydrophobic segments, and by performing soap-free emulsion polymerization in the presence of a hydrophobic polymerizable monomer together with the polypeptide from the middle of the polymerization reaction, thereby achieving the present invention.

[0019] That is, the present invention is configured as follows. [1] The present invention provides a method for producing a polymer particle comprising: a hydrophobized glycopolypeptide having a plurality of sugar chains and a plurality of hydrophobic groups in its side chains in one molecule, the hydrophobic groups being non-covalently immobilized to the polymer particle via the hydrophobic groups individually embedded at a plurality of different positions of the polymer particle from the surface toward the interior of the polymer particle; the polymer particles are polymers of one or more hydrophobic monomers selected from the group consisting of styrene and its derivatives, vinyl esters, and (meth)acrylic acid esters; the hydrophobized glycopolypeptide has a main chain that is a polypeptide having amino acid residues represented by the following formula (1) or (2), a side chain formed by binding a glycoside having a monosaccharide or oligosaccharide structure to the main chain via an amide bond, and a side chain formed by binding a linear or branched alkyl group having 3 to 15 carbon atoms as the hydrophobic group to the main chain via an amide bond; of Each one is multiple have It is characterized by Sugar chain-immobilized polymer particles are provided. [ka] [2] The present invention provides the sugar chain-immobilized polymer particles according to the above [1], characterized in that the sugar chain-immobilized polymer particles have an average particle size of 50 nm to 1 μm. [3] The present invention provides the sugar chain-immobilized polymer particle according to [2] above, characterized in that the hydrophobized glycopolypeptide is immobilized without spanning multiple polymer particles. [ 4The present invention provides the above-mentioned hydrophobized glycopolypeptide, characterized in that the hydrophobized glycopolypeptide is a polypeptide represented by the following formula (3): 3 The present invention provides a sugar chain-immobilized polymer particle according to the present invention. [ka] where R1 is hydrogen, an alkyl group, or a monovalent cationic metal, R2 is a monosaccharide glycoside or an oligosaccharide glycoside, and R3 is The aforementioned It is a hydrophobic group, a is an integer of 0 or 1 or more, and b and c are each independently an integer of 2 or more. [ 5 The present invention relates to the above-mentioned [ 4 The present invention provides a sugar chain-immobilized polymer particle according to the present invention. [ 6 The present invention provides the above-mentioned polymer particles, characterized in that the polymer particles are particles having a polymer of at least one hydrophobic monomer selected from the group consisting of styrene and its derivatives, and (meth)acrylic acid esters, the R1 is a cationic metal of sodium or potassium, the R2 is a glycoside containing at least one sugar structure selected from the group consisting of monosaccharides and disaccharide to heptasaccharide sugars represented by the following formula (4) or (5), and the R3 is a linear or branched alkyl group having 3 to 15 carbon atoms: 4 10. The sugar chain-immobilized polymer particle according to claim 1, [ka] Here, Y1 and Y2 are each independently an organic group having a structure in which 3 to 15 methylene groups are covalently bonded as a spacer group, m and n are each independently an integer of 0 to 2, and Z is a sugar chain containing at least one sugar structure selected from the group consisting of monosaccharides and disaccharides to heptasaccharides. [ 7The present invention provides a sugar chain-immobilized polymer particle according to any one of [1] to [3] above, characterized in that the sugar chain-immobilized polymer particle has a hydrophilic polyalkylene glycol or polyalkylene glycol derivative adsorbed on the surface of the polymer particle. [ 8 The present invention provides a sugar chain-immobilized polymer particle according to any one of [1] to [3] above, characterized in that the polymer particle has a fluorescent dye non-covalently fixed to the polymer particle by hydrophobic interaction or electrostatic interaction with the polymer particle. [ 9 The present invention comprises the steps of initiating polymerization by adding a water-soluble radical polymerization initiator into a polymerization vessel having an aqueous solvent in which a polymerizable monomer is dispersed, and adding, during the polymerization reaction, a hydrophobic glycopolypeptide having multiple sugar chains and multiple hydrophobic groups in its side chains in one molecule, and continuing the polymerization reaction in a state in which the hydrophobic glycopolypeptide is present in the polymerization vessel. death, The polymerizable monomer is one or more hydrophobic monomers selected from the group consisting of styrene and its derivatives, vinyl esters, and (meth)acrylic acid esters, and the hydrophobized glycopolypeptide has a main chain of a polypeptide having amino acid residues represented by the following formula (1) or (2), and has a plurality of side chains in which a glycoside containing a monosaccharide or oligosaccharide structure is bound to the main chain via an amide bond, and a plurality of side chains in which a linear or branched alkyl group having 3 to 15 carbon atoms is bound to the main chain via an amide bond as the hydrophobic group. The present invention provides a method for producing sugar chain-immobilized polymer particles. [ka] [ 10 The present invention is directed to a method for preparing a polymerization system in which the addition of the hydrophobized glycopolypeptide causes the polymerization of the polymerizable monomer in the polymerization tank to proceed, contained in the polymerization vessel The conversion rate of the polymerizable monomer by the polymerization reaction is , 3% by mass or more relative to the time of initiation of polymerization 95% by mass or less becomes Time Polymerization reaction time The method of the present invention is characterized in that 9 The present invention provides a method for producing a sugar chain-immobilized polymer particle according to the present invention. [ 11 The present invention provides the method for producing a polymerizable monomer in a polymerization vessel, characterized in that the hydrophobized glycopolypeptide is added during a polymerization reaction time when the conversion rate of the polymerizable monomer contained in the polymerization vessel due to the polymerization reaction is 3 to 50% by mass relative to the conversion rate at the start of polymerization. 10 The present invention provides a method for producing a sugar chain-immobilized polymer particle according to the present invention. [ 12The present invention provides the above-mentioned hydrophobized glycopolypeptide, characterized in that the hydrophobized glycopolypeptide is a polypeptide represented by the following formula (3): 11 The present invention provides a method for producing a sugar chain-immobilized polymer particle according to the present invention. [ka] where: R1 is hydrogen, an alkyl group, or a monovalent cationic metal, R2 is a monosaccharide glycoside or an oligosaccharide glycoside, and R3 is The aforementioned It is a hydrophobic group, a is an integer of 0 or 1 or more, and b and c are each independently an integer of 2 or more. [ 13 The present invention provides the above-mentioned [ 12 The present invention provides a method for producing a sugar chain-immobilized polymer particle according to the present invention. [ 14 The present invention provides the above-mentioned polymer particles, characterized in that the polymer particles are particles having a polymer of at least one hydrophobic monomer selected from the group consisting of styrene and its derivatives, and (meth)acrylic acid esters, the R1 is a cationic metal of sodium or potassium, the R2 is a glycoside containing at least one sugar structure selected from the group consisting of monosaccharides and disaccharide to heptasaccharide sugars represented by the following formula (4) or (5), and the R3 is a linear or branched alkyl group having 3 to 15 carbon atoms: 12 The present invention provides a method for producing a sugar chain-immobilized polymer particle according to the present invention. [ka] Here, Y1 and Y2 are each independently an organic group having a structure in which 3 to 15 methylene groups are covalently bonded as a spacer group, m and n are each independently an integer of 0 to 2, and Z is a sugar chain containing at least one sugar structure selected from the group consisting of monosaccharides and disaccharides to heptasaccharides. [ 15 The present invention relates to the [9]~

[11] The present invention provides a method for producing sugar chain-immobilized polymer particles, characterized in that the method for producing sugar chain-immobilized polymer particles according to any one of the above items (1) to (4) above comprises a step of adsorbing a hydrophilic polymer, such as polyalkylene glycol or a polyalkylene glycol derivative, onto the surface of the sugar chain-immobilized polymer particles during or after completion of the polymerization reaction of the sugar chain-immobilized polymer particles. [ 16 The present invention provides the method for producing a polymer particle having a hydrophilic polymer adsorbed on the surface thereof, characterized in that the step of adsorbing the hydrophilic polymer onto the surface of the polymer particle comprises either a step of adding the hydrophilic polymer to the polymerization vessel containing the sugar chain-immobilized polymer particles during or after the completion of the polymerization reaction of the sugar chain-immobilized polymer particles, or a step of transferring the sugar chain-immobilized polymer particles to a reaction vessel containing an aqueous medium, separate from the polymerization vessel, and then adding the hydrophilic polymer to the reaction vessel containing the sugar chain-immobilized polymer particles. 15 The present invention provides a method for producing a sugar chain-immobilized polymer particle according to the present invention. [ 17 The present invention provides a method for preparing a hydrophobic glycopolypeptide by adding a fluorescent dye to the polymerization vessel in the presence or absence of the polymerizable monomer before the step of initiating the polymerization, or by adding the hydrophobic glycopolypeptide at a polymerization time when the conversion rate of the polymerizable monomer due to the polymerization reaction is 95% by mass or less after the initiation of the polymerization. and The method is characterized in that the sugar chain-immobilized polymer particles in which the fluorescent dye is immobilized on the polymer particles are produced by adding the fluorescent dye to the polymerization vessel in the presence or absence of the fluorescent dye. [9]~

[11] The present invention provides a method for producing a sugar chain-immobilized polymer particle according to any one of the above items. [Effects of the invention] [Effects of the Invention]

[0020] In the glycan-immobilized polymer particles according to an embodiment of the present invention, two or more hydrophobic groups possessed by the hydrophobized glycopolypeptide are embedded inside the polymer particle at separate locations. This increases the contact area between the polymer particle and the hydrophobic group portion that functions as an anchor site, making it difficult for the glycan to come off the polymer particle. This allows the glycan to be firmly immobilized on the surface of the polymer particle. Furthermore, since two or more glycans possessed by the hydrophobized glycopolypeptide are densely introduced in a molecular arrangement on the outermost surface of the polymer particle, the specific adsorption of proteins and viruses adsorbed via the glycans can be significantly improved.

[0021] Furthermore, the glycan-immobilized polymer particles according to the present invention can be used as polymer particles with desired specific adsorption properties by varying the type and number of glycans on the hydrophobized glycopolypeptide depending on the type of protein or virus. Therefore, they are widely applicable as biosensors, separation agents, and purification agents, and are highly versatile. Furthermore, by incorporating a fluorescent dye, the glycan-immobilized polymer particles according to the present invention can be used not only as a highly accurate means for detecting proteins or viruses, but also as a fluorescent probe for imaging biological substances or a fluorescent label for cell imaging. For example, by measuring the fluorescence spectrum of the glycan-immobilized polymer particles incorporating the fluorescent dye in an aqueous solution or biological tissue by fluorescence spectroscopy or the like, and analyzing the absorption peak and intensity of the obtained fluorescence spectrum, it is possible to understand not only the dispersion state and behavior of the glycan-immobilized polymer particles before and after specific adsorption of proteins or viruses, but also the behavior of the proteins or viruses after specific adsorption, making them highly useful.

[0022] The method for producing a sugar chain-immobilized polymer of the present invention involves performing soap-free emulsion polymerization in the presence of a hydrophobic polymerizable monomer and a polypeptide from the middle of the polymerization reaction, which not only prevents aggregation of polymer particles but also enables efficient production of nano-level particles with an average particle size in the range of 50 nm to 1 μm, with a desired average particle size and a narrow particle size distribution. Therefore, the method is excellent for mass production of sugar chain-immobilized polymers with precisely controlled particle size and particle size distribution. [Brief explanation of the drawings]

[0023] [Figure 1] 1 shows a cross-sectional schematic diagram of an example of a sugar chain-immobilized polymer particle according to an embodiment of the present invention and an SEM photograph image observed with a scanning electron microscope (SEM). [Figure 2] FIG. 1 is a cross-sectional view showing an example of a sugar chain-immobilized polymer particle according to the prior art. [Figure 3] 1 is a schematic diagram showing the polymerization process of the method for polymerizing sugar chain-immobilized polymer particles according to the present invention and the polymerization process of a conventional soap-free emulsion polymerization method. FIG. [Figure 4] FIG. 1 is a diagram showing a schematic diagram of an example of the production of a precursor of a hydrophobic glycoside used in the present invention. [Figure 5] FIG. 1 is a schematic diagram showing an example of synthesis of a high molecular weight oligosaccharide chain from a low molecular weight sugar chain. [Figure 6] FIG. 1 is a diagram schematically showing an example of a method for producing a hydrophobized glycopolypeptide according to an embodiment of the present invention. [Figure 7] FIG. 1 is a cross-sectional view schematically showing the state in which a hydrophilic polymer is adsorbed on the surface of a sugar chain-immobilized polymer particle of the present invention and a conventional sugar chain-immobilized polymer particle. [Figure 8] FIG. 1 is a diagram showing the polymerization process when producing sugar chain-immobilized polymer particles in Examples and Comparative Examples of the present invention. [Figure 9] FIG. 1 is a view showing a scanning electron microscope (SEM) photograph of the sugar chain-immobilized polymer particles according to Example 1 of the present invention when observed with an SEM. [Figure 10]FIG. 1 shows scanning electron microscope (SEM) photographs of the sugar chain-immobilized polymer particles of Comparative Example 1 and Reference Examples 1 and 2. [Figure 11] FIG. 1 shows the results of measuring the amounts of proteins specifically adsorbed onto the sugar chain-immobilized polymer particles of Example 4 and Comparative Example 2 of the present invention. [Figure 12] FIG. 1 shows the results of measuring the amount of protein (WGA) specifically adsorbed to the sugar chain-immobilized PMMA particles of Example 5 of the present invention and the PMMA particles of Comparative Examples 3 and 4, on whose surfaces polyethylene glycol is adsorbed. [Figure 13] FIG. 1 shows the polymerization process when producing sialo-oligosaccharide-immobilized PMMA particles according to Example 6 of the present invention. [Figure 14] FIG. 1 shows the results of measuring the amount of protein (SSA) adsorbed onto sialo-oligosaccharide-immobilized PMMA particles and onto PMMA particles with no added sugar chains in Example 6 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] An example of a sugar chain-immobilized polymer particle according to an embodiment of the present invention is shown schematically in Figure 1. In Figure 1, (a) is a schematic cross-sectional view of a sugar chain-immobilized polymer particle of the present invention, and (b) is a scanning electron microscope (SEM) image of the sugar chain-immobilized polymer particle of the present invention observed with an SEM.

[0025] As shown in Figure 1(a), a hydrophobized glycopolypeptide 4 having multiple glycans 2 and multiple hydrophobic groups 3 per molecule is immobilized on the surface of a glycan-immobilized polymer particle 1. The hydrophobized glycopolypeptide 4 is immobilized non-covalently via multiple hydrophobic groups 3 embedded individually at multiple different positions in the polymer particle 5 from the surface toward the interior of the polymer particle 5. Here, the multiple hydrophobic groups 3 are not covalently bonded to the polymer particle 5 but are instead introduced into the side chains of the hydrophobized glycopolypeptide 4 to achieve an anchoring effect by embedding them in the polymer particle 5. Because the anchoring effect of the hydrophobized glycopolypeptide 4 is achieved at multiple positions in a single molecule of the hydrophobized glycopolypeptide 4, the immobilization can be strengthened as the contact area with the polymer particle increases. Furthermore, it is preferable that the hydrophobized glycopolypeptide 4 be immobilized only on one polymer particle 5, rather than across two or more polymer particles. As will be described later, when the hydrophobized glycopeptide 4 is immobilized across multiple polymer particles 5, the polymer particles are more likely to aggregate, resulting in insufficient embedding of the hydrophobic group 3 into each polymer particle 5. This results in a decrease in the strength of non-covalent immobilization. Furthermore, because fine particles of less than several tens of nanometers are also synthesized, as will be described later, the broadening of the particle size distribution leads to secondary problems such as a decrease in adsorption properties that affect the detection sensitivity of proteins and viruses, and a decrease in the efficiency of particle recovery from aqueous solvents.

[0026] FIG. 1(a) is a schematic diagram showing the cross section of a polymer particle 5 in which three molecules of hydrophobized glycopolypeptide 4 are immobilized on the polymer particle 5. The amount of hydrophobized glycopolypeptide 4 immobilized is adjusted depending on the amounts of polymerizable monomers and hydrophobized glycopolypeptide 4 supplied to a polymerization vessel in the polymerization reaction described below. Alternatively, a hydrophobized glycopolypeptide 4 having numerous glycan 2 and hydrophobic group 3 segments on its side chains may be synthesized and used. By maintaining the glycan 2 in a molecular arrangement on the outermost surface of the polymer particle 5, the glycan 2 can be densely introduced, significantly improving the specific adsorption of proteins or viruses adsorbed via the glycan 2. Furthermore, by selecting the type of glycan 2 or the number of glycosides having the glycan 2 according to the type of protein or virus, the selective and specific adsorption of these proteins or viruses can be simultaneously improved. In the present invention, a peptide is used in which a glycoside containing at least one sugar structure selected from the group consisting of monosaccharides and oligosaccharides (2-7 sugars) has been introduced into its side chain from among glycosides, which are compounds in which sugars are linked to various atomic groups via glycoxide bonds. The introduction of these glycosides to improve specific adsorption is practical in terms of the availability of raw materials and ease of production. Thus, the term "sugar chain" in the present invention refers collectively to not only molecular chains in which multiple monosaccharides are linked, but also molecular chains of a single monosaccharide.

[0027] Figure 1(b) shows a scanning electron microscope (SEM) image of an example of a sugar chain-immobilized polymer particle 1 obtained by an embodiment of the present invention. The example shown in Figure 1(b) shows that the sugar chain-immobilized polymer particles are monodisperse, with a particle size of less than 200 nm, a narrow particle size distribution, high particle size uniformity, and no aggregation.

[0028] The sugar chain-immobilized polymer particles 1 according to an embodiment of the present invention have an average particle size of 50 nm to 1 μm in the form of non-aggregated primary particles. Furthermore, the average particle size is preferably in the range of 50 nm to 500 nm. Proteins and viruses have average particle sizes in the ranges of several nm to several tens of nm and 10 to several hundreds of nm, respectively, assuming that the particles are spherical. Therefore, sugar chain-immobilized polymer particles can fully exhibit their adsorption properties when their average particle size is in the range of 50 nm to 1 μm, preferably 50 nm to 500 nm.

[0029] If the average particle size exceeds 1 μm, the total surface area of ​​the particles per unit mass decreases, resulting in a decrease in the amount of protein and virus adsorption and a significant decrease in detection sensitivity. In particular, agglomerated (secondary) particles formed by aggregation of glycan-immobilized polymer particles are likely to occur due to the hydrophobic glycopolypeptides immobilized on the glycan-immobilized polymer particles spanning between aggregated particles, resulting in an average particle size exceeding 1 μm and large, irregularly shaped particles. In such cases, the embedding of multiple hydrophobic groups 3 into the polymer particles 5 becomes insufficient, resulting in an insufficient anchoring effect due to the embedding, resulting in a decrease or variability in the immobilization strength. Therefore, in the present invention, it is necessary to avoid aggregation of glycan-immobilized polymer particles 1. Furthermore, if the average particle size is less than 50 nm, not only is it difficult to recover the particles from the aqueous solvent after adsorption of proteins and viruses, but detection sensitivity also decreases significantly.

[0030] The sugar chain-immobilized polymer particles 1 according to an embodiment of the present invention are practical because they have a narrow particle size distribution and high particle size uniformity. When the particle size distribution is defined as the dispersity (coefficient of variation) CV = (standard deviation of particle size) / (average particle size) × 100 (%), narrowing the particle size distribution so that the CV is 20% or less, preferably 10% or less, can improve the adsorption properties for proteins and viruses. If the CV exceeds 20%, the variation in the amount of adsorption between particles during protein and virus adsorption becomes large, resulting in a significant decrease in adsorption properties.

[0031] The average particle size of the sugar chain-immobilized polymer particles 1 can be determined by measuring the particle sizes of approximately 100 to 200 particles randomly selected from an SEM photograph and averaging these measurements, as shown in Figure 1(b). Alternatively, a particle size distribution curve can be created by plotting the measured particle sizes on the horizontal axis and the cumulative number of particles having each particle size on the vertical axis. The standard deviation of particle size can be calculated from the particle size distribution curve, and then divided by the average particle size to determine the particle size dispersity (CV). The average particle size and dispersity of the sugar chain-immobilized polymer particles are not limited to the above-mentioned methods and may also be determined using a particle size distribution curve measured using a laser diffraction / scattering particle size distribution analyzer.

[0032] Figure 2 shows a schematic diagram of an example of a glycan-immobilized polymer particle produced by conventional technology. The glycan-immobilized polymer particle 6 shown in Figure 2 was obtained by the conventional technology disclosed in Non-Patent Document 1. In the glycan-immobilized polymer particle 6 shown in Figure 2, a hydrophobized glycopolypeptide 9 containing only one glycan 7 and one hydrophobic group 8 per molecule is non-covalently immobilized to a polymer particle 10, and the immobilization of the hydrophobized glycopolypeptide 9 is achieved by embedding one hydrophobic group 8. In addition, there is a limit to how much the glycan immobilization density can be increased due to restrictions on the amount of hydrophobized glycopolypeptide 9 that can be charged during the production of the glycan-immobilized polymer particle 6 and its weak surface adsorption.

[0033] In contrast, in the case of a sugar chain-immobilized polymer particle 1 according to an embodiment of the present invention, as shown in Figure 1(a), when a hydrophobized glycopolypeptide 4 containing two or more sugar chains 2 and hydrophobic groups 3 in its side chains is physically adsorbed onto a polymer particle 5, the contact area between the hydrophobic groups 3 and the polymer particle 5 is increased by embedding the multiple hydrophobic groups 3 inside the polymer particle 5, thereby enhancing the anchoring effect. This not only significantly improves the immobilization strength of the hydrophobized glycopolypeptide 4, but also increases the density of sugar chains arranged on the surface of the polymer particle 5.

[0034] Furthermore, the present invention was made by not only using hydrophobized glycopolypeptide 4 but also by investigating a method for embedding multiple hydrophobic groups 3 inside polymer particles 5. One method for increasing the density of sugar chains arranged on the surface of polymer particles 5 is to form hydrophobic polymer particles 5 by soap-free emulsion polymerization, and then add hydrophobized glycopolypeptide 4 having multiple sugar chains 2 and multiple hydrophobic groups 3 in one molecule, thereby physically adsorbing the hydrophobic groups 3 onto the surface of the polymer particles 5. This method is simple because it does not require the procedure of embedding multiple hydrophobic groups 3 inside the polymer particles, but as will be explained in Comparative Example 4 below, the specific adsorption performance for proteins and viruses and the immobilization strength of the hydrophobized glycopolypeptide 4 were not sufficient, and the object of the present invention could not be achieved.

[0035] The sugar chain-immobilized polymer particles 1 of the present invention are produced by soap-free emulsion polymerization, which is known as a method for synthesizing monodisperse polymer particles, among the various polymerization methods used for polymer particles, such as emulsion polymerization, suspension polymerization, dispersion polymerization, and seed polymerization. Soap-free emulsion polymerization is commonly used to synthesize particles with clean particle surfaces without using surfactants, but the present invention is characterized not only by the use of hydrophobic glycopolypeptide 4 as a new component, but also by the method of adding hydrophobic glycopolypeptide 4 during soap-free emulsion polymerization, which is different from conventional polymerization processes.

[0036] The polymerization process actually employed as the soap-free emulsion polymerization method in the present invention will be explained in comparison with a conventionally commonly employed polymerization process using Figure 3. Figure 3(a) is a diagram schematically illustrating the polymerization process employed in the method for producing sugar chain-immobilized polymer particles 1 according to the present invention. Figure 3(b) is a diagram illustrating the conventionally commonly employed soap-free emulsion polymerization process, which was adopted in Non-Patent Document 1 as a polymerization process capable of narrowing the particle size distribution.

[0037] Soap-free emulsion polymerization is a method for synthesizing polymer particles by dispersing hydrophobic polymerizable monomers, such as styrene or methyl methacrylate, in an aqueous solvent 12 in a polymerization vessel 11 to form oil droplets 13 of the polymerizable monomer. A water-soluble radical polymerization initiator, such as potassium dihydrogen phosphate or a persulfate (e.g., potassium persulfate), is then added. The polymerization atmosphere (e.g., a nitrogen or argon atmosphere), temperature, and time are selected under predetermined conditions, and radical polymerization is performed to synthesize polymer particles. As shown in Figure 3(a), the production method of the present invention is characterized by adding a hydrophobized glycopolypeptide 4 during the polymerization reaction, i.e., during the polymerization reaction. Here, "polymer particles 14 during the polymerization reaction" refers to the state in which the polymer particles are swollen with the polymerizable monomer, as described below.

[0038] The polymerization process shown in Figure 3(a) overcomes the technical issues of polymer particle aggregation observed in the conventional polymerization process shown in Figure 3(b), as well as the tendency for fine particles of several tens of nanometers or less to be synthesized. In the conventional polymerization process shown in Figure 3(b), hydrophobized glycopolypeptide 4 is added to a polymerization vessel 11 containing hydrophobic polymerizable monomers before the radical polymerization initiator is added (before the start of polymerization), and then polymerization is allowed to proceed to form polymer particles. However, in this polymerization process, the use of hydrophobized glycopolypeptide 4 with a large molecular weight results in physical adsorption to the surface of the fine polymers 14 formed during the polymerization reaction, acting as bridges between the fine polymer particles. As a result, aggregation occurs between multiple polymer particles as the polymerization progresses, resulting in the formation of aggregated glycan-immobilized polymer particles 15. Furthermore, particle aggregation tends to create an inhomogeneous environment in the polymerization reaction, which tends to result in the synthesis of fine particles of several tens of nanometers or less. This also leads to a broad particle size distribution of the glycan-immobilized polymer particles, which must be taken into consideration.

[0039] In contrast, in the polymerization process of the present invention shown in Figure 3(a), when polymer particles 14 swollen with polymerizable monomers during polymerization grow large enough to allow multiple hydrophobic glycopolypeptides 4 to adsorb to their surfaces, the hydrophobic glycopolypeptides 4 are surface-adsorbed onto the polymer particles 14. This behavior prevents bridging between polymer particles due to physical adsorption of the hydrophobic glycopolypeptides 4, preventing aggregation of polymer particles 5 as the polymerization proceeds. During this process, the multiple hydrophobic groups 3 possessed by the hydrophobic glycopolypeptides 4 are embedded in the interior of the swollen polymer particles due to hydrophobic interactions, providing a significant anchoring effect for the polymer particles 5. In addition, because the polymerization reaction proceeds under substantially the same conditions between each particle, there is the advantage that the particle size distribution of the polymer particles 5 can be narrowed.

[0040] The technical problem of aggregation of glycan-immobilized polymer particles 15 and the production of fine particles shown in Figure 3(b) was first recognized when the unique hydrophobic glycopolypeptide of the present invention was used. Based on this technical problem, it was discovered that when a hydrophobic glycopolypeptide 4 having multiple glycans and multiple hydrophobic groups in the side chains per molecule is used, glycan-immobilized polymer particles 1 with the unique properties and physical properties of the present invention can be obtained by further optimizing the polymerization process in the production method, and this led to the present invention.

[0041] As described above, the sugar chain-immobilized polymer particle 1 according to the embodiment of the present invention has a structure in which a polymer particle 5, which is a polymer of a hydrophobic monomer, is used as a carrier, and a hydrophobized glycopolypeptide 4 having a plurality of sugar chains 2 and a plurality of hydrophobic groups 3 is immobilized on the surface of the polymer particle 5. Furthermore, the hydrophobized glycopolypeptide 4 has a plurality of hydrophobic groups 3 embedded inside the polymer particle 5, and is non-covalently immobilized to the polymer particle 5 by hydrophobic interactions of the plurality of hydrophobic groups 3. The polymer particle 5 and hydrophobized glycopolypeptide 4 used in the embodiment of the present invention are described below.

[0042] <Polymer particles> In an embodiment of the present invention, the polymer particles are polymers of hydrophobic monomers, and the hydrophobic monomers are one or more monomers selected from the group consisting of styrene and its derivatives, vinyl esters, and (meth)acrylic acid esters, more preferably one or more monomers selected from the group consisting of styrene and its derivatives, and (meth)acrylic acid esters in terms of ease of soap-free emulsion polymerization and past application history. These hydrophobic monomers are monomers commonly used in soap-free emulsion polymerization, which will be described later as a production method of the present invention.

[0043] Examples of styrene and its derivatives used as hydrophobic monomers include styrene, α-methylstyrene, methylstyrene, butylstyrene, t-butylstyrene, dimethylstyrene, divinylbenzene, etc., and these can be used alone or in combination. Among these, styrene is preferred from the viewpoints of ease of soap-free emulsion polymerization in the production of polymer emulsions, availability, and economy.

[0044] Examples of vinyl esters used as hydrophobic monomers include vinyl esters having an alkyl group or an alkenyl group, such as vinyl acetate, vinyl propionate, vinyl butyrate, vinyl hexanoate, vinyl octanoate, vinyl decanoate, vinyl laurate, vinyl palmitate, and vinyl stearate, and these can be used alone or in combination. Of these, vinyl acetate is preferred from the viewpoints of ease of soap-free emulsion polymerization in the production of polymer emulsions, availability, and economy.

[0045] As the (meth)acrylic acid ester used as the hydrophobic acrylic monomer, from the viewpoints of ease of soap-free emulsion polymerization in the production of the polymer emulsion, availability, and economy, a (meth)acrylic acid alkyl ester is preferred, and examples thereof include those whose homopolymer solubility in water at 20°C is 1% by mass or less. The number of carbon atoms in the alkyl of the (meth)acrylic acid alkyl ester is preferably 1 or more and 24 or less, more preferably 1 or more and 12 or less, even more preferably 1 or more and 8 or less, and still more preferably 1 or more and 6 or less. In this specification, "(meth)acrylic acid" means methacrylic acid or acrylic acid.

[0046] Specific examples of methacrylic acid esters include methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, sec-butyl methacrylate, tert-butyl methacrylate, n-hexyl methacrylate, 2-ethylhexyl methacrylate, isooctyl methacrylate, n-decyl methacrylate, isodecyl methacrylate, lauryl methacrylate, cyclohexyl methacrylate, benzyl methacrylate, and isobornyl methacrylate.

[0047] Among these, from the viewpoints of ease of soap-free emulsion polymerization in the production of polymer emulsions, availability, and economy, one or more selected from the group consisting of methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, sec-butyl methacrylate, tert-butyl methacrylate, n-hexyl methacrylate, 2-ethylhexyl methacrylate, isooctyl methacrylate, n-decyl methacrylate, isodecyl methacrylate, and lauryl methacrylate are preferred, and methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, and tert-butyl methacrylate are particularly preferred. More preferred is one or more selected from the group consisting of sec-butyl methacrylate, tert-butyl methacrylate, n-hexyl methacrylate, 2-ethylhexyl methacrylate, and isooctyl methacrylate, even more preferred is one or more selected from the group consisting of methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, sec-butyl methacrylate, tert-butyl methacrylate, and n-hexyl methacrylate, even more preferred is one or more selected from the group consisting of methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, and n-butyl methacrylate, and even more preferred is methyl methacrylate.

[0048] Specific examples of acrylic acid esters include methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, sec-butyl acrylate, tert-butyl acrylate, n-hexyl acrylate, 2-ethylhexyl acrylate, isooctyl acrylate, n-decyl acrylate, isodecyl acrylate, lauryl acrylate, cyclohexyl acrylate, benzyl acrylate, and isobornyl acrylate.

[0049] Among these, from the viewpoints of ease of soap-free emulsion polymerization in the production of polymer emulsions, availability, and economy, one or more selected from the group consisting of methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, sec-butyl acrylate, tert-butyl acrylate, n-hexyl acrylate, 2-ethylhexyl acrylate, isooctyl acrylate, n-decyl acrylate, isodecyl acrylate, and lauryl acrylate are preferred, and methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, More preferred is one or more selected from the group consisting of sec-butyl acrylate, tert-butyl acrylate, n-hexyl acrylate, and isooctyl acrylate, even more preferred is one or more selected from the group consisting of methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, sec-butyl acrylate, tert-butyl acrylate, and n-hexyl acrylate, even more preferred is one or more selected from the group consisting of methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, and n-butyl acrylate, and even more preferred is n-butyl acrylate.

[0050] <Hydrophobic glycopolypeptide with multiple sugar chains and multiple hydrophobic groups in the side chains in one molecule> In an embodiment of the present invention, a hydrophobized glycopolypeptide having multiple sugar chains and multiple hydrophobic groups in its side chains per molecule is used as the hydrophobized glycopolypeptide immobilized on the polymer particle. This hydrophobized glycopolypeptide has a main chain made of a polypeptide having amino acid residues represented by the following formula (1) or (2), and has multiple side chains in which glycosides containing monosaccharide or oligosaccharide structures are bound to the main chain via amide bonds, and multiple side chains in which hydrophobic groups are bound to the main chain via amide bonds.

[0051] [ka]

[0052] The amino acid residues represented by the above formula (1) are gamma-glutamic acid residues that constitute a polypeptide made of glutamic acid, a type of amino acid. Poly-gamma-glutamic acid (γ-PGA), which has gamma-glutamic acid residues in its main chain, is a biopolymer obtained by culturing bacteria such as Bacillus subtilis var. natto. Poly-gamma-glutamic acid (γ-PGA) can be obtained not only from biopolymers but also by artificial synthesis. Furthermore, the amino acid residues represented by the above formula (2) are aspartic acid residues that constitute a polypeptide made of aspartic acid. Polyaspartic acid, which has aspartic acid residues in its main chain, does not exist in nature and is therefore obtained by artificial synthesis.

[0053] As a hydrophobized glycopolypeptide having multiple sugar chains and multiple hydrophobic groups in its side chains in one molecule, it is preferable to use a polypeptide represented by the following formula (3) from the viewpoints of ease of synthesis, availability of raw materials, and economy.

[0054] [ka]

[0055] Here, R1 is hydrogen, an alkyl group, or a monovalent cationic metal, R2 is a monosaccharide glycoside or an oligosaccharide glycoside, R3 is a hydrophobic group, a is an integer of 0 or 1 or more, and b and c are each independently an integer of 2 or more.

[0056] As represented by the above formula (3), a polypeptide having polyglutamic acid residues in its main chain can be synthesized according to known methods using poly-γ-glutamic acid, which is also a biopolymer, as a raw material. Therefore, it is suitable for the present invention from the viewpoints of easy availability of poly-γ-glutamic acid and cost reduction.

[0057] When poly-γ-glutamic acid is used as a raw material, R1 is hydrogen, an alkyl group, or a monovalent cationic metal, and is represented by -COOH, -COOX (where X is an alkyl group), or -COO - M+ (wherein M is a monovalent cationic metal). In the present invention, when poly-γ-glutamic acid is used as a raw material, the synthesis of the polypeptide represented by the above formula (3) is easily carried out in an aqueous solvent, and since poly-γ-glutamic acid sodium salt or poly-γ-glutamic acid potassium salt is readily available commercially as a water-soluble compound of poly-γ-glutamic acid, the above R1 is preferably the above -COO - Na + , or -COO - K + Preferably, the cationic metal is sodium or potassium, which forms:

[0058] In the polypeptide represented by formula (3), from the viewpoints of ease of synthesis of the hydrophobized glycopolypeptide, dispersibility in aqueous solvents during soap-free emulsion polymerization, fixation of polymer particles, and high density of sugar chains arranged on the polymer particle surface, b and c in formula (3) are preferably independently 500 to 3000, more preferably 1000 to 2500. Furthermore, in the polypeptide represented by formula (3), a, b, and c are preferably contained in ratios of 0.05 to 0.9, 0.05 to 0.5, and 0.05 to 0.6 relative to (a + b + c), respectively. This allows polypeptide synthesis to be easily carried out under commonly employed conditions. When synthesizing a polypeptide in which a, b, and c are contained in ratios outside the above ranges, further dehydration condensation reaction is required, making it difficult to optimize the reaction mode, reaction conditions, etc. Furthermore, the content ratio of glycosides and hydrophobic groups contained in the polypeptide is greatly biased, and the effect of the present invention cannot be fully achieved.

[0059] In the polypeptide represented by the formula (3) above, R2 is a glycoside containing at least one sugar structure selected from the group consisting of monosaccharides and disaccharides to heptasaccharides, and from the viewpoint of improving specific adsorption, it is preferable that R2 is a glycoside represented by the following formula (4) or (5):

[0060] [ka]

[0061] Here, Y1 and Y2 each independently represent an organic group having a structure in which 3 to 15 methylene groups are covalently bonded as a spacer group, i.e., a group having a chain length ranging from -(CH2)3- to -(CH2) 15 -, m and n each independently represent an integer of 0 to 2, and Z is a sugar chain having at least one sugar structure selected from the group consisting of monosaccharides and disaccharides to heptasaccharides.

[0062] Examples of sugar chains containing at least one sugar structure selected from the group consisting of monosaccharides and disaccharides to heptasaccharides include those having the sugar chain structures (Z-) shown in Tables 1 to 4 below. The examples of sugar chain structures listed in Tables 1 to 4 below are compounds that have already been synthesized by the present inventors (see, for example, Bioorganic & Medical Chemistry, 15, 1383-1393 (2007), Bioconjugate Chemistry, vol. 20, No. 3, 538-549 (2009), Bioorganic & Medical Chemistry, 18, 621-629 (2007), Journal of Applied Glycoscience, 61, 1-7 (2014), Journal of Biotechnology, 209, 50-57 (2015), Bioscience Biotechnology and Biochemistry 81, 1520-1528 (2017)).

[0063] [Table 1]

[0064] [Table 2]

[0065] [Table 3]

[0066] [Table 4]

[0067] Among the sugar chains listed in Tables 1 to 4, five types (Nos. 5, 6, 27, 28, and 29) are sugar chains that may serve as receptors for the novel coronavirus (SARS-CoV-2). Therefore, the sugar chain-immobilized polymer particles of the present invention containing these sugar chains are expected to be applicable as biosensors for the novel coronavirus. Furthermore, Nos. 5, 6, 15, and 19 are known to exhibit specific adsorption properties for JC virus, dengue virus, and parainfluenza, respectively. Furthermore, Nos. 3, 5, 7, 9, 11, 13, 19, and 27 are known to exhibit specific adsorption properties for avian influenza viruses, Nos. 4, 6, 8, 10, 12, 14, 20, and 27 are known to exhibit specific adsorption properties for human influenza viruses, and Nos. 22 and 24 are known to exhibit specific adsorption properties for equine influenza viruses.

[0068] When the polypeptide represented by formula (3) is immobilized on a polymer particle, the sugar chains are arranged at high positions away from the surface of the polymer particle, thereby increasing the sensitivity to proteins and viruses and improving specific adsorption to them. In this case, the organic groups Y1 and Y2 of the glycoside represented by formula (4) or (5) function as spacer groups to elevate the arrangement position of the sugar chains. This function is most effectively exhibited when the number of methylene groups bound to Y1 and Y2 is 3 to 15, and, combined with the high-density arrangement of sugar chains on the surface of the polymer particle, it is possible to significantly improve specific adsorption to proteins and viruses. This effect cannot be achieved when the number of methylene groups contained in Y1 and Y2 is 2 or less, while synthesis and handling of the hydrophobized glycopolypeptide becomes difficult when the number of methylene groups exceeds 15. To lengthen the spacer group, the number m or n in formula (4) or (5) may be set to 1 or 2. However, if m or n is 3 or greater and the spacer group is too long, the mobility of the sugar chains located at high positions away from the surface of the polymer particle increases, allowing them to move freely, thereby eliminating the technical significance of improving specific adsorption by immobilizing the sugar chains on the polymer particle. Therefore, in the present invention, m or n is preferably 0 or 1. In addition, methylene groups contained in Y1 and Y2 may be used in embodiments of the present invention in which at least one hydrogen atom is substituted with a short-chain alkyl group such as a methyl group or an ethyl group.

[0069] Meanwhile, Patent Document 2 discloses a glycopolymer in which the main chain is linked to a polypeptide main chain via an amide bond via a hydrophobic group bonded to two methylene groups. However, a hydrophobic group with a short chain length (-CHCH-) is insufficient to function as a spacer group, making it difficult to achieve the same effect as the present invention. In contrast, the present invention can solve this technical problem by adopting a method for synthesizing a glycoside in which the length of the spacer group is optimized.

[0070] As an example of a precursor of the hydrophobized glycopolypeptide represented by formula (3), a glycoside in which R2 is represented by formula (4) is shown in Figure 4 (see, for example, Bioconjugate Chem, Vol. 20, No. 3, pp. 538-549). As shown in Figure 4, compound (III) is synthesized by enzymatic condensation of glycoside compound (I) represented by Z-OH with long-chain alcohol (II) having a trifluoroacetamide terminal. Although trifluoroacetamidopentanol is shown as an example of compound (II) in Figure 4, this compound is not limited to this compound in the present invention. The hydrolysis reaction uses a hydrolase such as cellulase from Trichoderma reesei as a catalyst. To synthesize a glycoside with a chain longer than compound (III), compound (IV) is synthesized by deacylation via dehydration condensation of compound (III) with a long-chain carboxylic acid having a trifluoroacetamide terminal in an alkaline solution. Here, a dehydration condensation agent such as a carbodiimide is used for the dehydration condensation reaction. Furthermore, by repeating a similar deacylation reaction, a long-chain compound (V) is synthesized. Compounds (III), (IV), and (V) correspond to m = 0, 1, and 2, respectively, in the above formula (4).

[0071] In an embodiment of the present invention, a sugar chain compound represented by Z-OH contains at least one sugar structure selected from the group consisting of a monosaccharide and a disaccharide to heptasaccharide as the sugar chain (Z-). The sugar chain compound may be synthesized according to the synthesis method shown in Figure 5. Figure 5 shows an example of synthesis when synthesizing a longer, high-molecular-weight oligosaccharide chain from a low-molecular-weight sugar chain. As shown in Figure 5, as an example of compound (IV) shown in Figure 4, a disaccharide glycoside [compound (VI)] in which Z1- has the chemical structure No. 1 or No. 2 shown in Table 1 is used, and a tetrasaccharide glycoside [compound (VII)] is synthesized by an addition reaction of Z1-OH, and a hexasaccharide glycoside [compound (VII)] is synthesized by further addition reaction. Details are disclosed in the aforementioned literature (Bioconjugate Chem, Vol. 20, No. 3, pp. 539-549). However, by using a monosaccharide glycoside as Z1-OH instead of a disaccharide glycoside, any glycoside having an odd number of sugars may be selected and synthesized.

[0072] Furthermore, a method (not shown) for synthesizing a glycoside in which R2 is represented by the above formula (5) as a precursor of a hydrophobized glycopolypeptide is, for example, synthesized by a dehydration condensation reaction between a glycoside compound represented by Z-NH2 and a long-chain carboxylic acid to which a trifluoroacetamide is attached at the end. A dehydration condensation agent such as a carbodiimide is used as a catalyst for the dehydration condensation reaction. The compound synthesized in this manner is subjected to a deacylation reaction by dehydration condensation with a long-chain carboxylic acid to which a trifluoroacetamide is attached at the end in an alkaline solution, according to essentially the same method as the dehydration condensation reaction shown in the middle and subsequent sections of Figure 4. Furthermore, by repeating the same deacylation reaction, a long-chain compound in which n = 1 or 2 is synthesized as a precursor of a hydrophobized glycopolypeptide in which R2 is a glycoside represented by the above formula (5). Here, n in the above formula (5) is an integer of 0 to 2, just like m in the above formula (4). Furthermore, the Z-glycan structure contained in the compound represented by Z-NH2 is essentially the same as that shown in Tables 1 to 4.

[0073] When R3 in the polypeptide represented by formula (3) is a linear or branched alkyl group having 3 to 15 carbon atoms, sufficient hydrophobic interactions can be obtained at different positions inside the polymer particle, allowing for strong non-covalent immobilization of the sugar chain. If the alkyl group has 2 or fewer carbon atoms, hydrophobic interactions with the polymer particle cannot be expected, and if the alkyl group has more than 15 carbon atoms, the polypeptide main chain is immobilized in a state where it is elevated above the surface of the polymer particle, resulting in a significant decrease in immobilization strength. In embodiments of the present invention, both linear and branched alkyl groups having 3 to 15 carbon atoms can be used, but linear alkyl groups are preferred in terms of penetration into the interior of the polymer particle, as they do not require consideration of steric hindrance due to substituents.

[0074] <Polymer particles encapsulating fluorescent dyes> Sugar chain-immobilized polymer particles according to an embodiment of the present invention may encapsulate a fluorescent dye. When the sugar chain-immobilized polymer particles are used as biosensors for proteins, viruses, etc., or as means for separating and purifying proteins, viruses, etc., in order to suppress or prevent a decrease in their functions and actions, it is necessary to immobilize the fluorescent dye to the polymer particles so that it does not migrate out of the sugar chain-immobilized polymer particles and become detached. Therefore, in an embodiment of the present invention, the fluorescent dye is immobilized to the polymer particles non-covalently by hydrophobic or electrostatic interaction between the sugar chain-immobilized polymer particles and the polymer particles.

[0075] The fluorescent dye may have a functional group, such as a double bond, that can covalently bond with the polymerizable monomer during the polymerization reaction to immobilize it to the sugar chain-immobilized polymer particle. However, the formation of a covalent bond with the polymerizable monomer can be one of the factors that restrict the migration of the fluorescent dye. Therefore, to promote migration into the sugar chain-immobilized polymer particle and achieve deeper penetration, it is preferable to use an organic fluorescent dye that has a higher affinity with the polymerizable monomer and a structure that allows it to be non-covalently immobilized with the sugar chain-immobilized polymer particle through hydrophobic or electrostatic interactions, rather than a fluorescent dye that forms a covalent bond by reacting with the polymerizable monomer. Examples of organic fluorescent dyes that can be used in embodiments of the present invention include coumarin-based dyes such as coumarin 6 [3-(2-benzothiazolyl)-7-(diethylamino)coumarin] and coumarin 7 [3-(2-benzimidazolyl)-7-(diethylamino)coumarin], and rhodamine-based dyes such as rhodamine B, rhodamine 6G, and rhodamine 123. Coumarin-based fluorescent dyes have low solubility in water but high solubility in alcohols such as ethanol and polymerizable monomers such as methyl methacrylate. Therefore, when adding coumarin-based fluorescent dyes to a polymerization vessel, they are used in the form of an aqueous solution in which the fluorescent dye is dispersed, a mixed solution of alcohol and water in which the fluorescent dye is dissolved, or an alcohol solution, or dissolved in a polymerizable monomer. In addition, rhodamine-based dyes are used as an aqueous solution or dissolved in a polymerizable monomer with high affinity.

[0076] Since the sugar chain-immobilized polymer particles encapsulating a fluorescent dye can be used as a fluorescent probe or fluorescent label, the fluorescent dye can be used not only to detect the dispersion state and location of the sugar chain-immobilized polymer particles in an aqueous solution or in biological tissues by fluorescence spectrum, but also to understand the behavior of the sugar chain-immobilized polymer particles before and after specific adsorption. Furthermore, this can be useful for elucidating and analyzing the behavior and specific adsorption mechanism of proteins or viruses specifically adsorbed to the sugar chain-immobilized polymer particles.

[0077] <Method for synthesizing hydrophobized glycopolypeptides> A method for synthesizing a hydrophobized glycopolypeptide according to an embodiment of the present invention is described with reference to Figure 6. As shown in Figure 6, compound (IX) is synthesized in an alkaline solution using compound (IV) or (V) shown in Figure 4 as an example of a glycoside. This is then subjected to a dehydration condensation reaction with polyglutamic acid or a derivative thereof [compound (X)], synthesizing compound (XI) in which a substituent represented by formula (4) above has been introduced into the side chain of compound (X) via an amide bond (Step 1). Compound (X) is then subjected to a dehydration condensation reaction with an alkylamine having a linear or branched alkyl group having 3 to 15 carbon atoms, synthesizing a hydrophobized glycopolypeptide [compound (XII)] in which a substituent R3 represented by formula (3) above has been introduced into the side chain of compound (X) via an amide bond (Step 2). Figure 6 illustrates 1) 1-aminodecane as an example of the alkylamine. As shown in Figure 6, the dehydration condensation reaction is generally carried out with the addition of a dehydration condensation agent, such as a phosphonium-based condensation agent, optionally in combination with an active ester or other additive. In an embodiment of the present invention, compounds (IV) and (V) and the alkylamine are not limited to the examples shown in Figure 6, but are selected from the group of compounds included in the scope specified by the present invention. Furthermore, synthesis is carried out according to essentially the same procedures in the case of a hydrophobized glycopolypeptide having a substituent R2 represented by the above formula (5). The bottom of Figure 6 also shows an example (XIII) of a hydrophobized glycopolypeptide having a substituent R2 represented by the above formula (5).

[0078] 6 does not specify the number of repetitions of a, b, and c shown in the above formula (3), but by adjusting the conditions (temperature, time) of the dehydration condensation reaction in steps 1 and 2, a hydrophobic polypeptide can be synthesized in which a is an integer of 1 or greater and b and c are each independently adjusted to 2 or greater, more preferably 500 to 3000. Similarly, the dehydration condensation reaction is adjusted so that the ratios of a, b, and c relative to (a+b+c) are 0.05 to 0.9, 0.05 to 0.5, and 0.05 to 0.6, respectively.

[0079] <Improved sugar chain adsorption performance> In the case of glycan-immobilized polymer particles according to an embodiment of the present invention, proteins or viruses may be nonspecifically adsorbed to surfaces to which hydrophobic glycopolypeptides are not adsorbed. To prevent this situation and improve the adsorption performance of the glycans, the surfaces to which the hydrophobic glycopolypeptides are not adsorbed may be blocked. One method for this is to adsorb a hydrophilic polyalkylene glycol or polyalkylene glycol derivative to the surface of the glycan-immobilized polymer particles. Figure 7 shows a schematic diagram of the effect of adding a hydrophilic polyalkylene glycol or polyalkylene glycol derivative to improve glycan adsorption.

[0080] 7(a) and 7(b) are schematic cross-sectional views showing a state in which a hydrophilic polymer 16 of polyalkylene glycol or a polyalkylene glycol derivative is surface-adsorbed on a glycan-immobilized polymer particle 1 obtained in an embodiment of the present invention and a conventional glycan-immobilized polymer particle 6 described in Non-Patent Document 1. In the glycan-immobilized polymer particle 1 shown in Fig. 7(a), the organic groups Y1 and Y2 function as spacer groups to elevate the arrangement position of the glycan, so that the glycan moiety of the hydrophobic glycopolypeptide 4 is retained at a position away from the surface of the polymer particle. By surface-adsorbing the hydrophilic polymer 16 at locations where the hydrophobic glycopolypeptide 4 is not surface-adsorbed, nonspecific adsorption of proteins or viruses is blocked at those locations.

[0081] In contrast, in the conventional sugar chain-immobilized polymer particle 6 shown in Figure 7(b), the sugar chain portion of the hydrophobized glycopolypeptide 9 produced by conventional technology is retained on the surface of the polymer particle 10, and the sugar chain portion 7 is therefore buried in the hydrophilic polymer 16. In this case, the effect of blocking nonspecific adsorption of proteins or viruses is weakened, and even if a hydrophilic polymer 16 such as polyalkylene glycol or a polyalkylene glycol derivative is adsorbed on the surface, it is not possible to improve the sugar chain adsorption performance.

[0082] Examples of hydrophilic polyalkylene glycols include polyethylene glycol, polypropylene glycol, and polyoxyethylene polyoxypropylene glycol, each having a weight-average molecular weight of less than 30,000, preferably less than 10,000. Furthermore, examples of hydrophilic polyalkylene glycol derivatives include ethers such as polyoxyethylene polyoxypropylene hexyl glycol ether and polyoxyethylene polyoxypropylene butyl ether, each having a weight-average molecular weight of less than 30,000, preferably less than 10,000. In an embodiment of the present invention, polyethylene glycol, polypropylene glycol, or polyoxyethylene polyoxypropylene glycol is preferred in terms of ease of adsorption to the surface of sugar chain-immobilized polymer particles and ease of material availability.

[0083] <Method for producing sugar chain-immobilized polymer particles> The sugar chain-solidifying polymer particles according to an embodiment of the present invention are basically produced by a soap-free emulsion polymerization method, but are particularly characterized by the addition of a polypeptide having multiple sugar chains and multiple hydrophobic groups in its side chains in one molecule during the polymerization reaction, as shown in Figure 3(a). Hereinafter, "polypeptide having multiple sugar chains and multiple hydrophobic groups in its side chains in one molecule" will be abbreviated as "hydrophobic sugar chain polypeptide" as above.

[0084] The general mechanism of polymer formation using soap-free emulsion polymerization is as follows. While the majority of polymerizable monomer molecules exist as monomer oil droplets in an aqueous solvent, a very small amount of monomer molecules dissolves in the aqueous solvent. Polymerization is initiated by a water-soluble radical polymerization initiator added in advance and polymerizable monomer molecules dissolved in the aqueous solvent, resulting in the precipitation of polymer particles that are no longer soluble in the aqueous solvent. Polymerization then proceeds by incorporating monomer or oligomer radicals supplied from the monomer oil droplets. Water is primarily used as the aqueous solvent, but water-miscible solvents such as lower alcohols (e.g., ethanol and propanol) may also be used in amounts up to 30% by weight of the total solvent. During the initial to middle stages of polymerization, the polymer particles being formed contain unreacted polymerizable monomer, resulting in the polymerization proceeding in a swollen form. After the monomer oil droplets disappear, polymerization of the polymerizable monomer contained in the polymer particles proceeds, ultimately resulting in the formation of spherical polymer particles with a desired particle size. The water-soluble radical polymerization initiator may be added not only at the beginning but also during the polymerization reaction, if necessary, in order to promote the polymerization.

[0085] Soap-free emulsion polymerization is commonly used to synthesize particles with clean particle surfaces without using surfactants. However, as mentioned above, the polymerization process of the present invention differs from conventional soap-free emulsion polymerization in that a hydrophobized glycopolypeptide is added during polymerization. Specifically, as shown in Figure 3(a), the soap-free emulsion polymerization method employed in the present invention comprises the steps of: initiating polymerization by adding a water-soluble polymerization initiator to a polymerization vessel containing an aqueous solvent in which polymerizable monomers are dispersed; and adding a hydrophobized glycopolypeptide during the polymerization reaction and continuing the polymerization reaction in the presence of the hydrophobized glycopolypeptide in the polymerization vessel. The amount of hydrophobized glycopolypeptide added is adjusted according to the amount of polymerizable monomer contained in the aqueous solvent, and is preferably adjusted to a concentration sufficient to sufficiently cover the surface of the resulting polymer particles.

[0086] The term "during polymerization" as defined in the present invention means at least the period from when polymerization of the polymerizable monomer starts in the polymerization tank, followed by the formation of polymer particle nuclei and the stable growth of the polymer particles, until the disappearance of the polymerizable monomer.

[0087] In the present invention, the conversion rate of the polymerizable monomer due to the polymerization reaction (hereinafter referred to as "monomer conversion rate") may be used as a physical quantity to specifically specify the period "from when stable growth of polymer particles is observed until the disappearance of the polymerizable monomer" and to define the range of the conversion rate. "From when stable growth of polymer particles is observed until the disappearance of the polymerizable monomer" comprehensively refers to a state in which the monomer conversion rate is greater than 0% by mass and less than or equal to 95% by mass. That is, a monomer conversion rate of 0% by mass corresponds to the case in which the hydrophobic glycopolypeptide is added before the start of polymerization. On the other hand, a monomer conversion rate of more than 95% by mass corresponds to the case in which the hydrophobic glycopolypeptide is added to the surface of the hydrophobic polymer particles after synthesis and physically adsorbed without embedding hydrophobic groups. Ideally, the disappearance of the monomer occurs when the conversion rate is 100% by mass, but it is known that in reality it is approximately 95% by mass. When this value is exceeded, the formation of the hydrophobic polymer particles is almost complete. As mentioned above, in the former case, aggregation of polymer particles was observed, and in the latter case, the immobilization strength of the hydrophobized glycopolypeptide could not be improved. Thus, when the monomer conversion rate was 0% by mass or more than 95% by mass, the object of the present invention could not be achieved. Therefore, in the soap-free emulsion polymerization process, the timing for adding the hydrophobized glycopolypeptide to achieve the effects of the present invention is when the monomer conversion rate is at least in the range of more than 0% by mass and not more than 95% by mass.

[0088] According to experimental verification by the present inventors, for example, when styrene is used as the polymerizable monomer, polymer particle nuclei begin to form when the monomer conversion rate is about a few mass % compared to the time at the start of polymerization, and furthermore, disappearance of the monomer oil droplets was confirmed when the conversion rate was about 40 mass %. When the monomer oil droplets disappear, the viscosity of the polymer particles increases, which is thought to make it difficult for the hydrophobic groups of the hydrophobized glycopolypeptide (hydrophobic groups contained in the substituents of the above-mentioned R3) to be embedded, so it is desirable to start before the disappearance of the monomer oil droplets.

[0089] The monomer conversion rate can be determined by stopping the polymerization at a predetermined time between the start and end of the polymerization and determining the content of the monomer in the aqueous solvent in the polymerization tank by weight or spectroscopic means such as infrared absorption. The presence of polymer particle nuclei and aggregation of polymer particles can be confirmed by observing and measuring the state of formation of polymer particles contained in the aqueous solvent, and, if necessary, the polymer particle size, etc., using an optical microscope, a transmission electron microscope, or a scanning electron microscope. Furthermore, since the polymerizable monomer conversion rate measured at each polymerization reaction time is correlated with the average particle size of polymer particles grown from the start of polymerization to each polymerization reaction time, the polymerizable monomer conversion rate at each polymerization reaction time can be estimated by determining the average particle size of polymer particles at each elapsed time from the start of polymerization and comparing it with the average particle size of polymer particles obtained after the end of polymerization.

[0090] Based on these experimental verifications, in terms of preventing polymer particle aggregation and the immobilization strength of the hydrophobized glycopolypeptide, in the present invention, the hydrophobized glycopolypeptide is added at a polymerization reaction time when the conversion rate of the polymerizable monomer contained in the polymerization vessel is greater than 0% by mass but not greater than 95% by mass relative to the time at the start of polymerization. A practical polymerization reaction time for adding the hydrophobized glycopolypeptide within the range of greater than 0% by mass but not greater than 95% by mass is preferably when the conversion rate of the polymerizable monomer contained in the polymerization vessel is 3 to 50% by mass relative to the time at the start of polymerization. Furthermore, the hydrophobized glycopolypeptide is preferably added at a polymerization reaction time when the conversion rate of the polymerizable monomer is 10 to 40% by mass. If the conversion rate of the polymerizable monomer is less than 3% by mass, polymer particles that are stably dispersed in the aqueous solvent are not formed, and aggregation occurs in the glycopolypeptide-immobilized polymer particles obtained after the polymerization reaction. If the conversion rate of the polymerizable monomer is 10% by mass or greater, stable growth of the polymer particles is reliably observed following the nucleation of the polymer particles. Here, stable growth of polymer particles means that the polymer particles contained in the aqueous solvent grow with a nearly uniform particle size, as described above. Furthermore, if the conversion rate of the polymerizable monomer exceeds 50%, the hydrophobic glycopolypeptide present on the surface of the polymer particles may promote particle aggregation. If the conversion rate of the polymerizable monomer is 40% by mass or less, not only can the hydrophobic groups of the hydrophobic glycopolypeptide be reliably embedded within the polymer particles, but also particle aggregation can be suppressed after polymer particle formation.

[0091] The "polymerization reaction time at which the conversion rate of the polymerizable monomer contained in the polymerization vessel reaches 3 to 50% by mass relative to the time at the start of polymerization" varies slightly depending on the polymerizable monomer used and the polymerization conditions, but it has been found that a polymerization reaction time of more than 15 minutes and less than 2 hours from the start of polymerization corresponds to a polymerization reaction time at which the conversion rate of the polymerizable monomer reaches 3 to 50% by mass. Furthermore, in order to ensure stable particle growth following nucleation of polymer particles, it is preferable to add the hydrophobic glycopolypeptide more than 30 minutes from the start of polymerization. Thus, in the production method of the present invention, it is preferable to add the hydrophobic glycopolypeptide when the polymerization time is more than 30 minutes and less than 2 hours.

[0092] The polymerizable monomer and hydrophobized glycopolypeptide used in the production method shown in Figure 3(a) are the same hydrophobic monomer and "polypeptide having multiple glycochains and multiple hydrophobic groups in the side chains in one molecule" as described above.

[0093] From the viewpoint of improving glycan adsorption performance, the method for producing a glycan-immobilized polymer according to an embodiment of the present invention may include a step of adsorbing a hydrophilic polymer 16, such as polyalkylene glycol or a polyalkylene glycol derivative, to the surface of the glycan-immobilized polymer particle 1 during or after the polymerization reaction of the glycan-immobilized polymer particle 1 to block the surface to which the hydrophobic glycopolypeptide 4 has not been adsorbed. Examples of the polyalkylene glycol or polyalkylene glycol derivative that can be used include the same compounds as those listed above in the section "Improvement of glycan adsorption performance." In the present invention, the hydrophilic polymer 16 may be added either during or after the polymerization reaction, and the addition time is selected taking into consideration the ease of control and management of the synthesis reaction of the glycan-immobilized polymer particle 1, the glycan adsorption performance, etc.

[0094] The adsorption step of the hydrophilic polymer 16 is carried out in one of the following two ways during the polymerization reaction of the sugar chain-immobilized polymer particles 1 or after the polymerization reaction is completed. One is a step of adding the hydrophilic polymer 16 to the polymerization tank 11 containing the sugar chain-immobilized polymer particles 1 in Figure 3(a). The other is a step of transferring the sugar chain-immobilized polymer particles to a reaction tank prepared separately from the polymerization tank 11 containing an aqueous medium, and then adding the hydrophilic polymer 16 to the reaction tank containing the sugar chain-immobilized polymer particles 1. In the present invention, by carrying out the former step, it is possible to simplify and save labor in the hydrophilic polymer addition step compared to the latter step.

[0095] <Method for producing sugar chain-immobilized polymers containing fluorescent dyes> A method for producing a sugar chain-immobilized polymer containing a fluorescent dye will be described below. In the synthesis of a glycan-immobilized polymer according to an embodiment of the present invention, polymer particle nuclei are first formed as the polymerization reaction progresses. Subsequently, as the polymer particles grow, swollen polymer particles are formed by unreacted polymerizable monomers. Finally, when the polymerization is nearly complete, spherical polymer particles with the desired particle size are obtained. The fluorescent dye added to the polymerization vessel migrates and penetrates into the swollen polymer particles and is incorporated into them. After the polymerization of the polymerizable monomer is nearly complete, the fluorescent dye is non-covalently immobilized to the glycan-immobilized polymer particles through hydrophobic or electrostatic interactions with the glycan-immobilized polymer particles. To achieve this, the fluorescent dye must be added at least while unreacted polymerizable monomers remain in the polymerization vessel during the polymerization reaction during the synthesis of the glycan-immobilized polymer particles. Therefore, the fluorescent dye is added to the polymerization vessel before the start of polymerization or between the start of polymerization of the polymerizable monomers and the middle of the polymerization reaction. When adding the dye before the start of polymerization, the fluorescent dye may be dissolved in the polymerizable monomer and added to the polymerization vessel together with the polymerizable monomer. Thus, in an embodiment of the present invention, the fluorescent dye is added to the polymerization tank in the presence or absence of the polymerizable monomer before the start of polymerization, or the fluorescent dye is added to the polymerization tank in the presence or absence of the hydrophobic glycopolypeptide at any polymerization reaction time during the polymerization reaction after the start of polymerization.

[0096] Here, "in the presence or absence of the polymerizable monomer" means that the fluorescent dye may be added to the polymerization layer together with the polymerizable monomer, or either before or after the addition of the polymerizable monomer. Similarly, "in the presence or absence of the hydrophobic glycopolypeptide" means that the fluorescent dye may be added to the polymerization vessel either before or after the addition of the hydrophobic glycopolypeptide. In other words, the fluorescent dye can be added regardless of the presence or absence of the polymerizable monomer or the hydrophobic glycopolypeptide in the polymerization vessel.

[0097] In the polymerization reaction of a polymerizable monomer, when the disappearance of the polymerizable monomer is almost complete, the conversion rate of the polymerizable monomer due to the polymerization reaction is approximately 95% by mass, as described above. Therefore, the "midway" of the polymerization reaction as defined in the present invention refers to any polymerization reaction time when the conversion rate of the polymerizable monomer is 95% by mass or less. Furthermore, the migration and penetration of the fluorescent dye is significantly promoted when only the polymerizable monomer is present in the polymerization vessel or when the sugar chain-immobilized polymer particles are swollen with the polymerizable monomer. This state is achieved when polymerizable monomer oil droplets are dispersed in the aqueous solution in the polymerization vessel. As described above in the section "Method for Synthesizing Hydrophobized Glycopolypeptides," polymerizable monomer oil droplets are observed when the conversion rate of the polymerizable monomer due to the polymerization reaction is 50% by mass or less compared to the start of polymerization. Furthermore, the presence of polymerizable monomer oil droplets is reliably confirmed when the conversion rate is 40% by mass or less. Therefore, it is practical to add the fluorescent dye by the time when the conversion rate of the polymerizable monomer due to the polymerization reaction is 95% by mass or less, and it is desirable to add the fluorescent dye by the time when the conversion rate is 50% by mass or less, and even 40% by mass or less.

[0098] As described above, the fluorescent dye can be immobilized on the sugar chain-immobilized polymer particles by adjusting the timing of adding the fluorescent dye to the polymerization reaction vessel. However, in the production of sugar chain-immobilized polymer particles, there is no need to change the method of adding other components other than the fluorescent dye or the polymerization conditions, and the production method is basically the same as that explained in the above section <Production method of sugar chain-immobilized polymer particles>.

[0099] Whether or not a fluorescent dye has been reliably immobilized on a glycan-immobilized polymer particle can be evaluated as follows, for example, when a hydrophobic fluorescent dye such as coumarin is used. Specifically, the glycan-immobilized polymer particles obtained after the polymerization reaction are removed from the polymerization vessel and immersed in ethanol for a predetermined time (e.g., 10 minutes). Only the fluorescent dye attached or adsorbed to the surface of the glycan-immobilized polymer particle but not immobilized inside the glycan-immobilized polymer particle is dissolved in the ethanol. Ethanol is a solvent that dissolves the fluorescent dye but not the glycan-immobilized polymer particle. The immersion solvent used here is not limited to ethanol, and any solvent that does not dissolve the glycan-immobilized polymer particle but has equal or greater solubility for the fluorescent dye than ethanol can be used. The glycan-immobilized polymer particle is then removed from the ethanol, and the fluorescence spectrum of the ethanol after removal of the polymer particle is measured. For example, when the fluorescent dye is coumarin 7, a peak in the fluorescence spectrum intensity can be observed in the wavelength range of 500 to 510 nm. As a result of measurement by fluorescence spectroscopy, if a large amount of fluorescent dye is extracted into ethanol without being fixed in the sugar chain-immobilized polymer particles, a large peak intensity is observed in the fluorescence spectrum at a wavelength specific to the fluorescent dye.

[0100] The above procedure was performed on glycan-immobilized polymer particles produced at different times when the fluorescent dye was added. The fluorescence spectra were measured for each ethanol solution after the glycan-immobilized polymer particles were immersed and removed. Preliminary studies showed that when the fluorescent dye was added before and immediately after the start of polymerization (Case A), the fluorescence spectrum of the ethanol obtained after the removal of the glycan-immobilized polymer particles obtained upon completion of polymerization showed very small peak intensities at wavelengths specific to the fluorescent dye. Furthermore, when the fluorescent dye was added shortly after the start of polymerization (Case B), the fluorescence spectrum showed similarly small peak intensities as in Case A. In contrast, when the fluorescent dye was added near the end of the polymerization reaction (Case C), a fluorescence spectrum with greater absorption intensity than Cases A and B was observed. It was confirmed that in Case C, a large amount of the fluorescent dye was present on the surface of the glycan-immobilized polymer particles, rather than being immobilized inside. Here, the conversion rate of the polymerizable monomer during the polymerization reaction was investigated for each of Cases A, B, and C. As a result, the conversion rates were approximately 0% by mass and approximately 25% by mass in the cases of A and B, respectively, while in the case of C the conversion rate was slightly above 95% by mass.

[0101] From the results of the above preliminary investigations, it was found that the timing of adding the fluorescent dye is affected by the polymerization reaction time when producing sugar chain-immobilized polymer particles, i.e., the conversion rate of the polymerizable monomer due to the polymerization reaction. Therefore, as described above, it is practical to add the fluorescent dye by the polymerization reaction time when the conversion rate of the polymerizable monomer due to the polymerization reaction is 95% by mass or less, and it is desirable to add the fluorescent dye by the polymerization reaction time when the conversion rate is 50% by mass or less, and even more preferably 40% by mass or less. This can improve the immobilization of the fluorescent dye to the sugar chain-immobilized polymer. [Example]

[0102] The present invention will be explained below using specific examples, but the present invention is not limited to these examples in any way.

[0103] -Synthesis of hydrophobized glycopolypeptides- Example 1 A hydrophobic glycopolypeptide having the structure shown in No. 24 of Table 1 as the glycan and a decanyl group (an alkyl group having 10 carbon atoms) as the hydrophobic group was synthesized as follows according to the method shown in Figure 6.

[0104] First, as compound (XI) shown in Figure 6, a compound corresponding to a hydrophobic glycopeptide in which a spacer group having m = 0 and a methylene group with five carbon atoms in the formula (4) above is amide-linked to the side chain of a poly-γ-glutamic acid residue was synthesized (see the synthesis method described in Bioconjugate Chemistry, Vol. 20, No. 3, 538-549 (2009)). In this synthesis, compound (X) shown in Figure 6 was synthesized using sodium γ-polyglutamate (manufactured by Meiji Food Materials Co., Ltd.) with a molecular weight of 990,000, and was subjected to dehydration condensation with compound (IX), which has the sugar chain represented by No. 24 in Table 1 above as the Z-structural formula and a methylene group with five carbon atoms in the spacer group. As a dehydration condensation agent, 1H-benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (hereinafter abbreviated as BOP) was used in combination with 1-hydroxybenzotriazole (hereinafter abbreviated as HOBt) as an additive.

[0105] Subsequently, according to the synthesis method shown in the following formula (6), 9.2 mg of compound (XI-1) was dissolved in 1.66 mL of 0.1 M carbonate / bicarbonate buffer (pH = 10), and then 3.3 mL of dimethyl sulfoxide (DMSO) containing 142 mg (0.32 mmol) of BOP as a dehydration condensation agent and 11.9 mg (0.09 mmol) of HOBt as an additive was added and stirred for 15 minutes at 25 ° C. Furthermore, 11.7 μL (0.06 mmol) of 1-aminodecane was added, and the mixture was stirred for another 24 hours under the same conditions to prepare a mixture containing compound (XII-1) in which a hydrophobic group (decanyl group) was introduced into the side chain of the poly-γ-glutamic acid residue via an amide bond.

[0106] [ka]

[0107] The mixture thus prepared was loaded onto a disposable Pd-10 desalting column pre-equilibrated with saline buffered to pH 7.4 with 10 mM phosphate and dialyzed for 3 days, then concentrated and lyophilized to extract the target hydrophobic glycopolypeptide [compound (XII-1)].

[0108] The compounds (XI-1) and (XII-1) shown in the above formula (6) are 1 H or 13 Using C NMR (2-channel 500 MHz Bruker AVIII), the compound was identified by peak analysis of the NMR spectrum shown below, and it was confirmed that the target compound had been synthesized. · 1 H-NMR (D2O,500.13 MHz at 318 K): δ4.55-4.13 (1H, α-methine, γ-PGA (polyglutamic acid)), 4.19 (s, 2H, HOCH2CONH-″),4.08-3.57 (21H, from sugar, H-α), 3.35-3.05 (H-ε, Hj), 2.85 (dd,1H, J3″ax,3″eq 12.5, J3″eq,4″ 4.5 Hz, H-3″eq), 2.45 (2H, γ-methylene, γ-PGA), 2.38-1.98 (2H, β-methylene, γ-PGA), 2.09 (s, 3H,CH3CONH-), 1.88 (t, 1H, J3″ax,3″eq 12.5, J3″ax,4″ 12.5 Hz, H-3″ax),1.55-1.00 (22H, H-β, H-γ, H-δ, Hb, Hc, Hd, He, Hf, Hg, Hh, Hi),0.90 (3H, Ha). · 13C-NMR (D2O,125.13 MHz at 318 K): δ 177-176(carbonyl carbon of amide groups), 175.7 (HOOC″-), 105.5 (C-1′),103.8 (C-1), 102.7 (C-2″), 81.6 (C-4), 78.3 (C-5′), 77.9 (C-3′), 77.5 (C-5), 75.4 (C-6″), 75.2 (C-3), 74.5 (C-8″), 72.9 (C-α), 72.2 (C-2′),71.0 and 70.8 (C-4″, C-7″), 70.2 (C-4′), 65.4 (C-9″), 63.7 (C-6′,HOCH2CONH''-), 63.0 (C-6), 57.9 (C-2), 56.5 and 53.6 (α-methine,γ-PGA), 54.3 (C-5″), 42.5 (C-3″), 42.1 (C-ε, Cj), 34.5 (Cc), 34.1 (γ-methylene, γ-PGA), 32.3 and 32.0 (Cd, Ce, Cf, Cg, Ci), 31.0 (C-β,C-δ), 30.0 (β-methylene, γ-PGA), 29.7 (Ch), 25.2 (C-γ, Cb,CH3CONH-), 16.4 (Ca).

[0109] Furthermore, the ratio of the number of each repeating unit shown in formula (3) to the total number of repeating units (e.g., a / (a+b+c)) can be determined from the characteristic substituent peaks contained in each unit in the NMR spectrum, and the repeating numbers a, b, and c of each unit can be calculated from these values ​​and the molecular weight of sodium polyglutamate. As a result, for compound (XII-1) obtained in this example, p = a + b + c = 6557, a = 2295, b = 1377, and c = 2885.

[0110] <Example 2> As shown in the following formula (7), a hydrophobic glycopolypeptide (compound XIII-1) was synthesized, in which n = 0 in formula (5) above, a spacer group having a methylene group with 5 carbon atoms is amide-linked to the side chain of a poly-γ-glutamic acid residue, a sialo-glycan having the structure shown in No. 25 in Table 3 above as the sugar chain, and a decanyl group (an alkyl group having 10 carbon atoms) as the hydrophobic group. Compound (XIII-1) differs structurally from compound (XII-1) in Example 1 above in that the sialo-glycan is amide-linked to the pentanyl spacer group. Synthesis was essentially carried out according to the method of Example 1 above, except that instead of the intraenzymatic condensation reaction between Z-OH and a long-chain alcohol bound to a trifluoroacetamide terminal (as in Example 1 above), a dehydration condensation reaction between Z-NH2 and a long-chain carboxylic acid bound to a trifluoroacetamide terminal was carried out. The compound (XIII-1) synthesized in this example had p=a+b+c=6662, a=2951, b=1252, and c=2458.

[0111] [ka]

[0112] -Synthesis of sugar chain-immobilized polymer particles- Example 3 Sugar chain-immobilized polymethyl methacrylate (PMMA) particles were synthesized by immobilizing compound (XIII-1), obtained as the hydrophobized glycopolypeptide in Example 2, onto polymer particles made of methyl methacrylate (MMA) using the method described below. An example of the synthesis method for sugar chain-immobilized PMMA particles is shown in Figure 8.

[0113] Deionized water 18 was supplied into a polymerization tank 17 with a lid, and nitrogen gas was bubbled through the water for 20 minutes while stirring. The temperature of the deionized water 18 in the polymerization tank 17 was then heated to 70°C. After confirming that the heating temperature had stabilized, MMA monomer (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the polymerization tank, and stirring was continued for 20 minutes. Deionized water containing potassium persulfate (KPS, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved therein as a radical polymerization initiator was then added to initiate polymerization. In an embodiment of the present invention, the start of the polymerization time is defined as the time when the radical polymerization initiator was added to the polymerizable monomer.

[0114] One hour after the start of polymerization (time point (A) indicated by the tip of the solid arrow in Figure 8), compound (XIII-1), obtained as a hydrophobic glycopolypeptide, was added. The amount of compound (XIII-1) added was adjusted to be approximately 1 / 1000 of the weight of the MMA monomer. Here, one hour after the start of polymerization, the conversion rate of the monomer contained in the deionized water was approximately 25% by mass. This conversion rate was determined by comparing the average particle size of the sugar chain-immobilized polymer particles produced one hour after the start of polymerization with the average particle size of the polymer particles obtained after the completion of polymerization.

[0115] The polymerization was continued for 2 hours, for a total polymerization time of 3 hours. The suspension containing the sugar chain-immobilized PMMA particles was then centrifuged three times with deionized water, followed by vacuum freeze-drying for 24 hours to obtain sugar chain-immobilized PMMA particles 19.

[0116] The sugar chain-immobilized PMMA particles 19 obtained in this manner were observed using a scanning electron microscope (SEM) and an SEM image is shown in Figure 9. Furthermore, the particle sizes of approximately 200 particles randomly selected from the SEM image shown in Figure 9 were measured, and the particle size and dispersity were calculated as the average values. As a result, it was confirmed that the sugar chain-immobilized PMMA particles of this example had an average particle size and dispersity of 176 nm and 8%, respectively, and were free of aggregation and had a nearly uniform particle size.

[0117] Example 4 Sugar chain-immobilized polymer particles were synthesized using the same method as in Example 3 above, using compound (XIII-2) represented by the following formula (8) as the hydrophobic glycopolypeptide. Compound (XIII-2) used in this example is a hydrophobic glycopolypeptide in which n = 0 in the above formula (5), a spacer group having a methylene group with 5 carbon atoms is amide-linked to the side chain of a poly-γ-glutamic acid residue, and the sugar chain is a chitobiose sugar chain having the structure shown in No. 17 in Table 2 above, and a decanyl group (an alkyl group with 10 carbon atoms) as the hydrophobic group. Compound (XIII-2) synthesized in this example had p = a + b + c = 6488, a = 3199, b = 1453, and c = 1836.

[0118] [ka]

[0119] The sugar chain-immobilized polymer particles obtained in this Example 4 had an average particle size and a dispersity of 188 nm and 10%, respectively, and it was confirmed that the particles were not aggregated and had a nearly uniform particle size.

[0120] <Comparative Example 1> Sugar chain-immobilized polymer particles were produced using the same hydrophobized glycopeptide, polymerizable monomer, and polymerization initiator as in Example 4. In Comparative Example 1, polymerization of MMA monomer was carried out using the same method and conditions as in Example 2, except that compound (XIII-2) was added as the hydrophobized glycopeptide at time point (B), indicated by the dotted arrow in Figure 8. Here, time point (B) in Figure 8 corresponds to "before the initiation of polymerization," before the radical polymerization initiator (KPS) was added.

[0121] <Reference example 1> Sugar chain-immobilized polymer particles were produced using the same hydrophobized glycopeptide, polymerizable monomer, and polymerization initiator as in Example 4 above. In this Reference Example 1, polymerization of MMA monomer was carried out using the same method and conditions as in Example 4 above, except that the addition of the above-mentioned compound (XIII-2) as the hydrophobized glycopeptide was carried out at time point (C) indicated by the dashed-dotted arrow in Figure 8. Here, time point (C) in Figure 8 corresponds to 15 minutes after the start of polymerization. Judging from the results of previous experiments involving soap-free emulsion polymerization, the monomer conversion rate at time point (C) in Figure 8 is estimated to be less than 3% by mass.

[0122] <Reference example 2> Sugar chain-immobilized polymer particles were produced using the same hydrophobized glycopeptide, polymerizable monomer, and polymerization initiator as in Example 4 above. In this Reference Example 2, polymerization of MMA monomer was carried out using the same method and conditions as in Example 4 above, except that compound (XIII-2) was added as the hydrophobized glycopeptide at time point (D) indicated by the two-dot chain arrow in Figure 8. Here, time point (D) shown in Figure 8 corresponds to 2 hours after the start of polymerization. D The monomer conversion at this point was determined by comparing the average particle size of the polymer particles in the same manner as in Example 3, and was found to be slightly more than 50% by mass.

[0123] FIG. 10 shows scanning electron microscope (SEM) images of the sugar chain-immobilized PMMA particles obtained in Comparative Example 1 and Reference Examples 1 and 2. In FIG. 10, (a), (b), and (c) correspond to Comparative Example 1, Reference Example 1, and Reference Example 2, respectively. As shown in FIG. 10(a), when added before the start of polymerization, many of the sugar chain-immobilized polymer particles aggregated, resulting in the morphology shown in FIG. 3(b). In contrast, when added 15 minutes after the start of polymerization in Reference Example 1, particle aggregation decreased, and the number of non-aggregated, single sugar chain-immobilized polymer particles increased. However, FIG. 10(b) shows that particle aggregation was not completely eliminated. Furthermore, as shown in FIG. 10(c), when added 2 hours after the start of polymerization in Reference Example 2, a large particle size mass formed due to aggregation of many polymer particles. In this case, the conversion rate of the polymerizable monomer contained in the polymerization tank 20 exceeds 50% by mass compared to the time of the start of polymerization, and instead of the polymerization of the monomer proceeding, it is thought that the addition of the hydrophobized glycopolypeptide caused association of polymer particles with slightly larger particle sizes.

[0124] As described above, in the embodiment of the present invention, when the polymer particles are PMMA particles, the addition of the hydrophobized glycopolypeptide is preferably carried out at a polymerization time of more than 15 minutes but less than 2 hours after the start of polymerization. Furthermore, when the polymer particles are polystyrene particles, although the timing of the addition of the hydrophobized glycopolypeptide varies slightly from that of PMMA particles, it has been confirmed that a polymerization time of more than 15 minutes but less than 2 hours after the start of polymerization results in the production of glycan-immobilized polymer particles with a nearly uniform particle size without any particle aggregation. A polymerization reaction time of more than 15 minutes but less than 2 hours can be considered to be the period from the time when stable growth of polymer particles is observed following nucleation of the polymer particles during the monomer polymerization process until the time when the oil droplets of the polymerizable monomer completely disappear. This range corresponds to the range in which the conversion rate of the polymerizable monomer contained in the polymerization vessel is 3 to 50% by mass relative to the time at the start of polymerization.

[0125] -Method for verifying specific adsorption performance for proteins- The specific protein adsorption properties of the sugar chain-immobilized polymer particles obtained in Example 4 above were examined by the following method.

[0126] Wheat germ agglutinin (WGA) with a molecular weight of 43,200 (J-Chemical Co., Ltd.) was used as the protein to which the chitobiose sugar chain of compound (XIII-2) exhibits specific adsorption. 0.0188 g of polymer particles with immobilized hydrophobic glycopolypeptides having the structure of compound (XIII-2) was suspended in 0.25 mL of deionized water. The suspension was mixed with 0.25 mg of WGA dissolved in 0.25 mL of phosphate-buffered saline (PBS) in a microtube and allowed to stand at 0°C for 30 minutes. The PBS used was prepared by dissolving NaCl, KCl, and potassium dihydrogen phosphate in deionized water. The sugar chain-immobilized polymer particles with WGA adsorbed on their surfaces were separated from the suspension by centrifugation for 30 minutes (conditions: 4°C, 14,000 rpm). The absorbance (A280) of the supernatant containing WGA that was not adsorbed to the surface of the sugar chain-immobilized polymer particles was then measured using an ultramicrospectrophotometer (Nano-100, manufactured by Bio Medical Science Co., Ltd.) The concentration of WGA adsorbed to the sugar chain-immobilized polymer particles was calculated using a calibration curve previously prepared for the relationship between absorbance and WGA concentration.

[0127] For comparison, polymer particles were synthesized by soap-free emulsion polymerization of MMA without the addition of the hydrophobized glycopolypeptide having the structure of compound (XIII-2), i.e., PMMA particles to which the hydrophobized glycopolypeptide had not been immobilized. The resulting polymer particles without added glycopolypeptides were also measured using the same method as above, using absorbance values ​​measured with an ultramicrospectrophotometer, and the amount of WGA adsorbed on the surface was calculated. The amount of WGA adsorbed thus calculated is shown in Figure 11(a).

[0128] As shown in Figure 11(a), the WGA adsorption amounts of the sugar chain-immobilized polymer particles and sugar chain-free polymer particles of this example were 73 μg and 25 μg per 0.0188 g of produced particles, respectively. Therefore, the adsorption amount of the protein (WGA) that specifically adsorbs to the sugar chain (chitobiose) contained in the sugar chain-immobilized polymer particles of this example can be seen from the difference between the two to be 48 μg. In this way, it was confirmed that the polymer particles on which hydrophobic glycopolypeptides were immobilized have the function of specific protein adsorption.

[0129] <Comparative Example 2> To compare the specific protein adsorption performance with that of Example 4, we synthesized sugar chain-immobilized polymer particles identical to those described in Non-Patent Document 1. These sugar chain-immobilized polymer particles consisted of a hydrophobic sugar chain, each containing glucose (Glc) with the structure shown in No. 26 in Table 3 as the sugar chain and an octyl group as the hydrophobic group, immobilized to a PMMA polymer particle via one octyl group. The glucose-immobilized PMMA particles were synthesized essentially according to the method shown in Figure 8, except for the addition of the hydrophobic sugar chain compound XIV-1 (octyl-β-D-glucopyranoside) represented by the following formula (9). Compound XIV-1 was added at the point (B) indicated by the dotted arrow in Figure 8, i.e., before the initiation of polymerization and before the addition of the radical polymerization initiator (KPS).

[0130] [ka]

[0131] The sugar chain-immobilized polymer particles obtained in Comparative Example 2 had an average particle size and a dispersity of 258 nm and 5%, respectively, and it was confirmed that the particles were not aggregated and had a nearly uniform particle size.

[0132] The specific adsorption performance for proteins was verified using essentially the same methods and analytical procedures as in Example 4, except that the glucose-immobilized PMMA particles and concanavalin A (ConA, J-Chemical Co., Ltd.) with a molecular weight of 104,000 were used as the sugar chain-immobilized polymer particles and protein, respectively. The amount of ConA adsorption determined in this manner is shown in Figure 11(b).

[0133] As shown in Figure 11(b), the sugar chain-immobilized polymer particles of Comparative Example 2 and the sugar chain-free polymer particles had ConA adsorption amounts of 38 μg and 21 μg per 0.0188 g of produced particles, respectively. Therefore, the adsorption amount of the protein (ConA) that specifically adsorbs to the sugar chain (glucose) contained in the sugar chain-immobilized polymer particles of Comparative Example 2 was found to be 17 μg, based on the difference between the two. In this way, it was confirmed that the sugar chain-immobilized polymer particles of Comparative Example 2 also have a certain degree of protein specific adsorption function, but the adsorption amount is presumed to be less than that of Example 4 above.

[0134] In Comparative Example 2, the types of sugars contained in the produced sugar chain-immobilized polymer particles and the types of adsorbed proteins were different from those in Example 4, so the amounts of sugars and proteins adsorbed to both sugar chain-immobilized polymers were converted into moles instead of weights to compare the specific adsorption performance of proteins. The results are shown in Table 5 below.

[0135] [Table 5]

[0136] From Table 5 above, it can be seen that the specific adsorption amount of protein and the specific adsorption amount of protein per substance amount (charged amount) of sugar in Example 4 are approximately 7 times and 100 times, respectively, compared to Comparative Example 2. From the above results, it was inferred that the sugar chain-immobilized polymer particles of Example 4 of the present invention have significantly superior specific adsorption properties for proteins compared to the conventional ones in Comparative Example 2.

[0137] <Example 5> In the case of the glycan-immobilized polymer particles according to an embodiment of the present invention, proteins and viruses may be nonspecifically adsorbed to the surface to which the hydrophobized glycopolypeptide is not adsorbed. To prevent or suppress this state and improve the glycan adsorption performance, in this example, we investigated blocking the surface to which the hydrophobized glycopolypeptide is not adsorbed with hydrophilic polyethylene glycol (PEG).

[0138] Hydrophilic polyethylene glycol (PEG) with a weight-average molecular weight of 4000 was used. Sugar chain-immobilized PMMA particles were synthesized in the same manner as in Example 4, except that 0.1 g of PEG was added to the polymerization vessel simultaneously with the addition of the hydrophobic glycopolypeptide (XIII-2). This procedure allowed PEG to be adsorbed onto the surface of the sugar chain-immobilized PMMA particles.

[0139] The sugar chain-immobilized PMMA particles thus obtained were examined for their specific adsorption performance toward proteins (WGA). Furthermore, to confirm that the hydrophobic glycopolypeptide was firmly immobilized on the PMMA polymer particles, the sugar chain-immobilized polymer particles of Example 5 were stored in water for one month, and the change in the amount of WGA adsorbed after storage was examined. Furthermore, to compare the specific adsorption performance of WGA and the immobilization of the hydrophobic glycopolypeptide with those of Example 5, PMMA particles without added sugar chains and PMMA particles with added sugar chains after polymerization were produced by the methods described below as Comparative Examples 3 and 4. Similar to Example 5, Comparative Examples 3 and 4 also used PMMA particles with PEG surface adsorbed as the verification sample.

[0140] <Comparative Example 3> Except for the fact that no hydrophobic glycopolypeptide was added during the polymerization of MMA, 0.1 g of polyethylene glycol (PEG) was added 1 hour after the start of polymerization in the same manner as in Example 5 above, to produce sugar chain-free PMMA particles with PEG adsorbed on their surfaces.

[0141] <Comparative Example 4> The hydrophobized glycopolypeptide (XIII-2) was surface-adsorbed onto PMMA particles in the same manner as in Example 4, except that the hydrophobized glycopolypeptide (XIII-2) was added after the completion of the PMMA polymerization process and 0.1 g of PEG was added simultaneously. This procedure produced PMMA particles containing glycochains with polyethylene glycol (PEG) adsorbed onto their surfaces. As mentioned above, the completion of the PMMA polymerization process corresponds to a time when the conversion rate of MMA in the polymerization reaction exceeds 95% by mass. Therefore, in Comparative Example 4, in which the hydrophobized glycopolypeptide was added after the completion of the PMMA polymerization process, the hydrophobic groups contained in the hydrophobized glycopolypeptide were surface-adsorbed without being embedded inside the polymer particles.

[0142] For the above-mentioned Example 5 (polymer particles with immobilized hydrophobic glycopolypeptides), Comparative Example 3 (polymer particles with no added sugar chains), and Comparative Example 4 (polymer particles with surface-adsorbed hydrophobic glycopolypeptides after the polymerization process), protein (WGA) was adsorbed using the same method as in Example 4, and the specific adsorption performance for WGA was verified immediately after the production of each particle using essentially the same method as in Example 4. The amount of WGA adsorption determined in this manner is shown in Figure 12(a). Furthermore, the amount of WGA adsorption determined using the same method immediately after the production of each particle after storing each particle in water for one month is shown in Figure 12(b).

[0143] As shown in Figure 12(a), the WGA adsorption amounts immediately after production for each of the polymer particles of Example 5 (sugar chain-immobilized polymer particles) and Comparative Example 3 (when no hydrophobic glycopolypeptide was added) were 129 μg and 23 μg per 0.0188 g of produced particles, respectively. Therefore, the adsorption amount of the protein (WGA) that specifically adsorbs to the sugar chain (chitobiose) contained in the sugar chain-immobilized polymer particles of this Example 5 can be seen from the difference between the two to be 106 μg. When the specific adsorption amount of WGA is converted to moles, since the molecular weight of WGA is 43,200 g / mol, the specific adsorption amount of WGA is 2.45 x 10 -9Furthermore, since the amount of sugar chain-immobilized polymer particles added for the verification was 0.0188 g, the specific adsorption amount of WGA per 1 g of the produced polymer particles (sugar chain-immobilized polymer particles) was 1.3 × 10 -7 On the other hand, as explained in Example 4 above, the sugar chain-immobilized polymer particles to which PEG was not added had a 5.9 × 10 mole per 1 g of produced particles (sugar chain-immobilized polymer particles). -8 molar (see Table 5 above).

[0144] Therefore, it was confirmed that the specific adsorption performance of proteins can be improved by approximately 2.2 times by surface adsorption of PEG to the sugar chain-immobilized polymer particles of Example 4. In this way, nonspecific adsorption of proteins and viruses can be prevented or suppressed by surface adsorption of a hydrophilic polymer to the sugar chain-immobilized polymer particles.

[0145] Furthermore, as shown in Figure 12(a), immediately after production of Comparative Example 4 (polymer particles with hydrophobized glycopolypeptides surface-adsorbed after the polymerization process), the WGA adsorption amount was 79 μg per 0.018 g of produced particles. The adsorption amount of the protein (WGA) that specifically adsorbs to the sugar chain (chitobiose) contained in the polymer particles with hydrophobized glycopolypeptides surface-adsorbed after the polymerization process was 56 μg, which is a difference from the 23 μg WGA adsorption amount immediately after production of polymer particles without added hydrophobized glycopolypeptides. Comparing the WGA adsorption amounts between Example 5 and Comparative Example 4, it was confirmed that Example 5 was able to improve the specific protein adsorption performance by nearly two-fold compared to Comparative Example 4. Thus, in order to improve the specific protein adsorption performance, it is effective to add the hydrophobized glycopolypeptide during the polymerization of the polymer particles.

[0146] 12(b), the sugar chain-immobilized polymer particles of Example 5 after storage in water for one month had a WGA adsorption amount of 119 μg per 0.018 g of produced particles, which, compared to the 23 μg WGA adsorption amount of polymer particles without the addition of hydrophobic glycopolypeptide, indicates that the adsorption amount of the protein (WGA) that specifically adsorbs to the sugar chain (chitobiose) contained in the sugar chain-immobilized polymer particles of Example 5 was 96 μg. This shows that the reduction in WGA adsorption amount was approximately 9.4% compared to 106 μg immediately after particle production. On the other hand, the polymer particles of Comparative Example 4, in which hydrophobized glycopolypeptides were surface-adsorbed after the polymerization process, had a WGA adsorption amount of 69 μg per 0.018 g of produced particles. This difference from the 23 μg WGA adsorption amount of polymer particles without the addition of hydrophobized glycopolypeptides indicates that the adsorption amount of the protein (WGA) that specifically adsorbs to the glycochain (chitobiose) contained in the polymer particles to which the hydrophobized glycopolypeptides were surface-adsorbed after the polymerization process was 46 μg. This indicates a reduction in the amount of WGA adsorption of approximately 17.9% compared to 56 μg immediately after particle generation. Therefore, it was confirmed that the reduction in the amount of WGA adsorption of the glycan-immobilized polymer particles of Example 5 could be reduced to approximately half that of Comparative Example 4, in which the hydrophobized glycopolypeptides were surface-adsorbed after the polymerization process. Thus, by adding the hydrophobized glycopolypeptides during the polymerization process and embedding the hydrophobic groups of the hydrophobized glycopolypeptides inside the polymer particles, the glycochains can be firmly immobilized to the polymer particles.

[0147] Example 6 To confirm whether or not the sugar chain-immobilized polymer particles of the present invention have specific adsorption properties for viruses, in this example, the specific adsorption properties of human influenza viruses were examined using hydrophobized sugar chain polypeptide polymer particles having sialo-sugar chains. In the actual verification experiment, instead of using influenza virus hemagglutinin, SSA (Sambucus Sieboldiana Lectin), which is known to function as a model for influenza virus hemagglutinin, was used (see, for example, M. Ogata, et al., Biomacromolecules, Vol. 10, pp. 1894-1903, 2009).

[0148] -Synthesis of PMMA particles immobilized with sialo-oligosaccharides- The hydrophobized glycopolypeptide having sialo-oligosaccharides was the same compound as used in Example 2 (compound (XIII-1) in the formula (7) above), and was immobilized on polymer particles of methyl methacrylate (MMA) using the method described below to synthesize sialo-oligosaccharide-immobilized polymethyl methacrylate (PMMA) particles. The synthesis method for sialo-oligosaccharide-immobilized PMMA particles is shown schematically in Figure 13.

[0149] Deionized water 18 was supplied to a lidded polymerization vessel 17, and nitrogen gas was bubbled through the water for 20 minutes while stirring. The deionized water in the vessel was then heated to 70°C. After confirming that the heating temperature had stabilized, an aqueous solution containing rhodamine B (RhB) as a fluorescent dye and MMA monomer were added to the vessel in that order. Stirring was continued for 5 minutes, and then deionized water containing potassium persulfate (KPS) dissolved as a radical polymerization initiator was added to initiate polymerization. In this embodiment of the present invention, the polymerization time begins when the radical polymerization initiator is added to the polymerizable monomer.

[0150] One hour after the start of polymerization, a predetermined amount of compound (XIII-1), obtained as a hydrophobized glycopolypeptide, and polyethylene glycol (PEG) as a hydrophilizing agent were added in that order. The amount of compound (XIII-1) added was adjusted to be approximately 1 / 1000 of the weight of the MMA monomer. Here, one hour after the start of polymerization, the conversion rate of the monomer contained in the deionized water was approximately 25% by mass. This conversion rate was determined by comparing the average particle size of the sialo-glycan-immobilized polymer particles 20 produced one hour after the start of polymerization with the average particle size of the polymer particles obtained after the completion of polymerization.

[0151] The polymerization was continued for two hours, resulting in a total polymerization time of three hours, after which a suspension containing sialoglycan-immobilized PMMA particles was obtained. The molar concentrations of the components added to the polymerization vessel during the polymerization reaction were: [sialoglycan (XIII-1)] = 50 nmol; [MMA] = 0.8 mol; [KPS (radical polymerization initiator)] = 8 mmol; [Rhodamine B (fluorescent dye)] = 0.5 mmol per 1 L of MMA, assuming a total volume of 5 mL. The amount of [PEG (hydrophilizing agent)] added was 25 mg by weight, calculated based on a total volume of 5 mL. The suspension containing the sialoglycan-immobilized PMMA particles was centrifuged three times with deionized water, followed by 24 hours of vacuum freeze-drying to obtain sialoglycan-immobilized PMMA particles 20.

[0152] The resulting sialo-glycan-immobilized PMMA particles were observed using a scanning electron microscope (SEM). Approximately 200 particles were randomly selected from the SEM images and their particle sizes were measured. The particle size and dispersity were calculated as the average values ​​of these measurements. The sialo-glycan-immobilized PMMA particles 20 contained a large number of small particles of approximately 100 nm in diameter, along with main particles of 250 nm or larger. This was likely due to the negative charge of the sialo-glycans contributing to the stability of the PMMA particles during polymerization, resulting in the generation of new PMMA particle nuclei. Therefore, the sialo-glycan-immobilized PMMA particles 20 were redispersed in water and centrifuged at 15,000 rpm for 2 minutes to separate the small particles. The sugar-immobilized PMMA particles of this example had an average particle size and dispersity of 270 nm and 4%, respectively, and were confirmed to have a nearly uniform particle size without any aggregation. Furthermore, the sialo-glycan-immobilized PMMA particles obtained in this example are polymer particles in which RhB (a fluorescent dye) has been encapsulated according to the method shown in Figure 13. By using RhB as a fluorescent probe or fluorescent label in fluorescence spectroscopic measurements, the presence of sialo-glycan-immobilized polymer particles before and after specific adsorption of proteins or viruses can be easily confirmed and verified.

[0153] -Verification of specific adsorption performance against viruses- The amount of viral protein adsorption was determined using essentially the same methods and analytical procedures as those used to examine the specific adsorption performance of the glycan-immobilized polymer particles obtained in Example 4 above, except that sialo-glycan-immobilized PMMA particles and SSA (Sambucus Sieboldiana Lectin), a model substance for human influenza virus protein (hemagglutinin), were used as the glycan-immobilized polymer particles and protein, respectively. Specifically, 0.025 g of polymer particles with sialo-hydrophobic glycopolypeptides immobilized thereon were suspended in 0.10 mL of deionized water, and the suspension was mixed with 0.10 mg of SSA dissolved in 0.10 mL of phosphate-buffered saline (PBS, pH 6.5) in a microtube and allowed to stand at 4°C for 1 hour. The PBS used was prepared by dissolving NaCl, KCl, and potassium dihydrogen phosphate in deionized water. The sialo-oligosaccharide-immobilized PMMA particles with SSA adsorbed on their surfaces were separated from the suspension by centrifugation for 90 minutes (conditions: 4°C, 15,000 rpm). The absorbance (A280) of the supernatant containing SSA that was not adsorbed to the sialo-oligosaccharide-immobilized PMMA particles was then measured using an ultramicrospectrophotometer (Nano Drop ONE, manufactured by Thermo Fisher Scientific Inc.). C The concentration of SSA adsorbed to the sugar chain-immobilized polymer particles was calculated using a calibration curve previously prepared for the relationship between absorbance and SSA concentration.

[0154] As a comparative example, we also measured the amount of SSA adsorbed per mg of sialoglycan-immobilized PMMA particles (Sia(α-2,6)+C10 / PEG / RhB / PMMA particles) and sialoglycan-immobilized PMMA particles (PEG / RhB / PMMA particles) using the same analytical method as above, except that the hydrophobized glycopolypeptide compound (XIII-1) was not added. Figure 14 shows the results of measuring the amount of SSA adsorbed per mg of particles for sialoglycan-immobilized PMMA particles (Sia(α-2,6)+C10 / PEG / RhB / PMMA particles) and sialoglycan-immobilized PMMA particles (PEG / RhB / PMMA particles).

[0155] As shown in Figure 14, the sialo-glycan-immobilized PMMA particles of Example 6 (Sia(α-2,6)+C10 / PEG / RhB / PMMA particles) had an SSA adsorption amount of 5.55 μg per mg of particles, with a standard deviation of 0.27 μg. In contrast, the sugar chain-free PMMA particles (PEG / RhB / PMMA particles) synthesized as a comparative example had an SSA adsorption amount of 0.75 μg per mg of particles, with a standard deviation of 0.50 μg. Comparing the SSA adsorption amounts of the two particles, it can be seen that the sialo-glycan-immobilized PMMA particles have an SSA specific adsorption performance that is approximately 7.4 times higher than that of sugar chain-free PMMA particles. Considering the specific adsorption ability of SSA, a model substance for the human influenza virus protein (hemagglutinin), it was confirmed that the sialo-glycan-immobilized PMMA particles of this example have high specific adsorption ability for human influenza viruses by fixing and arranging sialo-glycans on the surface.

[0156] As described above, in the sugar chain-immobilized polymer particles according to an embodiment of the present invention, two or more hydrophobic groups of the hydrophobic glycopolypeptide are embedded inside the polymer particle at independent locations, which increases the contact area between the polymer particle and the hydrophobic group portion that functions as an anchor site, making the sugar chain less likely to come off the polymer particle and firmly immobilizing it. Furthermore, since the two or more sugar chains of the hydrophobic glycopolypeptide are densely introduced in a molecular arrangement on the outermost surface of the polymer particle, the specific adsorption of proteins and viruses via the sugar chains can be significantly improved.

[0157] Furthermore, the sugar chain-immobilized polymer particles according to the embodiments of the present invention can be used as polymer particles with desired specific adsorption properties by changing the type and number of sugar chains of the hydrophobized glycopolypeptide depending on the type of protein or virus. Furthermore, the sugar chain-immobilized polymer particles according to the embodiments of the present invention are highly useful because, by incorporating a fluorescent dye, they can be used not only as a means for highly accurate detection of proteins or viruses, but also as a fluorescent probe for imaging biological substances or a fluorescent label for cell imaging.

[0158] The method for producing a sugar chain-immobilized polymer of the present invention involves performing soap-free emulsion polymerization in the presence of a hydrophobic polymerizable monomer in the coexistence of a polypeptide from the middle of the polymerization reaction, which not only prevents aggregation of polymer particles but also enables efficient production of fine particles with a nano-level average particle size and a desired average particle size and narrow particle size distribution, thereby providing excellent mass productivity for sugar chain-immobilized polymers with precisely controlled particle size and particle size distribution. [Industrial Applicability]

[0159] The sugar chain-immobilized polymer of the present invention can be used as a biosensor for proteins, viruses, etc., or as a versatile means for separating and purifying them by utilizing its affinity with them. [Explanation of symbols]

[0160] 1. Sugar chain-immobilized polymer particles 2,7···glycan 3,8···Hydrophobic group 4. Hydrophobized glycopolypeptide 5. Polymer particles 6. Sugar chain-immobilized polymer particles by conventional technology 9. Hydrophobized Glycopolypeptides by Conventional Techniques 10. Prior art polymer particles 11,17...Polymerization tank 12,18···Aqueous solvent 13···Monomer oil droplets 14...Polymer particles during polymerization reaction 15···Aggregated sugar chain-immobilized polymer particles 16···Hydrophilic polymer 19···Sugar chain-immobilized PMMA particles 20···Sialoglycan-immobilized PMMA particles

Claims

1. polymer particles; a hydrophobized glycopolypeptide having a plurality of sugar chains and a plurality of hydrophobic groups in its side chains in one molecule, the hydrophobized glycopolypeptide being non-covalently fixed to the polymer particle via the plurality of hydrophobic groups individually embedded from the surface of the polymer particle toward the inside at a plurality of different positions of the polymer particle, the polymer particles are polymers of one or more hydrophobic monomers selected from the group consisting of styrene and its derivatives, vinyl esters, and (meth)acrylic acid esters; The sugar chain-immobilized polymer particle is characterized in that the hydrophobized glycopolypeptide has a main chain made of a polypeptide having amino acid residues represented by the following formula (1) or the following formula (2), and has a plurality of side chains formed by binding a glycoside containing a monosaccharide or oligosaccharide structure to the main chain via an amide bond, and a plurality of side chains formed by binding a linear or branched alkyl group having 3 to 15 carbon atoms as the hydrophobic group to the main chain via an amide bond. 【Chemistry 1】

2. 2. The sugar chain-immobilized polymer particles according to claim 1, wherein the sugar chain-immobilized polymer particles have an average particle size of 50 nm to 1 μm.

3. 3. The sugar chain-immobilized polymer particle according to claim 2, wherein the hydrophobic glycopolypeptide is immobilized without spanning a plurality of the polymer particles.

4. 4. The sugar chain-immobilized polymer particle according to claim 3, wherein the hydrophobized sugar chain polypeptide is a polypeptide represented by the following formula (3): 【Chemistry 2】 Here, R １ is hydrogen, an alkyl group, or a monovalent cationic metal; R ２ is a monosaccharide glycoside or an oligosaccharide glycoside, and R ３ is the hydrophobic group, a is 0 or an integer of 1 or more, and b and c are each independently an integer of 2 or more.

5. The sugar chain-immobilized polymer particle according to claim 4, characterized in that b and c are each independently an integer of 500 to 3000, and the ratios of a, b, and c to (a+b+c) are 0.05 to 0.9, 0.05 to 0.5, and 0.05 to 0.6, respectively.

6. The polymer particles are particles having a polymer of at least one hydrophobic monomer selected from the group consisting of styrene and its derivatives, and (meth)acrylic acid esters, and the R １ is a cationic metal of sodium or potassium, and said R ２ is a glycoside containing at least one sugar structure selected from the group consisting of monosaccharides and disaccharides to heptasaccharides represented by the following formula (4) or (5), ３ 5. The sugar chain-immobilized polymer particle according to claim 4, wherein is a linear or branched alkyl group having 3 to 15 carbon atoms. 【Transformation 3】 Here, Y １ and Y ２ are each independently an organic group having a structure in which 3 to 15 methylene groups are covalently bonded as a spacer group, m and n are each independently an integer of 0 to 2, and Z is a sugar chain containing at least one sugar structure selected from the group consisting of monosaccharides and disaccharides to heptasaccharides.

7. The sugar chain-immobilized polymer particle according to any one of claims 1 to 3, characterized in that the sugar chain-immobilized polymer particle has a hydrophilic polyalkylene glycol or polyalkylene glycol derivative adsorbed on the surface of the sugar chain-immobilized polymer particle.

8. The sugar chain-immobilized polymer particle according to any one of claims 1 to 3, characterized in that the polymer particle has a fluorescent dye non-covalently fixed to the polymer particle by hydrophobic interaction or electrostatic interaction with the polymer particle.

9. a step of initiating polymerization by adding a water-soluble radical polymerization initiator into a polymerization tank containing an aqueous solvent in which a polymerizable monomer is dispersed; and adding, during the polymerization reaction, a hydrophobized glycopolypeptide having a plurality of sugar chains and a plurality of hydrophobic groups in its side chains in one molecule, and continuing the polymerization reaction in a state in which the hydrophobized glycopolypeptide is present in the polymerization vessel, the polymerizable monomer is one or more hydrophobic monomers selected from the group consisting of styrene and its derivatives, vinyl esters, and (meth)acrylic acid esters; The method for producing sugar chain-immobilized polymer particles is characterized in that the hydrophobized glycopolypeptide has a main chain made of a polypeptide having amino acid residues represented by the following formula (1) or the following formula (2), and has a plurality of side chains formed by glycosides having a monosaccharide or oligosaccharide structure bound to the main chain via amide bonds, and a plurality of side chains formed by linear or branched alkyl groups having 3 to 15 carbon atoms bound to the main chain via amide bonds as the hydrophobic group. 【Chemistry 4】

10. The method for producing sugar chain-immobilized polymer particles described in claim 9, characterized in that the addition of the hydrophobic glycopolypeptide is carried out at a polymerization reaction time when the polymerization of the polymerizable monomer progresses in the polymerization tank and the conversion rate of the polymerizable monomer contained in the polymerization tank due to the polymerization reaction becomes 3% by mass or more and 95% by mass or less compared to the time of the start of polymerization.

11. The method for producing sugar chain-immobilized polymer particles according to claim 10, characterized in that the hydrophobic glycopolypeptide is added during the polymerization reaction time when the conversion rate of the polymerizable monomer contained in the polymerization tank due to the polymerization reaction reaches 3 to 50 mass % relative to the mass at the start of polymerization.

12. The method for producing sugar chain-immobilized polymer particles according to claim 11, characterized in that the hydrophobized glycopolypeptide is a polypeptide represented by the following formula (3): 【Transformation 5】 Here, R １ is hydrogen, an alkyl group, or a monovalent cationic metal; R ２ is a monosaccharide glycoside or an oligosaccharide glycoside, and R ３ is the hydrophobic group, a is 0 or an integer of 1 or more, and b and c are each independently an integer of 2 or more.

13. The method for producing sugar chain-immobilized polymer particles according to claim 12, characterized in that b and c are each independently an integer of 500 to 3000, and the ratios of a, b, and c to (a+b+c) are 0.05 to 0.9, 0.05 to 0.5, and 0.05 to 0.6, respectively.

14. The polymer particles are particles having a polymer of at least one hydrophobic monomer selected from the group consisting of styrene and its derivatives, and (meth)acrylic acid esters, and the R １ is a cationic metal of sodium or potassium, and said R ２ is a glycoside containing at least one sugar structure selected from the group consisting of monosaccharides and disaccharides to heptasaccharides represented by the following formula (4) or (5), ３ 13. The method for producing sugar chain-immobilized polymer particles according to claim 12, wherein is a linear or branched alkyl group having 3 to 15 carbon atoms. 【Transformation 6】 Here, Y １ and Y ２ are each independently an organic group having a structure in which 3 to 15 methylene groups are covalently bonded as a spacer group, m and n are each independently an integer of 0 to 2, and Z is a sugar chain containing at least one sugar structure selected from the group consisting of monosaccharides and disaccharides to heptasaccharides.

15. The method for producing sugar chain-immobilized polymer particles according to any one of claims 9 to 11, characterized in that it comprises a step of adsorbing a hydrophilic polymer of polyalkylene glycol or a polyalkylene glycol derivative onto the surface of the sugar chain-immobilized polymer particles during or after the polymerization reaction of the sugar chain-immobilized polymer particles.

16. The step of adsorbing the hydrophilic polymer onto the surface of the polymer particles includes: During or after the polymerization reaction of the sugar chain-immobilized polymer particles is completed, adding the hydrophilic polymer to the polymerization vessel containing the sugar chain-immobilized polymer particles; a step of transferring the sugar chain-immobilized polymer particles to a reaction vessel containing an aqueous medium separately from the polymerization vessel, and then adding the hydrophilic polymer to the reaction vessel containing the sugar chain-immobilized polymer particles; 16. The method for producing sugar chain-immobilized polymer particles according to claim 15, characterized in that it comprises any one of the steps of:

17. a fluorescent dye is added to the polymerization tank in the presence or absence of the polymerizable monomer before the step of initiating the polymerization, or the fluorescent dye is added to the polymerization tank in the presence or absence of the hydrophobized glycopolypeptide at any polymerization time when the conversion rate of the polymerizable monomer due to the polymerization reaction is 95% by mass or less after the initiation of the polymerization, The method for producing sugar chain-immobilized polymer particles according to any one of claims 9 to 11, characterized in that sugar chain-immobilized polymer particles are produced in which the fluorescent dye is immobilized on the polymer particles.

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