JP2005315666A - Sugar bonded filler and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a sugar bonded filler, which is easy to handle and suitable for use in liquid chromatography, easy to measure molecular characteristics such as a medicament bonding capacity, a selective molecule recognizing capacity, solubility and the like or to set a separation condition. <P>SOLUTION: The sugar bonded filler constituted by fixing sugar to a carrier through a ligand, the sugar bonded filler wherein the ligand of a separation agent is a 4-12C alkyl group, the sugar bonded filler wherein the ligand and sugar are bonded through a sulfur bond, a nitrogen bond or an oxygen bond and the sugar bonded filler wherein sugar is a monosaccharide having an ion exchange capacity are provided. Further, the sugar bonded filler wherein mannose or glactose is fixed to a carrier through a propylamino group is provided. Furthermore, the sugar bonded filler is manufactured by bonding the ligand to the carrier and subsequently bonding sugar thereto or by bonding a sugar bonded silylating agent to the carrier. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a sugar bond filler and a method for producing the same. More specifically, it is useful as a separating agent for various physiologically active compounds, and also as a separating agent that can also have molecular recognition properties of glycoproteins, can be manufactured with good reproducibility, has a long life, has good separation ability, and is selected. The present invention relates to a highly sugar-bonding filler and a method for producing the same.

  Conventionally, a protein is directly coated on a carrier, a filler is synthesized by binding to a carrier with a propylamino group as a ligand, a drug binding constant is measured, and a drug is previously deproteinized in a physiological fluid. The direct quantification has been carried out without doing (Non-patent Documents 1 and 2). However, since these fillers are not stable in protein quality, it has been difficult to supply fillers with high reproducibility.

  There are two methods for investigating the characteristics of membrane proteins: a method in which a membrane protein extracted from a natural product is coated on a carrier or measured using particles fixed in a membrane, and the membrane protein extracted in a transfer solution is added Measurement methods based on adsorption and distribution phenomena in liquids have been performed. However, since these methods use extracts from natural products as raw materials, it is difficult to construct an easy-to-use system with a very small amount of membrane protein, poor purity, good reproducibility.

  As a filler to which sugar is bound, a filler in which glucose is bound to a propylamino group-bound filler using silica gel as a carrier has been developed (Non-patent Document 3). This filler was reported to be suitable for molecular sieve liquid chromatography. However, this filler has a shorter lifetime in the buffer than the conventionally used molecular sieve chromatography fillers and has not been widely used. Furthermore, a filler in which amylose is bonded to silica gel as a polysaccharide is widely used as a filler for optical isomer separation (Non-patent Document 4).

  Moreover, as a separating agent having basic molecular recognition of protein, guanidyl group (Patent Document 1) which is anion exchange group considered to be responsible for anion exchange ability of protein, and cation exchange ability of protein is assumed. For example, for the measurement of drug binding ability of human serum albumin (Non-patent Document 5) and for chromatography as an ion exchanger. It is used as a separating agent (Patent Document 2). It is also used as a sugar separation filler with good sugar recovery (Patent Document 1).

JP-A-10-160719 Japanese Laid-Open Patent Publication No. 2000-9708 H. Yoshida, I. Morita, G. Tamai, T. Masujima, Y. Tsuru, N. Takai and H. Imai, Chromatographia, 19 (1984) 466-472. N. Lammers, H.D.Bree, C.P.Groen, H.M.Ruijten, B.J.D.Jong, J. Chromatogr., 496 (1989) 291-300. R.E.Huisden, J.C.Krak and H.Poppe, J. Chromatogr., 508 (1990) 289-299. N. Enomoto, S. Furukawa, Y. Ogasawara, H. Akano, Y. Kawamura, E. Yashima and Y. Okamoto, Anal. Chem., 68 (1996) 2798-2804. T. Hanai, R. Miyazaki, T. Kinoshita, Anal. Chim. Acta, 378 (1999) 77-82. T. Hanai, Advances in Chromatography, 40 (2000) 315−357, T. Hanai, R. Miyazaki, T. Kinoshita, F. Ahmed, B. Modrek, J. Liq. Chromatogr. & Rel. Technol., 22 (1999) 2613-2625 '' J.L.Speier, J.A.Webster, J. Org.Chem., 21 (1956) 1044-46. T. Kinoshita, M. Miyayama, Y. Kyodo, Y. Kamitani, M. Miyamasu, N, Nimura, T. Hanai, Biomdical Chromatography, 7 (1993) 64-67. S. Ciccotosto, M.J.Kiefel, S. Abo, W. Stewart, K. Quelch, and M. von Itzstein, Glycoconjugate Journal, 15 (1998) 663-669, R.L.Whistler, M.L.Wolfrom eds.Methods in Carbohydrate Chemistry, Academic Press Inc.New York and London, 2 (1963) 211-220.)

  Fillers whose surfaces are treated with silica gel as a base are widely used because they have a high number of theoretical plates, are easily separated, and are easy to handle. For this reason, synthetic organic polymer fillers widely used in the early stage of high performance liquid chromatography (hereinafter referred to as HPLC) have been used less frequently. On the other hand, there are few types of surface-modifying groups in silica gel-based fillers, and it is said that fillers chemically modified with octadecyl groups are used in 50% or more of the separation. However, since this filler has a hydrophobic surface and little selectivity other than due to the difference in the pore size of silica gel, the characteristics of physiologically active compounds such as drug binding ability, selective molecular recognition ability, solubility, etc. are measured. In addition, it is necessary to devise difficult methods for setting the separation conditions.

  In order to facilitate the above-described measurement of molecular characteristics and setting of separation conditions using liquid chromatography, it is necessary to develop a packing material that has characteristics of biological components and is selective and easy to handle. The guanidyl group-bonded filler previously developed by the present inventors is not synthesized by paying attention to the ion exchange capacity and the ionic strength chemically like the conventional ion exchanger. A guanidyl group-bonded filler is a filler simulating an in vivo ion exchange function. further. In order to solve the environmental problem and the problem of reducing the required sample amount, it is necessary to reduce the column size. For this purpose, it is necessary to synthesize a packing material using silica gel having a high physical strength as a support. . In order to facilitate the setting of separation conditions for liquid chromatography, it is necessary to develop a selective and easy-to-handle filler.

  The present invention provides a sugar-binding filler characterized in that a sugar is immobilized on a carrier via a ligand. The present invention further provides a filler in which the ligand of the separating agent is an alkyl group having 4 to 12 carbon atoms. The present invention further provides the above filler in which the ligand and the sugar are bonded through a sulfur bond, a nitrogen bond or an oxygen bond. The present invention further provides the filler, wherein the sugar is a monosaccharide having ion exchange ability. The present invention further provides a sugar-binding filler useful as a separating agent having molecular recognition characteristics of protein or glycoprotein. Furthermore, the present invention provides a method for producing a protein or glycoprotein separating agent, characterized in that a ligand is bound to a carrier and then a sugar is bound. The present invention further provides a method for producing a sugar-binding filler by synthesizing a sugar-bonding silylating agent and then binding the silylating agent to silica gel. The present invention further provides a sugar-binding filler in which mannose or galactose is fixed to a carrier via a propylamino group.

  The sugar-bonded filler of the present invention is not a filler synthesized by paying attention to an alcoholic hydroxyl group like conventional diol conjugates, but a filler that mimics the function of glycoprotein in vivo. In particular, it has the property of being able to measure separation and purification of biological components and the molecular recognition ability of glycoproteins. The sugar-binding filler of the present invention needs to have a small column size in consideration of environmental problems and the problem of reducing the required sample amount. For this reason, silica gel having high physical strength can be used as a carrier. It is also possible to synthesize an organic synthetic polymer as a support in order to process a large amount of sample. The sugar bond filler of the present invention is also useful as a protein or glycoprotein separating agent.

When the bonding groups between the sugar-bonding filler presented in Non-Patent Document 2 and the sugar-bonding filler carrier and sugar provided by the present invention are compared by chemical formula, they are expressed as follows.
Non-patent document 2 Sugar-bonded filler Propylamino group-bonded glucose →-(CH ₂ ) ₃ N glucose Sugar-bonded filler of the present invention Hexyl group S-bonded glucose →-(CH ₂ ) ₆ S glucose Hexyl group S-bonded galactose → -(CH ₂ ) ₆ S galactose hexyl group S-linked glucosamine →-(CH ₂ ) ₆ S glucosamine This linked sugar not only has a higher polarity than the diol group, but also takes advantage of the characteristics of glycoprotein. It is a sugar bond filler. However, glucose is not a glycoprotein constituent sugar but an energy sugar, but it is cheaper and more useful as a raw material than other sugars. This filler can be synthesized using an organic synthetic polymer as a carrier and silica gel as a carrier. A packing material using an organic synthetic polymer as a carrier is chemically stable but is physically unstable and difficult to obtain a column having a high number of theoretical plates, but is suitable for a large amount of processing. Since the filler synthesized using silica gel as a carrier has high physical strength, it is suitable as a filler for high performance liquid chromatography. The above sugar bond filler is chemically stable because it uses a hexyl group as a ligand. The alkyl group bond is chemically stable, and the chemical stability increases when the chain length of the alkyl group is longer than pentyl. The length of the alkyl group is 4 to 12 carbon atoms, more preferably 6 to 12 carbon atoms. The bond between the alkyl chain and the sugar may be any of a direct bond, a bond via N, a bond via S, and a bond via O, but the bond via S is particularly preferable from the viewpoint of chemical stability. . In the present invention, the term “sugar” is used to include glucosamine, mannose, galactose, arabinose, fructose, glucose, fucose, glucuronic acid, sialic acid, xylose, galactosamine and derivatives thereof.

When the previously proposed guanidyl group (Patent Document 1) and a conventional ion exchange group are limited to an anion exchange group corresponding to the guanidyl group and compared by chemical formula, they are expressed as follows.
Propyl group →-(CH ₂ ) ₃ NH ₂
Diethylaminoethyl group →-(CH ₂ ) ₂ N (CH ₂ CH ₃ ) ₂
Trimethylammonium group → -CH ₂ N (CH ₃ ) ₃
Triethylammonium group → -CH ₂ N (CH ₂ CH ₃ ) ₃
Guanidyl _{→ -CH 2 -NH-C (NH} ) NH 2
Unlike other ion exchange groups, this guanidyl group has three nitrogen atoms, and is a substituent for arginine, which is one of the amino acids that constitute the protein. This guanidyl group has an anion exchange ability and is thought to be responsible for the anion exchange ability of proteins. This filler can be synthesized using an organic synthetic polymer as a carrier and an inorganic synthetic polymer typified by silica gel as a carrier. However, a column using an organic synthetic polymer as a carrier is chemically stable, but it is difficult to obtain a column that is physically unstable and has a high theoretical plate number. However, it is a filler suitable for mass processing. A material synthesized from an inorganic synthetic polymer typified by silica gel is suitable as a packing material for high performance liquid chromatography because of its high physical strength. The glucosamine-bonded filler used in the present invention can be synthesized using an organic synthetic polymer as a carrier and an inorganic synthetic polymer typified by silica gel as a carrier in the same manner as the guanidyl group-bonded filler. When compared by chemical formula, they are expressed as follows.
Glucosamino group → -S-C ₆ H ₁₂ O ₄ N
Unlike the ion exchange groups developed so far, this glucosamino group is a sugar having ion exchange ability, is an oligosaccharide constituent sugar of glycoprotein, and plays an important role in molecular recognition ability of oligosaccharide. It is believed that. This primary amino group of glucosamine has not only weak anion exchange but also hydrogen bonding ability.

  As the carrier for immobilizing the sugar-containing compound used in the present invention, silica, silica gel, porous alumina as an inorganic carrier, polystyrene, polystyrene hydroxyl gel, methacrylate gel, ethylene glycol, starch, vinyl as an organic carrier Examples include alcohol. These carriers may be those in which a ligand is immobilized in advance, and can be easily obtained as commercial products. As silica gel, "Spherisorb Si" (manufactured by Phase Separation), "YMS Gel Silicagel 60" (manufactured by Yamamura Chemical Co., Ltd.), and as porous alumina, "Alumina Alox 60" (manufactured by Nagel), "LiChrosorb Alox T" ( Merck & Co.) are commercially available.

  As the organic carrier, polystyrene gel, "Hitachi gel 3010, 3011, 3019" (Hitachi), "MCI GEL GHP 20P" (Mitsubishi Chemical), "Shodex polymer pak D" (Showa Denko) , "TSK GEL 110,111" (manufactured by Tosoh Corporation), "Shodex RS pak DE613" (manufactured by Showa Denko), "TSK GEL LS 120,130,140" (manufactured by Tosoh Corporation) as a methacrylate gel, "TSK GEL LS" as ethylene glycol 160 "(manufactured by Tosoh Corporation), TSK GEL LS 170" (manufactured by Tosoh Corporation) as starch, "Asahipak GS 220-620" (manufactured by Showa Denko) as vinyl alcohol, etc. These carriers are commercially available. Can be appropriately selected depending on the use of the separating agent.

  Next, an example of a method for producing a sugar-bonded filler in which a glucose group-containing compound of the present invention is immobilized on a carrier via an S bond will be described. In order to synthesize a sugar-binding silylating agent, first, the sugar is acetylated, then the 1-position is brominated, and further thioacetylated. Next, as a ligand, for example, 5-bromo-1-pentene is bound, and further, for example, trichlorosilane is bound to the silylating agent by the method of Speier et al. To do. Subsequently, this silylating agent and silica gel are reacted in, for example, toluene by a synthesis method similar to that for synthesizing an alkyl group-bonded filler to obtain a sugar-bonded silica gel.

  In order to synthesize a sugar-bonding filler by sequentially binding to a carrier without synthesizing a silylating agent having good reactivity but strong toxicity, first, a vinyl group bond is bound to the carrier. Next, after acetylating the sugar, the 1-position is brominated and further thioacetylated. A bromine acid is allowed to act on the vinyl group of the vinyl group-bonded carrier synthesized earlier, brominated, and then reacted with tetraacetylthioacetyl sugar to synthesize a sugar-bonded filler.

  Conventionally used carriers are represented by cellulose, agarose, sephadex, etc. made from polysaccharides, such as polyvinyl alcohol, poly (meth) acrylate, polystyrene, etc. made into petrochemical products in the form of spheres, tubes or nets. There are soft organic polymer systems and inorganic polymer systems represented by silica, alumina, titania and the like. These carriers are used as they are as adsorbent filtration agents or as packing materials for liquid chromatography. In addition, attempts have been made to change the properties of the carriers by extending the surface of these carriers with natural polymer proteins, polysaccharides, or synthetic polymers such as silicone rubber, carbowax, polysiloxane, etc., thereby expanding the range of application. .

  In order to enhance the properties of these carriers, a method of chemically treating the surface with a low molecular compound has also been widely adopted as a separating agent, for example, for a packing material for chromatography. That is, when the surface treatment is performed with a natural polymer, it is difficult to obtain a reproducible filler because the quality and purity are not constant even if the natural polymer is stably supplied. However, when a surface treatment of a carrier is performed by a chemical reaction using a low molecular compound, quality control is easy, and the product is supplied to the market as a product with stable quality and characteristics. In the present invention, a sugar-bonding filler can be produced using these carriers.

  In the case of a binder that binds neutral sugars such as mannose or glucose, the polar polymer is not blocked by the sugar immobilized on the binder surface and is not retained by the binder, but passes through the column. In principle, large molecules are eluted first by the so-called molecular sieving effect. The hydrophobic low molecular weight compound reaches the inner surface of the binder through the gap between the sugars on the surface, and is held by the binder carrier and the ligand to which the sugar is bound. Such a sugar-bonded filler can be used as a so-called chromatographic filler that is directly injected into a biological component and is suitable for analysis of a drug in the biological component. Depending on the polarity of the drug to be analyzed, a hydrophobic ligand alkyl can be used. By adjusting the chain length, impurities can be removed.

  In the ion exchange by the amino group of glucosamine having an anion exchange ability and an amino group, it is anion exchange, such as chloro ion, fluorine ion, brom ion, nitrate ion, sulfate ion, etc. that are contained in seawater. Various inorganic ions, oxalic acid, malic acid, and citric acid that are abundant in plants. Not only organic ions such as maleic acid but also various organic compounds that become anions when dissolved selectively adsorb anionic compounds from metal ions, cations such as amines and neutral compounds such as polycyclic aromatic compounds and saccharides. It has a function. In particular, it is a sugar-bonding filler suitable for the separation and purification of acidic metabolites and acidic drugs (anionic) in vivo, and it is suitable for examining anion exchange reactions and amino group hydrogen bonding ability in vivo. It is an agent. Furthermore, glucosamine is a sugar that is the basis of membrane proteins, and many oligosaccharides are linked to amino acids constituting the protein via glucosamine. This amino group also has hydrogen bonding ability and is thought to play an important role in molecular recognition of glycoprotein membranes. This also suggests that the glucosamine-bound filler has a specific selectivity.

  The sugar-bonded filler of the present invention can supply products with stable quality and characteristics to the market as compared with a filler in which a natural polymer is immobilized. Furthermore, by selecting an appropriate carrier according to the application, it can be made a product that can withstand acid and base, and a filler suitable for a wide range of applications can be supplied. In particular, it can be widely applied in the field of biochemistry.

  Hereinafter, the present invention will be described in more detail based on examples.

Production Example 1 Production of Organic Polymeric N-Mannose Filler 0.5 g of Asahipak NH2P-50 (Asahi Kasei Co., Ltd.), in which a propylamino group is introduced into a polyvinyl alcohol resin, is suspended in 25 mL of methanol, and 0.5 g of mannose and 5 mg of ammonium chloride and 200 mg of sodium cyanoborohydride were added and refluxed for 16 hours. After cooling, each was washed with 100 mL of methanol, water, acetone, and diethyl ether, and then air-dried to remove the solvent to obtain an N-mannose bond filler.

Production Example 2 Production of Silica N-Mannose Filler 0.5 g of propylamino group-bonded silica gel was suspended in 25 mL of methanol, 0.5 g of mannose, 5 mg of ammonium chloride and 200 mg of sodium cyanoborohydride were added thereto and refluxed for 16 hours. did. After cooling, each was washed with 100 mL of methanol, water, acetone, and diethyl ether, and then air-dried to remove the solvent to obtain an N-mannose bond filler. The use of mannose in the reactions of Production Examples 1 and 2 is based on experimental results (Non-patent Document 9) in which mannose is likely to cause a glycosyl reaction and is difficult to produce an Amadori transfer form.

Production Example 3 Production of silica-based S-glucose filler
After 5 g of glucose is added to a solution of 25 mL of glacial acetic acid and 0.4 mL of perchloric acid, the reaction solution is added to ice for crystallization, filtered and washed, and pentater O-acetylglucose is added. Obtained. Next, 1 g of penta-O-acetylglucose was added to 2 mL of an acetic acid solution of bromic acid to change the 1-position acetyl group to a bromine group. This was crystallized by ether extraction. Furthermore, brominated sugars and potassium thioacetate were condensed using a reaction similar to Williamson's ether synthesis. That is, 300 mg of tetra-O-acetylglucopyranosyl bromide was added to a solution of 430 mg of potassium thioacetate in 430 mg of anhydrous acetonitrile, and tetra-O-acetylglucopyranose thioacetate was extracted with ethyl acetate. (Non-Patent Document 10). On the other hand, 2.65 g of silica gel and 1.40 mL of hexenyltrichlorosilane are reacted in toluene to synthesize hexenyl group-bonded silica gel. Next, 2 g of hexenyl group-bonded silica gel is added to 20 mL of bromate / acetic acid solution to synthesize bromohexyl silica gel, and the reaction solution is filtered, washed with acetone and dried. 1 g of bromohexyl silica gel was added to a solution of 476 mg of tetra-O-acetylglucopyranose thioacetate dissolved in 24 mL of anhydrous dimethylformamide, and further reacted with anhydrous diethylamine to give 1-tetra-O-acetylglucose. Pyranose hexyl bonded silica gel was obtained. Finally, the acetyl group was removed to obtain 1-glucopyranose hexyl-bonded silica gel by washing with a solution of sodium methoxy in anhydrous methanol by the Twenprene method (Non-patent Document 11).

Production Example 4 Production of silica-based S-glucosamine filler
5 g of N-acetylglucosamine was added to 50 mL of acetyl chloride, extracted with ethyl acetate, washed with saturated sodium hydrogen carbonate solution, and then evaporated to obtain tri-0-acetyl-N-acetylglucosamine. . Next, 4 g of tri-0-acetyl-N-acetylglucosamine was added to a solution of 6 g of potassium thioacetate dissolved in 320 mL of anhydrous acetonitrile, and tri-0-acetyl-N-acetylglucosamine thioacetate was added with ethyl acetate. Obtained by extraction. On the other hand, 2.65 g of silica gel was reacted in toluene containing 1.40 mL of hexenyltrichlorosilane to synthesize hexenyl group-bonded silica gel. Next, 2 g of hexenyl group-bonded silica gel was added to 20 mL of bromate / acetic acid solution to synthesize bromohexyl silica gel, and the reaction solution was filtered, washed with acetone and dried. 435 mg of bromohexyl silica gel was added to a solution of 180 mg of tri-0-acetyl-N-acetylglucosamine thioacetate dissolved in 10 mL of anhydrous dimethylformamide, and further reacted with 1.6 mL of anhydrous diethylamine to give 1- Tri-0-acetyl-N-acetylglucosamine hexyl-bonded silica gel was obtained. Finally, the acetyl group was removed by washing with a solution of sodium methoxy in anhydrous methanol to obtain 1-glucosamine hexyl-bonded silica gel.

Example 1 Separation of Human Serum Drugs and Proteins Represented by Albumin Conventionally, in order to perform separation analysis by directly injecting human serum drugs, diol-binding fillers, protein-binding fillers, internal hydrophobic packings The agent has been used. However, when a neutral sugar-binding column using the silica-based S-glucose filler obtained in Production Example 3 of the present invention is used, the separation can be performed only with an aqueous solution whose pH is adjusted. Examples of analytical conditions and retention times of human serum albumin and acidic drugs are shown below. The results are shown in Table 1.
Column: Glucose-bonded silica gel, length 100 mm, inner diameter 2.1 mm,
Eluent: 50 mM sodium phosphate solution, pH 7.40,
Flow rate: 0.2mL / min,
Column temperature: 37 ° C
Detection: UV absorption 220-300 nm.

Example 2 Measurement of selectivity of glucosamine-binding column In order to measure the selectivity of the silica-based S-glucosamine filler obtained in Production Example 4, the chromatographic behavior of five chlorophenols was examined. The results are shown in FIG.
Column: Glucosamine-bonded silica gel, 50mm length, 2.1mm inner diameter,
Eluent: 50 mM sodium phosphate solution containing 33.3% methanol, pH 2.0-11.0,
Flow rate: 0.2 mL / min,
Column temperature: 37 ° C
Detection: UV absorption 254nm.

Example 3 Measurement of Selectivity of Propylaminomannose Group Binding Column The selectivity of the propylaminomannose filler obtained in Production Example 2 was tested in the same manner as in Example 2. The results are shown in FIG.
Column: Propylaminomannose-bonded silica gel column, length 100 mm, inner diameter 2.1 mm
Eluent: 50 mM sodium phosphate solution with 20% methanol, pH 4.0-7.0,
Flow rate: 0.2mL / min,
Column temperature: 37 ° C
Detection: UV absorption 254nm.
Comparative Examples 1-3

  Pentyl group-bonded silica gel column (Comparative Example 1), which is a packing for reverse phase liquid chromatography for investigating hydrophobic binding ability, Propylamino group-bonded silica gel column (Comparative Example 2) for comparing the characteristics of amino groups, The chromatographic behavior of a guanidino binding column (Comparative Example 3) for comparing ion exchange capacity was examined under the following conditions. The results are shown in FIGS.

Comparative Example 1
Column: Silica gel column bonded with pentyl group, length 50 mm, inner diameter 2.1 mm,
Eluent: 50 mM sodium phosphate solution containing 33.3% methanol, pH 2.0-11.0,
Flow rate: 0.2mL / min,
Column temperature: 37 ° C
Detection: UV absorption 254 nm.

Comparative Example 2
Column: Propylamino group-bonded silica gel column, length 50 mm, inner diameter 2.1 mm,
Eluent: 50 mM sodium phosphate solution containing 10% methanol, pH 3.0-10.0,
Flow rate: 0.2mL / min,
Column temperature: 37 ° C
Detection: UV absorption 254 nm.

Comparative Example 3
Column: Guanidyl group-bonded polyvinyl gel column, length 50 mm, inner diameter 4.6 mm,
Eluent: 50 mM sodium phosphate solution containing 50% methanol, pH 6.0-8.0,
Flow rate: 1.0mL / min,
Column temperature: 37 ° C
Detection: UV absorption 254 nm.

Table 1 Elution time of various compounds on glucose binding column
Elution time (min) Elution time (min)
Fructose 1.09 Mefenamic acid 4.51
Paraaminohippuric acid 1.10 Nalidixic acid 2.61
Amoxicillin 1.16 naproxen 2.05
Barbituric acid 1.04 Nicotinic acid 1.12
Benzoic acid 1.09 Phenylbutazone 2.80
Furosemide 2.04 Salicylic acid 1.12
Parahydroxybenzoic acid 2.57 Sulfamethoxazole 1.18
Ibuprofen 2.21 Tolazamide 3.22
Indomethacin 1.20 Tolbutamide 1.99
Iopanoic acid 7.08 Warfarin 2.09
Human serum albumin containing 48% by weight of glycated albumin 1.00
Human serum albumin containing 3% by weight of glycated albumin 1.18.

Example 4 Measurement of Drug Selectivity of Glucosamine Binding Column In order to measure drug selectivity of the glucosamine binding column of Example 2, the pentyl group binding column of Comparative Example 1 and the glucose binding column of Example 1 were used. The retention behavior of the summarized drugs was investigated. The column selected as the target used a pentyl group-bonded silica gel column, which is a packing material for reversed-phase liquid chromatography, and a glucose-bonded column for comparing the characteristics of sugars in order to examine the hydrophobic binding ability. The results are shown in Table 2.
Eluent: 50 mM sodium phosphate solution containing 50% methanol for pentyl group binding columns, 33.3% methanol for glucosamine and glucose binding columns, pH 4.0
Flow rate: 0.2 mL / min.
Column temperature: 37 ° C
Detection: UV absorption 220-300 nm.

Example 5 Measurement of Drug Selectivity of Glucosamine Binding Column In order to measure the ion exchange selectivity of the glucosamine binding column of Example 2, the pentyl group binding column of Comparative Example 1 and the guanidyl group binding column of Comparative Example 3 were used. The retention behavior of the drug was measured and compared based on the octanol / water partition constant (logP), which is one of the physical properties of the drug. The results are shown in FIG.
Eluent: 50 mM sodium phosphate solution, pH 4.0, containing 50% methanol for guanidyl group binding columns and 33.3% methanol for glucosamine binding columns. The pentyl group-binding column is 50 mM sodium phosphate solution containing 50% methanol, pH 2.0.
Flow rate: 0.2 mL / min.
Column temperature: 37 ° C
Detection: UV absorption 220-300 nm.

Table 2 Selectivity of glucosamine binding column (unit retention ratio)
Glucosamine glucose pentyl
Join column Join column Join column
Fructose 0.000 0.000 0.000
Paraaminohippuric acid 0.976 0.025 0.019
Amoxicillin 0.243 0.049 0.084
Barbituric acid 0.509 0.000 0.027
Benzoic acid 3.093 0.075 0.666
Furosemide 19.579 0.053 0.505
Parahydroxybenzoic acid 3.235 0.108 0.832
Ibuprofen 72.430 0.634 16.566
Indomethacin 4.952 0.113 0.613
Iopanoic acid 428.817 1.417 23.727
Mefenamic acid 273.526 1.167 20.687
Nalidixic acid 9.484 0.350 1.130
Naproxen 32.049 0.148 3.461
Nicotinic acid 1.066 0.033 0.055
Phenylbutazone 76.035 0.158 4.592
Salicylic acid 7.726 0.000 0.274
Sulfamethoxazole 2.102 0.064 0.224
Tolazamide 16.962 0.259 2.977
Tolbutamide 14.163 0.171 2.596
Warfarin 56.992 0.251 5.067.

The column in Example 1 was prepared by synthesizing silica gel as a support and packed in a semi-micro column with an inner diameter of 2.1 mm. The waste liquid treatment became easy and the chromatograph was able to operate economically friendly to the environment. Furthermore, it can be considered that the specific adsorption of protein was prevented by the quantitative recovery of human serum albumin. Since the alkyl chain of the ligand used for binding was hexyl, the effect of silanol groups on the silica gel surface was not observed, and a chromatogram in which tailing of the drug peak was suppressed was obtained. Compared to conventional commercially available fillers, the manufacturing method is easier and artificial synthetic raw materials are used, so that there is an advantage that the filler is supplied as a filler with good reproducibility and stable quality. Human serum albumin purified to 97% or more under physiological conditions and human serum albumin containing 48% glycated albumin were eluted by the molecular sieving effect, and the partially dissociated acidic drug was retained and eluted. This means that deproteinized and drug can be easily separated by passing human serum directly through this layer of filler. In order to retain a drug that is polar and weakly retained, ionization of the drug may be suppressed in order to strengthen the retention of the drug, or a sugar bond filler having a long alkyl group may be used. The sugar to be bound is not particularly limited to glucose, but glucose is suitable as a synthetic raw material from the viewpoint of price and purity.
As summarized in Table 1, many acidic drugs are dissociated at pH 7.40, and the retention on the reverse phase column is weak. Human serum containing furosemide, parahydroxybenzoic acid, ibuprofen, iopanoic acid, mefenamic acid, nalidixic acid, naproxen, phenylbutazone, tolazamide and warfarin, human serum albumin, and 48% glycated albumin It is possible to separate albumin. When these drugs are lowered in pH, dissociation is suppressed, retention is strong, and they can be separated from albumin.

  The properties of the glucosamine binding column of Example 2 and the mannose binding propylamino column of Example 3 can be understood by comparing the elution order of chlorophenol from the various columns. The column used as a control is an alkyl group bond, a pentyl group bond column (Comparative Example 1) used as an example of a hydrophobic column, an amino group of glucosamine and a conventional amino group bond (propylamine bond) column (Comparative Example 2), It is a guanidyl group coupling | bonding column (comparative example 3) used in order to compare ion exchange ability. The propylmannose-bonded silica gel column of Example 3 has very weak retention of phenols and seems to be usable for the same purpose as the glucose-bonded silica gel column used in Example 1, but is suitable for application to a highly hydrophobic compound. Yes. It is thought that the protein is eluted without being retained.

  As shown in Table 2, when comparing the retention ratio at the pH at which these drugs have the maximum retention, the glucosamine-binding column is different from the glucose-binding column, and the influence of the amino group appears strongly. Can be used.

  Comparing the results obtained with a pentyl group coupled column as standard chromatographic behavior, one can understand the selectivity of columns with different linking groups. In reverse phase liquid chromatography, chlorophenols are eluted in principle from a compound having a low hydrophobicity to a compound having a high hydrophobicity. However, as can be seen from the results of the pentyl group-bonded silica gel column of FIG. Although the retention on the propylamino group binding column is very weak, the elution order is similar to the elution order from the pentyl group binding column, and as shown in FIG. 4, the ion exclusion effect is observed at low and high pH. The As seen in FIG. 3, the order of elution of phenols was the same for the guanidyl group binding column. On the other hand, in the glucosamine binding column, the elution order is not necessarily the hydrophobic order, and as shown in FIG. Were eluted. This can be attributed to the stereospecificity of glucosamine. The retention of these phenols on the mannose-bound propylamino column is very weak as seen in FIG. 2, and it can be said that the elution order is similar to that of the pentyl group-bound column.

  FIG. 6 is a graph comparing retention values (log k) of retention ratios measured with various columns based on drug octanol / water partition constant (log P). The symbols in FIG. 6 represent the following meanings. 1: paraaminobenzoic acid, 2: nicotinic acid, 3: sulfamethoxazole, 4: nalidixic acid, 5: parahydroxybenzoic acid, 6: salicylic acid, 7: tolazamide, 8: benzoic acid, 9: furosemide, 10: Tolbutamide, 11: warfarin, 12: naproxen, 13: phenylbutazone, 14: indomethacin, 15: ibuprofen, 16: mefenamic acid.

  Comparing the selectivity of the glucosamine binding column from the elution behavior of acidic drugs summarized in FIG. 6, in principle, the retention ratio is proportional to the hydrophobicity of the drug in the pentyl group binding column, and even in the glucosamine binding column. Although it seems to correspond to the hydrophobicity of the drug, the retention of indomethacin was extremely weak. Furthermore, in order to compare the ion exchange ability of glucosamine, the retention of furosemide was very weak in the glucosamine binding column compared with the result of the guanidyl group binding column. Although it is difficult to quantitatively analyze the retention behavior of glucosamine, these chromatographic behaviors can be regarded as characteristic molecular recognition phases.

4 is a graph showing phenol retention behavior of the glucosamine group-binding column of Example 2. 4 is a graph showing phenol retention behavior of the propylmannose group-bonded column of Example 3. 3 is a graph showing phenol retention behavior of a pentyl group-bonded column of Comparative Example 1. 6 is a graph showing phenol retention behavior of the propylamino group-bonded column of Comparative Example 2. 5 is a graph showing phenol retention behavior of a guanidyl group-bonded polyvinyl gel column of Comparative Example 3. It is a graph which shows the comparison by the logarithm value (logk) of the retention ratio measured with various columns.

Claims (9)

A sugar-binding filler, wherein sugar is fixed to a carrier via a ligand. The sugar-bonding filler according to claim 1, wherein the ligand is an alkyl group having 4 to 12 carbon atoms. The sugar-bonding filler according to claim 3, wherein the ligand and the sugar are bonded through a sulfur bond, a nitrogen bond or an oxygen bond. The sugar-binding filler according to claim 1, wherein the sugar is a monosaccharide having ion exchange capacity. The sugar-bonding filler according to claim 1, wherein the carrier is silica gel. Use of the sugar-binding filler according to claim 1 as a separating agent having protein or glycoprotein molecular recognition properties. A method for producing a sugar-binding filler, comprising binding a ligand to a carrier and then binding a sugar. A method for producing a sugar bond filler, comprising synthesizing a sugar bond silylating agent and then bonding the silylating agent to silica gel. A sugar-bonding filler characterized in that mannose or galactose is fixed to a carrier via a propylamino group.

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