Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/08—Esters of oxyacids of phosphorus
  • C07F9/09—Esters of phosphoric acids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
  • B01J20/28004—Sorbent size or size distribution, e.g. particle size
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
  • B01J20/28016—Particle form
  • B01J20/28019—Spherical, ellipsoidal or cylindrical
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
  • B01J20/28057—Surface area, e.g. B.E.T specific surface area
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
  • B01J20/28078—Pore diameter
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
  • B01J20/289—Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/3092—Packing of a container, e.g. packing a cartridge or column
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
  • B01J20/3204—Inorganic carriers, supports or substrates
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3244—Non-macromolecular compounds
  • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3244—Non-macromolecular compounds
  • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
  • B01J20/3257—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one of the heteroatoms nitrogen, oxygen or sulfur together with at least one silicon atom, these atoms not being part of the carrier as such
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3244—Non-macromolecular compounds
  • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
  • B01J20/3257—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one of the heteroatoms nitrogen, oxygen or sulfur together with at least one silicon atom, these atoms not being part of the carrier as such
  • B01J20/3259—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one of the heteroatoms nitrogen, oxygen or sulfur together with at least one silicon atom, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulfur with at least one silicon atom
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/56—Labware specially adapted for transferring fluids
  • B01L3/569—Glassware
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F19/00—Metal compounds according to more than one of main groups C07F1/00 - C07F17/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
  • C07F7/02—Silicon compounds
  • C07F7/08—Compounds having one or more C—Si linkages
  • C07F7/10—Compounds having one or more C—Si linkages containing nitrogen having a Si-N linkage
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/08—Esters of oxyacids of phosphorus
  • C07F9/09—Esters of phosphoric acids
  • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2220/00—Aspects relating to sorbent materials
  • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
  • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2220/00—Aspects relating to sorbent materials
  • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
  • B01J2220/58—Use in a single column
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/16—Surface properties and coatings
  • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
  • B01L2300/163—Biocompatibility

Definitions

• the present invention relates to a phosphorylcholine group-containing compound and a surface modifier comprising the compound.
• the present invention provides a novel compound containing a phosphorylcholine group, a surface modifier comprising the compound, a modified powder modified with the surface modifier, and the modified powder as a carrier.
• the present invention also relates to a chromatography filler, a filter modified with the surface modifier, and a glass laboratory instrument.
• the surface modifier comprising a compound of the present invention imparts biocompatibility, moisture retention, and other various useful functions to an object.
• a polymer having a phosphorylcholine group has been studied as a biocompatible polymer, and a biocompatible material obtained by coating the polymer with various bases has been developed.
• Patent Document 1 discloses that a powder coated with a homopolymer or a copolymer of metachloroxyshethyl phosphorylcholine is used as a cosmetic powder to improve moisture retention and skin adhesion.
• the disclosed cosmetics are disclosed.
• Patent Documents 2 and 3 disclose a medical material and a separating agent coated with a polymer having a phosphorylcholine group.
• the above materials are obtained by reacting an acrylic monomer mainly having a hydroxyl group with 2-chloro-1,3,2-dioxaphosphorane 2-oxide, and further forming a quaternary ammonium with trimethylamine.
• Patent Document 4 discloses a copolymer of 2 methacryloxyshethyl phosphorylcholine and ester of methacrylic acid
• Patent Document 5 discloses a homopolymer of 2 methacryloxyshethyl phosphorylcholine. Being manufactured.
• GFC fillers for separating biological samples such as proteins and polypeptides having a smaller molecular weight than proteins by size exclusion.
• This GFC Fillers for use include fillers using a crosslinked hydrophilic polymer as a carrier and fillers using silica gel as a carrier.
• a filler containing a crosslinked hydrophilic polymer as a carrier has high versatility, as the pH range of the applicable mobile phase is wide.
• a filler using a polymer as a carrier is more difficult to obtain a high theoretical plate number than a filler using silica gel as a carrier because (1) the pore size is difficult to control.
• (2) the reproducibility is high due to the poor strength under high pressure conditions used in high performance liquid chromatography (HPLC) and the swelling of the particles by the mobile phase solvent. I can't get it! / Many things!
• the packing using silica gel as a carrier has a problem in that proteins and polypeptides are adsorbed on the surface of the silica gel carrier. Therefore, in order to suppress the adsorption of proteins and polypeptides in an analysis sample to silica gel, a filler using a silica gel whose surface is modified with a non-dissociable hydrophilic group is commercially available.
• Shodex P ROTEIN KW-803 (product name) is commercially available from Showa Denko KK as a silica gel GFC column.
• This silica gel column is described as a silica gel GFC mode column suitable for the analysis of proteins with molecular weights of several thousand to one million.
• YMC Co., Ltd. sells YMC-Pack Diol (product name).
• This is also a silica gel column for GFC, in which a functional group having a diol structure is chemically bonded to a silica gel carrier, and it is described that it can be applied to the separation of hundreds of thousands of proteins with a molecular weight of 10,000.
• Non-Patent Document 1 describes that phosphorylcholine groups chemically grafted on a carrier reduce adsorption of proteins.
• Patent Documents 6 and 7 disclose an organosilane-based surface modifier (silane coupling agent) which is known to exhibit excellent hydrophilicity and has a pervetine structure. hand! RU
• a silane cup having a sulfobetaine consisting of a quaternary ammonium-positive charge and a sulfonic acid-negative charge is obtained by reacting dimethylaminoalkylsilane with 1,3-propane sultone in an organic solvent. It is said that a ring agent can be obtained.
• Patent Document 7 discloses a silane cappuri having a quaternary ammonium and carboxybetaine which also has a carboxyl group power. A method for producing a coating agent is described.
• silane coupling agents can be applied to glass or the like and dried to modify the material surface.
• betaine with these structures can impart excellent hydrophilicity to the material surface, it does not become electrically neutral due to the bias of the positive and negative charges in betaine.
• sulfobetaine is negatively charged due to the strong acidity of sulfonic acid, and carboxybetaine exhibits a positive charge due to quaternary ammonium.
• Such a betaine structure causes too strong an ion exchange interaction with the protein, resulting in irreversible adsorption of the protein.
• silane coupling agents have been used as a filler for chromatography, a class of filters, and laboratory instruments for the purpose of suppressing biocompatibility and protein adsorption.
• Patent document 1 JP-A-7-118123
• Patent Document 2 Japanese Patent Application Laid-Open No. 2000-279512
• Patent Document 3 JP-A-2002-98676
• Patent Document 4 JP-A-9 3132
• Patent Document 5 JP-A-10-298240
• Patent Document 6 JP-A-5-222064
• Patent Document 7 JP-A-63-295593
• Non-patent literature l Jian R. Lu et al., Langmuir 2001, 17, 3382-3389
• amino groups when an amino group is first introduced into the surface of an object and then an aldehyde derivative of phosphorylcholine is reacted with an amino group on the surface of the object, many unreacted amino groups remain. These residual amino groups can be blocked to some extent by bonding another low molecular weight compound. It is difficult to maintain the hydrophilicity of the surface of the force object, and not all of them can be blocked.
• amino groups When amino groups remain on the surface of a substance, amino acids have strong basicity, so mainly acidic proteins show remarkably strong ion-exchange interactions, and most of them are adsorbed. . When it is used as a packing material for chromatography, it may cause a decrease in the recovery rate of the protein and a significant tailing of the peak. Furthermore, adsorption of protein causes its denaturation, and it is not preferable to use it as a biocompatible material because it causes inflammation and the like.
• the present invention provides a simple and highly versatile method for directly reacting a compound containing a phosphorylcholine group with an object having a functional group that reacts with the compound and having a functional group that forms a bond with the surface of the object. It has been found that any desired amount of phosphorylcholine groups can be added directly to the surface of an object.
• a modified powder, a packing material for chromatography using the modified powder as a carrier, a filter modified by the surface modifier, and a glass laboratory device are produced by the surface modifier comprising the compound. They have found that they can be easily manufactured, and have completed the present invention. Means for solving the problem
• the present invention provides a phosphorylcholine group-containing compound represented by the following formula (1).
• X, X and X each independently represent a methoxy group, an ethoxy group or a halogen.
• X, X, X are methyl, ethyl, propyl, isopropyl,
• R is any of the structures in the following formulas (2) to (4) (however, in the structures of the following formulas (2) to (4), the compound of the formula (1) is represented by A—R—B).
• L represents 1-6 and P represents 1-3.
• the present invention also provides a phosphorylcholine group-containing conjugate represented by the following formula (5) or (6).
• m is 2, 4, 6, and n is 1 to 4.
• X, X and X each independently represent a methoxy group, an ethoxy group or a halogen. However, up to two of X, X, X are methyl group, ethyl Group, propyl group, isopropyl group, butyl group and isobutyl group.
• the present invention also provides a surface modifier comprising the above-mentioned phosphorylcholine group-containing compound.
• the present invention also provides a compound having a phosphorylcholine group and a carboxyl group by an oxidation reaction of sodium periodate of glycerol phosphorylcholine with ruthenium trichloride, and an organosilane conjugate having an amino group and a phosphorylcholine.
• the present invention provides a method for producing a compound according to the above formula (6), which is synthesized from a compound having a group and a carboxyl group by a condensing agent.
• the present invention provides a modified powder treated with the above surface modifier.
• the present invention also provides a packing material for chromatography which is treated with the above-mentioned surface modifier and has a modified carrier strength.
• the present invention provides a filter treated with the above-mentioned surface modifier.
• the present invention also provides a glass laboratory tool surface-treated with the above surface modifier.
• a phosphorylcholine group with extremely low adsorption of proteins and polypeptides can be introduced easily and quantitatively without impairing the fine structure of the object surface. Further, since an unreacted functional group other than a phosphorylcholine group is not introduced, a material having extremely high biocompatibility can be provided.
• ion-exchange and hydrophobic can be for as possible exits can be adjusted depending on the salt concentration and P H of the mobile phase, a unique separation depending on the protein.
• proteins do not cause irreversible adsorption on the powder surface, separation, fractionation, and analysis are possible without denaturing or deactivating proteins.
• FIG. 1 is a structural formula and a 1H-NMR ⁇ vector of a compound prepared in Synthesis Example 1.
• FIG. 2 is a 13 C-CPMAS spectrum of the modified powder produced in Example 2.
• FIG. 3 is a 31 P-CPMAS spectrum of the modified powder produced in Example 2.
• FIG. 4 is an FT-IR ⁇ vector of the modified powder produced in Example 2.
• FIG. 5 is a calibration curve when the packing material for liquid chromatography produced in Example 3 is used in a GFC mode.
• FIG. 6 is a chromatogram obtained when a human serum protein was separated using the packing material for liquid chromatography produced in Example 3.
• FIG. 7 is a chromatogram obtained when human serum proteins were separated at a mobile phase salt concentration of 500 mM.
• A A filler synthesized using the surface modifier of the present invention.
• B Shodex PROTEIN KW803.
• FIG. 8 is a chromatogram obtained when human serum proteins were separated at a mobile phase salt concentration of 150 mM.
• A A filler synthesized using the surface modifier of the present invention.
• B Shodex PROTEIN KW803.
• FIG. 9 is a chromatogram obtained by separating five kinds of organic acids using a filler synthesized using the surface modifier of the present invention.
• FIG. 10 shows a human obtained by inserting a secondary amine into a part of a spacer of the surface modifier of the present invention. It is a chromatogram at the time of separating a serum protein.
• FIG. 11 is an FT-IR ⁇ vector of the modified powder synthesized in Example 9.
• FIG. 12 is a chromatogram of a packing material for liquid chromatography produced in Example 10.
• FIG. 13 is a chromatogram obtained by using a packing material for liquid chromatography produced in Example 3.
• FIG. 14 is a chromatogram of a filler for liquid chromatography, the surface of which has been modified by the conjugate shown in formula (14) produced in Comparative Example 2.
• FIG. 15 is a chromatogram of a liquid chromatography packing material surface-modified with the compound represented by the formula (15) produced in Comparative Example 2.
• FIG. 16 is a 1 H-NMR ⁇ vector of the compound prepared in Example 7.
• FIG. 17 is a Mass spectrum of the compound obtained in Example 7.
• FIG. 18 is a Mass spectrum of the compound prepared in Synthesis Example 1.
• the compound of the present invention can be surface-modified regardless of purification or non-purification, and can provide effects such as suppression of protein adsorption.
• the phosphorylcholine group-containing compound shown by the following formula (1) or (5) or (6) is a new compound.
• n is 1-4.
• X, X and X each independently represent a methoxy group, an ethoxy group or a halogen.
• up to two of X are methyl, ethyl, propyl, isopropyl,
• R is any of the structures in the following formulas (2) to (4) (however, in the structures of the following formulas (2) to (4), the compound of the formula (1) is represented by A—R—B).
• L represents 1-6 and P represents 1-3.
• X, X and X each independently represent a methoxy group
• a phosphorylcholine derivative represented by the following formula (7) is dissolved in distilled water.
• the phosphorylcholine derivative of the following formula (7) is a known compound and can be obtained as a commercial product. (7)
• the method for purifying the compound of the present invention is not limited to the following.
• the surface modifier comprising the compound of the present invention can be used as it is at the stage of the methanol solution before purification.
• methanol water, an alcohol such as ethanol, propanol, and butanol, and an aprotic solvent such as N, N-dimethylformamide / dimethyl sulfoxide can be used.
• an alcohol such as ethanol, propanol, and butanol
• an aprotic solvent such as N, N-dimethylformamide / dimethyl sulfoxide
• a dewatering solvent is preferable.
• the reaction is carried out by changing methanol to ethanol.
• the reaction is changed to dimethylformamide dimethyl sulfoxide.
• the compounds of the above formulas (5) and (6) are useful as surface modifiers for substances. That is, a desired amount of phosphorylcholine groups can be easily introduced into the surface of a substance for modification. Specifically, in the case of a substance having a hydroxyl group on the surface, a chemical bond is formed by a dehydration reaction between the hydroxyl group on the surface of the substance and the compound of the formulas (5) and (6) by Si-OCH force. This
• the chemical reaction proceeds very readily and quantitatively in most organic solvents at temperatures between 10 ° C and 250 ° C.
• surface modification with a phosphorylcholine group that is extremely stable chemically and physically can be performed.
• the dehydration reaction causes Si—O—Si bonds to form, which can cover the surface of the material.
• the reaction of dehydration between Si-OCHs to form Si—O—Si bonds is known.
• the film thus formed during the volatilization of methanol is traced on most material surfaces. Since a bond is generated in some places with an existing hydroxyl group, a surface modification method with good stability can be obtained. This method is an extremely effective surface modification method not only for substances that do not have hydroxyl groups, but also for substances that have hydroxyl groups.
• the substance (or raw material) modified by the surface modifier of the present invention is a material or molded article having excellent biocompatibility and hydrophilicity.
• a material having a biocompatible phosphorylcholine group directly on the surface it can be applied to a wide range of applications such as cosmetics, medical materials (artificial organs, surgical instruments, etc.), fillers for mouth mats, and paints.
• the surface modifier of the present invention is used as a method for modifying members that come into contact with a test liquid, such as pipes for separation or analysis equipment, pipe connection parts, one dollar for sampling, sample vials, and detector cells. It is useful, and particularly, materials such as connection pipes for HPLC, MS, and NMR and capillary pipes of an electrophoresis apparatus are preferably modified. Materials such as Teflon (registered trademark) pipes, Tefzel pipes, Peak resin pipes, and fused silica pipes.
• Phosphorylcholine derivatives are extremely hydrophilic and have extremely low solubility in organic solvents. Phosphorylcholine derivatives are synthesized using dioxaphospholane as a starting material, and glycerol phosphorylcholine obtained by hydrolysis of phosphatidylcholine, a phospholipid contained in soybeans, as a starting material. The law is roughly divided. The synthesis route for phosphorylcholine derivatives is limited by the limited amount of organic solvent in which they can be dissolved, and the production cost is high, which is a barrier to practical use. The problem of the complexity and cost of this synthesis is noticeable in the total synthesis method.
• a phosphorylcholine derivative having a carboxyl group can be produced very easily and in a high yield in a good solvent for the derivative of phosphorylcholine.
• the compound represented by the formula (6) can be obtained simply and at a high yield.
• Glycetophosphorylcholine, sodium periodate, and ruthenium trichloride (hydrate) are added to an aqueous solution of acetate nitrile. After stirring at room temperature, the mixture is filtered, and the solvent is removed from the filtrate. Gain The target substance is extracted from the obtained solid with methanol, and then methanol is distilled off to obtain a phosphorylcholine derivative having a carboxyl group represented by the following formula (9).
• FIG. 16 shows the structural formula and the NMR spectrum
• FIG. 17 shows the Mass spectrum.
• the reaction solvent can be water, and it is also possible to use other periodate or periodate other than periodate.Other than ruthenium trichloride, other divalent and Z or trivalent ruthenium compounds and hydrates thereof can also be used.
• the reaction solvent can be other than methanol, such as N, N-dimethylformamide, dimethylsulfoxide, and chloroform.
• DCC dicyclocarbodiimide
• CDI carboxydiimidazole
• the reaction is carried out by changing the solvent to ethanol, and in the case of C1, the reaction is changed to dimethylformamide / dimethyl sulfoxide.
• two or one of the methoxy group, ethoxy group, or C1 bonded to Si may be substituted with any of a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, and an isobutyl group. It can be manufactured in exactly the same manner as described above.
• Modified powder The surface modifier of the present invention can be preferably used to modify a powder having a hydroxyl group.
• the modified powder of the present invention is produced by the following method. According to this method, a modified powder having a phosphorylcholine group directly on the powder surface, that is, a modified powder in which the phosphorylcholine group is introduced into the powder surface by a chemical bond can be easily produced.
• the modified powder has an advantage that the phosphorylcholine group is not lost due to peeling of the polymer as compared with a powder in which the phosphorylcholine group is introduced by coating with a polymer having a phosphorylcholine group. Have. Also, since it is not coated with a polymer, there is an advantage that the fine structure of the powder itself is not impaired. Specifically, by using the surface modifier of the present invention, the surface can be coated with a phosphorylcholine group without filling the three-dimensional microstructure (micropores, etc.) of the powder surface of several nm. Is possible.
• a functional group capable of reacting with the phosphorylcholine group derivative is first introduced into the substance.
• unreacted functional groups remain on the object surface, which leads to a decrease in biocompatibility.
• an amino group is first introduced into the surface of an object and then an aldehyde derivative of phosphorylcholine is reacted with an amino group on the surface of the object, many unreacted amino groups remain.
• These residual amino groups can be blocked to some extent by binding another low molecular compound, but it is difficult to maintain the hydrophilicity of the surface of the object, and not all of them can be blocked.
• acidic proteins When many amino groups remain on the surface of a substance, acidic proteins mainly show remarkably strong electric interactions, and most of them are adsorbed because amino groups have strong basicity. . If it is regarded as a packing material for chromatography, it may cause deterioration of the recovery rate of the protein and cause tailing of peaks. Furthermore, protein adsorption causes its denaturation, and if it is considered as a biocompatible material, it may cause inflammation and the like, which is not preferable.
• an aldehyde derivative of phosphorylcholine is added in excess to the organosilane compound having an amino group during the synthesis, and both are reacted in a liquid phase.
• the reactivity between the amino group and the aldehyde group is extremely high. It is known that 100% of the amino groups react with the aldehyde. Therefore, unreacted amino groups were not detected in the surface modifier according to the present invention. Therefore, in the surface modification according to the present invention, only a phosphorylcholine group can be introduced without mixing unreacted amino groups on the object surface. This makes it possible to obtain a powder having extremely excellent biocompatibility and little protein adsorption as compared with a method in which a two-step reaction is performed on a solid phase.
• the powder used is not particularly limited. Depending on the application, it generally means any object with an average particle size of 0.01-m or 0.01-1000 m.
• Specific powders include, for example, inorganic powders (e.g., talc, kaolin, mica, sericite (sericite), muscovite, phlogopite, synthetic mica, rhodolite, biotite, permikilite, carbonate Magnesium, calcium carbonate, aluminum silicate, barium silicate, calcium silicate, magnesium silicate, strontium silicate, metal tungstate, magnesium, silica, zeolite, barium sulfate, calcined calcium sulfate (baked calcium sulfate), Organic powder (for example, polyamide resin); calcium phosphate, fluorine apatite, hydroxyapatite, ceramic powder, metal stone (eg, zinc myristate, calcium palmitate, aluminum stearate), boron nitrid
• a powder containing a hydrophilic phosphorylcholine group in an arbitrary amount can be easily obtained.
• the powder is a synthetic polymer
• its hydrophilic part includes carboxylic acid groups, hydroxyl groups, primary and tertiary amino groups, sulfonic acid groups, phosphoric acid groups, polyoxyethylene groups, ammonium groups, and amide groups.
• the function of the powder can be designed with these types and contents which may contain carboxybetaine, saccharides and the like.
• hydrophobic portion examples include linear or branched alkyl having 2 to 22 carbon atoms, cyclic alkyl such as cholesterol, alkyl groups containing unsaturated bonds such as oleyl, benzene ring, naphthalene ring, and pyrene. Depending on the intended use of the powder, it may contain hydrocarbon-based aromatics, pyridine rings, heteroaromatics such as imidazole, thiazole and indole, and hydrophobic groups such as perfluoroalkyl and polyalkylsiloxane. You can choose and design.
• the bonding form of the hydrophobic group of the synthetic polymer powder may be directly bonded to the polymer main chain by an ester, ether, amide, urethane, urea bond, or the like, or may be bonded to the main chain via a spacer. May be.
• the spacer include hydrophilic polyethylene oxide, hydrophobic polypropylene oxide, and linear alkyl (2 to 22 carbon atoms).
• the modified powder of the present invention is a powder excellent in hydrophilicity and moisture retention. As a biocompatible powder, it can be applied to a wide range of applications such as cosmetics, medical materials, chromatographic fillers, and paints. “Modified carrier that has been treated with a surface modifier to be used as a chromatographic filler” The surface modifier of the present invention modifies the surface of a carrier to easily have a desired amount of phosphorylcholine groups. A packing for chromatography can be manufactured.
• a phosphorylcholine group is introduced into the carrier surface by a dehydration reaction between a hydroxyl group present on the carrier surface and Si—OCH of the compounds of the formulas (5) and (6).
• the average particle diameter was 5 ⁇ m
• the average pore diameter was 300 ⁇
• the specific surface area was 100 m.
• the reaction solvent can be a water-methanol mixed solvent, a protic solvent such as water, ethanol, or 2-propanol, or an aprotic solvent such as dimethyl sulfoxide, dimethylformamide, toluene, or ethyl ether. Or it can be used in combination.
• a method of dissolving the compounds of formulas (5) and (6) in a volatile solvent, applying the solution to the surface of the substance, and then drying the solvent is effective. It is. Specifically, an appropriate amount of a methanol solution (0.3 mmol / mL) of the compound of formulas (5) and (6) is directly applied to the substance according to the specific surface area of the substance. Next, methanol is vaporized in the temperature range of 10 ° C to 250 ° C. At this time, the Si—OCH of the compounds of formulas (5) and (6)
• the biggest difference from the method using is that there is no unreacted amino group on the surface of the object. That is, when the surface modifier according to the present invention is used, only phosphorylcholine groups can be introduced without mixing unreacted amino groups on the surface of the substance. If an amino group is first introduced to the powder surface, the second step of introducing the phosphorylcholine group must react the aldehyde form of glycerol phosphorylcholine in the liquid phase with the amino group on the solid surface.
• reaction rate is low due to diffusion control, steric hindrance due to the steric structure of the solid phase surface, and steric properties of the phosphorylcholine group itself.
• a phosphorylcholine group can be introduced into only about 30% of the amino groups.
• the remaining amino group can be blocked to some extent by bonding another low molecular weight compound, but it is difficult to maintain the hydrophilicity of the object surface, and it is not possible to block all of the remaining amino groups.
• the amino group When a large number of amino groups remain on the surface of the substance, the amino group has a strong and basic property, so that mainly acidic proteins exhibit remarkably strong electric interaction, Most of them are absorbed. When it is used as a packing material for chromatography, it may cause poor recovery of the protein and remarkable tailing of peaks. In addition, protein adsorption leads to its denaturation and, if considered as a biocompatible material, causes inflammation and is not preferred.
• the surface modifier according to the present invention is prepared by adding an aldehyde derivative of phosphorylcholine excessively to an organosilane conjugate having an amino group at the time of synthesis, and combining both in a liquid phase. Let react.
• the reactivity between the amino group and the aldehyde group is very high. It is generally known that almost 100% of the amino group reacts with the aldehyde when the aldehyde is added in excess.
• the carrier used in the present invention includes an inorganic porous material such as silica, silica gel, activated carbon, zeolite, alumina, and clay mineral, and a porous organic polymer resin.
• the carrier is preferably a powder.
• spherical or crushed porous silica gel is used.
• the average particle size of the spherical porous silica gel is 11 to 200 ⁇ m, preferably 1 to 10 ⁇ m, and the average diameter of the pores of the spherical porous silica gel is 10 to 2000 ⁇ , preferably 80 to 1000 ⁇ .
• the area is 0.01-800 m 2 Zg, preferably 80-600 m 2 Zg.
• the packing material for chromatography of the present invention is used as a column for GFC, the adsorption capacity of proteins and polypeptides is extremely low and the separation ability is exhibited.
• the present invention can be applied to a mode in which proteins and polypeptides are separated based on differences in molecular weight (GFC mode).
• the packing material for chromatography of the present invention is based on not only the difference in the molecular weight of the sample due to the double charge of the phosphorylcholine group but also the difference in the weak charge of the sample! /, It is a column packing material with higher separation ability. As described above, there is no example in which a functional group having a bicharge is introduced to suppress the adsorption of proteins.
• the packing material for chromatography produced by the surface treatment agent of the present invention is a new colorant for GFC. Filler.
• the GFC mode can separate and purify proteins and enzymes without deactivating them, so the high separation power of the column packing material of the present invention is useful for the isolation of unknown biological samples and medical applications. Is expected.
• the packing material for chromatography of the present invention is, specifically, a ram packing material having a high separation ability with very little adsorption of proteins and polypeptides, for example, separation of proteins in human serum or protein separation. It is excellent for separation of polypeptides contained in a sample obtained by digesting Escherichia coli with trypsin, or separation and fractionation based on evaluation of the activity of an unknown protein contained in a living body.
• a phosphorylcholine group having an excellent ability to suppress the adsorption of water can be introduced.
• the compounds of formulas (5) and (6) are dissolved in a volatile solvent, the filter or the material is immersed in the solution, and then the solvent is removed. Drying and washing are effective.
• methanol of the compounds of formulas (5) and (6) The solution directly (0.3 mmol ZmL) into an appropriate amount according to the specific surface area of the substance. Next, methanol is vaporized in a temperature range of 10 ° C to 250 ° C. At this time, the Si—OCH of the compounds of the formulas (5) and (6) cause a dehydration reaction to form a Si—O—Si bond, and the material surface
• the glass laboratory device is a laboratory device such as a storage container, a measuring device, a cell, a dispensing tip, a sample dispensing syringe, and the like.
• a laboratory device such as a storage container, a measuring device, a cell, a dispensing tip, a sample dispensing syringe, and the like.
• Table 1 shows the elemental analysis values of the modified powder treated with the surface modifying agent of Example 1 in the above procedure.
• C% or N% in the table is the quality of carbon or nitrogen element contained in powder. % Is shown. From this value, the atomic ratio (CZN) of carbon and nitrogen in the powder after the treatment with the surface modifier of Example 1 was 5.08.
• the CZN of the surface modifiers of formulas (10) and (11) after all the methoxy group sites are bonded is 5, indicating that the surface modifier was introduced into the powder without being destroyed. Is shown.
• FIG. 2 shows the 13C-CPMAS spectrum and 13C-PSTMAS spectrum of this silica gel.
• the PSTMAS spectrum is a technique for selectively obtaining the spectrum of a free-moving molecular chain, and is widely used for analyzing modified chains on the powder surface.
• a spectrum due to carbon of the choline group is observed at 54.2 ppm.
• FIG. 4 shows an FT-IR spectrum of the modified powder synthesized in this example. An absorption characteristic of the amide bond was observed at around 1650 cm- 1 .
• Example 2 Using the modified powder produced in Example 2 as a carrier, by a usual slurry method, an inner diameter of 4.6 m m, packed into a 250 mm long empty column.
• the conditions for obtaining the chromatogram are as follows.
• FIG. 5 shows a calibration curve when the column of Example 3 was used under these conditions.
• the calibration curve shown in Figure 5 has extremely good linearity over the measurement range. It can be seen that the interaction between the surface of the packing material and the protein was extremely small due to the small amount of protein adsorption, and as a result, the separation was performed in the GFC mode, in which the molecular weight of the molecular weight was large and the elution was fast. Next, the results of separating human serum proteins under the above conditions are shown in FIG.
• Concealer N product name
• 2 ⁇ L was injected.
• Actual samples such as human serum were separated in GFC mode in order of molecular weight, demonstrating very high practicality.
• This filler is described as a filler for a size exclusion mode in which a hydrophilic group is bonded to the surface of porous silica gel. It has an average pore diameter of 300 angstroms and an average particle diameter of 5 m, and is suitable for comparison with the filler described in Example 1. It is described as suitable for separating proteins with a molecular weight of 10,000 to hundreds of thousands.
• Shodex PROTEIN KW803 uses a non-dissociative hydrophilic group, unlike the filler prepared using the surface modifier of the present invention, it has a charged functional group. . Therefore, it is considered that there is almost no ionic interaction with the protein.
• a 50 mmol Zl phosphate buffer (prepared from NaHPO and KHPO) was used. Using a mobile phase prepared by adding 500 mmol / l sodium salt to sodium chloride at a flow rate of 0.1 ml / min and a column oven temperature of 25 ° C. (Twofold dilution) was attempted. Detection was performed at UV280nm. The results are shown in FIG. 7 (b).
• FIG. 7 (a) shows the chromatogram of the same sample and the same condition of the filler prepared using the surface modifier of the present invention obtained in Example 3 under the same conditions.
• FIG. 8 (a) shows a chromatogram obtained when a filler prepared using the surface modifier of the present invention was applied at a salt concentration of 150 mM
• FIG. 8 (b) shows a salt of Shodex PROTEIN KW803 with salt. The chromatogram when applied at a concentration of 150 mM is shown. Conditions other than the salt concentration in FIG. 8 are the same as those in FIG.
• the molecular weights of anolebumin and tranferrin are approximately 69,000 and 75,000, respectively, and are always similar. With a conventional GFC column, it is not possible to separate a sample near the molecular weight of albumin and transferrin.
• the filler prepared using the surface modifier of the present invention has very low protein adsorption, and has a weak ionic interaction with the protein due to the dual charge of the phosphorylcholine group, which is not only a protein having a similar molecular weight. It can also be seen that the separation can be performed based on the difference between the isoelectric point and the hydrophobicity.
• the filler prepared using the surface modifier of the present invention has a weak ion exchange interaction. This can be confirmed by lowering the salt concentration of the mobile phase.
• transferrin is such that transferrin is recognized as a shoulder of the albumin peak even in the filler prepared using the surface modifier of the present invention.
• FIG. 8 (a) with the salt concentration reduced to 150 mM the peaks for transferrin and albumin achieve baseline separation.
• FIG. 8 (a) peaks due to many proteins can be confirmed.
• the filler prepared using the surface modifier of the present invention caused strong retention especially in albumin by reducing the salt concentration of the mobile phase, and achieved complete separation from transferrin. Since albumin, which is negatively charged at neutral pH as shown in Fig. 7 and Fig. 8, was maintained at a low salt concentration, the filler prepared using the surface modifier of the present invention was an iron-based filler. It is presumed that there is an exchange mode!
• the powder treated with the surface modifier of the present invention Can be said to be very effective as a filler for aeon exchange.
• the above-mentioned ion exchange properties are not strong enough to adsorb and denature proteins and are suitable for unique separation with high recovery.
• the filler prepared using the surface modifier of the present invention is a very unique filler that has an ion exchange mode in addition to the GFC mode (size exclusion mode) due to its excellent function of suppressing protein adsorption. . Since protein adsorption is extremely low, it is possible to separate and purify proteins while maintaining enzyme activity without denaturing proteins. Because of the coexistence of ion exchange mode as well as GFC mode, it is an extremely innovative packing material that can control separation according to the salt concentration and pH of the mobile phase.
• Fig. 10 (b) silica gel introduced with formulas (12) and (13) was filled by a usual slurry method, and a human serum sample (Consera N (product name) was doubled with distilled water). (Diluted) is shown.
• FIG. 10 (a) is a chromatogram when the powder prepared in Example 3 is used.
• Example 3 is different from Example 3 in that the number of secondary amines in a part of the spacer of the surface modifier is different.
• the silica gel used in FIGS. 10 (a) and 10 (b) is the same.
• FIG. 10 (b) having one more secondary amine between the silicon atom and the phosphorylcholine group than in FIG. 10 (a) it can be seen that the separation of transferrin and albumin is further improved. This is thought to be because the basicity of the modifying group was increased by the insertion of the secondary amine between the silicon atom and the phosphorylcholine group, and the acidic protein albumin was more strongly retained.
• the surface modifier of the present invention not only has the effect of suppressing protein adsorption by the phosphorylcholine group but also changes the properties of the spacer between the silicon atom and the phosphorylcholine group, thereby improving the ion exchange property and hydrophobicity. It is possible to impart an interaction such as hydrophilicity and hydrogen bonding.
• Example 5 borosilicate glass fiber filter material
• a 100 mL Erlenmeyer flask was charged with 20 g of distilled water and 1. OmL of a methanol solution containing the compounds of formulas (10) and (11) (about 0.4 mmol) produced in Example 1 and shaken.
• a borosilicate glass fiber filter (glass fiber filter grade GF / F, diameter 25mm ⁇ , about 0.070g per sheet) After adding 8 pieces, 100. Heated to C and reflux boiled for 5 hours. After cooling to room temperature, the filter was filtered, washed, and dried under reduced pressure at 80 ° C. for 3 hours to obtain a borosilicate glass fiber filter having a phosphorylcholine group directly on the surface.
• BSA Dissolve serum albumin
• the present invention can provide a filter material with extremely low adsorption of proteins and polypeptides.
• the filter material of the present invention is useful for a wide range of filtration of biological substances such as separation and concentration of antibodies and enzymes, hemodialysis, blood purification and analysis of blood filters, and the like.
• a 100 mL Erlenmeyer flask was charged with 20 g of distilled water and 1. OmL of a methanol solution containing the compounds of formulas (10) and (11) (about 0.4 mmol) produced in Example 1 and shaken. After adding 10 sets of glass vials made of Nippon Otters Co., Ltd., 12 ⁇ 32 mm glass vials, the mixture was heated to 100 ° C. and boiled under reflux for 5 hours. Vial after cooling to room temperature Was washed with methanol and dried under reduced pressure at 80 ° C. for 3 hours to obtain a glass vial having phosphorylcholine groups directly on the surface.
• the surface modifying agent of the present invention makes it possible to obtain a glass experimental device having very little protein adsorption. It is known that in a general vial in which protein adsorption cannot be suppressed, the protein sample concentration decreases with time during an experimental operation. Specifically, it is known that when a sample is injected into a liquid high-performance chromatography, the peak area decreases over time even when the same volume is injected each time. Since the glass vial manufactured in this example is excellent in suppressing protein adsorption, such a phenomenon can be avoided.
• Example 7 Production of phosphorylcholine derivative having a carboxyl group
• FIG. 16 shows the 1 H NMR spectrum
• FIG. 17 shows a Mass spectrum of the compound of the formula (9).
• the surface modifier of the present invention can fix a phosphorylcholine group on a substrate without purification. However, it can be purified, for example, by the following method.
• Capsule Pack SCX UG80 S—5 size: 4.6 mmi. D. X 250 mm
• a high-performance liquid chromatography column with hydrophobic interaction and cation exchange capacity was connected to an HPLC system.
• 0.2 mmol / L phosphate buffer (pH 3.5) in ImL After flowing at a flow rate of Z and equilibrating, 10 L of the sample is injected.
• a differential refractometer as a detector, a chromatogram can be obtained, and a target compound can be isolated.
• Example 9 Modified powder treated with an organosilane compound having an amide bond in a spacer and having a phosphorylcholine group at a terminal
• the elemental phosphorus in the modified powder having a phosphorylcholine group on the surface produced in Example 9 was quantified.
• the phosphorus element is an element peculiar to the phosphorylcholine group, and the introduction of the phosphorylcholine group can be actually confirmed by quantifying the phosphorus element present on the powder surface.
• the determination of the phosphorus element was performed by a molybdic acid coloring method.
• the quantification method will be described below.
• step 3 Centrifuge the solution in step 3 (3000 rpm, 5 minutes), and transfer the supernatant to an lmL sample tube.
• the phosphorus element of the modified powder having a phosphorylcholine group on the surface produced in Example 9 was 0.13 mmol / g. That is, 0.13 mmol / g of phosphorylcholine groups
• Fig. 11 shows an FT-IR spectrum of the modified powder synthesized in this example.
• Example 10 Filler for liquid chromatography treated with a surface modifier (silane coupling agent) having an amide bond in the spacer and having a phosphorylcholine group at the end”
• a surface modifier silane coupling agent
• an empty column having an inner diameter of 4.6 mm and a length of 250 mm was packed by a usual slurry method.
• the conditions for acquiring the chromatogram are as follows.
• a2 macroglobulin 0.67 mgZmL (molecular weight about 800,000, abbreviation ⁇ 2 ⁇ ), ⁇ -globulin 1.3 mgZmL (molecular weight about 160,000, abbreviation ⁇ G), human serum albumin 1.7 mgZmL ( Chromatography when 2 L of a water solution sample containing a mixture of 0.3 mgZmL of lysozyme (molecular weight of about 70,000, abbreviation HSA), 0.3 mgZmL of lysozyme (molecular weight of about 14,000, abbreviation LYZ), and 0.017 mgZmL of peracyl (molecular weight of 112, abbreviation U) was injected.
• the gram is shown in FIG. a 2M, ⁇ -GHSA and LYZ were obtained from Sigma-Aldrich Japan Co., Ltd., and Peracil was obtained from Nakarai Testa Co., Ltd. Five peaks were clearly identified, and these were identified from the elution time of a commercially available single sample, and found to be a2 ⁇ , ⁇ G, HSA, LYZ, and U in the order of elution order. Small peaks observed in addition to the five peaks are impurities contained in commercially available standard samples.
• Phosphorylcholine group is immobilized on silica gel via amide bond
• the interaction between the surface of the packing material and the protein was extremely small due to the small amount of the protein adsorbed, and as a result, the separation was performed in the GFC mode in which the molecular weight was large and the molecular force was quickly eluted.
• the amide bond has good hydrophilicity. From the viewpoint of suppressing protein adsorption, it is desirable to cover the material surface with a hydrophilic and non-ionic functional group. Phosphorylcholine groups have extremely low ionicity due to the extremely good hydrophilicity inherent in zwitterions and the balance of the charge balance of zwitterions, indicating that they are functional groups that are excellent at suppressing protein adsorption. I can say. While applying force, the spacer used for immobilizing the phosphorylcholine group on the surface of the substance has high hydrophobicity, and induces nonspecific irreversible adsorption of the protein. Therefore, the presence of an amide bond in the spacer is extremely important and realizes a more hydrophilic surface modification. The hydrophilicity including the spacer is extremely effective in suppressing protein adsorption.
• Japanese Patent Application Laid-Open No. 5222064 discloses a silane compound having a sulfobetaine represented by the formula (14). Further, Japanese Patent Application Laid-Open No. 63-295593 discloses a silani conjugate having carboxybetaine represented by the formula (15).
• silica gel having an average particle diameter of 5 m, an average pore diameter of 30 nm, and a specific surface area of 140 m 2 Zg is added to 5.Og. I got calo.
• This powder dispersion solution was refluxed at 80 ° C. for 5 hours, and the iridani compound shown in the formula (14) was fixed on the silica gel. After the reflux, the mixture was filtered and washed with 50 mL of methanol to obtain a silica gel surface-modified with the compound represented by the formula (14).
• each of the obtained surface-modified silica gels was packed into an empty column having an inner diameter of 4.6 mm and a length of 250 mm by a usual slurry method.
• the conditions for acquiring the chromatogram are as follows.
• FIG. 13 shows a chromatogram obtained by injecting 2; zL of an aqueous sample mixed with 017 mgZmL (molecular weight: 112, abbreviation: U).
• a 2M, yG, HSA and LYZ were obtained from Sigma-Aldrich Japan Co., Ltd., and Peracil was obtained from Nakarai Testa Co., Ltd.
• FIG. 14 shows a chromatogram obtained by injecting the same sample into a column filled with a filler in which sulfobetaine represented by the formula (14) was immobilized.
• FIG. 15 shows a chromatogram obtained by injecting the same sample into a column filled with a filler in which carboxybetaine represented by the formula (15) was immobilized.
• carboxybetaine represented by the formula (15) was immobilized.
• the five types of peaks eluted better than the column in which sulfobetaine was immobilized, and eluted in the order of ⁇ 2 ⁇ , ⁇ -glopurin, lysozyme, peracil, and human serum albumin.
• negatively charged human serum albumin under the neutral mobile phase was characteristically retained and eluted later than the small molecule peracyl.
• Betaine structures such as the sulfobetaines and carboxybetaines mentioned in the examples have excellent hydrophilicity, but induce protein adsorption by too strong ion exchange properties.
• the phosphorylcholine group has an appropriate balance between the charges of phosphoric acid and quaternary ammonium, and has excellent hydrophilicity inherent to the betaine structure, nonionicity, and a function of suppressing protein adsorption. You can see it.
• an organosilane-based surface modifying agent having a phosphorylcholine group (silane coupling agent), which has not been reported so far, is extremely effective in modifying the surface of a substance for the purpose of protein non-adsorption.
• silane coupling agent silane coupling agent
• the novel conjugate containing a phosphorylcholine group of the present invention is useful as a surface modifier.
• the surface modifier of the present invention imparts various useful functions to a substance, such as biocompatibility, moisture retention, and the like.
• a modified powder modified with a phosphorylcholine group by the surface modifier of the present invention, a chromatography filler using the modified powder as a carrier, a filter modified with the surface modifier, Glassware modified with a surface modifier can be easily manufactured.

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