KR100848308B1 - The application using non-covalent bond between a cucurbituril derivative and a ligand - Google Patents

Abstract

The present invention provides a kit comprising a) a first component of a compound of formula (1) bound to a first material, and b) a second component of a ligand bound to a second material (the first material and the second material are each independently As solid phase, biomolecule, antioxidant, chemotherapeutic agent, antihistamine, cucurbituril dendrimer, cyclodextrin derivative, crown ether derivative, calixarene derivative, cyclophan derivative, cyclic peptide derivative, metal ion, chromophore, fluorescent substance, phosphorescence Selected from the group consisting of a substance, a radioactive substance and a catalyst, wherein the ligand may be non-covalently bonded to the cucurbituril derivative; And separating and purifying the substance bound to the ligand using the compound of formula 1 bound to the solid phase; A sensor chip comprising a combination of a compound of Formula 1 bound to a first material, and a ligand bound to a second material; Provided is a solid catalyst binder consisting of a compound of Formula 1 bonded to a first material and a ligand bound to a second material.

Description

The application using non-covalent bond between a cucurbituril derivative and a ligand}

1 schematically shows the formation of a mutually non-covalent bond between cucurbitur [7] ryl bound to a solid phase and a ligand bound to a solid phase,

Figure 2 is a polystyrene-depicts the co-hydroxy-cooker cucurbit [7] The reaction for immobilizing a reel on -DVB-NCO, - co -DVB- NH polystyrene from a _{second-reaction} for producing the NCO and polystyrene-co -DVB

3 and 4 are FT-IR spectra for polystyrene- co- DVB-NH ₂ and polystyrene- co- DVB-NCO, respectively,

5 is an FT-IR spectrum of polystyrene- co- DVB-CO-hydroxy cookerbitu [7] ryl immobilized with hydroxy cookerbitu [7] reel in polystyrene- co- DVB-NCO,

6 shows the chloromethylation and dithiolation reaction of CombiGel XE-305,

7 is FT-IR spectrum of CombiGel XE-305,

8 is an FT-IR spectrum of chloromethylated CombiGel XE-305,

9 is an FT-IR spectrum of a chloromethylated CombiGel XE-305 with dithiol immobilized,

FIG. 10 illustrates a reaction for immobilizing allyloxy cookerbitu [7] yl in CombiGel XE-305, which was subjected to chloromethylation and dithiolation.

11 illustrates a process of binding a solid phase to ferrocene,

12A and 12B show purification of BSA proteins using a complex of a solid support to which hydroxy cucurbituril (FIG. 12A) and allyloxy cookerbitu [7] ryl (FIG. 12B) are linked and an anti-BSA antibody to which ferrocene is linked. Result of electrophoresis,

FIG. 13 shows electrophoresis results of purification of a protein solution containing lysozyme (lane 2) and a protein solution containing BSA (lane 3) into a complex of an allyloxy cucurbituril-bound solid phase and a ferrocene-linked anti-BSA. 1 is the result of loading the molecular weight indicator,

14 shows the immobilization reaction of allyloxy cookerbitu [7] ryl on the gold surface,

15 is an FT-IR spectrum of ferrocenemethylene trimethylammonium iodine,

16 is an FT-IR spectrum of allyloxy cucurbituril [7] bound to a gold surface,

FIG. 17 is an FT-IR spectrum of ferrocenemethylene trimethylammonium iodine entrapped in an allyloxy cucurbituril [7] molecule bound on a gold surface, FIG.

FIG. 18 is the UV-Vis absorption spectrum of o -Dianisidine. 2HCl showing the activity of a sensor in which glucose oxidase is linked to CB [7] by ferrocene.

FIG. 19 is a calibration curve measured in glucose solution between 10 and 120 mM using glucose oxidase-coupled sensor with CB [7] and ferrocene.

20 is treated with an annexin V-ferrocene derivative to KB cells (a) and doxorubicin-treated KB cells (b) that are not treated with doxorubicin, and then treated with FITC-cookerbituryl derivatives, followed by flow cytometry analysis of FITC levels ( Flow cytometry results are shown.

The present invention comprises a first component of the compound of formula (1) bound to a first material, and b) a second component of a ligand bound to a second material (the ligand may be non-covalently bonded to the cucurbituril derivative). Kits; Separating and purifying the substance bound to the ligand using the compound of formula 1 bound to the solid phase; A sensor chip comprising a combination of a compound of Formula 1 bound to a first material, and a ligand bound to a second material; It relates to a solid catalyst binder consisting of a compound of formula (1) bonded to a first material and a ligand bound to a second material.

Host molecule materials, such as cyclodextrins (US Pat. No. 4,53399) and Crownether (Korean Patent No. 026382), have the ability to entrap multiple guest molecule compounds, thereby separating and removing the material. Has been studied for. In order to apply the host molecular materials as column fillers, they must be covalently linked to a solid substrate selected from polymers such as silica gel, zeolite, titanium oxide, and cellulose. The host molecules covalently linked to the solid substrate are used as the stationary phase of column fillers of various types of column chromatography, and are used for separation of various samples to be measured.

Cucurbituril is a newly attracted main molecule, unlike cyclodextrin and the like, may form a non-covalent bond with not only hydrophobic compounds but also various kinds of hydrophilic compounds, particularly amine-substituted biochemical compounds, as guest molecules. ( J. Am. Chem. Soc. 2001, 123 , 11316; EP 1094065; J. Org. Chem . 1986, 51 , 1440.)

The present inventors have completed the present invention by establishing various application means using non-covalent bonds of cucurbituril and specific ligands.

It is an object of the present invention to provide a kit comprising components in which a cucurbituril derivative and a ligand capable of non-covalent binding thereto are each bound to a specific substance.

Another object of the present invention is to provide a method for separating and purifying a substance bound to a ligand capable of mutually non-covalent bonding with the cucurbituryl derivative using a cucurbituryl derivative bound to a solid phase.

Still another object of the present invention is to provide a sensor chip comprising a combination of a cucurbituril derivative and a ligand, each bound to specific substances.

It is another object of the present invention to provide a solid catalyst binder consisting of a cucurbituril derivative and a ligand, each bound to specific materials.

In order to achieve the above object of the present invention, a first aspect of the present invention provides a kit comprising the following a) and b) components:

a) a first component of a compound of formula 1 bonded to a first substance; And

b) a second component of the ligand bound to the second substance

(The first substance and the second substance are each independently a solid phase, a biomolecule, an antioxidant, a chemotherapeutic agent, an antihistamine, a cucurbituril dendrimer, a cyclodextrin derivative, a crown ether derivative, a calixarene derivative, a cyclophan derivative, a cyclic Selected from the group consisting of peptide derivatives, metal ions, chromophores, fluorescent materials, phosphors, radioactive materials and catalysts,

The ligand may be non-covalently bonded to the compound of Formula 1, and having one or more amine functional groups, a C ₁ -C ₂₀ alkyl group; C ₂ -C ₂₀ alkenyl group; C ₂ -C ₂₀ alkynyl group; C ₁ -C ₂₀ alkoxy group; C ₁ -C ₂₀ aminoalkyl group; C ₄ -C ₂₀ cycloalkyl group; C ₄ -C ₇ heteroarylcyclo group; C ₆ -C ₂₀ aryl group; C ₅ -C ₂₀ heteroaryl group; C ₁ -C ₂₀ alkylsilyl group; C ₆ -C ₂₀ aryl group; C ₅ -C ₂₀ heteroaryl group; Adamantane with a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Ferrocene or metallocene having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Carborane having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Fullerenes having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Cyclic or crown ethers having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Oxygen-protected amino acids having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Peptides having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Alkaloids having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Cisplatin having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Oligonucleotides having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Rhodamine having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; And nanoparticles having a C ₁ -C ₂₀ alkyl group, a C ₆ -C ₂₀ aryl group or a C ₅ -C ₂₀ heteroaryl group):

<Formula 1>

Where

n is an integer from 6 to 10,

X is O, S or NH,

A ₁ and A ₂ are each independently H, OR, SR, or NHR,

R is H, substituted or unsubstituted C ₁ -C ₃₀ alkyl, substituted or unsubstituted C ₂ -C ₃₀ alkenyl, substituted or unsubstituted C ₂ -C ₃₀ alkynyl, substituted or unsubstituted C ₂ -C ₃₀ carbonylalkyl, substituted or unsubstituted C ₁ -C ₃₀ thioalkyl, substituted or unsubstituted C ₁ -C ₃₀ alkylthiol, substituted or unsubstituted C ₁ -C ₃₀ hydroxyalkyl, substituted or unsubstituted Substituted C ₁ -C ₃₀ alkylsilyl, substituted or unsubstituted C ₁ -C ₃₀ aminoalkyl, substituted or unsubstituted C ₁ -C ₃₀ aminoalkylthioalkyl, substituted or unsubstituted C ₅ -C ₃₀ cycloalkyl , Substituted or unsubstituted C ₂ -C ₃₀ heterocycloalkyl group, substituted or unsubstituted C ₆ -C ₃₀ aryl group, substituted or unsubstituted C ₆ -C ₃₀ arylalkyl group, substituted or unsubstituted C ₄ -C ₃₀ heteroaryl, and a substituted or unsubstituted C ₄ -C ₃₀ heteroarylalkyl group, provided that A ₁ and A ₂ are not simultaneously hydrogen.

In another aspect of the present invention, a second aspect of the present invention is a substance bound to a ligand comprising the following steps (the substance is a biomolecule, an antioxidant, a chemotherapeutic agent, an antihistamine, a cucurbituril dendrimer, a cyclodextrin derivative, Crown ether derivatives, callix arene derivatives, cyclopan derivatives, cyclic peptide derivatives, metal ions, chromophores, fluorescents, phosphors, radioactive materials and catalysts).

a) providing an affinity chromatography column packed with a stationary phase of a compound of formula 1 bound to a solid phase;

b) providing to said column a mixture comprising a substance bound to said ligand;

c) washing the column with a wash solution; And

d) providing a mobile phase solvent to the column to separate and purify the substance bound to the ligand.

In order to achieve another object of the present invention, a third aspect of the present invention is a sensor chip comprising a compound of formula (1) bonded to a first material, and a conjugate of a ligand bound to a second material (the first material and One of the second substances is a solid phase and the other is an enzyme, substrate, substrate analog, inhibitor, coenzyme, antibody, antigen, virus, cell lectin, polysaccharide, glycoprotein, cell surface receptor, including histidine, cysteine or tryptophan. , Nucleic acids, complementary sequences, histones, nucleic acid polymerases, nucleic acid binding proteins, ATP, ADP, hormones, vitamins, receptors, transport proteins, glutathione, GST fusion proteins, metal ions, polyhis fusion proteins, natural proteins and combinations thereof Selected from the group consisting of:

In order to achieve another object of the present invention, a fourth aspect of the present invention is a solid catalyst binder composed of a compound of formula 1 bonded to a first material and a ligand bonded to a second material (in this case, the first material and One of the second materials is in the solid phase and the other is the catalyst).

Hereinafter, the present invention will be described in detail.

Kit according to the invention, a) a first component of the compound of formula (1) bonded to the first material; And b) a second component of the ligand bound to the second material.

Compounds of formula (1) and ligands included in the first component and the second component, respectively, form ligands in the pupil of the cucurbituryl derivatives of formula (1) to form non-covalent bonds. The ligand may be non-covalently bonded to the compound of Formula 1.

The first substance or the second substance is not only a solid phase which is a conventional solid support, but also a biomolecule, an antioxidant, a chemotherapeutic agent, an antihistamine, a cucurbituril dendrimer, a cyclodextrin derivative, a crown ether, which is a substance which performs a specific function such as a probe substance, and the like. Derivatives, callix arene derivatives, cyclophan derivatives, cyclic peptide derivatives, metal ions, chromophores, fluorescent materials, phosphors, radioactive materials and catalysts.

In the present invention, any one of the first material and the second material is a biomolecule, the other is a chromophore, a fluorescent material or a phosphor, the kit is used to detect a specific material that binds to or reacts with the biological molecule It may further be used to analyze the presence, location or extent of such specific substances. For example, the kit may use various binding methods such as immunochemical staining and flow cytometry using the binding force of the compound of Formula 1 in the first component and the ligand in the second component. flow cytometry, in-situ hybridization, and the like.

In the kit according to the present invention, the solid phase which can be used as the first material or the second material is independently a polymer, a resin, a magnetic material, a silica gel, a polymer or a gold-coated silica gel, zirconium oxide, a monolithic polymer, and Polymer coated magnetic particles, gold thin film, silver thin film, glass, glass coated with ITO, silicon, metal electrodes, nanorods, nanotubes, nanowires, curlran gum, cellulose, nylon membranes, Sepharose and Sephadex It is preferably a solid support selected from the group consisting of a group consisting of; Most preferred is polystyrene resin or silica gel coated with polymer.

When a halogen functional group (preferably a chloro functional group) is present on the surface of the solid phase, the solid phase and the cucurbituryl derivative or ligand are formed by reacting with a functional group (for example, an amine functional group, etc.) present in the cucurbituryl derivative or ligand to form a covalent bond. It can be connected with.

In the kit according to the present invention, the biomolecules that can be used as the first or second material are enzymes, nucleic acids, proteins, amino acids, antibodies, antigens, inhibitors, vitamins, cofactors, fatty acids, cells, cell membranes, substrates, Substrate analogs, inhibitors, coenzymes, viruses, lectins, polysaccharides, glycoproteins, receptors, histones, ATP, ADP, hormones, receptors, glutathione and the like.

Examples of the enzyme include cellulase, hemicellulase, peroxidase, protease, amylase, xylanase, lipase, esterase, cutinase, pectinase, keratinase, reductase, oxidase, phenoloxyda Azedes, lipoxygenases, ligninases, pullulases, arabinosidases, hyaluronidases, combinations thereof, and the like, but are not limited thereto.

In addition, examples of the catalyst include, but are not limited to, a Graves catalyst, a radical initiator, or a combination thereof.

In one embodiment of the present invention, the compound of Formula 1 may further include a functional group, and examples of such functional groups include an amine group, a carboxyl group, and the like, preferably (aminoethylsulfanyl) propyl, (carboxy) Ethylsulfanyl) propyl and the like.

Meanwhile, in the compound of Formula 1, which is a cucurbituril derivative, A ₁ and A ₂ are each independently H, OH, or OR, wherein R is substituted or unsubstituted C ₂ -C ₃₀ alkenyl.

In the compound of Formula 1, n is preferably 7 and X is O.

The cucurbituril derivative, which is a compound of Formula 1, of the first component included in the kit according to the present invention, and a specific ligand of the second component are determined according to pH. Specifically, an acidic condition is required to form a conjugate between a cucurbituril derivative and a specific ligand, and basic conditions are required to release the conjugate. More specifically, if pH of 8 or less acidic solution, the cucurbituril derivative with a specific ligand is about 10 ¹⁰ to 10 ^15, while as coupling non-covalently with a binding constant of ^-1 M, pH 8 or higher For the basic solution is about 10 ⁰ It remains undissociated with a binding constant of ^{˜10 4} M ⁻¹ . As the pH decreases and the solution turns acidic, the binding constant between the cucurbituril derivative and the specific ligand increases, and as the pH rises and the solution turns basic, the binding constant between the cucurbituril derivative and the specific ligand decreases.

A cucurbituril derivative, a compound of Formula 1, in the first component and a specific ligand in the second component may be non-covalently bound with a binding constant of about 10 ⁰ to 10 ¹⁵ M ⁻¹ depending on the pH of the solution. For example, a cucurbituril derivative having n of 7 and adamantanamine or ferrocene methylamine ligand are non-covalently bound at a binding constant of about 10 ¹² M ⁻¹ or higher when pH 8 or less, whereas about 10 when on pH 8 or higher. ^It exists in a dissociated state without binding with a binding constant of ⁴ M ⁻¹ or less.

If the coupling constant of the cucurbituril derivative, the compound of formula 1 in the first component, and the specific ligand in the second component is less than 10 ⁴ M ^−1, the binding is weak, and the bond may be dissociated when the mobile phase solvent is flowed. If the binding constant is ^greater than 10 ¹⁰ M ^−1, their bonds will be strong and it will be difficult to separate them.

In the kit according to the present invention, a ligand in the second component capable of forming a non-covalent bond with a cucurbituril derivative which is a compound of formula 1 in the first component is a C ₁ -C ₂₀ alkyl group having one or more amine functional groups; C ₂ -C ₂₀ alkenyl group; C ₂ -C ₂₀ alkynyl group; C ₁ -C ₂₀ alkoxy group; C ₁ -C ₂₀ aminoalkyl group; C ₄ -C ₂₀ cycloalkyl group; C ₄ -C ₇ heteroarylcyclo group; C ₆ -C ₂₀ aryl group; C ₅ -C ₂₀ heteroaryl group; C ₁ -C ₂₀ alkylsilyl group; C ₆ -C ₂₀ aryl group; C ₅ -C ₂₀ heteroaryl group; Adamantane with a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Ferrocene or metallocene having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Carborane having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Fullerenes having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Cyclic or crownethers having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Oxygen-protected amino acids having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Peptides having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Alkaloids having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Cisplatin having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Oligonucleotides having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Rhodamine having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; And nanoparticles having a C ₁ -C ₂₀ alkyl group, a C ₆ -C ₂₀ aryl group or a C ₅ -C ₂₀ heteroaryl group.

Preferably, the ligand is adamantane or ferrocene having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group having one or more amine functional groups or It is a compound which is a metallocene. The amine functional group is preferably a primary amine, secondary amine or hydrogenated amine.

Most preferably, the ligand is adamantanamine or ferrocene methylamine.

The present invention can also isolate / purify a substance bound to a ligand, by using a cucurbituril derivative and a ligand to form a non-covalent bond.

Specifically, the present invention also relates to a substance bound to a ligand comprising the following steps (the substance is a biomolecule, an antioxidant, a chemotherapeutic agent, an antihistamine, a cucurbituril dendrimer, a cyclodextrin derivative, a crown ether derivative, a calixarene derivative, a cyclo Plate derivatives, cyclic peptide derivatives, metal ions, chromophores, fluorescents, phosphors, radioactive materials and catalysts).

a) providing an affinity chromatography column packed with a stationary phase of a compound of formula 1 bound to a solid phase;

b) providing to said column a mixture comprising a substance bound to said ligand;

c) washing the column with a wash solution; And

d) providing a mobile phase solvent to the column to separate and purify the substance bound to the ligand.

As the column used in the above may be used that is commonly used in the art.

Biomolecules that can be used in the method according to the invention are enzymes, nucleic acids, proteins, amino acids, antibodies, antigens, inhibitors, vitamins, cofactors, fatty acids, cells, cell membranes, substrates, substrate analogs, inhibitors, coenzymes, viruses , Lectins, polysaccharides, glycoproteins, receptors, histones, ATP, ADP, hormones, receptors, glutathione and the like. Examples of the enzyme include cellulase, hemicellulase, peroxidase, protease, amylase, xylanase, lipase, esterase, cutinase, pectinase, keratinase, reductase, oxidase, phenoloxidase Lipoxygenase, ligninase, pullulase, arabinosidase, hyaluronidase and combinations thereof.

Ligands that can be used in the method for separating and purifying a substance bound to a ligand according to the present invention are those capable of mutually non-covalent bonding with a compound of Formula 1, C ₁ -C ₂₀ alkyl group having one or more amine functional groups; C ₂ -C ₂₀ alkenyl group; C ₂ -C ₂₀ alkynyl group; C ₁ -C ₂₀ alkoxy group; C ₁ -C ₂₀ aminoalkyl group; C ₄ -C ₂₀ cycloalkyl group; C ₄ -C ₇ heteroarylcyclo group; C ₆ -C ₂₀ aryl group; C ₅ -C ₂₀ heteroaryl group; C ₁ -C ₂₀ alkylsilyl group; C ₆ -C ₂₀ aryl group; C ₅ -C ₂₀ heteroaryl group; Adamantane with a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Ferrocene or metallocene having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Carborane having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Fullerenes having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Cyclic or crownethers having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Oxygen-protected amino acids having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Peptides having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group or C ₅ -C ₂₀ heteroaryl group; Alkaloids having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Cisplatin having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Oligonucleotides having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; Rhodamine having a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, C ₆ -C ₂₀ aryl group, or C ₅ -C ₂₀ heteroaryl group; And nanoparticles having a C ₁ -C ₂₀ alkyl group, a C ₆ -C ₂₀ aryl group or a C ₅ -C ₂₀ heteroaryl group.

In the method for separating and purifying a substance bound to a ligand according to the present invention, it is preferable to further include the step of combining the compound of Formula 1 with a solid phase prior to step a). Additional functional groups may be attached, and examples of such functional groups include amine groups, carboxyl groups, and the like, preferably (aminoethylsulfanyl) propyl, (carboxyethylsulfanyl) propyl, and the like.

In the method for separating and purifying a substance bound to a ligand according to the present invention, it is preferable to further include the step of binding the ligand and the substance between the steps a) and b).

It is preferable that the compound of Formula 1 and a solid phase or between a ligand and a solid phase are covalently linked.

In the method for separating and purifying a substance bound to a ligand according to the present invention, mobile phase solvents that can be used are methanol, trifluoroacetic acid, triethylamine, methylene chloride, chloroform, dimethylformamide, dimethyl sulfoxide, toluene, aceto Nitrile, xylene, chlorobenzine, tetrahydrofuran, diethyl ether, ethanol, diglycol ether, silicone oil, supercritical carbon dioxide, ionic liquids, N-methylpyrrolidine, pyridine, water, Ammonium hydroxide, dioxane, chloroform and the like.

In addition, the washing solution which can be used in the method for separating and purifying the substance bound to the ligand according to the present invention is methanol, trifluoroacetic acid, triethylamine, methylene chloride, chloroform, dimethylformamide, dimethyl sulfoxide, toluene, acetonitrile , Xylene, chlorobenzine, tetrahydrofuran, diethyl ether, ethanol, diglycol ether, silicone oil, supercritical carbon dioxide, ionic liquid, N-methylpyrrolidine, pyridine, water, ammonium hydroxide, dioxane, Chloroform and the like.

By carrying out the separation and purification method according to the present invention with a solution recovered from the column to perform an additional purification step such as dialysis (purification) can be obtained a more pure material.

The cucurbituril derivative of formula (1) bound to a solid phase or a specific material forms a stable complex with a ligand bound to the solid phase or a specific material in a solvent at room temperature, which is strong enough to form a complex even after the solvent is removed. Whether the complex of the Cucurbituril derivative of Formula 1 and the ligand is formed depends on the kind of solvent, pH, polarity, temperature, and electrical change.

Coupling of the cucurbituril derivative of Formula 1 with the ligand requires conditions under which the amine functional group of the ligand is hydrogenated or acidic, and conditions under which dehydrogenation or basic conditions are required to dissociate the bond.

In addition, the present invention uses mutual non-covalent bonds of the compound of Formula 1 with the ligand to separate and purify specific substance components bound to the ligand contained in the mixture using the compound of Formula 1 bound to the solid phase; Alternatively, the ligand bound to the solid phase may be used to separate a specific substance component bound to the compound of Formula 1 included in the mixture.

Specifically, the specific material component as described above may be easily separated by using a magnet or using a filter using a solid component as a magnetic material.

Furthermore, after performing the method using the magnet or the filter, it is possible to obtain a more purified material through a dialysis process.

The method using the magnet or the filter is methanol, trifluoroacetic acid, triethylamine, methylene chloride, chloroform, dimethylformamide, dimethyl sulfoxide, toluene, acetonitrile, xylene, chlorobenzine, tetrahydrofuran, Diethyl ether, ethanol, diglycol ether, silicone oil, supercritical carbon dioxide, or ionic liquids, N-methylpyrrolidine, pyridine, water, ammonium hydroxide, dioxane, chloroform, etc. Can be used.

The invention also relates to a sensor chip comprising a combination of a compound of formula 1 bound to a first material, and a ligand bound to a second material.

The sensor chip is a device for fixing a probe material (for example, biomolecules, etc.) that is a material that recognizes a desired material on a solid surface and detecting the desired material by using the same.

In the sensor chip according to the present invention, one of the first material and the second material is a solid phase, and the other is a probe material.

In the sensor chip, as the solid phase that can be used as the first material or the second material, a solid support such as a gold thin film, a silver thin film, or glass coated with ITO is preferable as the probe material.

In the sensor chip, the probe material that can be used as the first material or the second material may be an enzyme, a substrate, a substrate analog, an inhibitor, a coenzyme, an antibody, an antigen, a virus, a cell lectin, including histidine, cysteine or tryptophan. Polysaccharide, glycoprotein, cell surface receptor, nucleic acid, complementary sequence, histone, nucleic acid polymerase, nucleic acid binding protein, ATP, ADP, hormone, vitamin, receptor, transport protein, glutathione, GST fusion protein, metal ion, polyhis fusion protein , Natural proteins and combinations thereof, but is not limited thereto. The enzymes include cellulase, hemicellulase, peroxidase, protease, amylase, xylase, lipase, esterase, cutinase, pectinase, keratinase, reductase, oxidase, phenoloxidase, liposome. Poxygenase, ligninase, pullulase, arabinosidase, hyaluronidase, combinations thereof, and the like are preferred.

The sensor chip can confirm the detection of a desired substance by electrochemical method, photochemical method, fluorescence measurement, phosphorescence measurement, HPLC, gas chromatography, nuclear magnetic resonance method, EPR, method using isotope.

The sensor chip according to the present invention may be used as a biosensor or a bio-fuel cell electrode when the probe material is a biomolecule.

The present invention also relates to a solid catalyst binder comprising a compound of formula (1) bonded to a first material and a ligand bound to a second material (in this case, any one of the first and second materials is a catalyst and the other is a solid phase Is).

In the solid catalyst binder according to the invention, the catalyst used in the solid phase is preferably an enzyme, a Grabs catalyst, a radical initiator, an enzyme or a combination thereof.

In addition, in the solid catalyst binder, the catalyst is a cellulase, hemicellulase, peroxidase, protease, amylase, xylanase, lipase, esterase, curinase, pectinase, keratinase, reductase, oxidase , Enzymes such as phenol oxidase, lipoxygenase, ligninase, pullulase, arabinosidase, hyaluronidase or combinations thereof.

In addition, examples of those that can be used as a solid phase in the solid catalyst binder according to the present invention include polymers, resins, magnetic materials, silica gels, polymers or gold-coated silica gels, zirconium oxides, monolithic polymers, and polymer-coated magnetics. Particles, gold thin film, silver thin film, glass, ITO coated glass, silicon, metal electrodes, nanorods, nanotubes, nanowires, curlran gum, cellulose, nylon membranes, sepharose and sephadex And preferably, a polystyrene resin or a silica gel coated with a polymer.

The solid catalyst binder according to the present invention can be reused until the catalytic activity disappears, and can participate in an organic reaction at room temperature to perform catalysis. In catalysis, methanol, trifluoroacetic acid, triethylamine, methylene chloride, chloroform, dimethylformamide, dimethyl sulfoxide, toluene, acetonitrile, xylene, chlorobenzine, tetrahydrofuran, diethyl ether, ethanol, diglycol ether Various buffers suitable for the respective reactions, such as silicone oils, supercritical carbon dioxide, or ionic liquids, N-methylpyrrolidine, pyridine, water, ammonium hydroxide, dioxane or chloroform can be used.

Whether or not the cucurbituril derivatives and ligands each bound to specific substances formed a conjugate can be confirmed by Fourier transform infrared absorption experiment.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example  One: Cookerbitu [7] Reel's  synthesis

Glycouril (5.7 g, 40 mmol) was dissolved in 20 mL of sulfuric acid (9M), followed by addition of aqueous formaldehyde solution (7.0 mL, 91 mmol). The reaction mixture was stirred at 75 ° C. for 24 hours, the temperature was raised to 95-100 ° C., and the reaction was further proceeded for 7 hours. The reaction mixture was quickly poured into 200 mL of water, and 1 L of acetone was added thereto. The resulting precipitate was filtered off under reduced pressure filtration and washed with a water: acetone (1: 4) solution. 200 mL of a water: acetone (1: 1) solution was added to the filtered solid to make a solution, and the remaining material (Cookerbitu [6] Reel) still precipitated was removed by filtration under reduced pressure.

800 mL of acetone was added again to the filtered filtrate to obtain a mixture of cookerbitu [5] reel and cookerbitu [7] reel as a precipitate. A mixture of cucurbitur [5] ryl and cucurbitur [7] ryl in the precipitated state was dissolved in 40 mL of water, and then 25 mL of methanol was added to precipitate only cucurbitur [7] ryl as a precipitate. The Cucurbitur [7] reel obtained was filtered under reduced pressure and dried in vacuo, and the analysis results are as follows.

¹ H NMR (500 MHz, D ₂ O): δ = 5.56 (d, 14H), 5.35 (s, 14H), 4.11 (d, 14H).

Example  2: hydroxy Cookerbitu [7] Reel's  synthesis

Cookerbitu [7] reel (10 g, 8.6 mmol) and K ₂ S ₂ O ₈ (39 g, 145 mmol) obtained in Example 1 were added to 450 mL of distilled water and sonicated for 10 minutes. Then, the reaction was carried out for 12 hours by raising the temperature to 85 ℃. The reaction mixture was cooled to room temperature to give a white precipitate, which was removed by filtration under reduced pressure. The precipitate of the filtrate was evaporated to give a precipitate, which was hydroxy cookerbitur [7] ryl. The degree of substitution of hydroxy cucurbitu [7] ryl was about 0.8. The degree of substitution refers to the proportion of hydrogen (A ₁ and A ₂ in Formula 1) substituted with a hydroxyl group in cucurbitu [7] yl.

¹ H-NMR (500 MHz, DMSO-d ₆ ): δ = 7.83 (s, 11H), 5.68-5.12 (d, 17H), 4.42 (d, 14H);

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 152.7, 93.8, 40.2.

Example  3: Allyloxy Cookerbitu [7] Reel's  synthesis

The hydroxy cookerbitu [7] ryl (1.2 g, 0.92 mmol) prepared in Example 2 was added to anhydrous DMSO solution (150 mL) to dissolve, followed by addition of NaH (0.89 g, 22.4 mmol). The reaction mixture was stirred for 1 hour in an argon environment and room temperature, and allyl bromide (2.0 mL, 22.0 mmol) was slowly added at 0 ° C. while stirring continued. The reaction mixture was again stirred at room temperature for 12 hours, and excess water was added to give allyloxy cookerbitur [7] ryl as a precipitate. The degree of substitution was about 0.7.

Example  4: solid phase (polystyrene- co -DVD- NCO Manufacturing

Polystyrene having a polymer resin, an amine functional group - co - divinylbenzene (polystyrene- co -divinylbenzene (DVB), amine functionalized; polystyrene _{- co -DVB-NH 2) (} Aldrich Co., 30 to 60 mesh, 2.5mmol N / g , 5.0 g, 12.5 mmol) was added to 50 mL of dichloromethane, and triethylamine (9.6 mL, 69 mmol) and triphosphene (2.7 g, 9.1 mmol) were added thereto, followed by shaking at room temperature for 36 hours. Then, the mixture was filtered under reduced pressure, washed with dichloromethane, chloroform, ether, and tetrahydrofuran in that order, followed by vacuum drying for 24 hours, whereby polystyrene- co -divinylbenzene, an isocyanate methyl polymer resin having an amine group substituted with an isocyanate group ( Polystyrene- co- DVB-NCO) was obtained (FIG. 2).

Comparing the IR spectrum of FIG. 3 for polystyrene- co- DVB-NH ₂ and the IR spectrum of FIG. 4 for polystyrene- co- DVB-NCO, a new peak appears near 2264 cm ⁻¹ in the IR spectrum of FIG. 4. Corresponds to isocyanate groups (-NCO).

Specifically, the IR spectrum of the polystyrene- co- DVB-NCO of FIG. 4 is as follows.

IR (KBr): 3027, 2929, 2264, 1659, 1602, 1494, 1452, 1269, 1116, 1029 cm ^-1 .

Example  5: hydroxy in solid phase Cookerbitu [7] Reel's  Immobilization

Polystyrene- co- DVB-NCO polymer resin (2.0 g, about 2.5 mmol N / g) prepared in Example 4 was added to 8 mL of dimethyl sulfoxide and inflated, followed by dimethyl sulfoxide / pyridine (37 mL / 3 mL). Hydroxy cookerbitu [7] reel (1.44 g, 1.2 mmol) prepared in Example 2 in the solution was added and stirred for 120 hours at 60 ° C. under argon to mix uniformly (FIG. 2). The reaction mixture was then filtered under reduced pressure, washed several times in turn with dimethylsulfoxide, methanol, water and ether, followed by vacuum drying at 50 ° C. for 24 hours, whereby hydroxy cookerbitu [7] ryl was immobilized on the solid phase. The IR spectrum results are as follows (see FIG. 5).

IR (KBr): 3026, 2920, 1755, 1689, 1602, 1493, 1452, 1272, 1115, 1028 cm ^-1 .

According to FIG. 5, the peak corresponding to the isocyanate group of 2264 cm ⁻¹ shown in FIG. 4 disappears, and a new peak corresponding to the carbonyl carbon of hydroxy cookerbitu [7] yl at 1755 cm ⁻¹ can be identified. In addition, elemental analysis confirmed that 75-110 μmol of hydroxy cookerbitu [7] yl per 1 g of solid phase was connected.

Example  6: Preparation of solid phase

(1) Chloromethylation of porous polystyrene (crosslinked with 1% divinylbenzene)

After mixing 3.0 g of CombiGel XE-305 (Aldrich), porous polystyrene (crosslinked with 1% divinylbenzene), and chloromethyl methyl ether (29.90 g, 0.38 mmol), SnCl ₄ (0.90 mL) was added at 0 ° C. Add dropwise. The reaction mixture was refluxed at 59 ° C. for 4.5 h (FIG. 6). CombiGel XE-305 slowly changed from colorless to red. Aqueous methanol solution was added to neutralize the reaction mixture, and the red color disappeared. The polymer resin was filtered through a glass filter with methanol, tetrahydrofuran (THF) and water to obtain chloromethylated polystyrene (crosslinked with 1% divinylbenzene) as a white powder. The analysis results are as follows.

Elemental Analysis: C, 68.68; H, 6.01; Cl, 18.35%;

Elemental analysis shows that 5.2 mmol of chlorine is present per gram of chloromethylated polystyrene (crosslinked with 1% divinylbenzene).

Comparing the IR spectrum of FIG. 7 for CombiGel XE-305 and the IR spectrum of FIG. 8 for chloromethylated CombiGel XE-305, the absorption peak of 1265 cm ⁻¹ , which was not observed on the IR spectrum of FIG. This peak is equivalent to -C-Cl.

Specifically, the IR spectrum of the chloromethylated polystyrene of FIG. 8 is as follows.

IR spectrum: 2928, 1725, 1612, 1510, 1445, 1422, 1265 cm ⁻¹ .

(2) Chloromethylated  Polystyrene (1% With divinylbenzene Crosslinking Immobilization of Dithiol onto Phase

Chloromethylated polystyrene (crosslinked with 1% divinylbenzene) (2.0 g) obtained in the above (1) was added to 20 mL of toluene and inflated. Propane-1,3-dithiol (2 mL, 20 mmol) was added thereto, followed by slow shaking and stirring for 15 minutes. 1,8-diazabicyclo [5.4.0] undec-7-ene (1,8-diazabicyclo [5.4.0] undec-7-ene, DBU) (0.9 mL, 6 mmol) was added dropwise to the reaction mixture. And, the reaction mixture was stirred at room temperature for 24 hours (FIG. 6). The obtained resin was filtered under reduced pressure, and washed sequentially with CH ₂ Cl ₂ and DMF (dimethylformamide). The dithiol was immobilized on chloromethylated CombiGel XE-305 by vacuum drying for 12 h. As a result of elemental analysis, it was found that the degree of immobilization of the thiol group was 1.9 mmol / g (the thiol functional group per 1.9 g of solid phase was 1.9 mmol).

IR spectrum: 2918, 2850, 2563, 1727, 1509, 1423, 1238 cm ^-1

Looking at the IR spectrum of Figure 9, a -SH stretching peak near 2563 cm ^-1 was observed.

Example  7: solid phase Allyloxy Cookerbitu [7] Reel's  fixing

1.5 g of distyrene-immobilized polystyrene (crosslinked with 1% divinylbenzene) prepared in Example 6 and allyloxy cookerbit [7] ryl (1.1 g, 0.68 mmol) prepared in Example 3 were added to 16 mL methanol. Was added. Then, 300 nm ultraviolet rays were irradiated for 120 hours (5 days) in an argon environment (see FIG. 10). After completion of the reaction, the polymer resin to which allyloxy cucurbitu [7] reel was connected was filtered under reduced pressure, and washed sequentially with methanol, chloroform, acetone, and ether. Then vacuum dried for 12 hours.

IR spectrum: 2917, 2852, 1753, 1725, 1510, 1425, 1238 cm ⁻¹ .

On the IR spectrum, a peak corresponding to carbonyl of cucurbitur [7] ryl was found at 1753 cm ⁻¹ , and elemental analysis confirmed that 80-115 μmol of allyloxy cucurbitur [7] ryl was attached per gram of solid phase. .

Example  8: Ligands (ferrocene derivatives)  Binding of protein (bovine serum albumin)

(One) ( N Preparation of-(ferrocenylmethyl) -6-aminocaproic acid (Fc-aca)

Dissolve 1.38 g (6.4 mmol) ferrocene-aldehyde (Aldrich) in 20 ml DMF solution and mix with 0.75 g (5.7 mmol) 6-aminocaproic acid (Merck) in 5 ml NaOH (2M). To reflux at 80 ° C. for 2 hours. After cooling the reaction mixture to room temperature, NaBH ₄ (0.63 g, 16.5 mmol) in 5 ml of water was added little by little (see FIG. 11). Then, after 12 hours, an acetic acid aqueous solution (10%) was slowly added to bring the pH to 5. Through extraction with ethyl acetate, 6-aminocaproic acid, which was not reacted with ferrocene-CH ₂ OH dissolved in the organic layer, was removed, and the water layer was distilled under reduced pressure at 50 ° C. to obtain yellowish brown crystals. This yellowish-brown crystal was dissolved in hot ethanol and recrystallized to obtain N- (ferrocenylmethyl) -6-aminocaproic acid, which is a plate yellow crystal which is the title compound.

The resulting N- (ferrocenylmethyl) -6-aminocaproic acid was confirmed by HPLC, ¹ H-NMR and mass spectrometry. The recrystallized product was separated by high purity via HPLC. The ¹ H-NMR spectrum of N- (ferrocenylmethyl) -6-aminocaproic acid is as follows: ¹ H-NMR (D ₂ O, 300 MHz): δ = 4.05-4.43 (9H, m, ferrocene ring protons ), 3.46 (2H, s, CH ₂ ), 2.49 (2H, t, CH ₂ ), 1.36 (6H, m, CH ₂ ), 2.02 (2H, t CH ₂ ).

Mass spectrometry also yielded molecular peaks of m / e = 330 (Fc-acaH ⁺ ).

(2) N -( Ferrocenylmethyl ) -6- Aminocaproic acid (Fc-aca)  Binding of bovine serum albumin (BSA)

(1) Preparation of N in - (ferrocenyl methyl) -6-aminocaproic acid (Fc-aca) 15mg, N-hydroxy succinimide (succinimide) (NHS, Fluka Co.), 6mg, and N - (3 9 mg of N -ethylcarbodiimide hydrochloride (EDC, Fluka) was dissolved in 0.3 ml of anhydrous DMF solution, heated to 90 ° C. under a nitrogen atmosphere, and stirred for 30 minutes (see FIG. 11). The reaction was further performed for 30 minutes while maintaining the temperature of the solution at 80 ° C, and cooled to room temperature. 30 μL of the reaction mixture was added to a solution of bovine serum albumin (7 mg) in 1 ml of phosphate buffer (100 mM) at 2 minute intervals. After shaking for one day at room temperature, the precipitate was separated by centrifugation, and dialyzed in 50 mM phosphate buffer (pH 7.4) for 30 hours to obtain N- (ferrocenylmethyl) -6-aminocaproic acid (Fc-aca). Linked bovine serum albumin (ferrocene linked BSA) was prepared.

Example  9: Purification of Proteins

In this example, the BSA to which the ferrocene derivative is linked was purified using the solid phase in which the hydroxy cookerbitu [7] yl is prepared in Example 5.

First, 1.5 g of the hydroxy cookerbitu [7] reel-immobilized solid phase prepared in Example 5 was placed in a 10 mL disposable syringe, and CH ₂ Cl ₂ (40 mL) was continuously flowed at a flow rate of 1.3 mL / min. The solid phase was then washed with DMF (50 mL, flow rate: 0.7 mL / min).

Meanwhile, a protein mixture was prepared by dissolving BSA (2 mg), glucose oxidase (1 mg) and lipase (1 mg) linked to the ferrocene derivative prepared in Example 8 in 0.1 M HEPES buffer (pH 6.0).

The protein mixture was then slowly flowed into the disposable syringe at a flow rate of 0.7 ml / min, then the syringe was washed with 50 ml of water (flow rate: 2 ml / min), and then DMF / TEA (40 mL / 20 mL, flow rate). : 2 mL / min).

Then, the eluate was recovered from the syringe by flowing HEPES buffer solution (0.1 M, pH 8.5) (60 mL, flow rate: 2 mL / min).

The recovered eluate was treated at a temperature of 60 ° C. to denature the protein, and then loaded onto SDS-PAGE by adding Coomassie Blue to 150 μL of this eluate and operated at 10 mA for 5 hours. At this time, the molecular weight indicator was also loaded on the SDS-PAGE.

Then, the SDS-PAGE gel was removed from the electrophoresis and washed, then immersed in Coomassie blue for about 1 hour, and washed overnight with a washing solution.

Thereafter, the degree of staining of the gel was recorded by a gel scanner, and the result is shown in FIG. 12A. Lane 1 of Figure 12a is the molecular weight index, lane 2 is the result of loading the eluent.

As a result, lane 2 showed a single band of 66 kDa, and it was confirmed that hydroxy cookerbituryl bound to the solid phase separated and purified the ferrocene-linked BSA from the protein mixture.

The eluate was collected and lyophilized to obtain pure BSA protein linked with ferrocene derivatives.

Example  10: Purification of Protein

In this example, the cooker-linked protein was purified using a ferrocene-linked solid phase.

(One) Chloromethylated  Solid phase Ferrocenemethyl Amination

The chloromethylated Combigel XE-305 (1.5 g) prepared in Example 6 (1) was inflated by dipping in DMF (14 mL) for 2 hours. Then, to a solution of DMF-pyridine (1: 2 v / v, 9.0 mL) in which ferrocenemethylamine (1.35 g, 6.3 mmol) was dissolved, chloromethylated Combigel XE-305 in the DMF was added and the solution was heated to 55 ° C. Stirred for 43 h. The reaction mixture was filtered under reduced pressure, washed with a solvent such as DMF, CH ₂ Cl ₂ , methanol, ether, and dried under vacuum to give ferrocenemethylamined Combigel XE-305.

Elemental Analysis: C, 64.52; H, 6.72, N, 5.10%. As a result, it was confirmed that 3.64 mmol of ferrocene was linked per 1 g of the polymer. (It was found that 3.64 mmol of ferrocene was contained per gram of the polymer particles (ferrocenemethyl-amined CombiGel XE-305) obtained from this result.)

FT-IR: 3419, 3020, 2936, 1627, 1485, 1210, 1151, 1025 cm ^-1 .

(2) [3- (2-carboxyethylsulfanyl) propyl-O] ₁₄ Synthesis of Cookerbitu [7] Reels

Add DMF (2 mL) to the quartz tube, allyloxy cookerbitu [7] ryl ((allyl-O) ₁₄ -CB [7], 35 mg, 0,018 mmol) and 3-mercaptopropionic acid (70 mL, 0.78 mmol ) Was added and reacted for 36 hours under ultraviolet light (254 nm). Then, the DMF was removed by distillation under reduced pressure, and the reaction mixture was washed four times with 15 mL of diethyl ether, and then dried under reduced pressure to produce [3- (2-carboxyethylsulfanyl) propyl-O] ₁₄ cooker. Vitu [7] ryl was obtained in 37 mg, 60% yield.

The analysis results of the obtained [3- (2-carboxyethylsulfanyl) propyl-O] ₁₄ cookerbitu [7] yl were as follows.

¹ H NMR (300 MHz, DMSO-d ₆ ): δ = 5.56 (d, J = 14.3 Hz, 14H), 4.05 (d, J = 14.3 Hz, 14H), 3.52 (m, 28H), 2.62 (m, 56H) 2.48 (m, 28 H), 1.84 (t, 28 H);

¹³ C NMR (75 MHz, DMSO): δ = 170.0, 151.8, 95.5, 63.2, 40.8, 34.4, 29.2, 26.2; MS (MALDI-TOF): m / z 3454.1 [M + Na &lt; + &gt;].

(3) Cookerbitu [7] Reel  Combination of Derivatives with Bovine Serum Albumin (BSA)

37 mg of [3- (2-carboxyethylsulfanyl) propyl-O] ₁₄ cookerbitu [7] yl prepared in (2), 6 mg of N -hydroxy succinimide (NHS, Fluka) and N 9 mg of-(3-dimethylaminopropyl) -N -ethylcarbodiimide hydrochloride (EDC, Fluka) was dissolved in 0.3 ml of anhydrous DMF solution, and the temperature was raised to 90 ° C. under a nitrogen atmosphere and stirred for 30 minutes.

The reaction was further performed for 30 minutes while maintaining the temperature of the solution at 80 ° C., and cooled to room temperature. 30 μL of the reaction mixture was added to a solution of bovine serum albumin (BSA, Sigma) (7 mg) in 1 ml of phosphate buffer (100 mM) at 2 minute intervals. After shaking for 1 day at room temperature, the precipitate was separated by centrifugation, and dialyzed in 50 mM phosphate buffer (pH 7.4) for 30 hours to [3- (2-carboxyethylsulfanyl) propyl-O] ₁₄ CB [7 ] -Linked bovine serum albumin was prepared.

The BSA prepared by linking Cucurbitu [7] reel prepared in (2) was purified using the ferrocenemethyl aminated solid phase prepared in Example (1).

First, 1.5 g of the prepared ferrocenemethylamined solid phase was placed in a 10 mL disposable syringe, and CH ₂ Cl ₂ (40 mL) was continuously flowed at a flow rate of 1.3 mL / minute. Then the resin was washed with DMF (50 mL, flow rate: 0.7 mL / min).

Meanwhile, a protein mixture was prepared by dissolving BSA (2 mg), glucose oxidase (1 mg) and lipase (1 mg) linked to CB [7] prepared in phosphate buffer (0.1M pH 6.0).

Then, the protein mixture was slowly flowed into the column at a flow rate of 0.7 ml / min, then the syringe was washed with 50 ml of water (flow rate: 2 ml / min), and then DMF / TEA (40 mL / 20 mL, flow rate: 2 mL / Min).

Then, the protein mixture was slowly flowed into the column at a flow rate of 0.7 ml / min, then the syringe was washed with 50 ml of water (flow rate: 2 ml / min), and then DMF / TEA (40 mL / 20 mL, flow rate: 2 mL / Min).

Then, the eluate was recovered by flowing HEPES buffer solution (0.1 M, pH 8.5) (60 mL, flow rate: 2 mL / min).

The recovered eluate was denatured at a temperature of 60 ° C., and then loaded onto SDS-PAGE by adding Coomassie Blue to 150 μL of the eluent, and the SDS-PAGE was operated at 10 mA for 5 hours. At this time, the molecular weight indicator was also loaded.

Thereafter, the degree of staining of the gel was recorded by a gel scanner, and the result is shown in FIG. 12B. Lane 1 of Figure 12b is the molecular weight index, lane 2 is the result of loading the eluent.

As a result, a single band of 66 kDa appeared in lane 2, and it was confirmed that the solid phase linked to ferrocene separated and purified BSA bound to a cucurbituril derivative.

The eluate was collected and lyophilized to obtain a BSA protein to which a cucurbituryl derivative was bound.

Example  11: Purification of Antigens

In this example, the antigen specifically binding to the antibody was purified using an allyloxy cookerbitu [7] yl-bound solid phase prepared in Example 7 and an antibody bound to a ferrocene derivative.

(One) Cooker bituril  Derivatives Combined  Preparation of the solid phase

300 mg of the allyloxy cookerbitu [7] yl-immobilized solid phase prepared in Example 7 was centrifuged at 10,000 rpm for 5 minutes with an excess of 50 mM HEPES solution (pH 8.5 ) , followed by an upper layer using a 200 μL pipette. The solid phase was washed twice by removing the liquid.

(2) Ferrocene  Preparation of Anti-BSA Protein Solution Linked Derivatives

The anti-BSA protein solution linked to the ferrocene derivative was prepared by the following method.

Embodiment the N prepared in Example 8 (1) - (ferrocenyl methyl) -6-aminocaproic acid (Fc-aca) 15mg, N-hydroxy succinimide (succinimide) (NHS, Fluka Co.), and 6mg N - 9 mg of (3-dimethylaminopropyl) -N -ethylcarbodiimide hydrochloride (EDC, Fluka) was dissolved in 0.3 ml of anhydrous DMF solution, heated to 90 ° C. under a nitrogen atmosphere, and stirred for 30 minutes. The reaction was further performed for 30 minutes while maintaining the temperature of the solution at 80 ° C, and cooled to room temperature. 30 μL of the reaction mixture was added to a 7 mg solution of anti-bovine serum albumin (anti-BSA solution, Sigma) in 1 ml of phosphate buffer (100 mM) at 2 minute intervals. After shaking for one day at room temperature, the precipitate was separated by centrifugation, and dialyzed in 50 mM phosphate buffer (pH 7.4) for 30 hours to obtain N- (ferrocenylmethyl) -6-aminocaproic acid (Fc-aca). Linked anti-bovine serum albumin (ferrocene linked anti-BSA) was prepared.

The antibody protein solution of anti-BSA to which ferrocene derivatives were linked was diluted to a concentration of 100 μg / mL with 50 mM HEPES (pH 7.4) solution (500 μL in volume). (If the antibody protein solution contains foreign proteins, or other samples containing azide, glycine, tris, and primary amine groups, they were removed by dialysis or gel-filtration chromatography before use.)

(3) Allyloxy Cookerbitu [7] Reilly Combined Solid phase Ferrocene  Binding of Anti-BSA Proteins with Derivatives Linked

Suspended by adding 500 μL of 50 mM HEPES (pH 7.4) solution to the allyloxy cookerbitu [7] reel-bonded solid prepared in (1), and then ferrocene diluted to 100 μg / mL in (2). 500 μL of antibody protein solution of linked anti-BSA was added.

The mixture was stirred slowly in the dark at room temperature for 3 hours. Thereafter, the particles were washed twice with HEPES buffer (pH 7.4). 500 μL of blocking buffer (0.1 M ethanolamine and HEPES buffer) was added. The solid phase was then washed twice with 500 μL of HEPES buffer (pH 7.4).

(4) purification of antigen

Dividing the solid-phase conjugate of anti-BSA protein and allyloxy cookerbitu [7] reel to which ferrocene derivatives were linked, was added to two tubes, and one of the tubes added 500 μL of buffer solution containing lysozyme (1 mg / mL) To the other was added 500 μL of buffer containing BSA (1 mg / mL).

Two tubes, each of which was mixed with the solid phase and the protein solution, were slowly stirred in the dark at room temperature for 3 hours. The solid particles were washed twice with HEPES buffer (pH 7.4).

Each of the solid support treated with the buffer containing lysozyme as a control and the solid support treated with the buffer containing BSA were extracted using 300 μL of elution buffer (0.1 M glycine).

Proteins were denatured at a temperature of 60 ° C., then 150 μL of the eluent was added to Coomassie Blue and loaded on SDS-PAGE, and SDS-PAGE was operated at 10 mA for 5 hours. At this time, the molecular weight indicator was also loaded.

After operation, the SDS-PAGE gel was taken out of the electrophoresis and washed, then immersed in Coomassie blue dye for about 1 hour, and washed overnight with a washing solution.

Then, the degree of staining of the gel was recorded by a gel scanner, and the result is shown in FIG. 12C. Lane 1 of FIG. 13 is a molecular weight index, and lanes 2 and 3 are the results of treatment with a solution containing lysozyme and a solution containing BSA, respectively.

Referring to Figure 13, when treated with a solution containing lysozyme (lane 2), while the protein was not detected, when treated with a solution containing BSA, a band of 66kDa appeared, whereby BSA is a cucurbituril It was confirmed that the immobilized solid phase and the ferrocene derivative were separated by a conjugate of linked anti-BSA.

Example  12: Sensor chip

(1) Immobilization of Allyloxy Cookerbitu [7] Reel on Gold Surface

A flat gold surface (2 cm x 1 cm) was immersed in an ethanol solution containing 1 mM 1,8-octanedithiol for 2 hours to introduce thiol functional groups onto the gold surface. Allyl by immersing the gold surface in the DMF solution dissolved in allyloxy cookerbitu [7] ryl (1 mM) prepared in Example 3 and irradiating 254 nm and 300 nm UV light simultaneously for 1 hour, then removing the gold surface and washing with DMF, Oxy cookerbitu [7] ryl was covalently linked to a gold surface (see FIG. 14).

(2) connected to the gold surface Allyloxy Cookerbitu [7] Reel Ferrocene  Inclusion of Molecules

In this example, it was confirmed whether the ferrocene molecule can be included in the pupil of allyloxy cookerbitu [7] yl.

First, an allyloxy cookerbitu [7] ryl covalently bonded gold surface prepared in (1) was immersed in 0.2 M aqueous ferrocentrimethylammonium iodine solution (pH 6.2) and left for 1 hour. As a result, it was confirmed through Fourier transform infrared absorption experiment that the ferrocene trimethylammonium iodine is included in the pupil of allyloxy cookerbitu [7] reel (see FIGS. 15 and 16).

FTIR spectra for ferrocene trimethylammonium iodine (FIG. 15): 1483, 1473, 1459, 1445, 1409, 1386, 1247 cm ⁻¹ ;

FTIR spectra for allyloxy cookerbitu [7] ryl (FIG. 16): 1756, 1471, 1377, 1322, 1255 cm ⁻¹ ;

FTIR spectra for the reaction of allyloxy cucurbitu [7] yl covalently bonded gold surface with ferrocene trimethylammonium iodine (FIG. 17): 1755, 1483, 1473, 1459, 1409, 1386, 1317, 1247 cm ⁻¹ .

(3) On the gold surface  Connected Allyloxy Cookerbitu [7] Reel Ferrocene  Inclusion of Linked Glucose Oxidase and Determination of its Enzyme Activity

First, a ferrocene-linked glucose oxidase (Fc-GOx) enzyme solution was prepared. The preparation method was performed in the same manner as described in (8) of Example 8, except that glucose oxidase was used instead of BSA.

Then, 0.1 M phosphate buffer solution of a glucose electrode oxidase (Fc-GOx) enzyme solution (10 ml) in which allyloxy cucurbitu [7] yl is bound to the gold surface prepared in the above (1). , 200U ml ^-1 glucose oxidase containing, pH 6.3) and left at room temperature for 30 minutes. The gold electrode was removed and washed with 0.1 M phosphate buffer (pH 6.3). The activity of glucose oxidase encapsulated in allyloxy cucurbitu [7] reel was confirmed by immersing the gold electrode in 0.1 M glucose solution (10 ml of 0.1 M phosphate buffer, pH 6) and incubating at 25 ° C. for 1 minute. After removing the gold electrode, 100 μl peroxidase (50 purpurogallin units m1 ⁻¹ ) solution and 20 μl o- dianisidine. 2HCl ( o- dianisidine.2HCl) solution (1% m / v in water) were added and the mixture was transferred to a glass cell. Moved. Enzyme activity was confirmed by absorbance at 460 nm (FIG. 18).

Absorption at 460 nm indicates effective activity. When glucose reacts with an enzyme, hydrogen peroxide is produced, and peroxidase reacting with hydrogen peroxide reacts with hydrogen peroxide to oxidize o- dianisidine.2HCl to give 460 nm light. Will be absorbed. Therefore, the absorbance at 460nm can be seen the activity of the enzyme.

(4) electrochemical measurement

In order to measure the glucose concentration in the sample, the amount of H ₂ O _{2 that} is the result of the enzymatic reaction caused by consuming glucose was measured. The gold electrode prepared in (1) was used to measure the concentration of H ₂ O ₂ . Glucose oxidase was dissolved in 0.1 M phosphate buffer, and a solution of 10 to 120 mM concentration was prepared and measured. All experiments were conducted in a thermostat at 25 ° C.

Based on the results, a calibration curve was drawn (FIG. 19).

Example  13: solid catalyst

(One) N Preparation of Lipase Linked with-(ferrocenylmethyl) -6-aminocaproic acid (Fc-aca)

Example 8 Preparation of N (1) - (ferrocenyl methyl) -6-aminocaproic acid (Fc-aca) 15mg, N-hydroxy succinimide (succinimide) (NHS, Fluka Co.), 6mg, and N 9 mg of-(3-dimethylaminopropyl) -N -ethylcarbodiimide hydrochloride (EDC, Fluka) was dissolved in 0.3 ml of anhydrous DMF solution, and the temperature was raised to 90 ° C. under a nitrogen atmosphere and stirred for 30 minutes (FIG. 15). Reference). The reaction was further performed for 30 minutes while maintaining the temperature of the solution at 80 ° C, and cooled to room temperature. 30 μL of the reaction mixture was added to a solution of lipase (7 mg) in 1 ml of pulling buffer (100 mM) at 2 minute intervals. After shaking for one day at room temperature, the precipitate was separated by centrifugation, and dialyzed in 50 mM phosphate buffer (pH 7.4) for 30 hours to obtain N- (ferrocenylmethyl) -6-aminocaproic acid (Fc-aca). Linked lipases (ferrocene linked lipases) were prepared.

(2) Ferrocene  The linked lipase molecule is entrapped in hydroxy cookerbitu [7] yl immobilized on a solid support

1.0 g of hydroxy cookerbitu [7] ryl immobilized on the polymer resin prepared in Example 6 was placed in a 10 mL disposable syringe, and CH ₂ Cl ₂ (40 mL) was continuously flowed at a flow rate of 1.3 mL / min. Then, the resin was washed with DMF (50 mL, flow rate: 0.7 mL / min). 2 mg of pherase linked lipase prepared in (1) was dissolved in phosphate buffer solution (0.1M pH 6.0), slowly flowed through the column at a flow rate of 0.7 ml / min, and then 50 ml of water (flow rate: 2 ml / Minutes). Subsequently, hydroxy cooker bite [7] reel immobilized on the solid support in the syringe was collected and polymerized in the form of the lipase conjugated with lipase linked with ferrocene and vacuum dried.

A polymer resin in the form of a lipase molecule in which lipase molecules linked to hydroxy cookerbitu [7] yl immobilized on the solid support prepared in (2) was encapsulated was used as a catalyst.

(3) α- Pinene  Epoxidation epoxidation )

Α-pinene (2.38 mL, 15 mmol) and octanoic acid (2.37 mL, 15 mmol) were dissolved in 8 mL of toluene, and 150 mg of the catalyst obtained in (2) was added thereto. The reaction was started by gradually adding H ₂ O ₂ (23 mmol, 2.6 mL) by stirring at room temperature.

After 4 hours, the reaction mixture was filtered to recover catalyst particles. After the filtrate was evaporated under reduced pressure, the product was separated by column chromatography.

The product α-pinene oxide compound was confirmed by ¹ H NMR, ¹³ C NMR and mass spectrometry. A portion of the organic layer was removed at regular time intervals to identify the product.

α-pinene oxide: oil, boiling point 102-104 ° C / 50 mm;

¹ H-NMR (CDCl ₃ , 300 MHz): δ = 0.91 (3H, s, CH ₃ ), 1.30 (3H, s, CH ₃ ), 1.32 (3H, s, CH ₃ ), 1.59 (1H, m, CH), 1.72 (1H, m, CH), 1.90-2.05 (4H, m, CH ₂ ), 3.08 (1H, m, CH);

¹³ C-NMR (CDCl ₃ , 300 MHz): δ = 60.23, 56.7, 44.9, 40.4, 39.6, 27.64, 26.72, 25.87, 22.41, 20.18. MS (EI) m / z 152 (M ⁺ ).

(4) methyl With acetoacetate n - Butanol Transesterification  reaction

Methyl acetoacetate (2.1 mL, 20 mmol) and n -butanol (1.8 mL, 20 mmol) were added to 20 mL of toluene and stirred at 400 rpm for 15 minutes at 30 ° C. In addition, 115 mg of the catalyst (FerlipaseCB [7] polymer beads) prepared in (2) was added to initiate the reaction. The clear liquid sample was removed periodically to confirm the product n-butyl acetoacetate by GC.

¹ H-NMR (CDCl ₃ , 300 MHz): δ = 2.12 (3H, s, CH ₃ ), 3.46 (2H, s, CH ₂ ), 4.05 (2H, t, CH ₂ ), 1.53 (2H, m, CH ₂ ), 1.35 (2H, m, CH ₂ ), 1.06 (3H, t, CH ₃ ).

Example  14. Confirmation of apoptosis

Ferrocene derivatives bound to the protein Annexin V (annexin V-ferrocene derivatives), which are known to selectively bind to the surface of the killed cells, are treated on cells induced by apoptosis by an anticancer agent, and then cucurbituril derivatives bound to FITC. (FITC-cookerbituril derivatives) were treated and flow cytometry was used to confirm the degree of cell death.

(One) Anticancer  Induction of Apoptosis by Treatment

Two ⁶ -well cell culture dishes containing approximately 10 ⁶ KB cells (oral cancer cells) (Korea Cell Line Bank, KCLB) at 37 ° C. and 5% CO ₂ under 2 mL of RPMI-1640 medium (including 10% FCS). Incubated for 48 hours at.

Then, one of the 6-well cell culture dishes exchanges 2 mL of RPMT-1640 culture medium (with 10% FCS) only (control), and the other RPMT-1640 with 0.5 μM (0.3 μg / ml) of doxorubicin. After exchange with 2 mL of culture (including 10% FCS), the two plates were further incubated at 37 ° C., 5% CO ₂ for 3 days.

(2) in the cell Annexin  V- Ferrocene  Treatment of Derivatives

Example 8 Preparation of N (1) - (ferrocenyl methyl) -6-aminocaproic acid (Fc-aca) 15mg, N-hydroxy succinimide (succinimide) (NHS, Fluka Co.), 6mg, and N 9 mg of-(3-dimethylaminopropyl) -N -ethylcarbodiimide hydrochloride (EDC, Fluka) was dissolved in 0.3 ml of anhydrous DMF solution, and the temperature was raised to 90 ° C. under a nitrogen atmosphere and stirred for 30 minutes (FIG. 15). Reference).

The reaction was further performed for 30 minutes while maintaining the temperature of the solution at 80 ° C, and cooled to room temperature. 30 μL of the reaction mixture was added to a solution of Annexin V (Sigma) (9 mg) in 1 ml of phosphate buffer (100 mM) at 2 minute intervals. After shaking for one day at room temperature, the precipitate was separated by centrifugation, and dialyzed in 50 mM phosphate buffer (pH 7.4) for 30 hours to obtain N- (ferrocenylmethyl) -6-aminocaproic acid (Fc-aca). Linked Annexin V (annexin V-ferrocene derivative) was obtained.

Remove the existing culture solution in the two cell culture dish of (1) and wash twice with 2 mL with PBS buffer (pH 7.4, sigma) only, and then the ferrocene connected in (1) 2 mL RPMT-1640 medium (with 10% FCS) containing 100 μl of Nexin-V solution reagent (10 μl of 10 × binding buffer (Trevigen), 1 μl of Annexin-V with ferrocene linked, 89 μl of distilled water) After exchange with the two culture dish was again incubated for 2 hours at 37 ℃, 5% CO ₂ conditions.

Then, in order to fix the cells, the existing culture solution in the two cell culture dish was removed and washed twice with 2 mL with PBS buffer (pH 7.4, Sigma Co., Ltd.), followed by 2% formaldehyde (Sigma Co., Ltd.). ) Was added 2 mL of PBS buffer solution (pH 7.4, sigma) and soaked for 15 minutes.

(3) [3- (2- Aminoethylsulfanyl ) Profile-O] ₁₄ Cookerbitu [7] manufacture of reels

In a quartz tube, 3 mL of DMF, 50 mg (0.025 mmol) of allyloxy cookerbitu [7] ryl prepared in Example 3, 87 mg (0.77 mmol) of 2-aminoethanethiol hydrochloride and 0.32 mg (0.002 mmol) of AIBN were added. Drop and seal. The inside of the tube was made into a nitrogen environment using the freeze-pump-thaw degassing method, and the tube was placed in a reactor irradiated with ultraviolet rays of 254 nm and left for 36 hours. After completion of the reaction, the solvent was removed under reduced pressure, 0.5 mL of triethylamine was added, and the mixture was washed four times with 15 mL of diethyl ether. After washing, the compound was dried under reduced pressure to obtain a final compound at 48 mg, 62% yield.

1 H NMR (300 MHz, DMSO): δ = 5.56 (d, J = 14.3 Hz, 14H), 4.05 (d, J = 14.3 Hz, 14H), 3.52 (m, 28H), 2.91 (m, 28H), 2.62 (m, 56 H), 1.84 (t, 28 H); 13 C NMR (75 MHz, DMSO): δ = 151.8, 95.5, 63.2, 40.8, 38.1, 33.4, 32.2 26.2; MS (MALDI-TOF): m / z 3048.1 [M + Na &lt; + &gt;].

(4) FITC - Cooker bituril  Treatment of Derivatives

Add [3- (aminoethylsulfanyl) propyl-O] ₁₄ cookerbitu [7] yl (100 mg, 0.033 mmol) and 1 mL of 1 M NaOH to DMSO (5 mL) and dissolve in DMSO (8 mL) FITC (28.26 mg). , 0.072 mmol) was added, followed by stirring at room temperature for 12 hours.

Distilled water (10 mL) was added to the reaction vessel, followed by neutralization with 11 M hydrochloric acid solution. Then, after dialysis for 12 hours, the solvent was obtained through a dark distillation to give a final compound as a white solid at a yield of 60% (75 mg).

The final compound was stored in a 4 ° C. refrigerator without light.

¹ H NMR (300 MHz, DMSO): = 10.5 (s, 2H), 9.9 (br, 2H), 8.2 (s, 2H), 7.9 (m, 2H), 7.3 (m, 2H), 6.4 6.7 (m , 12H), 5.56 (d, J = 14.3 Hz, 14H), 4.05 (d, J = 14.3 Hz, 14H), 3.52 (m, 28H), 2.91 (m, 28H), 2.62 (m, 56H), 1.84 (t, 28H).

After removing the existing buffer solution in two cell culture dishes and washing twice with 9 mL of PBS buffer solution (simga), 45 μg of the FITC-cookerbituryl derivative obtained above was dissolved in two cell culture dishes. It was immersed in 9 mL of PBS buffer solution (sigma) for 15 minutes.

(5) Check the degree of cell death

The cells contained in the PBS buffer were each transferred to a centrifuge tube, and each cell was taken through centrifugation and then suspended again in 500 μl of PBS buffer.

Then, the degree of cell death was confirmed using flow cytometry (Becton Dickinson, FACSCalibur, Ex = 488 nm, Em = 530 nm (FITC detection)).

Doxorubicin treated cells show much stronger FITC signals than doxorubicin-treated cells, and most of the cells killed by doxorubicin can be confirmed by flow cytometry. (A) of FIG. 20 is for KB cells not treated with doxorubicin, and (b) is for KB cells treated with doxorubicin.

As described above, the present invention is variously divided into a system using a non-covalent property between a specific ligand, such as a ferrocene derivative, and a cucurbituryl derivative, and a pair of molecules represented by Formula 1, and separation / purification, sensing, and analysis of a specific material using the same. Can be used

Claims (24)

Kits comprising the following a) and b) components: a) a first component comprising a first material and a compound of formula 1 linked to the first material; And b) a second component comprising a second substance and a ligand linked to the second substance (The first material and the second material are each independently a polymer comprising a benzene ring, silica gel, the polymer or gold-coated silica gel, gold thin film, silver thin film, glass, glass coated with ITO, metal electrode, nano Solid phase selected from the group consisting of tubes, cellulose, Sepharose and Sephadex; biomolecules selected from the group consisting of enzymes, proteins, amino acids, antibodies, antigens, cells, membranes, coenzymes, lectins and receptors; fluorescent materials and phosphors Selected from the group consisting of The ligand is capable of mutually non-covalent coupling with a compound represented by the following Chemical Formula 1, having at least one amine functional group, having a C ₁ -C ₂₀ alkyl group, a C ₆ -C ₂₀ aryl group, or a C ₅ -C ₂₀ heteroaryl group burnt; Or ferrocene or metallocene having at least one amine functional group and having a C ₁ -C ₂₀ alkyl group, a C ₆ -C ₂₀ aryl group or a C ₅ -C ₂₀ heteroaryl group: <Formula 1>

Where n is an integer from 6 to 10, X is O, A ₁ and A ₂ are each independently H or OR, R is H, C ₁ -C ₃₀ alkyl, C ₂ -C ₃₀ alkenyl, C ₂ -C ₃₀ alkynyl, C ₂ -C ₃₀ carbonylalkyl, C ₁ -C ₃₀ thioalkyl, C ₁ -C ₃₀ Alkylthiol, C ₁ -C ₃₀ hydroxyalkyl, C ₁ -C ₃₀ alkylsilyl, C ₁ -C ₃₀ aminoalkyl, C ₁ -C ₃₀ aminoalkylthioalkyl, C ₅ -C ₃₀ cycloalkyl, C ₂ -C ₃₀ heterocycloalkyl group, C ₆ -C ₃₀ aryl group, C ₆ -C ₃₀ arylalkyl group, C ₄ -C ₃₀ heteroaryl, and C ₄ -C ₃₀ heteroarylalkyl group, provided that A ₁ and A ₂ is not hydrogen at the same time). The kit of claim 1, wherein one of the first material and the second material is the biomolecule, and the other is a fluorescent material or a phosphor. delete The kit of claim 1, wherein said solid phase is a polystyrene resin. delete The kit of claim 1, wherein the enzyme is selected from glucose oxidase or lipase. delete The kit of claim 1 wherein A ₁ and A ₂ of Formula 1 are each independently H, OH or OR, wherein R is C ₂ -C ₃₀ alkenyl. The kit of claim 1 wherein n in formula 1 is 7 and X is O. 7. delete delete The kit of claim 1, wherein the ligand is adamantanamine or ferrocene methylamine. A method for separating and purifying a substance bound to a ligand, wherein the substance is selected from the group consisting of an enzyme, a protein, an antigen, an antibody, a fluorescent substance, and a phosphor; a) providing an affinity chromatography column filled with a stationary phase of a compound of formula 1 bound to a solid phase; b) providing to said column a mixture comprising a substance bound to said ligand; c) washing the column with a wash solution; And d) providing a mobile phase solvent to the column to separate and purify the substance bound to the ligand: <Formula 1>

Wherein n, X, A ₁ and A ₂ are each as defined in claim 1. The method of claim 13, further comprising combining the compound of Formula 1 with the solid phase prior to step a). The method of claim 13, further comprising binding the ligand and a substance between steps a) and b). The method of claim 13, wherein the mobile phase solvent is methanol, trifluoroacetic acid, triethylamine, methylene chloride, chloroform, dimethylformamide, dimethyl sulfoxide, toluene, acetonitrile, xylene, chlorobenzine, tetrahydrofuran, diethyl ether , Ethanol, diglycol ether, silicone oil, supercritical carbon dioxide, ionic liquids, N-methylpyrrolidine, pyridine, water, ammonium hydroxide, dioxane and chloroform How to be. The method of claim 13, wherein the washing liquid is methanol, trifluoroacetic acid, triethylamine, methylene chloride, chloroform, dimethylformamide, dimethyl sulfoxide, toluene, acetonitrile, xylene, chlorobenzine, tetrahydrofuran, diethyl ether, Ethanol, diglycol ether, silicone oil, supercritical carbon dioxide, ionic liquid, N-methylpyrrolidine, pyridine, water, ammonium hydroxide, dioxane and chloroform. A sensor chip comprising a compound of formula (1) bound to a first substance, and a conjugate of a ligand bound to a second substance (one of the first substance and the second substance is the solid phase, and the other is an enzyme, a coenzyme , Antibody, antigen, cell lectin, receptor, protein and combinations thereof): <Formula 1>

Wherein n, X, A ₁ and A ₂ are each as defined in claim 1. 19. The sensor chip of claim 18, wherein the solid phase is a gold thin film, silver thin film or ITO glass. Solid catalyst binder consisting of a compound of formula 1 bonded to a first material and a ligand bound to a second material (in this case, any one of the first material and the second material is a polymer comprising a benzene ring, silica gel, the polymer Or a solid phase selected from the group consisting of gold coated silica gel, gold thin film, silver thin film, glass, ITO coated glass, metal electrodes, nanotubes, cellulose, sepharose and sefadex, and the other is lipase or glucose oxy. It is the catalyst of choice in multiplexes): <Formula 1>

Wherein n, X, A ₁ and A ₂ are each as defined in claim 1. delete The solid catalyst binder according to claim 20, wherein the solid phase is a polystyrene resin. delete delete

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