Classifications

• • G—PHYSICS
  • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
  • G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
  • G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
  • G03F7/16—Coating processes; Apparatus therefor
  • G03F7/165—Monolayers, e.g. Langmuir-Blodgett
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
  • B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
  • B05D1/00—Processes for applying liquids or other fluent materials
  • B05D1/18—Processes for applying liquids or other fluent materials performed by dipping
  • B05D1/185—Processes for applying liquids or other fluent materials performed by dipping applying monomolecular layers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y40/00—Manufacture or treatment of nanostructures
• • G—PHYSICS
  • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
  • G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
  • G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
  • G03F7/004—Photosensitive materials
  • G03F7/008—Azides
• • G—PHYSICS
  • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
  • G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
  • G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
  • G03F7/004—Photosensitive materials
  • G03F7/016—Diazonium salts or compounds
• • G—PHYSICS
  • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
  • G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
  • G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
  • G03F7/004—Photosensitive materials
  • G03F7/075—Silicon-containing compounds
  • G03F7/0755—Non-macromolecular compounds containing Si-O, Si-C or Si-N bonds
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
  • B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
  • B05D3/00—Pretreatment of surfaces to which liquids or other fluent materials are to be applied; After-treatment of applied coatings, e.g. intermediate treating of an applied coating preparatory to subsequent applications of liquids or other fluent materials
  • B05D3/06—Pretreatment of surfaces to which liquids or other fluent materials are to be applied; After-treatment of applied coatings, e.g. intermediate treating of an applied coating preparatory to subsequent applications of liquids or other fluent materials by exposure to radiation

Definitions

• the present invention relates to a method for immobilizing a low-molecular compound on a solid support, a low-molecular microarray obtained by the method, a method for detecting a substance that interacts with the low-molecular compound using the method, and a method for detecting a low-molecular compound.
• the present invention relates to a method for identifying an interaction site. Background art
• a low-molecular-weight microarray in which various kinds of low-molecular-weight compounds are introduced in small amounts into a substrate typified by a glass slide, and immobilized, is useful as a tool for developing inhibitors of proteins such as enzymes and understanding their functions.
• conventional methods for producing low-molecular-weight microarrays have required uniform functional groups such as hydroxyl groups and amino groups on the low-molecular side in order to immobilize low-molecular compounds.
• Only synthetic combinatorial library compounds could be arrayed (Non-Patent Document 1). In the small molecule compound, the partial structure containing these functional groups faces the binding surface with the substrate and is ignored in the binding experiment with the protein on the array.
• Patent Document 1 JP 2001-178472 A
• Non-Patent Document 1 MacBeath et al., J. Am. Cliem. Soc. 1999, 121, 7967 Disclosure of the invention
• the immobilizable small molecule is limited to a group of molecules having a specific functional group, and includes a functional group used for bonding to a substrate.
• the partial structure is neglected in the binding experiment with proteins and the like on the array.
• the present invention has been made under the above technical background, and an object of the present invention is to provide a means for producing a low-molecular-weight microarray that solves the above-mentioned problems.
• the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by fixing a low-molecular compound on a substrate using a photoreactive compound, a low-molecular compound can be obtained without depending on a functional group. They have found that they can be fixed, and have completed the present invention.
• the present invention is a method for immobilizing a low-molecular compound on a solid support, which comprises the following steps.
• the present invention is a low-molecular-weight microarray obtained by the above method. Furthermore, the present invention is a method for detecting a substance that interacts with a low-molecular compound, comprising the following steps.
• the present invention is a method for identifying an interaction site in a low-molecular compound, comprising the following steps.
• the method for immobilizing a low-molecular compound on a solid support of the present invention is characterized by including the following steps (A-1) to (A-3).
• step (A-1) a solution containing a small molecule compound is brought into contact with a solid support having a photoreactive compound bound to its surface.
• the photoreactive compound is not particularly limited as long as it can be activated by irradiation with light and can form a covalent bond with a low-molecular compound.
• a group of the following compounds (a) to (c) Things can be mentioned.
• Compounds that generate radicals include conjugated ketones such as benzophenones and phenones, aromatic halides, and olefins, and compounds that generate carbon electrophiles include aromatic diazonium salts, Nitrobenzenes, sulfonium salts, phosphonium salts, ammonium salts and the like.
• R 1 is water atom, an optionally substituted alkyl group, or a substituent Represents an aryl group which may have a substituent;
• R 2 represents an aryl group which may have a substituent;
• Z— represents an anion;
• 4 , 5 , and 5 represents a group capable of forming a covalent bond by reacting with a functional group supported on the surface of the solid support, and the other four independently represent a hydrogen atom or a halogen. Represents an atom.
• the group represented by R 1 is preferably an alkyl group which may have a substituent having 1 to 6 carbon atoms, and a group having 6 to 12 carbon atoms. And a phenyl group which may have a substituent, particularly preferably an alkyl group substituted with an electron-withdrawing group such as a fluorine atom.
• the group represented by R 2 is preferably a phenyl group.
• the anion represented by the formula (1) is preferably a halide ion, a boron tetrafluoride ion, or a hexafluorophosphate ion.
• ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4 may be any of Upsilon 5, it is preferably Upsilon 3 .
• Preferred as a group capable of forming a covalent bond by reacting with a functional group carried on the surface of the solid support are a lipoxyl group, A formyl group, an active ester group, a hydroxyl group, a thiol, a sulfide, an amino group, a halogen-substituted alkyl group, a trialkoxysilyl group, and a group having these substituents.
• Specific examples of the compound represented by the general formula (I) include the following compounds 1 to 27, but are not limited thereto.
• Means for binding the photoreactive compound to the surface of the solid support is not particularly limited.
• the method can be performed according to the method described in JP-A-2001-178472.
• the photoreactive compound is preferably bound directly to the surface of the solid phase carrier, but another compound may be bound to the surface of the solid phase carrier and bound via the compound.
• the precursor may be bound to the surface of the solid support, and then converted to a photoreactive compound by performing an appropriate reaction treatment.
• the solid phase carrier may be any as long as it can bind the photoreactive compound.
• the solid phase carrier should be a microarray substrate, beads, fibers, tubes, containers (test tubes or vials), etc. Of these, a microarray substrate is most preferred.
• the microarray substrate on which the low-molecular compound is immobilized can be used as a low-molecular microarray for detecting proteins and the like.
• the material of the solid support include ceramics or new ceramics such as glass, cement, and porcelain, polyethylene terephthalate, cellulose acetate, bisphenol A polystyrene, polystyrene, polymethyl methacrylate, and the like.
• Porous materials such as polymers, silicon, activated carbon, porous glass, porous ceramics, porous silicon, porous activated carbon, woven fabric, knitted fabric, nonwoven fabric, filter paper, staple fiber, membrane filter, etc.
• a conductive material such as gold can be fisted, and among these, glass, silicon, gold and the like are preferable.
• the surface of the solid support is coated with a polymer such as a polycation to introduce amino groups, carbonyl groups, hydroxyl groups, etc., or treated with a silane coupling agent having a substituent introduced on the solid support surface. May be applied.
• a reactive functional group may be introduced by plasma treatment.
• the low molecular weight compound in the present invention is an organic compound having carbon, hydrogen, oxygen, nitrogen, and sulfur as main components, and primary metabolites such as oligosaccharides and polypeptides, fatty acids, and polyketides.
• primary metabolites such as oligosaccharides and polypeptides, fatty acids, and polyketides.
• secondary metabolites such as isoprenoid, phenylpropanoide, and alkyloid
• synthetic organic compounds having a molecular weight of up to about 2000, including aromatic rings and heterocycles, or complexes of these compounds.
• the solvent used for preparing the low-molecular compound solution can be appropriately determined according to the type of the low-molecular compound, and examples thereof include water, dimethyl sulfoxide (DMS0), N, N-dimethylformamide (DMF), dioxane, and acetonitrile. , Black mouth form etc. can be used.
• the concentration of the low-molecular compound solution varies depending on the type of the low-molecular compound and the solvent, or the contacting method.For example, when a DMS0 solution of a polyketide having a molecular weight of about 500 is dropped on a solid support, 0.1 to 0.1% is used.
• the concentration is about 0.1 to 100 mM.
• the method for bringing the low-molecular compound solution into contact with the solid support may be any method that can fix the low-molecular compound to the solid support by light irradiation.
• a solution containing a low molecular weight compound may be dropped on this substrate. The dropping amount at this time varies depending on the type of the low molecular weight compound and the concentration of the solution, but is about 1 nL to 0.2.
• step (A-2) the solution containing the low molecular weight compound is dried while being in contact with the solid phase carrier.
• the method for drying the solution is not particularly limited, and the solution may be dried by natural drying. However, it is preferable to artificially dry the solution using a vacuum pump or the like.
• the low-molecular compound and the photoreactive group come into close contact, and the low-molecular compound binds to the photoreactive compound at various sites. This means that all parts of the low molecular weight compound are exposed on the surface of the solid support, and it is possible to examine the properties (such as binding ability) of the entire molecule of the low molecular weight compound.
• the molecular density of the low-molecular compound increases, and the effect of improving the reaction efficiency with the photoreactive compound can be obtained.
• the solid support is irradiated with light to form a covalent bond between the photoreactive compound and the low-molecular compound.
• the wavelength of the light to be irradiated may be determined according to the type of the photoreactive compound, and the light is usually irradiated with a wavelength in the range of 200 to 400 nm, preferably around 360 nm.
• the light source electric light such as solar light or mercury lamp, laser light (semiconductor laser, solid-state laser, gas laser), light emission of a light emitting diode, light emission of an electorescent luminescent element, and the like can be used.
• the light irradiation method can be such that light from a light source such as a mercury lamp can be uniformly applied to the surface of the solid support through an appropriate filter if necessary, or a pattern exposure of a desired shape using a so-called mask You may do.
• light may be condensed using a lens or a mirror and irradiated to a fine shape.
• the condensed light beam may be scanned and exposed.
• the irradiation time is not particularly limited.
• the solid support After the light irradiation, the solid support is washed with an appropriate solvent or buffer to remove unfixed low molecular weight compounds. Thereby, a solid support on which the low molecular weight compound is immobilized can be obtained.
• the immobilization method of the present invention can be applied to the following “method of detecting a substance that interacts with a low molecular compound” and “method of specifying an interaction site in a low molecular compound”.
• the method for detecting a substance that interacts with a low molecular weight compound of the present invention is characterized by comprising the following steps (B-1) to (B-3).
• step (B-1) a solution containing a labeled sample substance is brought into contact with the low-molecular-weight microarray obtained by the above-described fixing method of the present invention.
• sample substance is not particularly limited, a biological substance such as a protein is mainly used.
• the method of labeling the sample substance is not particularly limited, and the sample substance may be labeled by the RI method or may be labeled by the non-RI method (fluorescence method, biotin method, etc.).
• the solvent used for preparing the sample substance solution can be appropriately determined according to the type of the sample substance, and for example, water, a phosphate buffer, an acetate buffer, a Tris buffer, or the like can be used.
• concentration of the sample substance solution varies depending on the type of the low-molecular compound and the solvent. For example, in the case of a buffer solution of a fluorescently labeled protein having a molecular weight of about 160,000, it is about 10 to 100 g / mL.
• the method for bringing the sample substance solution into contact with the low-molecular microarray is not particularly limited.
• the sample substance solution may be dropped on the microarray.
• the drop amount at this time varies depending on the type of the sample substance and the concentration of the solution, but is about 0.1 to 1 ⁇ ⁇ mm— 2 .
• step (B-2) substances that do not bind to the low molecular weight compounds are removed.
• Removal of substances that do not bind to low molecular weight compounds is performed by washing with a suitable buffer or solvent.
• step (B-3) the label of the sample substance is detected.
• Detection of the label can be performed according to the type of the labeling substance. For example, when labeled with a fluorescent substance, it can be detected by a fluorescent slide scanner or the like.
• the sample substance can be determined to be a substance that interacts with the low-molecular compound, and if no label is detected, the sample substance interacts with the low-molecular compound. It can be determined that the substance is not used.
• a low-molecular compound is immobilized on a solid phase carrier by using a specific functional group.
• the partial structure containing the functional group faces the bonding surface with the solid phase carrier. It is not exposed on the surface of the phase carrier. Therefore, if the partial structure containing the functional group is involved in the interaction with the sample substance, even if it is a low molecular compound that actually interacts with the sample substance, There is a possibility that it will be judged as not carrying out.
• the detection method of the present invention uses a microarray in which all parts of the low-molecular compound are exposed on the substrate surface, there is no possibility of making an erroneous determination as described above, and the interaction with the low-molecular compound can be performed with high accuracy. Can be detected.
• the method for identifying the interaction site in the low molecular weight compound of the present invention comprises the following steps (C-1 1) to (C-16).
• step (C-11) a photoreactive compound and a low-molecular compound that interacts with a specific substance are mixed.
• photoreactive compound regardless of its form, whether powdery or oily, the same ones as used in the immobilization method of the present invention can be used.
• the same compounds as those used in the immobilization method of the present invention can be used. In this method, among such compounds, those that have already been confirmed to interact with a specific substance have been used.
• the mixture is irradiated with light to form a covalent bond between the photoreactive compound and the low-molecular compound.
• This step can be performed in the same manner as the step (A-3) of the fixing method of the present invention.
• the step (C-13) the complex of the photoreactive compound and the low-molecular compound is separated based on the difference in the binding site of the low-molecular compound.
• the method for fractionating the complex is not particularly limited, and for example, it can be fractionated by high performance liquid chromatography.
• the separated conjugates are respectively fixed on a microarray substrate.
• the conjugate can be immobilized on the microarray substrate according to a conventional method, and can be immobilized using a functional group carried on the surface of the solid support.
• a solution containing the labeled specific substance is brought into contact with the complex fixed on the substrate.
• This step can be performed in the same manner as step (B-1) of the detection method of the present invention.
• the substance to be brought into contact with the substrate is not a substance that does not know whether it interacts with a low-molecular compound (sample substance), but a substance that has been confirmed to interact with a low-molecular compound.
• step (C-17) of the complexes immobilized on the substrate, those in which no label is detected are selected, and the binding site between the low-molecular compound and the photoreactive compound of the complex is identified.
• Methods for specifying the binding site include, for example, mass spectrum, nuclear magnetic resonance This can be done by a kutor or the like.
• the method for identifying the interaction site in the low-molecular compound described above will be described with reference to FIG. 1.
• the low-molecular compound 1 in the figure has an SH group, and this group is involved in the interaction with the specific substance 3. I have.
• the low-molecular compound 1 binds to the photoreactive compound 2.
• the low molecular compound 1 binds to the photoreactive compound 2 not only at the SH group but also at various sites (Fig. 1b).
• the conjugate group of the low-molecular compound 1 and the photoreactive compound 2 is separated by the steps (C-3) to (C-4) and fixed on the substrate 4.
• FIG. 1 is a diagram schematically showing a method for identifying an interaction site in a low-molecular compound of the present invention
• FIG. 2 is a diagram showing an outline of a glass substrate modification step
• FIG. , 488 nm, 532 nm, and 635 nm are images of a low-molecular-weight immobilized slide irradiated with a fluorescent scanner.
• the treated slide glass was sequentially washed with ethanol, deionized water, ethanol and deionized water for 10 minutes each, dehydrated with a dehydration centrifuge (400 ⁇ g) for 1 minute, and further dried under vacuum to obtain a succinimidylated slide.
• Each DMS0 solution of low-molecular compounds prepared in advance to 100, 10, 1, 0.1, 0.1 OlmM on a slide with a photoaffinity group introduced at 0. Spotted.
• the slide was dried in a 35 ° C incubator for 3 hours, and then dried for 20 hours using a vacuum pump. After irradiating this slide with ultraviolet light of 365 nm for 30 minutes, excess low molecular weight compounds not immobilized on the slide were washed away with ethanol.
• the slide was further immersed in ethanol, wisteria, THF, ethanol, and deionized water (one hour each) and washed with shaking to obtain a slide on which low molecules were immobilized.
• Protein solution (162 g / mL anti-digoxin monoclonal antibody clone DI-22-FITC conjugate, 3. T g / mL streptavidin-Alexa Fluor 633 conjugate, 1% (w / v) Skim milk, 77 mM NaCK 0.05% (w / v) Tween 20, 50 mM Tris-HCK pH 7.5) were added at a rate of 0.18 mL ⁇ mm- 2 and treated at room temperature for 1 hour.
• the slide is washed three times with a washing buffer (77 mM NaCl, 0.05% (w / v) Tween 20, 50 mM Tris-HCK pH 7.5) three times, rinsed with deionized water, and then centrifuged with a dehydration centrifuge ( (400xg) for 1 minute.
• the slide was excited with light having wavelengths of 488, 532, and 635im, respectively, and the resulting fluorescence was observed using a fluorescence slide scanner.
• Figure 3 shows the results.
• the image in the left column is obtained by observing the slide before immersion in the protein solution with a fluorescence scanner
• the image in the right column is obtained by observing the slide with the protein solution after treatment with the protein solution.
• the immobilization method of the present invention can immobilize a low-molecular-weight compound independently of a functional group, and thus can immobilize more various compounds than conventional immobilization methods.
• the immobilized low-molecular compound is bonded to the solid support at various sites in the molecule, all parts of the low-molecular compound are exposed on the surface of the solid support. As a result, it becomes possible to examine the binding properties of the entire molecule of the low molecular weight compound.

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• Physics & Mathematics (AREA)
• General Physics & Mathematics (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Nanotechnology (AREA)
• Crystallography & Structural Chemistry (AREA)
• Condensed Matter Physics & Semiconductors (AREA)
• Manufacturing & Machinery (AREA)
• Spectroscopy & Molecular Physics (AREA)
• Composite Materials (AREA)
• Materials Engineering (AREA)
• Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
• Physical Or Chemical Processes And Apparatus (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)

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• 2003
  • 2003-04-09 JP JP2003104928A patent/JP3901120B2/ja not_active Expired - Fee Related
• 2004
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