WO2020250998A1 - Composé fluorescent à rétention intracellulaire et procédé de coloration de cellules et procédé de détection de cellules à haute sensibilité l'utilisant - Google Patents

Composé fluorescent à rétention intracellulaire et procédé de coloration de cellules et procédé de détection de cellules à haute sensibilité l'utilisant Download PDF

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WO2020250998A1
WO2020250998A1 PCT/JP2020/023120 JP2020023120W WO2020250998A1 WO 2020250998 A1 WO2020250998 A1 WO 2020250998A1 JP 2020023120 W JP2020023120 W JP 2020023120W WO 2020250998 A1 WO2020250998 A1 WO 2020250998A1
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cell
enzyme
antibody
general formula
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大内雄也
野口克也
石山宗孝
片山佳樹
森健
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株式会社同仁化学研究所
国立大学法人九州大学
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/02—Coumarine dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

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  • the present invention relates to an improvement of a cell staining technique having high specificity for a specific cell, and more specifically to a cell-retaining fluorescent compound, a cell staining method using the same, and a highly sensitive detection method.
  • Non-Patent Document 1 a method in which an antibody modified with a fluorescent group is used, a fluorescence-modified antibody is bound to a target marker protein, and the antibody is read out using a fluorescence detection method is widely known.
  • the disadvantage of this method is that sensitivity is low, the detection limit for 10 3 copies per cell, there is a problem that application target is limited.
  • One of the proposed high-sensitivity detection methods using an antibody and a fluorescent dye is a method called the CARD method (CAtalyzed Reporter Deposition method) (see, for example, Non-Patent Document 2).
  • CARD method CAtalyzed Reporter Deposition method
  • an antibody modified with HRP Hydrophilicity-Propane
  • HRP Hydrophilicity-Propane
  • the tyramide pigment is oxidized using HRP and hydrogen peroxide to generate the tyrosine radical and the tyrosine residue of the protein.
  • the effect of signal amplification by this method is about 10 times that when a fluorescently labeled antibody is used, and there remains a problem that the sensitivity is still low.
  • CARP method has been proposed (see Patent Document 1).
  • a fluorescent dye (which has hydrophilicity and therefore does not have cell membrane permeability) to which a hydrophilic group is bound approaches cells to which an enzyme-modified antibody is bound to a target marker protein, and the hydrophilic group is cleaved by the action of the enzyme. Then, the fluorescent dye becomes permeable to the cell membrane, and is accumulated inside the cell by diffusing on the cell membrane or penetrating the cell membrane, enabling detection with higher sensitivity than the conventional method (lower). See formula).
  • the fluorescent dye used in the CARP method has low intracellular retention, causing leakage from cells and concomitantly non-specific fluorescent staining for other cells that do not express the target cell surface marker protein. there is a possibility.
  • the present invention has been made in view of such circumstances, and a cell-retaining fluorescent compound that enables specific and highly sensitive detection of only cells expressing a target cell surface protein, and cells using the same. It is an object of the present invention to provide a staining method and a highly sensitive detection method.
  • the first aspect of the present invention in line with the above object is to solve the above problem by providing a cell-retaining fluorescent compound represented by the following general formula (I).
  • A is a monovalent hydrophilic group cleaved by an enzymatic reaction.
  • R 1 , R 2 , R 3 , R 4 and R 5 are atoms or groups of atoms or atomic groups independently selected from the group consisting of the following (a), (b) and (c), respectively.
  • One or more hydrogen atoms are replaced with other atoms or atomic groups.
  • R 1 , R 3 or R 5 is an alkyl group having one or two fluorine atoms at the benzyl position. Any one of R 1 , R 2 , R 3 , R 4 and R 5 contains a hydrophobic fluorescent group.
  • two of R 1 , R 2 , R 3 , R 4 and R 5 adjacent to each other except for an alkyl group having one or two fluorine atoms at the benzyl position share an atom or an atomic group and form a ring. It may be formed and a hydrophobic fluorescent group may be formed together with the benzene ring to which the ring is bonded.
  • the hydrophobic fluorescent group has cell membrane permeability.
  • A may be a ⁇ -D-galactoxyl group or a phosphoric acid group.
  • R 1 or R 5 is a fluoromethyl group or a difluoromethyl group.
  • cell-retaining fluorescent compound according to the first aspect of the present invention is represented by any of the following formulas (1) to (10).
  • a second aspect of the present invention is a step of binding an enzyme-labeled antibody in which an enzyme is bound to an antibody having specificity for an antigen protein expressed on the surface of a target cell to the target cell, and the binding of the enzyme-labeled antibody.
  • the above problem is solved by providing a method for staining cells, which comprises a step of contacting the target cells with a cell-retaining fluorescent compound represented by the following general formula (I).
  • A is a monovalent hydrophilic group cleaved by an enzyme-catalyzed reaction bound to the enzyme-labeled antibody.
  • R 1 , R 2 , R 3 , R 4 and R 5 are atoms or groups of atoms or atomic groups independently selected from the group consisting of the following (a), (b) and (c), respectively.
  • One or more hydrogen atoms are replaced with other atoms or atomic groups.
  • R 1 , R 3 or R 5 is an alkyl group having one or two fluorine atoms at the benzyl position. Any one of R 1 , R 2 , R 3 , R 4 and R 5 contains a hydrophobic fluorescent group.
  • two of R 1 , R 2 , R 3 , R 4 and R 5 adjacent to each other except for an alkyl group having one or two fluorine atoms at the benzyl position share an atom or an atomic group and form a ring. It may be formed and a hydrophobic fluorescent group may be formed together with the benzene ring to which the ring is bonded.
  • the antigen protein expressed in the target cell is epidermal growth factor receptor (EGFR), HER2, PDL-1, PDL-2, WT-1, PD-. 1.
  • EGFR epidermal growth factor receptor
  • HER2 PDL-1, PDL-2, WT-1, PD-. 1.
  • CCR4, CD33, CD24, CD29, CD40, CD44, CD80, CD86, CD105, CD133, CD166, CD200, ESA, CXCR4, Stro-1, EpCAM, Protein12b1, BMI-1 may be used.
  • the enzyme contained in the enzyme-labeled antibody is ⁇ -galactosidase, and in the general formula (I), A is a ⁇ -D-galactoxyl group. You may.
  • the enzyme contained in the enzyme-labeled antibody may be alkaline phosphatase, and in the general formula (I), A may be a phosphate group. ..
  • R 1 or R 5 is a fluoromethyl group or a difluoromethyl group. ..
  • the cell-retaining fluorescent compound is represented by any of the following formulas (1) to (10).
  • a third aspect of the present invention is a step of staining a target cell using the cell staining method according to the second aspect of the present invention, and a step of detecting the target cell stained by a fluorescence method.
  • flow cytometry or fluorescence imaging may be used to detect the stained target cells.
  • the cell-retaining fluorescent compound of the present invention represented by the general formula (I) has a hydrophilic group A, it does not have cell membrane permeability as it is, but when the hydrophilic group A is cleaved by an enzymatic reaction. Increases hydrophobicity and develops cell membrane permeability. Further, at the same time as the cleavage of the hydrophilic group A, hydrogen fluoride is eliminated from R 1 , R 3 or R 5 , which is an alkyl group having one or two fluorine atoms at the benzyl position, to generate quinone methide. Quinone methide is an electrophilic Michael adsorbent that reacts with amino groups, sulfhydryl groups, etc.
  • the cell-retaining fluorescent compound of the present invention is, for example, a protein targeted by the enzyme-modified antibody by combining with an enzyme-modified antibody modified with an enzyme having an activity of catalyzing a reaction for cleaving the hydrophilic group A.
  • the cells expressed on the cell surface can be specifically fluorescently stained.
  • a cell-retaining fluorescent compound that enables specific and highly sensitive detection of only cells expressing a target cell surface protein, a cell staining method using the same, and highly sensitive detection. A method is provided.
  • 6 is a histogram showing the results of flow cytometric analysis of HeLa cells using CD44 as a surface antigen.
  • 6 is a histogram showing the results of flow cytometric analysis of HeLa cells using CD44 as a surface antigen.
  • 6 is a histogram showing the results of flow cytometric analysis of HeLa cells using CD44 as a surface antigen.
  • 3 is a fluorescence micrograph showing the result of fluorescence staining of HeLa cells using CD44 as a surface antigen.
  • cell-retaining fluorescent compound is represented by the following general formula (I).
  • A is a monovalent hydrophilic group in which the bond between A and phenolic oxygen is cleaved by an enzymatic reaction (reaction catalyzed by an enzyme).
  • any one that can be a substrate for a hydrolase can be used without particular limitation.
  • Specific examples of A include a phosphoric acid group, a sulfate group, a glucosyl group, a galactoxyl group, a xylosyl group, a glucuronic acid group and the like.
  • R 1 , R 2 , R 3 , R 4 and R 5 are independently selected atoms from the group consisting of the following (a), (b) and (c), respectively. Or it is an atomic group.
  • R represents a hydrogen atom, an alkyl group (for example, 1 to 10 carbon atoms) or a fluorescent group (the other examples will be described later), and when a plurality of Rs are present, they represent each other. It may be the same or different.
  • Hydrogen atom (-H), hydroxyl group (-OH), alkoxyl group (-OR), halogen atom (-F, -Cl, -Br, -I), amino group (-NR 2 ), nitro group (-NO 2), a sulfonic acid group (-SO 3 H or a salt thereof), sulfonamide group (-SO 2 NR 2), a cyano group (-CN)
  • One or more hydrogen atoms may be substituted with other atoms or atomic groups, and double bonds, triple bonds, amino groups (-NR-), oxygen atoms (-O) in the carbon skeleton.
  • Branched chain alkyl group for example, 1 to 10 carbon atoms
  • cycloalkyl group for example, 3 to 10 carbon atoms
  • alkenyl group containing a double bond at the end, for example, 2 to 10 carbon atoms
  • alkynyl groups containing triple bonds at the ends, for example, hydrocarbon groups having 2 or more and 10 or less carbon atoms
  • aryl groups including one or more monocyclic or fused ring aromatic rings.
  • a hydrocarbon group) and a heteroaryl group containing one or more monocyclic or fused ring aromatic rings, one or more carbon atoms substituted with heteroatoms such as oxygen, nitrogen or sulfur atoms.
  • R 1 , R 3 or R 5 is an alkyl group having one or two fluorine atoms at the benzyl position (for example, having 1 or more and 10 or less carbon atoms), and R 1 , R 2 , R 3 , R 4 and R 5 contain a hydrophobic fluorescent group, or one of R 1 , R 2 , R 3 , R 4 and R 5 has a fluorine atom at the benzyl position. Adjacent two, excluding one or two alkyl groups, may share an atom or atomic group to form a ring and form a hydrophobic fluorescent group together with the benzene ring to which the ring is bonded.
  • the hydrophobic fluorescent group any known group having a desired emission wavelength and excitation wavelength can be used without particular limitation.
  • the hydrophobic fluorescent group preferably has cell membrane permeability.
  • the hydrophobic fluorescent group may be contained in any one of R 1 , R 2 , R 3 , R 4 and R 5 of the general formula (I), or R 1 , R 2 , ,.
  • Two of R 3 , R 4 and R 5 adjacent to each other except for an alkyl group having one or two fluorine atoms at the benzyl position form a ring by sharing an atom, and together with a benzene ring to which the ring is bonded. It may form a hydrophobic fluorescent group.
  • hydrophobic fluorescent groups include coumarin derivatives, fluorescein derivatives, borondipyrromethene derivatives, rhodamine derivatives, rodol derivatives, cyanine derivatives, xanthene derivatives, resorphin derivatives, pyrene derivatives, naphthalimide derivatives, benzoxazole derivatives, and DDAO derivatives. , Dansyl derivative, squalene derivative and the like.
  • R 1 , R 3 or R 5 is an alkyl group having one or two fluorine atoms at the benzyl position, which is a condition for producing o- or p-quinone methide by cleavage of A. It is preferably a fluoromethyl group.
  • preferable compounds include those represented by any of the following formulas (1) to (10).
  • the cell-retaining fluorescent compound can be synthesized by using any known method. Some specific examples will be described in the examples.
  • the cell staining method according to the second embodiment of the present invention is a step of binding an enzyme-labeled antibody in which an antibody is bound to an antibody having specificity for an antigen protein expressed on the surface of the target cell to the target cell. And, the step of contacting the target cell to which the enzyme-labeled antibody is bound with the cell-retaining fluorescent compound represented by the above general formula (I) is included.
  • any antibody having a protein expressed on the surface of the target cell preferably a protein specifically expressed on the surface of the target cell as an antigen can be used without particular limitation.
  • the antigen protein include epidermal growth factor receptor (EGFR), HER2, PDL-1, PDL-2, WT-1, PD-1, CCR4, CD33, CD24, CD29, CD40, CD44, CD80, CD86. , CD105, CD133, CD166, CD200, ESA, CXCR4, Stro-1, EpCAM, Antigen12b1, BMI-1, and the like.
  • EGFR epidermal growth factor receptor
  • HER2 PDL-1, PDL-2, WT-1, PD-1, CCR4, CD33, CD24, CD29, CD40, CD44, CD80, CD86.
  • CD105, CD133, CD166, CD200 ESA, CXCR4, Stro-1, EpCAM, Antigen12b1, BMI-1, and the like.
  • a monoclonal antibody produced using a hybridoma a polyclonal antibody obtained by administration of an antigen to an animal (mouse, rat, goat, etc.), and an Fc region of an animal-derived polyclonal antibody are used for human-derived immunoglobulin. Examples thereof include substituted humanized antibodies.
  • the enzyme used for labeling the antibody is any hydrolase having an activity of catalyzing the reaction of the cell-retaining fluorescent compound represented by the general formula (I) to break the bond between A and phenolic oxygen. Can be appropriately selected and used according to the type of A used for the cell-retaining fluorescent compound. Specific examples of the enzyme include alkaline phosphatase, ⁇ -galactosidase and the like.
  • Labeling an antibody with an enzyme means binding the enzyme to the antibody via a binding group, and the labeling site of the enzyme and the antibody and the binding group used are not particularly limited as long as they do not inhibit their respective functions. Covalent binding via a linker, binding of a primary antibody to a secondary antibody, and formation of a complex of biotin and avidin are preferably used for labeling an antibody with an enzyme. Binding of the enzyme-labeled antibody to the surface of the target cell may be carried out by directly binding the enzyme-labeled antibody to the surface of the target cell, or binding the primary antibody or biotin-labeled antibody to the surface of the target cell, and then the enzyme-labeled antibody. This may be carried out by binding the secondary antibody or avidin-labeled enzyme to the primary antibody or biotin-labeled antibody previously bound to the surface of the target cell.
  • the hydrophilic group A of the cell-retaining fluorescent compound is cleaved by hydrolysis due to the action of the enzyme bound to the antibody.
  • the cell-retaining fluorescent compound expresses cell membrane permeability and produces highly reactive quinone methide. The binding of quinone methide to proteins on the surface or inside of the target cells causes the target cells to be specifically fluorescently stained.
  • the high-sensitivity detection method for cells according to the third embodiment of the present invention includes a step of staining target cells and a fluorescence method using the cell staining method according to the second embodiment of the present invention. It comprises the step of detecting the target cells stained using.
  • the target cell may be any cell that expresses the protein to be recognized by the enzyme-labeled antibody on the surface.
  • target cells include epidermal growth factor receptor (EGFR), HER2, PDL-1, PDL-2, WT-1, PD-1, CCR4, CD33, CD24, CD29, CD40, CD44, CD80, CD86.
  • EGFR epidermal growth factor receptor
  • HER2 PDL-1, PDL-2, WT-1, PD-1, CCR4, CD33, CD24, CD29, CD40, CD44, CD80, CD86.
  • the cleavage of the hydrophilic group A of the cell-retaining fluorescent compound by the action of the enzyme bound to the antibody triggers the cell-retaining fluorescent compound to express cell membrane permeability and to produce quinonemethide having high reactivity.
  • the target cells are fluorescently stained by producing and binding to proteins on the surface or inside of the target cells. Any known method such as a fluorescence imaging method or a flow cytometry method can be used for the detection of the fluorescently stained cells, and for example, the flow cytometry method is preferably used.
  • cells expressing a specific surface protein can be specifically fluorescently stained and detected with high sensitivity. Therefore, a highly sensitive diagnostic method for a specific disease using the same, the above-mentioned cell-retaining fluorescent compound and enzyme labeling.
  • a sensitive detection kit for specific cells containing an antibody is also provided. They may also be included in embodiments of the present invention.
  • Example 1 Synthesis of Compound 1 Compound 1 was synthesized according to the following scheme.
  • Hexamethylenetetramine (20 g, 143 mmol), 4-methylcoumarin (10 g, 57 mmol), and acetic acid (75 mL) were placed in a 300 mL eggplant flask.
  • the oil bath was set at 95 ° C. and reacted for 4 hours while stirring with a stirrer. After returning to room temperature, 140 mL of dilute hydrochloric acid was added. The mixture was heated to 95 ° C. and further reacted for 1 hour. Then, 100 mL of ethyl acetate was added and an extraction operation was performed. The ethyl acetate layer was concentrated and then purified by column chromatography to obtain 720 mg (3.5 mmol, 2.5%) of intermediate A as a slightly yellowish white powder.
  • Example 3 Synthesis of Compound 3 Compound 3 was synthesized according to the following scheme.
  • Example 5 Synthesis of compound 10 Compound 10 was synthesized according to the following scheme.
  • Example 6 Examination of fluorescence emission characteristics of compound 1 Compound 1 (5 ⁇ M) is mixed with ⁇ -galactosidase (5 U / mL) in a phosphate buffer solution (10 mM, pH 7.4) and enzymatically reacted at room temperature for 10 minutes. Was done. Then, the UV-VIS spectrum (UV-2450, Shimadzu Corporation) and the fluorescence spectrum (FP-6300, JASCO Corporation) were measured. For comparison, the UV-VIS spectrum and the fluorescence spectrum were also measured without the enzymatic reaction.
  • Example 7 Specific fluorescent staining of A549 cells targeting CD44 CD44 is known as a marker expressed on the surface of cancer cells such as A549 cells. In this example, fluorescent staining of A549 cells targeting CD44 was performed.
  • A549 cells were contacted with an anti-CD44 antibody (Abcam: rabbit monoclonal antibody) (MEM medium) and left in a CO 2 incubator (37 ° C.) for 1 hour. After washing, ⁇ -galactosidase-labeled anti-rabbit antibody (Abcam) (MEM medium) was contacted and left in a CO 2 incubator (37 ° C.) for 1 hour. After washing, compound 1 (HBSS) was added, and after incubation in a CO 2 incubator (37 ° C.) for 30 minutes, fluorescence observation of cells was performed using a fluorescence microscope (KEYENCE BZ-X710). For comparison, a similar experiment was performed using 4-methylumbelliferyl ⁇ -D-galactopyranoside (MUG), which does not form quinone methide.
  • MEM medium ⁇ -galactosidase-labeled anti-rabbit antibody
  • HBSS compound 1
  • fluorescence observation of cells was performed using a fluorescence microscope (
  • Example 8 Specific Fluorescent Staining of PD-L1 Expressing HepatG2 Cells
  • PD-L1 is a ligand for PD-1 expressed on the surface of activated T cells and is involved in the cancer immune cycle. Although it is known, it has been attracting attention as a marker for hepatocellular carcinoma cells in recent years. In this example, specific staining with compound 1 was attempted using HepG2 cells derived from human hepatocellular carcinoma in which PD-L1 was induced by interferon ⁇ (IFN- ⁇ ).
  • IFN- ⁇ interferon ⁇
  • HepG2 cells in which PD-L1 was induced were contacted with an anti-PD-L1 antibody (Abcam: rabbit monoclonal antibody) (DMEM medium) and left in a CO 2 incubator (37 ° C.) for 1 hour. After washing, ⁇ -galactosidase-labeled anti-rabbit antibody (Abcam) (DMEM medium) was contacted and left in a CO 2 incubator (37 ° C.) for 1 hour. After washing, compound 1 (HBSS) was added, and after incubating in a CO 2 incubator (37 ° C.) for 30 minutes, fluorescence observation of cells was performed using a fluorescence microscope (KEYENCE BZ-X710).
  • Abcam rabbit monoclonal antibody
  • Example 9 Flow cytometric analysis of HeLa cells using CD44 as a surface antigen (1)
  • the suspended HeLa cells detached from the incubator using trypsin / EDTA were contacted with an anti-CD44 antibody (Abcam: rabbit monoclonal antibody) (MEM medium) and left in a CO2 incubator (37 ° C.) for 1 hour. .. After washing, ⁇ -galactosidase-labeled anti-rabbit antibody (Abcam) (MEM medium) was contacted and left in a CO 2 incubator (37 ° C.) for 1 hour.
  • Abcam rabbit monoclonal antibody
  • HBSS 20 ⁇ M solution
  • HBSS 20 ⁇ M solution
  • CO 2 incubator 37 ° C.
  • a flow cytometer Becton Dickinson, Fortessa X-20
  • Fluorescence intensity (excitation 405 nm, fluorescence 450/40 nm BP) was measured. ..
  • As a general secondary antibody method an experiment was conducted using an anti-CD44 antibody and a commercially available fluorescent dye (Alexa405) -labeled anti-rabbit antibody.
  • FIGS. 6 and 7 The results of flow cytometry analysis using Compound 1 and Compound 8 are shown in FIGS. 6 and 7, respectively.
  • FIGS. 6 and 7 The results of flow cytometry analysis using Compound 1 and Compound 8 are shown in FIGS. 6 and 7, respectively.
  • HeLa cells were treated with anti-CD44 antibody, ⁇ -galactosidase-labeled antibody and compound 1 or 8, they showed higher fluorescence intensity than anti-CD44 antibody and Alexa405-labeled antibody. This indicates that the sensitivity is insufficient by the method using a general fluorescently labeled antibody.
  • Example 10 Flow cytometric analysis of HeLa cells using CD44 as a surface antigen (2) Suspended HeLa cells detached from the incubator using trypsin / EDTA were fixed with 4% PFA / PBS and subjected to membrane permeation treatment with 1% Triton-X100 / PBS.
  • Anti-CD44 antibody (Abcam: rabbit monoclonal antibody) (10% Blocking One / PBS) was contacted and left at room temperature for 1 hour. After washing, ⁇ -galactosidase-labeled anti-rabbit antibody (Abcam) (0.1% Tween20 / PBS) was contacted and allowed to stand at room temperature for 1 hour.
  • a 20 ⁇ M solution (PBS) of compound 1 or compound 8 is added, incubated in a CO 2 incubator (37 ° C.) for 30 minutes, and then cells are used using a flow cytometer (Becton Dickinson, Fortessa X-20). The fluorescence intensity (excitation 405 nm, fluorescence 450/40 nm BP) was measured for each.
  • a general secondary antibody method an experiment was conducted using an anti-CD44 antibody and a commercially available fluorescent dye (Alexa405) -labeled anti-rabbit antibody.
  • FIGS. 8 and 9 The results of flow cytometry analysis using Compound 1 and Compound 8 are shown in FIGS. 8 and 9, respectively.
  • FIGS. 8 and 9 The results of flow cytometry analysis using Compound 1 and Compound 8 are shown in FIGS. 8 and 9, respectively.
  • HeLa cells were treated with anti-CD44 antibody, ⁇ -galactosidase-labeled antibody and compound 1 or 8, they showed higher fluorescence intensity than anti-CD44 antibody and Alexa405-labeled antibody. This indicates that the sensitivity is insufficient by the method using a general fluorescently labeled antibody.
  • Example 11 Fluorescent staining of HeLa cells using CD44 as a surface antigen HeLa cells were contacted with an anti-CD44 antibody (Abcam: rabbit monoclonal antibody) (MEM medium) and left in a CO 2 incubator (37 ° C.) for 1 hour. did. After washing, ⁇ -galactosidase-labeled anti-rabbit antibody (Abcam) (MEM medium) was contacted and left in a CO 2 incubator (37 ° C.) for 1 hour. After washing, compound 3 (HBSS) was added, and after incubating in a CO 2 incubator (37 ° C.) for 30 minutes, fluorescence observation of cells was performed using a fluorescence microscope (KEYENCE BZ-X710). As a general secondary antibody method, an experiment was conducted using an anti-CD44 antibody and a commercially available fluorescent dye (Alexa488) -labeled anti-rabbit antibody.

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Abstract

Le problème à la base de l'invention concerne un composé fluorescent à rétention intracellulaire représenté par la formule générale (I) en tant que composé fluorescent à rétention intracellulaire qui permet la détection d'une seule cellule présentant une protéine de surface cellulaire cible exprimée sur celle-ci d'une manière spécifique et avec une sensibilité élevée. Dans la formule générale (I), A représente un groupe hydrophile monovalent qui peut être dissocié par une réaction enzymatique ; R1, R3 ou R5 représente un groupe alkyle présentant un ou deux atomes de fluor en une position benzyle ; un quelconque groupe parmi R1, R2, R3, R4 et R5 contient un groupe fluorescent hydrophobe ; et deux groupes adjacents parmi R1, R2, R3, R4 et R5 peuvent partager un atome ou un groupe atomique l'un avec l'autre pour former un cycle et peuvent former un groupe fluorescent conjointement avec un cycle benzénique.
PCT/JP2020/023120 2019-06-14 2020-06-12 Composé fluorescent à rétention intracellulaire et procédé de coloration de cellules et procédé de détection de cellules à haute sensibilité l'utilisant WO2020250998A1 (fr)

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