WO2018150450A1 - Composé aminocoumarine, et particules de résine avec composé aminocoumarine encapsulé - Google Patents

Composé aminocoumarine, et particules de résine avec composé aminocoumarine encapsulé Download PDF

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Publication number
WO2018150450A1
WO2018150450A1 PCT/JP2017/005284 JP2017005284W WO2018150450A1 WO 2018150450 A1 WO2018150450 A1 WO 2018150450A1 JP 2017005284 W JP2017005284 W JP 2017005284W WO 2018150450 A1 WO2018150450 A1 WO 2018150450A1
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aminocoumarin compound
compound
aminocoumarin
resin particles
formula
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PCT/JP2017/005284
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English (en)
Japanese (ja)
Inventor
健作 高梨
中山　慎
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コニカミノルタ株式会社
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Priority to PCT/JP2017/005284 priority Critical patent/WO2018150450A1/fr
Priority to PCT/JP2018/004803 priority patent/WO2018151072A1/fr
Priority to EP18754496.0A priority patent/EP3553136A4/fr
Priority to US16/477,433 priority patent/US11434376B2/en
Priority to JP2018568516A priority patent/JP7151489B2/ja
Publication of WO2018150450A1 publication Critical patent/WO2018150450A1/fr

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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

Definitions

  • the present invention relates to an aminocoumarin compound which is a novel pigment and aminocoumarin compound-encapsulating resin particles obtained by encapsulating the aminocoumarin compound in a resin.
  • the antigen is fluorescently labeled by specifically binding a fluorescently labeled antibody to an in vivo molecule (antigen) whose expression level increases or decreases depending on the presence or absence of the disease. The quantity is quantified.
  • CCR4 Inspection As an example of a recent clinical test developed to provide an indication of whether to administer to adult T-cell leukemia (ATL), the target of CCR4 Inspection to perform detection was required.
  • the detection of CCR4 has been developed by FACS method for immune reaction to collected blood samples and IHC method for immune reaction to collected tissue samples.
  • the former quantifies (scores) CCR4 by fluorescence detection
  • the latter quantifies (scores) CCR4 by color detection.
  • the need to obtain a lot of information at one time is not limited to immunohistochemical methods, and there is a strong need for medical personnel, and many methods for detecting different targets simultaneously with a plurality of fluorescent dyes are being developed. For example, preparing dyes that exhibit fluorescence in each of the green region, red region, orange region, and far-infrared region, and detecting four types of targets is possible in addition to the FACS method using the above immune reaction.
  • the ELISA method and the FISH method can be used and commercialized.
  • a sulfonated coumarin dye is known as a dye emitting in the green region.
  • the sulfonated coumarin dye include sulfonated coumarin dyes described in Patent Document 1. Since sulfonated coumarin dyes have excellent solubility in water, they are adaptable to fluorescence detection tools based on various immune reactions. However, on the other hand, the fluorescence noise derived from the specimen as described below has been a major factor for inaccurate fluorescence detection and quantification.
  • the present invention is intended to solve the problems associated with the prior art as described above, and a sulfonated coumarin dye having a maximum wavelength of an excitation spectrum in a wavelength region of 475 nm or more, for example, a wavelength region of 475 to 510 nm.
  • the purpose is to provide.
  • the aminocoumarin compound of the present invention has a structure represented by the following formula (1).
  • each R independently represents a hydrogen atom or a methyl group
  • Q represents a sulfur atom, an oxygen atom or N—R 1
  • R 1 represents a hydrogen atom or a methyl group.
  • the aminocoumarin compound is an aminocoumarin compound in which Q in the formula (1) is a sulfur atom, an aminocoumarin compound that is an oxygen atom, or an aminocoumarin compound that is N—R 1 .
  • the aminocoumarin compound-encapsulating resin particles of the present invention have the aminocoumarin compound and resin particles that encapsulate the aminocoumarin compound.
  • the resin particles are preferably amino resin particles.
  • the aminocoumarin compound-containing resin particles preferably have an excitation spectrum maximum wavelength of 475 to 510 nm and a fluorescence spectrum maximum wavelength of 510 to 540 nm.
  • the aminocoumarin compound of the present invention has an excitation wavelength longer than that of the conventionally known sulfonated coumarin compound described in Patent Document 1, and has a wavelength of 475 or more, particularly about 500 nm. It is a fluorescent dye that is effectively excited.
  • the aminocoumarin compound of the present invention is a fluorescent dye that can be excited in the wavelength range of 475 to 510 nm and observed in the green light emission region of 510 to 540 nm by utilizing this wavelength characteristic. It can be expected to be used appropriately.
  • the aminocoumarin compound of the present invention has a structure represented by the following formula (1).
  • R's each independently represent a hydrogen atom or a methyl group.
  • Q represents a sulfur atom, an oxygen atom or N—R 1 .
  • R 1 represents a hydrogen atom or a methyl group.
  • the aminocoumarin compound of the present invention has a benzothiazole structure when Q in formula (1) is a sulfur atom, has a benzoxazole structure when Q is an oxygen atom, and has benzoxazole structure when it is N—R 1. It will have an imidazole structure.
  • the sulfonic acid group SO 3 H contained in the formula (1) is bonded to any of the four carbon atoms that can be bonded to the benzene ring contained in the benzothiazole structure, benzoxazole structure or benzimidazole structure. May be.
  • the nitrogen atom bonded to the coumarin structure forms two 6-membered rings together with the four carbon atoms of the benzene ring contained in the coumarin structure, that is, the amino group of aminocoumarin is
  • the structure is different from known sulfonated coumarin compounds described in Patent Document 1 and the like in that it has a julolidine structure.
  • the aminocoumarin compound of the present invention having such a structure has an excitation wavelength longer than that of a known sulfonated coumarin compound, and has an excitation spectrum maximum wavelength of 475 nm or more, for example, 475 to 510 nm.
  • the aminocoumarin compound of the present invention has a longer emission wavelength than known sulfonated coumarin compounds, and has a maximum fluorescence spectrum wavelength of 510 nm or more, for example, 510 to 540 nm.
  • the “excitation spectrum maximum wavelength” means a wavelength exhibiting a maximum in the excitation spectrum.
  • “Fluorescence spectrum maximum wavelength” means a wavelength exhibiting a maximum in a fluorescence spectrum.
  • the aminocoumarin compound of the present invention represented by the formula (1) has a feature that the emission intensity is higher than that of an aminocoumarin compound formed by substituting the sulfone group of the aminocoumarin compound with a hydrogen atom.
  • the aminocoumarin compound of the present invention can be produced, for example, by a method of sulfonating a coumarin compound having a structure represented by the following formula (2). Specifically, the aminocoumarin compound of the present invention is produced by adding 1 ml of fuming sulfuric acid to 0.1 g of the coumarin compound represented by the formula (2) and reacting at 0 to 140 ° C. for 1 to 8 hours. be able to.
  • the aminocoumarin compound-encapsulating resin particles of the present invention have the aminocoumarin compound and resin particles that encapsulate the aminocoumarin compound.
  • the aminocoumarin compound-encapsulating resin particles of the present invention can be suitably used for immunostaining and the like.
  • the aminocoumarin compound encapsulated in the resin particles is as described above.
  • the resin particles encapsulating the aminocoumarin compound are not particularly limited as long as the aminocoumarin compound can be encapsulated, but is preferably a thermosetting resin. Since the thermosetting resin has a three-dimensional network structure, the dye encapsulated in the thermosetting resin is not easily detached from the resin particles, and is suitable for immunostaining and the like.
  • the thermosetting resin include melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, and furan resin.
  • amino resins such as melamine resin and urea resin are particularly preferable because they can more effectively suppress the release of the pigment from the resin particles.
  • the amount of the aminocoumarin compound encapsulated in the resin particles is not particularly limited, and when the aminocoumarin compound-encapsulated resin particles are used for immunostaining, it may be an amount that can ensure a detectable luminance.
  • the average particle diameter of the aminocoumarin compound-encapsulating resin particles is not particularly limited, but when used for immunostaining, it is usually 40 to 500 nm, preferably 50 to 200 nm. If the average particle size of the aminocoumarin compound-encapsulating resin particles exceeds 500 nm, there may be a problem with dyeability, and if it is less than 40 nm, there may be a problem with visibility.
  • the average particle diameter is calculated as an average value obtained by measuring the particle diameter of 1000 aminocoumarin compound-encapsulated particles by SEM observation.
  • aminocoumarin compound-encapsulating resin particles of the present invention can be produced according to known methods for producing dye-encapsulating resin particles.
  • the aminocoumarin compound-encapsulating resin particles of the present invention can be produced by polymerizing a monomer raw material for resin synthesis in the presence of an aminocoumarin compound. Specifically, a monomer raw material for resin synthesis is added to an aqueous solution containing an aminocoumarin compound and a surfactant, and usually at 55 to 75 ° C., preferably 68 to 72 ° C., for about 10 minutes, preferably 10 to 15 Stir vigorously for minutes. Thereafter, a polymerization initiator is added, and emulsion polymerization is carried out by stirring vigorously at 55 to 75 ° C., preferably 68 to 72 ° C. for 4 to 24 hours, preferably 4 to 5 hours.
  • the liquid temperature is raised to 80 to 90 ° C., preferably 80 to 82 ° C., and the mixture is vigorously stirred for 30 to 60 minutes, preferably 30 to 40 minutes.
  • the reaction solution is usually divided into agglomerates and a particle dispersion as a supernatant.
  • the particle dispersion is recovered from the reaction solution.
  • This particle dispersion is centrifuged to remove the dispersion medium that is a supernatant, and then ultrapure water is added to the precipitate for ultrasonic dispersion.
  • the steps of centrifugation, addition of ultrapure water to the precipitate, and ultrasonic dispersion are repeated several more times. As a result, an aqueous dispersion of aminocoumarin compound-encapsulating resin particles is obtained.
  • the monomer raw material for resin synthesis is a raw material capable of forming the resin particles by polymerization.
  • the amount of aminocoumarin compound added is usually 1 to 50 mg with respect to 1 g of monomer raw material for resin synthesis.
  • the surfactant is not particularly limited, and a surfactant that is used in a usual emulsion polymerization reaction can be used.
  • a surfactant that is used in a usual emulsion polymerization reaction can be used.
  • all anionic, nonionic and cationic surfactants can be used.
  • the anionic surfactant include sodium dodecylbenzenesulfonate.
  • nonionic surfactants include polyethylene glycol and polyoxyethylene alkyl ether.
  • Examples of the cationic surfactant include dodecyltrimethylammonium bromide.
  • Surfactant may be used individually by 1 type, or may use 2 or more types together.
  • Emulgen registered trademark, manufactured by Kao Corporation
  • Neoperex registered trademark, manufactured by Kao Corporation
  • the active ingredient of emulgen is polyoxyethylene alkyl ether
  • the active ingredient of neoperex is sodium dodecylbenzenesulfonate.
  • the addition amount of the surfactant is usually 1 to 3 mg per 1 g of the monomer raw material for resin synthesis.
  • polymerization initiator examples include thermal polymerization initiators such as azo compounds and peroxides that generate radicals by heat.
  • V-50 (2,2′-Azobis (2-methylpropionamide) dihydrochloride is preferred as the azo compound.
  • the peroxide is preferably ammonium persulfate.
  • the polymerization initiator may be a redox polymerization initiator.
  • the addition amount of the polymerization initiator is usually 0.1 to 1.5 mg, preferably 0.3 to 0.45 mg per 1 g of the monomer raw material for resin synthesis.
  • the resin particles are preferably amino resin particles.
  • the aminocoumarin compound-containing resin particles preferably have an excitation spectrum maximum wavelength of 475 to 510 nm and a fluorescence spectrum maximum wavelength of 510 to 540 nm.
  • the aminocoumarin compound-encapsulating resin particle of the present invention produced by encapsulating the aminocoumarin compound represented by the formula (1) in the resin is formed by substituting the sulfo group of the aminocoumarin compound with a hydrogen atom.
  • the emission intensity tends to be strong. This is because the aminocoumarin compound represented by the formula (1) is more easily included in the resin than the aminocoumarin compound formed by replacing the sulfone group of the aminocoumarin compound with a hydrogen atom. It is presumed that it is taken in.
  • Example 1 In a 20 mL vial tube, 600 mg of the compound (3) represented by the following formula (3) was added, 6 mL of fuming sulfuric acid was added, and the mixture was stirred at 25 ° C. for 4 hours to carry out the reaction. Progress of the reaction was confirmed by TLC. Specifically, a part of the reaction solution was neutralized with an aqueous NaOH solution, ethanol was added to the reaction solution, and TLC was performed using a solution in which CHCl 3 was mixed at a ratio of 2 and MeOH at a ratio of 3. The Rf value of the target product was 0.73 against the Rf value of 0.88 of the raw material, and the convergence of the reaction and the generation of the target product were confirmed from the TLC data.
  • the recovered precipitate was dispersed with ethanol, the dispersion was centrifuged, and the supernatant was removed to obtain an aminocoumarin compound I represented by the following formula (I) as a precipitate.
  • the yield of aminocoumarin compound I was 80%.
  • the obtained powder was added to pure water, then neutralized with an aqueous NaOH solution to dissolve the precipitate, and the pH of the solution was adjusted to 7-8.
  • This solution was dried with a freeze dryer to obtain Na salt of aminocoumarin compound I. It was confirmed that the aminocoumarin compound I dissolves quickly in water by using Na salt, whereas the sulfonic acid form has poor solubility in water.
  • the aminocoumarin compound I was dissolved in a solvent obtained by mixing double distilled water (DDW) 10 and ethanol at a ratio of 1 to prepare a 10 nM solution.
  • the fluorescence intensity at the excitation spectrum maximum wavelength, the fluorescence spectrum maximum wavelength, and the excitation wavelength 490 nm was measured for this solution using a spectrofluorophotometer F-7000 (manufactured by Hitachi, Ltd.). The results are shown in Table 1.
  • the excitation spectrum was measured by fixing the fluorescence wavelength to be detected and scanning the wavelength of the excitation light. In the fluorescence spectrum, the fluorescence intensity was measured with the wavelength of excitation light fixed.
  • Example 2 An aminocoumarin compound II represented by the following formula (II) was prepared in the same manner as in Example 1 except that the compound (4) represented by the following formula (4) was used instead of the compound represented by the formula (3). Got. The yield of aminocoumarin compound II was 82%.
  • Example 3 The aminocoumarin compound III represented by the following formula (III) was prepared in the same manner as in Example 1 except that the compound (5) represented by the following formula (5) was used instead of the compound represented by the formula (3). Got. The yield of aminocoumarin compound III was 80%.
  • Excitation spectrum maximum wavelength and fluorescence spectrum maximum wavelength were measured in the same manner as in Example 1 using aminocoumarin compound III. The results are shown in Table 1.
  • Example 4 The aminocoumarin compound IV represented by the following formula (IV) was prepared in the same manner as in Example 1 except that the compound (6) represented by the following formula (6) was used instead of the compound represented by the formula (3). Got. The yield of aminocoumarin compound IV was 75%.
  • Example 5 The aminocoumarin compound V represented by the following formula (V) was prepared in the same manner as in Example 1 except that the compound (7) represented by the following formula (7) was used instead of the compound represented by the formula (3). Got. The yield of aminocoumarin compound V was 70%.
  • Example 6 The aminocoumarin compound VI represented by the following formula (VI) was prepared in the same manner as in Example 1 except that the compound (8) represented by the following formula (8) was used instead of the compound represented by the formula (3). Got. The yield of aminocoumarin compound VI was 75%.
  • Example 7 14.4 mg of aminocoumarin compound I was added to 22 mL of water and dissolved. To this solution, 2 mL of a 5% aqueous solution of an emulsion (registered trademark) 430 (polyoxyethylene oleyl ether, manufactured by Kao Corporation) of an emulsifier for emulsion polymerization was added.
  • an emulsion (registered trademark) 430 polyoxyethylene oleyl ether, manufactured by Kao Corporation
  • the solution was heated to 70 ° C. while stirring on a hot stirrer, and then 0.65 g of melamine resin raw material Nicalak MX-035 (manufactured by Nippon Carbide Industries Co., Ltd.) was added to the solution.
  • melamine resin raw material Nicalak MX-035 manufactured by Nippon Carbide Industries Co., Ltd.
  • To this solution was added 1000 ⁇ L of a 10% aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Chemical Co., Ltd.) as a reaction initiator, and the mixture was heated and stirred at 70 ° C. for 50 minutes, then heated to 90 ° C. and stirred for 20 minutes.
  • aminocoumarin compound-encapsulating resin particles I were obtained.
  • the resulting dispersion of aminocoumarin compound-encapsulating resin particles I was washed with pure water to remove excess resin raw materials and impurities such as aminocoumarin compounds. Specifically, the mixture was centrifuged at 20000 G for 15 minutes in a centrifuge (Kubota Micro Cooling Centrifuge 3740), and after removing the supernatant, ultrapure water was added and ultrasonically irradiated to redisperse. Centrifugation, supernatant removal, and washing by redispersion in ultrapure water were repeated 5 times.
  • the average particle size of the aminocoumarin compound-encapsulating resin particles I was calculated from the particle diameter of the SEM image. The results are shown in Table 2.
  • Aminocoumarin compound-encapsulating resin particle I is diluted to 0.0189 mg / mL, 100 uL is taken, put into a fluorescence microcell, and using a spectrofluorometer F-7000 (manufactured by Hitachi, Ltd.) at an excitation wavelength of 490 nm. The fluorescence intensity was measured. The results are shown in Table 2.
  • the aminocoumarin compound-encapsulating resin particles I were subjected to the following FACS (Fluorescence Activated Cell Sorting) and FISH (Fluorescence in situ hybridization).
  • FACS evaluation Maleimide was introduced into the end of the aminocoumarin compound-encapsulating resin particle I using NHS-PEG (polyethylene glycol) -maleimide reagent, and a thiolated anti-HER2 antibody was bound thereto.
  • the frozen cells of COLO201 obtained from ATCC are thawed in a 37 ° C constant temperature bath, and about 1 ⁇ 10 5 cells / mL are suspended in the medium, and the first culture is performed in a 25 cm 2 flask. Carried out. Trypsinize the culture medium with about 1 ⁇ 10 6 cells / mL and dilute a part of the cells detached from the flask with Cell bunker solution (10% DMSO). did. The remaining cells were subcultured and cultured to a target amount of about 5 ⁇ 10 7 cells. After floating and recovering with a scraper, a part was used for FACS measurement.
  • HER2 protein present on the surface of cultured cells was measured with FACSCalibur (trade name; manufactured by Becton Dickinson).
  • FACSCalibur (trade name; manufactured by Becton Dickinson).
  • a mixture of the PBS solution of aminocoumarin compound-encapsulating resin particles I and COLO201 cultured as described above was incubated at 4 ° C. for 30 minutes, 100 ⁇ l of the mixture was taken into a measuring tube, and Assay Diluent (trade name, Becton It was diluted to 2 ml with Deckson Corporation and injected into FACSCalibur.
  • the results are shown in Table 2.
  • the diagram shown as a result of FACS in Table 2 is a histogram with the horizontal axis representing the fluorescence intensity of 530 nm by 490 nm excitation light and the vertical axis representing the count number.
  • the fluorescence intensity is stronger, that is, brighter as the histogram is shifted to the right side.
  • the result of Example 7 is shown in comparison with the result of Comparative Example 5 using the following aminocoumarin compound-encapsulating resin particles i. The same applies to other examples and comparative examples 6 to 8.
  • a histogram having a peak near the fluorescence intensity 90 is the histogram of Comparative Example 5
  • a histogram having a peak near the fluorescence intensity 500 is the histogram of Example 7. It can be seen that the histogram of Example 7 is considerably shifted to the right from the histogram of Comparative Example 5. From this result, it can be seen that the aminocoumarin compound-encapsulating resin particles I of Example 7 have a stronger fluorescence intensity and brighter than the aminocoumarin compound-encapsulating resin particles i of Comparative Example 5.
  • FISH evaluation [Preparation of BAC probe] CellBiochemBiophys. In accordance with the method described in 2006; 45 (1) 59, a nucleic acid molecule was prepared as follows.
  • dTTP of the HER2 DNA clone was replaced with DNP-labeled dUTP by the nick translation method.
  • a solution containing 25 ⁇ L (concentration 1 ⁇ g / 250 ⁇ L) of a DNP-labeled BAC probe by nick translation and 1.0 ⁇ L (50 nmol / 50 ⁇ L) of aminocoumarin compound-encapsulating resin particles surface-modified with an anti-DNP antibody was mixed to room temperature. Then, a binding reaction was performed for 30 minutes to obtain a DNA probe reagent for HER-2 detection.
  • Hybridization buffer (25% deionized formamide, 2 ⁇ SSC, 200 ng / ⁇ L salmon sperm DNA, 5 ⁇ Denhardt's solution, 50 mM sodium phosphate, pH 7.0, 1 mM EDTA) Diluted to a final concentration of 1-5 ng / ⁇ L. If necessary, free ligand was removed with an S300 size spin column (Amersham Biosciences, Piscataway, NJ). If not used immediately, the DNA probe was stored frozen at -20 ° C.
  • FISH includes deparaffinization, specimen slide pretreatment, enzyme treatment, specimen fixing treatment, probe preparation, specimen slide DNA denaturation treatment, hybridization treatment, slide glass washing treatment, and DAPI. The staining process was performed in this order.
  • Deparaffinization was performed by treating a specimen slide of a HER2-positive stained control specimen (“HER2-FISH Control Slide Code PS-09006” manufactured by Pathology Laboratories) in the order of (1) to (4) below. .
  • HER2-FISH Control Slide Code PS-09006 manufactured by Pathology Laboratories
  • (1) Immerse in Hemo-De for 10 minutes at room temperature.
  • (2) Immerse the specimen slide in new Hemo-De for 10 minutes at room temperature. Repeat the same operation three times.
  • the specimen slide is immersed in 100% ethanol at room temperature for 5 minutes, washed twice, and dehydrated.
  • the specimen slide is air-dried or dried on a slide warmer at 45 to 50 ° C.
  • the specimen slide was pretreated in the following order (1) to (6) to remove proteins from the cell membrane and the nuclear membrane.
  • (1) Treat the specimen slide with 0.2 mol / L HCl at room temperature for 20 minutes.
  • (2) Immerse the specimen slide in purified water for 3 minutes.
  • (3) The specimen slide is immersed in a washing buffer solution (2 ⁇ SSC: standard saline citrate) for 3 minutes.
  • (4) The specimen slide is immersed in a pretreatment solution (1N NaSCN) at 80 ° C. for 30 minutes.
  • (6) The specimen slide is immersed in a washing buffer solution (2 ⁇ SSC) for 5 minutes, and this immersion operation is repeated twice.
  • the sample treatment that had been pretreated was subjected to enzyme treatment by performing the following treatments (1) to (4) in this order.
  • (2) The specimen slide is immersed in a protease solution heated to 37 ° C. for 10 to 60 minutes. This immersion treatment is performed with 25 mg protease (2500-3000 Units / mg) [pepsin] / 1M NaCl [pH 2.0] in 50 mL at 37 ° C. for 60 minutes) in order to decompose cell membrane and nuclear membrane proteins, particularly collagen. It is desirable to process.
  • (4) The specimen slide is air-dried or dried on a slide warmer at 45 to 50 ° C. for 2 to 5 minutes.
  • specimen fixation As the specimen fixing process, the following processes (1) to (3) were performed on the specimen slide that had been pretreated.
  • the specimen slide is immersed in 10% neutral buffered formalin (“4% paraformaldehyde / phosphate buffer” manufactured by Wako Pure Chemical Industries, Ltd., product number 163-1004) at room temperature for 10 minutes.
  • 10% neutral buffered formalin (“4% paraformaldehyde / phosphate buffer” manufactured by Wako Pure Chemical Industries, Ltd., product number 163-1004)
  • the specimen slide is air-dried or dried on a slide warmer at 45 to 50 ° C. for 2 to 5 minutes.
  • DAPI staining was performed as follows. First, 10 ⁇ L of DAPI counterstain was added to the hybridization area of the specimen slide. Next, after hybridization treatment, DAPI staining (2 ⁇ g / mL PBS) is performed at 25 ° C. for 10 minutes in order to count the number of cells. The cell nucleus is stained, covered with a cover glass, and the specimen slide is covered until signal measurement. Stored protected from light. DAPI (4 ′, 6-Diamidino-2-phenylindole, Dihydrochloride) used Molecular Probes (D1306).
  • Table 2 shows the result of measuring the number of bright spots from the obtained fluorescence image.
  • the difference in the number of measured starting points was observed in the comparative example, while it was relatively difficult to find the green bright spot (signal) from the green background (noise). This is because in the case of the example, since the luminance is high, detection can be performed without being affected by noise.
  • aminocoumarin compound-encapsulating resin particles II to VI were prepared in the same manner as in Example 7 except that aminocoumarin compounds II to VI were used in place of aminocoumarin compound I, respectively.

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Abstract

L'invention concerne un composé aminocoumarine possédant une structure représentée par la formule (1), et des particules de résine avec composé aminocoumarine encapsulé qui possèdent ce composé aminocoumarine, et des particules de résine enveloppant le composé aminocoumarine. Dans la formule (1), R représentent chacun indépendamment un atome d'hydrogène ou un groupe méthyl, Q représente un atome de soufre, un atome d'oxygène ou Ｎ－Ｒ1, et Ｒ1 représente un atome d'hydrogène ou un groupe méthyl. Le composé aminocoumarine de l'invention consiste en un colorant fluorescent qui présente une longueur d'onde d'excitation plus longue qu'un composé coumarine sulfoné tel que dans l'art antérieur, et qui produit une excitation efficace vis-à-vis d'une longueur d'onde supérieure ou égale à 475nm, et particulièrement vis-à-vis d'une longueur d'onde voisine de 500nm. En outre, le composé aminocoumarine de l'invention consiste en un colorant fluorescent produisant une excitation à une longueur d'onde comprise entre 475 et 510nm, par exemple, à l'aide de ses caractéristiques de longueurs d'ondes, et permettant une observation dans une région de luminescence verte de 510 à 540nm, et permet d'envisager une application adéquate dans le cadre de l'immunocoloration, ou similaire.
PCT/JP2017/005284 2017-02-14 2017-02-14 Composé aminocoumarine, et particules de résine avec composé aminocoumarine encapsulé WO2018150450A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
PCT/JP2017/005284 WO2018150450A1 (fr) 2017-02-14 2017-02-14 Composé aminocoumarine, et particules de résine avec composé aminocoumarine encapsulé
PCT/JP2018/004803 WO2018151072A1 (fr) 2017-02-14 2018-02-13 Composé aminocoumarine, et particules de résine avec composé aminocoumarine encapsulé
EP18754496.0A EP3553136A4 (fr) 2017-02-14 2018-02-13 Composé aminocoumarine, et particules de résine avec composé aminocoumarine encapsulé
US16/477,433 US11434376B2 (en) 2017-02-14 2018-02-13 Aminocoumarin compound, and aminocoumarin compound-containing resin particles
JP2018568516A JP7151489B2 (ja) 2017-02-14 2018-02-13 アミノクマリン化合物およびアミノクマリン化合物内包樹脂粒子

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WO2021182228A1 (fr) * 2020-03-10 2021-09-16 コニカミノルタ株式会社 Particule contenant un pigment émettant de la lumière, son procédé de production et agent de marquage pour diagnostic pathologique l'utilisant

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