WO2013075285A1 - Colorant fluorescent à base de bi-benzyle pentaméthyle cyanine de classe i et procédés de préparation et d'application associés - Google Patents

Colorant fluorescent à base de bi-benzyle pentaméthyle cyanine de classe i et procédés de préparation et d'application associés Download PDF

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WO2013075285A1
WO2013075285A1 PCT/CN2011/082619 CN2011082619W WO2013075285A1 WO 2013075285 A1 WO2013075285 A1 WO 2013075285A1 CN 2011082619 W CN2011082619 W CN 2011082619W WO 2013075285 A1 WO2013075285 A1 WO 2013075285A1
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Prior art keywords
dye
compound
cells
fluorescent dye
group
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PCT/CN2011/082619
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English (en)
Chinese (zh)
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樊江莉
李·安德鲁
彭孝军
强新新
王倩
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大连理工大学
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Priority to PCT/CN2011/082619 priority Critical patent/WO2013075285A1/fr
Publication of WO2013075285A1 publication Critical patent/WO2013075285A1/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
    • 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
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
    • C09B23/083—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines five >CH- groups
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1007—Non-condensed systems
    • 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
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom

Definitions

  • the present invention relates to a class of bisbenzylpentaphthalocyanine fluorescent dyes, a process for their preparation and their use as specific fluorescent probes in living cells and fixed cell markers. Background technique
  • Mitochondria are organelles that are ubiquitous in eukaryotic cells. Most of the energy required for various life activities is provided by ATP synthesized in mitochondria. Therefore, it is known as the "power plant" of cells.
  • Currently available commercial mitochondrial fluorescent probes are: JC-1, Rhodamine 123 and MitoTracker® series of probes. Among them, JC-1 is the most widely used. When the concentration or mitochondrial membrane potential is low, it exists as a monomer with an excitation wavelength of 490 nm and an emission wavelength of 527 nm, which is green fluorescent.
  • Rhodamine 123 is a fluorescent probe that penetrates into cells and carries cations. It has an excitation wavelength of 505 nm and an emission wavelength of 534 nm. Living cells uptake Rhodamine 123 reaches equilibrium at a faster rate. It takes only 5 minutes to observe the mitochondria stained green by Rhodamine 123 by laser scanning confocal microscopy.
  • Rhodamine 123 When cells are washed repeatedly, Rhodamine 123 is usually not retained by cells, but many cancer cells retain this dye for a long time, so it contributes to the diagnosis of certain cancers. However, once the membrane potential of the mitochondria is lost, they are easily washed away. This limits some applications.
  • the MitoTracker® range of probes, designed by Invitrogen for mitochondria, is cell-permeable and passively penetrates the plasma membrane and accumulates in active mitochondria. Although some MitoTracker probes can achieve mitochondrial labeling in fixed cells, the entire cell is evenly stained in a short period of time, so these dyes are recommended only for living cells.
  • the cyanine dye has a large molar extinction coefficient, the absorption and emission wavelengths are adjustable with the length of the dye conjugated chain, and the fluorescence is stronger after binding with biomolecules, and the cytotoxicity is small. It has become the main fluorescent probe used in analytical assays such as DNA, proteins and nucleic acids.
  • the absorption and emission wavelength of the pentamethine cyanine dye Cy5 is above 600 nm, close to the near-infrared region, and is the shortest wavelength near-infrared Cyanine dyes, as a new generation of commercial fluorescent labeling agents, have been widely used in protein labeling, DNA sequencing, ion neutral small molecule recognition, cell and living tissue imaging.
  • no pentamethine cyanine dye has been reported for fluorescence-specific labeling of intracellular mitochondria.
  • An object of the present invention is to provide a probe molecule based on pentamethine cyanine dye which has a spectral range in the near-infrared region, which is capable of fluorescently labeling mitochondria in living cells and fixed cells, and having a simple structure and excellent performance.
  • One of the objects of the present invention is a class of bisbenzylpentaphthalocyanine fluorescent dyes having the following structural formula I:
  • X- is selected from the group consisting of halogen anions, ClO 4 ⁇ PF 6 " , CF 3 -, BF 4 R!C , R2SO or OTs",
  • R 2 are each independently selected from a fluorenyl or aryl group of d- 12 .
  • X- is selected from the group consisting of halogen anions, most preferably Cl-, Br-, and fluorene.
  • Another object of the present invention is to provide a process for producing the above-mentioned bisbenzylpentaphthalocyanine fluorescent dye of the present invention, which comprises reacting a compound of the formula II with a compound III (condensing agent malondialdehyde aniline salt) in the presence of a base.
  • the reaction solvent is an acid anhydride, and the reaction temperature is 20 to 150 ° C, and the reaction time is 10 to 100 minutes. The preferred reaction time is 30 to 50 minutes.
  • X is selected from the group consisting of halogen anions, ClO 4 ⁇ PF 6 " , CF 3 -, BF 4 -, R!C, R 2 SO 3 - or OTs- preferably halogen anions, most preferably Cl-, Br ⁇ ⁇ .
  • R n are each independently selected from a fluorenyl or aryl group of d 12 , preferably a fluorenyl group of d 6 .
  • the molar ratio of the compound II to the compound III is 1: 0.1 to 1;
  • the molar ratio of the compound II to the base is 1:0.5 to 2.
  • the molar ratio of the compound II to the compound III is preferably 1:0 ⁇ 4 to 0.6.
  • the condensation reaction of the compound II and the compound III is carried out in the presence of a base.
  • a base used in the reaction and its amount.
  • sodium acetate, potassium acetate, sodium phosphate, sodium formate, sodium propionate, potassium propionate, sodium oxalate or potassium oxalate is selected as the base. Most preferred is sodium acetate.
  • the amount of the base is 0.9 to 1.1 times the molar amount of the compound II.
  • the condensation reaction of the compound II and the compound III is carried out in a reaction system in which an acid anhydride is a solvent, and preferred acid anhydrides as a solvent include acetic anhydride, propionic anhydride, and dibutyl hydride. Anhydride and glutaric anhydride. Most preferred is acetic anhydride.
  • the bisbenzylpentaphthalocyanine dye of the structural formula I provided by the above invention has the most absorption in DMSO at 654/662 nm and the maximum emission spectrum at 672 nm; the maximum absorption and maximum emission spectra in water are respectively 647 Nm and 662 nm; specific fluorescent labeling of mitochondria in fixed cells, independent of mitochondrial membrane potential, is therefore particularly useful for pathogenic cells; cell viability IC 5 () after incubation for 24 hours at room temperature with 10 nM concentration I Can reach 60%. Mitotracker Green TM I and mitochondria in living cells staining position identical mitochondria in living cells by specific fluorescent label.
  • the mitochondria described therein include living cell mitochondria and fixed cell mitochondria.
  • fluorescent labeling with the bisbenzylpentaphthalocyanine fluorescent dye of the present invention necessarily includes a compound of one of the above dyes of the present invention, a conjugate thereof, or a dye containing the same.
  • Figure 1 (a) is the absorption spectrum of dye 1-1 in DMSO and water, respectively.
  • the abscissa is the wavelength and the ordinate is the absorption intensity
  • Figure 1 (b) is the fluorescence emission spectrum of dye 1-1 in DMSO and water, respectively.
  • the abscissa is the wavelength (nm).
  • the ordinate is the relative fluorescence intensity.
  • the instrument used was an ultraviolet-visible spectrophotometer, model: Hp8453 ; fluorescence spectrophotometer, model: FP-6500.
  • the concentration of the dye 1-1 was 5 ⁇ .
  • Figure 2 is a fluorescent staining image of live cell staining of different concentrations of dye 1-1 in MCF-7:
  • the final concentrations of dye 1-1 in the medium were a) 10 nM ; b) 100 nM; c) 500 nM and d) ⁇ ; dye and MCF-7 live cells at 37 ° C, 5% CO 2 After incubation for 30 minutes, photographs were taken using a fluorescence inverted microscope (Olympus 1X81), Cy5 filter (excitation wavelength: 628/40 nm), magnified 20 times.
  • Figure 3 (a) is a picture of live cell staining of Mito-Tracker GreenTM in MCF-7;
  • Figure 3 (b) is a photograph of live cell staining of dye 1-1 in MCF-7;
  • Figure 3 (c) is a superimposed photograph of a and b;
  • Figure 3 (d) is an enlarged view of the fluorescent staining area of the box portion in Figure c;
  • Figure 4 is the result of analyzing Example 5 using the Slidebook (Olympus) software, i.e., staining of living cells by the two dyes in Figure 3d.
  • the yellow line indicates the range of cell analysis selected by the software.
  • the abscissa is micron, the left ordinate is the relative fluorescence intensity of dye 1-1, and the right ordinate is the relative fluorescence intensity of the dye Mito-Tracker GreenTM.
  • Figure 7 is the cell viability of dye 1-1 in the presence of MCF-7 cells.
  • the final concentration of the dye in the medium was 0 M (TCP), 0.01 ⁇ , 0.1 ⁇ , 1 ⁇ and 10 ⁇ , 5% CO 2 cells at 37 ° C.
  • Dye 1-1 was dissolved in DMSO and water, respectively, to prepare a solution having a final concentration of 5 ⁇ M.
  • the test results are shown in Figure 1.
  • the maximum absorption of dye 1-1 in DMSO is 654/662 nm, and the maximum emission spectrum is 672 nm .
  • the maximum absorption and maximum emission spectra in water are at 647 nm and 662 nm, respectively. It can be seen that the maximum absorption and emission spectra of dye 1-1 are in the near-infrared region. It can also be seen from Figure 1 that the probe absorbs and emits more in a hydrophobic environment than in an aqueous environment, so when the probe interacts with mitochondrial lipids, it exhibits strong fluorescence.
  • the instrument used was an ultraviolet-visible spectrophotometer, model: Hp8453; fluorescence spectrophotometer, model: FP-6500.
  • Example 4 Observation of dyes under fluorescence microscope 1-1 staining of MCF-7 live cells at different concentrations
  • the passaged MCF-7 cells were seeded in a 12-well plate, cultured in a cell culture incubator at 37 ° C, 5% CO 2 for 24 hours, and dye 1-1 solution (dissolved in DMSO) was added. Incubate for 10 min at 37 nC for 10 nM, 100 nM, 500 nM and 1 ⁇ , aspirate the medium, rinse twice with PBS, and add fresh medium without phenol red, observe under a fluorescent inverted microscope (Olympus 1X81) Cell morphology.
  • a Cy5 filter (excitation wavelength: 628/40 nm) was used to structure the dye, which was amplified (20x) and repeated three times.
  • Mitotracker GreenTM is a commercial mitochondrial green fluorescent probe that can be used for mitochondrial-specific fluorescent staining in live cells.
  • the structural 1-1 dye and Mitotracker GreenTM were respectively stained for MCF-7 live cells, and their staining effects on living cells were compared to further determine the specific fluorescent labeling of DBCy5 on mitochondria.
  • the passageable MCF-7 cells were seeded in a 12-well glass well plate (the thickness of the well plate was about 0.13-0.17 ⁇ m), and cultured in a cell culture incubator at 37 ° C, 5% CO 2 for 24 hours, respectively.
  • Structure 1-1 dye and Mitotracker GreenTM, final concentration 500 nM incubate at 37 °C for 30 minutes, aspirate the medium, rinse 2 times with PBS, and add fresh medium without phenol red in an inverted microscope (Olympus 1X81) Under observation of cell morphology.
  • the Cy5 filter (excitation wavelength: 628/40 nm) was used to excite the 1-1 dye, and the GFP filter (excitation wavelength: 472/30 nm) was used to excite the Mitotracker GreenTM, and the corresponding exposure time was adjusted (500 sec-1000 Leap seconds) Observe with oil mirror (60x) and repeat three times.
  • Figure 3a is a photograph of live cell staining of Mito-Tracker GreenTM (green)
  • Figure 3b is a photograph of live cells staining of dye 1-1 (red)
  • Figure 3c is a superimposed photograph of Figures 3a and 3b.
  • Mito-Tracker GreenTMiP Dye 1-1 completely overlaps the staining position of living cells.
  • Figure 3d is an enlarged view of the partially fluorescent stained area in Figure 3c (inside the white box), and the position of the staining of the living cells by the two dyes in Figure 3d was analyzed using the Slidebook (Olympus) software. The results are shown in Fig. 4.
  • DAPI is a blue fluorescent dye that penetrates cell membranes. When combined with double-stranded DNA, it produces more than 20-fold more fluorescence than DAPI itself, so it is often used directly to fix cell nuclei in cells or tissues.
  • the DAPI dye and dye 1-1 were counterstained on fixed cells to observe the staining area of dye 1-1 in the cells.
  • the passageable MCF-7 cells were seeded on coverslips and cultured in a cell culture incubator at 37 ° C under 5% CO 2 for 24 hours. Add dyes 1-1 and DAPI at the same time, the final concentration is 500 nM, incubate at 37 °C for 30 min, aspirate the medium, rinse 2 times with PBS, fix the cells with 10% formaldehyde solution for 2 min, aspirate the formaldehyde solution, rinse 3 times with PBS. . Immediately unfold, seal, and observe cell morphology under an inverted microscope (Olympus 1X81).
  • the C5 filter (excitation wavelength: 628/40 nm) was used to excite the probe dye 1-1
  • the DAPI filter (excitation wavelength: 377/50 nm) was used to excite the DAPI dye.
  • the gray (blue) fluorescent region indicates DAPI stained MCF-7 nuclei
  • the white (red) fluorescent region is dye 1-1 specific fluorescent labeled mitochondria.
  • Figure 6 a cell was randomly selected, and the staining of DAPI and Dye 1-1 on the fixed cells was analyzed by Slidebook (Olympus) software.
  • CellTiter-BlueTM Cell Activity Assays A homogeneous, fluorescent method is used to estimate the number of active cells in a cell population.
  • This system comprises a CellTiter-Blue TM reagent, i.e. resazurin was dissolved in a buffer solution of high purity.
  • Resazurin is a redox indicator that can be added directly to cell culture.
  • the cells convert the dark blue oxidative dye (Resazurin) into a red, reduced dye (Querone). Since the inactive cells quickly lose their metabolic ability and cannot restore resazurin, they cannot produce a fluorescent signal, so the system specifically detects active cells.
  • the experimental results can be recorded using a fluorometer or a spectrophotometer.
  • the same number of cells were seeded into 96-well plates at a final volume of 100 L per well.
  • Each group of dyes was inoculated with 3 wells at a cell density of 7 x 10 3 cells/well, 37 ° C, and 5% CO 2 in the cells. Incubate for 24 h in the incubator.

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Abstract

La présente invention concerne un colorant fluorescent à base de bi-benzyle pentaméthyle cyanine de classe I et un procédé de préparation et d'application associé. Le colorant fluorescent a la structure de formule générale I. Dans la formule I, X- est choisi parmi un anion d'halogène, ClO4 -, PF6 -, CF3 -, BF4 -, R1CO2 -, R2SO3 - et OTs-, R1 et R2 étant respectivement et indépendamment choisis parmi alkyle et aryle en C1-12. Le composé peut être utilisé pour le marquage fluorescent spécifique de cellules mitochondriales cellulaires vivantes ou de cellules mitochondriales fixées.
PCT/CN2011/082619 2011-11-22 2011-11-22 Colorant fluorescent à base de bi-benzyle pentaméthyle cyanine de classe i et procédés de préparation et d'application associés WO2013075285A1 (fr)

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PCT/CN2011/082619 WO2013075285A1 (fr) 2011-11-22 2011-11-22 Colorant fluorescent à base de bi-benzyle pentaméthyle cyanine de classe i et procédés de préparation et d'application associés

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PCT/CN2011/082619 WO2013075285A1 (fr) 2011-11-22 2011-11-22 Colorant fluorescent à base de bi-benzyle pentaméthyle cyanine de classe i et procédés de préparation et d'application associés

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104744453A (zh) * 2014-12-05 2015-07-01 大连理工大学 用于检测线粒体极性的半菁类化合物

Citations (3)

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US5426015A (en) * 1993-10-18 1995-06-20 Eastman Kodak Company Metallized azo dianion with two cationic dye counter ions for optical information recording medium
US6291203B1 (en) * 1995-11-13 2001-09-18 Molecular Probes, Inc. Cyanine dyes that stain cells and mitochondria
CN102146215A (zh) * 2011-01-31 2011-08-10 大连理工大学 一类五甲川菁荧光染料、制备方法及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5426015A (en) * 1993-10-18 1995-06-20 Eastman Kodak Company Metallized azo dianion with two cationic dye counter ions for optical information recording medium
US6291203B1 (en) * 1995-11-13 2001-09-18 Molecular Probes, Inc. Cyanine dyes that stain cells and mitochondria
CN102146215A (zh) * 2011-01-31 2011-08-10 大连理工大学 一类五甲川菁荧光染料、制备方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OUSHIKI, D. ET AL.: "Development and Application of a Near-Infrared Fluorescence Probe for Oxidative Stress Based on Differential Reactivity of Linked Cyanine Dyes", J. AM. CHEM. SOC., vol. 132, no. 8, 5 February 2010 (2010-02-05), pages 2795 - 2801, XP055069810 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104744453A (zh) * 2014-12-05 2015-07-01 大连理工大学 用于检测线粒体极性的半菁类化合物

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