WO2016108316A1 - Sonde fluorescente à deux photons, son procédé de préparation et procédé d'imagerie du ph l'utilisant - Google Patents

Sonde fluorescente à deux photons, son procédé de préparation et procédé d'imagerie du ph l'utilisant Download PDF

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WO2016108316A1
WO2016108316A1 PCT/KR2014/013130 KR2014013130W WO2016108316A1 WO 2016108316 A1 WO2016108316 A1 WO 2016108316A1 KR 2014013130 W KR2014013130 W KR 2014013130W WO 2016108316 A1 WO2016108316 A1 WO 2016108316A1
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formula
photon
fluorescent probe
photon fluorescent
fluorescence
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김환명
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아주대학교 산학협력단
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    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G01N21/80—Indicating pH value
    • 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
    • G01N33/88—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving prostaglandins or their receptors

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  • the present invention relates to a two-photon fluorescent probe for imaging the pH, and more particularly, to a two-photon fluorescent probe for measuring and imaging the pH by quantifying the concentration of hydrogen ions in the acidic region in the cell or cell tissue, a method of manufacturing the same and pH using the same It relates to an imaging method.
  • Two-photon microscopy offers several advantages over single-photon microscopy because it uses two near-infrared photons with low excitation energy as excitation sources, specifically with increased transmission depth, localized excitation, and long-term imaging. have.
  • Non-Patent Document 1 Recently, studies have been reported that a variable color two-photon fluorescent probe containing 2-aminoflorene can be used in a region close to pH 7 (Non-Patent Document 1).
  • small molecule two-photon fluorescence probes such as DND-160 have been reported to measure pH in living tissues through variable color two-photon fluorescence microscopy.
  • the two-photon fluorescent probe comprising pyridine as a proton binding site has a problem in that the fluorescence expression rate is low, especially in the acidic region, to 0.1 or less.
  • the two-photon fluorescent probe in order to measure the pH of the acidic region, the two-photon fluorescent probe must have a pKa value at 4.5 to 6.5 pH. There is also the inconvenience of having to be able to easily modify it to bind the target material to measure the pH in a particular organelle, and to find a dye that is sensitive to pH.
  • the first problem to be solved by the present invention is the pH of the acidic region in the cell, in particular the acid organelle of the endosomes or lysosomes present in living cells and selectively located inside and outside the oligomer or protein, so that the pH can be directly and in real time. It is to provide a two-photon fluorescent probe to measure and image it concretely and clearly.
  • a second problem to be solved by the present invention provides a method for manufacturing the two-photon fluorescent probe.
  • the last problem to be solved by the present invention provides a method for quantifying the intracellular pH by the two-photon fluorescence microscope using the two-photon fluorescence probe to image.
  • the present invention provides a two-photon fluorescent probe represented by the following formula (1) to in order to solve the above problems:
  • X 1 and X 2 are each independently H, F, Cl, Br, OCH 3 , OH, NH 2 , N (CH 3 ) 2, NHCH 3, NCS, or CO 2 H
  • R 1 and R 2 Are each independently H, CH 3 , CH 2 (CH 2 ) n CH 3 , CH 2 (CH 2 ) n CO 2 H, CH 2 (CH 2 ) n OH, or CH 2 (CH 2 ) n SO 3 H, n is an integer of 0-10, and the same also applies below.
  • the present invention provides a method for producing a two-photon fluorescent probe represented by the formula (1), comprising the step of mixing and reacting a compound represented by the formula (A), a compound represented by the formula (B) and p-toluenesulfonic acid monohydrate It is characterized by.
  • X 1 and X 2 are each independently H, F, Cl, Br, OCH 3 , OH, NH 2 , N (CH 3 ) 2, NHCH 3, NCS, or CO 2 H
  • R 1 and R 2 Are each independently H, CH 3 , CH 2 (CH 2 ) n CH 3 , CH 2 (CH 2 ) n CO 2 H, CH 2 (CH 2 ) n OH, or CH 2 (CH 2 ) n SO 3 H, which is also the same below.
  • the present invention provides a method for producing a two-photon fluorescent probe represented by the formula (2) comprising the following steps:
  • step (b) preparing a compound represented by the following Chemical Formula E by mixing and reacting the compound represented by Chemical Formula D of step (a) with an aqueous LiOH solution;
  • step (c) reacting the compound represented by the formula (E) of step (b) with N, N-dimethylethylenediamine.
  • X 1 and X 2 are each independently H, F, Cl, Br, OCH 3 , OH, NH 2 , N (CH 3 ) 2, NHCH 3, NCS, or CO 2 H
  • R 1 and R 2 Are each independently H, CH 3 , CH 2 (CH 2 ) n CH 3 , CH 2 (CH 2 ) n CO 2 H, CH 2 (CH 2 ) n OH, or CH 2 (CH 2 ) n SO 3 H, which is also the same below.
  • the present invention also provides a method of imaging the intracellular pH using the two-photon fluorescent probe.
  • the imageable depth may be 100-200 ⁇ m.
  • the cells may be living cells, organelles or cellular tissues of pH 4-7.
  • the two-photon fluorescent probe according to the present invention selectively stains inside living cells and biological tissues and simultaneously reacts with protons to show strong color change in fluorescence. Due to its high solubility in water and its low molecular weight, it can be easily loaded into cells and can also selectively detect pH in living cells and in 100-200 ⁇ m deep tissues over a time period of 60 minutes or more. The distribution and activity of pH within can be observed by imaging through a two-photon microscope. This enables quantitative analysis of pH and comparative analysis of different samples, which can greatly contribute to pH-related life science research, early diagnosis of disease, and development of diagnostic reagents and therapeutics.
  • Figure 1 (a) and (b) is the intensity of the pH change of the photon fluorescent spectrum of BH1 (a) and BH1L (b) in the standard buffer, (c) is BH1, BH2, BH3 in the photon mode And pH of BH1L versus I green / I iso (excitation wavelength is 360 nm).
  • (A) is a two-photon activity spectrum of BH1 in standard buffers (pH 3.5, 7.2 and 10)
  • (b) is a graph showing the change in two-photon excitation fluorescence spectrum of BH1 with pH
  • (c) is a two-photon mode in a graph showing the pH versus green I / I iso of BH1, BH2, BH3 and BH1L (excitation wavelength is 470 nm).
  • FIG. 1 Survival graph of HeLa cells through MTS (Micro Technology Services) analysis.
  • Figure 4 (a) is a two-photon excitation fluorescence spectrum of ion-transmitter-Hell cells treated with BH1 3 ⁇ M, (b) is a two-photon I green / I IR titration curve according to the pH of the HeLa cells, (c) is Variable color TPM image of HeLa cells treated with BH1 3 ⁇ M, (d) is an enlarged photograph (arrow number is pH of measurement site, scale bar is (c) 30 ⁇ m, (d) 5 ⁇ m).
  • (a) and (b) are two-photon fluorescence microscope (a) and one-photon fluorescence microscope (b) images of HeLa cells treated with BH1L and LysoTracker Red.
  • (c) is a composite photograph of (a) and (b) (excitation wavelength is 740 nm in a two-photon fluorescence microscope, 543 nm in a one-photon fluorescence microscope, 20 micrometers in a scale bar).
  • Figure 6 (a) is a two-photon excitation fluorescence spectrum of ion permeate-Hell cells treated with BH1 3 ⁇ M.
  • (b) is two-photon I green / I IR titration curves according to pH of HeLa cells.
  • (c) and (d) are variable color TPM images (c) and enlarged photographs (d) of HeLa cells treated with BH1 3 ⁇ M.
  • the number of arrows is the pH of the measurement site, the scale bar is 30 ⁇ m in (c), (d) 5 ⁇ m)
  • (c) and (e) are variable color TPM images of HeLa cells treated with BH1 3 ⁇ M
  • ( c) is before NH 4 Cl addition and (e) is after addition.
  • (d) and (f) are composite pictures corresponding to DIC images.
  • (g) is an enlarged photograph of (c) showing the pH change with time (excitation wavelength 740 nm, scale bar is 20 micrometers in (c), 8 micrometers in (g)).
  • Figure 9 is a graph showing the absorption intensity (right) according to the photon spectra (left) and probe concentration in standard buffer (pH 7.2, 3 mL).
  • (a) and (b) are BH1
  • (c) and (d) are BH2
  • (e) and (f) are BH3
  • (g) and (h) are BH1L
  • (i) and (j) are P1 .
  • (a), (b) and (c) are BH2, (d), (e) and (f) are BH3, (g), (h) and (i) are BH1L.
  • the excitation wavelength is (a), (d) and (g) is 360 nm, (b), (e) and (h) is 740 nm, and the results at (c), (f) and (i) are maximum. Standardized at I green / I iso .
  • (a), (c), (e) and (g) are TPM images of HeLa cells treated with BH1 (a), BH2 (c), BH3 (e), and BH1L (g).
  • (b), (d), (f) and (h) are relative two-photon excitation fluorescence (TPEF; two-photon) over time in the AC region shown in (a), (c), (e) and (g). excited fluorescence) Intensity is a graph showing intensity intensity (numerical intensity measured every 2 seconds for 1 hour in xyt mode, and two-photon excitation fluorescence was collected in femtosecond pulses at 400-600 nm with an excitation wavelength of 740 nm Scale bar is 75 ⁇ m).
  • TPM images of HeLa cells treated with BH1 3 ⁇ M Two-photon excitation fluorescence was collected as femtosecond pulses at (a) 440-460 nm ( I IR ) and (b) 500-550 nm ( I green ) when the excitation wavelength was 740 nm. (c) is pseudocolored variable color two-photon fluorescence microscopy image ( I green / I IR ) (scale bar is 30 ⁇ m).
  • TPM image of rat hippocampal sections (a), (c) and (e) were treated with BH1L for 1 hour, and (b), (d) and (f) were treated with BH1 for 1 hour.
  • Two-photon excitation fluorescence is collected with femtosecond pulses at 440 nm excitation wavelength, (a) and (b) at 440-460 nm ( I IR ), (c) and (d) at 500-550 nm ( I green ) It became.
  • 120 TPM images were accumulated to visualize the pH along the Z-direction at a penetration depth of 90-180 ⁇ m.
  • the present invention relates to a two-photon fluorescence probe for measuring the pH in the cell, and because it uses a low energy excitation source can be measured by quantifying the real-time pH of the cell in real time without destroying the cell.
  • Histidine is an essential amino acid containing imidazole groups that have buffering capacity in the living body, and benzimidazole is a derivative of imidazole having about 5.5 pKa in acidic organelles.
  • the benzimidazole group is used as a proton acceptor, and is introduced at position 6 of 2-aminonaphthalene, which is a phosphor, to change the pH 4-7 region in the cell through color change (blue-green) according to the reaction with hydrogen ions. It provides a two-photon fluorescent probe that can be quantitatively measured and monitored in 0.001 pH units.
  • the present invention provides a two-photon fluorescent probe represented by the following Chemical Formulas 1 to 6.
  • X 1 and X 2 are each independently H, F, Cl, Br, OCH 3 , OH, NH 2 , N (CH 3 ) 2, NHCH 3, NCS, or CO 2 H
  • R 1 and R 2 Are each independently H, CH 3 , CH 2 (CH 2 ) n CH 3 , CH 2 (CH 2 ) n CO 2 H, CH 2 (CH 2 ) n OH, or CH 2 (CH 2 ) n SO 3 H, n is an integer of 0-10, and the same also applies below.
  • the two-photon fluorescent probe represented by Chemical Formula 1 may image pH while reacting with hydrogen ions in an internal pH 4-7 region of a cell, a cell organelle, or a living tissue to exhibit fluorescence.
  • the probe has a pKa 4-6.5 range by substituting an electron donor or electron acceptor with a benzimidazole group.
  • Figure 8 is a schematic diagram showing the fluorescence color change of the two-photon fluorescent probe according to an embodiment of the present invention and the image showing the intracellular pH observed through a two-photon fluorescence microscope.
  • the two-photon fluorescence probe When the cells are irradiated with a low excitation wavelength (740 nm), the two-photon fluorescence probe is protonated to cause a color change from blue to green, and the two-photon fluorescence microscope can observe the change and quantify the pH.
  • the two-photon fluorescent probe represented by Formula 2 and Formula 3 introduces a tertiary amine group to react with protons in an acidic organ to form a quaternary amine salt, and has about pKa 10. In particular, it can be selectively positioned inside the endosome or lysosome to sense the pH.
  • the two-photon fluorescent probe represented by Chemical Formulas 4 to 6 may introduce a maleimide group to selectively covalently bond with a thiol group of a biomolecule or a succinimdyl ester group to selectively introduce an amine group into a covalent bond.
  • pH By selectively labeling oligomers or proteins, pH can be measured.
  • FIG. 2a a graph showing the two-photon active cross-sectional area ( ⁇ ) versus the excitation wavelength of a two-photon fluorescent probe according to an embodiment of the present invention in buffers of pH 3.5, 7.2 and 10. It can be seen that the cross-sectional area is the largest at pH 3.5, which means that the two-photon fluorescent probe of the present invention has high pH measuring activity in the acidic region.
  • FIG. 2C is a calibration graph showing an isotropic point ( I green ) reference emission ( I iso ) ratio ( I green / I iso ) according to pH of a two-photon fluorescent probe according to an embodiment of the present invention. As shown in FIG. 2C, when plotting the plots, the plots show linearity at pH 4.5 to pH 7, which can be obtained from the pH 4.5 to pH 7 region representing a linear section through I green / I iso . Means.
  • Two-photon fluorescence probes of the present invention exhibit high fluorescence intensity two-photon emission spectra for regions with pH 4-7 in cells (FIGS. 4, 6).
  • pH can be imaged in real time in the pH 4-7 region of the cell. More preferably, the pH can be imaged by quantitatively measuring in real time in the pH 4.5-7 region.
  • the present invention is a two-photon fluorescent probe represented by the formula (1) prepared by mixing the compound represented by the formula (A), the compound represented by the formula (B) and p-toluenesulfonic acid monohydrate It provides a method of manufacturing.
  • X 1 and X 2 are each independently H, F, Cl, Br, OCH 3 , OH, NH 2 , N (CH 3 ) 2, NHCH 3, NCS, or CO 2 H
  • R 1 and R 2 Are each independently H, CH 3 , CH 2 (CH 2 ) n CH 3 , CH 2 (CH 2 ) n CO 2 H, CH 2 (CH 2 ) n OH, or CH 2 (CH 2 ) n SO 3 H, which is also the same below.
  • the present invention provides a method for producing a two-photon fluorescent probe represented by the formula (2) comprising the following steps:
  • step (b) preparing a compound represented by the following Chemical Formula E by mixing and reacting the compound represented by Chemical Formula D of step (a) with an aqueous LiOH solution;
  • step (c) reacting the compound represented by the formula (E) of step (b) with N, N-dimethylethylenediamine.
  • X 1 and X 2 are each independently H, F, Cl, Br, OCH 3 , OH, NH 2 , N (CH 3 ) 2, NHCH 3, NCS, or CO 2 H
  • R 1 and R 2 Are each independently H, CH 3 , CH 2 (CH 2 ) n CH 3 , CH 2 (CH 2 ) n CO 2 H, CH 2 (CH 2 ) n OH, or CH 2 (CH 2 ) n SO 3 H, which is also the same below.
  • the two-photon fluorescent probes represented by Chemical Formulas 1 to 6 of the present invention irradiate the cells with a low energy excitation wavelength (740 ⁇ m), the distribution of pH is selectively distributed to living cells and biological tissues of 100-200 ⁇ m depth without cell destruction. It can be seen and measured in real time for more than 60 minutes, so the pH change and activity can be imaged through a two-photon fluorescence microscope.
  • the two-photon fluorescent probe represented by Chemical Formula 1 of the present invention can be prepared as shown in Scheme 1 below, but is not limited thereto.
  • BH2 4,5-difluoro-1,2-phenylenediamine was used instead of o-phenyyenediamine, and a light yellow solid was obtained.
  • BH2 4,5-difluoro-1,2-phenylenediamine
  • BH3 4,5-dimethoxy-1,2-phenylenediamine was used instead of o-phenyyenediamine, and a pale yellow solid was obtained.
  • BH3 4,5-dimethoxy-1,2-phenylenediamine
  • Two-photon fluorescent probe represented by the formula (2) can be prepared as shown in the following schemes 2 to 4, it is not limited thereto.
  • Compound 2 was prepared by the method described in Document 2 (Document 2: Masanta, G .; Lim, CS; Kim, HJ; Han, JH; Kim, HM; Cho, BRJ Am. Chem. Soc. 2011, 133, 5698).
  • Compound 3 was the same as Example 1, except that Compound 2 was used instead of Compound 1, and a pale yellow solid was obtained at a yield of 51%.
  • Compound 4 (6-bromo-N-methyl-2-naphthylamine) was prepared by the method described in Document 1 (Document 1: Kim, HM; Jeong, BH; Hyon, Ju-Y .; An, MJ, Seo, MS; Hong, JH; Lee, KJ; Kim, CH; Joo, T .; Hong, Seok-C .; Cho, BRJ Am. Chem. Soc. 2008, 130, 4246).
  • R is the observed ratio of isotropic point ( I iso ) and 500-550 nm ( I green ) at a given pH.
  • R max and R min are the maximum and minimum limit values of R, and C is the slope.
  • I a / I b is the fluorescence intensity ratio of pH 3.5 to pH 10 at the selected wavelength to find the denominator of R. In this case, the correction is extinguished using the correct isotropic point.
  • the two-photon excitation fluorescence spectra were shown on a DDC meter (Monora 320 spectrograph set with Andor iDus DV401A-BV).
  • Two-photon fluorescent probes are excited at a 740 nm wavelength, 2510 mW (200 mW equivalent in focus) on a model-locked titanium-sapphire laser light source device (Mai Tai HP, Spectra Physics, 80 MHz pulses per second, 100 fs pulse width) It became.
  • Two-photon cross-sectional area ( ⁇ ) was measured using femto second (fs) fluorescence measurement.
  • Probes 1.0 ⁇ 10 ⁇ 6 M
  • two photons indicating fluorescent soils were measured using rhodamine 6G at 720-880 nm.
  • Two-photon excited fluorescence spectral intensities of the reference and the sample were determined at the same excited wavelength.
  • the cross section of the two-photon fluorescent probe was calculated using Equation 1 below.
  • Equation 1 s and r represent the sample and reference molecules, S represents the signal intensity collected from the CCD detector, ⁇ represents the fluorescence quantum yield, f represents the total fluorescence collection efficiency of the experimental instrument, c Represents the molecular density in the solution and ⁇ r represents the cross-sectional area of the two-photon fluorescent probe of the reference molecule.
  • HeLa cervical cancer cells (ATCC, Manassas, VA, USA) were treated with 10% FBS (WelGene), penicillin (100 units / mL) and streptomycin (100 ⁇ g / mL) in DMEM (WelGene Inc, Seoul, Korea). Incubated while feeding. Two days prior to imaging, cells were transferred to a glass bottom dish (NEST). All cells were grown in a humidified environment with an air / CO 2 ratio of 95: 5 at 37 ° C. Growth medium was removed for labeling and changed to serum-free DMEM. The cells were injected with 3 ⁇ M probe and incubated at 37 ° C. for 30 minutes.
  • FBS FelGene
  • penicillin 100 units / mL
  • streptomycin 100 ⁇ g / mL
  • Two-photon fluorescence probe microscopy images excite two-photon fluorescence probes to 2510 mW output at 740 nm wavelength using a model-locked titanium-sapphire laser light source device (Mai Tai HP, Spectra Physics, 80 MHz pulses per second, 100 fs pulse width) Observed by DMI6000B microscope (Leica).
  • a model-locked titanium-sapphire laser light source device Main Tai HP, Spectra Physics, 80 MHz pulses per second, 100 fs pulse width
  • DMI6000B microscope Leica
  • the pH calibration curve is represented by I green / I IR of HeLa cells treated with ion permeate carriers and BH1 or BH1L.
  • Cells were incubated for 30 minutes at 37 ° C., 5% CO 2 in DMSO stock containing 3.0 ⁇ L of 1 mM BH1 or 1 mM BH1L, and the extracellular media was 1 mL of calibration buffer (125 mM KCl, 20 mM NaCl, 0.5 mM CaCl 2 , 0.5 mM MgCl 2 , 5 ⁇ nigericin, 5 ⁇ monensin, and 25 mM buffer; acetates at pH 3.5, 4.0, 4.3, 5.0, 5.2; MES at pH 5.5, 6.0; pH 6.5, 7.0 , HEPES of 8.0).
  • calibration buffer 125 mM KCl, 20 mM NaCl, 0.5 mM CaCl 2 , 0.5 mM MgCl 2
  • the photostability of BH 1-3 and BH1L was shown by monitoring the change in two-photon excitation fluorescence intensity over time at three designated points in the probe-labeled HeLa cells (FIG. 13). Two-photon excitation fluorescence intensity showed high light stability while maintaining almost the same value for one hour.
  • Hippocampus fragments were prepared from hippocampus of 2 week old rats.
  • the coronal fragments were in artificial cerebrospinal fluid (ACSF; 138.6 mM NaCl, 3.5 mM KCl, 21 mM NaHCO 3 , 0.6 mM NaH 2 PO 4 , 9.9 mM D-glucose, 1 mM CaCl 2 , and 3 mM MgCl 2 ).
  • ADF cerebrospinal fluid
  • 138.6 mM NaCl 3.5 mM KCl, 21 mM NaHCO 3 , 0.6 mM NaH 2 PO 4 , 9.9 mM D-glucose, 1 mM CaCl 2 , and 3 mM MgCl 2
  • the pieces were incubated with 10 mM BCa1 and 20 mM BH1 and BH1L in ACSF
  • a certain amount of the two-photon fluorescent probe prepared in the above example was dissolved in DMSO (dimethyl sulfoxide, 1.0 ⁇ 10 ⁇ 2 M) and stored. Dilute the solution from 6.0 ⁇ 10 ⁇ 3 to 6.0 ⁇ 10 ⁇ 5 and buffer solution ((0.1 M citric acid, 0.1 M KH 2 PO 4 , 0.1 M Na 2 B 4 O 7 , 0.1 M Tris, 0.1 M KCl , pH 7.2) was added by micro syringe to a cuvette containing 3 mL The concentration of DMSO in the buffer solution was maintained at 0.2%.
  • FIG. 9 is a graph showing the absorption rate according to the concentration of the two-photon fluorescent probe prepared according to the Examples and Comparative Examples of the present invention. Plots in the graph were linear at low concentrations and downward curves at higher concentrations.
  • the maximum point in the straight line means solubility.
  • the solubility of Examples (BH1, BH2, BH3, BH1L) and Comparative Example (P1) of the present invention is 6, 2, 10, 10 and 2 ⁇ M, respectively, in pH 7.2 buffer solution, which is sufficient for cell staining. .
  • PKa and photophysical experimental results of the two-photon fluorescent probes prepared in Examples and Comparative Examples are shown in Table 1 below. All measurements were performed in standard buffer solutions (0.1 M citric acid, 0.1 M KH 2 PO 4 , 0.1 M Na 2 B 4 O 7 , 0.1 M tris (hydroxymethyl) aminomethane, 0.1 M KCl).
  • e pKa measured in daylight mode. In parentheses are pKa. Measured in two-photon mode.
  • the pKa of the conjugate acid of BH1 to 3 was obtained from a titration curve of the isotropic point ( I iso ) and the emission ratio ( I green / I iso ) of 500-550 nm ( I green ).
  • the pKa 4.92-6.11 range means that acidic regions can be detected.
  • the pKa shift is due to electron withdrawing (F in BH2) or electron donating (OMe in BH3).
  • the pKa value of BH1L was found to be almost the same as BH1.
  • the pH measuring capability in the two-photon mode of the two-photon fluorescent probe of the present invention was evaluated.
  • the two-photon fluorescence spectra ( ⁇ ) of BH1 at pH 7.2 and 3.5 showed 40 and 140 GM, respectively (Table 1 and FIG. 2A).
  • the maximum two-photon fluorescence spectrum ( ⁇ max ) of BH1-H + with electron withdrawing groups is 3.5 times larger than BH1, which enhances molecular charge transfer (ICT) between the electron acceptor and electron donor.
  • ICT molecular charge transfer
  • Intracellular pH was measured by TPM using BH1.
  • BH1 was injected into HeLa cells treated with ionopheres at 740 nm two-photon excitation (TP exctation).
  • TP exctation two-photon excitation
  • the two-photon emission fluorescence spectra were emitted at lambda fl values of 445 and 490 nm, respectively. This value almost coincides with the buffer and shift values in and around the 10 nm difference.
  • the emission spectrum of BH1 shows a gradual shift with increasing solvent polarity, which means that the probes of the invention are more homogeneous and hydrophobic in the intracellular buffer.
  • variable color imaging ( I green / I IR ) results were obtained using an internal reference window of 440-460 nm ( I IR ) and a pH recognition window of 500-550 nm ( I green ). It was obtained by (Fig. 4a).
  • the imaging values ( I green / I IR ) of HeLa cells treated with BH1 appeared in the pH range of 5.6-7.4 across the various compartments in the cells (FIGS. 4D and S7).
  • the slope of the graph is almost linear, which means that the pH can be quantified using variable color imaging ( I green / I IR ) in the pH 4-7 region.
  • BH1L a lysosomal target two-photon fluorescent probe.
  • LTR Lysotracker Red DND-99
  • 5 is a two-photon fluorescence microscopy image (FIG. 5A) and a one-photon fluorescence microscopy image (FIG. 5B) and superimposed images (FIG. 5C) (Pearson colocalization coefficient: 0.095).
  • Variable color imaging ( I green / I IR ) of HeLa cells treated with BH1L shows various pH values in lysosomes (FIG. 6). This means that BH1L can be used to measure the pH in each zone in the pH 4.6-5.9 range ( Figure 16).
  • the micro flakes of the hippocampus of rats which are responsible for learning and memory, were monitored. Since the structure of the brain tissue is heterogeneous, 120 variable color imaging ( I green / I IR ) were measured at a depth of 90-180 ⁇ m (FIG. 18).
  • I green / I IR 120 variable color imaging
  • the two-photon fluorescent probe of the present invention can clearly measure the pH value of the acidic compartment in the cells in living cells and tissues using two-photon microscopy.

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Abstract

La présente invention concerne une sonde fluorescente à deux photons pour imagerie du pH. La sonde fluorescente à deux photons de la présente invention détecte le pH dans une zone acide, ainsi qu'une variation de celui-ci, à l'aide d'un dérivé à structure benzimidazole. La sonde présente un point isotrope intrinsèque, une faible toxicité et une grande photostabilité et elle peut mesurer le pH en millièmes d'unité de pH tout en présentant un changement de couleur de la fluorescence à deux photons à un pH de 4 à 7. Selon la présente invention, la sonde fluorescente à deux photons est colorée de manière sélective à l'intérieur des cellules et des tissus biologiques vivants et présente, simultanément, un changement de couleur (bleu-vert) à forte fluorescence par réaction avec un pH acide. En outre, la sonde fluorescente à deux photons peut être facilement chargée sur des cellules grâce à sa grande solubilité dans l'eau et à son un faible poids moléculaire. En outre, la distribution du pH dans des cellules biologiques ou des tissus biologiques délicats et l'activité de celui-ci peuvent faire l'objet d'une étude faisant intervenir une image proportionnelle, car le pH peut être sélectivement détecté dans des cellules biologiques et des tissus biologiques à une profondeur de 100 à 200 µm pendant au moins 60 minutes. Par conséquent, la présente invention peut contribuer de façon importante aux recherches en sciences du vivant associées au pH, au diagnostic précoce de maladies, au développement d'un réactif diagnostique et d'un agent thérapeutique, et équivalent, étant donné que le pH peut être analysé de façon quantitative et que différents échantillons peuvent être comparés et analysés.
PCT/KR2014/013130 2014-12-31 2014-12-31 Sonde fluorescente à deux photons, son procédé de préparation et procédé d'imagerie du ph l'utilisant WO2016108316A1 (fr)

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CN109928949A (zh) * 2018-09-12 2019-06-25 兰州大学 一种新型荧光探针的制备及其长期稳定成像溶酶体的应用
CN113831291A (zh) * 2021-09-29 2021-12-24 山西大学 基于苯并咪唑的多功能溶酶体pH探针及其制备方法和应用
CN116987033A (zh) * 2023-07-28 2023-11-03 重庆医科大学 一种监测溶酶体微酸性环境轻微碱化的荧光探针及其制备方法和应用
CN117263915A (zh) * 2023-11-23 2023-12-22 山东海化集团有限公司 一种磺酸盐类三联吡啶衍生物及其制备方法和应用

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Publication number Priority date Publication date Assignee Title
CN109928949A (zh) * 2018-09-12 2019-06-25 兰州大学 一种新型荧光探针的制备及其长期稳定成像溶酶体的应用
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CN116987033A (zh) * 2023-07-28 2023-11-03 重庆医科大学 一种监测溶酶体微酸性环境轻微碱化的荧光探针及其制备方法和应用
CN117263915A (zh) * 2023-11-23 2023-12-22 山东海化集团有限公司 一种磺酸盐类三联吡啶衍生物及其制备方法和应用
CN117263915B (zh) * 2023-11-23 2024-04-05 山东海化集团有限公司 一种磺酸盐类三联吡啶衍生物及其制备方法和应用

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