CN116621814A - Fluorescent probe for detecting peroxynitroso anions, preparation method and application thereof - Google Patents

Fluorescent probe for detecting peroxynitroso anions, preparation method and application thereof Download PDF

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CN116621814A
CN116621814A CN202310627342.5A CN202310627342A CN116621814A CN 116621814 A CN116621814 A CN 116621814A CN 202310627342 A CN202310627342 A CN 202310627342A CN 116621814 A CN116621814 A CN 116621814A
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probe
fluorescent probe
preparation
reaction
niml
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CN116621814B (en
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田明刚
葛伟
张启龙
王志远
孙封凯
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University of Jinan
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • 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
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    • 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
    • C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6443—Fluorimetric titration
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides a fluorescent probe for detecting peroxynitroso anions, a preparation method and application thereof, wherein the probe is formed by connecting naphthalimide with indole salt through piperazine groups, the naphthalimide is a common fluorophore, and the structural characteristics of the indole salt enable the naphthalimide to have certain affinity with RNA. Spectral results demonstrate that probe NIML has a certain response to ONOO-and that the probe has a significant change in fluorescence signal in a biological imaging experiment with respect to apoptosis. The fluorescence probe NIML provided by the invention can detect the change of the ONOO concentration in a ratio, has low detection limit and short response time, has good response effects on viscosity and RNA, and can effectively detect the apoptosis process.

Description

Fluorescent probe for detecting peroxynitroso anions, preparation method and application thereof
Technical Field
The invention belongs to the technical field of organic material science, and particularly relates to a fluorescent probe for detecting peroxynitroso anions, a preparation method and application thereof.
Background
Peroxynitrite (ONOO-) is a strong oxidant as active oxygen. It has been found that such strong oxidants are typically generated by coupling nitric oxide with superoxide anions. ONOO-levels are normal and are able to defend against the invasion of some cytotoxins; when ONOO-levels are abnormal, they act as toxic substances, destroying various biomolecules due to their high reactivity, which can cause the destruction of the cell structure and thus the loss of function. Therefore, efficient detection of ONOO-is of great importance.
Cell death comprises three forms: apoptosis, cell necrosis, autophagy. Apoptosis, among other things, is a tightly controlled manner of apoptosis. Apoptosis plays a very important role both physiologically and pathologically. Physiologically, apoptosis is actively involved in various physiological processes in biological systems, such as embryonic development, immune processes, etc.; pathologically, damaged or infected cells are eliminated. This feature becomes one of the approaches to treat cancer: the preparation of a medicament can induce apoptosis to destroy cancer cells. Therefore, monitoring apoptosis is of great importance. Currently, there are several methods for detecting apoptosis. For example, annexin V probes are constructed to target phosphatidylserine on the cell surface so as to monitor apoptosis, but the probes have short shelf life, long detection time and low detection sensitivity.
Therefore, constructing fluorescent probes to detect apoptosis with high sensitivity and fast visualization is a problem to be solved.
Disclosure of Invention
The invention constructs a novel fluorescent probe NIML. The probe is formed by connecting naphthalimide, which is a common fluorophore, with indole salt through piperazine groups, and the structural characteristics of the indole salt enable the naphthalimide to have certain affinity with RNA. Spectral results demonstrate the ability of probe NIML to respond to ONOO-; meanwhile, the probe can have obvious fluorescence signal change in a biological imaging experiment on apoptosis.
The invention discloses a fluorescent probe for detecting peroxynitroso anions, the code of the fluorescent probe is NIML, and the fluorescent probe has the following structure:
the invention also provides a preparation method of the probe NIML, which comprises the following steps:
preparation of intermediate 1: adding 4-bromo-1, 8-naphthalene anhydride and N, N-dimethyl ethylenediamine into ethanol, heating to react, cooling to separate out solid, filtering to obtain intermediate 1 crude product, and recrystallizing with organic solvent to obtain pure intermediate 1;
preparation of intermediate 2: adding piperazine and p-fluorobenzaldehyde into ethylene glycol monomethyl ether for heating reaction, cooling for crystallization, filtering, adding an organic solvent for recrystallization to obtain an intermediate 2;
preparation of intermediate 3: adding the intermediate 1 and the intermediate 2 into a reaction bottle, adding the reaction bottle into ethylene glycol monomethyl ether, heating for reaction, cooling, adding water and an organic solvent, layering, concentrating an organic phase to obtain a crude product of the intermediate 3, and purifying by a silica gel chromatographic column to obtain a pure intermediate 3;
preparation of intermediate 4: adding 2, 3-trimethyl-3H-indole and methyl iodide into a reaction bottle, adding ethanol, heating for reaction for 7-10 hours, cooling for crystallization, filtering and washing to obtain an intermediate 4;
preparation of probe NIML: adding the intermediate 3, the intermediate 4 and the piperazine into ethanol, heating to react, cooling to crystallize, filtering, and purifying by a silica gel column to obtain the probe NIML.
Further preferably, the reaction temperature of the preparation process of the intermediate 1 is 70-90 ℃, the reaction time is 4-6h, and the organic solvent adopted by recrystallization is one or more of methanol, ethanol, isopropanol and ethyl acetate.
Further preferably, the reaction temperature of the preparation process of the intermediate 2 is 110-130 ℃ and the reaction time is 5-8h. The organic solvent used for recrystallization is one or more of methanol, ethanol, isopropanol and ethyl acetate. 5. The method for preparing a fluorescent probe according to claim 2, wherein the reaction temperature of the preparation process of the intermediate 3 is 110-130 ℃, the reaction time is 15-20h, and the organic solvent is one of dichloromethane and DMF.
Further preferably, the reaction temperature of the preparation process of the probe NIML is 75-85 ℃ and the reaction time is 5-6h.
The invention also discloses application of the fluorescence probe for detecting peroxynitroso anions in cell imaging visualization. In particular, the cell imaging is apoptosis imaging, cell activity imaging and cytotoxicity imaging, and the main reasons that the fluorescent probe can be used for cell imaging are that the fluorescent probe can detect the change of ONOO-concentration in cells, and the detection sensitivity and the detection limit are only 50 mu M. Meanwhile, the fluorescent probe has positive response to intracellular viscosity and RNA.
The invention has the beneficial effects that:
(1) The fluorescence probe NIML can detect the change of the concentration of the ONOO in a ratio, the detection limit is low, and the concentration of the ONOO saturation reaction is only 50 mu M.
(2) The response time is short, the reaction can be completed in about three minutes, and the response capability is good.
(3) The probe NIML has good response effects on viscosity and RNA.
(4) The probe NIML can well detect the apoptosis process.
(5) The fluorescent probe has simple preparation method and low preparation cost.
Drawings
FIG. 1 is a diagram of the compound NIML 1 H NMR spectrum;
FIG. 2 is a diagram of the compound NIML 13 C NMR spectrum;
FIG. 3 is a chart of a probe NIML spectral test;
(A) 10 mu M NIML for ONOO - Is a graph of the absorption spectrum of (2); (B) 10 mu M NIML under 450nm excitation, as for ONOO - Is a fluorescence spectrum of (2); (C, D) 10. Mu.M NIML at 450nm excitationAbout 50 μm ONOO - Is a dynamic fluorescence spectrum of (1);
FIG. 4 shows the NIML viscosity, RNA concentration titration ultraviolet and fluorescence spectroscopy,
(A) Fluorescence spectra of 10 μm NIML in mixed solutions of methanol and glycerol at different ratios under excitation at 450 nm; (B) absorbance spectra of 10 μm NIML with respect to RNA titration concentration; (C) Fluorescence spectra for RNA titration concentration at 450nm excitation with 10 μm NIML;
FIG. 5 is a fluorescence image of living cells incubated with NIML and MTDR for 30 minutes;
FIG. 6 is an image of fixed cells incubated with NIML for 30 minutes and fixed cells treated with DNase or RNase;
FIG. 7 is a fluorescent image of probe NIML in fixed cells and in fixed cells treated with 500. Mu.M ONOO.
Detailed Description
The present invention will be further described with reference to examples for the purpose of making the objects and technical aspects of the present invention more apparent, but the scope of the present invention is not limited to these examples, which are only for explaining the present invention. It will be understood by those skilled in the art that variations or equivalent substitutions that do not depart from the spirit of the invention are intended to be included within the scope of the invention. The present invention is further described below with reference to examples, but the present invention is not limited to the examples.
The medicines used in the invention are purchased from outsourcing, and 4-bromo-1, 8-naphthalene anhydride is purchased from Shanghai Honghao biological medicine technology Co., ltd; n, N-dimethylethylenediamine and 4-fluorobenzaldehyde are purchased from Shanghai Jizhui Biochemical technologies Co., ltd; piperazine was purchased from Shanghai national medicine control chemical reagent Co., ltd; 2, 3-trimethyl-3H-indole was purchased from Anhui Zernike technologies Co., ltd; methyl iodide was purchased from the scientific and technological company of belgium. Reagents required for biological imaging: mitochondrial deep red is purchased from molecular probes, inc; DNA and RNA were purchased from Sigma Aldrich trade Co., ltd (project number: DNA is D1501, RNA is R6625), which are natural DNA and RNA isolated from calf thymus and yeast.
The specific manufacturers of the organic synthesis instrument are as follows: magnetic stirrers, oil baths, and vacuum pumps (R5614Y-1) were purchased from the company of the instrument limited, incorporated; rotary evaporator (N-1100) was purchased from tokyo physicochemical company; electronic balance (LE 204E/02) purchased from Metler Shanghai Co., ltd; biological imaging instrument: nikon A1MP confocal microscope was purchased from Nikon corporation; carbon dioxide incubators are purchased from pine appliances limited.
Example 1:
synthesis of intermediate 1: 4-bromo-1, 8-naphthalene anhydride (7 mmol) was added to the reaction flask, dissolved in a small amount of ethanol solvent, and N, N-dimethylethylenediamine (21 mmol) was added thereto, dissolved in a large amount of ethanol solvent, and the mixed solution was refluxed for 4 hours and cooled to room temperature. The precipitate was separated out and filtered to give a crude product which was recrystallized from ethanol to give a solid with 86% yield.
Synthesis of intermediate 2: piperazine (17.4 mmol) was added to a reaction flask containing 25mL of methoxyethanol and 18mL of water, and after stirring at room temperature for 15 minutes, a mixed solution containing 5mL of water and 4-fluorobenzaldehyde (4.64 mmol) was slowly dropped into the reaction flask. The mixture was stirred and refluxed for 24 hours, cooled and then taken up in CH 2 Cl 2 Extracting. At Na (Na) 2 SO 4 After the organic layer was dried, the solvent was removed by evaporation. The yellow product was obtained after recrystallisation from ethanol in 67% yield.
Synthesis of intermediate 3: in a reaction flask, compound 1 (10 mmol) and Compound 2 (10 mmol) were added, and the mixture was refluxed with ethylene glycol monomethyl ether for 24 hours and cooled with CH 2 Cl 2 Extracting, removing the solvent by evaporation, using CH 2 Cl 2 /CH 3 The crude product was purified by chromatography on silica gel using an OH mixture (from 35:1 to 30:1, v/v) as eluent to give a yellow solid in 62% yield.
Synthesis of intermediate 4: 2, 3-trimethyl-3H-indole (5 mmol) was added to a reaction flask, dissolved with a small amount of ethanol solvent, methyl iodide (10 mmol) was added to the flask, dissolved with 2ml of ethanol solvent, heated to reflux for 9 hours, then solid was precipitated in the solution, the mixed solution was cooled to room temperature, filtered and washed with petroleum ether and ethanol several times to give a reddish-purple product with a yield of 85%.
Synthesis of probe NIML: to a reaction flask charged with compound 3 (13 mmol) and compound 4 (13 mmol) was added ethanol (3 mL) and stirred for 10 min, followed by several drops of piperidine. Reacting at 85deg.C for 12h, cooling to room temperature, removing solvent by evaporation, using CH 2 Cl 2 /CH 3 The crude product was purified by chromatography on silica gel with an OH mixture (from 15:1 to 10:1, v/v) as eluent to give a red solid in 65% yield. 1 H NMR(400MHz,DMSO-d 6 )δ8.60(d,J＝8.5Hz,1H),8.53(d,J＝7.2Hz,1H),8.46(d,J＝8.0Hz,1H),8.35(d,J＝15.8Hz,1H),8.16(d,J＝8.7Hz,2H),7.88(t,J＝7.9Hz,1H),7.82(d,J＝7.3Hz,1H),7.77(d,J＝7.9Hz,1H),7.59(t,J＝7.6Hz,1H),7.53(t,J＝7.4Hz,1H),7.47–7.35(m,2H),7.23(d,J＝8.8Hz,2H),4.18(t,J＝6.8Hz,2H),4.04(s,3H),3.88(d,J＝5.8Hz,4H),3.43(d,J＝5.6Hz,4H),3.38(t,J＝6.8Hz,2H),2.27(s,6H),1.78(s,6H).
13 C NMR(151MHz,DMSO-d 6 ) Delta 180.81,164.26,163.71,155.69,154.72,154.14,143.31,142.45,132.76,131.35,131.18,129.69,129.26,128.56,126.72,125.75,124.44,123.14,123.10,122.78,116.38,115.77,114.50,114.23,114.02,107.49,55.36,52.60,51.69,47.03,46.79,44.93,33.89,26.47 nuclear magnetic hydrogen spectrum and carbon spectrum are shown in figure 1 and figure 2.
Example 2: probe NIML spectroscopic testing
Preparing a probe mother solution: 3.7mg of probe was weighed and dissolved in 1mL of DMSO to prepare a 5mM stock solution.
Preparation of sodium bisulfite: 10.4mg of sodium bisulphite was weighed out and dissolved in 10mL of deionized water to prepare a 10mM solution.
ONOO - Is prepared from the following steps: under ice bath condition, stirring vigorously to contain NaNO 2 HCl (0.6M, 10 mL) was added to a mixture of (0.6M, 10 mL) and hydrogen peroxide (0.7M, 10 mL), followed by rapid addition of sodium hydroxide (1.5M, 20 mL). Finally, a very small amount of manganese dioxide was added and filtered. Note that the solutions described above were all prepared from deionized water, and that ONOO was prepared - It should be preserved at-20deg.C in dark place. ONOO (oxide-nitride-oxide) - And (3) concentration calibration: firstly, use2mL of 0.1M sodium hydroxide was scanned across the baseline, and X. Mu.L of prepared ONOO was added to the V mL of 0.1M sodium hydroxide - Solutions were subjected to UV measurements, calculated as follows, wherein A 302 nm Is ONOO - Absorbance at 302nm, V is total volume of solution, X is ONOO - Volume of solution.
With respect to ONOO - Is characterized by comprising the following steps of: firstly preparing a blank, adding 4 mu L of 5mM probe solution into 2mL of mixed solution of PBS buffer solution and ethanol (volume ratio is 1 to 1), wherein the working concentration of the probe is 10 mu M, and then adding ONOO with a certain concentration - The solution was subjected to ultraviolet or fluorescence spectroscopic testing. (excitation wavelength in fluorescence Spectrum test is 450 nm)
Method for measuring absorption and fluorescence spectrum of sodium bisulphite: a blank sample is prepared, 4 mu L of a probe solution with the concentration of 5mM is added into 2mL of a mixed solution of PBS buffer solution and ethanol (the volume ratio is 1 to 1), the working concentration of the probe is 10 mu M, and then a sodium bisulphite solution with a certain concentration is added for ultraviolet or fluorescence spectrum test. (excitation wavelength in fluorescence Spectrum test is 450 nm)
Fluorescence spectrum test method for viscosity: mixed solutions of glycerol and methanol with different volume ratios are prepared, and 2 mu L of NIML probe with the concentration of 5mM and NIML probe solution are respectively added into 2mL of the mixed solution with different proportions, so that fluorescence spectrum test can be carried out. (excitation wavelength in fluorescence Spectrum test is 450 nm)
Fluorescence spectrum test method for RNA and DNA: mu.L of NIML probe and NIML probe solution with concentration of 5mM are respectively added into 2mL of PBS buffer solution, and then a certain amount of DNA and RNA solution are respectively added for fluorescence spectrum test. (excitation wavelength 450nm in fluorescence spectroscopy test) DNA and RNA molar concentration determination: DNA and RNA solutions were initially prepared at a concentration of 1mg/mL, and a portion of the stock was diluted in order to quantify their molar concentration. The DNA solution was diluted 200-fold and the RNA solution was diluted 250-fold. The absorbance spectra of the diluted DNA, RNA solutions were then measured.
Regarding the selective absorption, fluorescence spectrum test method:
cysteine, glutathione, glycine, serine, proline, alanine, glutamic acid, threonine, histidine, valine, asparagine, phenylalanine, arginine, glucose, sucrose, sodium chloride, potassium nitrate, hydrogen peroxide and VC (working concentration is 50 mu M) are selected as interference substances to be detected, and the analysis substances are weighed and dissolved by deionized water. mu.L of a 5mM probe solution was added to 2mL of a mixed solution of PBS buffer and ethanol (volume ratio: 1 to 1), and the above interfering substances were added to each solution, respectively, to perform ultraviolet or fluorescence spectroscopic measurement. (excitation wavelength in fluorescence Spectrum test is 450 nm)
(1) ONOO-concentration titration ultraviolet spectroscopy test:
first, the probe pair ONOO is performed - Is measured by concentration titration ultraviolet. As shown in FIG. 3A, no ONOO is added - When the absorption peak appears in the range of 450nm and 600 nm; with the continuous addition of ONOO - (0-5 eq) where the absorption peak was changed, was continuously decreased, and another absorption peak was gradually appeared in the range of 350nm and 450nm, and had a tendency to be increased. The UV spectrum test results indicated that the probe was responsive to ONOO-.
(2) ONOO-concentration titration fluorescence spectroscopy test
ONOO of probes - As shown in fig. 3B. Similar to the above ultraviolet test results, the emission peak of the blank probe solution was around 608nm under 450nm excitation; ONOO when added to solution - At higher and higher concentrations (0-5 eq), the fluorescence intensity of the probe at this point correspondingly decreases and an emission peak appears around 545nm, which peak increases continuously; when ONOO is formed - When 5 equivalents were added, the fluorescence intensity at 545nm reached a maximum. The fluorescence spectrum test results show that the probe and the ONOO - The reaction is continuously carried out andthere will be a major material formation.
(3) ONOO-kinetic Spectrometry test
To further understand the probe pair ONOO - Dynamic testing was performed in response to the conditions of the test. As shown in FIG. 3C, when 5 equivalents of ONOO are added under 450nm excitation - At this time, the fluorescence intensity of the probe at 608nm was drastically reduced, the emission peak at 545nm was rapidly developed, and the peak was rapidly reached. As is clearly shown in FIG. 3D, this process is performed with the probe pair ONOO - Only about 3 minutes is required for the response time of (c). The test results show that it is specific to ONOO - The response is quicker.
(4) Viscosity fluorescence Spectrometry test
Probe NIML is viscosity responsive due to limited rotation within the molecule. Fluorescence spectroscopy of the viscosity system was performed on probe NIML. Mixed solutions of methanol and glycerin in different ratios were used as viscosity test systems. The more glycerol content means the greater the viscosity of the whole mixed solution. As shown in FIG. 4A, the greater the ambient viscosity at which the probe is exposed to excitation at 450nm, the greater the fluorescence intensity. When the viscosity is 0.6cp, the fluorescence of the probe at 611nm is very weak; when the viscosity was increased to 945.3cp, the fluorescence of the probe at this point was significantly enhanced, increasing the fluorescence intensity from 139.4 to 4248 by nearly 30-fold. The above results indicate that the probe responds well to viscosity.
(6) RNA concentration titration ultraviolet and fluorescence spectrum test
From previous measurements of the probe's emission spectrum with respect to viscosity, it was determined that the probe has a certain response to viscosity, and that the probe will generally have a certain affinity for RNA since RNA will also provide an environment similar to viscosity such that intramolecular rotation of the probe is limited. To verify the above hypothesis, spectroscopic testing of the probe with respect to RNA was performed. First, the ultraviolet spectrum test, as shown in FIG. 4B, shows that the absorbance peak slightly shifted when 80 equivalents of RNA were added. As shown in FIG. 4C, the fluorescence of the blank probe solution at 605nm is weak under 450nm excitation; the fluorescence intensity gradually increases as the concentration of added RNA increases; the fluorescence intensity was maximized until 80 equivalents of RNA were added. The results of the two spectrum experiments show that the probe has stronger binding capacity to RNA.
Example 3: cell imaging experiments
Co-localization experiments Using the commercial probe MitoTracker Deep-Red (MTDR) as a co-localization reagent, cell images were obtained under a Nikon A1MP confocal microscope after incubation of cells for half an hour with 2. Mu.M NIML and 50nM MTDR. The fluorescence signal of NIML is collected in the red channel (570-620 nm,561nm excitation), while the emission signal of MTDR is collected in the deep red channel (665-730 nm,647nm excitation). Firstly, living cells and co-localization imaging experiments are carried out, as shown in fig. 5, the probe is expressed in a filiform form in a green channel fluorescent signal, the staining position of the probe is presumed to be mitochondria, then the probe and MTDR are stained together, and a green channel fluorescent image and a red channel fluorescent image are well overlapped.
The cell immobilization experiment and the RNA hydrolase and DNA hydrolase experiment steps are as follows: cells were treated with 4% paraformaldehyde for 1 hour, washed with PBS, and then treated with 0.5% Triton X-100 for 30 minutes to give dead cells. The fixed cells were incubated with 2. Mu.M probe NIML for half an hour, and the fixed cell imaging experiment was observed under a confocal laser microscope. During the RNA hydrolase assay, the fixed cells were treated with 20. Mu.g/mL RNA hydrolase for two hours, then with 2. Mu.M NIML for 30 minutes, and then with Nikon A1MP confocal microscopy to obtain imaging assay data. In the DNA hydrolase test, 10 mu L of DNA hydrolase with the product specification of 1000u is added into dead cells and treated for half an hour, and then 2 mu M of NIML is used for incubation for 30 minutes, so that fluorescent signals of the probe in the cells can be collected under a microscope. Cells were imaged with red (collected at 570-620nm, excitation wavelength 561 nm) channels. In order to understand the staining and distribution of probe NIML in fixed cells, a related cell imaging experiment was performed. As shown in FIG. 6, in living cells, the probe emits strongly in the red channel, mainly distributed in the cytoplasm, and the fluorescent signal is expressed in a filiform form. In fixed cells, the fluorescent signal of the probe in the red channel is enhanced and distributed in the cytoplasm and nucleus. When the immobilized cells were treated with DNase, the fluorescent signal of the probe in the red channel was distributed in the cytoplasm and nucleolus, indicating that the probe was not bound to DNA. When the fixed cells were treated with RNase, red fluorescent signals were distributed in the nuclei, indicating binding of the probe to RNA.
ONOO-cell experiments fixed cells were used with 500 μm ONOO - After a period of treatment, the fluorescent signal of the probe in the cell can be collected under a microscope. Cells were imaged with two channels, green (500-550 nm for excitation wavelength 488 nm) and red (570-620 nm for excitation wavelength 561 nm). In order to understand the staining and distribution of probe NIML after ONOO treatment in fixed cells, a related cell imaging experiment was performed.
As shown in FIG. 7, in the absence of ONOO treatment, probe NIML produces a distinct fluorescent signal in the red channel, which is localized in the nucleus; when cells were treated with ONOO-, the fluorescent signal of the probe in the red channel was significantly reduced and barely detectable; while generating a distinct fluorescent signal in the green channel. The results show that the probe added ONOO in the immobilized cells - And then has a good response.

Claims (10)

1. A fluorescent probe for detecting peroxynitroso anions, code NIML, characterized in that the fluorescent probe has the following structure:
2. a method for preparing a fluorescent probe for detecting peroxynitroso anions according to claim 1, wherein the preparation method comprises the steps of:
preparation of intermediate 1: adding 4-bromo-1, 8-naphthalene anhydride and N, N-dimethyl ethylenediamine into ethanol, heating to react, cooling to separate out solid, filtering to obtain intermediate 1 crude product, and recrystallizing with organic solvent to obtain pure intermediate 1;
preparation of intermediate 2: adding piperazine and p-fluorobenzaldehyde into ethylene glycol monomethyl ether for heating reaction, cooling for crystallization, filtering, adding an organic solvent for recrystallization to obtain an intermediate 2;
preparation of intermediate 3: adding the intermediate 1 and the intermediate 2 into a reaction bottle, adding the reaction bottle into ethylene glycol monomethyl ether, heating for reaction, cooling, adding water and an organic solvent, layering, concentrating an organic phase to obtain a crude product of the intermediate 3, and purifying by a silica gel chromatographic column to obtain a pure intermediate 3;
preparation of intermediate 4: adding 2, 3-trimethyl-3H-indole and methyl iodide into a reaction bottle, adding ethanol, heating for reaction for 7-10 hours, cooling for crystallization, filtering and washing to obtain an intermediate 4;
preparation of probe NIML: adding the intermediate 3, the intermediate 4 and the piperazine into ethanol, heating to react, cooling to crystallize, filtering, and purifying by a silica gel column to obtain the probe NIML.
3. The method for preparing a fluorescent probe according to claim 2, wherein the reaction temperature of the preparation step of the intermediate 1 is 70-90 ℃, the reaction time is 4-6 hours, and the organic solvent used for recrystallization is one or more of methanol, ethanol, isopropanol and ethyl acetate.
4. The method for preparing a fluorescent probe according to claim 2, wherein the reaction temperature of the intermediate 2 is 110-130 ℃ and the reaction time is 5-8h. The organic solvent used for recrystallization is one or more of methanol, ethanol, isopropanol and ethyl acetate.
5. The method for preparing a fluorescent probe according to claim 2, wherein the reaction temperature of the intermediate 3 is 110-130 ℃ and the reaction time is 15-20h, and the organic solvent is one of dichloromethane and DMF.
6. The method of preparing a fluorescent probe according to claim 2, wherein the reaction temperature is 75-85 ℃ and the reaction time is 5-6h.
7. Use of a fluorescent probe for detecting peroxynitroso anions according to claim 1 in cell imaging visualization.
8. The use of cell imaging visualization of claim 7, wherein the cell imaging is apoptosis imaging, cell activity imaging, and cytotoxicity imaging.
9. The use of the cell imaging visualisation of claim 7, wherein the fluorescent probe is capable of detecting changes in ONOO-concentration in cells, wherein the ONOO-detection limit is 50 μm.
10. The use of cell imaging visualization of claim 7 wherein the fluorescent probe responds positively to intracellular viscosity, RNA.
CN202310627342.5A 2023-05-31 Fluorescent probe for detecting peroxynitroso anions, preparation method and application thereof Active CN116621814B (en)

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