CN110437283B - Potassium ion fluorescent probe and preparation method and application thereof - Google Patents
Potassium ion fluorescent probe and preparation method and application thereof Download PDFInfo
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- CN110437283B CN110437283B CN201910849629.6A CN201910849629A CN110437283B CN 110437283 B CN110437283 B CN 110437283B CN 201910849629 A CN201910849629 A CN 201910849629A CN 110437283 B CN110437283 B CN 110437283B
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- potassium ion
- potassium
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6596—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having atoms other than oxygen, sulfur, selenium, tellurium, nitrogen or phosphorus as ring hetero atoms
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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
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention relates to a potassium ion fluorescent probe and a preparation method and application thereof. The potassium ion fluorescent probe NK1 provided by the invention has the advantages of short synthesis steps and simple structure, wherein ACLE is used as K+The identification unit, the BODIPY derivative as a fluorophore and the TPP as a mitochondrion targeting group are matched, so that the potassium ion fluorescent probe has high selectivity and sensitivity in the process of identifying potassium ions. NK1 has little toxic and side effect on human cervical cancer cells (HeLa), has high biocompatibility and can also target mitochondria. In addition, the NK1 provided by the invention can qualitatively monitor the potassium ion inflow or outflow in the cell mitochondria.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a potassium ion fluorescent probe and a preparation method and application thereof, and particularly relates to a mitochondrion targeted potassium ion fluorescent probe and a preparation method and application thereof.
Background
Mitochondrial Potassium (K)+) Channels are a class of transporters located in the inner mitochondrial membrane. Mitochondria K+Channel-mediated K between cytoplasm and mitochondria+The influx can regulate mitochondrial membrane potential, maintain mitochondrial volume homeostasis, regulate the concentration of reactive oxygen species, and prevent Ca in the matrix2+Overload. In addition, mitochondria K+The channel plays an important role in a cell protection mechanism in the process of cerebral hypoxia or myocardial infarction and can also be used as an effective target point for treating cancer. Unfortunately, due to the lack of efficient techniques, such as mitochondrial targeting of fluorescence K+The molecular identification and localization of these transporters is still not fully understood. Therefore, to monitor mitochondrial transmembrane K+Correlation between flux and other biological parameters in biological pathwaysIs, there is an urgent need for mitochondrially targeted fluorescent K+Development and application of sensors.
In 2003, He et al reported that Na is hardly accepted+Ion, pair K+Ion-highly specific potassium ion ligand TAC with triazacyclonoether TAC as K+Ion ligand, 4-aminonaphthalimide as chromophore to synthesize novel potassium ion fluorescence sensor (A fluorescent sensor with high selectivity and sensitivity for potassium in water [ J)]Journal of the American Chemical Society,2003,125(6):1468-+In the presence of ions, the K content of the active component is 2-10mM+Has large fluorescence response and can be used for detecting extracellular K clinically+And (5) detecting the ion concentration. Because the TAC ligand has excellent performance, a plurality of excellent sensors based on the TAC ligand are successfully developed. This is K+A major breakthrough in the research history of ion sensors is that the developed potassium ion ligand TAC is used up to now and is considered as the best potassium ion binding ligand. But the synthesis is extremely complex, the reaction conditions are harsh, and the overall yield is extremely low. And mitochondrial targeting cannot be achieved.
Therefore, there is a need in the art to develop a mitochondrial targeting K with simple synthesis, good selectivity and high sensitivity+Sensors to monitor intracellular mitochondrial K+The concentration changes.
Disclosure of Invention
In view of the defects of the prior art, an object of the present invention is to provide a potassium ion fluorescent probe, and in particular, to provide a mitochondria-targeted potassium ion fluorescent probe. The potassium ion fluorescent probe can target mitochondria, has higher selectivity and sensitivity for detecting potassium ions, can qualitatively monitor the inflow or outflow of potassium ions in cells, and has simple synthesis method.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a potassium ion fluorescent probe (NK1), which has a structure shown in a formula (I);
in the formula (I), X is halogen.
In the potassium ion fluorescent probe NK1 provided by the invention,
(ACLE) as K+The identification unit, the BODIPY derivative as a fluorophore and the triphenylphosphine salt (TPP) as a mitochondrion targeting group are matched, so that the potassium ion fluorescent probe has high selectivity and sensitivity in the process of identifying potassium ions, can target the mitochondrion, and can qualitatively monitor the potassium ion inflow or outflow in cells.
In addition, the NK1 provided by the invention basically has no toxic or side effect on human cervical cancer cells (HeLa) and human normal liver cells (L02), the biocompatibility is high, and the ACLE synthesis method is simpler than TAC, so that the synthesis step of NK1 is simplified.
The recognition mechanism of the potassium ion fluorescent probe NK1 on potassium ions is shown in FIG. 1, before NK1 is not combined with potassium ions, electrons of chromophore molecules cannot return to a ground state and release fluorescence after being excited by light due to the electron donating effect on an N atom in NK1, and a Photoinduced Electron Transfer (PET) phenomenon occurs, so that fluorescence quenching is caused; when potassium ions are bound, the action of the PET with lone pair electrons is hindered, and fluorescence is restored.
Preferably, X is bromine, iodine or chlorine, preferably bromine.
The second object of the present invention is to provide a method for preparing the potassium ion fluorescent probe according to the first object, comprising the steps of (c): reacting a compound ACLE-CHO with a compound BODIPY-TPP to prepare the potassium ion fluorescent probe, wherein the reaction formula is as follows:
and X is halogen.
In the present invention, the compound BODIPY-TPP is prepared according to the literature (A highly selective mitochondria-targeting fluorescent K)+sensor[J]Angew Chem Int Ed,2015,54, 12053-12057).
Preferably, in step (c), the reaction is carried out in the presence of a catalyst, which is piperidine.
Preferably, in step (c), the reaction is carried out under reflux.
Preferably, in step (c), the solvent of the reaction comprises ethanol and/or tert-butanol.
Preferably, in step (c), the reaction time is 20-25h, such as 21h, 21.5h, 22h, 23h, 23.5h, 24h, 24.5h, etc.
Preferably, step (b) is further included before step (c): the compound ACLE-CHO is prepared by reacting the compound ACLE with phosphorus oxychloride, and the reaction formula is as follows:
preferably, in step (b), the reaction time is 2-4 h.
Preferably, in step (b), the solvent of the reaction comprises N, N-dimethylformamide and/or dichloromethane.
Preferably, step (a) is performed before step (b): reacting compound 8 with compound 7 to give compound ACLE, of the formula:
preferably, in step (a), the compound ACLE is obtained by precipitating the reaction product of compound 8 and compound 7 with a precipitating agent.
In a preferable scheme, the compound ACLE is obtained by adopting a precipitant precipitation method, so that the operation is simple, and the yield is up to more than 60%.
Preferably, in step (a), the precipitating agent comprises sodium perchlorate monohydrate and/or sodium perchlorate.
Preferably, in step (a), the solvent of the reaction comprises tetrahydrofuran and/or acetonitrile.
Preferably, in step (a), the reaction is carried out under reflux.
Preferably, in step (a), the reaction time is 20-25h, such as 21h, 21.5h, 22h, 23h, 23.5h, 24h, 24.5h, etc.
Preferably, the preparation method of the compound 8 comprises the following steps:
(1) reacting compound 1 with
The reaction gives compound 2, which has the formula:
(2) reacting compound 2 with NH2NH2·H2Reaction of O gives compound 3, which has the following reaction formula:
(3) reacting compound 3 with 2-bromoethanol to give compound 8, having the formula:
preferably, in step (1), the reaction is carried out in the presence of a catalyst comprising potassium iodide and/or sodium iodide.
Preferably, in step (1), the reaction is carried out in the presence of an acid scavenger comprising potassium carbonate and/or triethylamine.
Preferably, in step (1), the solvent of the reaction comprises acetonitrile and/or N, N-dimethylformamide.
Preferably, in step (1), the reaction time is 20-25h, such as 21h, 21.5h, 22h, 23h, 23.5h, 24h, 24.5h, and the like.
Preferably, in step (2), the reaction is carried out in the presence of a catalyst comprising Pd/C.
Preferably, in step (2), the solvent of the reaction comprises ethanol and/or methanol.
Preferably, in step (2), the reaction time is 2-3h, such as 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, etc.
Preferably, in step (3), the solvent for the reaction comprises water and/or 1, 4-dioxane.
Preferably, in step (3), the solvent for the reaction comprises water and 1, 4-dioxane, and the volume ratio of the water to the 1, 4-dioxane is 1-1.5:1, such as 1.1:1, 1.2:1, 1.3:1, 1.4:1, and the like.
Preferably, in step (3), the reaction is carried out in the presence of a catalyst comprising potassium iodide and/or sodium iodide.
Preferably, in step (3), the reaction is carried out in the presence of an acid scavenger comprising potassium carbonate and/or calcium carbonate.
Preferably, in step (3), the reaction is carried out for a period of 5 to 7 days, e.g., 5.2 days, 5.3 days, 5.5 days, 5.8 days, 6 days, 6.3 days, 6.8 days, etc.
Preferably, the preparation method of the compound 7 comprises the following steps:
(1') reacting tetraethylene glycol with p-toluenesulfonyl chloride to obtain compound 7, which has the following reaction formula:
preferably, in step (1'), the solvent of the reaction comprises dichloromethane and/or acetone.
Preferably, in step (1'), the reaction is carried out in the presence of an acid scavenger comprising sodium hydroxide and/or potassium hydroxide.
Preferably, in step (1'), the reaction time is 3 to 5h, such as 3.2h, 3.4h, 3.6h, 3.8h, 4h, 4.2h, 4.4h, 4.6h, 4.8h, and the like.
The invention also aims to provide the application of the potassium ion fluorescent probe in potassium ion detection.
Compared with the prior art, the invention has the following beneficial effects:
in the potassium ion fluorescent probe NK1 provided by the invention, ACLE is used as K+The potassium ion fluorescent probe has high selectivity and sensitivity in the process of identifying potassium ions, can target mitochondria and can qualitatively monitor the inflow or outflow of potassium ions in cells.
In addition, the NK1 provided by the invention basically has no toxic or side effect on human cervical cancer cells (HeLa) and human normal liver cells (L02), has high biocompatibility and is simple in synthesis method.
Drawings
FIG. 1 is a diagram of the recognition mechanism of potassium ions by the potassium ion fluorescent probe provided by the present invention.
FIG. 2 is a graph showing the UV-VIS absorption spectrum of NK1 according to K in test example 1 of the present invention+Graph of the change in concentration.
FIG. 3a shows the fluorescence emission spectrum of NK1 with K in test example 2 of the present invention+Graph of the change in concentration.
FIG. 3b is a graph showing the fluorescence intensity at 572nm of NK1 with K in test example 2 of the present invention+Graph of the change in concentration.
FIG. 4a is a graph showing the change in fluorescence intensity of NK1 with other physiologically relevant ions in test example 3 of the present invention.
FIG. 4b is a graph showing the change in fluorescence intensity at 572nm of NK1 with different ion concentrations in test example 3 of the present invention.
FIG. 5 is a graph showing the effect of NK1 treatment for 4h, 8h and 16h on HeLa cell proliferation activity in test example 4 of the present invention.
FIG. 6a is a combined graph of NK1, Mito-tracker Green and bright field in test example 4 of the present invention.
FIG. 6b is a distribution diagram of the fluorescence intensity area of the area indicated by the straight line (inside the oval frame) in FIG. 6a according to the present invention.
FIG. 7a is L02 intracellular K induced by NK1 on niger in test example 5 of the present invention+Monitoring of inflow or outflow.
FIG. 7b is L02 intracellular K induced by NK1 on ionomycin in test example 5 of the present invention+Monitoring of inflow or outflow.
FIG. 7c is HeLa intracellular K induced by NK1 on nigericin in test example 5 of the present invention+Monitoring of inflow or outflow.
FIG. 7d is the induction of HeLa intracellular K by NK1 on ionomycin in test example 5 of the present invention+Monitoring of inflow or outflow.
Detailed Description
For the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
This example provides a process for the preparation of compound ACLE, as follows:
(1) synthesis of Compound 7
Tetraethylene glycol (9.7g,50mmol), and potassium hydroxide (5.6g,100mmol) were dissolved in 100mL of dichloromethane, cooled to 0 ℃ in an ice bath, and p-toluenesulfonyl chloride (38.0g,200mM) was added dropwise under nitrogen. After dropping, the reaction was continued for 4 hours. After the reaction was completed, the reaction mixture was washed with saturated NaCl 3 times, extracted with dichloromethane, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and separated by silica gel column chromatography (PE: EA ═ 1:1) to obtain 17.5g of a colorless transparent liquid, with a yield of 70%.
1H NMR(400MHz,CDCl3)δ7.76(d，J＝8.2Hz，4H)，7.32(d，J＝8.1Hz，4H)，4.15–4.10(m，4H)，3.67–3.62(m，4H)，3.53(m，8H)，2.41(s，6H)。
13C NMR(101MHz，CDCl3)δ144.87(s)，132.89(s)，129.86(s)，127.92(s)，77.52(s)，77.20(s)，76.88(s)，70.57(d，J＝16.7Hz)，69.34(s)，68.62(s)，21.61(s)。
(2) Synthesis of Compound 8
2-Nitrophenol (11.2g,80.0mmol), 1-bromo-2-methoxyethane (16.4g, 120mmol), potassium iodide (6.72g, 40.0mmol) and potassium carbonate (12.0g, 88.0mmol) were dissolved in a 500mL round-bottom flask, dissolved in 200mL and heated to 90 deg.C for reflux overnight. TLC was used to monitor the completion of the reaction, and after completion of the reaction, the solvent was distilled off under reduced pressure. The residue was dissolved in 100mL CH2Cl2In 3 times saturated NaCl (3X 100mL), the aqueous phase was washed with CH2Cl2Two times (2X 100 mL). The organic phases were combined, anhydrous MgSO4Drying, suction filtration, concentration and silica gel column chromatography (PE: EA ═ 2:1) gave 15.73g of yellow solid (compound 2) in 94% yield.
1H NMR(400MHz，Chloroform-d)δ7.84(dd，J＝8.1，1.5Hz，1H)，7.56–7.49(m，1H)，7.13(d，J＝8.4Hz，1H)，7.05(t，J＝7.8Hz，1H)，4.29–4.24(m，2H)，3.84–3.78(m，2H)，3.47(s，3H)。
Compound 2(12.8g, 62.9mmol) and 10% Pd/C (1.3g) were dissolved in 100mL of anhydrous EtOH and cooled below 0 ℃ in an ice bath. Then slowly dropping N by using a constant-pressure dropping funnel2H4·H2O (10 mL). After the addition was complete, the ice bath was removed and the temperature was slowly raised to reflux. After 2h the reaction was complete, cooled to room temperature, filtered, the filtrate was distilled under reduced pressure to remove the solvent and the residue was dissolved in 100mL CH2Cl2In 3 times saturated NaCl (3X 100mL), the aqueous phase was washed with CH2Cl2Two times (2X 100 mL). The organic phases were combined, anhydrous MgSO4Drying, filtering, concentrating and directly using for the next reaction without purification. 10g of a pale yellow liquid (Compound 3) was obtained in 94% yield.
1H NMR(400MHz，Chloroform-d)δ6.86–6.81(m，2H)，6.77–6.70(m，2H)，4.19–4.14(m，2H)，3.80–3.76(m，2H)，3.53(s，2H)，3.47(s，3H)。
Compound 3(11.0g, 65.8mmol), 2-bromoethanol and CaCO3(13.16g, 131.6mmol) was dissolved in 200mL of a mixed solution of water and 1, 4-dioxane (1:1), and the reaction was refluxed for 6 days. After completion of the reaction, calcium carbonate was removed by filtration, most of the solvent was removed by distillation under the reduced pressure, washed with saturated NaCl for 3 times, extracted with dichloromethane, the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and separated by silica gel column chromatography (PE: EA ═ 1:2) to obtain 12.0g of a dark red liquid with a yield of 71.4%.
1H NMR(400MHz，CDCl3)δ7.14(dd，J＝7.8，1.6Hz，1H)，7.03(td，J＝7.8，1.6Hz，1H)，6.93–6.82(m，2H)，4.09–4.02(m，2H)，3.72–3.67(m，2H)，3.46–3.42(m，4H)，3.37(s，3H)，3.15–3.09(m，4H)。
(3) Synthesis of Compound ACLE
100mL of anhydrous THF and NaH (60% paraffin oil suspension, 2.5g) were added to a 500mL two-necked flask, evacuated and reloaded with nitrogen, and after 1 hour of reflux, a THF solution (100mL) of Compounds 8 and (8.9g, 35.0mmol) and Compound 7(17.6g, 35.0mmol) was slowly added to the solution over 3 hours and added dropwise. Reflux was continued for 24 hours. After cooling, filtration and washing with THF, the filtrate was concentrated under reduced pressure. The residue was then dissolved in 5mL of methanol. To this solution was added 15mL of sodium perchlorate monohydrate (4.9g, 35.0mmol) dissolved in methanol. The mixture was heated under reflux for 1 hour, the solvent was evaporated, and the residue was recrystallized from ethyl acetate. Repeated crystallization was repeated 2 times to obtain ACLE-sodium perchlorate complex as a white solid. The solid was then dissolved in a mixture of dichloromethane and water (1:1) and stirred overnight. Then the mixture is treated with CH2Cl2Diluted (50 mL. times.3) and washed with saturated NaCl (50 mL. times.3). The organic layers were combined and dried over anhydrous MgSO4After drying, filtration and concentration, 9.0g of a brown oil was obtained in 62.4% yield and used in the next reaction without further purification.
1H NMR(400MHz，Chloroform-d)δ7.07–6.99(m，1H)，6.89–6.77(m，3H)，4.11–4.04(m，2H)，3.72–3.53(m，22H)，3.42(t，J＝5.9Hz，4H)，3.38(s，3H)。
13C NMR(101MHz，Chloroform-d)δ152.16，140.09，121.28，113.73，71.10，70.73，70.60，70.58，70.31，69.98，67.54，58.96，52.70。
Example 2
The embodiment provides a preparation method of a potassium ion fluorescent probe NK1, which comprises the following specific steps:
(1) synthesis of Compound ACLE-CHO
ACLE (4.49g, 12mmol) was dissolved in 30mL DMF, cooled to-20 ℃ and POCl was added slowly dropwise3(18.5g, 120mmol) and stirred at room temperature for 30min after dropping. Then heated to 70 ℃ for reaction for 1 h. The reaction mixture was added dropwise to 250g of ice-water mixture, extracted 3 times with dichloromethane, the organic phases were combined, dried, concentrated and chromatographed on silica gel to give 2.87g of a pale yellow viscous liquid in 54% yield.
1H NMR(CDCl3，300MHz):d＝9.69(s，1H)，7.30(dd，1H，J＝8.3Hz，1.8Hz)，7.26(d，1H，J＝1.8Hz)，6.93(d，1H，J＝8.3Hz)，4.11–4.08(m，2H)，3.71–3.55(m，26H)，3.36ppm(s，3H)；13C NMR(CDCl3，75MHz):d＝190.14，149.69，146.08，128.22，126.73，116.46，111.01，70.70，70.61，70.55，70.48，70.42，69.95，67.46，58.65，52.61。
(2) Synthesis of Potassium ion fluorescent Probe NK1
ACLE-CHO (180mg, 0.396mmol), BODIPY-TPP (333mg, 0.436mmol) and 1 drop piperidine were dissolved in 10mL absolute ethanol and refluxed for 24 h. Spin-drying ethanol, extracting with dichloromethane, washing with saturated saline solution for three times, mixing organic phases, drying with anhydrous magnesium sulfate, vacuum-filtering, spin-drying the filtrate by reduced pressure distillation, and separating by alkaline alumina column chromatography with DCM as the fluidity: MeOH-50: 1, developing solvent DCM: MeOH 25:2 gave 76.0mg of a dark blue solid in 18.6% yield.
1H NMR(400MHz，Chloroform-d)δ7.93–7.71(m，15H)，7.50(d，J＝16.2Hz，1H)，7.21–7.07(m，5H)，6.98(t，J＝8.2Hz，3H)，6.59(s，1H)，5.99(s，1H)，4.23–4.17(t，2H)，3.99(s，2H)，3.73–3.59(m，26H)，3.46(s，3H)，3.43(s，2H)，2.59(s，3H)，1.75–1.68(m，7H)，1.57–1.52(m，2H)，1.48(s，3H)，1.44(s，3H)。
13C NMR(101MHz，Chloroform-d)δ163.14，159.59，142.70，134.93，134.90，133.79，133.69，130.51，130.39，129.42，127.03，122.07，119.03，118.17，117.52，115.00，88.03，77.26，71.18，71.06，70.69，70.55，70.26，70.14，69.53，67.90，67.77，59.01，58.94，56.24，52.64，47.15，30.20，30.04，29.70，28.95，26.46，25.75，25.33，23.58，22.69，22.17，14.92，14.59，14.12。
HR-MS(ESI+)C65H78O8N3BF2P+Calculating the value: 1108.55822 theoretical value 1108.55762,. DELTA. 0.54030 ppm.
Test example 1
Testing the change of the ultraviolet-visible absorption spectrum of NK 1:
NK1 solid was dissolved in DMSO to make up a stock solution (2.5mM), 6 μ L of NK1 stock solution was added to 150 μ L of cetyltrimethylammonium bromide (CTAB) aqueous solution (10mM), and 2844 μ L of HEPES buffer (pH 7.4, 5mM) was added to give a final NK1 concentration of 5 μ M and a final CTAB concentration of 0.5 mM. The mixed solution was added to a cuvette (1cm), and the ultraviolet-visible absorption spectrum was measured in the range of potassium ion concentration from 0 to 1000mM (FIG. 2).
As can be seen from FIG. 2, when no potassium ion is added, the maximum absorption wavelength of NK1 is 580nm, and in a certain range of not less than 580nm, the absorbance decreases with the increase of potassium ion concentration (for example, the position indicated by the arrow on the right side of FIG. 2), and as the potassium ion concentration increases, the maximum absorption wavelength blue shifts to about 570nm, and there is an isoabsorption point at 575nm, and between 570nm and the isoabsorption point, the absorbance increases with the increase of potassium ion concentration (for example, the position indicated by the arrow on the left side of FIG. 2). It is shown that the electron donating ability of the benzazepine-18-crown ether of NK1 is weakened and the PET effect is weakened after the benzazepine-18-crown ether is combined with potassium ions, so that the ultraviolet visible absorption has blue shift.
Test example 2
Test K+Effect of concentration on fluorescence of NK 1:
mu.L of NK1 stock solution was added to 150. mu.L of CTAB aqueous solution (10mM), and 2844. mu.L of HEPES buffer (pH 7.4, 5mM) was added to give a final concentration of NK1 of 5. mu.M and CTAB of 0.5 mM. The mixed solution was added to a fluorescence cuvette (1cm), and the fluorescence emission spectrum was measured at a potassium ion concentration in the range of 0 to 1000mM, with an excitation wavelength of 540nm and an Ex/Em slit width of 5/5 nm.
The fluorescence emission spectrum of NK1 with K shown in FIG. 3a is obtained by testing+Graph of concentration change, and fluorescence intensity at 572nm of NK1 with K shown in FIG. 3b+A graph of the change in concentration; as can be seen from FIG. 3a, NK1 has two emission peaks at 620nm and 572nm, wherein the emission maximum is at 620nm, the fluorescence intensity of NK1 increases gradually with the increasing concentration of potassium ions, and the wavelength of the maximum fluorescence emission shifts from blue to 572 nm. As can be seen from FIG. 3b, the change in fluorescence intensity at 572nm is significant, and the maximum kinetic Range F/F0≈160。F0Is not bound with K+Fluorescence intensity at the previous 572nm, F is the binding corresponding concentration K+Followed by fluorescence intensity at 572 nm.
Test example 3
Potassium ion selectivity study of NK 1:
(1) add appropriate stock solution to HEPES buffer (10mM, pH 7.4) to give a final concentration of 5. mu.M NK1, and a working solution volume of 3 mL. The working solution is filled in a quartz dish with 1cm light transmission on four sides, and Na of different metal ions is measured+(15mM)，Mg2+(2mM)，Ca2+(2mM)，Zn2+(2mM)，Mn2+(50μM)，Cu2+(50μM)，Fe2+(50μM)，Fe3+(50. mu.M) effect on NK1 fluorescence intensity to test the selectivity and specificity of sensor NK 1.
The fluorescence intensity of NK1 was tested as a function of other physiologically relevant ions as shown in FIG. 4 a. As shown in FIG. 4a, Na is added+、Mg2+、Ca2+、Zn2+、Mn2+、Cu2+、Fe2+And Fe3+The fluorescence intensity of the post-NK 1 at 572nm substantially agreed with that before addition of no ion, 5mM K was added+Then the fluorescence amplification is increased by about 2 times, and K of 150mM is added+This can result in an approximately 60-fold increase in fluorescence. Illustrating NK1 vs. Na at physiological concentrations+、Mg2+、Ca2+、Zn2+、Mn2+、Cu2+、Fe2+And Fe3+Basically insensitive and has large specificity to potassium ions.
(2) To continue to investigate the selectivity and specificity of potassium ions, we also focused on Li of the same main group as potassium ions and with similar ionic radius+，Na+，Rb+And Cs+Curve of change in NK1 fluorescence. The fluorescence intensity at 572nm of NK1 was plotted against the ion concentration for each sample as shown in FIG. 4 b.
As shown in FIG. 4b, where Li+And Cs+Curve of (d) and Na+Higher degrees of overlap lead to no resolution, in particular, when the concentrations are all 150mM, K is added+F/F of0Value is addition of Li+29 times of that of Na+23 times of that of Rb is added+11 times of that of the total amount of the additive Cs+24-fold higher, indicating that NK1 has high potassium specificity.
Test example 4
Cytotoxicity and cell distribution testing of NK 1:
(1) the MTT colorimetric assay was used to determine the potential cytotoxicity of NK1 on living cells as follows:
human cervical cancer cells (HeLa cells) were cultured in DMEM medium containing 10% heat-inactivated Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin at 37 ℃ and 5% CO2Culturing in an incubator. HeLa cells (1X 10)4Individual cells/well) were seeded in 96-well plates at 37 ℃. After 24 hours of incubation, cells were treated with different doses of NK1 for 4 hours, 8 hours and 16 hours, respectively.Cells treated with fresh medium served as negative control group. The medium was removed from each well, washed 3 times with PBS, and then 10. mu.L of MTT solution and 100. mu.L of medium were added. After further incubation for 4 hours, the formazan was dissolved in DMSO solution (150. mu.L) and absorbance intensities at 490nm and 510nm were recorded using a microplate reader (BioTek Synergy H4, USA). All experiments were performed in triplicate and relative cell viability (%) was expressed as a percentage relative to untreated control cells.
The effect profile on HeLa cell proliferation activity after 4h, 8h and 16h of NK1 treatment as shown in figure 5 was tested. As can be seen from FIG. 5, after treatment for 4h, 8h, and 16h with the working concentration of NK1 (2 μ M), the cell survival rates of HeLa cells were all above 90%, so NK1 had no significant toxic side effects on HeLa cells.
(2) The subcellular organelle co-localization experiment carried out by using the single photon laser confocal microscope verifies the mitochondrial targeting property of the NK1, which comprises the following steps:
HeLa cells (20,000 cells/well) were seeded on a glass-bottomed petri dish dedicated to a single photon laser confocal microscope (TCS-SP8, Leica, Germany), and after 24h of culture, the medium was removed and replaced with fresh medium containing 2. mu.M NK 1. After 10min incubation, the medium was removed and replaced with fresh medium containing 100nM mitochondrial Green fluorescent Probe (Mito-Tracker Green, Thermo Fisher Scientific, USA). After incubation for 10min, removing the culture medium, washing for 3 times by Phosphate Buffered Saline (PBS), and finally adding 1mL of PBS into a culture dish, and observing under a laser confocal microscope, wherein the excitation wavelength of NK1 is 554nm, and the emission wavelength interval is 560-610 nm; the excitation wavelength of the Mito-Tracker Green is 488nm, and the emission wavelength interval is 490-540 nm.
The test results are shown in fig. 6a and 6b, where fig. 6a is a combined graph of NK1, Mito-tracker Green and bright field. FIG. 6b is a distribution diagram of the fluorescence intensity area of the area indicated by the straight line (inside the oval frame) in FIG. 6 a. The Pearson fitting coefficient of red fluorescence and Green fluorescence calculated by software Image Pro is 0.9, the Mander fitting coefficient is 0.99, and the fluorescence fitting coefficient of NK1 and Mito-tracker Green is high, so that the NK1 can be proved to have mitochondrial targeting.
Test example 5
NK1 for detecting dynamic change of potassium ions in mitochondria of cells
Verification of NK1 with a fluorescent microplate reader (BioTek Synergy H4, USA) qualitatively monitored K induced by nigericin (20. mu.M) or ionomycin (10. mu.M)+The internal flow or the external flow is specifically as follows:
inoculating HeLa cells (6000 cells/well) or L02 cells (12,000 cells/well) into a 96-well plate, culturing for 24h, adding 100 μ L of fresh medium containing 2 μ M NK1, culturing for 30min, washing with PBS for 3 times, and adding blanks containing a) respectively; b)200mM K+；c)nigericin；d)nigericin+200mM K+；e)ionmycin；f)ionmycin+200mM K+The culture medium is placed on a pre-preheated enzyme-labeling instrument for detection, the excitation wavelength is 540nm, the detection emission wavelength is 572nm, the detection is carried out once every 30s, and the detection lasts for 40 min.
The results are shown in FIGS. 7a-7d, and FIG. 7a shows that NK1 induces NIGERicin-induced L02 intracellular K+FIG. 7b is a graph of NK1 versus ionomycin induced L02 intracellular K+FIG. 7c is a graph of monitoring of influx or efflux, and NK1 vs. nigericin-induced HeLa intracellular K+FIG. 7d is a graph of NK1 versus ionomycin induced HeLa intracellular K+Monitoring of inflow or outflow, wherein0Is not bound with K+Fluorescence intensity at the previous 572nm, I is the binding corresponding concentration K+The experiment was repeated three times for each set of experiments at 572nm after which the individual experimental data consisted of mean ± variance.
As can be seen in FIGS. 7a-7d, the fluorescence intensity of the blank was substantially unchanged with 200mM K added+Can cause the fluorescence of NK1 to be enhanced. Either nigericin or ionmycin caused a decrease in fluorescence of NK1, indicating intracellular potassium efflux, whereas both nigericin and ionmycin caused intracellular potassium efflux. When 200mM K was added simultaneously+And nigericin or ionomycin, the fluorescence of NK1 increased and then decreased, indicating that potassium ions in the cell mitochondria flowed in and out first. Furthermore, we have found that L02 cells are more sensitive to potassium ion concentration than HeLa cells. NK1 can qualitatively monitor induction of intracellular status by either nigericin or ionomycinK+The result of the internal flow or the external flow can be further used for screening out the medicaments related to the potassium ion channel in high flux, and has great promotion effect on the research and development of new medicaments.
And (4) analyzing results:
NK1 is a mitochondrial targeting potassium ion fluorescence sensor synthesized by using ACLE as a potassium ion specific ligand, BODIPY as a chromophore and triphenylphosphine salt (TPP) as a mitochondrial targeting group. Through the performance test, the following conclusion is obtained:
(1) NK1 has a more sensitive response to potassium ion (30-400mM) and a wider kinetic range (F/F)0160) with higher brightness (QY of 5.5% in the presence of 150mM potassium ion) and physiological pH (5.5-9.0) versus K of NK1+The sensing is not influenced;
(2) NK1 not only for Na+(15mM)，Mg2+(2mM)，Ca2+(2mM)，Zn2+(2mM)，Mn2+(50μM)，Cu2+(50μM)，Fe2+(50μM)，Fe3+(50. mu.M) and the like, and also has little difference in ionic radius from rubidium (Rb) which is the same as the main group+) And cesium (Cs)+) Is also substantially insensitive;
(3) the laser confocal microscope is used for carrying out cell subcellular organelle co-localization experiments, so that the NK1 can be specifically enriched into mitochondria of the HeLa cells, and the material has good targeting property on the mitochondria;
(4) MTT screening shows that NK1 basically has no toxic or side effect on human cervical cancer cells (HeLa) and has high biocompatibility;
(5) NK1 also qualitatively monitored the influx and efflux of cellular potassium ions induced by nigericin and ionomycin. The enzyme-labeling instrument is used for the first time to realize the high-flux rapid monitoring of the potassium ion flow.
The applicant states that the present invention is illustrated by the above examples to show the detailed process equipment and process flow of the present invention, but the present invention is not limited to the above detailed process equipment and process flow, i.e. it does not mean that the present invention must rely on the above detailed process equipment and process flow to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (34)
1. A potassium ion fluorescent probe is characterized by having a structure shown in a formula (I);
in the formula (I), X is bromine, iodine or chlorine.
2. The potassium ion fluorescent probe of claim 1, wherein X is bromine.
3. A method for preparing a potassium ion fluorescent probe according to claim 1 or 2, comprising the step (c): reacting a compound ACLE-CHO with a compound BODIPY-TPP to prepare the potassium ion fluorescent probe, wherein the reaction formula is as follows:
and X is bromine, iodine or chlorine.
4. The method according to claim 3, wherein in the step (c), the reaction is carried out in the presence of a catalyst, and the catalyst is piperidine.
5. The method according to claim 3, wherein in the step (c), the reaction is carried out under reflux.
6. The method according to claim 3, wherein in the step (c), the solvent for the reaction comprises ethanol and/or t-butanol.
7. The method according to claim 3, wherein the reaction time in the step (c) is 20 to 25 hours.
8. The method of claim 3, further comprising, before step (c), step (b): the compound ACLE-CHO is prepared by reacting the compound ACLE with phosphorus oxychloride, and the reaction formula is as follows:
9. the method according to claim 8, wherein the reaction time in the step (b) is 2 to 4 hours.
10. The method according to claim 8, wherein in the step (b), the solvent for the reaction comprises N, N-dimethylformamide and/or dichloromethane.
11. The method of claim 8, wherein step (a) is performed before step (b): reacting compound 8 with compound 7 to give compound ACLE, of the formula:
12. the method according to claim 11, wherein in the step (a), the compound ACLE is obtained by precipitating the reaction product of the compound 8 and the compound 7 with a precipitant.
13. The method according to claim 11, wherein in step (a), the precipitating agent comprises sodium perchlorate monohydrate and/or sodium perchlorate.
14. The method according to claim 11, wherein in the step (a), the solvent for the reaction comprises tetrahydrofuran and/or acetonitrile.
15. The method according to claim 11, wherein the reaction is carried out under reflux in step (a).
16. The method according to claim 11, wherein the reaction time in the step (a) is 20 to 25 hours.
17. The method of claim 11, wherein the compound 8 is prepared by the steps of:
(1) reacting compound 1 with 2-bromoethyl methyl ether to give compound 2, having the formula:
(2) reacting compound 2 with NH2NH2·H2Reaction of O gives compound 3, which has the following reaction formula:
(3) reacting compound 3 with 2-bromoethanol to give compound 8, having the formula:
18. the production method according to claim 17, wherein in the step (1), the reaction is carried out in the presence of a catalyst comprising potassium iodide and/or sodium iodide.
19. The method according to claim 17, wherein in step (1), the reaction is carried out in the presence of an acid scavenger comprising potassium carbonate and/or triethylamine.
20. The method according to claim 17, wherein in the step (1), the solvent for the reaction comprises acetonitrile and/or N, N-dimethylformamide.
21. The method according to claim 17, wherein the reaction time in step (1) is 20 to 25 hours.
22. The method according to claim 17, wherein in the step (2), the reaction is carried out in the presence of a catalyst comprising Pd/C.
23. The method according to claim 17, wherein in the step (2), the solvent for the reaction comprises ethanol and/or methanol.
24. The method according to claim 17, wherein the reaction time in the step (2) is 2 to 3 hours.
25. The method according to claim 17, wherein in the step (3), the solvent for the reaction comprises water and/or 1, 4-dioxane.
26. The method according to claim 17, wherein in the step (3), the solvent for the reaction comprises water and 1, 4-dioxane, and the volume ratio of the water to the 1, 4-dioxane is 1-1.5: 1.
27. The production method according to claim 17, wherein in the step (3), the reaction is carried out in the presence of a catalyst comprising potassium iodide and/or sodium iodide.
28. The method according to claim 17, wherein in the step (3), the reaction is carried out in the presence of an acid scavenger comprising potassium carbonate and/or calcium carbonate.
29. The method according to claim 17, wherein in the step (3), the reaction is carried out for 5 to 7 days.
30. The method of claim 11, wherein the compound 7 is prepared by the steps of:
(1') reacting tetraethylene glycol with p-toluenesulfonyl chloride to obtain compound 7, which has the following reaction formula:
31. the method according to claim 30, wherein in step (1'), the solvent for the reaction comprises dichloromethane and/or acetone.
32. The process of claim 30, wherein in step (1'), the reaction is carried out in the presence of an acid scavenger comprising sodium hydroxide and/or potassium hydroxide.
33. The method according to claim 30, wherein in the step (1'), the reaction time is 3 to 5 hours.
34. Use of the potassium ion fluorescent probe according to claim 1 or 2 in potassium ion detection.
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| A Fluorescent Sensor with High Selectivity and Sensitivity for Potassium in Water;Huarui He等;《J. AM. CHEM. SOC.》;20030212;1468-1469 * |
| A Highly K+-Selective Phenylaza-[18]crown-6-Lariat-Ether-Based Fluoroionophore and Its Application in the Sensing of K+ Ions with an Optical Sensor Film and in Cells;Sandra Ast等;《Chem. Eur. J.》;20130917;14911-14917 * |
| A Highly Selective Mitochondria-Targeting Fluorescent K+ Sensor;Xiangxing Kong等;《Angew. Chem. Int. Ed.》;20150821;12053-12057 * |
| Red- to NIR-Emitting， BODIPY-Based， K + -Selective Fluoroionophores and Sensing Materials;Bernhard J等;《Adv. Funct. Mater.》;20160914;7697-7707 * |
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