WO2021047212A1

WO2021047212A1 - Potassium ion fluorescent probe, preparation method and application thereof

Info

Publication number: WO2021047212A1
Application number: PCT/CN2020/093849
Authority: WO
Inventors: 田颜清; 宁爵伟
Original assignee: 南方科技大学
Priority date: 2019-09-09
Filing date: 2020-06-02
Publication date: 2021-03-18
Also published as: CN110437283B; CN110437283A

Abstract

The present application relates to a potassium ion fluorescent probe, a preparation method, and an application thereof. The potassium ion fluorescent probe has a structure represented by formula (I). The potassium ion fluorescent probe NK1 provided in the present application has short synthesis steps and a simple structure. Therein, ACLE is used as a K ⁺ recognition unit, a BODIPY derivative is used as a fluorophore, and TPP is used as the mitochondrial targeting group. Cooperation between these three enables the potassium ion fluorescent probe to have higher selectivity and sensitivity in the potassium ion identification process. The NK1 has few toxic side effects with respect to human cervical cancer cells (HeLa), high biocompatibility, and can also target mitochondria. Furthermore, the NK1 provided in the present application can qualitatively monitor the influx or outflow of potassium ions in cell mitochondria.

Description

Potassium ion fluorescent probe and preparation method and application thereof

Technical field

This application relates to the technical field of biological materials, in particular to a potassium ion fluorescent probe and a preparation method and application thereof, and in particular to a mitochondrial targeted potassium ion fluorescent probe and a preparation method and application thereof.

Background technique

Mitochondrial potassium (K ⁺ ) channels are a type of transporter located in the inner mitochondrial membrane. Mitochondrial K ⁺ channel-mediated K ⁺ influx between cytoplasm and mitochondria can regulate mitochondrial membrane potential, maintain mitochondrial volume stability, regulate the concentration of reactive oxygen species, and prevent Ca ²⁺ overload in the matrix. In addition, mitochondrial K ⁺ channels play an important role in cytoprotective mechanisms during cerebral hypoxia or myocardial infarction, and can also be used as effective targets for cancer treatment. Unfortunately, due to the lack of effective technologies, such as mitochondrial targeted fluorescent K ⁺ sensors, the molecular identification and localization of these transporters is still not completely clear. Therefore, in order to monitor ^{the relationship between mitochondrial transmembrane K +} flux and other biological parameters in biological pathways, there is an urgent need for the development and application of ^{mitochondrial-targeted fluorescent K + sensors.}

In 2003, He et al. reported a ^{potassium ion ligand TAC that is hardly affected by Na +} ions and ^{highly specific to K +} ions. It uses triaza cryptate TAC as the K ⁺ ion ligand and 4-aminonaphthalene A new type of potassium ion fluorescent sensor (A fluorescent sensor with high selectivity and sensitivity for potassium in water) synthesized with imide as a chromophore[J].Journal of the American Chemical Society,2003,125(6):1468-1469), The results show that the sensor still has a large fluorescence response to 2-10 mM K ^{+ in the} ^{presence of 160 mM Na +} ions, and can be used to detect the concentration of ^{extracellular K + ions in clinics.} Due to the excellent performance of the TAC ligand, some excellent sensors based on the ligand have been successfully developed. This is ^{a major breakthrough in the history of K +} ion sensor research. The potassium ion ligand TAC developed by it has been used until now and is considered the best potassium ion binding ligand. However, the synthesis is extremely complicated, the reaction conditions are harsh, and the overall yield is extremely low. And can not achieve mitochondrial targeting.

Therefore, the field urgently needs to develop a mitochondrial targeted K ⁺ sensor with simple synthesis, good selectivity and high sensitivity to monitor changes in the intracellular mitochondrial K ^{+ concentration.}

Summary of the invention

The present application provides a potassium ion fluorescent probe, and particularly provides a potassium ion fluorescent probe targeted by mitochondria. The potassium ion fluorescent probe can target mitochondria, has high selectivity and sensitivity for potassium ion detection, can qualitatively monitor the inflow or outflow of potassium ions in the cell, and has a simple synthesis method.

This application provides a potassium ion fluorescent probe (referred to herein as "NK1"), which has a structure represented by formula (I):

Wherein in formula (I), the X is halogen.

In the potassium ion fluorescent probe NK1 provided in this application,

(Referred to herein as "ACLE") as the K ⁺ recognition unit, fluoroboron dipyrrole (referred to herein as "BODIPY") derivative as the fluorophore and triphenylphosphine salt (referred to as "TPP" herein) as the mitochondria Targeting group, the three are coordinated, so that the potassium ion fluorescent probe not only has high selectivity and sensitivity in the process of recognizing potassium ions, but also can target mitochondria, and can qualitatively monitor the potassium ion influx in the cell Or outflow.

In addition, the NK1 provided in this application has basically no toxic side effects on human cervical cancer cells (HeLa) and human normal liver cells (L02), and has high biocompatibility. The synthesis method of ACLE is simpler than that of TAC, thus simplifying Synthesis steps of NK1.

The recognition mechanism of potassium ion fluorescent probe NK1 for potassium ions is shown in Figure 1. Before NK1 binds to potassium ions, the electrons of the chromophore molecules cannot return to them after being excited by light due to the electron donating effect on the N atom in NK1. The ground state releases fluorescence, and the phenomenon of photoinduced electron transfer (PET) occurs, which leads to fluorescence quenching; when potassium ions are combined, the PET effect of the lone pair of electrons is hindered, and the fluorescence is restored.

Preferably, the X is bromine, iodine or chlorine, preferably bromine.

This application also provides a method for preparing the potassium ion fluorescent probe of the present application. The preparation method includes step (c): reacting compound ACLE-CHO and compound BODIPY-TPP to prepare a potassium ion fluorescent probe. The reaction formula is as follows :

Wherein said X is halogen.

In this application, the compound BODIPY-TPP is prepared according to the method described in 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, and the catalyst is piperidine.

Preferably, in step (c), the reaction is carried out under reflux.

Preferably, in step (c), the reaction is carried out in a solvent, and the solvent includes 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 is reacted with phosphorus oxychloride to prepare the compound ACLE-CHO, and the reaction formula is as follows:

Preferably, in step (b), the reaction time is 2-4 hours.

Preferably, in step (b), the reaction is carried out in a solvent, and the solvent includes N,N-dimethylformamide and/or dichloromethane.

Preferably, step (a) is further included before step (b): reacting compound 8 with compound 7 to obtain compound ACLE, the reaction formula is as follows:

Preferably, in step (a), the reaction product of compound 8 and compound 7 is precipitated with a precipitation agent to obtain compound ACLE.

In a preferred embodiment, the present application adopts the precipitant precipitation method to obtain compound ACLE, which is not only simple in operation, but also has a yield of more than 60%.

Preferably, in step (a), the precipitating agent includes sodium perchlorate monohydrate and/or sodium perchlorate.

Preferably, in step (a), the reaction is carried out in a solvent, and the solvent includes 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 compound 8 includes the following steps:

(1) Make compound 1 and

Compound 2 is obtained by the reaction, and the reaction formula is as follows:

(2) Reacting compound 2 with NH ₂ NH ₂ ·H ₂ O to obtain compound 3, the reaction formula is as follows:

(3) Compound 3 is reacted with 2-bromoethanol to obtain compound 8. The reaction formula is as follows:

Preferably, in step (1), the reaction is carried out in the presence of a catalyst, and the catalyst includes potassium iodide and/or sodium iodide.

Preferably, in step (1), the reaction is carried out in the presence of an acid binding agent, and the acid binding agent includes potassium carbonate and/or triethylamine.

Preferably, in step (1), the reaction is carried out in a solvent, and the solvent includes 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, etc.

Preferably, in step (2), the reaction is carried out in the presence of a catalyst, and the catalyst includes Pd/C.

Preferably, in step (2), the reaction is carried out in a solvent, and the solvent includes 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 reaction is carried out in a solvent, and the solvent includes water and/or 1,4-dioxane.

Preferably, in step (3), the reaction is carried out in a solvent, the solvent includes water and 1,4-dioxane, and the volume ratio of the water to 1,4-dioxane is 1. -1.5:1, such as 1.1:1, 1.2:1, 1.3:1, 1.4:1, etc.

Preferably, in step (3), the reaction is carried out in the presence of a catalyst, and the catalyst includes potassium iodide and/or sodium iodide.

Preferably, in step (3), the reaction is carried out in the presence of an acid binding agent, and the acid binding agent includes potassium carbonate and/or calcium carbonate.

Preferably, in step (3), the reaction time is 5-7 days, for example, 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 compound 7 includes the following steps:

(1') The compound 7 is obtained by reacting tetraethylene glycol with p-toluenesulfonyl chloride. The reaction formula is as follows:

Preferably, in step (1'), the reaction is carried out in a solvent, and the solvent includes dichloromethane and/or acetone.

Preferably, in step (1'), the reaction is carried out in the presence of an acid binding agent, and the acid binding agent includes sodium hydroxide and/or potassium hydroxide.

Preferably, in step (1'), the reaction time is 3-5h, such as 3.2h, 3.4h, 3.6h, 3.8h, 4h, 4.2h, 4.4h, 4.6h, 4.8h, etc.

The application also provides the application of the potassium ion fluorescent probe of the application in potassium ion detection.

Compared with the prior art, this application has the following beneficial effects:

In the potassium ion fluorescent probe NK1 provided in this application, ACLE is used as the K ⁺ recognition unit, BODIPY derivative is used as the fluorophore, and TPP is used as the mitochondrial targeting group, so that the potassium ion fluorescent probe is not only in the process of recognizing potassium ions With high selectivity and sensitivity, it can also target mitochondria, and can qualitatively monitor the inflow or outflow of potassium ions in the cell.

In addition, the NK1 provided in this application has basically no toxic and side effects on human cervical cancer cells (HeLa) and human normal hepatocytes (L02), has high biocompatibility, and has a simple synthesis method.

Description of the drawings

FIG. 1 is a diagram of the recognition mechanism of potassium ion by the potassium ion fluorescent probe provided in this application.

Fig. 2 is a graph showing the variation of the ultraviolet-visible absorption spectrum of NK1 with the K ^{+ concentration in Test Example 1 of the present application.}

Fig. 3a is a graph showing the variation of the fluorescence emission spectrum of NK1 with the K ^{+ concentration in Test Example 2 of the present application.}

Fig. 3b is a graph showing the variation of the fluorescence intensity of NK1 at 572 nm with the K ^{+ concentration in Test Example 2 of the present application.}

Fig. 4a is a graph showing the variation of the fluorescence intensity of NK1 with other physiologically relevant ions in Test Example 3 of the present application.

Fig. 4b is a graph showing the variation of the fluorescence intensity of NK1 at 572 nm with different ion concentrations in Test Example 3 of the present application.

Figure 5 is a graph showing the effect of NK1 treatment on the proliferation activity of HeLa cells in Test Example 4 of the present application after 4h, 8h and 16h.

Fig. 6a is a combined diagram of NK1, Mito-tracker Green, and bright field in Test Example 4 of the present application.

Fig. 6b is an area distribution diagram of the fluorescence intensity of the area indicated by the straight line (in the elliptical frame) in Fig. 6a of the present application.

Fig. 7a is a monitoring diagram ^{of NK1 on the influx or efflux of K +} in L02 cells induced by nigericin in Test Example 5 of the present application.

^{Fig. 7b is a monitoring diagram of NK1 on K+} influx or efflux in L02 cells induced by ionomycin in Test Example 5 of the present application.

^{Fig. 7c is a monitoring diagram of NK1 on Nigericin-induced K +} influx or efflux in HeLa cells in Test Example 5 of the present application.

^{Figure 7d is a monitoring diagram of NK1 on K +} influx or efflux in HeLa cells induced by ionomycin in Test Example 5 of the present application.

detailed description

In order to facilitate the understanding of this application, the following examples are listed in this application. It should be understood by those skilled in the art that the described embodiments are only to help understand the application, and should not be regarded as specific limitations to the application.

Example 1

This embodiment provides a preparation method of compound ACLE, which is specifically as follows:

(1) Synthesis of compound 7

Dissolve tetraethylene glycol (9.7g, 50mmol) and potassium hydroxide (5.6g, 100mmol) in 100mL of dichloromethane. After cooling to 0℃ in an ice bath, add p-toluenesulfonyl chloride dropwise under nitrogen protection (38.0g, 200mM). After dripping, continue to react for 4h. After the reaction is completed, washed with saturated NaCl 3 times, extracted with dichloromethane, combined the organic phases, dried over anhydrous magnesium sulfate, filtered, concentrated, and separated by silica gel column chromatography (PE:EA=1:1) to obtain a colorless transparent liquid 17.5 g, the yield is 70%.

¹ H NMR (400MHz, CDCl ₃ ) δ 7.76 (d, J = 8.2 Hz, 4H), 7.32 (d, J = 8.1 Hz, 4H), 4.15-4.10 (m, 4H), 3.67-3.62 (m, 4H), 3.53 (m, 8H), 2.41 (s, 6H).

¹³ C NMR (101MHz, CDCl ₃ ) 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=16.7 Hz), 69.34 (s), 68.62 (s), 21.61 (s).

(2) Synthesis of compound 8

Combine 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) Dissolve in a 500ml round bottom flask, dissolve in 200mL, and heat to 90°C to reflux and react overnight. TLC monitors whether the reaction is complete, and after the reaction is completed, the solvent is distilled off under reduced pressure. The residue was dissolved in 100 mL CH ₂ Cl ₂ , washed with saturated NaCl (3×100 mL) 3 times, and the aqueous phase was _{extracted twice with CH 2} Cl ₂ (2×100 mL). The organic phases were combined, dried with anhydrous MgSO ₄ , filtered with suction, concentrated, and separated by silica gel column chromatography (PE:EA=2:1) to obtain 15.73 g of a yellow solid (compound 2) with a yield of 94%.

¹ H NMR (400MHz, Chloroform-d) δ 7.84 (dd, J = 8.1, 1.5 Hz, 1H), 7.56-7.49 (m, 1H), 7.13 (d, J = 8.4 Hz, 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 (12.8 g, 62.9 mmol) and 10% Pd/C (1.3 g) were dissolved in 100 mL of anhydrous EtOH, and cooled in an ice bath to below 0°C. Then use a constant pressure dropping funnel to slowly drop N ₂ H ₄ ·H ₂ O (10 mL). After the addition is complete, remove the ice bath and slowly heat to reflux. The reaction was finished after 2h. After cooling to room temperature, it was filtered. The filtrate was distilled under reduced pressure to remove the solvent. The residue was dissolved in 100 mL CH ₂ Cl ₂ and washed with saturated NaCl (3×100 mL) for 3 times. The aqueous phase was then reused with CH ₂ Cl ₂ (2×100 mL) was extracted twice. The organic phases were combined, dried with anhydrous MgSO ₄ , filtered with suction, concentrated, and used directly in the next step without purification. 10 g of light yellow liquid (Compound 3) was obtained, and the yield was 94%.

¹ H NMR (400MHz, Chloroform-d) δ 6.86–6.81(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 (11.0 g, 65.8 mmol), 2-bromoethanol and CaCO ₃ (13.16 g, 131.6 mmol) were dissolved in 200 mL of water and 1,4-dioxane mixed solution (1:1), and the reaction was refluxed for 6 day. After the reaction, the calcium carbonate was removed by filtration, most of the solvent was removed by distillation under reduced pressure, 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=1:2) 12.0 g of dark red liquid was obtained, and the yield was 71.4%.

¹ H NMR (400MHz, CDCl ₃ ) δ 7.14 (dd, J = 7.8, 1.6 Hz, 1H), 7.03 (td, J = 7.8, 1.6 Hz, 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 double-necked flask, evacuated and reloaded with nitrogen, refluxed for 1 hour, and then compound 8 and (8.9g, 35.0mmol) and A THF solution (100 mL) of compound 7 (17.6 g, 35.0 mmol) was slowly added to the above solution, and the addition was completed after 3 hours. Continue to reflux for 24 hours. After cooling, it was filtered, washed with THF, and the filtrate was concentrated under reduced pressure. The residue was then dissolved in 5 mL methanol. To this solution was added 15 mL of sodium perchlorate monohydrate (4.9 g, 35.0 mmol) dissolved in methanol. The mixture was heated to reflux for 1 hour, the solvent was evaporated and the residue was recrystallized from ethyl acetate. Repeat the crystallization twice to obtain the 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. The mixture was diluted with CH ₂ Cl ₂ (50 mL×3) and washed with saturated NaCl (50 mL×3). The organic layers were combined, dried over anhydrous MgSO ₄ , filtered, and concentrated to obtain 9.0 g of a brown oil with a yield of 62.4%. It was directly used in the next step without further purification.

¹ H 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=5.9 Hz, 4H), 3.38 (s, 3H).

¹³ C NMR (101 MHz, Chloroform-d) δ 152.16, 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

This embodiment provides a method for preparing potassium ion fluorescent probe NK1, which is specifically as follows:

(1) Synthesis of compound ACLE-CHO

ACLE (4.49g, 12mmol) was dissolved in 30mL DMF, cooled to -20°C, and then POCl ₃ (18.5g, 120mmol) was slowly added dropwise, after dripping, stirred at room temperature for 30min. Then it was heated to 70°C and reacted for 1 hour. The reaction solution was added dropwise to 250 g of ice-water mixture, extracted with dichloromethane 3 times, the organic phases were combined, dried, concentrated, and separated by silica gel column chromatography to obtain 2.87 g of a light yellow viscous liquid with a yield of 54%.

¹ H NMR (CDCl ₃ , 300MHz): d = 9.69 (s, 1H), 7.30 (dd, 1H, J = 8.3 Hz, 1.8 Hz), 7.26 (d, 1H, J = 1.8 Hz), 6.93 (d, 1H, J=8.3Hz), 4.11-4.08 (m, 2H), 3.71-3.55 (m, 26H), 3.36ppm (s, 3H); ¹³ C NMR (CDCl ₃ , 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 of piperidine were dissolved in 10mL of absolute ethanol and refluxed for 24h. Rotate to dry the ethanol, extract with dichloromethane, wash with saturated brine three times, combine the organic phases, dry with anhydrous magnesium sulfate, and filter with suction. The filtrate is spin-dried by vacuum distillation and separated by basic alumina column chromatography. : MeOH=50:1, the developing solvent is DCM:MeOH=25:2, 76.0 mg of dark blue solid is obtained, and the yield is 18.6%.

¹ H NMR (400MHz, Chloroform-d) δ 7.93–7.71 (m, 15H), 7.50 (d, J = 16.2 Hz, 1H), 7.21–7.07 (m, 5H), 6.98 (t, J = 8.2 Hz) , 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).

¹³ C NMR (101MHz, Chloroform-d) δ 163.14, 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, 14.92, 14.92 14.12.

HR-MS (ESI ⁺ ) C ₆₅ H ₇₈ O ₈ N ₃ BF ₂ P ⁺ , calculated value: 1108.55822, theoretical value 1108.55762, Δ=0.54030 ppm.

Test case 1

Change test of NK1's UV-Vis absorption spectrum:

Dissolve NK1 solid in DMSO to prepare a stock solution (2.5mM), take 6μL of NK1 stock solution and add to 150μL of cetyltrimethylammonium bromide (CTAB) aqueous solution (10mM), then add 2844μL of HEPES buffer Solution (pH=7.4, 5mM) so that the final concentration of NK1 is 5μM and the final concentration of CTAB is 0.5mM. The mixed solution was added to a cuvette (1cm), and the ultraviolet-visible absorption spectrum of the potassium ion concentration in the range of 0-1000 mM was measured (Figure 2).

It can be seen from Figure 2 that when potassium ions are not added, the maximum absorption wavelength of NK1 is 580nm. Within a certain range ≥580nm, the absorbance decreases with the increase of potassium ion concentration (for example, the position indicated by the arrow on the right of Figure 2). As the potassium ion concentration increases, the maximum absorption wavelength blue shifts to about 570nm, and there is an isoabsorption point at 575nm. Between 570nm and the isoabsorption point, the absorbance increases as the potassium ion concentration increases (for example, the left arrow in Figure 2 Referred location). It shows that after NK1's benzodiazepine-18-crown ether is combined with potassium ions, the electron donating ability is weakened, and the PET effect is weakened, resulting in a blue shift in UV-visible absorption.

Test case 2

Test ^{the influence of K +} concentration on the fluorescence of NK1:

Take 6μL of NK1 stock solution and add it to 150μL of CTAB aqueous solution (10mM), and then add 2844μL of HEPES buffer (pH=7.4, 5mM), so that the final concentration of NK1 is 5μM and the final concentration of CTAB is 0.5mM. Add the mixed solution to a fluorescence cuvette (1cm), measure the fluorescence emission spectrum of the potassium ion concentration in the range of 0-1000mM, the excitation wavelength is 540nm, and the width of the Ex/Em slit is 5/5nm.

The test obtains the graph of the fluorescence emission spectrum of NK1 shown in Figure 3a as a function of K ⁺ concentration, and the graph of the fluorescence intensity of NK1 at 572 nm shown in Figure 3b as a function of K ⁺ concentration; it can be seen from Figure 3a that NK1 has The two emission peaks are at 620nm and 572nm, of which the maximum emission is at 620nm. As the potassium ion concentration increases, the fluorescence intensity of NK1 gradually increases, and the maximum fluorescence emission wavelength shifts from blue to 572nm. It can be seen from Figure 3b that the fluorescence intensity at _{572nm changes significantly, and the maximum dynamic range F/F 0} ≈160. F ₀ is the fluorescence intensity at 572 nm before the unbound K ⁺ , and F is the fluorescence intensity at 572 nm after the ^{corresponding concentration of K + is bound.}

Test case 3

Study on the potassium ion selectivity of NK1:

(1) Add an appropriate amount of stock solution to HEPES buffer solution (10mM, PH=7.4), so that the final concentration of NK1 is 5μM and the volume of the working solution is 3mL. Put the working solution in a 1cm transparent quartz dish, measure different metal ions Na ⁺ (15mM), Mg ²⁺ (2mM), Ca ²⁺ (2mM), Zn ²⁺ (2mM), Mn ²⁺ (50μM) ), Cu ²⁺ (50μM), Fe ²⁺ (50μM), Fe ³⁺ (50μM) on the fluorescence intensity of NK1 to test the selectivity and specificity of the sensor NK1.

The test obtains the graph of the change of the fluorescence intensity of NK1 with other physiologically relevant ions shown in Fig. 4a. As shown in Figure 4a, the fluorescence intensity of NK1 at 572nm after ^{adding Na +} , Mg ²⁺ , Ca ²⁺ , Zn ²⁺ , Mn ²⁺ , Cu ²⁺ , Fe ²⁺ and Fe ^{3+ is compared with that before adding ions} The fluorescence intensity is basically the same. The addition of 5mM K ⁺ can cause about a 2-fold increase in fluorescence, and the addition of 150 mM K ⁺ can cause about a 60-fold increase in fluorescence. ^{It shows that NK1 is basically insensitive to Na +} , Mg ²⁺ , Ca ²⁺ , Zn ²⁺ , Mn ²⁺ , Cu ²⁺ , Fe ²⁺ and Fe ^{3+ at} physiological concentrations, and has a high specificity for potassium ions.

(2) In order to continue to study the selectivity and specificity of potassium ions, we also focused on the changes of Li ⁺ , Na ⁺ , Rb ⁺ and Cs ⁺ with the same main group as potassium ions and similar ionic radius on the fluorescence of NK1. The fluorescence intensity of NK1 at 572 nm as shown in Fig. 4b is obtained as a function of different ion concentrations.

As shown in Figure 4b, the curve ^{of Li +} and Cs ⁺ ^{in the figure overlaps with Na +} to a high degree, which makes it impossible to distinguish. Specifically, when the concentration is 150 mM, the F/F ₀ ^{value of adding K +} is that of adding Li ⁺ 29 times, which is 23 times that of ^{Na +} , 11 times that of ^{Rb +} ^{, and 24 times that of Cs +} , indicating that NK1 has a high degree of potassium ion specificity.

Test case 4

Cytotoxicity and cell distribution test of NK1:

(1) Use MTT colorimetry to determine the potential cytotoxicity of NK1 to living cells, as follows:

Culture human cervical cancer cells (HeLa cells) in DMEM medium containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C and 5% CO ₂ in an incubator . Then HeLa cells (1×10 ⁴ cells/well) were seeded in a 96-well plate at 37°C. After 24 hours of incubation, the cells were treated with different doses of NK1 for 4 hours, 8 hours and 16 hours, respectively. Cells treated with fresh medium served as a negative control group. The medium of each well was removed, and after washing with PBS three times, 10 μL of MTT solution and 100 μL of medium were added. After incubating for another 4 hours, the obtained formazan body was dissolved in DMSO solution (150 μL), and the absorbance intensity at 490 nm and 510 nm was recorded with a microplate reader (BioTek Synergy H4, USA). All experiments were performed in triplicate, and the relative cell survival rate (%) was expressed as a percentage relative to untreated control cells.

The test obtained the graph of the effect of NK1 treatment on the proliferation activity of HeLa cells after 4h, 8h and 16h as shown in FIG. 5. It can be seen from Fig. 5 that after 4h, 8h, and 16h treatment with the working concentration of NK1 (2μM), the cell survival rate of HeLa cells is more than 90%, so NK1 has no obvious toxic and side effects on HeLa cells.

(2) Use single-photon laser confocal microscope to verify the mitochondrial targeting of NK1 in sub-organelle co-localization experiments, as follows:

Inoculate HeLa cells (20,000 cells/well) into a special glass-bottom culture dish for a single-photon laser confocal microscope (TCS-SP8, Leica, Germany). After culturing for 24 hours, remove the medium and replace it with a fresh culture containing 2μM NK1 Basic training. After incubating for 10 min, the medium was removed and replaced with a fresh medium containing 100 nM mitochondrial green fluorescent probe (Mito-Tracker Green, Thermo Fisher Scientific, USA). After incubating for 10 minutes, remove the medium, wash with phosphate buffered saline (PBS) 3 times, and finally add 1 mL of PBS to the petri dish. Observe under a laser confocal microscope. The excitation wavelength of NK1 is 554 nm, and the emission wavelength range is 560. -610nm range; Mito-Tracker Green's excitation wavelength is 488nm, and emission wavelength range is 490-540nm.

The test results are shown in Figures 6a and 6b, among which Figure 6a is the combined image of NK1, Mito-tracker Green and bright field. Fig. 6b is an area distribution diagram of the fluorescence intensity in the area indicated by the straight line (in the elliptical frame) in Fig. 6a. The Pearson fitting coefficient of red fluorescence and green fluorescence calculated by the 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, which proves that NK1 has mitochondrial targeting.

Test case 5

NK1 is used to detect the dynamic changes of potassium ion in cell mitochondria

Use a fluorescence microplate reader (BioTek Synergy H4, USA) to verify that NK1 can qualitatively monitor the K ⁺ influx or outflow induced by nigericin (20μM) or ionomycin (10μM), as follows:

Inoculate HeLa cells (6000 cells/well) or L02 cells (12,000 cells/well) into a 96-well plate. After culturing for 24 hours, add 100 μL of fresh medium containing 2 μM NK1 to culture. After incubating for 30 minutes, wash with PBS for 3 times, then Add the medium containing a) blank; b) 200mM K ⁺ ; c) nigericin; d) nigericin+200mM K ⁺ ; e) ionomycin; f) ionomycin+200mM K ⁺ and place it in the pre-heated microplate reader In the upper detection, the excitation wavelength is 540nm, and the detection emission wavelength is 572nm. The detection is performed every 30s, and the detection is continued for 40 minutes.

FIG flow monitoring or K ⁺ efflux inner results shown in L02 cells, as shown in FIG. 7a 7a-7d NK1 induced on nigericin, and Figure 7b is of the NK1 ionomycin induced in L02 cells or K monitored FIG outflow stream within ⁺ FIG 7c within NK1 of nigericin-induced HeLa cells K ⁺ monitored FIG flow or efflux, and Figure 7d is a monitor of FIG flow or outflow of K within NK1 for ionomycin-induced HeLa cells ⁺ in, where I ₀ is the unbound K ⁺ Fluorescence intensity at 572 nm before, I is the fluorescence intensity ^{at 572 nm after combining the corresponding concentration K +} , each group of experiments are repeated three times, and a single experimental data is composed of the mean ± variance.

It can be seen from Figures 7a-7d that the fluorescence intensity of the blank group is basically unchanged, adding 200mM K ⁺ can cause the fluorescence of NK1 to increase. Both nigericin or ionomycin can cause the fluorescence of NK1 to decrease, which indicates the outflow of potassium ions in the cell, and both nigericin and ionomycin can cause the outflow of potassium ions in the cell. When 200 mM K ⁺ and nigericin or ionomycin were added at the same time, the fluorescence of NK1 first increased and then decreased, indicating that potassium ions in the cell mitochondria first flowed in and then flowed out. And we also found that L02 cells are more sensitive to potassium ion concentration than HeLa cells. NK1 can qualitatively monitor the intracellular K ⁺ influx or efflux induced by nigericin or ionomycin. This result can be further used for high-throughput screening of potassium ion channel-related drugs, which will greatly promote the development of new drugs.

Result analysis:

NK1 is a mitochondrial targeted potassium ion fluorescent sensor synthesized with ACLE as the potassium ion specific ligand, fluoroboron dipyrrole (BODIPY) as the chromophore, and triphenylphosphonium salt (TPP) as the mitochondrial targeting group. After the above performance test, the following conclusions are obtained:

(1) NK1 has a more sensitive response to potassium ions (30-400mM), a large dynamic range (F/F ₀ ≈160), and high brightness (QY 5.5% in the presence of 150mM potassium ions), and Physiological pH (5.5-9.0) has no effect ^{on K + sensing of NK1;}

(2) NK1 is not only for Na ⁺ (15mM), Mg ²⁺ (2mM), Ca ²⁺ (2mM), Zn ²⁺ (2mM), Mn ²⁺ (50μM), Cu ²⁺ (50μM), Fe ²⁺ (50μM), Fe ³⁺ (50μM), etc. are basically non-responsive, and they are also basically insensitive to ^{rubidium (Rb +} ) and cesium (Cs ^{+) with the same main group and small difference in ion radius;}

(3) The colocalization experiment of cell subcellular organelles with laser confocal microscope showed that NK1 can be specifically enriched in the mitochondria of HeLa cells, and this material has a good targeting ability to mitochondria;

(4) MTT screening shows that NK1 has basically no toxic side effects on human cervical cancer cells (HeLa), and has high biocompatibility;

(5) NK1 can also qualitatively monitor the influx and outflow of potassium ions in cells induced by nigericin and ionomycin. For the first time, a microplate reader was used to realize high-throughput rapid monitoring of potassium ion flow.

The applicant declares that this application uses the above-mentioned embodiments to illustrate the detailed process equipment and process flow of this application, but this application is not limited to the above-mentioned detailed process equipment and process flow, which does not mean that this application must rely on the above-mentioned detailed process equipment and process flow. The process flow can be implemented. Those skilled in the art should understand that any improvement to this application, the equivalent replacement of each raw material of the product of this application, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of this application.

Claims

A potassium ion fluorescent probe, which has the structure shown in formula (I):

Wherein, in formula (I), the X is halogen.
The potassium ion fluorescent probe according to claim 1, wherein the X is bromine, iodine or chlorine.
The potassium ion fluorescent probe according to claim 2, wherein the X is bromine.
A method for preparing a potassium ion fluorescent probe according to any one of claims 1 to 3, which comprises step (c): reacting compound ACLE-CHO and compound BODIPY-TPP to prepare a potassium ion fluorescent probe, The reaction formula is as follows:

Wherein, the X is halogen.
The preparation method according to claim 4, wherein, in step (c), the reaction is carried out in the presence of a catalyst, and the catalyst is piperidine;

Preferably, in step (c), the reaction is carried out under reflux;

Preferably, in step (c), the reaction is carried out in a solvent, and the solvent includes ethanol and/or tert-butanol;

Preferably, in step (c), the reaction time is 20-25h.
The preparation method according to claim 4 or 5, which further comprises step (b) before step (c): reacting compound ACLE with phosphorus oxychloride to prepare compound ACLE-CHO, the reaction formula is as follows:
The preparation method according to claim 6, wherein in step (b), the reaction time is 2-4h;

Preferably, in step (b), the reaction is carried out in a solvent, and the solvent includes N,N-dimethylformamide and/or dichloromethane.
The preparation method according to any one of claims 4 to 7, wherein before step (b), it further comprises the step

(a): The compound ACLE is obtained by reacting compound 8 with compound 7, and the reaction formula is as follows:
The preparation method according to claim 8, wherein in step (a), the reaction product of compound 8 and compound 7 is precipitated with a precipitating agent to obtain compound ACLE;

Preferably, in step (a), the precipitating agent includes sodium perchlorate monohydrate and/or sodium perchlorate;

Preferably, in step (a), the reaction is carried out in a solvent, and the solvent includes 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.
The preparation method according to claim 8 or 9, wherein the preparation method of compound 8 comprises the following steps:

(1) Compound 1 is reacted with 2-bromoethyl methyl ether to obtain compound 2. The reaction formula is as follows:

(2) Reacting compound 2 with NH 2 NH 2 ·H 2 O to obtain compound 3, the reaction formula is as follows:

(3) Compound 3 is reacted with 2-bromoethanol to obtain compound 8. The reaction formula is as follows:
The preparation method according to claim 10, wherein, in step (1), the reaction is carried out in the presence of a catalyst, and the catalyst includes potassium iodide and/or sodium iodide;

Preferably, in step (1), the reaction is carried out in the presence of an acid binding agent, and the acid binding agent includes potassium carbonate and/or triethylamine;

Preferably, in step (1), the reaction is carried out in a solvent, and the solvent includes acetonitrile and/or N,N-dimethylformamide;

Preferably, in step (1), the reaction time is 20-25h.
The preparation method according to claim 10 or 11, wherein, in step (2), the reaction is carried out in the presence of a catalyst, and the catalyst includes Pd/C;

Preferably, in step (2), the reaction is carried out in a solvent, and the solvent includes ethanol and/or methanol;

Preferably, in step (2), the reaction time is 2-3 hours.
The preparation method according to any one of claims 10 to 12, wherein in step (3), the reaction is carried out in a solvent, and the solvent includes water and/or 1,4-dioxane;

Preferably, in step (3), the reaction is carried out in a solvent, the solvent includes water and 1,4-dioxane, and the volume ratio of the water to 1,4-dioxane is 1. -1.5:1;

Preferably, in step (3), the reaction is carried out in the presence of a catalyst, and the catalyst includes potassium iodide and/or sodium iodide;

Preferably, in step (3), the reaction is carried out in the presence of an acid binding agent, and the acid binding agent includes potassium carbonate and/or calcium carbonate;

Preferably, in step (3), the reaction time is 5-7 days.
The preparation method according to any one of claims 8 to 13, wherein the preparation method of compound 7 comprises the following steps:

(1') The compound 7 is obtained by reacting tetraethylene glycol with p-toluenesulfonyl chloride. The reaction formula is as follows:

Preferably, in step (1'), the reaction is carried out in a solvent, and the solvent includes dichloromethane and/or acetone;

Preferably, in step (1'), the reaction is carried out in the presence of an acid binding agent, and the acid binding agent includes sodium hydroxide and/or potassium hydroxide;

Preferably, in step (1'), the reaction time is 3-5h.
An application of the potassium ion fluorescent probe according to any one of claims 1 to 3 in potassium ion detection.