Spontaneous-flickering visible light activated fluorophore and preparation method and application thereof

Abstract

The invention discloses a spontaneous flickering visible light activated fluorophore as well as a preparation method and application thereof, and belongs to the field of spontaneous flickering optical switch molecular materials. The fluorophore is a visible light activation molecule based on a rhodamine dye mother nucleus; the preparation method comprises a substitution reaction and an oxidation reaction. The fluorophore molecules designed by the invention can be activated one by one to generate fluorescence in the process of using exciting light to irradiate, can release nitric oxide free radicals while being activated, and can be used for super-resolution imaging of cells and biological nitric oxide treatment.

Description

Technical Field

[0001] The present invention belongs to the field of self - blinking light - switch molecular materials, and particularly relates to a visible - light - activated fluorophore with self - blinking behavior, a preparation method thereof, and an application thereof. Background Art

[0002] Photo - activated fluorescent molecules have attracted much attention in biological detection and molecular imaging because of the significant increase in fluorescence intensity before and after activation. When a photocage group is attached to a fluorescent molecule, it shows a non - fluorescent state. After the photocage group is removed by using activation light, the fluorescence is restored, which is beneficial for imaging under specific conditions, in specific regions, and at specific times, and is widely used in super - resolution imaging based on single - molecule localization. Super - resolution imaging based on single - molecule localization requires the fluorescent molecule to switch back and forth between the bright state and the dark state. By reconstructing the signals at different positions on different time scales, a super - resolution image can be obtained, which can image and study the fine structures in the biological microenvironment. This puts forward requirements for the structure of the fluorescent molecule, which needs to be able to switch between the bright state and the dark state. Cyanine dyes with the addition of thiols and spirolactone - type rhodamine dyes can achieve blinking by regulating the solution pH, but the additional imaging conditions limit their application in living cells. Achieving self - blinking of dyes in the physiological environment through structural regulation has also been the focus of recent research. Introducing a photocage group on the basis of self - blinking endows the dye with the property of photo - activation, enabling its signal to be further sparsely distributed during the imaging process to better collect signals and complete image reconstruction. Most traditional photocage groups such as o - nitrobenzyl, diazo, and tetrazine need to be activated by light in the ultraviolet band, and the high phototoxicity and low penetrability of ultraviolet light to biological tissues limit the further application of such probes. Summary of the Invention

[0003] Object of the Invention: The object of the present invention is to provide a visible - light - activated fluorophore with self - blinking behavior. During the process of irradiating with excitation light, such fluorophore molecules will be activated one by one to generate fluorescence and be accompanied by self - blinking behavior. At the same time of activation, nitric oxide free radicals can be released; Another object of the present invention is to provide a preparation method of a visible - light - activated fluorophore with self - blinking behavior; Another object of the present invention is to provide an application of a visible - light - activated fluorophore.

[0004] Technical Solution: The visible - light - activated fluorophore with self - blinking behavior of the present invention has the following chemical structure:

[0005]

[0006] On the other hand, the present invention provides a preparation method of the above - mentioned visible - light - activated fluorophore with self - blinking behavior, including the following steps:

[0007]

[0008] Step 1: Trimellitic anhydride reacts with 3 - diethylaminophenol to obtain compound R - 1;

[0009] Step 2: Compound R - 1 reacts with m - bromophenol to obtain compound R - 3;

[0010] Step 3: Compound R - 3 is mixed with an acid - binding agent, a catalyst, and a coupling agent to obtain a mixed solution; the mixed solution reacts with aniline to obtain compound R - 4;

[0011] Step 4: Compound R - 4 is added to a solvent to obtain a mixed solution. Under ice - bath conditions, the mixed solution reacts with sodium nitrite to obtain the target compound RN0 - 2, and compound RN0 - 2 is a visible - light - activated fluorophore that spontaneously flashes.

[0012] Further, in Step 1, trimellitic anhydride and 3 - diethylaminophenol are added to a solvent, and heated under reflux for reaction to obtain compound R - 1.

[0013] Further, in Step 1, trimellitic anhydride and 3 - diethylaminophenol are added to a solvent, and the solvent is toluene.

[0014] Further, in Step 2, compound R - 1 and m - bromophenol are added to a solvent, and heated under oil - bath for reaction to obtain compound R - 3.

[0015] Further, in Step 2, compound R - 1 and m - bromophenol are added to a solvent, and the solvent is methanesulfonic acid.

[0016] Further, in Step 3, the acid - binding agent is cesium carbonate, the catalyst is tris(dibenzylideneacetone)dipalladium, and the coupling agent is 2 - dicyclohexylphosphino - 2′,4′,6′ - triisopropylbiphenyl.

[0017] Further, in Step 3, compound R - 3, cesium carbonate, tris(dibenzylideneacetone)dipalladium, and 2 - dicyclohexylphosphino - 2′,4′,6′ - triisopropylbiphenyl are added to toluene, stirred at room temperature under nitrogen protection to obtain a mixed solution, and aniline is added to the mixed solution, and heated under oil - bath for reaction to obtain compound R - 4.

[0018] Further, in Step 4, compound R - 4 is added to a solvent to obtain a mixed solution, and the solvent is a mixed solution containing acetic acid and dichloromethane.

[0019] On the other hand, the present invention provides an application of the above - mentioned visible - light - activated fluorophore that spontaneously flashes in single - molecule localization, super - resolution imaging of cell mitochondria, or preparation of a nitric oxide therapeutic agent.

[0020] Further, the light - activation condition of the visible - light - activated fluorophore that spontaneously flashes: activation is carried out using light with a wavelength in the visible - light region.

[0021] Furthermore, in single-molecule localization or super-resolution imaging of cell mitochondria, the above-mentioned visible-light-activated fluorophore is used as a photoactivated fluorescence probe. The specific method is as follows:

[0022] Cells are stained with RNO-2 dye, incubated, and washed to obtain a sample. The sample is placed on the stage of a total internal reflection fluorescence microscope (TIRFM), and a 532 nm laser is used to excite the dye. After processing the image, a super-resolution image is obtained.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: Most traditional photoactivated fluorophores are activated by light in the ultraviolet band, which has a relatively high phototoxicity to biological samples. The nitroso photocage group adopted by the present invention has a small molecular weight, and can be activated by a broad spectrum of visible light, with relatively low requirements for the imaging system. Moreover, the nitric oxide free radicals released upon activation can also be used for biological therapy applications, and the signal of the synergistic fluorophore can be used as a diagnostic and therapeutic integrated probe; in addition, most of the structural modifications of the spontaneously blinking rhodamine molecules focus on the spiro ring. The present invention modifies the nitrogen atom on one side of the xanthene luminescence center, and can achieve spontaneous blinking while retaining the rhodamine spiro lactone structure. Description of the Drawings

[0024] Figure 1 It is the schematic diagram of the spontaneous blinking of the compound RNO-2 of the present invention.

[0025] Figure 2 It is the time-of-flight mass spectrum of compound R-1.

[0026] Figure 3 It is the nuclear magnetic resonance hydrogen spectrum of compound R-1 in deuterated methanol.

[0027] Figure 4 It is the high-resolution mass spectrum of compound R-3.

[0028] Figure 5 It is the nuclear magnetic resonance hydrogen spectrum of compound R-3 in deuterated chloroform.

[0029] Figure 6 It is the high-resolution mass spectrum of compound R-4.

[0030] Figure 7 It is the nuclear magnetic resonance hydrogen spectrum of compound R-4 in deuterated DMSO.

[0031] Figure 8 It is the high-resolution mass spectrum of compound RNO-2.

[0032] Figure 9 It is the nuclear magnetic resonance hydrogen spectrum of compound RNO-2 in deuterated DMSO.

[0033] Figure 10 The photoactivated fluorescence spectrum of compound RNO-2 in methanol.

[0034] Figure 11 The typical intensity-time trace of compound RNO-2.

[0035] Figure 12 The super-resolution image and diffraction-limited image of U2OS cells of compound RNO-2. Detailed implementation mode

[0036] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings.

[0037] The structure of the self-blinking visible-light-activated fluorophore of the present invention is based on the rhodamine nucleus. Rhodamine dyes can switch between the bright state and the dark state due to the ring-opening and closing at the spirolactone ring, and some external conditions or modifications can make this ring-opening and closing process repeat continuously to achieve self-blinking. The present invention modifies one side of the nitrogen atom of the rhodamine xanthene luminescence center, which can achieve self-blinking while retaining the rhodamine spirolactone structure, and then introduces a nitroso photocage group to endow the molecule with photoactivation properties. The specific photoactivation principle is as Figure 1 shown. When the photocage group is on the molecule, the protonation of the xanthene luminescence center of the molecule is inhibited, showing a non-fluorescent dark state. Subsequently, the molecule is irradiated with light, the photocage group leaves, the fluorescence of the molecule is restored and self-blinking can occur, which can be used for super-resolution imaging based on single-molecule localization.

[0038] In the present invention, the reagents, consumables, etc. used in the experiments are all obtained through commercial channels without special instructions. The experimental methods used are all conventional methods without special instructions.

[0039] Example 1: Preparation of R-1

[0040]

[0041] Trimellitic anhydride (0.698 g, 3.636 mmol) and 3-diethylaminophenol (0.5 g, 3.03 mmol) were added to 20 mL of toluene and heated under reflux at 100 °C overnight. After the reaction was completed, the reaction solution was cooled to room temperature, the solvent was removed by rotary evaporation under reduced pressure, and then an appropriate amount of methanol was added to the solid residue, heated to 60 °C and stirred for half an hour. After cooling, the yellow solid R-1 was obtained by filtration, with a total of 110.1 mg and a yield of 10%. The product was confirmed by 1 1H NMR and MS: 11H NMR (600 MHz, MeOD) δ 8.29 (dd, J = 8.1, 1.7 Hz, 1H), 8.24 (d, J = 8.1 Hz, 1H), 8.10 (d, J = 1.6 Hz, 1H), 6.98 (d, J = 9.0 Hz, 1H), 6.25 - 6.20 (m, 2H), 3.52 (q, J = 7.1 Hz, 4H), 1.29 (t, J = 7.1 Hz, 6H); MALDI-TOF-MS m / z calcd for C 19 H 19 NO 6 [M + H] + : 358.1212; found: 358.1403。

[0042] Example 2: Preparation of R-3

[0043]

[0044] R-1 (100.0 mg, 0.279 mmol) and m-bromophenol (145.0 mg, 0.838 mmol) were added to 4 mL of methanesulfonic acid, and the reaction was heated in an oil bath at 90 °C for 4 hours. After the reaction was completed, the reaction solution was cooled to room temperature, extracted three times with 50 ml of dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate and concentrated, and purified by column chromatography (mobile phase: dichloromethane:methanol = 10:1) to obtain 87.0 mg of red solid R-3, with a yield of 63%. The product was confirmed by 1 1H NMR and MS: 1 1H NMR (400 MHz, CDCl3) δ 8.31 (dd, J = 8.0, 1.3 Hz, 1H), 8.10 (dd, J = 8.0, 0.7 Hz, 1H), 7.87 (dd, J = 1.3, 0.8 Hz, 1H), 7.45 (d, J = 1.9 Hz, 1H), 7.11 (dd, J = 8.5, 2.0 Hz, 1H), 6.61 (d, J = 8.5 Hz, 1H), 6.53 (d, J = 8.9 Hz, 1H), 6.45 (d, J = 2.6 Hz, 1H), 6.37 (dd, J = 9.0, 2.6 Hz, 1H), 3.36 (q, J = 7.1 Hz, 4H), 1.17 (t, J = 7.1 Hz, 6H); HR-MS m / z calcd for C 25 H 21 BrNO 5 + [M + H] + : 494.0598; found: 494.0609。

[0045] Example 3: Preparation of R-4

[0046]

[0047] R-3 (50.0 mg, 0.101 mmol), cesium carbonate (98.5 mg, 0.303 mmol), tris(dibenzylideneacetone)dipalladium(0) (9.2 mg, 0.011 mmol), and 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (15.3 mg, 0.032 mmol) were added to 2 mL of toluene. After stirring at room temperature for 30 minutes under nitrogen protection, aniline (28 mg, 0.303 mmol) was added thereto, and the reaction was carried out in an oil bath at 100 °C for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature, and the reaction solution was extracted with a dichloromethane / water system. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated, and purified by column chromatography (mobile phase: dichloromethane:methanol:formic acid = 10:1:0.5) to obtain a purple metallic luster solid R-4, 24.1 mg in total, with a yield of 47%. The product was confirmed by 1 1H NMR and MS: 1H NMR (400 MHz, DMSO) δ 8.65 (s, 1H), 8.17 (s, 1H), 7.91 (d, J = 7.5 Hz, 1H), 7.66 (s, 1H), 7.28 (t, J = 7.7 Hz, 2H), 7.16 (d, J = 7.8 Hz, 2H), 6.93 (d, J = 7.2 Hz, 1H), 6.90 (d, J = 2.2 Hz, 1H), 6.75 (d, J = 8.6 Hz, 1H), 6.56 (d, J = 8.5 Hz, 1H), 6.46 (d, J = 3.2 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 3.33 (t, J = 7.1 Hz, 4H), 1.07 (t, J = 6.9 Hz, 6H); HR-MS m / z calcd for C 31 H 27 N 2 O 5 [M + H] + : 507.1920; found: 507.1924.

[0048] Example 4: Preparation of RNO-2

[0049]

[0050] Dissolve R-4 (15.0 mg, 0.028 mmol) in a mixed solution of acetic acid and dichloromethane (v / v = 1:1), stir for 10 minutes in an ice bath, then add sodium nitrite (19.3 mg, 0.28 mmol) and react at room temperature for 30 minutes. After the reaction, add water to quench the reaction, extract three times with 50 ml of dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate and concentrate, and purify by column chromatography (mobile phase: dichloromethane:methanol = 20:1) to obtain 3.5 mg of red solid RNO-2 with a yield of 22%. The product was confirmed by 1 1H NMR and MS: 1H NMR (400 MHz, DMSO) δ 8.65 (s, 1H), 8.17 (s, 1H), 7.90 (d, J = 7.4 Hz, 1H), 7.66 (s, 1H), 7.28 (t, J = 7.6 Hz, 2H), 7.16 (d, J = 7.8 Hz, 2H), 6.94 (d, J = 7.2 Hz, 1H), 6.90 (d, J = 2.2 Hz, 1H), 6.75 (d, J = 8.6 Hz, 1H), 6.56 (d, J = 8.5 Hz, 1H), 6.48 - 6.46 (m, 1H), 6.43 (d, J = 2.4 Hz, 1H), 3.34 (q, J = 7.0 Hz, 4H), 1.08 (t, J = 6.9 Hz, 6H); HR-MS m / z calcd for C 31 H 25 N 3 O 6 [M + H] + : 535.1743; found: [M - NO + H] + 506.1868.

[0051] Example 5: Photocatalytic performance test of RNO-2

[0052] Dissolve 1 mg of RNO-2 in methanol and dilute to a 2 mL methanol solution with a concentration of about 1 μM, place it in a cuvette, place the cuvette on a magnetic stirring table, irradiate with a 532 nm laser (MGL-III-532, CNI Tech) while stirring, and measure its spectra at different time points. The spectral results are as Figure 9 shown. It can be seen that within 120 min of irradiation time, an absorption peak appears significantly near 550 nm and its intensity gradually increases with the irradiation time. In the fluorescence spectrum, it is also observed that its emission intensity increases significantly with the irradiation time. From the spectral changes, it can be seen that RNO2 can gradually activate and release the nitric oxide photocage group during light irradiation and gradually turn on fluorescence, and can be used as a photocatalytic fluorophore in photocatalytic single molecule imaging experiments.

[0053] Example 6: Single-molecule imaging of RNO-2

[0054] Assemble the pre-treated coverslip and slide to form multiple chambers with a volume of about 2 - 4 μL. Dilute RNO-2 with PBS buffer to a concentration of about 10 nM, and then inject 10 μL of the RNO-2 solution into the chambers. After waiting for 10 min, rinse the chambers three times with 200 μL of PBS buffer to complete sample preparation. Place the prepared sample on the stage of a total internal reflection fluorescence microscope (TIRFM), and use a 532 nm laser (Cobolt 06-DPL, Photonics) to simultaneously activate and excite RNO-2. The laser excites the sample through a 100× oil objective lens (NA 1.49, Nikon), and the emission of the sample is received by an EMCCD (iXon 897, Andor) connected to the microscope through the same objective lens. An emission filter (ZET532 / 640, Chroma) is installed in the optical path to filter the excitation light, and the laser power density is 1.0 kW / cm 2 , with the exposure time fixed at 100 ms, and a total of 5000 frames of data are collected. The data is processed by self-built code in Matlab software. After being activated by the 532 nm laser, RNO-2 exhibits good spontaneous blinking behavior in the single-molecule state, and the blinking signals are sparsely distributed throughout the sampling process, indicating its potential for super-resolution imaging based on single-molecule localization.

[0055] Example 7: Super-resolution imaging of RNO-2 based on single-molecule localization

[0056] Place U2OS cells in a culture dish and grow them in an incubator at 37 °C for 12 hours. When in use, the cell density is about 80%. Stain the cells with 10 μM RNO-2 (diluted from a 5 mM stock solution to DMEM to obtain a final concentration of 10 μM), and incubate them in an incubator at 37 °C for 20 minutes. Then wash the cells 3 times with PBS. Place the prepared sample on the stage of a total internal reflection fluorescence microscope (TIRFM), and use a 532 nm laser (Cobolt 06-DPL, Photonics) to excite the dye, with the laser power density of 1.0 kW / cm 2 , the exposure time fixed at 100 ms, and a total of 5000 frames of data are collected. Use the ThunderStorm plugin to reconstruct the obtained image data to obtain a super-resolution image.

Claims

1. A spontaneously blinking visible light activated fluorophore, characterized in that It has the chemical structure shown below:

2. A method for preparing the spontaneously blinking visible light activated fluorophore as claimed in claim 1, characterized in that: The following steps are involved: Step 1, reacting trimellitic anhydride with 3-diethylaminophenol to obtain compound R-1; Step 2, compound R-1 reacts with m-bromophenol to obtain compound R-3; Step 3, compound R-3 is mixed with an acid binding agent, a catalyst and a coupling agent to obtain a mixed solution; the mixed solution is reacted with aniline to obtain compound R-4; Step 4: Compound R-4 is added to a solvent to obtain a mixed solution. The mixed solution is reacted with sodium nitrite under ice bath conditions to obtain the target compound RN0-2. Compound RN0-2 is a spontaneously blinking visible light-activated fluorophore.

3. The method for preparing a spontaneously blinking visible light activated fluorophore according to claim 2, characterized in that: In step 1, trimellitic anhydride and 3-diethylaminophenol are added to a solvent and heated under reflux to react to obtain compound R-1.

4. The method for preparing a spontaneously blinking visible light activated fluorophore according to claim 3, characterized in that: In step 1, trimellitic anhydride and 3-diethylaminophenol are added into a solvent, wherein the solvent is toluene.

5. The method for preparing a spontaneously blinking visible light activated fluorophore according to claim 2, characterized in that: In step 2, compound R-1 and m-bromophenol are added to a solvent and heated in an oil bath to react to obtain compound R-3.

6. The method for preparing a spontaneously blinking visible light activated fluorophore according to claim 5, characterized in that: In step 2, compound R-1 and m-bromophenol are added to a solvent, wherein the solvent is methanesulfonic acid.

7. The method for preparing a spontaneously blinking visible light activated fluorophore according to claim 2, characterized in that: In step 3, the acid binding agent is cesium carbonate, the catalyst is tris(dibenzylideneacetone)dipalladium, and the coupling agent is 2-dicyclohexylphosphine-2′,4′,6′-triisopropylbiphenyl; and / or, in step 4, compound R-4 is added to a solvent to obtain a mixed solution, and the solvent is a mixed solution containing acetic acid and dichloromethane.

8. The method for preparing a spontaneously blinking visible light activated fluorophore according to claim 2, characterized in that: In step three, compound R-3, cesium carbonate, tris(dibenzylideneacetone)dipalladium and 2-dicyclohexylphosphine-2′,4′,6′-triisopropylbiphenyl are added to toluene, and stirred at room temperature under nitrogen protection to obtain a mixed solution. Aniline is added to the mixed solution, and the reaction is heated in an oil bath to obtain compound R-4.

9. Use of the spontaneously blinking visible light-activated fluorophore as claimed in claim 1 in single-molecule localization, cell mitochondrial super-resolution imaging or the preparation of nitric oxide therapeutic agents.

10. The use according to claim 9, characterized in that: Photoactivation conditions for spontaneously blinking visible light-activated fluorophores: Activation is performed using light with a wavelength in the visible light region.

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