Design and synthesis of acridine fluoroboradiazaindacene fluorescent dyes with large stokes shift

Abstract

The application relates to design and synthesis of acridine fluoroboron dyes with large Stokes shift, and belongs to the technical field of preparation of organic functional materials. The molecular structure is as follows: wherein R=Me, HNCOCH3, Br, CN. Compared with traditional fluoroboron dyes, the new fluoroboron dyes have long emission wavelength, large Stokes shift and high fluorescence quantum yield, and have good application prospect in biological imaging and the like.

Description

[0001] This invention relates to the design and synthesis of acridine-based fluorinated boron dyes with large Stokes shifts, belonging to the field of organic functional materials preparation technology. Background Technology

[0002] Classic fluorine-boron dyes, such as BODIPY, have been widely used in various fields, including photodynamic therapy, laser materials, and bioimaging, due to their advantages such as good photostability, high molar extinction coefficient, and high fluorescence quantum yield. However, classic BODIPY dyes have drawbacks such as short emission wavelength, small Stokes shift, and fluorescence quenching due to π-π stacking in aggregated and solid states, which limit their biological applications. Therefore, developing dyes with long-wavelength emission and large Stokes shift is of great significance. Boranil is an asymmetric fluoroboron dye with a larger Stokes shift compared to BODIPY. However, previously reported Boranil dyes suffer from drawbacks such as short emission wavelengths and low fluorescence quantum yields. For example, diethylamino, as the donor moiety of Boranil dyes, emits in the blue light region in dichloromethane with a fluorescence quantum yield of only 0.07. The low fluorescence quantum yield is mainly due to the formation of a torsional intramolecular charge transfer (TICT) state by the diethylamino moiety. Therefore, to improve the photophysical properties of the dyes, we need to introduce a structure with a higher charge density as the donor moiety of Boranil dyes to enhance the ICT effect, thereby increasing the emission wavelength and Stokes shift of the fluorescent dye. Furthermore, the donor moiety should have a certain degree of rigidity to suppress nonradiative transitions caused by the rotation of the dye molecule. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing research by developing an acridine-based fluoroboron dye with a large Stokes shift. Its structural formula is as follows: Where R = Me, HNCOCH3, Br, CN. The synthesis steps are as follows: (a) Compound MA-CHO and the corresponding aniline were dissolved in anhydrous ethanol, and p-toluenesulfonic acid was added as a catalyst. The mixture was heated under reflux overnight. The reaction solution was poured into water and extracted with DCM. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to give crude products 2a / 2b / 2c / 2d. These crude products were directly used for the next reaction without purification. (b) Dissolve 2a / 2b / 2c / 2d in 1,2-dichloroethane, add N,N-diisopropylpropylamine and stir at room temperature for 10 min, then add boron trifluoride diethyl ether and heat to 70 °C for 6 h. Quench the reaction in a saturated sodium bicarbonate solution, then extract with DCM. Dry the organic phase with anhydrous sodium sulfate and evaporate to dryness to obtain the crude product. Analyze the crude product MABF-Me / MABF-NHAc / MABF-Br / MABF-CN by column chromatography. In step (a), the molar ratio of MA-CHO to the corresponding aniline is 1:1.2, and the amount of p-toluenesulfonic acid added to the reaction system is catalytically sufficient (1 crystal). In step (b), the volume ratio of N,N-diisopropylpropylamine to boron trifluoride diethyl ether is 1:1. The eluents used in the column chromatography in step (b) are (V petroleum ether / V dichloromethane = 2 / 1).

[0004] The fluorescent dye testing method of the present invention is as follows: the probe molecule is dissolved in dichloromethane and ethanol, and the test is performed at room temperature. Specific implementation methods are described in detail in the implementation examples.

[0005] The asymmetric fluorinated boron dyes MABF-Me, MABF-NHAc, MABF-Br, and MABF-CN of this invention exhibit bright fluorescence in dichloromethane, ethanol, and solid states. Their photophysical properties are shown in the table below. Wherein λ abs λ represents the maximum absorption wavelength of the dye. em Δ represents the maximum emission wavelength of the dye. ss Φ represents the Stokes shift of the dye, ε represents the molar extinction coefficient of the dye, and Φ represents the Stokes shift of the dye. f This indicates the fluorescence quantum yield of the dye.

[0006] The purpose of this invention is to synthesize a series of novel asymmetric fluoroboron fluorescent dyes by introducing a strong electron-donating group, 10-ethyl-9,9-dimethyl-9,10-dihydroacridine (MA), as the donor moiety of the fluorescent dye and introducing different substituents as acceptor moieties. Compared with traditional fluoroboron dyes, the new fluoroboron dyes have longer emission wavelengths and larger Stokes shifts, and also exhibit higher fluorescence quantum yields. Attached Figure Description

[0007] Figure 1 The image shows the 1H NMR spectrum of the fluorescent dye MABF-Me in deuterated chloroform, with the horizontal axis representing chemical shift and the vertical axis representing intensity.

[0008] Figure 2The image shows the 1H NMR spectrum of the fluorescent dye MABF-HNAc of this invention in deuterated chloroform, with the horizontal axis representing chemical shift and the vertical axis representing intensity.

[0009] Figure 3 The image shows the 1H NMR spectrum of the fluorescent dye MABF-Br in deuterated chloroform, with the horizontal axis representing chemical shift and the vertical axis representing intensity.

[0010] Figure 4 The image shows the 1H NMR spectrum of the fluorescent dye MABF-CN in deuterated chloroform, with the horizontal axis representing chemical shift and the vertical axis representing intensity.

[0011] Figure 5 The image shows the absorption spectrum of the fluorescent dye of the present invention in dichloromethane, with the horizontal axis representing the absorption wavelength and the vertical axis representing the absorbance.

[0012] Figure 6 The image shows the fluorescence spectrum of the fluorescent dye of the present invention in dichloromethane, with the horizontal axis representing the emission wavelength and the vertical axis representing the fluorescence intensity.

[0013] Figure 7 The image shows the absorption spectrum of the fluorescent dye of this invention in ethanol, with the horizontal axis representing the absorption wavelength and the vertical axis representing the absorbance.

[0014] Figure 8 The image shows the fluorescence spectrum of the fluorescent dye of the present invention in ethanol, with the horizontal axis representing the emission wavelength and the vertical axis representing the fluorescence intensity.

[0015] Figure 9 The image shows the fluorescence spectrum of the fluorescent dye of the present invention in the solid state, with the horizontal axis representing the emission wavelength and the vertical axis representing the fluorescence intensity.

[0016] Figure 10 This is a cell imaging image of the fluorescent dye MABF-Me of the present invention.

[0017] Specific implementation examples Example 1: Synthesis of the dye MABF-Me Compound MA-CHO (50 mg, 0.178 mmol) and p-toluidine (23 mg, 0.214 mmol) were dissolved in 5 mL of EtOH, and a trace amount of p-toluenesulfonic acid was added. The mixture was heated to 80 °C and refluxed overnight. The reaction solution was poured into 50 mL of water and extracted with DCM (3 x 30 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to obtain crude product 2b. This crude product was not purified and proceeded directly to the next step of the reaction. 2a was dissolved in 5 mL of 1,2-dichloroethane, and 200 μL of N,N-diisopropylpropylamine was added and stirred at room temperature for 10 min. Then, 200 μL of boron trifluoride diethyl ether was added, and the mixture was heated to 70 °C and reacted for 6 h. The reaction solution was poured into 100 mL of saturated sodium bicarbonate solution and extracted with DCM (3 x 50 mL). The organic phase was dried with anhydrous sodium sulfate, and after evaporation, it was subjected to column chromatography (V petroleum ether / V dichloromethane = 2 / 1) to give 20 mg of yellow solid. Yield: 27%. 1 H NMR(400MHz,Chloroform-d)δ8.21(s,1H),7.44(d,J=9.3Hz,2H),7.41(s,1H),7.38(d,J=4.9Hz,1H),7.30(d,J=6.0Hz,1H),7.24(s,1H),7.11 (t,J=3.3Hz,1H),7.08(dd,J=10.5,3.0Hz,1H),6.62(d,J=16.1Hz,1H),4.08(q,J=7.1Hz,2H),2.39(s,3H),1.53(s,6H),1.48(t,J=7.0Hz,3H). Example 2: Synthesis of dye MABF-HNAc Compound MA-CHO (60 mg, 0.214 mmol) and p-aminoacetanilide (38 mg, 0.256 mmol) were dissolved in 5 mL of PET H₂O, and a trace amount of p-toluenesulfonic acid was added. The mixture was heated to 80 °C and refluxed overnight. The reaction solution was poured into 50 mL of water and extracted with DCM (3 x 30 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to give crude product 2b. This crude product was not purified and proceeded directly to the next step. 2b was dissolved in 5 mL of 1,2-dichloroethane, and 300 μL of DIEA was added. The mixture was stirred at room temperature for 10 min, and then 300 μL of boron trifluoride diethyl ether was added. The mixture was heated to 70 °C and reacted for 6 h. The reaction solution was poured into 50 mL of saturated sodium bicarbonate solution and extracted with DCM (3 x 30 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. The mixture was then subjected to column chromatography (V petroleum ether / V dichloromethane = 2 / 1) to give 51 mg of an orange-yellow solid, with a yield of 52%. 1H NMR(400MHz,Chloroform-d)δ8.20(s,1H),7.58(s,1H),7.55(d,J=7.4Hz,2H),7.45(d,J=7.5Hz,3H),7.40(d,J=4.3Hz,1H),7.30–7.27(m,1 H),7.11(d,J=1.9Hz,1H),7.10–7.05(m,1H),6.60(d,J=16.3Hz,1H),4.09(q,J=7.0Hz,2H),2.20(s,3H),1.54(s,6H),1.48(t,J=7.0Hz,3H). Example 3: Synthesis of the dye MABF-Br Compound MA-CHO (80 mg, 0.285 mmol) and p-bromoaniline (58 mg, 0.342 mmol) were dissolved in 5 mL of EtOH. A trace amount of p-toluenesulfonic acid was added, and the solution immediately turned red. The mixture was heated to 80 °C and refluxed overnight. The reaction solution was poured into 50 mL of water and extracted with DCM (3 x 30 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to give crude product 2c. This crude product was not purified and proceeded directly to the next step. 2c was dissolved in 5 mL of 1,2-dichloroethane, and 400 μL of DIEA was added and stirred for 5 min. Then, 400 μL of boron trifluoride diethyl ether was added, and the mixture was heated to 70 °C and reacted for 6 h. The reaction solution was poured into 50 mL of saturated sodium bicarbonate solution and extracted with DCM (3 x 30 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. The mixture was then subjected to column chromatography (V petroleum ether / V dichloromethane = 2 / 1) to give 63 mg of an orange-yellow solid, with a yield of 46%. 1 H NMR(400MHz,Chloroform-d)δ8.20(s,1H),7.59(s,1H),7.56(s,1H),7.44(d,J=5.0Hz,2H),7.41(s,1H),7.39(d,J=5.0Hz,1 H),7.30–7.28(m,1H),7.13–7.09(m,2H),6.60(d,J=16.1Hz,1H),4.09(q,J=7.1Hz,2H),1.53(s,6H),1.49(t,J=7.1Hz,3H). Example 4: Synthesis of dye MABF-CN Compound MA-CHO (90 mg, 0.320 mmol) and p-aminobenzonitrile (45 mg, 0.381 mmol) were dissolved in 5 mL of LEtOH. A trace amount of p-toluenesulfonic acid was then added, and the solution immediately turned red. The mixture was heated to 80 °C and refluxed overnight. The reaction solution was poured into 50 mL of water and extracted with DCM (3 x 30 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to obtain crude product 2d. This crude product was not purified and proceeded directly to the next reaction. 2d was dissolved in 5 mL of 1,2-dichloroethane, and 400 μL of N,N-diisopropylpropylamine was added and stirred for 5 min. Then, 400 μL of boron trifluoride diethyl ether was added, and the mixture was heated to 70 °C and reacted for 6 h. The reaction solution was poured into 50 mL of saturated sodium bicarbonate solution and extracted with DCM (3 x 30 mL). The organic phase was dried with anhydrous sodium sulfate, and after evaporation, it was subjected to column chromatography (V petroleum ether / V dichloromethane = 2 / 1) to give 68 mg of yellow solid, with a yield of 50%. 1 HNMR(400MHz,Chloroform-d)δ8.23(s,1H),7.75(d,J=8.3Hz,2H),7.67(d,J=8.4Hz,2H),7.48–7.44(m,1H),7.41(d,J=4.9Hz,1 H),7.32–7.28(m,1H),7.13(d,J=7.9Hz,2H),6.60(d,J=15.2Hz,1H),4.11(q,J=7.1Hz,2H),1.54(s,6H),1.50(t,J=7.1Hz,3H). Example 5: Photophysical property testing of dyes Weigh out a certain mass of MABF dye and dissolve it in dichloromethane and anhydrous ethanol respectively to prepare 10 -5 The solution of M was prepared, and the UV-Vis absorption and fluorescence spectra of the test dye were then measured. Additionally, a suitable amount of MABF dye was prepared into a thin film on a glass slide, and the emission spectrum of the dye in the solid state was measured using a solid-state fluorescence apparatus. The molar extinction coefficient of the dye was calculated based on the measured absorbance, and the molar extinction coefficient can be obtained from the following formula: Where A represents the absorbance at the maximum absorption wavelength, b represents the thickness of the cuvette, and c represents the concentration of the dye. JuloBF-Me(Φ) was selected in the experiment. f =0.20, solvent: dichloromethane) was used as the reference dye to determine the fluorescence quantum yield of the above fluorescent dye. The fluorescence quantum yield can be calculated using the following formula: Where, Φ fThe fluorescence quantum yield is expressed as x, s, and s, where x and s represent the analyte and reference dye, respectively. x and A s F represents the absorbance of the analyte and the reference dye at a specific absorption wavelength, respectively. x and F s n represents the integrated area of ​​the fluorescence correction spectrum of the analyte and the reference dye at a specific excitation wavelength, respectively. x and n s These represent the refractive indices of the solvents used for the analyte and the reference dye, respectively. Example 6: Cell Imaging Experiment with Dyes The dye MABF-Me was dissolved in DMSO to prepare a 10 -3 Add 990 μL of culture medium to a confocal dish pre-incubated with HeLa cells for 24 h, then add 10 μL of LABF-Me stock solution to the confocal dish to stain the cells for 20 min. Wash three times with PBS, then add 1 mL of PBS solution. Excitation light is set at 400 nm, and the collection channel is set at 500-550 nm. Bright green fluorescence can be observed under a confocal microscope.

Claims

1. A class of acridine-based asymmetric fluorinated boron fluorescent dyes, MABF-R, with a large Stokes shift, has the following structural formula: Where R = Me, HNCOCH3, Br, CN.

2. The method for preparing the acridine-based asymmetric fluorine-boron fluorescent dye MABF-R with a large Stokes shift as described in claim 1, characterized in that, The steps are as follows: (a) Dissolve the compound MA-CHO and the corresponding aniline in anhydrous ethanol, add p-toluenesulfonic acid as a catalyst, heat under reflux overnight, pour the reaction solution into water, extract with DCM, dry the organic phase with anhydrous sodium sulfate, and evaporate to dryness to obtain the crude product; (b) Dissolve the crude product from the previous step in 1,2-dichloroethane, add N,N-diisopropylpropylamine and stir at room temperature for 10 min, then add boron trifluoride diethyl ether, heat to 70 °C and react for 6 h, pour the reaction solution into saturated sodium bicarbonate solution to quench the reaction, then extract with DCM, dry the organic phase with anhydrous sodium sulfate, evaporate to dryness to obtain the crude product, and obtain the target product MABF-R by column chromatography; .

3. The method for preparing a class of acridine-based asymmetric fluorine-boron fluorescent dyes MABF-R with large Stokes shifts according to claim 2, characterized in that: In step (a), the molar ratio of MA-CHO to the corresponding aniline is 1:1.2, and p-toluenesulfonic acid is added to the reaction system in a catalytic amount of 1 crystal.

4. The method for preparing a type of acridine-based asymmetric fluorine-boron fluorescent dye MABF-R with a large Stokes shift according to claim 2, characterized in that: In step (b), the volume ratio of N,N-diisopropylpropylamine to boron trifluoride diethyl ether is 1:

1.

5. The method for preparing a class of acridine-based asymmetric fluorine-boron fluorescent dyes MABF-R with large Stokes shifts according to claim 2, characterized in that: In step (b), the eluent used in the column chromatography is V. 石油醚 :V 二氯甲烷 =2:

1.

6. The test method for a class of acridine-based asymmetric fluorinated boron fluorescent dyes MABF-R with large Stokes shifts as described in claim 1, characterized in that: At room temperature, fluorescent dyes MABF-Me, MABF-NHCOCH3, MABF-Br, and MABF-CN were dissolved in dichloromethane and anhydrous ethanol to prepare 10 μM solutions. Their absorption and emission spectra were measured using a UV-Vis spectrophotometer and a fluorescence spectrophotometer. In addition, an appropriate amount of MABF-R dye was prepared into a thin film on a glass slide, and the emission spectrum of the dye in the solid state was measured using a solid-state fluorescence device.

7. The application of the reagent for preparing cell imaging using the acridine-based asymmetric fluorine-boron fluorescent dye MABF-R with a large Stokes shift as described in claim 1, characterized in that: At room temperature, the fluorescent dye MABF-Me was dissolved in DMSO to prepare a stock solution. Culture medium was added to a confocal dish incubated with HeLa cells for 24 h. The MABF-Me stock solution was added to the confocal dish and incubated for 20 min. The excitation light was 400 nm and the collection channel was 500-550 nm. Bright green fluorescence could be observed under a confocal microscope.

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