A dehydroabietic acid-based aggregation-inducing red light compound, preparation method thereof and use thereof

Abstract

The present invention discloses a dehydroabietic acid-based aggregation-inducing red light compound, a preparation method thereof, and its use. The structural formula of the dehydroabietic acid-based aggregation-inducing red light compound is: #imgabs0# The dehydroabietic acid-based aggregation-inducing red light compound is prepared by reacting 12-[N,N-p-methoxy-phenyl]amino-dehydroabietic acid methyl ester and 2,3-bis(4-bromophenyl)-2-butene dinitrile. The dehydroabietic acid-based aggregation-inducing red light compound 2DTPA-CN of the present invention has excellent spectral properties, effectively extends the fluorescence emission wavelength of the dehydroabietic acid-based compound, has low cytotoxicity and strong anti-photobleaching performance, can be used as a fluorescent probe in cell imaging, and can also be used as an anti-counterfeiting material in colored fluorescent anti-counterfeiting, etc.; and is simple to prepare and easy to operate.

Description

Technical Field

[0001] The invention relates to a dehydroabietic acid-based aggregation-inducing red light compound, a preparation method and application thereof, and belongs to the technical field of organic synthesis. Background Art

[0002] Biomass-based luminescent materials have the advantages of natural availability, sustainability, biodegradability, and biocompatibility, and their design and preparation have become a hot topic of research. However, the emission wavelengths of most biomass-based luminescent materials are relatively short, and aggregation quenching (ACQ) caused by π-π stacking in aggregates or solid states limits their practical applications. In 2001, Professor Tang Benzhong proposed the concept of aggregation-induced emission (AIE), which has led to a new understanding of aggregated and solid-state organic luminescent materials.

[0003] Dehydroabietic acid is purified from rosin through catalytic disproportionation. Its backbone contains an aromatic ring. Based on the aromatic ring and carboxylic acid groups, a variety of fluorescent derivatives can be developed, opening up new avenues for the high-value utilization of rosin. However, its ACQ effect and short emission wavelength result in poor cell imaging, limiting its application in bioimaging. To address this issue, the present invention combines dehydroabietic acid with cyano-substituted diphenylethylene, a compound with AIE properties, into a single molecule. Through specific structural design, the dehydroabietic acid derivative not only extends its emission from high-energy blue light to low-energy red light, but also achieves strong red fluorescence emission in an aggregated state. The synthetic route is simple and easy to operate. Summary of the Invention

[0004] The present invention provides a dehydroabietic acid-based aggregation-induced red light compound, a preparation method and use thereof. The dehydroabietic acid-based AIE compound has aggregation-induced emission enhancement characteristics and red light emission, and has strong resistance to photobleaching. It can be used as a fluorescent probe in biological imaging and can also be used for colored fluorescent anti-counterfeiting.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] A dehydroabietic acid-based aggregation-inducing red light compound, the structural formula of which is:

[0007]

[0008] The above-mentioned cyano-containing red light-emitting compound with aggregation-induced emission (AIE) characteristics is named 2DTPA-CN. It effectively extends the fluorescence emission wavelength of the dehydroabietic acid compound to 632nm in THF solution and 683nm in the solid state. It has a narrow band gap value that meets the band gap characteristics of red light molecules and exhibits red light emission. It also has a twisted molecular structure, which hinders the π-π stacking of molecules in the aggregated state, thereby enhancing fluorescence emission and exhibiting obvious AIE characteristics. At the same time, it has strong resistance to photobleaching and low cytotoxicity, and can be used as a fluorescent probe in biological imaging; it can also be used for colored fluorescent anti-counterfeiting.

[0009] The synthesis route of the above-mentioned dehydroabietic acid-based aggregation-induced red light-emitting compound is as follows:

[0010]

[0011] The preparation method of the above-mentioned dehydroabietic acid aggregation-induced red light compound is as follows: under nitrogen protection, 12-[N,N-p-methoxy-phenyl]amino-dehydroabietic acid methyl ester and 2,3-bis(4-bromophenyl)-2-butenedinitrile are used as raw materials, cesium carbonate (Cs2CO3), tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) and 2-dicyclohexylphosphine-2',6'-diisopropoxybiphenyl (RuPhO3) are added, and a CN coupling reaction is carried out in anhydrous o-xylene. After the reaction is completed, the mixture is cooled and purified to obtain the compound 2DTPA-CN.

[0012] To ensure product purity, the following purification method was used: after cooling, extraction with dichloromethane and water was performed, the organic phase was collected, dried over anhydrous magnesium sulfate, purified by column chromatography, rotary evaporation, and dried to obtain the AIE red-emitting compound 2DTPA-CN. During column chromatography, the eluent used was preferably a mixture of petroleum ether and dichloromethane in a volume ratio of (3-5):1.

[0013] In order to improve the product yield, the molar ratio of 12-[N,N-p-methoxy-phenyl]amino-dehydroabietic acid methyl ester and 2,3-bis(4-bromophenyl)-2-butenediconitrile is (2-2.5):1.

[0014] In order to further improve the product yield, the molar ratio of 2,3-bis(4-bromophenyl)-2-butenedinitrile, cesium carbonate, tris(dibenzylidene-base acetone)dipalladium(0) and 2-dicyclohexylphosphine-2',6'-diisopropoxybiphenyl is 1:(2.8~3.3):(0.1~0.2):(0.1~0.2).

[0015] To improve the reaction efficiency, the anhydrous organic solvent is anhydrous o-xylene or anhydrous toluene. When the anhydrous organic solvent is anhydrous o-xylene, the reaction temperature is 110-140°C and the reaction time is 12-24 hours. When the anhydrous organic solvent is anhydrous toluene, the reaction temperature is 90-110°C and the reaction time is 12-24 hours.

[0016] In order to reduce loss, lower costs and ensure reaction efficiency, the organic solvent is preferably anhydrous o-xylene.

[0017] The above-mentioned 12-[N,N-p-methoxy-phenyl]amino-dehydroabietic acid methyl ester can be prepared by the following method: 12-bromodehydroabietic acid methyl ester and p-methoxyaniline are dissolved in anhydrous o-xylene, nitrogen is introduced, sodium tert-butoxide, tri-tert-butylphosphine and palladium acetate are added, and the mixture is reacted at a temperature of 130-140° C. for 10-12 hours. The mixture is cooled and extracted with ether and saturated brine. The organic phase is collected, dried over anhydrous magnesium sulfate, separated and purified by column chromatography, the solvent is removed by rotary evaporation, and vacuum drying is performed to obtain 12-[N,N-p-methoxy-phenyl]amino-dehydroabietic acid methyl ester as a light yellow solid.

[0018] The molar ratio of the above-mentioned 12-bromodehydroabietic acid methyl ester, p-methoxyaniline, sodium tert-butoxide, tri-tert-butylphosphine and palladium acetate is preferably 1:(1.1-1.3):(1.4-1.5):(0.10-0.13):(0.02-0.04).

[0019] The technologies not mentioned in this invention are all referred to the prior art.

[0020] Beneficial effects

[0021] (1) The synthesis route of the dehydroabietic acid-based aggregation-inducing red-emitting compound provided by the present invention is simple, using 12-[N,N-p-methoxy-phenyl]amino-dehydroabietic acid methyl ester as the raw material, and only requires a one-step CN coupling reaction for synthesis.

[0022] (2) The dehydroabietic acid-based aggregation-induced red light compound 2DTPA-CN provided by the present invention has excellent spectral properties. In THF solution, the maximum ultraviolet absorption wavelength and the maximum fluorescence emission wavelength are 500 nm and 632 nm, respectively. The maximum fluorescence emission wavelength in the solid state is 683 nm, which effectively extends the fluorescence emission wavelength of the dehydroabietic acid-based compound. The long-wavelength red light probe can effectively overcome the absorption, spectral overlap and autofluorescence interference of biological tissues, and can significantly improve the signal-to-noise ratio of biological imaging (Wu Qian, Cheng Dan, Lu Yun, etc. Large Stokes shift far-red to near-infrared fluorescent probe for detecting the dynamic changes of peroxynitrite during liver injury (English) [J]. Journal of Chemistry in Universities, 2020, 41 (11): 2426-2434.). Experiments have shown that the water-soluble nanoparticles of 2DTPA-CN (2DTPA-CN NPs) have low cytotoxicity and obvious effects in cell imaging. They can be used as fluorescent probes in cell imaging, have strong anti-photobleaching properties, and can be stably stored for a long time.

[0023] (3) The dehydroabietic acid-based aggregation-induced red light compound 2DTPA-CN provided by the present invention has obvious aggregation-induced emission enhancement properties, a twisted molecular structure and a band gap value that conforms to the band gap characteristics of red light molecules. It has strong resistance to photobleaching and can be used as an anti-counterfeiting material in colored fluorescent anti-counterfeiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the 2DTPA-CN of the present invention 1 H NMR spectrum;

[0025] Figure 2 is the 2DTPA-CN of the present invention 13 C NMR spectrum;

[0026] Figure 3 is the fluorescence emission spectrum (excitation wavelength e) of 2DTPA-CN in the solid state of the present invention. x =488nm);

[0027] Figure 4 The UV absorption spectrum of 2DTPA-CN in THF solution (1×10 -5 mol / L);

[0028] Figure 5 The fluorescence emission spectrum of 2DTPA-CN in THF solution (1×10 -5 mol / L);

[0029] Figure 6 The 2DTPA-CN of the present invention has different water volume fractions (f w) in H2O / THF mixed solvent (1×10 -5 mol / L);

[0030] Figure 7 The relationship between the fluorescence intensity I / I0 of 2DTPA-CN and the water content of the present invention (I0 is the compound at f w = 0% solution fluorescence emission intensity, I is the compound in the corresponding f w = fluorescence emission intensity at 0%, 10%, 30%, 50%, 70%, 90%, 95%);

[0031] Figure 8 2DTPA-CN fluorescence quantum yield and different water contents;

[0032] Figure 9 The optimized structure of 2DTPA-CN obtained by density functional theory calculation;

[0033] Figure 10 The lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) diagrams of 2DTPA-CN of the present invention are shown in FIG.

[0034] Figure 11 2DTPA-CN NPs of the present invention are shown in the transmission electron microscope image;

[0035] Figure 12 The nanoparticle size (DLS) diagram of 2DTPA-CN NPs of the present invention;

[0036] Figure 13 Figure 2 is the cytotoxicity graph of 2DTPA-CN NPs of the present invention (the six groups of bar graphs correspond to six different concentrations of 0, 2.5, 5, 10, 20, 40 and 80 μg / mL, respectively);

[0037] Figure 14 Fluorescence confocal imaging of 2DTPA-CN NPs of the present invention in A549 lung cancer cells (20 μg / ml, 4 hours). (I is the confocal imaging of A549 cells with 2DTPA-CN NPs and the nuclear dye Hoechst 33342 added; II is the confocal imaging of cells with 2DTPA-CN NPs added; III is the confocal imaging of cells with only the nuclear dye Hoechst 33342 added).

[0038] Figure 15 The fluorescence emission spectra of the 2DTPA-CN solution of the present invention when irradiated with ultraviolet light for different durations;

[0039] Figure 16 This is a colored fluorescent anti-counterfeiting effect diagram of the 2DTPA-CN solution of the present invention; DETAILED DESCRIPTION

[0040] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the examples, but the content of the present invention is not limited to the following examples.

[0041] Example 1

[0042] A dehydroabietic acid-based aggregation-induced red light compound, the synthesis route is:

[0043]

[0044] The preparation process is as follows:

[0045] 1 mmol of 12-bromodehydroabietic acid methyl ester (Ⅰ) (Tan Guanni, et al. Synthesis and spectral properties of dehydroabietic acid triarylamine compounds containing bis-naphthalene [J]. Chemistry and Industry of Forest Products, 2019, 39(1): 61-66.) and 1.2 mmol of p-methoxyaniline (Ⅱ) were dissolved in 10 mL of anhydrous o-xylene. After deoxygenation for 15 minutes under nitrogen atmosphere, 1.44 mmol of sodium tert-butoxide (t-BuONa), 0.12 mmol of tri-tert-butylphosphine (P(t-Bu)3) and 0.03 mmol of palladium acetate (Pd(OAc)2) were added. The mixture was heated to reflux at 140 ° C for 12 hours, cooled, extracted with ether and saturated brine, and the organic phase was collected, dried over anhydrous magnesium sulfate, and purified by column chromatography (eluent, V 石油醚 :V 乙酸乙酯 =20:1), the solvent was removed by rotary evaporation, and the mixture was dried in a vacuum oven at 65°C for 12 h to obtain a light yellow solid 12-[N,N-p-methoxy-phenyl]amino-dehydroabietic acid methyl ester (III), which was stored in a desiccator for later use.

[0046] 2.5 mmol of 12-[N,N-p-methoxy-phenyl]amino-dehydroabietic acid methyl ester (III), 1 mmol of 2,3-bis(4-bromophenyl)-2-butenediconitrile (IV), and 3 mmol of cesium carbonate (Cs2CO3) were dissolved in 50 mL of anhydrous o-xylene. Under a nitrogen atmosphere, 0.1 mmol of tris(dibenzylidene-base acetone)dipalladium(0) (Pd2(dba)3, Aladdin) and 0.1 mmol of 2-dicyclohexylphosphine-2',6'-diisopropoxybiphenyl (RuPhO3, Aladdin) were added. The mixture was heated to reflux at 140°C for 24 h, cooled, and extracted with dichloromethane and water. The organic phase was collected, dried over anhydrous magnesium sulfate, and purified by column chromatography (eluent, V 石油醚 :V 二氯甲烷 =10:1), the solvent was removed by rotary evaporation, and then the mixture was placed in a vacuum drying oven at 60°C for 12 hours to obtain red powder 2DTPA-CN.

[0047] like Figure 1-2 As shown, the characterization data: 1 H NMR (500MHz, CDCl3) δ7.64(d,J=8.8Hz,4H),7.10(dd,J=39.8,9.3Hz,8H),6.88(d,J=8.8Hz,4H),6.82(s,4H),3.84(s,6H),3.72(s, 6H),3.11-2.87(m,6H),2.24(dd,J=35.8,12.6Hz,4H),1.95-1.53(m,14H),1.52-1.34(m,6H),1.32(d,J=9.4Hz,10H),1.23(s,8H). 13 C NMR (126MHz, CDCl3) δ179.18,156.41,149.29,144.09,140.51,134.72,129.87,128.12,126.20,125.70,122.79,119.92,118.25,114 .79,77.41,77.16,76.90,55.64,52.08,47.77,44.83,38.08,37.22,36.77,31.66,29.86,27.45,25.35,23.74,21.78,18.55,16.63.

[0048] like Figure 3-5 As shown, the maximum ultraviolet absorption wavelength and maximum fluorescence emission wavelength of the dehydroabietic acid aggregation-induced red light compound 2DTPA-CN in THF solution are 500 nm and 632 nm, respectively, and the maximum fluorescence emission wavelength in the solid state is 683 nm.

[0049] like Figure 6-8 As shown, 2DTPA-CN has a typical aggregation-induced emission enhancement effect, that is, with the increase of water content, its fluorescence emission intensity increases and the fluorescence quantum yield increases.

[0050] like Figure 9 As shown, 2DTPA-CN has a twisted structure, and its π-π stacking is restricted in the aggregated and solid states.

[0051] like Figure 10 As shown, the band gap value (ΔE) of 2DTPA-CN is 2.51 eV, indicating that it has a narrow band gap value that is consistent with the band gap characteristics of red light molecules.

[0052] Application Example 1

[0053] 2 mg of 2DTPA-CN and 18 mg of MPEG-PLGA (methoxy polyethylene glycol poly (glycolide lactide, Mw: 2000-2000) were added to 1 mL of THF, and then 9 mL of ultrapure water was added and continuously sonicated (720W) for 3 min. The mixture was stirred at room temperature for 12 h to remove THF in the mixture, and further purified by dialysis and then filtered through a 0.22 μm filter membrane to finally obtain water-soluble nanoparticles of 2DTPA-CN (such as 2DTPA-CN NPs). Figure 11 and Figure 12 As shown, the formation of nanoparticles was demonstrated, with a nanoparticle size of 80.56 nm and a concentration of 2.0 mg / mL.

[0054] The cytotoxicity of 2DTPA-CN NPs was tested by digesting the cells, counting them, and preparing them to a concentration of 5×10 4 100 μL of cell suspension was added to each well of the cell culture plate (96 wells). The 96-well cell culture plate was placed in a 37°C, 5% CO2 incubator for 24 hours to adhere to the wall. 100 μL of culture medium containing 2DTPA-CN NPs at concentrations of 0, 2.5, 5, 10, 20, 40 and 80 μg / mL was added. At the same time, a negative control group was set up and the cell culture plate was placed in a 37°C, 5% CO2 incubator for further culturing for 24 hours. The well plate was then stained with CCK-8, and the OD value was measured at a wavelength of 450 nm on a microplate reader to calculate the cell survival rate. Figure 13 As shown in the figure, the cell viability in the culture medium containing 80 μg / mL 2DTPA-CN NPs can still reach 82.27%, indicating that 2DTPA-CN NPs have low cytotoxicity.

[0055] Cells in the logarithmic growth phase (Jiangsu KeyGen Biotech Co., Ltd., A549 cells) were digested and inoculated into confocal microplates, with 1×10 4 cells; the next day, the cells adhered to the wall and were incubated with 2DTPA-CN NPs (20 μg / mL) for 4 hours. A blank control group was set up and washed 3 times with PBS. Hoechst33342, a nuclear dye, was diluted at a ratio of 1:1000 and added to the confocal microplate. After incubation in the dark for 15 minutes, the cells were washed 3 times with PBS. The expression was observed under a confocal microscope, and the high expression area was photographed and preserved (600X). The excitation wavelength of 488 nm was selected for the 2DTPA-CN NPs group, and the nuclear dye was Hoechst33342. The results of cell imaging are shown in Figure 2. Figure 14As shown in the figure, under an excitation wavelength of 488 nm, the fluorescence signal of 2DTPA-CN NPs in the 550-750 nm band was collected. Bright green fluorescence of 2DTPA-CN NPs can be observed in the cytoplasm, and the fluorescence intensity is weak in the cell nucleus, which can be used as a cytoplasmic fluorescent probe.

[0056] In summary, it can be clearly seen that 2DTPA-CN has excellent optical properties, its nanoparticles have low toxicity in cells, and have obvious in vitro imaging effects, and can be used as a fluorescent probe for cell imaging.

[0057] Application Example 2

[0058] Take 1.0965 mg of 2DTPA-CN and place it in a 10 ml volumetric flask, dilute it with dichloromethane to a concentration of 1×10 -3 mol / L stock solution; pipette 1000 μL of the stock solution into a 10 ml volumetric flask, and then dilute to 10 ml with dichloromethane to obtain a concentration of 1×10 -4 ml / L test solution. Take an appropriate amount of solution and place it in a fluorescence cuvette. Fix the UV lamp on an iron stand and irradiate the test solution continuously for 36 minutes in a dark environment. The fluorescence emission spectrum (excitation at 488nm) of the test solution is obtained at irradiation times of 0, 4, 9, 16, 25, and 36 minutes. The test results are as follows: Figure 15 As shown in the figure, continuous UV irradiation only caused the fluorescence emission intensity of the 2DTPA-CN solution to slightly weaken, indicating that 2DTPA-CN has strong anti-photobleaching properties.

[0059] Take the above tested solution with a 2mm diameter capillary spotting tube, write the words 2DTPA-CN on a paper without fluorescent background, and take pictures of the words under natural light, 365nm ultraviolet irradiation for 0h, 365nm ultraviolet irradiation for 0.5h, 365nm ultraviolet irradiation for 1h, 365nm ultraviolet irradiation after 15 days in natural environment, and 365nm ultraviolet irradiation after 20 days in natural environment. The results are as follows. Figure 16 As shown, the text appears non-fluorescent red under natural light, but emits bright orange-red fluorescence under 365nm ultraviolet irradiation. Even after continuous UV irradiation, relatively bright fluorescence can still be observed. After 15 days of exposure to UV light, it still emits strong, bright fluorescence under UV light, and the fluorescence intensity under 365nm UV irradiation after 20 days is no different from that at 15 days, indicating that 2DTPA-CN has strong resistance to photobleaching. In summary, 2DTPA-CN can be used as a fluorescent anti-counterfeiting material in colored fluorescent anti-counterfeiting.

Claims

1. A dehydroabietic acid-based aggregation-inducing red light compound, characterized in that: Its structural formula is:

2. The dehydroabietic acid-based aggregation-inducing red light-emitting compound according to claim 1, wherein It has resistance to photobleaching and aggregation-induced emission enhancement effect; the fluorescence emission wavelength in the solid state is 683nm.

3. A method for preparing the dehydroabietic acid-based aggregation-inducing red light compound according to claim 1 or 2, characterized in that: The synthetic route is as follows: Under nitrogen protection, 12-[N,N-p-methoxy-phenyl]amino-dehydroabietic acid methyl ester and 2,3-bis(4-bromophenyl)-2-butenediconitrile were used as raw materials, cesium carbonate, tris(dibenzylideneacetone)dipalladium(0) and 2-dicyclohexylphosphine-2',6'-diisopropoxybiphenyl were added, and a CN coupling reaction was carried out in an anhydrous organic solvent. After the reaction was completed, the mixture was cooled and purified to obtain the compound 2DTPA-CN.

4. The preparation method according to claim 3, wherein The purification process is as follows: the cooled material is extracted with dichloromethane and water in sequence, the organic phase is collected, dried over anhydrous magnesium sulfate, separated and purified by column chromatography, rotary evaporated, and dried to obtain the AIE red-light compound 2DTPA-CN; the eluent used for column chromatography separation and purification is: a mixture of petroleum ether and dichloromethane with a volume ratio of (3 to 5):

1.

5. The preparation method according to claim 3 or 4, characterized in that The molar ratio of 12-[N,N-p-methoxy-phenyl]amino-dehydroabietic acid methyl ester and 2,3-bis(4-bromophenyl)-2-butenediconitrile is (2-2.5):1; the molar ratio of 2,3-bis(4-bromophenyl)-2-butenediconitrile, cesium carbonate, tris(dibenzylideneacetone)dipalladium(0) and 2-dicyclohexylphosphine-2',6'-diisopropoxybiphenyl is 1:(2.8-3.3):(0.1-0.2):(0.1-0.2).

6. The preparation method according to claim 3 or 4, characterized in that The anhydrous organic solvent is anhydrous o-xylene, the reaction temperature is 110-140° C., and the reaction time is 12-24 hours.

7. The preparation method according to claim 3 or 4, characterized in that The anhydrous organic solvent is anhydrous toluene, the reaction temperature is 90-110° C., and the reaction time is 12-24 hours.

8. Use of the dehydroabietic acid-based aggregation-inducing red light compound according to claim 1 or 2, characterized in that: As a cytoplasmic fluorescent probe, it is used for cell imaging for non-disease diagnosis and treatment purposes.

9. Use of the dehydroabietic acid-based aggregation-inducing red light compound according to claim 1 or 2, characterized in that: Used for colored fluorescent anti-counterfeiting.

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