Preparation and application of a polar fluorescent probe based on triphenylamine-malonitrile
By designing a triphenylamine-malononitrile fluorescent probe, the sensitivity and interference problems of existing probes in detecting polarity in vivo were solved, achieving highly sensitive detection and selective response to polarity, which is suitable for biological imaging.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XIANGTAN UNIV
- Filing Date
- 2026-03-24
- Publication Date
- 2026-05-29
Smart Images
Figure CN122103110A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to the preparation and application of a polar fluorescent probe based on triphenylamine-malononitrile. Background Technology
[0002] Polarity, as an important parameter of the cellular microenvironment, participates in various physiological processes, including protein denaturation, enzyme catalysis, peptide aggregation, membrane fusion, and signal transduction (Wang Y, Wang G, Zhang H, et al. A lysosomes-targeted ratio fluorescent probe for real-time monitoring of micropolarity in cancer cells [J]. Sensors & Actuators: B. Chemical, 2018, 261: 210-217). Furthermore, abnormal polarity is closely associated with various diseases such as Alzheimer's disease, diabetes, cirrhosis, and cancer (Zhu LL, Zhang TG, Lin WY, et al. Real-time detection of attenuated blood polarity in mouse models of circulating tumor based on a fluorescent probe [J]. Sensors & Actuators: B. Chemical, 2021, 348: 130664). Therefore, accurate detection of polarity is very important in biological systems (Chao LM, Gerile Aodeng, Ai J, et al. Design and synthesis of a highly polarity-sensitive fluorescent probe and its application in tumor cellimaging [J]. Bioorganic Chemistry, 2025, 159: 108399).
[0003] However, due to the complexity of its influencing factors, the detection of polarity in organisms faces severe challenges. Traditional methods for detecting polarity include electrochemical analysis, infrared spectroscopy, and chromatography (Santos A, Bueno PR, Davis JJ. A dual marker label free electrochemical assay for Flavivirus dengue diagnosis [J]. Biosensors and Bioelectronics, 2018, 100: 519-525; MariMC, SalvadorG, MiguelDLG. Variable selection for the determination of total polar materials in fried oils by near infrared spectroscopy [J]. Journal of Near Infrared Spectroscopy, 2019, 27: 812884; Wei TL, Mei YX, Xiao TH, et al. Rapid synergistic cloud point extraction for simultaneous determination of five polar phenols in environmental water samples via high-performance liquid chromatography with fluorescence detection [J]. Microchemical Journal, 2021, 164: 105963). Despite their rapid development, it remains difficult to monitor polarity in biological samples.Fluorescent probes, due to their high sensitivity, non-invasiveness, real-time monitoring, and excellent spatiotemporal resolution (Tian M, Ma Y, Lin W. Fluorescent probes for the visualization of cell viability[J]. Accounts of Chemical Research, 2019, 52: 2147-2157), are gradually becoming important research tools in the fields of chemistry, biology, and medicine (Xiao HB, Li P, Tang B, et al. Recent progresses in fluorescent probes for detection of polarity [J]. Coordination Chemistry Review, 2021, 427: 213582; Qian M, Wang JY, Peng XJ, et al. A mitochondria-targeting and polarity-sensitive fluorescent probe for cancer diagnosis [J]. Sensors & Actuators: B. Chemical, 2021, 344: 130261).Currently, several fluorescent probes for monitoring polarity have been reported (Zhou R, Sha H, Lu GY, et al. Hyperspectral Fluorescence Imaging with a New Polarity-Ultrasensitive Fluorescent Probe [J]. Advanced Science, 2025, 12: e08792; Zhang T, Huo FJ, Yin CX, et al. Development of near-infrared mitochondrial polarity fluorescent probe for evaluating mitophagy in mice heart and potential cancer diagnosis [J]. Chemical Engineering Journal, 2022, 437: 135397; Afzal MW, Yasmin Iram, Mouni L, et al. Rhodol-based fluorescent probe for polarity detection in living cells and application in bioimaging [J]. Results in Chemistry, 2025, 16: 102427). However, these polar fluorescent probes have short emission wavelengths, limiting their biological applications (Meng FF, Yu XQ, Gao L, et al. Intraoperative pathological diagnosis for breast cancer with a polarityfluorescent probe targeting lipid droplets [J]. Sensors & Actuators: B. Chemical, 2025, 443:138305).
[0004] Triphenylamine-malononitrile, as a novel fluorescent dye, possesses advantages such as large Stokes shift and high sensitivity. In particular, due to its near-infrared emission, it exhibits deep tissue penetration and is less susceptible to interference from autofluorescence, making it more advantageous for bioimaging. Studies have found that fluorescent probes using triphenylamine derivatives have been successfully applied to detect some targets, such as nitroreductase and hydrazine (Jia HG, Juan Z, Wen JB, et al. Atriphenylamine-based near-infrared fluorescence turn-off probe for nitroreductase imaging [J]. Dyes and Pigments, 2024, 221:111807; Jun JH, YanH Z, Wen XW, et al. A fluorescent probe based on triphenylamine with AIE and ICT characteristics for hydrazine detection [J]. Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy, 2023, 286:122011). However, to date, there is no fluorescent probe based on triphenylamine-malononitrile dyes for polarity detection. Therefore, it is essential to design and synthesize a fluorescent probe based on triphenylamine-malononitrile dyes for polarity detection. Summary of the Invention
[0005] Based on the requirements, the inventors conducted in-depth research and, after a great deal of creative work, provided a polar fluorescent probe based on triphenylamine-malononitrile.
[0006] The technical solution of this invention is a polar fluorescent probe based on triphenylamine-malononitrile, the structural formula of which is as follows:
[0007] .
[0008] A method for preparing a polar fluorescent probe based on triphenylamine-malononitrile. The steps are as follows:
[0009] In a 50 mL round-bottom flask, 1.0 equivalent of 5-[4-[bis[4-methoxyphenyl]amino]phenyl]thiophene-2-carboxaldehyde and 1.0–1.5 equivalents of 2-[3-cyano-4,5,5-trimethylfuran-2-yl]malonitrile were dissolved in acetonitrile, and then 0.1–0.3 mL of piperidine was added. The reaction mixture was refluxed at 70–80 °C for 12–18 h. After the reaction was stopped, the solvent was removed by vacuum distillation. The crude product was purified by column chromatography using dichloromethane:methanol = 100:1–50:1 as the eluent, finally yielding a blue solid compound TM, which is the fluorescent probe.
[0010] The beneficial effect of this invention is the excellent spectral response performance of a polar fluorescent probe based on triphenylamine-malononitrile. Solutions of different polarities were obtained by adjusting the ratio of water and 1,4-dioxane, and evaluated using the Lippert-Matagall polarity parameter Δƒ. Fluorescence spectra at different polarities showed that the fluorescence intensity at 760 nm gradually increased as the solvent polarity increased from Δƒ = 0.021 (99% 1,4-dioxane) to Δƒ = 0.319 (0% 1,4-dioxane). A slight redshift occurred in the maximum emission wavelength. Simultaneously, the fluorescence intensity exhibited a good linear relationship with polarity (Δƒ) between 0.021 and 0.319. These results indicate that the probe is highly sensitive to polarity. Subsequently, the selectivity of the probe was investigated, examining various analytes, such as cations (K... + Ca 2+ Mg 2+ Zn 2+ Co 2+ , Cr 3+ Al 3+ Cd 2+ Ni 2+ Cu 2+ ), anion (F) - , Br - HCO3 - CO3 2- SO4 2- SO3 2- NO3 - HS - , S 2-The fluorescence response of the probe in biothiols (Cys, Hcy, GSH) and 1,4-dioxane was investigated. The results showed that 1,4-dioxane caused a change in the fluorescence spectrum, while other substances had no significant effect on the probe's fluorescence spectrum. This indicates that the probe can be used for polarity detection without interference from other substances. Next, the UV-Vis absorption spectrum was measured. The initial absorption peak of TM was located at 610 nm; as the solvent polarity decreased, the absorption peak of TM blue-shifted to 590 nm. Then, the effect of pH on the polarity determination of the fluorescent probe was studied. In aqueous solutions containing 50% or 0% 1,4-dioxane, the probe fluorescence intensity remained essentially unchanged within the pH range of 3–10, indicating that the probe can be used for polarity determination under physiological conditions. Furthermore, the fluorescent probe exhibits excellent photostability, ensuring its practicality.
[0011] Application of a triphenylamine-malononitrile-based polar fluorescent probe. 4T-1 cells were used in the experiment. After centrifugation and discarding the supernatant, the cells were washed three times with PBS and resuspended in PBS. When the fluorescent probe was added to the PBS solution of the cells, no obvious fluorescence signal was observed in the control group. Pretreatment of cells with different concentrations of oleic acid (OA) followed by incubation with the probe resulted in a significant increase in fluorescence in the OA-induced cells, indicating that the probe responds to polarity in living cells. Attached Figure Description
[0012] Figure 1 This is the synthetic route for the fluorescent probe.
[0013] Figure 2 The images show the fluorescence spectra of the fluorescent probe in solutions of different polarities.
[0014] The x-axis represents wavelength, and the y-axis represents fluorescence intensity. The concentration of the fluorescent probe was 10 μM, and the percentages of 1,4-dioxane in water were: 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%. The corresponding Δƒ values were 0.319, 0.300, 0.294, 0.287, 0.277, 0.262, 0.228, 0.222, 0.215, 0.207, 0.197, 0.185, 0.169, 0.149, 0.122, 0.082, and 0.021. The emission wavelength is 765~840 nm, and the corresponding excitation wavelength is 590 nm.
[0015] Figure 3 This is a graph showing the relationship between the logarithm of the fluorescence intensity of the fluorescent probe and the logarithm of Δƒ.
[0016] The x-axis represents Δƒ, and the y-axis represents fluorescence intensity. The concentration of the fluorescent probe is 10 μM.
[0017] Figure 4 This shows the UV-Vis absorption spectra of the fluorescent probe in solutions of different polarities.
[0018] Figure 5 This is a selectivity diagram of the fluorescent probe.
[0019] The concentration of the fluorescent probe was 10 μM, and the concentration of other analytes was 200 μM.
[0020] Figure 6 This is a graph showing the effect of pH on fluorescent probes.
[0021] Figure 7 This is a photostability graph of the fluorescent probe.
[0022] Figure 8 This is a graph from a cytotoxicity assay. The horizontal axis represents the concentration of the fluorescent probe, and the vertical axis represents cell viability.
[0023] Figure 9 Cellular imaging of the fluorescent probe.
[0024] Figure 10 Relative fluorescence intensity diagram. Detailed Implementation
[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but is not limited thereto.
[0026] Example 1:
[0027] Synthesis of fluorescent probes
[0028] Synthetic routes such as Figure 1 In a 50 mL round-bottom flask, 1.0 equivalent of 5-[4-[bis[4-methoxyphenyl]amino]phenyl]thiophene-2-carboxaldehyde and 1.2 equivalent of 2-[3-cyano-4,5,5-trimethylfuran-2-yl]malonitrile were dissolved in acetonitrile, and then 0.2 mL of piperidine was added. The reaction mixture was refluxed at 75 °C for 13 h. After the reaction was stopped, the solvent was removed by vacuum distillation. The crude product was purified by column chromatography using dichloromethane:methanol = 100:1 as the eluent, and finally a blue solid compound TM was obtained, which is the fluorescent probe. Yield: 0.35 g (58%). 1H NMR (400 MHz, CDCl3) δ 7.80(d, J = 15.6 Hz, 1H), 7.54 – 7.43 (m, 3H), 7.28 (s, 1H), 7.11 (d, J = 8.4 Hz,4H), 6.88 (dq, J = 9.8, 2.8 Hz, 6H), 6.59 (d, J = 15.6 Hz, 1H), 3.82 (s, 6H), 1.76 (s, 6H). MS for [C 36 H 28 N4O3S] + : 596.1894.
[0029] Example 2:
[0030] Preparation of fluorescent probes and solutions of 1,4-dioxane in different proportions and calculation of Δƒ
[0031] Preparation of probe solution: Weigh a certain amount of probe and dissolve it in dimethyl sulfoxide to prepare a 1×10⁻⁶ solution. -4 A spare solution of mol / L was prepared. 1.0 mL of the probe's spare solution was added to a 10 mL volumetric flask, and the volume was adjusted to 1.0 × 10⁻⁶ mol / L with PBS buffer to obtain a concentration of 1.0 × 10⁻⁶ mol / L. -5 Fluorescent probe solutions of mol / L were prepared. Solutions with different percentages of 1,4-dioxane in water were prepared (0%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%). The corresponding Δƒ values were 0.319, 0.300, 0.294, 0.287, 0.277, 0.262, 0.228, 0.222, 0.215, 0.207, 0.197, 0.185, 0.169, 0.149, 0.122, 0.082, and 0.021, respectively.
[0032] Example 3:
[0033] Determination of fluorescence spectra of fluorescent probes in solutions of different polarities
[0034] Figure 2The fluorescence spectra of the fluorescent probe in solutions of different polarities are shown. The concentration of the fluorescent probe was 10 μM, and the proportion of 1,4-dioxane in the solution decreased from 99% to 0%. The excitation wavelength used in the experiment was 590 nm, and the emission wavelength range was 765–840 nm. The fluorescence spectrometer used was a Hitachi F-4600 fluorescence spectrometer. As can be seen from the figure, when the probe is in aqueous solution, there is almost no emission peak; as the proportion of 1,4-dioxane increases, that is, as the polarity of the solution decreases, the fluorescence gradually increases. Figure 3 This is a linear response graph of the probe to different polarities. Fluorescence intensity and Δƒ show a linear relationship, ranging from 0.021 (99% 1,4-dioxane) to 0.319 (0% 1,4-dioxane). This probe has a wide detection range and can meet the detection requirements for different polarities.
[0035] Example 4:
[0036] Determination of UV-Vis absorption spectra of fluorescent probes in solutions of different polarities
[0037] Figure 4 The images show the UV-Vis absorption spectra of a fluorescent probe at different polarities. The probe concentration was 10 μM, and the solutions were water and 1,4-dioxane. The UV-Vis absorption spectra were measured using an Agilent Cary 60 UV-Vis spectrophotometer. Figure 4 As can be seen, the maximum absorbance of the probe in water is at 610 nm, while the maximum absorbance wavelength in 1,4-dioxane is blue-shifted to 590 nm.
[0038] Example 5:
[0039] Selectivity of fluorescent probes for polarity determination
[0040] Figure 5 To demonstrate the polarity selectivity of the fluorescent probe, an excitation wavelength of 590 nm was used in the experiment to investigate the interaction between TM and various analytes, such as cations (K). + Ca 2+ Mg 2+ Zn 2+ Co 2+ , Cr 3+ Al 3+ Cd 2+ Ni 2+ Cu 2+ ), anion (F) - ,Br - HCO3 - CO3 2- SO4 2- SO3 2-NO3 - HS - , S 2- The study investigated the probe's response in the presence of biothiols (Cys, Hcy, GSH) and 1,4-dioxane. The results showed that only 1,4-dioxane caused a change in the fluorescence spectrum; other interfering substances had no significant effect on the probe's fluorescence spectrum, indicating that the fluorescent probe exhibits excellent polarity selectivity.
[0041] Example 6:
[0042] The effect of solution pH on the fluorescence properties of fluorescent probes that determine polarity
[0043] The effect of pH on the fluorescence spectrum of a fluorescent probe determining polarity was investigated, and the results are as follows: Figure 6 The pH range we studied was 3–10, the concentration of the fluorescent probe was 10 μM, and the excitation wavelength used in the experiment was 590 nm. As can be seen from the figure, the probe can maintain a good response to polarity within the pH range of 3–10, which is very beneficial for the use of this probe to determine the polarity in biological samples.
[0044] Example 7:
[0045] Determination of photostability of fluorescent probes in 1,4-dioxane
[0046] The photostability of the fluorescent probe in 1,4-dioxane solution was investigated. Measurements were started upon addition of the probe to the solution and continued until 120 min. The results are as follows: Figure 7 As can be seen from the figure, the probe maintains its polarity response in 1,4-dioxane for 120 min, exhibiting good photostability, which meets the requirements for monitoring in actual samples.
[0047] Example 8:
[0048] Application of fluorescent probes in living cells
[0049] First, cytotoxicity tests were performed, such as... Figure 8 As shown in the figure, when 0-30 μM of the probe was added, the survival rate of 4T-1 cells was over 90%. This indicates that the fluorescent probe has low toxicity and can be used to detect polarity in live cells. Then, the application of the fluorescent probe in live cells was investigated. Near-infrared polarity imaging was performed on 4T-1 cells, and the results are shown in the figure. Figure 9 As shown, when the fluorescent probe was added to the PBS solution of the cells, a weak fluorescence could be clearly observed. Pretreatment of cells with different concentrations of OA, followed by incubation with the probe, resulted in a significant increase in fluorescence in the OA-induced cells. Figure 10The results show the relative fluorescence intensities of four groups of cells, demonstrating that the probe can detect intracellular polarity with high sensitivity.
Claims
1. A polar fluorescent probe based on triphenylamine-malononitrile, namely TM, characterized in that, Its structure is as follows: 。 2. The method for preparing a polar fluorescent probe based on triphenylamine-malononitrile according to claim 1, characterized in that, The reaction steps are as follows: In a 50 mL round-bottom flask, 1.0 equivalent of 5-[4-[bis[4-methoxyphenyl]amino]phenyl]thiophene-2-carboxaldehyde and 1.0–1.5 equivalents of 2-[3-cyano-4,5,5-trimethylfuran-2-yl]malonitrile were dissolved in acetonitrile, and then 0.1–0.3 mL of piperidine was added. The reaction mixture was refluxed at 70–80 °C for 12–18 h. After the reaction was stopped, the solvent was removed by vacuum distillation. The crude product was purified by column chromatography using dichloromethane:methanol = 100:1–50:1 as the eluent, and finally a blue solid compound TM was obtained, which is the fluorescent probe.
3. The application of the triphenylamine-malononitrile-based polar fluorescent probe according to claim 1, characterized in that, The fluorescent probe can be used to detect polarity in living cells.