Preparation and application of a diphenylamine-benzoindole-based viscosity fluorescent probe

Abstract

The application relates to preparation and application of a diphenylamine-benzoindole-based viscosity fluorescent probe, and the structural formula of the probe is shown in the figure; the application provides a preparation method for synthesizing the fluorescent probe by taking 5-((4-methoxyphenyl)(phenyl)amino)thiophene-2-methyl formaldehyde and 1-ethyl-2-methylbenzo[cd]indole-1-ium as raw materials; the fluorescent probe is a near-infrared viscosity fluorescent probe with high sensitivity and high selectivity; firstly, the fluorescent probe shows response to viscosity in a linear range of 0.7 cP to 1012 cP, and has good sensitivity; secondly, the fluorescent probe is not affected by various ions, active oxygen, biological thiols and amino acids, and only shows good selectivity to viscosity; and when the pH value is between 7 and 8, the fluorescent probe has good response to viscosity, so that the viscosity can be detected under physiological conditions; in addition, the fluorescent probe has small cytotoxicity, and can be applied to detection of viscosity in living cells.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to the preparation and application of a viscous fluorescent probe based on diphenylamine-benzoindole. Background Technology

[0002] As one of the important physiological parameters in biology, viscosity is involved in many cellular processes, directly affecting membrane fluidity, protein-protein interactions, and signal transduction (C. Liu, D. Zhang, S. Ye, T. Chen, R. Liu, D-π-A structure fluorophore: NIR emission, response to viscosity, detection cyanide and bioimaging of lipid droplets, Spectrochim. Acta Part A: Mol. Biomol. Spectrosc., 2022, 267, 120593). Abnormal viscosity can lead to various diseases, such as inflammation, fatty liver, Alzheimer's disease, and Parkinson's syndrome (Y. Zhang, Z. Li, W. Hu, Z. Liu, Amitochondrial-targeting near-infrared fluorescent probe for visualizing and monitoring viscosity in live cells and tissues, Anal. Chem. 2019, 91, 10302–10309; H. Song, W. Zhang, Y. Zhang, C. Yin, F. Huo, Viscosity activated NIRfluorescent probe for visualizing mitochondrial viscosity dynamic and fattyliver mice, Chem. Eng. J., 2022, 445, 136448). Therefore, achieving the visualization and detection of viscosity in cells or in vivo microenvironments is of great significance.

[0003] Traditional viscosity measurement methods mainly include capillary viscometer, falling ball viscometer, and rotational viscometer (S. Yuan, Q. Zheng, B. Yao, M. Wen, W. Zhang, J. Yuan, H. Lei, Bio-compatible miniature viscosity sensor based on optical tweezers, Biomed. Opt. Express, 2022, 13, 1152-1160). These methods are well-established for detecting large volumes of solutions in vitro, but they are difficult to implement for real-time and in-situ monitoring of viscosity in living cells or within living organisms. Fluorescent probes are becoming increasingly important research tools in the fields of chemistry, biology, and medicine due to their high sensitivity, non-invasiveness, real-time monitoring capabilities, and excellent spatiotemporal resolution (X. Yang, D. Zhang, Y. Ye, Y. Zhao, Recent advances in multifunctional fluorescent probes for viscosity and analytes, Coord. Chem. Rev., 2022, 453, 214336). (H. Xiao, P. Li, B. Tang, Recent progresses in fluorescent probes for detection of viscosity, Coordin. Chem. Rev. 2020, 424: 213565).To date, various fluorescent probes have been developed for measuring viscosity (Y. Wu, W. Shu, C. Zeng, B. Guo, J. Shi, J. Jing, X. Zhang, A mitochondria targetable and viscosity sensitive fluorescent probe and its applications for distinguishing cancerous cells, Dyes Pigm., 2019, 168, 134-139; Y. Baek, SJ Park, X. Zhou, G. Kim, HM Kim, J. Yoon, Aviscosity sensitive fluorescent dye for real-time monitoring of mitochondria transport in neurons, Biosens. Bioelectron., 2016, 86, 885-889; Z. Yang, Y. He, J.-H. Lee, N. Park, M. Suh, W.-S. Chae, J. Cao, X. Peng, H. Jung, C. Kang, JS Kim, A self-calibrating bipartite viscosity sensor for mitochondria, J. Am. Chem. Soc., 2013, 135, 9181-9185. However, most of these reported probes suffer from short emission wavelengths, resulting in limited penetration depth into biological tissues and susceptibility to background autofluorescence interference, thus limiting their application in in vivo imaging (J. Cheng, Z. Li, W. Lin, Development of a one-step synthesized red emission fluorescent probe for sensitive detection of vision in vitro and in vivo, Spectrochim. Acta Part A: Mol. Biomol. Spectrosc., 2021, 258, 119808). Therefore, it is necessary to design high-performance fluorescent probes with long emission wavelengths.

[0004] Diphenylamine-benzoindole, as a novel fluorescent dye backbone, possesses advantages such as long emission wavelength and good photostability. In particular, its derivatives often exhibit near-infrared emission characteristics, thus possessing deep tissue penetration depth and being less susceptible to interference from biological autofluorescence, making it more advantageous for in vivo imaging. However, to date, no fluorescent probe based on diphenylamine-benzoindole dye has been used for viscosity detection. Therefore, this invention designs and synthesizes a fluorescent probe based on the diphenylamine-benzoindole backbone to achieve specific viscosity detection, which has significant research value and application prospects. 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 viscosity fluorescent probe based on diphenylamine-benzoindole.

[0006] The technical solution of this invention is a viscosity-fluorescent probe based on diphenylamine-benzoindole, the structural formula of which is as follows:

[0007] .

[0008] A method for preparing a viscosity-fluorescent probe based on diphenylamine-benzoindole. The steps are as follows:

[0009] In a 50 mL round-bottom flask, add 1.0 equivalent of 5-((4-methoxyphenyl)(phenyl)amino)thiophene-2-carboxaldehyde, 4.0-6.0 equivalents of 1-ethyl-2-methylbenzene[cd]indole-1-onium, and 10-20 mL of ethanol; reflux the reaction mixture for 20-24 h; after the reaction is stopped, remove the solvent by vacuum distillation; the crude product obtained is purified by column chromatography using dichloromethane:methanol = 200:1-10:1 as the eluent, and finally obtains the black-red solid compound DB, which is the fluorescent probe.

[0010] The beneficial effect of this invention is the excellent spectral response performance of a viscosity-fluorescent probe based on diphenylamine-benzoindole. First, the fluorescence spectral properties of the probe were investigated. The probe itself did not exhibit a significant near-infrared emission peak in aqueous solution; however, when added to a mixed solution of glycerol and water with a certain viscosity, a significant near-infrared emission peak appeared at 755 nm. Furthermore, its near-infrared fluorescence intensity continuously increased with increasing viscosity. The linear detection range of this probe was from 0.7 cP to 10¹² cP, indicating that the probe has high sensitivity. Next, the absorption spectrum of the probe was studied. In aqueous solution, the probe itself has an absorption peak at 680 nm, while in high-viscosity glycerol, the maximum absorption peak red-shifts to 696 nm. Then, the selectivity of the probe was studied, examining its interaction with various metal ions (Na₂O₃, Na ... + , K + Mg2+ Cu 2+ Zn 2+ Ca 2+ Fe 3+ ), anion (Cl - NO3 - CO3 2- CH3COO - HSO3 - SO4 2- ), reactive oxygen species (ClO) - The fluorescence response of the fluorescent probe to H2O2, amino acids (Thr, Met, Trp, Lle, Phe, Val), and biothiols (Cys, Hcy, GSH) as well as viscosity was investigated. The results showed that only viscosity caused a change in the fluorescence spectrum; other interfering substances had no significant effect on the probe's fluorescence spectrum. Finally, the effect of pH on the viscosity determination by the fluorescent probe was studied. In aqueous solution, the probe fluorescence intensity remained essentially constant with pH changes; in a 50% glycerol solution, the fluorescence response signal reached its maximum at pH = 7.0–8.0. This indicates that within the physiological pH range, the probe maintains a good response to viscosity and can be used for viscosity determination in biological samples. Furthermore, the fluorescent probe exhibits excellent photostability, remaining essentially stable within 60 min, ensuring its practicality.

[0011] An application of a diphenylamine-benzoindole-based viscous fluorescent probe was conducted. Human normal hepatocytes AML-12 were used in the experiment. When the fluorescent probe was added to the cells, weak fluorescence was observed; however, the fluorescence significantly increased after the addition of nystatin and monensin to induce an increase in cell viscosity. This demonstrates that the probe can monitor changes in viscosity in living cells, showcasing its practicality. 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 viscosities.

[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 glycerol in water were: 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 100%. The corresponding viscosities were 0.7 cp, 0.86 cp, 1.62 cp, 3.17 cp, 5.55 cp, 10.9 cp, 17.8 cp, 35.8 cp, 63.6 cp, 103.3 cp, 197.2 cp, 313.3 cp, and 1012 cp. The emission wavelength range was 716-900 nm, and the corresponding excitation wavelength was 696 nm.

[0015] Figure 3 This is a graph showing the relationship between the logarithm of fluorescence intensity and the logarithm of viscosity of a fluorescent probe.

[0016] The x-axis represents the logarithm of viscosity, and the y-axis represents the logarithm of fluorescence intensity. The concentration of the fluorescent probe is 10 μM.

[0017] Figure 4 This is the absorption spectrum of the fluorescent probe for solutions of different viscosities.

[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 This is a cell imaging image of a fluorescent probe.

[0024] Figure 10 This is a 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 1In a 50 mL round-bottom flask, 1.0 equivalent of 5-((4-methoxyphenyl)(phenyl)amino)thiophene-2-carboxaldehyde, 5.0 equivalents of 1-ethyl-2-methylbenzene[cd]indole-1-onium, and 10-20 mL of ethanol were added; the reaction mixture was refluxed for 24 h; after the reaction was stopped, the solvent was removed by vacuum distillation; the crude product was purified by column chromatography using dichloromethane:methanol = 200:1~10:1 as the eluent, finally yielding a blackish-red solid compound DB, which is the fluorescent probe. Yield: 58%. 1 H NMR (400 MHz, DMSO-d6) δ 8.95 (d, J = 7.5 Hz, 1H), 8.91 – 8.82 (m,1H), 8.36 (d, J = 8.1 Hz, 1H), 8.18 (s, 1H), 7.94 (t, J = 7.9 Hz, 1H), 7.91(d, J = 8.0 Hz, 1H), 7.72 (d, J = 7.0 Hz, 2H), 7.60 – 7.54 (m, 6H), 7.48 –7.43 (m, 1H), 7.13 (d, J = 8.8 Hz, 2H), 6.84 (s, 1H), 6.54 (d, J = 4.1 Hz,1H), 4.45 (q, J = 6.6 Hz, 2H), 3.83 (s, 3H), 1.32 (t, J = 7.0 Hz, 3H). MS for[C 32 H 27 N2OS] + : 487.64.

[0029] Example 2:

[0030] Preparation of fluorescent probes and glycerol solutions in different proportions and determination of viscosity

[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 M's spare solution. Preparation of solutions with different viscosities: Add 1 mL of probe solution to a 10 mL volumetric flask, then add different percentages of glycerol (0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), and dilute to volume with buffer solution to obtain a concentration of 1.0 × 10⁻⁶. -5The fluorescent probe M was mixed with glycerol solutions of varying viscosities. Viscosities were measured using an NDJ-1 pointer-digital rotational viscometer, yielding viscosities of 0.7 cp, 0.86 cp, 1.62 cp, 3.17 cp, 5.55 cp, 10.9 cp, 17.8 cp, 35.8 cp, 63.6 cp, 103.3 cp, 197.2 cp, 313.3 cp, and 1012 cp.

[0032] Example 3:

[0033] Determination of fluorescence spectra of fluorescent probes in solutions of different viscosities

[0034] Figure 2 The fluorescence spectra of the fluorescent probe in solutions of different viscosities are shown. The concentration of the fluorescent probe was 10 μM, and the percentages of glycerol in the solutions were 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 100%, respectively. The excitation wavelength used in the experiment was 696 nm, and the emission wavelength range was 716-900 nm. The slit width was 10.0 nm / 10.0 nm, and the fluorescence measurement instrument used was a Hitachi F4600 fluorescence spectrophotometer. The figures show that when the probe is in aqueous solution, the emission peak is relatively low; as the percentage of glycerol increases, the solution viscosity increases, and the fluorescence intensity gradually increases. Figure 3 This is a linear response graph of the probe to different logarithmic viscosities. The logarithm of fluorescence intensity and the logarithm of viscosity show a linear relationship, with a linear range of 0.7 cP to 10¹² cP. This probe has a wide detection range and can meet the requirements for viscosity detection.

[0035] Example 4:

[0036] Determination of absorption spectra of fluorescent probes in solutions of different viscosities

[0037] Figure 4 The images show the UV-Vis absorption spectra of the fluorescent probe at different viscosities. The concentration of the fluorescent probe was 10 μM, and the glycerol content of the solutions was 0% and 100%, respectively. The UV-Vis absorption spectra were measured using an Agilent Cary 60 UV-Vis spectrophotometer. Figure 4 As can be seen, in aqueous solution, the probe itself has an absorption peak at 680 nm, and when the viscosity of the solution increases to 10¹² cp, the absorption peak red-shifts to 696 nm.

[0038] Example 5:

[0039] Selectivity of fluorescent probes for viscosity measurement

[0040] Figure 5To investigate the viscosity selectivity of the fluorescent probe, the interactions between the probe and various metal ions (Na+, Na ... + , K + Mg 2+ Cu 2 + Zn 2+ Ca 2+ Fe 3+ ), anion (Cl - NO3 - CO3 2- CH3COO - HSO3 - SO4 2- ), reactive oxygen species (ClO) - The fluorescence response of the probe to H2O2, amino acids (Thr, Met, Trp, Lle, Phe, Val), biothiols (Cys, Hcy, GSH), and viscosity was investigated. The results showed that only viscosity caused a significant change in the fluorescence spectrum, while other interfering substances had almost no effect on the probe's fluorescence spectrum. This indicates that the fluorescent probe exhibits good selectivity in response to viscosity.

[0041] Example 6:

[0042] Effect of solution pH on the fluorescence properties of viscosity measured by fluorescent probe

[0043] The effect of pH on the fluorescence spectrum of viscosity measured by the fluorescent probe was observed, and the results are as follows: Figure 6 Our study investigated a pH range of 4.0–10.0, a fluorescent probe concentration of 10 μM, and solution viscosities of 0.7 cp (aqueous solution) and 10.9 cp (50% glycerol). The figures show that in the aqueous solution, the probe fluorescence intensity remained essentially constant with pH changes; however, in the 50% glycerol solution, the fluorescence response signal reached its maximum at pH 7.0–8.0. This indicates that within the physiological pH range, the probe maintains a good response to viscosity and can be used for viscosity determination in biological samples.

[0044] Example 7:

[0045] Determination of photostability of fluorescent probes in 50% glycerol

[0046] To investigate the photostability of the fluorescent probe in water and 50% glycerol solution, the probe was added to both solutions of varying viscosities and measurements were taken for 60 minutes. The results are as follows: Figure 7 As shown, the probe maintains a good response to viscosity within 60 minutes in water and 50% glycerol, indicating that it has good photostability and can meet 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 probe was added, the survival rate of normal human hepatocytes AML-12 was over 90%. This indicates that the fluorescent probe has low toxicity and can be used to detect viscosity in living cells. Then, the application of the fluorescent probe in living cells was investigated. Confocal microscopy was performed on normal human hepatocytes AML-12, and the results are shown in the figure. Figure 9 As shown, weak fluorescence was observed after the probe was added; the fluorescence was significantly enhanced after the addition of nystatin and monensin to induce an increase in cell viscosity. Figure 10 The relative fluorescence intensities of the three groups of cells were shown, demonstrating that the probe can sensitively detect intracellular viscosity and showcasing its practicality.

Claims

1. A diphenylamine-benzoindole-based viscosity fluorescence probe, characterized by, Its structure is as follows: 。 2. A method for preparing a diphenylamine-benzoindole based viscosity fluorescent probe according to claim 1, characterized by, The reaction steps are as follows: In a 50 mL round-bottom flask, add 1.0 equivalent of 5-((4-methoxyphenyl)(phenyl)amino)thiophene-2-carboxaldehyde, 4.0-6.0 equivalents of 1-ethyl-2-methylbenzene[cd]indole-1-onium, and 10-20 mL of ethanol; reflux the reaction mixture for 20-24 h; after the reaction is stopped, remove the solvent by vacuum distillation; the crude product obtained is purified by column chromatography using dichloromethane:methanol = 200:1-10:1 as the eluent, and finally obtains the black-red solid compound DB, which is the fluorescent probe.

3. The use of a diphenylamine-benzoindole-based viscosity fluorescent probe according to claim 1, characterized in that, The fluorescent probe can be used to detect viscosity in living cells.

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