A viscosity-fluorescent probe based on indole derivatives, its preparation method and application

Abstract

This invention discloses a viscosity-fluorescent probe based on indole derivatives, its preparation method, and its application, belonging to the field of fluorescent materials and biodetection technology. The molecular formula of the probe in this invention is C1. 34 H 29 N2 + ·ClO4 ‑ The structural formula is as follows: The indole derivative-based fluorescent probe for viscosity detection designed in this invention has a short synthesis path and a simple post-processing procedure. It exhibits strong response specificity, high sensitivity, and good selectivity, with a large Stokes shift and fluorescence enhancement factor, reducing background fluorescence interference during imaging and enabling rapid, visualized, and selective detection of viscosity.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent materials and biological detection technology, specifically relating to a viscosity fluorescent probe based on indole derivatives, its preparation method and application. Background Technology

[0002] As the basic unit of life, the stability of the cell's internal physiological state depends on a variety of regulatory factors. Viscosity, as a key physical parameter of the microenvironment, directly affects the rate of intracellular substance diffusion, molecular interactions, and the efficiency of biochemical reactions. Important processes in normal physiological activities, such as signal transduction, protein folding, and enzyme catalysis, all depend on maintaining the dynamic equilibrium of viscosity. When cells are in a pathological state (such as cancer), abnormal viscosity significantly alters the viscoelastic characteristics of the cytoskeleton, leading to changes in cytoplasmic fluidity, which in turn affects metabolic processes and molecular transport efficiency.

[0003] From a biomechanical perspective, cell structure can be divided into three parts: cytoplasm, cell membrane, and cytoskeleton. While the cytoskeleton exhibits rigid characteristics, the viscoelastic system formed by the cell membrane and cytoplasm is extremely sensitive to changes in the microenvironment. In cancer cells, due to cytoskeleton remodeling and abnormal aggregation of intracellular macromolecules, viscosity often increases abnormally. This change not only interferes with mitochondrial energy metabolism but also hinders the diffusion of signaling molecules, creating a vicious cycle of pathological microenvironment.

[0004] Furthermore, abnormal mitochondrial viscosity may contribute to diseases such as cancer, Alzheimer's disease, Parkinson's disease, and diabetes. Meanwhile, cell viscosity detection has become a crucial entry point for cancer research. Quantitative analysis of viscosity changes can reveal the mechanical mechanisms of abnormal tumor cell proliferation and provide biophysical markers for early diagnosis. More importantly, in-depth research into viscosity regulation mechanisms may open new avenues for developing therapeutic strategies targeting the tumor microenvironment. However, the intracellular microenvironment is complex and contains numerous interfering factors. Developing high-precision research tools to analyze the mechanisms of intracellular viscosity changes and their clinical translational value still faces significant technical bottlenecks. Therefore, studying viscosity changes is a highly valuable endeavor.

[0005] The indole derivative-based fluorescent probe for viscosity detection designed in this invention is simple to prepare and has a mature synthetic route. As the viscosity increases, the fluorescence intensity of the probe is significantly enhanced, exhibiting a large Stokes shift and fluorescence enhancement factor. It can effectively reduce background fluorescence interference during the imaging process and achieve rapid, visual, and selective detection of viscosity. Summary of the Invention

[0006] This invention addresses the current problems faced by viscosity fluorescent probe detection. Through molecular design, this invention synthesizes a viscosity fluorescent probe with a large Stokes shift, rapid visualization, and excellent selectivity.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0008] A viscosity-fluorescent probe based on indole derivatives, with the molecular formula C 34 H 29 N2 + ·ClO4 - The structural formula is as follows: The probes in this article are all referred to as TPA-WY.

[0009] The fluorescence emission mechanism of the fluorescent probe TPA-WY designed in this invention is as follows:

[0010] Based on the TICT principle, which states that different viscosities inhibit the free rotation of molecular rotors to varying degrees, the probe can respond to changes in the viscosity of the surrounding environment. In non-viscous solvents, since free rotation within molecules is not inhibited, it exhibits weak fluorescence; however, as viscosity increases, double bond rotation is restricted, and energy tends to return to the ground state in the form of radiative transitions, resulting in strong fluorescence.

[0011] A method for preparing a viscosity fluorescent probe based on indole derivatives includes the following preparation steps:

[0012] (1) 11g of compound 1 was added to 35mL of 20% HClO4 aqueous solution under stirring. The mixture was stirred for several minutes until crystals precipitated. After filtration, the crystals were recrystallized using a mixed solution of carbon tetrachloride and 1,2-dichloroethane. The crystals were then filtered under reduced pressure and dried under vacuum to obtain compound 2. The structural formula of compound 2 is as follows: ;

[0013] (2) Take 3.18 g of compound 2 and 0.024 mol of crotonaldehyde, dissolve them in 50 mL of ethanol, and heat under reflux at 75 °C for 12 h; pour the reaction product into 40 mL of water, filter quickly under reduced pressure, slowly add 20 mL of 48% HClO4 aqueous solution to the filtrate while stirring, filter under reduced pressure, wash with deionized water and dry under vacuum to obtain compound 3. The structural formula of compound 3 is: ;

[0014] (3) Take 210 mg of compound 3 and 273 mg of compound 4, dissolve them in 15 mL of ethanol, add 0.2 mL of piperidine while stirring, react at room temperature for 20 min, and then heat at 75 °C under reflux for 3 h. After the reaction is complete, separate and purify the target probe by column chromatography.

[0015] Furthermore, the structural formula of compound 1 in step (1) is as follows: In step (1), the volume ratio of carbon tetrachloride to 1,2-dichloroethane in the mixed solution is 4:1.

[0016] Furthermore, the structural formula of compound 4 in step (3) is as follows: .

[0017] The synthetic route for the target fluorescent probe is as follows:

[0018] .

[0019] An application of a viscosity fluorescent probe based on indole derivatives is disclosed for sensing and detecting changes in fluid viscosity. The sensing and detection includes fluorescence detection and visual qualitative detection.

[0020] Beneficial effects:

[0021] (1) The synthesis route is short and the process is simple. The synthesis of the probe can be completed in only three steps, and the post-processing is relatively simple;

[0022] (2) It exhibits strong specificity, high sensitivity, and good selectivity in its response. It can achieve a specific response to viscosity under competition from multiple interfering ions;

[0023] (3) This probe has a large Stokes shift and strong fluorescence stability, which can effectively reduce background fluorescence interference during imaging.

[0024] (4) It possesses colorimetric sensing capabilities, providing strong indicative results. The color change as the solution viscosity increases can be observed with the naked eye, and the fluorescence color change can also be observed under ultraviolet light. It is a fluorescent probe with colorimetric sensing capabilities, serving as an indicator of fluid viscosity changes, and enabling real-time qualitative and quantitative visual colorimetric detection. Therefore, this invention is a simple, rapid, and sensitive viscosity-specific detection reagent with broad application prospects in the field of biomolecular detection. Attached Figure Description

[0025] Figure 1 For probe TPA-WY 1 H NMR spectrum;

[0026] Figure 2 For probe TPA-WY 13 C NMR spectrum;

[0027] Figure 3 This shows the change in the fluorescence spectrum of the probe TPA-WY with increasing viscosity, where... Figure 3 In the middle (a), the UV absorption spectra of probe TPA-WY (25 μmol / L) are shown using different ratios of PBS and glycerol as solvents. Figure 3In (b), the fluorescence spectrum of probe TPA-WY (25 μmol / L) changes with increasing viscosity using different ratios of PBS and glycerol as solvents and 505 nm as excitation light.

[0028] Figure 4 This is a graph showing the change in fluorescence color of the probe TPA-WY solution after irradiation with a UV lamp in solutions of different viscosities (0% glycerol and 99% glycerol).

[0029] Figure 5 This describes the Stockholm shift and fluorescence stability of the probe TPA-WY, among which... Figure 5 In the middle (a), the absorption and emission spectra of probe TPA-WY (25 μmol / L) in a mixture of PBS and glycerol at a ratio of (PBS:glycerol = 20:80) are shown. Figure 5 In the middle (b), the fluorescence emission intensity of probe TPA-WY (25 μmol / L) in a mixture of PBS and glycerol with a ratio of (PBS:glycerol = 20:80) changes over time.

[0030] Figure 6 This is a fluorescence change graph of probe TPA-WY (25 μmol / L) in a mixture of PBS and glycerol in a ratio of (PBS:glycerol = 20:80) in the presence of various interfering substances (60 μM). Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0032] Example 1

[0033] Synthesis steps of the TPA-WY viscosity fluorescent probe:

[0034] The route for synthesizing compound 2 is as follows:

[0035] .

[0036] Compound 1 (11 g, 0.07 mol, 1 eq) was added to 35 mL of 20% HClO4 aqueous solution under stirring. Crystals precipitated during the reaction. The mixture was stirred for several minutes, filtered, and the product was recrystallized from CCl4-ClCH2CH2Cl (4:1). The product was filtered under reduced pressure and dried under vacuum to obtain compound 2, which weighed 15.9 g, with a yield of 88.0%.

[0037] The route for synthesizing compound 3 is as follows:

[0038] .

[0039] Compound 2 (3.18 g, 0.02 mol, 1 eq) and crotonaldehyde (1.96 ml, 0.024 mol, 1.2 eq) were dissolved in 50 mL of ethanol and reacted under reflux at 75 °C for 12 h. The product was poured into 40 mL of water, filtered quickly under reduced pressure, and 20 mL of 48% HClO4 aqueous solution was slowly added to the filtrate with stirring. The mixture was filtered under reduced pressure, washed with deionized water, and dried under vacuum to give compound 3, weighing 1.61 g, with a yield of 39%.

[0040] The synthesis route of the compound probe TPA-WY is as follows:

[0041] .

[0042] Compound 3 (210 mg, 1 mmol, 1 eq) and compound 4 (273 mg, 1 mmol, 1 eq) were dissolved in 15 mL of ethanol. 0.2 mL of piperidine was added while stirring, and the reaction was carried out at room temperature for 20 min, followed by reflux at 75 °C for 3 h. The reaction was monitored using a TCL plate. After the reaction was complete, the organic matter was extracted with 1,2-dichloroethane, washed three times with 50 mL of water, and once with 50 mL of saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and the organic solvent was evaporated under reduced pressure. Separation was then performed using a silica gel column with silica gel particles of 200-300 mesh. The eluent ratio was methanol / 1,2-dichloroethane = 1:20, yielding probe TPA-WY, weighing 298 mg, with a yield of 65%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.7599 (d, J = 6.70 Hz, 1H), 8.7920(d, J = 1.83 Hz, 1H), 8.4100 (dd, J = 6.76, 2.14 Hz, 1H), 8.2498 – 8.1623 (m,2H), 7.9171 – 7.8740 (m, 1H), 7.6771 – 7.6511 (m, 3H), 7.4505 – 7.3574 (m,6H), 7.1532 (ddd, J = 9.73, 7.70, 1.30 Hz, 6H), 6.9839 – 6.9306 (m, 2H), 1.7149(s, 6H). 13 C NMR (101 MHz, DMSO- d6) δ 162.6217, 153.7526, 149.5981, 146.1571,141.6785, 141.1161, 138.9075, 135.0920, 130.4278, 129.9363, 129.8121,129.1637, 128.0694, 125.5990, 124.6708, 124.2877, 122.4660, 121.0957,120.4979, 117.9534, 114.7134, 47.3313, 25.3362.

[0043] Example 2

[0044] UV absorption and fluorescence spectra of compound TPA-WY in response to viscosity:

[0045] The TPA-WY viscosity fluorescent probe prepared in Example 1 was weighed and dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mmol / L stock solution. 50 μL of the stock solution was added to a 2 mL centrifuge tube, and different viscosity values ​​were prepared using PBS and glycerol. Its UV absorption and fluorescence emission properties were then measured. Figure 3 As shown in (a), the probe TPA-WY exhibits an absorption peak at 480 nm in a low-viscosity system (PBS:glycerol = 100:1). When the solvent is a high-viscosity system (PBS:glycerol = 1:100), the absorption peak red-shifts from 480 nm to 508 nm; Figure 3 As can be seen in (b), when the solution viscosity changes, the fluorescence intensity of the probe at 661 nm increases with the increase of viscosity and has a good linear relationship.

[0046] Example 3

[0047] Visual detection of viscosity using a TPA-WY fluorescent probe.

[0048] Take 50 μL of the fluorescent probe stock solution from Example 2 and add it to 12 3 mL sample tubes. Add 1.5 mL of a mixture of PBS and glycerol in different ratios to each tube. Under UV light irradiation, it is visible to the naked eye that as the viscosity increases, the bright red fluorescence emitted by the fluorescent probe also becomes stronger. Figure 4 This indicates that compound TPA-WY is a fluorescent probe with chromogenic sensing function.

[0049] Example 4

[0050] Stokes shift and fluorescence stability of probe TPA-WY

[0051] Take 50 μL of the fluorescent probe stock solution from Example 2 and add it to a 2 mL centrifuge tube. Then add 2 mL of a PBS and glycerol mixture (PBS:glycerol = 20:80). Measure the UV absorption and fluorescence emission spectra of the probe solution at room temperature. Figure 5 As shown in (a), the Stokes shift of the probe is 153 nm. 50 μL of the fluorescent probe stock solution from Example 2 was added to a 2 mL centrifuge tube, followed by 2 mL of a PBS and glycerol mixture (PBS:glycerol = 20:80). The emission spectrum of the probe solution was measured every 10 min. Figure 5 As shown in (b), the emission intensity of the probe has good time stability.

[0052] Example 5

[0053] Selectivity and anti-interference spectral data of probe TPA-WY solution

[0054] Take 50 μL of the fluorescent probe stock solution from Implementation Case 2 and add it to a 2 mL centrifuge tube. Then add 2 mL of a PBS and glycerol mixture (PBS:glycerol = 20:80). Add an equal volume of 60 μM competing substance to each centrifuge tube and then detect the change in the fluorescence emission spectrum of the solution. Figure 6 As shown, the addition of all interfering ions and molecules induced only minimal perturbation in the fluorescence intensity spectrum of probe TPA-WY in PBS and glycerol solutions, with the fluorescence intensity remaining almost unchanged. However, when HClO was present simultaneously with these interfering substances, the fluorescence intensity of probe TPA-WY at 661 nm was significantly enhanced. This indicates that TPA-WY exhibits good selectivity and anti-interference properties to viscosity.

[0055] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A viscosity fluorescence probe based on indole derivatives, characterized in that, Probe molecule formula is C 34 H 29 N2 + ·ClO4 - , structure formula is as follows: .

2. A method for preparing the viscosity fluorescence probe based on indole derivatives according to claim 1, characterized by, The preparation includes the following steps: (1) 11 g of compound 1 was added to 35 mL of a 20% mass concentration HClO4 aqueous solution under stirring, and stirred for several minutes until crystals were precipitated, filtered, recrystallized using a carbon tetrachloride and 1,2-dichloroethane mixed solution, filtered under reduced pressure, and vacuum dried to obtain compound 2, the structural formula of which is: ; and the structural formula of compound 1 is ; (2) 3.18 g of compound 2 and 0.024 mol of crotonaldehyde were dissolved in 50 mL of ethanol, and heated to reflux at 75°C for 12 h; the reaction product was poured into 40 mL of water, and rapidly filtered under reduced pressure; 20 mL of a 48% HClO4 aqueous solution was slowly added to the filtrate under stirring, and filtered under reduced pressure, washed with deionized water, and dried under vacuum to obtain compound 3, the structural formula of which is: ; (3) Take 210 mg of compound 3 and 273 mg of compound 4, dissolve in 15 mL of ethanol, add 0.2 mL of piperidine during stirring, react at room temperature for 20 min, then heat to reflux at 75°C for 3 h. After the reaction is completed, the target probe is obtained by column chromatography separation and purification; the structural formula of compound 4 is .

3. Use of the viscosity fluorescence probe based on indole derivatives according to claim 1, characterized in that, The probe is applied to sensing detection of fluid viscosity change, and the application is not for the purpose of diagnosis and treatment of diseases.

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