Viscosity fluorescent probe based on indole derivatives as well as preparation method and application of viscosity fluorescent probe

Abstract

The invention discloses a viscosity fluorescent probe based on indole derivatives as well as a preparation method and application of the viscosity fluorescent probe, and belongs to the technical field of fluorescent materials and biological detection. The indole derivative-based fluorescence probe for detecting the viscosity, which is designed by the invention, has the advantages of short synthesis path, simple post-treatment process, strong response specificity, high sensitivity, good selectivity, large Stokes shift and fluorescence enhancement multiple, reduction of background fluorescence interference in an imaging process, high sensitivity and high sensitivity, and can be used for detecting the viscosity. And the rapid, visual and selective detection of the viscosity can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent materials and biological detection, and specifically relates to a viscosity fluorescent probe based on indole derivatives, a preparation method thereof, and applications thereof. Background Art

[0002] Cells, as the basic units of life activities, the stability of their internal physiological states depends on various regulatory factors. Viscosity, as a key physical parameter of the microenvironment, directly affects the diffusion rate of substances within cells, molecular interactions, and the efficiency of biochemical reactions. Important processes such as signal transduction, protein folding, and enzyme catalysis in normal physiological activities all rely on the maintenance of viscosity dynamic balance. When cells are in a pathological state (such as cancer), abnormal viscosity will significantly change the viscoelastic characteristics of the cytoskeleton, leading to changes in cytoplasmic fluidity, and further affecting the metabolic process and molecular transport efficiency.

[0003] From a biomechanical perspective, the cell structure can be divided into three parts: cytoplasm, cell membrane, and cytoskeleton. Among them, although the cytoskeleton presents rigid characteristics, the viscoelastic system composed of 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, the viscosity value often increases abnormally. This change will not only interfere with mitochondrial energy metabolism but also hinder the diffusion of signaling molecules, forming a vicious cycle of pathological microenvironment.

[0004] In addition, abnormal mitochondrial viscosity may lead to some diseases, such as cancer, Alzheimer's disease, Parkinson's disease, diabetes, etc. At the same time, the detection of cell viscosity has become an important entry point for cancer research. By quantitatively analyzing the characteristics of viscosity changes, not only can the mechanical mechanism of abnormal proliferation of tumor cells be revealed, but also biophysical markers can be provided for early diagnosis. More importantly, in-depth research on the viscosity regulation mechanism may open up new ways for the development of treatment strategies targeting the tumor microenvironment. However, the intracellular microenvironment is complex and there are many interfering factors. There are still significant technical bottlenecks in developing high-precision research tools for analyzing the mechanism of intracellular viscosity changes and their clinical translation value. Therefore, studying the changes in viscosity is a very meaningful work.

[0005] The indole derivative-based fluorescence probe for detecting viscosity designed in the present invention is simple to prepare and has a mature synthesis route. As the viscosity increases, the fluorescence intensity of the probe increases significantly, with a large Stokes shift and fluorescence enhancement multiple, which can effectively reduce the background fluorescence interference during imaging and achieve rapid, visual, and selective detection of viscosity. Summary of the Invention

[0006] Aiming at the current situation of the problems faced by the detection of viscosity fluorescence probes, the present invention synthesizes a viscosity fluorescence probe with a large Stokes shift, rapid visualization, and excellent selectivity through molecular design.

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

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

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

[0010] Based on the TICT principle, that is, different viscosities have different degrees of inhibition on the free rotation of molecular rotors, the probe can respond to the viscosity change of the surrounding environment. In non-viscous solvents, due to the lack of inhibition of intramolecular free rotation, weak fluorescence is exhibited; while as the viscosity increases, the rotation of double bonds is restricted, and the energy tends to return to the ground state in the form of radiative transition, and strong fluorescence can be observed.

[0011] A preparation method of a viscosity fluorescent probe based on indole derivatives, comprising the following preparation steps:

[0012] (1) Add 11 g of compound 1 to 35 mL of an aqueous solution of HClO4 with a mass concentration of 20% under stirring, stir for several minutes until crystals precipitate, filter, perform recrystallization using a mixed solution of carbon tetrachloride and 1,2-dichloroethane, filter under reduced pressure, and dry in vacuum to obtain compound 2. The structural formula of compound 2 is:

[0013] (2) Take 3.18 g of compound 2 and 0.024 mol of crotonaldehyde, dissolve them in 50 mL of ethanol, heat and reflux at 75 °C for 12 h; pour the reaction product into 40 mL of water, quickly filter under reduced pressure, slowly add 20 ml of an aqueous solution of HClO4 with a mass concentration of 48% to the filtrate under stirring, filter under reduced pressure, wash with deionized water and dry in 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 during stirring, react at room temperature for 20 min, then heat and reflux at 75 °C for 3 h. After the reaction is completed, separate and purify the target probe by column chromatography.

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

[0016] Furthermore, the structural formula of Compound 4 in step (3) is

[0017] The synthesis route of the target fluorescent probe is as follows:

[0018]

[0019] The application of a viscosity fluorescent probe based on indole derivatives is used for sensing and detecting the change of fluid viscosity. The said sensing and detecting 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 only requires three steps to complete, and the post-treatment process is relatively simple;

[0022] (2) The response is highly specific, sensitive and selective. Specific response to viscosity can be achieved under the competition of various interfering ions;

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

[0024] (4) It has a colorimetric sensing function and the detection result is highly indicative. The change of color with the increase of solution viscosity can be observed with the naked eye, and the change of fluorescence color can also be observed under ultraviolet light. It is a fluorescence probe with a colorimetric sensing function, which can be used as an indicator for showing the change of fluid viscosity and can perform real-time qualitative and quantitative visual colorimetric detection. Therefore, the present invention is a simple, rapid and sensitive viscosity-specific detection reagent, which has broad application prospects in the field of biomolecule detection. Description of the Drawings

[0025] Figure 1 It is the 1 1H NMR spectrum of the probe TPA-WY;

[0026] Figure 2 It is the 13 13C NMR spectrum of the probe TPA-WY;

[0027] Figure 3 It shows the change of the fluorescence spectrum of the probe TPA-WY with the increase of viscosity. Among them, Figure a is the ultraviolet absorption spectrum of the probe TPA-WY (25 μmol / L) with different ratios of PBS and glycerol as the solvent; Figure b is the change of the fluorescence spectrum of the probe TPA-WY (25 μmol / L) with the increase of viscosity with 505 nm as the excitation light and different ratios of PBS and glycerol as the solvent;

[0028] Figure 4 It is a diagram showing the change in fluorescence color of the probe TPA-WY solution after irradiation with an ultraviolet lamp in solutions of viscosity (0% glycerol) and viscosity (99% glycerol);

[0029] Figure 5 It shows the Stokes shift and fluorescence stability of the probe TPA-WY. Among them, Figure a is the absorption and emission spectra of the probe TPA-WY (25 μmol / L) in a mixed solution of PBS and glycerol with a ratio of (PBS:glycerol = 20:80), and Figure b is the diagram showing the change in fluorescence emission intensity over time of the probe TPA-WY (25 μmol / L) in a mixed solution of PBS and glycerol with a ratio of (PBS:glycerol = 20:80);

[0030] Figure 6 It is a diagram showing the fluorescence change of the probe TPA-WY (25 μmol / L) in a mixed solution of PBS and glycerol with a ratio of (PBS:glycerol = 20:80) in the presence of various interfering substances (60 μM). Specific embodiments

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

[0032] Example 1

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

[0034] Synthesize compound 2, and its route is as follows:

[0035]

[0036] Compound 1 (11 g, 0.07 mol, 1 eq) was added to 35 mL of an aqueous solution of 20% perchloric acid (HClO4) with stirring. Crystals precipitated during the reaction. Stirring was continued for several minutes, followed by filtration. The product was recrystallized with CCl4 - ClCH2CH2Cl (4:1), filtered under reduced pressure, and dried in vacuo to obtain compound 2, weighing 15.9 g with a yield of 88.0%.

[0037] Synthesize compound 3, and its route 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 the mixture was heated under reflux at 75 °C for 12 h. The product was poured into 40 mL of water, and the mixture was quickly filtered under reduced pressure. While stirring, 20 mL of an aqueous solution of 48% HClO4 was slowly added to the filtrate. The mixture was filtered under reduced pressure, washed with deionized water, and dried in vacuo to obtain Compound 3, weighing 1.61 g, with a yield of 39%.

[0040] The synthesis 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 during stirring, and the reaction was carried out at room temperature for 20 min, and then heated under reflux at 75 °C for 3 h. The reaction was monitored by TLC plate. After the reaction was completed, 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. The residue was separated by silica gel column chromatography. The silica gel particle size was 200 - 300 mesh, and the eluent ratio was methanol / 1,2-dichloroethane = 1:20 to obtain the probe TPA-WY, weighing 298 mg, with a yield of 65%. 1 H NMR (400 MHz, DMSO-d6) 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). 1313C 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 spectrum and fluorescence spectrum of compound TPA-WY in response to viscosity:

[0045] Weigh the TPA-WY viscosity fluorescence probe prepared in Example 1 and dissolve it in dimethyl sulfoxide (DMSO) to make a 1 mmol / L stock solution. Take out 50 μL from the stock solution and add it to a 2 mL centrifuge tube, and prepare viscosity values with different ratios using PBS and glycerol, and measure its UV absorption and fluorescence emission properties. As Figure 3 shown in a, the probe TPA-WY has 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 redshifts from 480 nm to 508 nm; as Figure 3 shown 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 compound TPA-WY fluorescence probe for viscosity

[0048] Take out 50 μL each from the fluorescence probe stock solution in Example 2 and add it to 12 3 mL sample tubes, and add 1.5 mL of a mixed solution of PBS and glycerol with different ratios to each. Along with the increase in viscosity that can be visually observed under ultraviolet lamp irradiation, the bright red fluorescence emitted by the fluorescence probe also becomes stronger ( Figure 4 ), indicating that compound TPA-WY is a fluorescence probe with chromogenic sensing function.

[0049] Example 4

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

[0051] Take out 50 μL from the fluorescent probe stock solution of Example 2 and add it into a 2 mL centrifuge tube, then add 2 mL of the mixed solution of PBS and glycerol with a ratio of (PBS:glycerol = 20:80). Measure the ultraviolet absorption spectrum and fluorescence emission spectrum of the probe solution at room temperature. As Figure 5 shown in a, the Stokes shift of the probe is 153 nm. Take out 50 μL from the fluorescent probe stock solution of Example 2 and add it into a 2 mL centrifuge tube, then add 2 mL of the mixed solution of PBS and glycerol with a ratio of (PBS:glycerol = 20:80). Measure the emission spectrum of the probe solution every 10 min. As Figure 5 shown in b, the emission intensity of the probe has good time stability.

[0052] Example 5

[0053] Spectral data of the selectivity and anti-interference of the probe TPA-WY solution

[0054] Take out 50 μL from the fluorescent probe stock solution of Example 2 and add it into a 2 mL centrifuge tube, then add 2 mL of the mixed solution of PBS and glycerol with a ratio of (PBS:glycerol = 20:80). Add an equal volume of 60 μM competitive substances to each centrifuge tube and then detect the change in the fluorescence emission spectrum of the solution. As Figure 6 shown, the addition of all interfering ions and molecules only induces a minimal perturbation in the fluorescence intensity spectrum of the probe TPA-WY in the PBS and glycerol solution, and the fluorescence intensity remains almost unchanged; while when HClO coexists with these interfering substances, the fluorescence intensity of the probe TPA-WY at 661 nm increases significantly. This indicates that TPA-WY has good selectivity and anti-interference for viscosity

[0055] It should be noted that the above embodiments are only some of the preferred embodiments for implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Claims

1. A viscosity fluorescent probe based on indole derivatives, characterized in that: The probe molecular formula is C 34 H 29 N2 + , the structural formula is as follows:

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

3. The method for preparing a viscosity fluorescent probe based on indole derivatives according to claim 2, characterized in that: The structural formula of compound 1 in step (1) is 4. The method for preparing a viscosity fluorescent probe based on indole derivatives according to claim 2, characterized in that: The structural formula of compound 4 in step (3) is 5. An application of the viscosity fluorescent probe based on indole derivatives according to claim 1, characterized in that: The probe is used to sense the change of fluid viscosity.

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