Polarity response fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a polarity-responsive fluorescent probe and a preparation method and application thereof, and relates to the technical field of fluorescent probes, the polarity-responsive fluorescent probe has a structure as shown in a formula I and # imgabs0 # formula I. The fluorescent probe shows remarkable response under environmental polarity change instead of traditional viscosity dependence, and the polarity-responsive fluorescent probe has a good application prospect. The application range of the fluorescent probe is expanded.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent probes, and in particular to a polarity-responsive fluorescent probe and a preparation method and application thereof. Background Art

[0002] Fluorescent probes are highly valued for their wide applications in chemical and biological sensing. In particular, probes that utilize the internal conversion and torsional excited state (TICT) mechanism are able to respond sensitively to changes in environmental viscosity. Traditionally, the working principle of such probes relies on the movement within the molecule, especially in a high viscosity environment, the fluorescence emission intensity of the probe changes significantly, thus providing effective detection of viscosity changes. In contrast, the common strategy for constructing small molecule polarity-sensitive probes is to introduce molecules containing polar functional groups (such as amino, carboxyl, hydroxyl, etc.) and molecules with conjugated double bonds or aromatic rings, which are particularly sensitive to polarity changes due to changes in their electron cloud distribution.

[0003] However, in recent years, research on fluorescent probes has gradually expanded to detect other physicochemical parameters, such as polarity. Advances in this field have made it possible to develop new probes, especially those that are sensitive to changes in environmental polarity. However, there are relatively few studies on the polarity response mechanism of fluorescent probes, and most probes still focus on viscosity response. Therefore, the performance of probes in polar environments has not been fully understood and utilized. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects of the prior art, provide a polarity-responsive fluorescent probe and its preparation method and use, and propose a new type of small molecule fluorescent probe with a twistable bond structure, which shows a significant response under environmental polarity changes, rather than the traditional viscosity dependence. The present invention not only expands the application range of fluorescent probes, but also provides a new perspective for further studying the behavior of fluorescent probes in complex environments.

[0005] In order to achieve the above and other purposes, the present invention is implemented by including the following technical solutions: The present invention first provides a polarity-responsive fluorescent probe XZTU-Pola, wherein the fluorescent probe has a structure as shown in Formula I:

[0006]

[0007] The present invention also provides a method for preparing the polarity-responsive fluorescent probe XZTU-Pola as described above.

[0008] The present invention further provides a use of the polarity-responsive fluorescent probe XZTU-Pola as described above. The polarity-responsive fluorescent probe XZTU-Pola can be used as a biosensor.

[0009] As described above, the fluorescent probe of the present invention has a wide range of application potentials in the sensitive detection of environmental polarity changes, which is completely different from the polarity response mechanism of conventional probes with similar structures. Due to its unique response mechanism, the probe can play an important role in the fields of biological imaging and chemical sensing. Specifically, in biological imaging, the probe can be used to study the polarity changes of the intracellular microenvironment, thereby revealing the dynamic process of cell metabolism and signal transduction.

[0010] Due to the response characteristics of the probe in polar environments, its structure can be further optimized in the future to achieve higher sensitivity and selectivity in detection, which will provide a basis for the development of new sensors and promote their widespread application in scientific research and industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Shown is the molecular formula of the fluorescent probe XZTU-Pola of the present invention.

[0012] Figure 2 Shown is a diagram of the synthesis steps of the fluorescent probe XZTU-Pola of the present invention.

[0013] Figure 3 The H NMR spectrum of compound 1 of the present invention is shown.

[0014] Figure 4 Shown is the H NMR spectrum of compound 2 of the present invention.

[0015] Figure 5 Shown is the H NMR spectrum of compound 3 of the present invention.

[0016] Figure 6 Shown is the H NMR spectrum of compound 4 of the present invention.

[0017] Figure 7 Shown is the nuclear magnetic hydrogen spectrum of the fluorescent probe XZTU-Pola of the present invention.

[0018] Figure 8 Shown is the nuclear magnetic carbon spectrum of the fluorescent probe XZTU-Pola of the present invention.

[0019] Fig. 9 Shown is the mass spectrum of the fluorescent probe XZTU-Pola of the present invention.

[0020] Fig.10 Shown is the rotation energy barrier diagram of the fluorescent probe XZTU-Pola of the present invention.

[0021] Fig.11It shows the fluorescence intensity diagram of the fluorescent probe XZTU-Pola of the present invention, wherein a is the fluorescence spectrum diagram of the probe XZTU-Pola in different solvents; b is the linear relationship diagram between the maximum emission wavelength of the probe XZTU-Pola in different polar solvents and the empirical solvent.

[0022] Fig.12 Shown are some performance standard result graphs of the fluorescent probe XZTU-Pola of the present invention, wherein a is the absorption spectrum graph of the probe XZTU-Pola in tetrahydrofuran (red line) and methanol (blue line); b is the fluorescence spectrum graph of the probe XZTU-Pola in tetrahydrofuran (red line) and methanol (blue line); c is the fluorescence spectrum graph of the probe XZTU-Pola in tetrahydrofuran and methanol in different volume ratios; d is the dielectric constant-fluorescence intensity relationship graph of the probe XZTU-Pola in tetrahydrofuran and methanol in different volume ratios; e is the fluorescence intensity graph of the probe XZTU-Pola to different interferents in tetrahydrofuran and methanol, respectively.

[0023] Fig.13 Shown are cell experiment diagrams of the fluorescent probe XZTU-Pola of the present invention, wherein a is the fluorescence imaging diagram of 3T3-L1 cells, 4T1 cells, HL-7702 cells, HepG2 cells, LX-2 cells, LM-3 cells, HeLa cells, B16-F10 cells, SMMC-7721 cells, A549 cells, HEK-293 cells, and Raw264.7 cells co-incubated with the probe XZTU-Pola; b is the cell imaging diagram of the probe XZTU-Pola under different oleic acid concentrations, different low-density lipoprotein (LDL) incubation times, different temperatures, DXMS, and different pH conditions; c is the fluorescence intensity histogram of Figure a; d is the fluorescence intensity histogram of Figure b. DETAILED DESCRIPTION

[0024] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0025] like Figures 1 to 13 As shown, the present invention provides a polarity-responsive fluorescent probe, wherein the fluorescent probe has a structure as shown in Formula I:

[0026]

[0027] The present invention also provides a method for preparing a polarity-responsive fluorescent probe, the preparation method comprising the following steps:

[0028] S1: reacting 4-ethynylaniline and bis(2-chloroethyl)ether under alkaline conditions to obtain compound 1;

[0029] S2: reacting 3-iodoaniline and methyl iodide under alkaline conditions to obtain compound 2;

[0030] S3: reacting compound 2, phosphorus oxychloride and N,N-dimethylformamide to obtain compound 3;

[0031] S4: Compound 3 and 2-(3,5,5-trimethylcyclohex-2-ene-1-ylidene)malononitrile are reacted to obtain Compound 4; S5: Compound 1 and Compound 4 are reacted to obtain a polar responsive fluorescent probe having a structure shown in Formula I;

[0032]

[0033] The present invention is further described below with reference to the examples.

[0034] Example 1

[0035] (a) Synthesis of Compound 1

[0036] 4-ethynylaniline (1.3 g, 11.4 mmol) and K2CO3 (4.8 g, 34.2 mmol) were dissolved in anhydrous acetonitrile, followed by addition of bis(2-chloroethyl) ether (3.9 g, 13.7 mmol). The mixture was stirred at 80 °C for 12 h. The solvent was evaporated under reduced pressure and the crude product was purified by column chromatography (1:10 v / v dichloromethane / petroleum ether). Yellow oily product 1.8 g (yield: 84.3%) was obtained.

[0037] like Figure 3 As shown, the H NMR spectrum of compound 1: 1 H NMR (500MHz, Chloroform-d) δ7.42–7.37(m,2H),6.85–6.79(m,2H),3.80(ddd,J=9.4,6.1,3.4Hz,4H),3.21(ddd,J=6.4,4.9,3.4Hz,4H),3.04(s,1H).

[0038] (b) Synthesis of Compound 2

[0039] 3-iodoaniline (5g, 22.8mmol) and K2CO3 (9.5g, 68.4mmol) were dissolved in anhydrous acetonitrile, followed by addition of iodomethane (7.8g, 55mmol). The mixture was stirred at 80°C for 12h. The solvent was evaporated under reduced pressure and the crude product was purified by column chromatography (1:10v / v dichloromethane / petroleum ether). Yellow oily product 4.9g (yield: 87.4%) was obtained.

[0040] like Figure 4 As shown, the H NMR spectrum of compound 2: 1 H NMR (500MHz, Chloroform-d) δ7.58(ddd,J=7.7,2.2,1.2Hz,1H),7.15(t,J=2.2Hz,1H),7.03(t,J=7.4Hz,1H),6.64(ddd,J=7.1,2.2,1.1Hz,1H),2.96(s,6H).

[0041] (c) Synthesis of Compound 3

[0042] Phosphorus oxychloride POCl3 (11 mL) and DMF (10 mL) were added to a flask, mixed and stirred at 0°C for 0.5 h, and then compound 2 (4.5 g, 18.3 mmol) dissolved in dichloroethane was added, and the mixture was stirred at 80°C for 12 hours. In this reaction, phosphorus oxychloride first reacts with DMF to form an intermediate, which then reacts with compound 2. The solvent was evaporated under reduced pressure and the crude product was purified by column chromatography (1:4 v / v dichloromethane / petroleum ether). 3.88 g of yellow oily product was obtained (yield: 77.3%).

[0043] like Figure 5 As shown, the H NMR spectrum of compound 3: 1 HNMR(500MHz,Chloroform-d)δ10.00(s,1H),7.72(d,J=7.9Hz,1H),7.40(d,J=2.2Hz,1H),6.75(dd,J=7.8,2.2Hz,1H),2.96(s,6H).

[0044] (d) Synthesis of Compound 4

[0045] Compound 3 (1.4 g, 5 mmol), 2-(3,5,5-trimethylcyclohex-2-ene-1-ylidene) malononitrile (930 mg, 5 mmol) and piperidine (catalyst, 10 drops) were dissolved in anhydrous ethanol (20 mL). The mixture was refluxed at 80 ° C for 9 hours under an inert argon environment. After cooling to room temperature, the red solid was filtered out. Then, the crude solid was purified by column chromatography (1: 6 v / v ethyl acetate / dichloromethane) to obtain a red solid product 746.5 mg (yield: 33.7%).

[0046] like Figure 6 As shown, the H NMR spectrum of compound 4: 1 HNMR(500MHz,Chloroform-d)δ7.40(d,J=7.6Hz,1H),7.27(s,1H),7.16–7.06(m,2H),6.67(dd,J=7 .6,2.2Hz,1H),6.45(q,J=1.0Hz,1H),2.96(s,6H),2.43(d,J=1.0Hz,2H),2.39(s,2H),1.03(s,6H).

[0047] (e) Synthesis of the polarity-responsive fluorescent probe XZTU-Pola

[0048] Pd(PPh3)2Cl2(63mg, 0.089mmol), CuI(17mg, 0.089mmol), PPh3(48mg, 0.18mmol) and compound 4(394.3mg, 0.89mmol) were added to compound 1(166.8g, 0.89mmol). Then, acid binding agent triethylamine NEt3(5mL) and tetrahydrofuran THF(25mL) were added, and the reaction mixture was stirred at room temperature 25°C under an inert Ar environment for 6h. After the reaction, the solvent was removed and the crude residue was purified by column chromatography (1:6v / v ethyl acetate / petroleum ether) to obtain 210.9mg of a dark red solid product (yield: 47.2%).

[0049] like Figure 7 and Figure 8 As shown, the H / C NMR spectrum of compound 4: 1H NMR(500MHz,Chloroform-d)δ7.46(d,J=6.9Hz,1H),7.39–7.28(m,3H),7.11–7.03(m,2H),6.82–6.77(m,2H),6.73(dd,J=7.1,2.2Hz,1H),6.45(q ,J=1.2Hz,1H),3.80(ddd,J=9.4,6.1,3.4Hz,4H),3.21(ddd,J=6.4,4.9, 3.4Hz, 4H), 2.96 (s, 6H), 2.43 (d, J = 1.0Hz, 2H), 2.39 (s, 2H), 1.03 (s, 6H). 13 C NMR(125MHz,Chloroform-d)δ166.99,151.30,150.47,141.08,132.53,128.99,128.98,127.74,127.07,122.76,117.68,11 4.28,113.75,113.29,113.13,112.46,112.13,93.88,88.21,80.15,66.74,48.53,42.94,41.33,40.41,34.37,28.29.HRMS m / z:calcd for C 44 H 42 N4O3[M+H] + :503.2806found:503.2764.

[0050] Example 2

[0051] Example 2 is similar to Example 1, except that the dichloroethane in (c) is replaced by solvent DMF, and the yield of the obtained compound 3 is 51.1%.

[0052] Example 3

[0053] Example 3 is similar to Example 1, except that the stirring time of the mixture in (c) is changed to 6 hours, and the yield of the obtained compound 3 is 69.8%.

[0054] Example 4

[0055] Example 3 is similar to Example 1, except that the stirring time of the mixture in (c) is changed to 24 hours, and the yield of the obtained compound 3 is 46.5%.

[0056] Example 5

[0057] Example 5 is similar to Example 1, except that CuI is not added in (e), and no dark red solid product can be obtained after the reaction.

[0058] Example 6

[0059] Example 6 is similar to Example 1, except that PPh3 is not added in (e), and the yield of XZTU-Pola obtained is 17.3%.

[0060] like Fig. 9 As shown, Fig. 9 This is the mass spectrum of XZTU-Pola in Example 1.

[0061] The performance of the XZTU-Pola prepared in Example 1 was evaluated.

[0062] like Figure 1 The present invention relates to a novel small molecule fluorescent probe XZTU-Pola, in particular a molecular structure having two rotatable bonds ( Figure 1 Although these two rotatable bonds have been reported in the prior art to have viscosity responsiveness, the probe of the present invention failed to exhibit this property in the fluorescence detection experiment. On the contrary, the probe exhibited a significant response to changes in environmental polarity, especially in polar solvents.

[0063] like Fig.10 As shown in the figure, through theoretical calculations, there is an energy barrier for the rotation of the alkyne bond in the XZTU-Pola molecule. In the polar solvent methanol, the energy barrier is 2.95eV, which indicates that the ability of the molecule to rotate is relatively strong and the energy is mainly consumed in the form of mechanical energy. In the non-polar solvent tetrahydrofuran, the energy barrier is 3.99eV, indicating that the molecular rotation is significantly restricted and the energy release is mainly carried out in the form of fluorescence. This phenomenon highlights the unique behavior of the probe in different polar environments and reveals its potential in detecting changes in environmental polarity.

[0064] The probe of the present invention provides a new fluorescence sensing mechanism, opening up a new direction for the design and application of fluorescent probes, especially in complex chemical environments, and can be used for quantitative detection of polarity changes.

[0065] like Fig.11 As shown in a, XZTU-Pola has no viscosity response and no fluorescence in glycerol and methanol, but has strong fluorescence in non-polar solvents and has a typical polar response, such as Fig.11 a and 11b.

[0066] Fig.10 It was demonstrated that in the polar solvent methanol, the energy barrier is 2.95 eV, which indicates that the ability of molecular rotation is relatively strong and the energy is mainly consumed in the form of mechanical energy. In the non-polar solvent tetrahydrofuran, the energy barrier is 3.99 eV, indicating that the molecular rotation is significantly restricted and the energy release is mainly carried out in the form of fluorescence.

[0067] In order to study the correlation between XZTU-Pola and polarity, the present invention uses methanol and tetrahydrofuran as solvents with high polarity and low polarity, respectively. The absorption spectrum of XZTU-Pola in tetrahydrofuran shows that the maximum absorption wavelength is 480nm ( Fig.12 a and 12b). The fluorescence spectrum of XZTU-Pola in methanol shows weak fluorescence emission with very low quantum yield. However, the fluorescence quantum yield of XZTU-Pola in tetrahydrofuran is 0.24. Polar titration experiment (see Fig.12 c) shows that the fluorescence intensity of XZTU-Pola in tetrahydrofuran is about 150 times higher than that in methanol. Fig.12 d shows the correlation between the solvent dielectric constant and the fluorescence intensity. These results confirm that XZTU-Pola is highly sensitive to solvent polarity.

[0068] Fig.12 The results also investigated the effects of potential intracellular perturbations on XZTU-Pola. Fig.12 e shows that the fluorescence emitted by the probe is confirmed in the presence of metal ions (Fe 2+ , Fe 3 + , K + 、Na + , Ca 2+ and Zn 2+ ), 200 μM active sulfur species (SO2), 200 μM active oxygen species (H2O2, ·OH and HClO), 200 μM active nitrogen species (NO, NO2 – and ONOO – ) and 200μM biomacromolecules (GSH) had no effect on the fluorescence intensity of XZTU-Pola. However, the fluorescence of XZTU-Pola was significantly enhanced in tetrahydrofuran, but not in methanol. These results show that XZTU-Pola is highly resistant to interference and can be used for precise in situ imaging analysis of cell polarity.

[0069] like Fig.13 As shown, the same results were obtained in cell imaging. The fluorescence intensity was significantly different in cells with different polarities. Adding different concentrations of oleic acid and different incubation times of low-density lipoprotein can also change the cell polarity, and it was found that the fluorescence intensity changed significantly. However, by changing the incubation temperature and adding dexamethasone to change the cell viscosity, the fluorescence change was not obvious, and different pH would not change the fluorescence signal.

[0070] Therefore, the present invention effectively overcomes various shortcomings in the prior art and has a high industrial utilization value. The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology can modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A polarity-responsive fluorescent probe, characterized in that: The fluorescent probe has a structure as shown in Formula I:

2. A method for preparing a polarity-responsive fluorescent probe, characterized in that: The preparation method comprises the following steps: S1: reacting 4-ethynylaniline and bis(2-chloroethyl)ether under alkaline conditions to obtain compound 1; S2: reacting 3-iodoaniline and methyl iodide under alkaline conditions to obtain compound 2; S3: reacting compound 2, phosphorus oxychloride and N,N-dimethylformamide to obtain compound 3; S4: Compound 3 and 2-(3,5,5-trimethylcyclohex-2-ene-1-ylidene)malononitrile are reacted to obtain compound 4; S5: reacting compound 1 with compound 4 to obtain a polar-responsive fluorescent probe having a structure shown in formula I; 3. The preparation method according to claim 2, characterized in that: In step S1 and step S2, the alkaline condition is potassium carbonate alkaline condition.

4. The preparation method according to claim 2, characterized in that: In steps S1, S2 and S4, the reaction temperature is 70-80°C, and the reaction time is 9-12 hours.

5. The preparation method according to claim 2, characterized in that: In step S3, the phosphorus oxychloride first reacts with the N,N-dimethylformamide to obtain an intermediate, and the intermediate then reacts with the compound 2.

6. The preparation method according to claim 2, characterized in that: In step S1, the molar ratio of 4-ethynylaniline to bis(2-chloroethyl)ether is 1:(1-1.2); and / or in step S2, the molar ratio of 3-iodoaniline to methyl iodide is 1:(2-2.5); and / or in step S4, the molar ratio of the compound 3 to the 2-(3,5,5-trimethylcyclohex-2-ene-1-ylidene)malononitrile is 1:(1-1.2); And / or in step S5, the molar ratio of compound 1 to compound 4 is 1:(1-1.2).

7. The preparation method according to claim 5, characterized in that: In step S3, the reaction time of the intermediate and the compound 2 is 6 to 12 hours, and the solvent for the reaction of the intermediate and the compound 2 is dichloroethane.

8. The preparation method according to claim 2, characterized in that: In step S5, the reaction temperature is 25-30° C., and the reaction time is 6-10 hours.

9. The preparation method according to claim 2, characterized in that: In step S4, the catalyst of the reaction is piperidine; And / or in step S5, the catalyst for the reaction includes Pd(PPh3)2Cl2, CuI and PPh3.

10. A biosensor, characterized in that: The biosensor comprises the polarity-responsive fluorescent probe according to claim 1 or comprises the polarity-responsive fluorescent probe prepared by the preparation method according to any one of claims 2 to 9.

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