Aggregation-induced emission molecular rotor and synthesis and application thereof

Abstract

The invention discloses an aggregation-induced emission molecular rotor and synthesis and application thereof. The structure of the molecular rotor is shown as a formula (I). The invention provides a synthesis method of the molecular rotor, which comprises the following steps: carrying out dehydration condensation reaction on a compound shown in a formula (II) and a compound shown in a formula (III) to prepare the molecular rotor shown in a corresponding formula (I). The invention provides application of the molecular rotor as an AIE fluorescent probe for quantitatively detecting viscosity. The invention provides a novel molecular rotor with large Stokes shift, AIE characteristics and sensitive viscosity responsiveness, and the novel molecular rotor can realize quantitative detection of viscosity. # imgabs0 #

Description

Technical Field

[0001] The invention relates to an aggregation-induced luminescence molecular rotor, a synthesis method thereof and an application thereof as an AIE fluorescent probe for quantitatively detecting viscosity. Background Art

[0002] Viscosity has an important influence on biological systems at both the microscopic level (such as cells) and the macroscopic level. From the microscopic level, viscosity, as a typical and extremely important biological microenvironment parameter, plays a vital role in controlling physiological processes such as diffusion rate, signal transduction, and metabolite transport. Abnormal viscosity levels in organisms are associated with a variety of diseases, such as malignant tumors, atherosclerosis, Alzheimer's disease, lysosomal storage diseases, and neurodegenerative diseases. Therefore, detecting viscosity changes at the cellular level is of great significance for the diagnosis of diseases. However, traditional viscometers are only applicable to macroscopic fluids. More importantly, the viscosity of the cell microenvironment is uneven. Different components at different locations in the cell, such as the cytoplasm, organelles, or membrane systems, have different viscosities, which makes traditional viscometers unusable for cell microenvironment viscosity detection. At present, fluorescence technology shows great potential in cell microenvironment viscosity detection. Molecular rotors are special fluorescent substances, and the changes in their fluorescence signals usually come from the competition between the radiative transition of the excited state and the non-radiative transition of intramolecular rotation. In non-viscous media, the rotation of the molecular rotor itself is an effective fluorescence quenching pathway. However, when the molecular rotor is in a viscous medium, the rotation of the molecule itself will be restricted, reducing the energy consumption of the non-radiative pathway, resulting in enhanced fluorescence emission. Therefore, molecular rotors are widely used as viscosity fluorescent probes to detect changes in the viscosity of the cell microenvironment. In recent years, people have done a lot of work in developing new viscosity fluorescent probes, but there are still some problems to be solved, such as the Stokes shift needs to be improved. When imaging, the viscosity fluorescent probe with a small Stokes shift has a large overlap between the emission peak and the excitation peak due to the small difference between the emission light wavelength and the excitation light wavelength, which easily produces background interference and affects the accuracy of the detection results. Therefore, it is very necessary to develop a new viscosity fluorescent probe with a large Stokes shift.

[0003] It is also worth noting that most fluorescent molecules usually have obvious fluorescence emission in a uniformly dispersed solution, but the fluorescence will be significantly weakened or even quenched in the aggregated state. This phenomenon is called aggregation-induced quenching (ACQ). The ACQ effect of fluorescent molecules greatly limits their application in living cells. Scholars have adopted many strategies to solve this phenomenon, such as reducing the concentration of fluorescent molecules and thus reducing the degree of aggregation. However, the aggregation of molecules is a spontaneous process and it is difficult to inhibit it by various physical methods. Therefore, as a common phenomenon of traditional fluorescent molecules, the ACQ effect urgently needs scientific and convenient means to solve the breakthrough. With the deepening of research on fluorescent substances, the concept of aggregation-induced emission (AIE) was proposed in 2001, that is, fluorescent molecules show strong fluorescence emission in the aggregated state, but emit weak or almost no light in a uniformly dispersed solution. AIE fluorescent molecules usually have high sensitivity and strong resistance to photobleaching, and can solve the problem of fluorescence quenching caused by aggregation in biological applications, which gives them significant advantages in the field of biological analysis and imaging. However, most reported viscosity fluorescent probes do not have AIE characteristics.

[0004] In summary, the design and synthesis of a fluorescent probe that has both large Stokes shift and AIE properties and can quantitatively detect viscosity has both theoretical and practical significance. Summary of the invention

[0005] The primary purpose of the present invention is to provide a novel molecular rotor which has a large Stokes shift, AIE properties and sensitive viscosity responsiveness.

[0006] The second object of the present invention is to provide a method for synthesizing a molecular rotor.

[0007] The third object of the present invention is to provide an application of the molecular rotor as an AIE fluorescent probe for quantitatively detecting viscosity.

[0008] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0009] In the first aspect, the present invention provides a molecular rotor, whose structure is shown in formula (I). The chemical name of the compound of formula (I) is 3-(2-hydroxyethyl)-2-[(1E)-2-[4-[[4-(1H-imidazo[4,5-f][1,10]phenanthroline-2-yl)phenyl]phenylamino]phenyl]vinyl]-1-methyl-1H-benzimidazolium bromide:

[0010]

[0011] In a second aspect, the present invention provides a method for synthesizing the molecular rotor according to the first aspect, comprising the following steps:

[0012] The compound of formula (II) undergoes a dehydration condensation reaction with the compound of formula (III) to prepare the corresponding molecular rotor represented by formula (I);

[0013]

[0014] The dehydration condensation reaction of the present invention is specifically implemented as follows: add the compound of formula (II), the compound of formula (III) and the solvent into the reaction bottle, stir and dissolve, then add the base, and then react at 30-150°C (preferably reflux temperature) for 15-30h (preferably 18-25h). After the reaction is completed, the reaction mixture is separated and purified to obtain the target compound of formula (I). The base used is generally piperidine, triethylamine or potassium hydroxide, and the molar amount of the base is 1.2-3 times the molar amount of the compound of formula (II). The solvent is generally methanol, ethanol, chloroform, dichloromethane, acetonitrile, DMF or a mixture of several thereof, and the molar amount of the solvent is 200-700 times the molar amount of the compound of formula (II). The molar ratio of the compound of formula (II) to the compound of formula (III) is 1:1-2. After the reaction is completed, the separation and purification method is preferably: cooling the reaction mixture to room temperature, filtering with suction, and separating and purifying the obtained solid by alumina column chromatography, and the eluting reagent is dichloromethane and methanol (volume ratio is 10 to 40:1).

[0015] Preferably, the dehydration condensation reaction is carried out as follows:

[0016] Add the compound of formula (II), the compound of formula (III), chloroform and methanol into the reaction flask, stir to dissolve, then add piperidine, and then heat to reflux to react for 18 to 25 hours, then cool to room temperature and filter. The obtained solid is separated and purified by alumina column chromatography (eluting reagents are dichloromethane and methanol) to obtain the target compound of formula (I).

[0017] In the present invention, the compounds represented by formula (II) and formula (III) can be synthesized by methods reported in the literature, and the recommended synthetic routes are as follows:

[0018]

[0019] The compound of formula (I) provided by the present invention uses triphenylamine with a propeller structure as an electron donor, and introduces an imidazophenanthroline group and a benzimidazolium group as electron acceptors at both ends of the triphenylamine, so that the whole molecule forms an AD-π-A' configuration (A is an electron acceptor, D is an electron donor, and π is a conjugated bridge). The imidazophenanthroline electron acceptor and the triphenylamine electron donor are connected by a σ-bond, so that the two groups are easy to rotate freely around the CC single bond. The benzimidazolium electron acceptor is connected to the triphenylamine electron donor through an ethylene conjugated bridge that can also rotate. Therefore, the compound of formula (I) can become an effective fluorescent molecular rotor. In a low viscosity environment or a uniformly dispersed solution, the intramolecular rotation of the molecular rotor causes the energy of the excited state to be consumed more by non-radiative transition, resulting in weak fluorescence emission; while in a high viscosity environment or an aggregated state, the intramolecular free rotation is suppressed, and the energy of the excited state is released more by radiative transition, thereby enhancing the fluorescence emission. In addition, the benzimidazolium group carries a positive charge, and the introduction of the hydroxyethyl group also helps to form hydrogen bonds in the biological microenvironment, thereby increasing the water solubility of the compound of formula (I) and enabling it to be better applied to the detection of viscosity in biological systems.

[0020] Therefore, in a third aspect, the present invention provides the use of the molecular rotor described in the first aspect as an AIE fluorescent probe for quantitatively detecting viscosity.

[0021] Compared with the prior art, the beneficial effect of the present invention is that the present invention provides a new molecular rotor with large Stokes shift, AIE characteristics and sensitive viscosity responsiveness, which can realize the practical application of quantitative viscosity detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The fluorescence emission spectra of the compound of formula (I) in glycerol-water systems with different mass fractions are shown in Figure 1. The ordinate represents the fluorescence intensity and the abscissa represents the wavelength.

[0023] Figure 2 The absorption and fluorescence emission spectra of the compound of formula (I) in a 99% glycerol-water system are shown in Figure 1. The left ordinate represents absorbance, the right ordinate represents fluorescence intensity, and the abscissa represents wavelength.

[0024] Figure 3 The linear relationship between the logarithm of the fluorescence intensity of the compound of formula (I) and the logarithm of the viscosity in the glycerol-water system is fitted. The ordinate represents the logarithm of the fluorescence intensity, and the abscissa represents the logarithm of the viscosity.

[0025] Figure 4 The fluorescence emission spectra of the compound of formula (I) in different proportions of methanol-PBS mixed solvents are shown in Figure 1. The ordinate represents the fluorescence intensity and the abscissa represents the wavelength. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme will be further described clearly and completely by examples below. The materials, reagents or instruments used in the examples without indicating the manufacturer are all conventional products that can be purchased commercially. If the specific conditions are not indicated in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0027] Example 1 Compound of formula (I)

[0028] 0.49 g (1 mmol) of the compound of formula (II), 0.33 g (1.2 mmol) of the compound of formula (III), 20 mL of chloroform and 5 mL of methanol were added to the reaction flask, stirred to dissolve, and then 0.17 g (2.0 mmol) of piperidine was added, and then heated under reflux for 24 h, then cooled to room temperature, filtered, and the obtained solid was separated and purified by alumina column chromatography (eluting reagent was V 二氯甲烷 :V 甲醇 =15:1), to obtain 0.41 g of the yellow target compound of formula (I). 1 HNMR (DMSO-d6, 500MHz) δ: 14.23 (s, 1H), 9.22 (dd, J1 = 8.2Hz, J2 = 1.6Hz, 1H), 9.03 (dd, J1=4.1Hz, J2=1.6Hz, 1H), 9.02 (dd, J1=4.1Hz, J2=1.5Hz, 1H), 8.91 (dd, J1=8. 0Hz, J2=1.5Hz, 1H), 8.40 (d, J=8.7Hz, 2H), 8.03-8.05 (m, 2H), 7.86 (d, J=8.6Hz, 2 H), 7.84 (d, J = 16.3Hz, 1H), 7.83 (dd, J1 = 8.2Hz, J2 = 4.1Hz, 1H), 7.81 (dd, J1 = 8.0Hz ,J2=4.1Hz,1H), 7.67(td,J1=7.5Hz,J2=1.3Hz,1H), 7.64(td,J1=7.4Hz,J2=1.2H z,1H), 7.46(t,J=7.9Hz,2H), 7.43(d,J=16.3Hz,1H), 7.27(d,J=8.6Hz,2H), 7.26( t,J=7.5Hz,1H), 7.22(d,J=7.9Hz,2H), 7.14(d,J=8.7Hz,2H), 5.29(t,J=5.6Hz,1H ), 4.70 (t, J = 4.5Hz, 2H), 4.16 (s, 3H), 3.86 (q, J = 5.2Hz, 2H); HRMS (ESI): m / zcalcd forC 43 H 35 N7O[M-Br+H] 2+:332.6446; found:332.6445.

[0029] Example 2 Compound of formula (I)

[0030] 0.49 g (1 mmol) of the compound of formula (II), 0.30 g (1.1 mmol) of the compound of formula (III) and 20 mL of chloroform were added to the reaction flask, and then 0.13 g (1.5 mmol) of piperidine was added, and then heated under reflux for 20 h, then cooled to room temperature, filtered, and the obtained solid was separated and purified by alumina column chromatography (elution reagent was V 二氯甲烷 :V 甲醇 =15:1), to obtain 0.36 g of the yellow target compound of formula (I).

[0031] Example 3 Fluorescence response test of viscosity

[0032] In the fluorescence response test of the compound of formula (I) to viscosity, a glycerol-water system was used to simulate the viscosity environment. Glycerol-water was prepared into 11 gradient solutions with different viscosities (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%) according to different mass fractions. The constant sample concentration was 1×10 -5 mol L -1 , the test temperature is 20°C. The fluorescence emission spectrum is measured using a RF-5301PC fluorescence spectrophotometer. Figure 1 The fluorescence emission spectra of the compound of formula (I) in different mass fractions of glycerol-water systems. As can be seen from the figure: with the increase of the glycerol ratio, that is, the increase of viscosity, the fluorescence emission intensity of the compound of formula (I) continues to increase, and the fluorescence intensity increases by more than 40 times from the pure water system to the 99% glycerol system. This result shows that: in low-viscosity media, the compound of formula (I) can undergo free intramolecular rotation to form a "twisted intramolecular charge transfer (TICT)" state, so that the energy of the excited state is consumed more by non-radiative transition, resulting in a very low fluorescence emission intensity; while in high-viscosity media, the intramolecular rotation will be inhibited, and the energy of the excited state will be released more by radiative transition, which ultimately leads to a significant increase in the fluorescence emission intensity. Therefore, the compound of formula (I) has a very sensitive response to viscosity.

[0033] Figure 2 The absorption and fluorescence emission spectra of the compound of formula (I) in a 99% glycerol-water system are shown in the figure. As can be seen from the figure, the Stokes shift of the compound of formula (I) is very large, reaching about 150nm, so that its emission spectrum and absorption spectrum overlap very little, which can significantly reduce the fluorescence self-absorption phenomenon and increase the signal-to-noise ratio during biological imaging, thereby improving the accuracy and sensitivity of imaging.

[0034] The relationship between the fluorescence intensity of the compound of formula (I) and the environmental viscosity is expressed by -Hoffmann formula, that is, formula (1) is used for fitting:

[0035] lgI=C+xlgη Formula (1)

[0036] Where: I is the fluorescence intensity, C is a constant related to concentration and temperature, x is a constant related to the fluorescent dye, and η is the viscosity of the solvent system.

[0037] Figure 3 The linear relationship between the logarithm of the fluorescence intensity of the compound of formula (I) and the logarithm of the viscosity in the glycerol-water system is fitted. As can be seen from the figure: the fluorescence intensity (lgI) of the compound of formula (I) and the viscosity (lgη) show a good linear relationship, and the linear correlation coefficient reaches 0.9937, which can realize the quantitative detection of viscosity.

[0038] Example 4 AIE effect test

[0039] The fluorescence emission spectrum of the compound of formula (I) in a methanol-PBS mixed solvent system was tested, wherein methanol and PBS are its good solvent and poor solvent, respectively. This is a commonly used method to verify the AIE behavior of fluorescent chromophores, because when the poor solvent in the mixed solvent system reaches a certain appropriate ratio, the solubility of the molecule in the mixed solution will change, thereby promoting the formation of aggregates.

[0040] Figure 4 The fluorescence emission spectra of the compound of formula (I) in different proportions of methanol-PBS mixed solvents are shown in Figure 1. In the good solvent methanol, its fluorescence is very weak. With the addition of the poor solvent PBS, the fluorescence intensity gradually changes significantly: when the volume fraction of PBS is lower than 70%, the fluorescence intensity is not improved and is so weak that it is almost invisible; but when the volume fraction of PBS is equal to 70%, the fluorescence intensity is significantly enhanced due to the generation of aggregates; when the volume fraction of PBS is further increased to 95%, the peak fluorescence intensity is more than 50 times that of pure methanol; but when the volume fraction of PBS is further increased, the fluorescence intensity becomes weaker due to the particle size of the aggregates. This result shows that in a solvent system with a low PBS content, the molecules are in a highly dispersed state of dissolution, and the energy of the excited state is released by free rotation within the molecules, resulting in very weak fluorescence; as the proportion of the poor solvent PBS gradually increases, the molecules aggregate in the solution, which inhibits the free rotation process within the molecules, and greatly enhances the fluorescence emission intensity. Therefore, the compound of formula (I) has a significant AIE effect.

[0041] The above description is only an embodiment of the present application and is not intended to limit the protection scope of the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A molecular rotor, whose structure is shown in formula (I), and whose chemical name is 3-(2-hydroxyethyl)-2-[(1E)-2-[4-[[4-(1H-imidazo[4,5-f][1,10]phenanthroline-2-yl)phenyl]phenylamino]phenyl]vinyl]-1-methyl-1H-benzimidazolium bromide:

2. A method for synthesizing a molecular rotor as claimed in claim 1, characterized in that: The synthesis method comprises the following steps: The compound of formula (II) undergoes a dehydration condensation reaction with the compound of formula (III) to prepare the corresponding molecular rotor represented by formula (I); 3. Use of the molecular rotor as claimed in claim 1 as an AIE fluorescent probe for quantitatively detecting viscosity.

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