Aggregation-induced emission molecules rotor and synthesis and application thereof

Abstract

This invention discloses an aggregation-induced emission (AIE) molecular rotor, its synthesis, and its applications. The structure of the molecular rotor is shown in formula (I). This invention provides a method for synthesizing the molecular rotor, comprising the following steps: a compound of formula (II) undergoes a dehydration condensation reaction with a compound of formula (III) to obtain the corresponding molecular rotor shown in formula (I). This invention provides the application of the molecular rotor as an AIE fluorescent probe for quantitative detection of viscosity. This invention provides a novel molecular rotor that combines large Stokes shift, AIE characteristics, and sensitive viscosity response, enabling quantitative detection of viscosity.

Description

TECHNICAL FIELD

[0001] The present application relates to an aggregation-induced emission molecule rotor, a synthesis method thereof and application thereof as an AIE fluorescent probe for quantitatively detecting viscosity. BACKGROUND

[0002] Viscosity has important influence on biological systems, both at microscale (e.g. cell) and macroscale. At microscale, viscosity, as a typical and extremely important biological microenvironment parameter, plays a crucial role in controlling diffusion rate, signal transduction, metabolite transport and other physiological processes. Abnormal viscosity level in vivo is related to a variety of diseases, such as malignant tumor, atherosclerosis, Alzheimer's disease, lysosomal storage disease and neurodegenerative disease, etc. Therefore, detecting viscosity change at cell level has important significance for disease diagnosis. However, traditional viscometer is only suitable for macroscopic fluid, and more importantly, the viscosity of cell microenvironment is not uniform, and different components at different positions in cells, such as cytoplasm, organelles or membrane system, have different viscosities, which makes traditional viscometer unable to be used for cell microenvironment viscosity detection. At present, fluorescence technology shows great potential in cell microenvironment viscosity detection. Molecular rotor is a special fluorescent substance, and the change of its fluorescence signal usually comes from the competition between excited state radiative transition and intramolecular rotation, a non-radiative transition. In non-viscous medium, the rotation of the molecular rotor itself is an effective fluorescence quenching path. However, when the molecular rotor is in viscous medium, the rotation of the molecule itself will be restricted, reducing the consumption of non-radiative path energy, thus leading to fluorescence emission enhancement. Therefore, molecular rotor is widely used as a viscosity fluorescent probe to detect the change of cell microenvironment viscosity. In recent years, a lot of work has been done in developing new viscosity fluorescent probes, but there are still some problems to be solved, such as the Stokes shift to be improved. Viscosity fluorescent probes with small Stokes shift have small difference between emission wavelength and excitation wavelength when imaging, resulting in large area overlap between emission peak and excitation peak, which is easy to produce background interference and affect the accuracy of detection results. Therefore, it is very necessary to develop new viscosity fluorescent probes with large Stokes shift.

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

[0004] In summary, it is of both theoretical and practical significance to design and synthesize a fluorescent probe with large stokes shift and AIE characteristics for quantitative detection of viscosity. SUMMARY

[0005] The primary object of the present application is to provide a novel molecular rotor which has large stokes shift, AIE characteristics and sensitive viscosity response.

[0006] The second object of the present application is to provide a synthesis method of the molecular rotor.

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

[0008] To achieve the above objects, the technical scheme adopted by the present application is as follows:

[0009] In a first aspect, the present application provides a molecular rotor, which has the structure shown in formula (I), and 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 application provides a synthesis method of the molecular rotor of the first aspect, comprising the following steps:

[0012] The compound of formula (II) is subjected to a dehydration condensation reaction with the compound of formula (III) to produce the corresponding molecular rotor of formula (I).

[0013]

[0014] The dehydration condensation reaction is carried out according to the following procedure: the compound of formula (II), the compound of formula (III) and a solvent are added to a reaction flask, stirred and dissolved, a base is then added, and the reaction is carried out at 30-150°C (preferably at reflux temperature) for 15-30 hours (preferably for 18-25 hours). After the reaction is completed, the obtained reaction mixture is separated and purified to obtain the 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 used is generally methanol, ethanol, chloroform, dichloromethane, acetonitrile, DMF or a mixture of several solvents, and the molar amount of the solvent is 200-700 times the molar amount of the compound of formula (II). The molar amount 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 as follows: the reaction mixture is cooled to room temperature, filtered, and the obtained solid is separated and purified by column chromatography on an alumina column using dichloromethane and methanol (volume ratio of 10-40:1) as the eluent.

[0015] As a preferred embodiment, the dehydration condensation reaction is carried out according to the following procedure:

[0016] The compound of formula (II), the compound of formula (III), chloroform and methanol are added to a reaction flask, stirred and dissolved, piperidine is then added, and the reaction is carried out at reflux temperature for 18-25 hours. After the reaction is completed, the reaction mixture is cooled to room temperature, filtered, and the obtained solid is separated and purified by column chromatography on an alumina column using dichloromethane and methanol as the eluent to obtain the compound of formula (I).

[0017] In the present application, the compounds of formula (II) and formula (III) can be synthesized by the methods reported in the literature, and the recommended synthesis route is as follows:

[0018]

[0019] The compound of formula (I) provided by the present application is a triphenylamine with a propeller structure as an electron donor, and imidazophenanthroline groups and benzimidazolium groups are introduced at both ends as electron acceptors, so that the whole molecule forms an A-D-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 sigma bond, so that the two groups are easy to rotate around the C-C single bond. The benzimidazolium electron acceptor is connected to the triphenylamine electron donor through a vinyl conjugated bridge which 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, intramolecular rotation of the molecular rotor causes the energy of the excited state to be consumed more through non-radiative transition, resulting in weak fluorescence emission; while in a high viscosity environment or an aggregated state, intramolecular free rotation is inhibited, and the energy of the excited state is more released in the form of radiation transition, thereby enhancing the fluorescence emission. In addition, the benzimidazolium group is positively charged, and the introduction of hydroxyethyl also helps to form hydrogen bonds in the biological microenvironment, thereby increasing the water solubility of the compound of formula (I), so that it can be better applied to the detection of viscosity in biological systems.

[0020] Therefore, in a third aspect, the present application provides the use of the molecular rotor of the first aspect as an AIE fluorescent probe for quantitative detection of viscosity.

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

[0022] Figure 1 Figure 4 is the fluorescence emission spectrum of the compound of formula (I) in different mass fraction glycerol-water systems. The vertical coordinate represents the fluorescence intensity, and the horizontal coordinate represents the wavelength.

[0023] Figure 2 Figure 5 is the absorption and fluorescence emission spectrum of the compound of formula (I) in a 99% glycerol-water system. The left vertical coordinate represents the absorbance, the right vertical coordinate represents the fluorescence intensity, and the horizontal coordinate represents the wavelength.

[0024] Figure 3 Figure 6 is a linear relationship fitting between the logarithm of the fluorescence intensity and the logarithm of the viscosity of the compound of formula (I) in a glycerol-water system. The vertical coordinate represents the logarithm of the fluorescence intensity, and the horizontal coordinate represents the logarithm of the viscosity.

[0025] Figure 4 Figure 7 is the fluorescence emission spectrum of the compound of formula (I) in different proportions of methanol-PBS mixed solvents. The vertical coordinate represents the fluorescence intensity, and the horizontal coordinate represents the wavelength. DETAILED DESCRIPTION

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions will be further described clearly and completely below through embodiments. Materials, reagents, or instruments used in the embodiments, unless otherwise specified, are all commercially available conventional products. Where specific conditions are not specified in the embodiments, they are performed under conventional conditions or conditions recommended by the manufacturer.

[0027] Example 1: Compound of Formula (I)

[0028] 0.49 g (1 mmol) of compound (II), 0.33 g (1.2 mmol) of compound (III), 20 mL of chloroform, and 5 mL of methanol were added to a reaction flask and stirred to dissolve. Then, 0.17 g (2.0 mmol) of piperidine was added, and the mixture was heated under reflux for 24 h. After cooling to room temperature, the mixture was filtered, and the resulting solid was purified by alumina column chromatography (eluting reagent: V). 二氯甲烷 :V 甲醇 =15:1), yielding 0.41 g of the yellow target compound (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 compound (II), 0.30 g (1.1 mmol) of compound (III), and 20 mL of chloroform were added to a reaction flask, followed by 0.13 g (1.5 mmol) of piperidine. The mixture was then heated under reflux for 20 h, cooled to room temperature, and filtered. The resulting solid was purified by alumina column chromatography (eluting reagent: V). 二氯甲烷 :V 甲醇 =15:1), yielding 0.36 g of the yellow target compound (Ⅰ).

[0031] Example 3: Fluorescence response test to viscosity

[0032] In the fluorescence response test of compound (I) to viscosity, a glycerol-water system was used to simulate the viscosity environment. Eleven gradient solutions of different viscosities (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%) were prepared with glycerol-water at different mass fractions, and the constant sample concentration was 1×10⁻⁶. -5 mol L -1 The test temperature was 20℃. The fluorescence emission spectrum was measured using an RF-5301PC fluorescence spectrophotometer. Figure 1 The figure shows the fluorescence emission spectra of compound (I) in glycerol-water systems with different mass fractions. As can be seen from the figure, the fluorescence emission intensity of compound (I) continuously increases with the increase of the glycerol content, i.e., the increase of viscosity. From the pure water system to the 99% glycerol system, the fluorescence intensity increases by more than 40 times. This result indicates that in low-viscosity media, compound (I) can undergo intramolecular free rotation, forming a "twisted intramolecular charge transfer (TICT)" state, which causes more of the energy of the excited state to be consumed through non-radiative transitions, resulting in very low fluorescence emission intensity. In high-viscosity media, intramolecular rotation is suppressed, and more of the energy of the excited state is released through radiative transitions, ultimately leading to a significant increase in fluorescence emission intensity. Therefore, compound (I) has a very sensitive response to viscosity.

[0033] Figure 2 The figure shows the absorption and fluorescence emission spectra of compound (I) in a 99% glycerol-water system. As can be seen from the figure, compound (I) exhibits a large Stokes shift, reaching approximately 150 nm, resulting in very little overlap between its emission and absorption spectra. In bioimaging, this significantly reduces fluorescence self-absorption, increases the signal-to-noise ratio, and thus improves imaging accuracy and sensitivity.

[0034] The relationship between the fluorescence intensity of compound (I) and environmental viscosity is obtained through... - Fit the Hoffmann formula, i.e., formula (1):

[0035] lgI=C+xlgη Formula (1)

[0036] In the formula: 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 To fit the linear relationship between the logarithm of the fluorescence intensity and the logarithm of the viscosity of compound (I) in the glycerol-water system. As can be seen from the figure, the fluorescence intensity (lgI) and viscosity (lgη) of compound (I) show a good linear relationship, with a linear correlation coefficient of 0.9937, which enables quantitative detection of viscosity.

[0038] Example 4: AIE Effect Test

[0039] The fluorescence emission spectra of compound (I) in a methanol-PBS mixed solvent system were tested, where methanol and PBS are its good and bad solvents, respectively. This is a commonly used method for verifying the AIE behavior of fluorescent chromophores, because when the bad solvent in the mixed solvent system reaches a suitable proportion, the solubility of the molecule in the mixed solution changes, thereby promoting the formation of aggregates.

[0040] Figure 4 The fluorescence emission spectra of compound (I) in methanol-PBS mixed solvents with different proportions are shown. In the good solvent methanol, the 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 below 70%, the fluorescence intensity is not improved and is almost invisible; however, when the volume fraction of PBS is equal to 70%, the fluorescence intensity is significantly enhanced due to the formation of aggregates; when the volume fraction of PBS is further increased to 95%, the fluorescence peak intensity is more than 50 times that in pure methanol; however, when the volume fraction of PBS is further increased, the fluorescence intensity weakens due to the particle size of the aggregates. This result indicates that in solvent systems with low PBS content, the molecules are in a highly dispersed dissolved state, and the energy of the excited state is released through intramolecular free rotation, resulting in very weak fluorescence; as the proportion of the poor solvent PBS gradually increases, the molecules aggregate in the solution, inhibiting the intramolecular free rotation process, thus significantly enhancing the fluorescence emission intensity. Therefore, compound (I) exhibits a significant AIE effect.

[0041] The above description is merely an embodiment of this application and is not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A molecular rotor having the structure shown in formula (Ⅰ) and the chemical name 3-(2-hydroxyethyl)-2-[(1 E )-2-[4-[[4-(1 H -imidazo[4,5-f][1,10]phenanthroline-2-yl)phenyl]phenylamino]phenyl]vinyl]-1-methyl-1 H -Benzimidazolium bromide: (Ⅰ)。 2. A method for synthesizing a molecular rotor as described in claim 1, characterized in that: The synthesis method includes the following steps: Compound (II) undergoes a dehydration condensation reaction with compound (III) to obtain the molecular rotor shown in formula (I); (Ⅱ) (Ⅲ)。 3. The application of the molecular rotor as described in claim 1 in the preparation of an AIE fluorescent probe for quantitative detection of viscosity.

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