Strong push-pull electron type fluorescent compound as well as synthesis and application thereof

Abstract

The invention discloses a strong push-pull electronic fluorescent compound as well as synthesis and application thereof. The structure of the compound is shown as a formula (I). The synthesis method of the compound comprises the following steps: (1) carrying out nucleophilic substitution reaction on a compound in a formula (II) and a compound in a formula (III) to prepare 1, 2-dimethyl-3-[3-(triphenylphosphonium) propyl]-1H-benzimidazolium dibromide, namely a corresponding compound in a formula (IV); and (2) carrying out dehydration condensation reaction on the compound shown in the formula (IV) and a compound shown in a formula (V) to prepare the corresponding compound shown in the formula (I). The invention provides application of the compound as a fluorescent probe for quantitatively detecting RNA (Ribonucleic Acid) and / or viscosity. The compound provided by the invention has effective RNA (Ribonucleic Acid) recognition capability, sensitive viscosity responsiveness, large Stokes shift and good water solubility. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a strong push-pull electron type fluorescent compound, a synthesis method thereof, and application of the compound as a fluorescent probe for quantitatively detecting RNA and / or viscosity. Background Art

[0002] Ribonucleic acid (RNA), as an indispensable molecule in life activities, plays a core role in maintaining the precision and complexity of life activities. It is not only a key bridge between deoxyribonucleic acid (DNA) and protein, but also participates in and regulates the transcription and translation of genetic information, directly guiding protein synthesis and gene expression. Fluorescent probe technology has become an important method for detecting RNA due to its high sensitivity, high specificity and good biocompatibility. However, traditional fluorescent probes that only recognize RNA have gradually shown limitations in the study of complex physiological processes, that is, they are unable to monitor the synergy and mutual correlation between RNA and its surrounding microenvironment. Therefore, it is crucial to develop multifunctional fluorescent probes that can not only specifically recognize the response RNA, but also sense changes in the surrounding microenvironment.

[0003] Signal transmission between biomacromolecules in cells depends on the viscosity of the microenvironment, and appropriate viscosity helps the diffusion and transmission of signal molecules. The distribution of RNA in cells is mainly concentrated in the cytoplasm. The cytoplasm is a highly crowded and dynamically changing biochemical environment, in which various organelles are densely distributed and filled with a large number of biomacromolecules such as proteins, nucleic acids, and sugars, which together construct a highly "crowded" and dynamically changing biochemical environment. Therefore, different regions in the cell exhibit different microviscosity characteristics. Viscosity, as an important physical parameter reflecting the resistance to fluid flow, plays an important role in the stability and functional regulation of the cell microenvironment. Traditional viscosity measurement methods include: rotating barrel method, falling ball method, damped vibration method, cup viscometer method, capillary method, etc., but the above methods are only applicable to the measurement of viscosity changes in the macroscopic range, and are not applicable to viscosity detection in the microscopic range such as biological samples. At present, the development of fluorescent probes has provided an opportunity for the visualization of biological environments. Viscosity detection can be achieved by using the fluorescence change from dark state to bright state of molecular rotor fluorescent probes in the process of changing from non-viscous environment to viscous environment and the regular characteristic spectrum presented. Studies have shown that changes in viscosity are often closely related to the state of RNA. For example, processes such as RNA folding, assembly, and damage may cause changes in local viscosity. Therefore, the development of fluorescent probes with dual detection functions of RNA and viscosity will help deepen the understanding of the biological functions of RNA and its interaction with the cell microenvironment.

[0004] Fluorescent probes with large Stokes shifts can effectively reduce the interference of background excitation light on fluorescence in fluorescence measurement. In complex and changeable biological environments, the presence of background excitation light often becomes a major challenge for fluorescence measurement. These background lights may come from the spontaneous fluorescence of the sample, scattering or reflection of the excitation light source, etc. They are mixed with the fluorescence signal of the probe and can easily mask or interfere with the true signal of the target molecule. However, when the fluorescent probe has a large Stokes shift, there will be a clear spectral separation between its emission spectrum and the excitation spectrum. This means that the background excitation light is effectively filtered out and only the fluorescence signal of the probe is retained, making it possible to accurately capture the fluorescence signal of the target molecule in a complex biological environment.

[0005] Water solubility, as a key indicator of the degree of solubility of a substance in water, affects the activity of fluorescent probes in vivo. Fluorescent probes with good water solubility can pass through the intercellular space more smoothly, so that they can better locate the target location, providing a solid foundation for subsequent fluorescent signal detection.

[0006] In summary, the design and synthesis of a bifunctional fluorescent probe with large Stokes shift and good water solubility that can quantitatively detect RNA and viscosity has both theoretical and practical significance. Summary of the invention

[0007] The primary purpose of the present invention is to provide a new strong push-pull electron type compound, which has effective RNA recognition ability, sensitive viscosity responsiveness, large Stokes shift and good water solubility.

[0008] The second object of the present invention is to provide a method for synthesizing the compound.

[0009] The third object of the present invention is to provide the use of the compound as a bifunctional fluorescent probe for quantitative detection of RNA and / or viscosity.

[0010] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0011] In the first aspect, the present invention provides a compound, the structure of which is shown in formula (I), and the chemical name of the compound of formula (I) is 2-[(1E)-2-[4-(hexylphenylamino)phenyl]vinyl]-1-methyl-3-[3-(triphenylphosphonium)propyl]-1H-benzimidazolium dibromide:

[0012]

[0013] In a second aspect, the present invention provides a method for synthesizing the compound of formula (I), comprising the following steps:

[0014] (1) The compound of formula (II) undergoes a nucleophilic substitution reaction with the compound of formula (III) to obtain 1,2-dimethyl-3-[3-(triphenylphosphonium)propyl]-1H-benzimidazolium dibromide, i.e., the corresponding compound of formula (IV);

[0015]

[0016] (2) the compound of formula (IV) undergoes a dehydration condensation reaction with the compound of formula (V) to obtain the corresponding compound of formula (I);

[0017]

[0018] The nucleophilic substitution reaction described in step (1) 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 flask, then react at 80-150°C (preferably reflux temperature) for 10-30h (preferably 15-20h), and after the reaction is completed, the obtained reaction mixture is separated and purified to obtain the compound of formula (IV). The solvent used is generally toluene, benzene or N,N-dimethylformamide, and the molar amount of the solvent is 30-80 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 1-1.5:1. After the reaction is completed, the separation and purification method is preferably: the reaction mixture is cooled to room temperature, filtered, and the obtained solid is separated and purified by silica gel column chromatography, and the eluting reagent is dichloromethane and methanol (volume ratio is 10-35:1).

[0019] Preferably, the step (1) is implemented as follows:

[0020] Add the compound of formula (II), the compound of formula (III) and N,N-dimethylformamide into the reaction flask, and then heat under reflux to react for 15 to 20 hours, then cool to room temperature and filter. The obtained solid is separated and purified by silica gel column chromatography (eluents are dichloromethane and methanol) to obtain the compound of formula (IV).

[0021] The dehydration condensation reaction described in step (2) of the present invention is specifically implemented as follows: add the compound of formula (IV), the compound of formula (V) and a solvent into a reaction flask, stir and dissolve, then add a base, and then react at 30-150°C (preferably reflux temperature) for 10-40h (preferably 20-25h). 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 1.2-3.5 times the molar amount of the compound of formula (IV). 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 (IV). The molar ratio of the compound of formula (IV) to the compound of formula (V) is 1:1-2. After the reaction is completed, the separation and purification method is preferably: cooling the reaction mixture to room temperature, separating and purifying by alumina column chromatography, and the eluting reagent is dichloromethane and methanol (volume ratio is 20-50:1).

[0022] Preferably, step (2) is implemented as follows:

[0023] Add the compound of formula (IV), the compound of formula (V) and ethanol into the reaction flask, stir to dissolve, then add piperidine, and then heat to reflux to react for 20 to 25 hours, then cool to room temperature, and separate and purify the reaction mixture by alumina column chromatography (eluting reagents are dichloromethane and methanol) to obtain the target compound of formula (I).

[0024] In the present invention, the compounds represented by formula (II), formula (III) and formula (V) can be synthesized by methods reported in the literature, and the recommended synthetic routes are as follows:

[0025]

[0026] The compound of formula (I) provided by the present invention uses diphenylamine with hexyl as an electron donor with excellent electron-donating ability, and uses benzimidazolium and triphenylphosphonium groups connected by propyl groups as electron acceptors with excellent electron-withdrawing ability, thereby forming a strong push-pull electron system that can produce significant intramolecular charge transfer. The introduction of the double cation not only leads to very good water solubility of the compound of formula (I), but also enables it to more effectively combine with the negatively charged phosphate skeleton in RNA through electrostatic adsorption, thereby enhancing its targeted recognition ability for RNA. The benzimidazolium electron acceptor and the hexyldiphenylamine electron donor are connected by a rotatable ethylene conjugated bridge, which helps the compound of formula (I) to become a fluorescent molecular rotor. In an environment with low viscosity, the intramolecular rotation of the fluorescent molecular rotor causes it to undergo non-radiative transition, thereby quenching fluorescence; while in an environment with high viscosity, the intramolecular rotation is inhibited, and then the fluorescence is restored. This "twisted intramolecular charge transfer (TICT)" effect can achieve a sensitive fluorescence response of the compound of formula (I) to viscosity. In addition, the conjugated unit of the compound of formula (I) adopts a benzimidazole group and its entire conjugated system is moderate, so it will not cause a significant red shift in its maximum absorption wavelength, and the introduced dicationic electron acceptor can effectively reduce the LUMO energy level and cause its emission wavelength to undergo a significant red shift, thus ultimately being beneficial for the compound of formula (I) to produce a large Stokes shift.

[0027] Therefore, in a third aspect, the present invention provides the use of the compound of formula (I) as a fluorescent probe for quantitative detection of RNA and / or viscosity.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a new strong push-pull electron type fluorescent compound with effective RNA recognition ability, sensitive viscosity responsiveness, large Stokes shift and good water solubility, which can realize the practical application of quantitative detection of RNA and viscosity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The fluorescence emission spectra of the compound of formula (I) acting on yeast RNA of different molar concentrations are shown in FIG. The Z-axis coordinate represents the fluorescence intensity, the X-axis coordinate represents the wavelength, and the Y-axis coordinate represents the molar concentration ratio of RNA to the compound of formula (I).

[0030] Figure 2 The linear relationship between the logarithm of the fluorescence intensity of the compound of formula (I) and the logarithm of the yeast RNA molar concentration is fitted. The ordinate represents the logarithm of the fluorescence intensity, and the abscissa represents the logarithm of the RNA molar concentration.

[0031] Figure 3 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.

[0032] Figure 4 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.

[0033] Figure 5 The absorption and fluorescence emission spectra of the compound of formula (I) in Tris-HCl buffer solution are shown in Figure 1. The left ordinate represents absorbance, the right ordinate represents fluorescence intensity, and the abscissa represents wavelength. DETAILED DESCRIPTION

[0034] 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.

[0035] Example 1 Compound of formula (IV)

[0036] 0.35 g (2.4 mmol) of the compound of formula (II), 0.93 g (2.0 mmol) of the compound of formula (III) and 10 mL of N,N-dimethylformamide were added to the reaction flask, and then heated under reflux for 15 h, then cooled to room temperature, filtered, and the obtained solid was separated and purified by silica gel column chromatography (eluent: V 二氯甲烷 :V 甲醇 =20:1), to obtain 0.73 g of a white compound of formula (IV). 1 H NMR (DMSO-d6, 500MHz) δ: 8.02 (d, J = 7.5Hz, 2H), 7.91 (td, J1 = 7.1Hz, J2 = 1.4Hz, 3H), 7.73-7.83 (m, 12H), 7.65 (t, J = 7.5 Hz,1H), 7.61(t,J=7.5Hz,1H), 4.75(t,J=7.0Hz,2H), 3.99(s,3H), 3.85-3.91(m,2H), 2.89(s,3H), 2.06-2.13(m,2H).

[0037] Example 2 Compound of formula (IV)

[0038] 0.29 g (2.0 mmol) of the compound of formula (II), 0.93 g (2.0 mmol) of the compound of formula (III) and 20 mL of N,N-dimethylformamide were added to the reaction flask, and then heated under reflux for 20 h, then cooled to room temperature, filtered, and the obtained solid was separated and purified by silica gel column chromatography (eluent: V 二氯甲烷 :V甲醇 =20:1), to obtain 0.61 g of a white compound of formula (IV).

[0039] Example 3 Compound of formula (I)

[0040] 0.61 g (1.0 mmol) of the compound of formula (IV), 0.31 g (1.1 mmol) of the compound of formula (V) and 20 mL of ethanol 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 20 h, and then cooled to room temperature. The reaction mixture was separated and purified by alumina column chromatography (eluting reagent was V 二氯甲烷 :V 乙醇 =40:1), to give 0.45 g of the orange-yellow target compound of formula (I). 1 H NMR (DMSO-d6, 500MHz) δ: 8.04 (dd, J1=7.4Hz, J2=1.3Hz, 2H), 7.83 (d, J=8.8Hz, 2H), 7.82 (td, J1=7.4Hz, J2= 1.9Hz,3H), 7.64-7.78(m,14H), 7.62(td,J1=7.4Hz,J2=1.3Hz,1H), 7.48(t,J=7.9Hz,2H), 7.25-7.29(m,4H) , 6.84(d,J=8.8Hz,2H), 4.92(t,J=7.1Hz,2H), 4.12(s,3H), 3.90-3.97(m,2H), 3.80(t,J=7.5Hz,2H), 2.06- 2.14(m,2H), 1.59-1.65(m,2H), 1.33-1.37(m,2H), 1.25-1.28(m,4H), 0.85(t,J=7.0Hz,3H); HRMS(ESI): m / z calcd for C 49 H 52 N3P[M-2Br] 2+ :356.6944; found:356.6938.

[0041] Example 4 Compound of formula (I)

[0042] 0.61 g (1.0 mmol) of the compound of formula (IV), 0.34 g (1.2 mmol) of the compound of formula (V) and 15 mL of methanol were added to the reaction flask, stirred to dissolve, and then 0.13 g (1.5 mmol) of piperidine was added, and then heated under reflux for 15 h, and then cooled to room temperature. The reaction mixture was separated and purified by alumina column chromatography (eluting reagent was V 二氯甲烷 :V 乙醇 =40:1), to obtain 0.40 g of the orange-yellow target compound of formula (I).

[0043] Example 5 Fluorescence spectroscopy test of RNA interaction

[0044] The molar concentration of the compound of formula (I) in the Tris-HCl buffer solution is 5×10 -6 mol L -1 , gradually increasing the concentration of yeast RNA, and using RF-5301PC fluorescence spectrophotometer to measure the fluorescence emission spectra of the compound of formula (I) acting on RNA of different molar concentrations. The specific results are shown in Figure 1 The compound of formula (I) hardly fluoresces before reacting with RNA. After reacting with RNA, the fluorescence intensity gradually increases with the increase of RNA concentration. When saturation is reached, the fluorescence intensity increases by more than 30 times. Figure 2 The linear relationship between the logarithm of the fluorescence intensity of the compound of formula (I) and the logarithm of the RNA molar concentration is fitted. It can be seen from the figure that the fluorescence intensity (lgI) of the compound of formula (I) and the molar concentration (lgc RNA ) showed a good linear relationship, with a linear correlation coefficient of 0.9947, which can realize the quantitative detection of RNA.

[0045] Example 6 Fluorescence response test of viscosity

[0046] 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 3 The fluorescence emission spectra of the compound of formula (I) in different mass fractions of glycerol-water systems. It can be seen from the figure that as the glycerol ratio increases, that is, the viscosity increases, the fluorescence emission intensity of the compound of formula (I) continues to increase. From the pure water system to the 99% glycerol system, the fluorescence intensity increases by more than 40 times. Therefore, the compound of formula (I) has a very sensitive response to viscosity.

[0047] 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:

[0048] lgI=C+xlgη Formula (1)

[0049] 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.

[0050] Figure 4 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 0.9934, which can realize the quantitative detection of viscosity.

[0051] Example 7 Absorption and fluorescence emission spectrum test

[0052] Cells in organisms live in water environments, and the pH value of the extracellular fluid is maintained at 7.35-7.45. Therefore, the absorption and fluorescence emission spectra of the compound of formula (I) in Tris-HCl buffer solution (pH = 7.4) were measured. The absorption spectrum was measured using a Shimadzu UV-2550 ultraviolet-visible spectrophotometer, and the fluorescence emission spectrum was measured using a RF-5301PC fluorescence spectrophotometer. The specific results are shown in Figure 5 The Stokes shift of the compound of formula (I) in Tris-HCl buffer solution is very large, reaching about 130nm, 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.

[0053] 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 compound, whose structure is shown in formula (I), the chemical name of the compound of formula (I) is 2-[(1E)-2-[4-(hexylphenylamino)phenyl]vinyl]-1-methyl-3-[3-(triphenylphosphonium)propyl]-1H-benzimidazolium dibromide:

2. A method for synthesizing the compound according to claim 1, characterized in that: The synthesis method comprises the following steps: (1) The compound of formula (II) undergoes a nucleophilic substitution reaction with the compound of formula (III) to obtain 1,2-dimethyl-3-[3-(triphenylphosphonium)propyl]-1H-benzimidazolium dibromide, i.e., the corresponding compound of formula (IV); (2) the compound of formula (IV) undergoes a dehydration condensation reaction with the compound of formula (V) to obtain the corresponding compound of formula (I); 3. Use of the compound as claimed in claim 1 as a fluorescent probe for quantitative detection of RNA and / or viscosity.

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