Push-pull electron-type fluorescent compounds and synthesis and application thereof

Abstract

The application discloses a strong push-pull electronic fluorescent compound and synthesis and application thereof. The structure of the compound is shown as formula (I). The synthesis method of the compound comprises the following steps: (1) a nucleophilic substitution reaction of a compound of formula (II) and a compound of formula (III) is carried out to prepare 1,2-dimethyl-3-[3-(triphenylphosphoniumyl)propyl]-1H-benzimidazolium dibromide, i.e. a corresponding compound of formula (IV); (2) a dehydration condensation reaction of the compound of formula (IV) and a compound of formula (V) is carried out to prepare a corresponding compound of formula (I). The application provides application of the compound as a fluorescent probe for quantitative detection of RNA and / or viscosity. The compound provided by the application has effective RNA recognition ability, sensitive viscosity responsiveness, a large Stokes shift and good water solubility.

Description

Technical Field

[0001] This invention relates to a strong push-pull electron fluorescent compound, its synthesis method, and its application as a fluorescent probe for the quantitative detection of RNA and / or viscosity. Background Technology

[0002] Ribonucleic acid (RNA), an indispensable molecule in life activities, plays a central role in maintaining the precision and complexity of these activities. It is not only a crucial bridge between deoxyribonucleic acid (DNA) and proteins, but also participates in and regulates the transcription and translation of genetic information, directly guiding protein synthesis and gene expression. Fluorescent probe technology, with its high sensitivity, high specificity, and good biocompatibility, has become an important method for RNA detection. However, traditional single-RNA fluorescent probes are gradually showing limitations in studying complex physiological processes, namely, their inability to monitor the synergistic effects and interrelationships between RNA and its surrounding microenvironment. Therefore, developing multifunctional fluorescent probes that can not only specifically recognize and respond to RNA but also sense changes in the surrounding microenvironment is crucial.

[0003] Signal transduction between intracellular biomolecules depends on the viscosity of the microenvironment; appropriate viscosity facilitates the diffusion and transmission of signaling molecules. RNA is primarily concentrated in the cytoplasm. The cytoplasm is a highly crowded and dynamically changing biochemical environment, densely populated with various organelles and abundant with biomolecules such as proteins, nucleic acids, and carbohydrates, collectively constructing a highly "crowded" and dynamically changing biochemical environment. Therefore, different regions within the cell exhibit varying microviscosity characteristics. Viscosity, as an important physical parameter reflecting fluid flow resistance, plays a crucial role in the stability and functional regulation of the cellular microenvironment. Traditional viscosity measurement methods include the rotating barrel method, falling ball method, damped vibration method, cup viscometer method, and capillary method. However, these methods are only suitable for measuring viscosity changes within a macroscopic range and are not applicable to viscosity detection in the microscopic range of biological samples. Currently, the development of fluorescent probes has provided an opportunity for the visualization of the biological environment. By utilizing the fluorescence changes from dark to bright states and the regular characteristic spectra exhibited by molecular rotor fluorescent probes as they transition from a non-viscous to a viscous environment, viscosity can be detected. Studies have shown that changes in viscosity are often closely related to the state of RNA; processes such as RNA folding, assembly, and damage can all cause changes in local viscosity. Therefore, developing fluorescent probes with dual RNA and viscosity detection capabilities will help deepen our understanding of the biological functions of RNA and its interaction with the cellular microenvironment.

[0004] Fluorescent probes with large Stokes shifts can effectively reduce the interference of background excitation light on fluorescence measurements. In complex and variable biological environments, the presence of background excitation light often poses a significant challenge to fluorescence measurements. This background light may originate from the sample's autofluorescence, scattering or reflection from the excitation source, etc., and it mixes with the probe's fluorescence signal, easily masking or interfering with the true signal of the target molecule. However, when a fluorescent probe has a large Stokes shift, a significant spectral separation is formed between its emission and excitation spectra. This means that background excitation light can be effectively filtered out, retaining only the probe's fluorescence signal, thus making it possible to accurately capture the fluorescence signal of target molecules in complex biological environments.

[0005] Water solubility, a key indicator of a substance's solubility in water, affects the activity of fluorescent probes within organisms. Fluorescent probes with good water solubility can more easily traverse intercellular spaces, allowing them to better locate their targets and providing a solid foundation for subsequent fluorescence signal detection.

[0006] In summary, the design and synthesis of a bifunctional fluorescent probe that combines large Stokes shift and good water solubility for the quantitative detection of RNA and viscosity is of both theoretical and practical significance. Summary of the Invention

[0007] The primary objective of this invention is to provide a novel push-pull electron compound that combines effective RNA recognition, sensitive viscosity response, large Stokes shift, and good water solubility.

[0008] A second objective of this invention is to provide a method for synthesizing a compound.

[0009] A third objective of this invention is to provide the application of the compound as a bifunctional fluorescent probe for the quantitative detection of RNA and / or viscosity.

[0010] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

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

[0012]

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

[0014] (1) Compound (II) undergoes a nucleophilic substitution reaction with compound (III) to prepare 1,2-dimethyl-3-[3-(triphenylphosphonium)propyl]-1H-benzimidazolium dibromide, which is the corresponding compound (IV);

[0015]

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

[0017]

[0018] The nucleophilic substitution reaction described in step (1) of this invention is specifically carried out as follows: Compound (II), compound (III), and solvent are added to a reaction flask, and then the reaction is carried out at 80–150 °C (preferably reflux temperature) for 10–30 h (preferably 15–20 h). After the reaction is completed, the resulting reaction mixture is separated and purified to obtain compound (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 compound (II). The molar ratio of compound (II) to compound (III) is 1–1.5:1. 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 silica gel column chromatography, with dichloromethane and methanol (volume ratio of 10–35:1) as the eluent.

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

[0020] Compound (II), compound (III), and N,N-dimethylformamide were added to a reaction flask, and the mixture was heated under reflux for 15–20 h. The mixture was then cooled to room temperature and filtered. The resulting solid was purified by silica gel column chromatography (eluents were dichloromethane and methanol) to obtain compound (IV).

[0021] The dehydration condensation reaction described in step (2) of this invention is carried out as follows: Compound (IV), compound (V), and solvent are added to a reaction flask, stirred to dissolve, and then a base is added. The reaction is then carried out at 30–150°C (preferably reflux temperature) for 10–40 h (preferably 20–25 h). After the reaction is completed, the resulting reaction mixture is separated and purified to obtain the target compound (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 compound (IV). The solvent is generally methanol, ethanol, chloroform, dichloromethane, acetonitrile, DMF, or a mixture thereof, and the molar amount of the solvent is 200–700 times the molar amount of compound (IV). The molar ratio of compound (IV) to compound (V) is 1:1–2. After the reaction is completed, the preferred separation and purification method is as follows: the reaction mixture is cooled to room temperature and purified by alumina column chromatography, with dichloromethane and methanol (volume ratio of 20-50:1) as the eluent.

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

[0023] Compound (IV), compound (V), and ethanol were added to a reaction flask and stirred to dissolve. Piperidine was then added, and the mixture was heated under reflux for 20–25 h. The mixture was then cooled to room temperature, and the reaction mixture was purified by alumina column chromatography (eluents were dichloromethane and methanol) to obtain the target compound (I).

[0024] In this invention, the compounds represented by formulas (II), (III), and (V) can all be synthesized using methods reported in the literature. The recommended synthetic routes are as follows:

[0025]

[0026] The compound of formula (I) provided by this invention uses hexyl diphenylamine as an electron donor with excellent electron-donating ability and benzimidazolium and triphenylphosphine groups linked by propyl groups as electron acceptors with excellent electron-withdrawing ability, thus forming a strong push-pull electron system capable of generating significant intramolecular charge transfer. The introduction of the two cations not only gives the compound of formula (I) very good water solubility, but also enables it to bind more effectively to the negatively charged phosphate backbone of RNA through electrostatic adsorption, thereby enhancing its targeting recognition ability for RNA. The benzimidazolium electron acceptor and the hexyl diphenylamine electron donor are connected by a rotatable ethylene conjugated bridge, which helps the compound of formula (I) to become a fluorescent molecular rotor. In a low-viscosity environment, the intramolecular rotation of the fluorescent molecular rotor leads to nonradiative transitions, thereby quenching fluorescence; while in a high-viscosity environment, the intramolecular rotation is suppressed, and the fluorescence is restored. This "twisted intramolecular charge transfer (TICT)" effect enables the compound of formula (I) to achieve a sensitive fluorescence response to viscosity. In addition, the conjugated unit of compound (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. The introduced dual cation electron acceptor can effectively reduce the LUMO energy level and cause a significant red shift in its emission wavelength. Therefore, it is ultimately beneficial for compound (I) to produce a large Stokes shift.

[0027] Therefore, in a third aspect, the present invention provides the application of the compound of formula (I) as a fluorescent probe for the 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 novel fluorescent compound with strong push-pull electron type that has effective RNA recognition ability, sensitive viscosity response, large Stokes shift and good water solubility, which can realize practical applications for quantitative detection of RNA and viscosity. Attached Figure Description

[0029] Figure 1 The fluorescence emission spectra are those of the compound of formula (I) reacting with yeast RNA at different molar concentrations. The Z-axis represents fluorescence intensity, the X-axis represents wavelength, and the Y-axis represents the molar ratio of RNA to the compound of formula (I).

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

[0031] Figure 3 The graph shows the fluorescence emission spectra of compound (I) in glycerol-water systems with different mass fractions. The vertical axis represents fluorescence intensity, and the horizontal axis represents wavelength.

[0032] Figure 4 To fit a linear relationship between the logarithm of fluorescence intensity and the logarithm of viscosity of compound (I) in the glycerol-water system. The ordinate represents the logarithm of fluorescence intensity, and the abscissa represents the logarithm of viscosity.

[0033] Figure 5 The graph shows the absorption and fluorescence emission spectra of compound (I) in Tris-HCl buffer solution. The left ordinate represents absorbance, the right ordinate represents fluorescence intensity, and the abscissa represents wavelength. Detailed Implementation

[0034] 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.

[0035] Example 1: Compound of Formula (IV)

[0036] 0.35 g (2.4 mmol) of compound (II), 0.93 g (2.0 mmol) of compound (III), and 10 mL of N,N-dimethylformamide were added to a reaction flask. The mixture was then heated under reflux for 15 h, cooled to room temperature, and filtered. The resulting solid was purified by silica gel column chromatography (eluent: V). 二氯甲烷 :V 甲醇 =20:1), yielding 0.73 g of white compound (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 = 7.5 Hz, 1H), 4.75 (t, J = 7.0 Hz, 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 compound (II), 0.93 g (2.0 mmol) of compound (III), and 20 mL of N,N-dimethylformamide were added to a reaction flask. The mixture was then heated under reflux for 20 h, cooled to room temperature, and filtered. The resulting solid was purified by silica gel column chromatography (eluent: V). 二氯甲烷 :V甲醇 =20:1), yielding 0.61 g of white compound (IV).

[0039] Example 3 Compound of Formula (I)

[0040] 0.61 g (1.0 mmol) of compound (IV), 0.31 g (1.1 mmol) of compound (V), and 20 mL of ethanol 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 20 h. After cooling to room temperature, the reaction mixture was purified by alumina column chromatography (eluting reagent V). 二氯甲烷 :V 乙醇 =40:1), yielding 0.45g of the orange-yellow target compound (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 (I)

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

[0043] Example 5: Fluorescence spectroscopy test of interaction with RNA

[0044] The molar concentration of compound (I) in Tris-HCl buffer solution is 5 × 10⁻⁶. -6 mol L -1 The concentration of yeast RNA was gradually increased, and the fluorescence emission spectra of the reaction between compound (I) and RNA of different molar concentrations were measured using an RF-5301PC fluorescence spectrophotometer. Specific results are shown in [link to results]. Figure 1 The compound of formula (I) showed almost no fluorescence before reacting with RNA. After reacting with RNA, the fluorescence intensity gradually increased with the increase of RNA concentration, and the fluorescence intensity increased more than 30 times when it reached saturation. Figure 2 The figure shows a linear relationship between the logarithm of the fluorescence intensity of compound (I) and the logarithm of the RNA molar concentration. As can be seen from the figure, the fluorescence intensity (lgI) of compound (I) is linearly related to the RNA molar concentration (lgc). RNA A strong linear relationship was observed between the two, with a linear correlation coefficient of 0.9947, enabling quantitative detection of RNA.

[0045] Example 6: Fluorescence response test to viscosity

[0046] 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 3 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. Therefore, compound (I) exhibits a very sensitive response to viscosity.

[0047] The relationship between the fluorescence intensity of compound (I) and environmental viscosity is obtained through... - Fit the Hoffmann formula, i.e., formula (1):

[0048] lgI=C+xlgη Formula (1)

[0049] 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.

[0050] Figure 4 A linear relationship was fitted between the logarithm of the fluorescence intensity and the logarithm of the viscosity of compound (I) in the glycerol-water system. The figure shows a good linear relationship between the fluorescence intensity (lgI) and viscosity (lgη) of compound (I), with a linear correlation coefficient of 0.9934, enabling quantitative detection of viscosity.

[0051] Example 7: Absorption and Fluorescence Emission Spectroscopy Tests

[0052] Cells in living organisms live in an aquatic environment, and the pH of the extracellular fluid is maintained between 7.35 and 7.45. Therefore, the absorption and fluorescence emission spectra of compound (I) in Tris-HCl buffer solution (pH = 7.4) were measured. The absorption spectrum was measured using a Shimadzu UV-2550 UV-Vis spectrophotometer, and the fluorescence emission spectrum was measured using an RF-5301PC fluorescence spectrophotometer. Specific results are shown in [link to results]. Figure 5 The compound of formula (I) exhibits a large Stokes shift in Tris-HCl buffer solution, reaching approximately 130 nm, resulting in very little overlap between its emission and absorption spectra. In bioimaging, this can significantly reduce fluorescence self-absorption, increase the signal-to-noise ratio, and thus improve the accuracy and sensitivity of imaging.

[0053] 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 compound having the structure shown in formula (I), wherein the chemical name of the compound of formula (I) is 2-[(1 E )-2-[4-(hexylphenylamino)phenyl]vinyl]-1-methyl-3-[3-(triphenylphosphonium)propyl]-1 H -Benzimidazolium dibromide: (Ⅰ)。 2. A method for synthesizing the compound as described in claim 1, characterized in that: The synthesis method includes the following steps: (1) Compound (II) undergoes a nucleophilic substitution reaction with compound (III) to prepare 1,2-dimethyl-3-[3-(triphenylphosphonium)propyl]-1 H - Benzimidazolium dibromide, i.e., the corresponding compound of formula (IV); (Ⅱ) (Ⅲ) (Ⅳ) (2) Compound (Ⅳ) undergoes a dehydration condensation reaction with compound (Ⅴ) to obtain the corresponding compound (Ⅰ); (Ⅴ)。 3. The use of the compound as described in claim 1 in the preparation of fluorescent probes for quantitative detection of RNA and / or viscosity.

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