Naphthalene anhydride-based hypochlorous acid fluorescent probe with large Stokes shift, preparation method and application thereof

Abstract

The present invention discloses a naphthalene anhydride-based hypochlorous acid fluorescent probe with a large Stokes shift, its preparation method, and application. The structural formula of the naphthalene anhydride-based hypochlorous acid fluorescent probe with a large Stokes shift is: The present invention also specifically discloses a preparation method of the naphthalene anhydride-based hypochlorous acid fluorescent probe and its application in the selective detection of hypochlorous acid. The fluorescent probe S-NAD of the present invention has high sensitivity and selectivity in response to HClO and can be used to determine the hypochlorous acid content in actual water samples.

Description

Technical Field

[0001] The invention belongs to the technical field of hypochlorous acid fluorescent probes, and particularly relates to a naphthalene anhydride-based hypochlorous acid fluorescent probe with a large Stokes shift, a preparation method thereof, and an application thereof. Background Art

[0002] Reactive oxygen species (ROS) include hydrogen peroxide (H2O2), hydroxyl radical (·OH), superoxide anion (·O2 - ), singlet oxygen ( 1 O2), peroxynitrite (ONOO - ) and hypochlorous acid (HClO) have attracted widespread attention due to their importance in various physiological and pathological processes. Among these ROS, hypochlorous acid (HClO) is an important oxidant produced by myeloperoxidase in cells. It can eliminate invading bacteria and pathogens and plays a vital role in the human immune system. However, increasing evidence shows that excessive HClO can cause oxidative stress, which is associated with many diseases such as Parkinson's disease and neurodegenerative diseases, inflammation, and even cancer. Therefore, it is of great significance to develop a highly specific and sensitive detection method for HClO in biological systems.

[0003] In recent decades, fluorescence imaging techniques based on small molecule probes have attracted widespread attention due to their high sensitivity, real-time detection, ease of use, and noninvasive imaging capabilities in living systems. To date, researchers have developed numerous fluorescent probes using naphthalimide, fluorescein, phenothiazine, coumarin, and cyanine as characteristic HClO chromophores through specific HClO-triggered oxidation reactions. Unfortunately, the Stokes shifts of most of these reported fluorescent probes are less than 100 nm, making them susceptible to interference from self-absorption and autofluorescence. In contrast, fluorescent probes with long-wavelength emission characteristics and large Stokes shifts largely avoid these drawbacks and exhibit enhanced sensitivity due to the longer distance between excitation and emission wavelengths. In summary, developing fluorescent probes with long emission wavelengths and large Stokes shifts is crucial to making them more suitable for quantitatively tracking HClO in complex cellular environments.

[0004] Typically, in the design of fluorescent probe molecules, strong electron-donating or electron-withdrawing groups are introduced into the molecular structure via π-bridges to expand π-conjugation, red-shift the fluorescence emission of the fluorescent probe molecule, and increase the Stokes shift. Based on this, the present invention designs a hypochlorous acid fluorescent probe with long-wavelength emission and a large Stokes shift. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a naphthalene anhydride-based hypochlorous acid fluorescent probe with a large Stokes shift and a preparation method thereof. To obtain a hypochlorous acid fluorescent probe with long-wavelength emission and a large Stokes shift, two naphthaleneimide-based fluorescent probes, S-NAD and B-NAD, were developed by introducing different π-conjugated groups onto the 1,8-naphthalene unit. A lipophilic thiophene bridge is introduced into the fluorescent probe S-NAD, and the C=N bond portion serves as a reactive group. In the presence of HClO, this group is easily oxidized and cleaved, causing the fluorescence of the fluorescent probe to switch from "off" to "on," thereby enabling the detection of hypochlorous acid.

[0006] The present invention adopts the following technical solution to solve the above technical problems: a naphthalene anhydride-based hypochlorous acid fluorescent probe with a large Stokes shift, characterized in that the structural formula of the anhydride-based hypochlorous acid fluorescent probe is:

[0007]

[0008] The preparation method of the naphthenic anhydride-based hypochlorous acid fluorescent probe with a large Stokes shift of the present invention is characterized in that the synthesis route is:

[0009]

[0010] The invention discloses an application of a naphthalene anhydride-based hypochlorous acid fluorescent probe with a large Stokes shift in the selective detection of hypochlorous acid.

[0011] The invention discloses an application of the naphthenic anhydride-based hypochlorous acid fluorescent probe with a large Stokes shift in the determination of hypochlorous acid content in actual water samples.

[0012] Compared with existing technologies, this invention offers the following advantages and benefits: A novel fluorescent probe with a large Stokes shift is constructed by using a naphthalene anhydride group with excellent fluorescent properties as a fluorophore and introducing a thiophene group with extended π conjugation. Experimental results show that the introduction of the lipophilic thiophene bridge into the fluorescent probe S-NAD significantly increases the Stokes shift and red-shifts the fluorescence emission wavelength. The C=N bond in the fluorescent probe is easily oxidized and cleaved in the presence of HClO, producing a turn-on fluorescence signal. Furthermore, the fluorescent probe S-NAD exhibits high sensitivity and selectivity in response to HClO. Finally, this method has been demonstrated to be applicable to the determination of hypochlorous acid in water samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 H spectrum of the fluorescent probe B-NA-CDCl3.

[0014] Figure 2 H spectrum of the fluorescent probe S-NA-CDCl3.

[0015] Figure 3 H spectrum of the fluorescent probe S-NAD in DMSO.

[0016] Figure 4 Mass spectrometry of the fluorescent probe S-NAD.

[0017] Figure 5 Fluorescence spectra of fluorescent probe B-NA (10 μM) and fluorescent probe S-NA (10 μM), λex = 370 nm.

[0018] Figure 6 DFT theoretical calculations for the fluorescent probes B-NA and S-NA.

[0019] Figure 7 (A) and (B) are the fluorescence probes S-NAD and B-NAD in ClO - Fluorescence spectra before and after addition of (250 μM), λex=370 nm.

[0020] Figure 8 This study is about the response time and pH of the fluorescent probe S-NAD.

[0021] Figure 9 Standard curve for the determination of HClO using the fluorescent probe S-NAD.

[0022] Figure 10 is the effect of interference on the fluorescent probe S-NAD, and the analytes are blank, F - 、Cl - 、SO3 2- 、H2O2、ONOO - .O2 - PO4 3- 、HPO4 2- 、SO4 2- 、CO3 2- 、Ac - 、Lys、Gly、Cys、Arg、K + Mg 2+ 、Fe 3+ and ClO - ,λex=370nm.

[0023] Figure 11 This study investigates the response mechanism of the fluorescent probe S-NAD to HClO. DETAILED DESCRIPTION

[0024] The above contents of the present invention are further described in detail below through examples, but this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0025] Example

[0026] 1.1 Reagents and Instruments

[0027] All reagents used in the experiments were of analytical grade and purchased from commercial suppliers, including 4-bromo-1,8-naphthoic anhydride, n-propylamine, anhydrous ethanol, N,N-dimethylformamide, dimethyl sulfoxide, and 2,4-dinitrofluorobenzene, and were used without further purification. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectra were recorded using a Bruker AmaZon SL spectrometer and a Bruker DMX-400 spectrometer, respectively. Absorption and fluorescence measurements were performed on a U-4100 spectrophotometer and an Edinburgh FS5 spectrophotometer, respectively. High-performance liquid chromatography experiments were performed using an Agilent Technologies 1260 Infinity.

[0028] 1.2 Synthesis and characterization of each compound

[0029]

[0030] Synthesis routes of fluorescent probes B-NAD and S-NAD

[0031] 1.3 Synthesis of NA

[0032] 4-Bromo-1,8-naphthoic anhydride (1.385 g, 5 mmol) and n-propylamine (822 μL, 10 mmol) were dissolved in 30 mL of ethanol and heated under reflux overnight. After the reaction, the solvent was evaporated to obtain a crude yellow solid. The product was further purified by silica gel column chromatography (dichloromethane:petroleum ether = 2:1, v / v) to afford NA (1.347 g) as a white solid.

[0033] Synthesis of 1.4B-NA

[0034] NA (792 mg) and 4-formylphenylboronic acid pinacol ester (780 mg) were accurately weighed and placed in a 100 mL three-necked flask. 30 mL of tetrahydrofuran and 10 mL of a 2 M K2CO3 aqueous solution were then added. Tetraphenylphosphine palladium was then added to the mixture, and the mixture was refluxed at 70°C under nitrogen for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, extracted with dichloromethane, and purified by silica gel column chromatography (petroleum ether:dichloromethane = 1:2, v / v) to obtain the final product, B-NA.

[0035] Synthesis of 1.5B-NAD

[0036] Accurately weigh B-NA (95 mg) and 2,4-dinitrophenylhydrazine (100 mg) into a 100 mL round-bottom flask. Add 20 mL of anhydrous ethanol and 2 drops of glacial acetic acid. Reflux at 80°C until the reaction is complete. Cool to room temperature, rotary evaporate, and recrystallize from ethanol to obtain the final product, B-NAD.

[0037] Synthesis of S-NA: The synthesis method of compound S-NA refers to the synthesis of B-NA, except that 4-formylphenylboronic acid pinacol ester is replaced by 5-formyl-2-thiopheneboronic acid.

[0038] Synthesis of S-NAD: The fluorescent probe S-NAD was synthesized according to the method of B-NAD, except that the raw material B-NA was replaced by S-NA.

[0039] 1.6 Solution preparation and experimental operation methods

[0040] The stock solutions of fluorescent probe B-NAD (2.0 mM) and fluorescent probe S-NAD (2.0 mM) were prepared in dimethyl sulfoxide (DMSO). Amino acids (Lys, Gly, Cys, Arg), cations (K + Mg 2+ 、Fe 3+ ), anion (F - 、Cl - 、SO3 2- 、H2O2、ONOO - .O2 - PO4 3- 、HPO4 2- 、SO4 2- 、CO3 2- 、Ac - , and ClO - ) and other stock solutions were prepared with deionized water and stored until needed. Spectral measurements were performed in PBS buffer (10 mM, pH 7.4, containing 50% DMSO, v / v). At room temperature, the spectral measurements were performed with a slit width of 4 nm / 4 nm and λex = 370 nm.

[0041] 2.1 Fluorescence spectrum test

[0042] At room temperature, the fluorescence performance of the fluorescent probe B-NA and the fluorescent probe S-NA was first tested. Figure 5As shown in Figure 2, the maximum fluorescence emission peaks of the fluorescent probe B-NA and the fluorescent probe S-NA under 370nm light excitation are located at around 368nm and 452nm respectively. Compared with the fluorescent probe B-NA, the fluorescence emission of the fluorescent probe S-NA is significantly red-shifted. In order to study the relationship between the spectral properties and chemical structures of the two parent materials, Gaussian (DFT) theoretical calculations were performed, and the results are shown in Figure 2. Figure 6 As shown in Figure 2, the HOMO-LUMO energy gaps of the fluorescent probe B-NA and the fluorescent probe S-NA are 3.75eV and 3.49eV respectively. The introduction of 5-formyl-2-thiopheneboronic acid causes the fluorescence emission of the molecule to be significantly red-shifted. This result is also consistent with Figure 5 The observed fluorescence red shift from B-NA to S-NA is consistent.

[0043] 2.2 Optimization of experimental conditions

[0044] Firstly, the fluorescence spectroscopy of the fluorescent probe B-NAD and the fluorescent probe S-NAD was used to investigate the effects of ClO on the - Reactivity. Figure 7 As shown in (A) and (B), under 370nm light excitation, the fluorescent probe B-NAD and the fluorescent probe S-NAD almost did not produce fluorescence emission at 457nm and 530nm respectively. However, when ClO was added to the test system, - After adding HCl (250 μM), the fluorescence intensity of the fluorescent probe B-NAD and the fluorescent probe S-NAD at 457 nm and 530 nm was significantly enhanced. This is because before the addition of hypochlorous acid, the fluorescent probes had no fluorescence due to the PET interaction between 2,4-dinitrophenylhydrazine and naphthalene anhydride fluorophores. - Afterwards, due to the interruption of PET, the fluorescent probe emits significantly enhanced fluorescence. Among them, the Stokes shift of the fluorescent probe S-NAD after reacting with HClO reaches 160nm. - The increase in fluorescence intensity before and after the action and the size of the Stokes shift were compared, and the fluorescent probe S-NAD was selected for subsequent experiments.

[0045] Then, the pH range and response time of the fluorescent probe S-NAD before and after the reaction with HClO were tested, and the results were as follows: Figure 8As shown, under 370nm light excitation, with the addition of the fluorescent probe S-NAD (250μM) solution, the fluorescence intensity of the fluorescent probe at 530nm gradually increased and reached saturation within 3 minutes, indicating that the fluorescent probe S-NAD can rapidly respond to HClO. In addition, through the fluorescence spectrum response test of the fluorescent probe to HClO at different pH values, it was found that it showed a good response to HClO in the pH range of 7.4, indicating that the fluorescent probe S-NAD can be used for the detection of HClO in biological environments.

[0046] 2.3 Detection performance experiment

[0047] By optimizing the above experimental conditions, the detection performance of the fluorescent probe S-NAD for different concentrations of HClO was studied. Figure 9 As shown in the results, the response of the fluorescent probe S-NAD to HClO showed concentration dependence, that is, the fluorescence intensity value of the fluorescent probe at 530 nm had a good linear correlation with the concentration of HClO (0-200 μM), and the linear correlation coefficient reached 0.994. According to the 3σ / k formula, the detection limit was 45 nM, which proved that the fluorescent probe had a sensitive response to HClO.

[0048] 2.4 Impact of Interfering Substances

[0049] The experiment investigated the effect of possible interferences on HClO determination. Different types of analytes were added to the fluorescent probe S-NAD solution. The results were as follows: Figure 10 The results showed that among various analytes, only HClO had a significant effect on the fluorescence system, while other analytes did not significantly enhance the fluorescence intensity of the fluorescent probe at 530 nm, indicating that the fluorescent probe S-NAD can selectively detect HClO.

[0050] 2.5 Detection mechanism research

[0051] pass Figure 11 As can be seen from a, the fluorescence spectrum of the fluorescent probe S-NAD after reacting with HClO is basically consistent with that of the fluorescent probe S-NA. It is speculated that the product of the reaction of the fluorescent probe S-NAD with HClO is S-NA. - After the reaction, fluorescence emission was generated at 530 nm compared to Figure 5 Although the fluorescence emission of S-NA at 452 nm is red-shifted, this may be due to the inner filter effect between 2,4-dinitrophenylhydrazine generated after the reaction of the fluorescent probe with hypochlorous acid and the released fluorophore. Subsequently, the mixture after the reaction of the fluorescent probe S-NAD with HClO was subjected to mass spectrometry analysis. The results are shown in Figure 2. Figure 11As shown in b, the product S-NA and the fluorescent probe S-NAD correspond to 347.87 ([M+H] + ) and 528([MH] - ), which further verifies the speculated detection mechanism.

[0052] 2.6 Actual water sample analysis

[0053] Laboratory tap water was filtered and then a trace amount of HClO solution was added to prepare a simulated sample. A spike recovery experiment was then conducted using the same test conditions as in the previous experiment. The results are shown in Table 1. The results in Table 1 show that the spike recovery rates of the tap water samples ranged from 98.6% to 102.4%.

[0054] Table 1 Analysis results of real water samples (n=3)

[0055]

[0056] This study uses a naphthalene anhydride group with excellent fluorescence properties as a fluorophore and introduces a thiophene group with extended π conjugation to construct a novel fluorescent probe with a large Stokes shift. Experimental results show that the introduction of a lipophilic thiophene bridge into the fluorescent probe S-NAD significantly increases the Stokes shift and red-shifts the fluorescence emission wavelength. The C=N bond in the fluorescent probe is easily oxidized and cleaved in the presence of HClO, producing a turn-on fluorescence signal. Furthermore, the fluorescent probe S-NAD exhibits high sensitivity and selectivity in response to HClO. Finally, this method has been demonstrated to be applicable to the determination of hypochlorous acid in water samples.

[0057] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A naphthalene anhydride-based hypochlorous acid fluorescent probe, characterized in that The structural formula of the naphthalene anhydride-based hypochlorous acid fluorescent probe is:

2. A method for preparing the naphthalene anhydride-based hypochlorous acid fluorescent probe according to claim 1, characterized in that The synthetic route is:

3. Use of the naphthylic anhydride-based hypochlorous acid fluorescent probe according to claim 1 in the selective detection of hypochlorous acid, wherein the detection is for non-therapeutic or diagnostic purposes.

4. Application of the naphthylic anhydride-based hypochlorous acid fluorescent probe according to claim 1 in the determination of hypochlorous acid content in an actual water sample, wherein the determination is for non-therapeutic or diagnostic purposes.

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