A mercury ion ratio fluorescent probe based on hemicyanine and preparation and application thereof

Abstract

The application relates to a mercury ion ratio fluorescent probe based on a hemicyanine and preparation and application of the fluorescent probe, the fluorescent probe has a molecular formula of C 26 H 23 INOPS; the preparation of the fluorescent probe comprises the following steps: (1) 4-hydroxybenzaldehyde is reacted with diphenylthiophosphonic dichloride to obtain O-(4-formylphenyl)diphenylthiophosphonate, i.e. compound 3, (2) 4-methylpyridine is reacted with methyl iodide to obtain 1,4-dimethylpyridine iodide, i.e. compound 2, (3) compound 3 is reacted with compound 2 to obtain a mercury ion ratio fluorescent probe 4-(4-(diphenylthiophosphonate)styryl)-1-methylpyridine iodide, i.e. compound 1; the application of the fluorescent probe is that the fluorescent probe is reacted with mercury ions in a buffer solution, and the existence of the mercury ions is detected by using the ratio (F 504nm / F 450nm ) change of the fluorescence intensity at two different emission wavelengths. The probe preparation process of the application is simple, and the fluorescent probe molecule exhibits high selectivity and sensitivity in a coexisting system of mercury ions and other cations.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology for metal ion detection, specifically relating to a mercury ion ratio fluorescent probe based on hemicyanine and its preparation and application. Background Technology

[0002] Heavy metal pollution has become a global environmental problem. Among them, mercury (Hg) 2+ Mercury has received considerable attention due to its toxic and harmful effects on the environment and human health. Sources of mercury are diverse, including thermometers, barometers, coal-fired power plants, and gold mines, inevitably diffusing into the human environment, such as air, water, and land, making it easily accessible to the human body through respiration, skin, and the food chain. Furthermore, mercury not only accumulates and damages the kidneys, liver, and other organs, but it can also cause serious diseases such as motor disorders, cognitive impairment, damage to the nervous and immune systems, Minamata disease, cancer, and even death. Therefore, developing effective Hg... 2+ Detection methods are of great significance to human health and environmental protection. Fluorescent probes have advantages such as high sensitivity, good selectivity, low cost, small sample volume, and simple equipment, and have become an effective means of detecting heavy metal ions.

[0003] Currently regarding Hg 2+ In fluorescent probe research, most studies rely on single-wavelength fluorescence emission intensity changes, such as fluorescence quenching or enhancement. These single-wavelength emission-based fluorescent probes are susceptible to interference from instrument and probe solubility. Ratiometric fluorescent probes, however, overcome these factors. They detect metal ions by recording the ratio of two fluorescence emission peaks, effectively eliminating or reducing interference from the aforementioned factors and making the detection more accurate. However, ratiometric Hg... 2+ Fluorescent probes are indeed scarce, therefore ratiometric Hg probes are being developed. 2+ Fluorescent probes are a challenging area of ​​work. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a mercury ion ratio fluorescent probe based on hemicyanine that is highly selective, sensitive, rapid, and easy to use. The invention also provides a method for preparing and applying this mercury ion ratio fluorescent probe based on hemicyanine.

[0005] The technical problem solved by this invention is achieved through the following technical solution:

[0006] A mercury ion ratio fluorescent probe based on hemicyanine, with the molecular formula C 26 H 23 INOPS, the structure is as follows:

[0007]

[0008] A method for preparing a mercury ion ratio fluorescent probe based on hemicyanine includes the following steps:

[0009] (1) 4-hydroxybenzaldehyde reacts with diphenylthiophosphonochloride to give O-(4-formylphenyl)diphenylthiophosphonate, namely compound 3, whose structural formula is as follows:

[0010]

[0011] (2) 4-Methylpyridine reacts with iodomethane to give 1,4-dimethylpyridine iodide, i.e., compound 2, whose structural formula is as follows:

[0012]

[0013] (4) The compound 3 obtained in step (1) is reacted with the compound 2 obtained in step (2) to obtain the mercury ion ratio fluorescent probe 4-(4-(diphenylthiophosphate)styryl)-1-methylpyridine iodide, i.e., compound 1, whose structural formula is as follows:

[0014]

[0015] Moreover, the specific method for obtaining compound 3 in step (1) is as follows: 4-hydroxybenzaldehyde and triethylamine are dissolved in anhydrous dichloromethane. Diphenylthiophosphonyl chloride is diluted with anhydrous dichloromethane and slowly added to the solution under ice bath argon protection. The mixture is then heated to room temperature and reacted overnight. After the reaction is completed, the mixture is washed with water and brine respectively. The organic phase is dried with anhydrous sodium sulfate, filtered, and the solvent is evaporated to obtain a crude solid. The solid is purified by silica gel chromatography and collected as compound 3.

[0016] Moreover, the ratio of 4-hydroxybenzaldehyde to diphenylthiophosphonic chloride and triethylamine is 1:1:3.

[0017] Moreover, the specific method for obtaining compound 2 in step (2) is as follows: 4-methylpyridine and iodomethane are reacted in solvent-free form at room temperature for 6 hours. After the reaction is complete, a solid is precipitated. The solid is filtered and washed with ethyl acetate. The collected solid is compound 2.

[0018] Furthermore, the ratio of 4-methylpyridine to iodomethane is 1:1.5.

[0019] Moreover, the specific method for obtaining compound 1 in step (3) is as follows: dissolve compound 3 and compound 2 in anhydrous ethanol, add piperidine, heat under argon protection and reflux for 6 hours. After the reaction is complete, cool to room temperature and let stand overnight. A solid precipitates out. Filter and wash with a small amount of cold anhydrous ethanol. Collect the solid as compound 1.

[0020] Moreover, the ratio of compound 3 to compound 2 and piperidine is 1:1.5:0.6.

[0021] An application of a hemicyanine-based mercury ion ratio fluorescent probe involves reacting the fluorescent probe with mercury ions in a buffer solution, utilizing the ratio (F) of fluorescence intensity at two different emission wavelengths. 504nm / F 450nm The presence or absence of mercury ions is detected by observing changes in these changes.

[0022] Furthermore, before the addition of mercury ions, the maximum fluorescence emission wavelength of the reaction solution was located at 450 nm. After the addition of mercury ions, the fluorescence at 450 nm of the reaction solution weakened, and the maximum fluorescence emission wavelength red-shifted to 504 nm. The detection limit for mercury ions was 0.68 μM.

[0023] Advantages and effects of the present invention

[0024] 1. The synthesis of the probe of the present invention can be completed in only three steps, and the post-processing process is simple.

[0025] 2. The fluorescent probe molecules of this invention exhibit high selectivity and sensitivity in systems where mercury ions coexist with other cations. Attached Figure Description

[0026] Figure 1 To implement compound 3 in Case 1 1 H NMR spectrum;

[0027] Figure 2 To implement compound 3 in Case 1 13 C NMR spectrum;

[0028] Figure 3 To obtain the ESI-MS spectrum of compound 3 in Case 1;

[0029] Figure 4 To implement compound 2 in Case 1 1 H NMR spectrum;

[0030] Figure 5 To implement compound 2 in Case 1 13 C NMR spectrum;

[0031] Figure 6 To obtain the ESI-MS spectrum of compound 2 in Case 1;

[0032] Figure 7 To implement compound 1 in Case 1 1 H NMR spectrum;

[0033] Figure 8 To implement compound 1 in Case 113 C NMR spectrum;

[0034] Figure 9 To obtain the ESI-MS spectrum of compound 1 in Case 1;

[0035] Figure 10 (a) represents the ratio of fluorescence intensity (F) of compounds 1 and 2 to the effect of pH value. 504nm / F 450nm (a) shows the effect of pH on compound 1 and Hg. (b) shows the effect of pH on compound 1 and Hg. 2+ Fluorescence intensity ratio before and after reaction (F) 504nm / F 450nm Schematic diagram illustrating the impact of )

[0036] Figure 11 (a) shows the reaction of compound 1 with Hg under optimal test conditions. 2+ Schematic diagram of the absorption spectrum changes of the reaction, (b) shows the reaction of compound 1 with Hg 2+ The absorbance ratio of the reaction (A) 376nm / A 351nm Schematic diagram showing the change over time;

[0037] Figure 12 (a) shows the reaction of compound 1 with Hg under optimal test conditions. 2+ Schematic diagram of the fluorescence spectrum changes of the reaction, (b) shows the reaction of compound 1 with Hg 2+ The fluorescence intensity ratio of the reaction (F) 504nm / F 450nm Schematic diagram showing the change over time;

[0038] Figure 13 (a) shows compound 1 reacted with different concentrations of Hg. 2+ The fluorescence intensity ratio of the reaction (F) 504nm / F 450nm (a) is a schematic diagram showing the change over time, and (b) is the fluorescence intensity ratio (F) of compound 1. 504nm / F 450nm ) and Hg 2+ A schematic diagram illustrating the linear relationship between concentrations;

[0039] Figure 14 The ratio of absorbance before and after the reaction of compound 1 with different metal ions (A) 376nm / A 351 nm The diagram illustrates the changes (gray bars represent probes treated only with the labeled metal ions, black bars represent probes treated with the labeled metal ions followed by the addition of Hg). 2+ );

[0040] Figure 15 The ratio of fluorescence intensity (F) before and after the reaction of compound 1 with different metal ions.504nm / F 450nm The diagram illustrates the changes (gray bars represent probes treated only with the labeled metal ions, black bars represent probes treated with the labeled metal ions followed by the addition of Hg). 2+ );

[0041] Figure 16 This is a schematic diagram of the synthetic route for mercury ratio fluorescent probe compound 1. Detailed Implementation

[0042] The embodiments of the present invention will be further described in detail below. It should be emphasized that the embodiments described in the present invention are illustrative and not limiting. Therefore, the present invention is not limited to the embodiments described in the specific implementation. Other implementations derived by those skilled in the art based on the technical solution of the present invention also fall within the scope of protection of the present invention.

[0043] Example

[0044] (I) Preparation of the target compound mercury ion fluorescent probe

[0045] (1) Synthesis of compound 3

[0046]

[0047] 4-Hydroxybenzaldehyde (7.94 g, 36 mmol) and triethylamine (15 mL, 108 mmol) were dissolved in anhydrous dichloromethane (15 mL) and cooled to 0 °C in an ice bath. Diphenylthiophosphonyl chloride (9.10 g, 36 mmol) was diluted with anhydrous dichloromethane (15 mL) and slowly added to the solution under argon protection. The mixture was then heated to room temperature and reacted overnight. After the reaction was complete, the mixture was washed with water and brine, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated to obtain a crude solid, which was purified by silica gel chromatography. The solid was washed with ethyl acetate / petroleum ether (1:4-1:2 v / v) and collected in 68% yield.

[0048] Compound 3 1 H NMR spectrum as follows Figure 1 As shown, 1 H NMR (400MHz, d6-DMSO, ppm) δ9.91 (s, 1H), 8.03-7.97 (m, 4H), 7.88 (d, J = 8.4Hz, 2H), 7.67-7.63 (m, 2H), 7.61-7.57 (m, 4H), 7.36-7.34 (m, 2H).

[0049] Compound 3 13 C NMR spectra as follows Figure 2 As shown, 13C NMR(100MHz,d6-DMSO,ppm)δ191.65,154.95,154.87,133.57,132.92,132.7 7,132.75,132.48,131.22,131.08,130.97,129.02,128.88,121.92,121.87.

[0050] The ESI-MS spectrum of compound 3 is as follows: Figure 3 As shown, C 19 H 16 O2PS + ([M+H)) + ):calcd:339.1,found:339.0.

[0051] (2) Synthesis of Compound 2

[0052]

[0053] 4-Methylpyridine (0.93 g, 10 mmol) and iodomethane (0.94 mL, 15 mmol) were reacted in solvent-free solution at room temperature for 6 hours. After the reaction was complete, a solid precipitated out. The solid was filtered and washed with ethyl acetate, and the yield was 85%.

[0054] Compound 2 1 H NMR spectrum as follows Figure 4 As shown, 1 HNMR (400MHz, d6-DMSO, ppm) δ8.86 (d, J = 6.8 Hz, 2H), 7.97 (d, J = 6.4 Hz, 2H), 3.85 (s, 3H), 4.31 (s, 3H), 2.59 (s, 3H).

[0055] Compound 2 13 C NMR spectra as follows Figure 5 As shown, 13 C NMR (100MHz, d6-DMSO, ppm) δ158.01, 144.27, 127.87, 127.79, 47.20, 21.32.

[0056] The ESI-MS spectrum of compound 2 is as follows: Figure 6 As shown, ESI-MS m / z for C7H 10 N + ([MI)) + ):calcd:108.1,found:108.2.

[0057] (3) Synthesis of Compound 1

[0058]

[0059] Compound 3 (761.4 mg, 2.25 mmol) and compound 2 (352.7 mg, 1.5 mmol) were dissolved in anhydrous ethanol (10 mL), and piperidine (0.1 mL, 1 mmol) was added. The mixture was heated to 98 °C for 6 hours under argon protection. After the reaction was complete, the mixture was cooled to room temperature and allowed to stand overnight. A solid precipitated out. The solid was filtered and washed with a small amount of cold anhydrous ethanol. The solid was collected, with a yield of 40%.

[0060] Compound 1 1 H NMR spectrum as follows Figure 7 As shown, 1 H NMR(400MHz,d6-DMSO,ppm)δ8.85(d,J=6.8Hz,2H),8.18(d,J=6.8Hz,2H),8.02-7.93(m,5H),7.7 1(d,J=8.4Hz,2H),7.68-7.58(m,6H),7.43(d,J=16.4Hz,1H),7.23(d,J=7.6Hz,2H),4.25(s,3H).

[0061] Compound 1 13 C NMR spectra as follows Figure 8 As shown, 13 C NMR(100MHz,d6-DMSO,ppm)δ172.13,152.41,151.81,151.72,145.19,139.49,133.79,132.86,132.84,1 32.70,132.14,131.22,131.11,129.60,129.13,128.99,123.59,123.32,122.06,122.01,46.97,21.21.

[0062] The ESI-MS spectrum of compound 1 is as follows: Figure 9 As shown, ESI-MS m / z for C 26 H 23 NOPS + ([MI)) + ):calcd:428.1,found:428.1.

[0063] (II) Effects of pH changes on the fluorescence intensity of compound 1 before and after its reaction with mercury ions and on the fluorescence intensity of compound 2

[0064] Compounds 1 and 2 synthesized in Example (I) were dissolved in dimethyl sulfoxide to prepare 2 mmol / L stock solutions. The fluorescence properties of compounds 1 and 2 were measured at room temperature with excitation light at 363 nm at different pH values. The results are as follows: Figure 10 As shown. (a) is the ratio of fluorescence intensity (F) of compounds 1 and 2 to pH value. 504nm / F 450nm (a) Effect of pH on compound 1 and Hg, (b) Effect of pH on compound 1 and Hg 2+ Fluorescence intensity ratio before and after reaction (F) 504nm / F 450nm The effect of ) . Experimental results show that the fluorescence intensity ratio (F) of compound 1 504nm / F 450nm The fluorescence intensity ratio (F) of compound 2 remained essentially unchanged from pH 4.0 to 9.0, but decreased from pH 9.0 to 10.0. 504nm / F 450nm The pH remains almost unchanged between 4.0 and 10.0; when Hg is added... 2+ Subsequently, the fluorescence intensity ratio (F) of compound 2 was... 504nm / F 450nm The Hg level remained almost constant at pH 4.0–6.0, increased rapidly from 6.0–7.0, and remained constant within the range of 7.0–10.0, indicating that it is suitable for physiological pH levels. 2+ Conduct an inspection.

[0065] (III) Absorption and fluorescence spectra of compound 1

[0066] Under optimal testing conditions (10 mM HEPES, H₂O / EtOH, 4:1, v / v, pH 7.0, 25 °C), the absorption and fluorescence spectra of compound 1 were studied, and the results are as follows: Figure 11 and Figure 12 As shown. Figure 11 (a) Compound 1 and Hg under optimal test conditions 2+ The absorption spectrum changes of the reaction, (b) is the reaction of compound 1 with Hg 2+ The absorbance ratio of the reaction (A) 376nm / A 351nm (Changes over time) Figure 12 (a) Compound 1 and Hg under optimal test conditions 2+ The fluorescence spectrum changes of the reaction, (b) is the reaction of compound 1 with Hg 2+ The fluorescence intensity ratio of the reaction (F) 504nm / F 450nm (This varies over time.) Experimental results demonstrate that compound 1 reacts with Hg... 2+ The reaction will cause the hydrolysis of diphenylthiophosphonate. Therefore, compound 1 is suitable for Hg. 2+Rapid detection.

[0067] (iv) Detection of Hg by Compound 1 2+ Sensitivity study

[0068] To test compound 1 for Hg detection 2+ Sensitivity, different Hg 2+ Under concentration (0-60 μM) conditions, compound 1 reacts with Hg 2+ The fluorescence intensity ratio of the reaction (F) 504nm / F 450nm Changes over time (e.g.) Figure 13 (a) and the fluorescence intensity ratio of compound 1 (F 504nm / F 450nm ) and Hg 2+ Linear relationship of concentration (e.g.) Figure 13 (b)) was studied. Figure 13 (a) It can be seen that Hg 2+ Higher concentrations will increase the fluorescence intensity ratio (F) 504nm / F 450nm The changes are faster and greater. Figure 13 (b) It can be deduced that compound 1 can detect Hg. 2 + The detection limit was 0.68 μM, indicating that compound 1 can detect Hg. 2+ Perform a sensitivity check.

[0069] (V) Compound 1 on Hg 2+ Selective studies

[0070] To test the effect of compound 1 on Hg 2+ The selectivity of compound 1 before and after reaction with different heavy metal ions (A) 376nm / A 351nm ) and fluorescence intensity ratio (F 504nm / F 450nm Experimental studies were conducted on the changes in ) Figure 14 and Figure 15 ).Depend on Figure 14 and Figure 15 It can be seen that compound 1 affects Hg 2+ The detection is unaffected by other potentially competing metal ions, and is effective for Hg. 2+ The detection is highly selective.

Claims

1. A mercury ion ratio fluorescent probe based on hemicyanine, characterized in that: Its molecular formula is C 26 H 23 INOPS, the structure is as follows: 。 2. A method for preparing a mercury ion ratio fluorescent probe based on hemicyanine as described in claim 1, characterized in that... The steps include the following: (1) 4-hydroxybenzaldehyde reacts with diphenylthiophosphonochloride to give O-(4-formylphenyl)diphenylthiophosphonate, namely compound 3, whose structural formula is as follows: ； (2) 4-Methylpyridine reacts with iodomethane to give 1,4-dimethylpyridine iodide, i.e., compound 2, whose structural formula is as follows: ； (3) The compound 3 obtained in step (1) is reacted with the compound 2 obtained in step (2) to obtain the mercury ion ratio fluorescent probe 4-(4-(diphenylthiophosphonate)styryl)-1-methylpyridine iodide, i.e., compound 1, whose structural formula is as follows: 。 3. The method for preparing a mercury ion ratio fluorescent probe based on hemicyanine according to claim 2, characterized in that: The specific method for obtaining compound 3 in step (1) is as follows: 4-hydroxybenzaldehyde and triethylamine are dissolved in anhydrous dichloromethane. Diphenylthiophosphonyl chloride is diluted with anhydrous dichloromethane and slowly added to the solution under ice bath argon protection. The mixture is then heated to room temperature and reacted overnight. After the reaction is completed, the mixture is washed with water and brine respectively. The organic phase is dried with anhydrous sodium sulfate, filtered, and the solvent is evaporated to obtain a crude solid. The solid is purified by silica gel chromatography and collected as compound 3.

4. The method for preparing a mercury ion ratio fluorescent probe based on hemicyanine according to claim 3, characterized in that, The ratio of 4-hydroxybenzaldehyde to diphenylthiophosphonic chloride and triethylamine is 1:1:

3.

5. The method for preparing a mercury ion ratio fluorescent probe based on hemicyanine according to claim 2, characterized in that, The specific method for obtaining compound 2 in step (2) is as follows: 4-methylpyridine and iodomethane are reacted in solvent-free form at room temperature for 6 hours. After the reaction is complete, a solid is precipitated. The solid is filtered and washed with ethyl acetate. The collected solid is compound 2.

6. The method for preparing a mercury ion ratio fluorescent probe based on hemicyanine according to claim 5, characterized in that, The ratio of 4-methylpyridine to iodomethane is 1:1.

5.

7. The method for preparing a mercury ion ratio fluorescent probe based on hemicyanine according to claim 2, characterized in that, The specific method for obtaining compound 1 in step (3) is as follows: Dissolve compound 3 and compound 2 in anhydrous ethanol, add piperidine, heat under argon protection and reflux for 6 hours. After the reaction is complete, cool to room temperature and let stand overnight. A solid will precipitate out. Filter and wash with a small amount of cold anhydrous ethanol. Collect the solid as compound 1.

8. The method for preparing a mercury ion ratio fluorescent probe based on hemicyanine according to claim 7, characterized in that, The ratio of compound 3 to compound 2 and piperidine is 1:1.5:0.6.

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