Bis-cyano isophorone derivative fluorescent probe and application thereof in Hg < 2 + > detection
By designing a fluorescent probe HPQ-Hg, a dicyanoisophorone derivative fluorescent probe, which significantly improves the fluorescence intensity after reacting with Hg2+, the problem of low sensitivity of the existing Hg2+ detection methods is solved, and the detection effect of high sensitivity and selectivity is achieved.
Patent Information
- Application Number
- CN202510256912.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-05
- Publication Date
- 2025-05-27
AI Technical Summary
The existing Hg2+ detection methods have problems such as low sensitivity, expensive equipment and complex operation. The fluorescent probe law has limited its application due to problems such as low fluorescence quantum yield and small Stokes displacement.
A fluorescent probe HPQ-Hg of dicyanoisophorone derivative was designed and synthesized. By introducing the double-identification group thiocarbonate, the probe significantly increased its fluorescence intensity after responding to Hg2+.
Ultra-high sensitivity and selective detection of Hg2+ is achieved, with a detection limit of 0.52 nM, and the fluorescence intensity shows a good linear relationship with the mercury ion concentration, providing a fast and effective detection tool.
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Figure CN120040357A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of organic chemistry, and particularly relates to a dicyanoisophorone derivative fluorescent probe and its application in the detection of Hg 2+ . Background Art
[0002] Mercury ions (Hg 2+ ) are extremely dangerous heavy metal ions that pose a serious threat to the ecosystem and biosafety. Due to rapid industrialization, mercury pollution has become a global public health problem. When Hg 2+ enters the human body, due to its electron-deficient nature, it will interact with active substances such as enzymes, DNA, and glutathione (GSH), resulting in serious toxic effects. Long-term exposure to Hg 2+ in the environment can lead to serious pathological conditions in humans, such as neurodegenerative changes, cardiovascular and renal dysfunctions, autoimmune dysfunctions, and tumors. Therefore, there is an urgent need to develop a highly sensitive and selective technology for the rapid detection of Hg 2+ .
[0003] Traditional methods for detecting heavy metals mainly include electrochemical analysis, gas chromatography, atomic emission / absorption spectroscopy, and inductively coupled plasma mass spectrometry. Although these methods are very sensitive for detecting and quantifying heavy metals, they require expensive equipment, have a cumbersome sample processing procedure, and require highly skilled operators. In contrast, the fluorescence probe method has been developed due to its high measurement efficiency, high sensitivity, excellent spatiotemporal resolution, etc., and is suitable for environmental monitoring and biological sample analysis. Many Hg 2+ -recognizing fluorescence probes have been reported and applied in many aspects such as environmental monitoring and in vivo research, but these fluorescence probes are limited in their application due to problems such as low fluorescence quantum yield, small Stokes shift, and relatively high detection limit. Summary of the Invention
[0004] In view of the above deficiencies in the prior art, the present invention provides a dicyanoisophorone derivative fluorescent probe and its application in the detection of Hg 2+ .
[0005] The present invention designed and synthesized a Hg 2+ -detecting fluorescence probe HPQ-Hg. By introducing a double recognition group, thiocarbonate, the fluorescence intensity of HPQ-Hg can be significantly increased after reacting with Hg 2+ . After a series of analytical tests, the probe HPQ-Hg shows ultra-high sensitivity and selectivity, and the detection limit is only 0.52 nM. In addition, the fluorescence intensity of the probe shows a good linear relationship with the mercury ion concentration (R 2= 0.9962). The development of the fluorescent probe HPQ-Hg provides a powerful tool for the rapid detection of Hg content in e-cigarette aerosols. 2+ The present invention relates to a dicyanoisophorone derivative fluorescent probe, which is a fluorescent probe HPQ-Hg based on 2-(2'-hydroxyphenyl)-4(3H)-quinazolinone-modified dicyanoisophorone derivative, with the molecular formula C
[0006] H 41 H 29 ClN 4 O 4 S 2 , and its structure is as follows:
[0007] .
[0008] The preparation method of the dicyanoisophorone derivative fluorescent probe of the present invention includes the following steps:
[0009] Step 1: Dissolve isophorone (3.79 g, 27.6 mmol) and malononitrile (1.8 g, 27.6 mmol) in anhydrous ethanol (20 mL), then add piperidine (272 μL, 2.76 mmol), and reflux and stir the mixture at 80 °C for 8 h; after the reaction is completed, cool to room temperature, concentrate under reduced pressure and pass through a column, and the crude product is purified by silica gel column chromatography (eluent is petroleum ether) and then dried in vacuo to obtain a white solid compound 1.
[0010] Step 2: Dissolve the compound 1 (289 mg, 1.5 mmol) obtained in Step 1 and 4-hydroxybenzaldehyde (183 mg, 1.5 mmol) in anhydrous ethanol (10 mL), then add piperidine (10 μL, 0.15 mmol), and reflux and stir overnight at 80 °C; after cooling to room temperature, concentrate under reduced pressure and pass through a column, and the crude product is purified by silica gel column chromatography (eluent is dichloromethane) to obtain a red solid compound 2 (159 mg, yield 54%).
[0011] Step 3: Add compound 2 (1.49 g, 5 mmol) and HMTA (840 mg, 6 mmol) to trifluoroacetic acid (10 mL), and reflux and stir the mixture at 75 °C for 6 h; after cooling to room temperature, add hydrochloric acid to the mixed solution and stir at room temperature for 1 h, extract the mixed solution with dichloromethane to obtain an organic phase, wash with saturated sodium chloride solution, then dry with anhydrous sodium sulfate, and finally concentrate under reduced pressure to obtain a crude product, and the crude product is purified by silica gel column chromatography (eluent is V 石油醚 : V 乙酸乙酯 = 10:1) to obtain an orange solid compound 3 (398 mg, yield 24%).
[0012] Step 4: Dissolve compound 3 (95.5 mg, 0.3 mmol) in ethanol (10 mL), add 2-amino-5-chlorobenzamide (51 mg, 0.3 mmol), after refluxing at 80 °C for 30 min, add p-toluenesulfonic acid monohydrate (15 mg), and continue the reaction at 80 °C for 1 h; cool the reaction mixture to room temperature, add 2,3-dichloro-5,6-dicyanobenzoquinone (68 mg, 0.3 mmol), stir the reaction mixture at room temperature overnight, filter the precipitate, wash it three times with ethanol (10 mL) and twice with cold dichloromethane (10 mL), and dry to obtain a dark red powder compound 4 (105 mg, yield 75%).
[0013] Step 5: Synthesis of the fluorescent probe HPQ-Hg
[0014] Dissolve compound 4 (140 mg, 0.3 mmol) in anhydrous dichloromethane (10 mL), add triethylamine (100 μL, 0.8 mmol), stir for 5 min in an ice bath, then slowly add phenyl chloroformate (138 mg, 0.8 mmol), stir the reaction mixture at room temperature for 3 h; after the reaction is completed, rotary evaporate the solvent under reduced pressure, and purify the crude product by silica gel column chromatography (the eluent is V 石油醚 :V 二氯甲烷 = 5:1) to obtain a pale yellow solid fluorescent probe HPQ-Hg (93 mg, yield 42%).
[0015] The synthetic route is as follows:
[0016] 。
[0017] The application of the dicyanoisophorone derivative fluorescent probe of the present invention in the preparation of Hg 2+ detection reagent.
[0018] When the concentration of Hg 2+ is in the range of 0 - 10 μM, the fluorescence intensity of the detection reagent at 659 nm shows a linear relationship with the concentration of Hg 2+ .
[0019] The detection reagent can achieve rapid detection of the content of Hg 2+ in electronic cigarette aerosols.
[0020] In the present invention, dicyanoisophorone derivative dyes are selected as the fluorophore mother nucleus due to their large Stokes shift, and the modification of the HPQ group improves the quantum yield of the probe.
[0021] In summary, the present invention designs a fluorescent probe HPQ-Hg based on a dicyanoisophorone derivative modified with 2-(2'-hydroxyphenyl)-4(3H)-quinazolinone. By introducing a dual recognition group, thiocarbonate, this compound can rapidly and specifically react with Hg 2+ and produce a "turn-on" type change in fluorescence signal. Through the enhancement of the fluorescence signal, Hg 2+ can be quantitatively detected. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe HPQ-Hg. As can be seen from Figure 1 , the number of hydrogens in HPQ-Hg is consistent with that of the designed compound.
[0023] Figure 2 is the nuclear magnetic resonance carbon spectrum of the fluorescent probe HPQ-Hg. As can be seen from Figure 2 , the number of carbons in HPQ-Hg is consistent with that of the designed compound.
[0024] Figure 3 is the high-resolution mass spectrum of the fluorescent probe HPQ-Hg. As can be seen from Figure 3 , the relative molecular mass of HPQ-Hg (741.1361) is consistent with that of the designed compound (741.1352), indicating that HPQ-Hg has been successfully synthesized.
[0025] Figure 4 is the ultraviolet-visible absorption spectrum of the fluorescent probe HPQ-Hg before and after reaction with Hg 2+ . As can be seen from Figure 4 , the structure of HPQ-Hg changes after reaction with Hg 2+ , and a new absorption peak is generated near 480 nm.
[0026] Figure 5 is the fluorescence spectrum of the fluorescent probe HPQ-Hg after reaction with Hg 2+ (0 - 30 μM). As can be seen from Figure 5 , the fluorescence emission of HPQ-Hg increases at 659 nm after reaction with Hg 2+ .
[0027] Figure 6 is the curve graph of the fluorescence intensity change at 659 nm of the fluorescent probe HPQ-Hg after reaction with Hg 2+ (0 - 30 μM). As can be seen from Figure 6 , the fluorescence emission of HPQ-Hg increases gradually after reaction with Hg 2+ (0 - 10 μM) and reaches saturation at 10 μM.
[0028] Figure 7 is the linear fitting graph of the fluorescence intensity at 659 nm after the reaction of the fluorescent probe HPQ-Hg with Hg 2+ (0 - 10 μM). As can be seen from Figure 7 it, the fluorescence intensity after the reaction of HPQ-Hg with Hg 2+ (0 - 10 μM) has a good linear correlation with the Hg 2+ concentration, and the correlation coefficient R 2 = 0.993.
[0029] Figure 8 is the curve graph of the fluorescence intensity change at 659 nm with time after the reaction of the fluorescent probe HPQ-Hg with Hg 2+ (10 μM). As can be seen from Figure 8 it, the reaction of HPQ-Hg with Hg 2+ (10 μM) reaches saturation at about 600 s.
[0030] Figure 9 is the selectivity test graph of the fluorescent probe HPQ-Hg. As can be seen from Figure 9 it, the reaction of HPQ-Hg with Hg 2+ is not affected by potential interfering substances.
[0031] Figure 10 is the interference test graph of the fluorescent probe HPQ-Hg. As can be seen from Figure 10 it, when Hg 2+ coexists with potential interfering substances, the reaction of HPQ-Hg with Hg 2+ is not affected. Specific Embodiments
[0032] The technical solution of the present invention will be further analyzed and described below through specific embodiments.
[0033] Example 1: Preparation of the Fluorescent Probe HPQ-Hg
[0034] Step 1: Dissolve isophorone (3.79 g, 27.6 mmol) and malononitrile (1.8 g, 27.6 mmol) in anhydrous ethanol (20 mL), then add piperidine (272 μL, 2.76 mmol), and reflux and stir the mixture at 80 °C for 8 h. After cooling to room temperature, concentrate under reduced pressure and pass through a column. The crude product is purified by silica gel column chromatography (the eluent is petroleum ether) and then dried in vacuo to obtain a white solid compound 1 (3.78 g, yield 73%).
[0035] 11H NMR (600 MHz, Chloroform-d) δ 6.63 (q, J = 1.4 Hz, 1H), 2.53 (s, 2H), 2.19 (dd, J = 1.8, 1.0 Hz, 2H), 2.05 (d, J = 1.2 Hz, 3H), 1.03 (s, 6H). 13 13C NMR (151 MHz, Chloroform-d) δ 170.44, 159.86, 120.57, 113.21, 112.43, 45.66, 42.63, 32.38, 27.82, 25.33.
[0036] Step 2: Dissolve the obtained compound 1 (289 mg, 1.5 mmol) and 4-hydroxybenzaldehyde (183 mg, 1.5 mmol) in anhydrous ethanol (10 mL), then add piperidine (10 μL, 0.15 mmol), and reflux and stir at 80 °C overnight. After cooling to room temperature, concentrate under reduced pressure and pass through a column. The crude product is purified by silica gel column chromatography (eluent: dichloromethane) to obtain the red solid compound 2 (159 mg, yield 54%).
[0037] 1 1H NMR (600 MHz, Chloroform-d) δ 7.37 - 7.33 (m, 2H), 6.94 (d, J = 16.0 Hz, 1H), 6.83 - 6.75 (m, 3H), 6.72 (s, 1H), 2.52 (s, 2H), 2.39 (s, 2H), 1.00 (s, 6H). 13 13C NMR (151 MHz, Chloroform-d) δ 169.56, 157.38, 154.52, 136.95, 129.44, 128.57, 127.00, 122.74, 116.13, 113.71, 112.97, 43.04, 39.24, 32.06, 28.05.
[0038] Step 3: Add compound 2 (1.49 g, 5 mmol) and HMTA (840 mg, 6 mmol) to trifluoroacetic acid (10 mL), and reflux and stir the mixture at 75 °C for 6 h. After cooling to room temperature, add hydrochloric acid to the mixed solution and stir at room temperature for 1 h. Extract the mixed solution with dichloromethane to obtain the organic phase, wash it with saturated sodium chloride solution, dry it with anhydrous sodium sulfate, and finally concentrate under reduced pressure to obtain the crude product. The crude product is purified by silica gel column chromatography (eluent: V石油醚 : V 乙酸乙酯 = 10: 1) Purification gave orange solid compound 3 (398 mg, yield 24%).
[0039] 1 H NMR (600 MHz, Chloroform-d) δ 11.10 (s, 1H), 9.87 (s, 1H), 7.64 (d, J = 7.1 Hz, 2H), 6.99 - 6.93 (m, 2H), 6.85 (d, J = 16.1 Hz, 1H), 6.78 (s, 1H), 2.54 (s, 2H), 2.40 (s, 2H), 1.02 (s, 6H). 13 C NMR (151 MHz, Chloroform-d) δ 196.33 (d, J = 1.9 Hz), 169.20, 162.61, 153.45, 135.39, 134.83, 133.01, 128.40, 128.07, 123.59, 120.73, 118.77, 115.97, 113.44, 112.70, 42.98, 39.23, 32.07, 28.05.
[0040] Step 4: Dissolve compound 3 (95.5 mg, 0.3 mmol) in ethanol (10 mL), add 2-amino-5-chlorobenzamide (51 mg, 0.3 mmol), heat to 80 °C and reflux for 30 min, then add p-toluenesulfonic acid monohydrate (15 mg), and continue the reaction at 80 °C for 1 h. Cool the reaction mixture to room temperature, add 2,3-dichloro-5,6-dicyanobenzoquinone (68 mg, 0.3 mmol), and stir the reaction mixture at room temperature overnight. Filter the precipitate, wash it three times with ethanol (10 mL) and twice with cold dichloromethane (10 mL), and dry to obtain dark red powder compound 4 (105 mg, yield 75%).
[0041] 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.54 (s, 1H), 8.08 (d, J = 2.0 Hz, 1H), 7.88 - 7.69 (m, 3H), 7.27 (s, 2H), 6.94 (s, 1H), 6.78 (s, 1H), 2.61 (s, 2H), 2.56 (s, 2H), 1.03 (d, J = 4.2 Hz, 6H).13 C NMR (151 MHz, DMSO-d 6 ) δ 137.57, 135.55, 134.61, 131.85, 125.53, 119.09, 114.38, 113.69, 76.16, 42.71, 32.13, 27.92.
[0042] Step 5: Synthesis of the fluorescent probe HPQ-Hg
[0043] Dissolve compound 4 (140 mg, 0.3 mmol) in anhydrous dichloromethane (10 mL), add triethylamine (100 μL, 0.8 mmol), stir for 5 min in an ice bath, then slowly add phenyl chlorothionoformate (138 mg, 0.8 mmol), and stir the reaction mixture at room temperature for 3 h. After completion of the reaction, rotate and evaporate the solvent under reduced pressure. The crude product is purified by silica gel column chromatography (eluent V 石油醚 :V 二氯甲烷 = 5:1) to obtain the pale yellow solid fluorescent probe HPQ-Hg (93 mg, yield 42%).
[0044] 1 H NMR (600 MHz, Chloroform-d) δ 8.51 (s, 1H), 8.39 (s, 1H), 7.79 (s, 2H), 7.73 (d, J = 8.5 Hz, 1H), 7.43 - 7.29 (m, 4H), 7.26 (t, J = 7.9 Hz, 4H), 7.19 (t, J = 7.5 Hz, 1H), 7.11 - 7.00 (m, 2H), 6.92 (s, 1H), 6.71 (d, J = 7.9 Hz, 2H), 2.64 (s, 2H), 2.49 (s, 2H), 1.11 (s, 6H). 1313C NMR (151 MHz, Chloroform-d) δ 206.26, 169.10, 158.20, 153.13, 152.87, 152.10, 151.10, 144.53, 143.90, 135.79, 134.70, 133.51, 132.94, 132.19, 129.92, 129.68, 129.56, 129.03, 126.93, 126.85, 126.67, 126.47, 124.19, 122.93, 122.35, 121.55, 117.08, 116.02, 113.60, 113.32, 112.65, 79.39, 42.98, 39.20, 32.08, 28.05. HRMS (ESI, m / z): Calcd for C 41 H 29 ClN 4 O 4 S 2 [M+H] + , 741.1397; found, 741.1361.
[0045] Example 2: Fluorescence Detection
[0046] The mercury ion fluorescence probe detection method provided by the present invention can achieve efficient quantitative analysis of the mercury ion concentration in the analyte through the configured Hg²⁺ test system. First, the fluorescence probe is dissolved into a 1 mM stock solution in N,N-dimethylformamide (DMF), and the mercury ion standard solution is obtained by dissolving mercury chloride in deionized water. After condition optimization, the optical properties of the fluorescence probe are finally studied in a system of N,N-dimethylformamide:deionized water = 3:7, where the final concentration of the fluorescence probe is 10 μM.
[0047] To achieve quantitative analysis, the present invention uses a pre-constructed concentration standard curve. The standard curve consists of mercury ion standard solutions with known concentrations and corresponding fluorescence intensity data points. By comparing the fluorescence intensity of the sample with the relationship of the standard curve, the concentration of mercury ions in the analyte can be accurately calculated. During the construction and testing of the standard curve, the linear relationship between the fluorescence signal and the mercury ion concentration is fully considered, and through multiple calibrations and verifications, the high precision and high reliability of the measurement results are ensured.
[0048] When Hg 2+When (10 μM) was added to the probe solution (10 μM), the absorption peak at 420 nm disappeared, and at the same time, a new absorption peak appeared at 488 nm. When observed with the naked eye, the color of the solution changed from light yellow to dark red. In the case of Hg 2+ After addition, the reaction between the probe and Hg 2+ was completed within 600 s, accompanied by a change in the color of the solution. Subsequently, the fluorescence intensity (λ 2+ : 488 nm, λ ex = 659 nm) after the reaction of the probe (10 μM) with 0 - 30 μM Hg em was recorded. It was found that the fluorescence intensity of the probe (10 μM) at 659 nm after the addition of Hg 2+ showed a linear relationship with the concentration of Hg 2+ in the concentration range of 0 - 10 μM. When the concentration exceeded 10 μM, the fluorescence intensity of the probe no longer increased significantly. The fluorescence probe HPQ - Hg (10 μM) reached saturation in the response to Hg 2+ (10 μM) at 600 s. The probe HPQ - Hg could specifically recognize Hg 2+ (10 μM), showed almost no response to competitive substances (100 μM), and had good anti - interference ability.
[0049] Subsequently, the present invention used the standard addition method. By adding Hg 2+ standards to the e - cigarette oil and diluting it to different concentrations (0, 2.5, 5, 7.5, 10.0 μM), the fluorescence intensity after its reaction with the fluorescence probe HPQ - Hg was detected in the optimized reaction system using a fluorescence spectrophotometer. According to the obtained linear fitting equation, the corresponding Hg 2+ concentration was calculated. The results are shown in Table 1. The recovery rates ranged from 96.4% - 100.4%, indicating that HPQ - Hg could accurately detect Hg 2+ in the e - cigarette oil in the concentration range of 0 - 10 μM.
[0050]
Claims
1. A fluorescent probe of a dicyanoisophorone derivative, referred to as HPQ-Hg, characterized in that Its structure is as follows: 。 2. The method for preparing the dicyanoisophorone derivative fluorescent probe according to claim 1, characterized in that The steps include: Step 1: Dissolve isophorone and malononitrile in anhydrous ethanol, then add piperidine, and reflux and stir to react at 80°C; after the reaction is completed, cool to room temperature, concentrate under reduced pressure and pass through a column, purify the crude product by silica gel column chromatography, and vacuum dry to obtain a white solid compound 1; Step 2: Dissolve the compound 1 obtained in step 1 and 4-hydroxybenzaldehyde in anhydrous ethanol, then add piperidine, and reflux and stir to react at 80°C; after the reaction is completed, cool to room temperature, concentrate under reduced pressure, and purify the crude product by silica gel column chromatography to obtain a red solid compound 2; Step 3: Compound 2 and HMTA were added to trifluoroacetic acid, and the mixture was heated to reflux and stirred for reaction; after the reaction was completed, the mixture was cooled to room temperature, hydrochloric acid was added to the mixed solution and stirred at room temperature for 1 h, and the mixed solution was extracted with dichloromethane to obtain an organic phase, which was washed with a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate, and finally concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to obtain an orange solid compound 3; Step 4: Compound 3 was dissolved in ethanol, 2-amino-5-chlorobenzamide was added, and the mixture was refluxed at 80°C for 30 min. Then, p-toluenesulfonic acid monohydrate was added and the reaction was continued at 80°C for 1 h. The reaction mixture was cooled to room temperature, 2,3-dichloro-5,6-dicyanobenzoquinone was added, and the reaction mixture was stirred at room temperature overnight. The precipitate was filtered, washed with ethanol and dichloromethane, and dried to obtain a dark red powder compound 4. Step 5: Compound 4 was dissolved in anhydrous dichloromethane, triethylamine was added, and after stirring in an ice bath for 5 min, phenyl chlorothioformate was slowly added, and the reaction mixture was stirred at room temperature for reaction; after the reaction was completed, the solvent was rotary evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a light yellow solid fluorescent probe HPQ-Hg; The synthetic route is as follows: 。 3. The dicyanoisophorone derivative fluorescent probe according to claim 1 is used in the preparation of Hg 2+ Application in detection reagents.
4. The use according to claim 3, characterized in that: When Hg 2+ When the concentration is in the range of 0-10 μM, the fluorescence intensity of the detection reagent at 659 nm is similar to that of Hg 2+ The concentration showed a linear relationship.
5. The use according to claim 4, characterized in that: The detection reagent can detect Hg in electronic cigarette aerosols 2+ Detection of content.