A nosegomalkylcarbazole enhanced fluorescent probe for efficient detection of HSO3 - and its preparation method and application

Abstract

The application discloses a norpinane alkyl carbazole enhanced fluorescent probe for efficiently detecting HSO3- and a preparation method and application thereof. The fluorescent probe is THCB-T-CAE. The THCB-T-CAE is prepared by the following steps: taking THC-B as raw material, carrying out substitution reaction with 1-bromobutane to obtain a compound THCB-B; carrying out coupling reaction of the compound THCB-B with (5-formylthiophene-2-yl)boronic acid to obtain a compound THCB-TC; and carrying out condensation reaction of the compound THCB-TC with ethyl 2-cyanoacetate to obtain the fluorescent probe THCB-T-CAE. The probe can specifically detect HSO3-. Under the irradiation of ultraviolet light with a wavelength of 365 nm, after HSO3- is added into the probe solution, the fluorescence color of the solution changes from yellow to orange red, the response time is 4.6 min, the detection limit of the probe for HSO3- is 50 nM, and the linear detection range of the probe for HSO3- is 0-100 muM. The probe has the advantages of high sensitivity, fast response speed, good selectivity, wide detection range and the like, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of fine organic synthesis technology, and relates to a method for efficient detection of HSO3. - Nopinel carbazole-based enhanced fluorescent probes, their preparation methods, and applications. Background Technology

[0002] HSO3 - Sodium bisulfite (primarily sodium bisulfite) is a common food additive and industrial bleaching agent, widely used in food processing, brewing, papermaking, and pharmaceuticals. As a reducing bleaching agent and preservative, sodium bisulfite and other salts effectively inhibit enzymatic browning and microbial growth, extending the shelf life of food. They are also used to prevent browning of fruits and vegetables and to bleach sucrose. However, when illegally or excessively added, it can cause serious harm to human health. The International Agency for Research on Cancer (IARC) classifies sulfites as Group 3 carcinogens. Excessive intake can induce asthma, allergic reactions, headaches, and gastrointestinal discomfort; long-term exposure is also associated with nervous system damage and respiratory diseases. my country's GB 2760-2024 "National Food Safety Standard for the Use of Food Additives" clearly stipulates the maximum permitted usage of sulfites in various foods (calculated as SO2 residue), such as no more than 0.35 g / kg in candied fruit. Therefore, rapid and sensitive detection of bisulfite residues in food and environmental samples is of great significance for ensuring public food safety and ecological health.

[0003] Traditional HSO3 - The main detection methods include iodometric titration, hydrochloric acid pararosaniline spectrophotometry, ion chromatography, and high-performance liquid chromatography. While these methods offer high accuracy and precision, they generally suffer from drawbacks such as complex sample pretreatment, long analysis times, reliance on large instruments, and specialized operation, making them unsuitable for market supervision and rapid on-site screening. In contrast, fluorescent probe technology, due to its fast response, high sensitivity, good selectivity, ease of operation, and lack of complex equipment, has demonstrated significant advantages in food analysis, environmental monitoring, and bioimaging. For the detection of bisulfite, numerous fluorescent probes based on different recognition mechanisms have been reported, primarily including aldehyde nucleophilic addition, carbon-carbon double bond Michael addition, and ester deprotection. However, the choice of fluorophore is crucial to the probe's optical performance; most probes utilize common fluorophores such as coumarin, hemicyanine, and dicyanoisophorone. A method for detecting HSO3 was synthesized using nopinene, a natural renewable raw material, as the backbone. - The novel fluorescent probe THCB-T-CAE has not been reported in the literature. This probe has dual detection functions, including colorimetric and enhanced detection, and boasts a wide detection range, high sensitivity, and good selectivity, particularly for HSO3-. - In-situ rapid detection is of great significance. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the technical problem to be solved by this invention is to provide a method for efficient detection of HSO3. - A nopinel carbazole-enhanced fluorescent probe that specifically recognizes HSO3. - It can meet the requirements for detecting HSO3. - The invention addresses the usage requirements of [the present invention]. Another technical problem to be solved by the present invention is to provide a method for the efficient detection of HSO3. - This invention relates to a method for preparing a nopinel carbazole-enhanced fluorescent probe. Another technical problem this invention addresses is providing a method for the efficient detection of HSO3. - Nopinel carbazole-enhanced fluorescent probes for detecting HSO3 - Applications.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for efficient detection of HSO3 - The nopinelide-based enhanced fluorescent probe has the following structural formula:

[0007]

[0008] Its chemical name is ethyl 3-(5-(9-butyl-2,2-dimethyl-2,3,4,9-tetrahydro-1H-1,3-bridged methylenecarbazole-6-yl)thiophen-2-yl)-2-cyanoacrylate, abbreviated as THCB-T-CAE.

[0009] The aforementioned method for efficient detection of HSO3 - The preparation method of the nopinelide carbazole-enhanced fluorescent probe includes the following steps:

[0010] (1) Using 6-bromo-2,2-dimethyl-2,3,4,9-tetrahydro-1H-1,3-bridged methylene carbazole (THC-B) as a raw material, a substitution reaction was carried out with 1-bromobutane to obtain the compound 6-bromo-9-butyl-2,2-dimethyl-2,3,4,9-tetrahydro-1H-1,3-bridged methylene carbazole (THCB-B);

[0011] (2) Compound THCB-B undergoes a coupling reaction with (5-formylthiophene-2-yl)boronic acid to give compound 5-(9-butyl-2,2-dimethyl-2,3,4,9-tetrahydro-1H-1,3-bridged methylenecarbazole-6-yl)thiophene-2-carboxaldehyde (THCB-TC);

[0012] (3) Compound THCB-TC undergoes a condensation reaction with ethyl 2-cyanoacrylate to obtain the fluorescent probe ethyl 3-(5-(9-butyl-2,2-dimethyl-2,3,4,9-tetrahydro-1H-1,3-bridged methylene carbazole-6-yl)thiophen-2-yl)-2-cyanoacrylate (THCB-T-CAE).

[0013] In step (1), the specific preparation method of compound THCB-B is as follows:

[0014] 1) Dissolve 1 mmol of compound THC-B in 1-3 mL of DMF, under nitrogen protection, add 5-10 mmol of NaH at 0 °C and stir for 0.5-1 h, then add 1-3 mmol of 1-bromobutane and gradually increase the temperature to react at 40-80 °C for 10-15 h;

[0015] 2) After the reaction is complete, the reaction solution is cooled to room temperature, 10-20 mL of distilled water is added, and the solution is extracted three times with ethyl acetate. The combined extracts are then washed with distilled water and saturated saline solution until neutral. The solvent is evaporated to obtain the crude THCB-B product.

[0016] 3) The crude THCB-B product was purified by silica gel column chromatography to obtain the compound THCB-B.

[0017] In step (2), the specific preparation method of compound THCB-TC is as follows:

[0018] 1) Add 1 mmol of compound THCB-B, 1-3 mmol of (5-formylthiophene-2-yl)boric acid, 3-6 mmol of K2CO3, 0.15-0.45 mmol of 1,1′-bis(diphenylphosphino)ferrocene palladium(II) dichloromethane complex, and 10-30 mL of toluene and methanol mixed solvent (1:1, v / v) to a reaction flask, purge with nitrogen, and react at 60-80 °C for 30-60 min;

[0019] 2) After the reaction is complete, toluene and methanol are evaporated to obtain crude THCB-TC product;

[0020] 3) The crude THCB-TC product was purified by silica gel column chromatography to obtain the compound THCB-TC.

[0021] In step (3), the specific preparation method of compound THCB-T-CAE is as follows:

[0022] 1) Add 1 mmol of compound THCB-TC, 1-3 mmol of ethyl 2-cyanoacetate, 5-30 mL of ethanol and 0.5-2 mL of piperidine to a reaction flask and reflux for 3-6 h;

[0023] 2) After the reaction is complete, the ethanol is evaporated off, the residue is dissolved in toluene, and then washed with distilled water and saturated brine until neutral. The toluene is then evaporated off to obtain the crude THCB-T-CAE product.

[0024] 3) The crude THCB-T-CAE product was purified by silica gel column chromatography to obtain the compound THCB-T-CAE.

[0025] The aforementioned method for efficient detection of HSO3 - Nopinel carbazole-enhanced fluorescent probes for detecting HSO3 - Applications in [the study]. Under ultraviolet light irradiation at a wavelength of 365 nm, HSO3 was added to the THCB-T-CAE probe solution. - Subsequently, the fluorescence color of the solution changed from yellow to orange-red, demonstrating its ability to specifically recognize HSO3. - The response time is 4.6 minutes for HSO3. - The detection limit for HSO3 is 50 nM. - The linear detection range is 0–100 μM.

[0026] Beneficial effects: Compared with the prior art, the compound THCB-T-CAE prepared by the present invention using THC-B as a raw material can specifically recognize HSO3. - It can quickly and sensitively detect HSO3 in solution. - The method has the characteristics of high sensitivity, fast response speed, good selectivity and wide detection range, and has good application prospects. Attached Figure Description

[0027] Figure 1 It is at different concentrations of HSO3 - Fluorescence spectrum of the probe THCB-T-CAE in presence;

[0028] Figure 2 The probe THCB-T-CAE reacts with different concentrations of HSO3 - Fluorescence intensity after treatment and HSO3 - Linear relationship graph between concentrations;

[0029] Figure 3 This is the fluorescence spectrum of the probe THCB-T-CAE after interaction with other interfering analytes (at: blank, Na). + Ca 2 + Zn 2+ Mg 2+ K + Cu 2+ Fe 3+ HCO3 - CO3 2-NO2 - O2 - Cl - , Br - I - , Cys, Gln, Gly, Glu, Trp);

[0030] Figure 4 HSO3 was added to the THCB-T-CAE probe solution. - The graph shows the relationship between the fluorescence intensity of the probe solution and time after the reaction. Detailed Implementation

[0031] The specific embodiments of the present invention will be further described below with reference to specific examples.

[0032] Example 1

[0033] The synthetic reaction formula for the fluorescent probe THCB-T-CAE is as follows:

[0034]

[0035] (1) Preparation of compound THCB-B:

[0036] 289.05 mg (1 mmol) of compound THC-B was dissolved in 2 mL of DMF under nitrogen protection. 240 mg (10 mmol) of NaH was added at 0 °C and stirred for 1 h. Then, 411.1 mg (3 mmol) of 1-bromobutane was added and the temperature was gradually increased to 60 °C for 10 h. After the reaction was completed, the reaction solution was cooled to room temperature, 15 mL of distilled water was added, and the mixture was extracted three times with ethyl acetate. The combined extracts were washed with distilled water and saturated brine until neutral. After evaporating the solvent, the crude THCB-B product was purified by silica gel column chromatography to obtain 251.96 g of compound THCB-B, with a yield of 73%.

[0037] (2) Preparation of compound THCB-TC:

[0038] 1 mmol (345.11 mg) of compound THCB-B, 1 mmol (155.97 mg) of (5-formylthiophene-2-yl)boric acid, 3.5 mmol (483.74 mg) of K2CO3, and 0.15 mmol (122.50 mg) of 1,1′-bis(diphenylphosphino)ferrocene palladium(II) dichloromethane complex and 10 mL of a toluene and methanol mixture (1:1, v / v) were added to a reaction flask. The mixture was purged with nitrogen and reacted at 62 °C for 45 min. After the reaction was completed, the toluene and methanol were evaporated, and the crude THCB-TC product was purified by silica gel column chromatography to obtain 301.75 mg of compound THCB-TC, with a yield of 80%.

[0039] (3) Preparation of compound THCB-T-CAE:

[0040] 1 mmol (377.18 mg) of compound THCB-TC, 1.2 mmol (135.73 mg) of ethyl 2-cyanoacetate, 10 mL of ethanol, and 1 mL of piperidine were added to a reaction flask and refluxed for 4 h. After the reaction was completed, the ethanol was evaporated off, the residue was dissolved in toluene, and then washed with distilled water and saturated brine until neutral. The toluene was then evaporated off, and the crude THCB-T-CAE product was purified by silica gel column chromatography to obtain 401.39 mg of compound THCB-T-CAE, with a yield of 85%.

[0041] Example 2

[0042] The fluorescent probe THCB-T-CAE was prepared into a 10 μM aqueous solution (containing 1% DMSO), and HSO3 was added. - It was dissolved in water to prepare aqueous solutions with concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μM. The concentrations of HSO3 at different concentrations were measured using a fluorescence spectrophotometer via fluorescence spectrophotometry. - The fluorescence emission spectrum of the probe THCB-T-CAE is present, and the results are as follows: Figure 1 As shown. The results indicate that with the increase of HSO3 in the solution... - With increasing concentration, the fluorescence emission wavelength of the fluorescent probe gradually red-shifted from 580 nm to 610 nm, and the fluorescence emission intensity gradually increased. Next, the fluorescence intensity of the probe was compared with that of HSO3. - A linear fit was performed on the concentration change relationship to obtain the following results: Figure 2 The linear relationship shown is (y = 5.731 × 10). 3 x + 1.46114 × 10 5 R 2 =0.99853), and further calculations (LOD = 3Sb / m) yielded HSO3 - The detection limit is 50 nM. Meanwhile, under ultraviolet light irradiation at a wavelength of 365 nm, the fluorescence color of the solution changes from yellow to orange-red. This indicates that compound THCB-T-CAE can be used as a sensitive detector for HSO3 in solution. - Fluorescent probes with varying concentrations.

[0043] Example 3

[0044] THCB-T-CAE was prepared into a 10 μM aqueous solution, and different metal ions, anions, and amino acids were dissolved in water to prepare 100 μM solutions. The fluorescence emission spectra of THCB-T-CAE in the presence of different metal ions, anions, and amino acids were measured using a fluorescence spectrophotometer via fluorescence spectrometry. Figure 3 As shown. The results indicate that HSO3 - The addition of [a specific substance] causes a red shift in the fluorescence emission peak of the probe and a significant increase in intensity, while the addition of other analytes (blank, Na) [increases / distributions]. + Ca2 + Zn 2+ Mg 2 + K + Cu 2+ Fe 3+ CO3 2- NO2 - HCO3 - O2 - Cl - , Br - I - Compared with the control group (Cys, Gln, Gly, Glu, Trp), the fluorescence spectrum of the probe did not change significantly. This indicates that the probe THCB-T-CAE can specifically recognize HSO3. - The time dependence of the probe's fluorescence intensity was determined using fluorescence spectroscopy, and the results are as follows: Figure 4 As shown. This indicates the addition of HSO3. - Afterward, the fluorescence emission intensity gradually increased and reached a stable level after 4.6 minutes.

Claims

1. A method for efficient detection of HSO3 - The nopinel carbazole-enhanced fluorescent probe, its preparation method, and its application are characterized by, The fluorescent probe is THCB-T-CAE, and its structural formula is:

2. The method for efficient detection of HSO3 as described in claim 1 - A method for preparing a nopinel carbazole-enhanced fluorescent probe, characterized in that, Includes the following steps: (1) Using 6-bromo-2,2-dimethyl-2,3,4,9-tetrahydro-1H-1,3-bridged methylene carbazole (THC-B) as a raw material, a substitution reaction was carried out with 1-bromobutane to obtain the compound 6-bromo-9-butyl-2,2-dimethyl-2,3,4,9-tetrahydro-1H-1,3-bridged methylene carbazole (THCB-B); (2) Compound THCB-B undergoes a coupling reaction with (5-formylthiophene-2-yl)boronic acid to give compound 5-(9-butyl-2,2-dimethyl-2,3,4,9-tetrahydro-1H-1,3-bridged methylenecarbazole-6-yl)thiophene-2-carboxaldehyde (THCB-TC); (3) Compound THCB-TC undergoes a condensation reaction with ethyl 2-cyanoacrylate to obtain the fluorescent probe ethyl 3-(5-(9-butyl-2,2-dimethyl-2,3,4,9-tetrahydro-1H-1,3-bridged methylene carbazole-6-yl)thiophen-2-yl)-2-cyanoacrylate (THCB-T-CAE).

3. A method for efficient detection of HSO3 according to claim 2 - A method for preparing a nopinel carbazole-enhanced fluorescent probe, characterized in that, In step (1), the specific preparation method of compound THCB-B is as follows: 1) Dissolve 1 mmol of compound THC-B in 1-3 mL of DMF, under nitrogen protection, add 5-10 mmol of NaH at 0 °C and stir for 0.5-1 h, then add 1-3 mmol of 1-bromobutane and gradually increase the temperature to react at 40-80 °C for 10-15 h. 2) After the reaction is complete, the reaction solution is cooled to room temperature, 10-20 mL of distilled water is added, and the solution is extracted three times with ethyl acetate. The combined extracts are then washed with distilled water and saturated saline solution until neutral. The solvent is evaporated to obtain the crude THCB-B product. 3) The crude THCB-B product was purified by silica gel column chromatography to obtain the compound THCB-B.

4. A method for efficient detection of HSO3 according to claim 2 - A method for preparing a nopinel carbazole-enhanced fluorescent probe, characterized in that, In step (2), the specific preparation method of compound THCB-TC is as follows: 1) Mix 1 mmol of compound THCB-B, 1-3 mmol of (5-formylthiophene-2-yl)boronic acid, 3-6 mmol of K2CO3 and 0.15-0.45 mmol of 1,1′-bis(diphenylphosphino)ferrocene palladium(II) dichloromethane complex thoroughly, purge with nitrogen for protection, then add 10-30 mL of toluene and methanol mixed solvent (1:1, v / v), and react at 60-80 °C for 30-60 min; 2) After the reaction is complete, toluene and methanol are evaporated to obtain crude THCB-TC product; 3) The crude THCB-TC product was purified by silica gel column chromatography to obtain the compound THCB-TC.

5. A method for efficient detection of HSO3 according to claim 2 - A method for preparing a nopinel carbazole-enhanced fluorescent probe, characterized in that, In step (3), the specific preparation method of compound THCB-T-CAE is as follows: 1) Add 1 mmol of compound THCB-TC, 1-3 mmol of ethyl 2-cyanoacetate, 5-30 mL of ethanol and 0.5-2 mL of piperidine to a reaction flask and reflux for 3-6 h; 2) After the reaction is complete, the ethanol is evaporated off, the residue is dissolved in toluene, and then washed with distilled water and saturated brine until neutral. The toluene is then evaporated off to obtain the crude THCB-T-CAE product. 3) The crude THCB-T-CAE product was purified by silica gel column chromatography to obtain the compound THCB-T-CAE.

6. The method for efficient detection of HSO3 as described in claim 1 - Nopinel carbazole-enhanced fluorescent probes for detecting HSO3 - Applications in [the context of the text].

7. The application according to claim 6, characterized in that, The fluorescent probe THCB-T-CAE can specifically detect HSO3. - HSO3 was added to the probe solution under ultraviolet light irradiation at a wavelength of 365 nm. - Afterwards, the fluorescence color of the solution changed from yellow to orange-red, with a response time of 4.6 min, for HSO3. - The detection limit for HSO3 is 50 nM. - The linear detection range is 0–100 μM.

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