Fluorescent probe based on benzo[b]phosphole-1-oxide molecular structure and preparation method and application thereof

Abstract

The application discloses a fluorescent probe based on a benzo[b]phosphole-1-oxide molecular structure and a preparation method and application thereof, and belongs to the technical field of analytical chemistry. The compound structure is shown in the following formula: the probe synthesized by the application has high sensitivity and high selectivity for NO2 recognition, and after the probe contacts with the detected substance NO2, the probe emits strong fluorescence intensity at 505 nm. In addition, after the probe contacts with the detected substance NO2, the probe solution also gradually changes from the previous yellow into colorless, and accurate judgment can be made on whether NO2 exists or not through the color change. The application can be applied to meat food preservation detection, water quality detection and three-waste pollution detection in heavy industry.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically a fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, its preparation method, and its application. Background Technology

[0002] Nitrites are inorganic salts widely found in nature and human activities. In the field of food safety, many foods produce NO2 during processing and storage. Excessive NO2 intake It may lead to various diseases, such as infantile methemoglobinemia, congenital malformations, congenital abnormalities of the central nervous system, and malignant tumors. The tolerable daily intake of NO2 in food... The amount was 0.06 mg / kg. -1 day -1 .

[0003] In the environment, NO2 Also a significant water pollutant, NO2 in rivers, lakes, and even drinking water sources is increasing due to the random discharge of domestic sewage, industrial wastewater, and other untreated wastewater. The concentration rises rapidly. This causes significant harm to aquatic life, such as fish deaths due to oxygen depletion. NO2 in water or food Excessive concentrations of NO2 pose an increasingly serious threat to human health and the environment. Therefore, NO2... Nitrite has become an important indicator of environmental and food safety. Under the aforementioned strict limits, developing rapid, sensitive, and reliable methods for detecting nitrite is of great significance for ensuring food safety.

[0004] Currently, the methods for detecting nitrite can be mainly divided into the following categories: (1) Traditional instrumental analysis methods Traditional detection methods include the Griess reagent colorimetric method, UV-Vis spectrophotometry, ion chromatography, and gas chromatography-mass spectrometry. Among these, the Griess reagent colorimetric method is the national standard recommended method, and its principle is based on the diazotization-coupling reaction to generate a colored azo compound. This type of method has high sensitivity; the detection limit for nitrite using ion chromatography can reach 0.01 mg / kg. -7 However, these methods generally rely on large, precision instruments, require professional operators, involve cumbersome sample pretreatment, and have high testing costs, making them difficult to meet the practical needs of rapid on-site testing and resource-limited areas.

[0005] (2) Electrochemical analysis method Electrochemical analysis methods, based on the redox properties of nitrite on electrode surfaces, offer advantages such as high sensitivity and rapid response. However, these methods are susceptible to interference from other electroactive substances in the complex matrices of food, and electrode modification and reproducibility remain technical challenges.

[0006] (3) Fluorescence spectroscopy Fluorescence spectroscopy has gained widespread attention in the field of nitrite detection in recent years due to its advantages such as high sensitivity, good specificity, and fast response time. Fluorescent probes induce changes in fluorescence signals through specific chemical reactions with nitrites (such as diazotization and nitrosation), thereby achieving quantitative detection. However, most reported fluorescent probes still suffer from problems such as cumbersome preparation processes, insufficient selectivity, long response times, and the need for reactions in strongly acidic media, which limit their practical applications.

[0007] To overcome the limitations of single-mode detection methods, colorimetric / fluorescence dual-mode probe strategies have gradually become a research hotspot in recent years. This strategy integrates visual colorimetric detection (facilitating on-site semi-quantitative screening) with highly sensitive fluorescence detection (achieving accurate quantification), combining the advantages of simple operation and high sensitivity. Existing studies have reported colorimetric / fluorescence dual probes based on gold nanorod / gold nanosphere composite systems, as well as color / fluorescence dual-signal test strips based on carbon dot-neutral red composite systems. However, existing dual-mode probes still have the following shortcomings: (1) Probe preparation involves multi-step nanomaterial synthesis and modification, which is complex and costly; (2) Some probe systems have poor stability, and signal drift occurs during storage and use; (3) The selectivity for interfering ions in actual food matrices needs to be improved; (4) The response time and detection sensitivity still need further optimization.

[0008] Therefore, it is necessary to develop a method that is simple to prepare, has a rapid response, good selectivity, and high sensitivity for detecting and monitoring NO2 in food. Colorimetric fluorescent probes are extremely important. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, its preparation method, and its applications. The probe synthesized in this invention is effective against NO2. The identification exhibits high sensitivity and selectivity; when the probe comes into contact with the analyte NO2... Upon contact, it emits a strong fluorescence intensity at 505 nm. Furthermore, when the probe reacts with the analyte NO2... After contact, the probe solution gradually changed from yellow to colorless; this color change can also indicate the presence of NO2. Make accurate judgments. It can be applied to meat product preservation testing, water quality testing, and the detection of pollution from waste gas, wastewater, and solid waste in heavy industry.

[0010] To achieve the above-mentioned objectives, the specific technical solution of this invention is as follows: This invention first provides a fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, the structure of which is shown in formula (1):

[0011] Equation (1).

[0012] This invention also provides a synthetic route for fluorescent probe compounds based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, specifically as follows: .

[0013] This invention also provides a method for preparing the fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide as described above, comprising the following steps: (1) Di(4-bromophenyl)yne, AgOAc and diphenylphosphine oxide were added to a container containing DMF and heated in N2 atmosphere. After the reaction was completed, the mixture was cooled to room temperature and extracted with EtOAc and H2O. The extracted substances were combined and the organic phase was dried with anhydrous magnesium sulfate. The mixture was then concentrated by rotary evaporator and column chromatography was performed using PE and EtOAc as eluents to obtain compound B. (2) Compound B, B2pin2, Pd(dppf)Cl2 and KOAc were added to a dry container, the air was replaced with N2, 1,4-dioxane was added and refluxed. After the reaction was completed, DCM and H2O were used for extraction. The extracted substances were combined and the organic phase was dried with anhydrous magnesium sulfate. Then it was concentrated by rotary evaporator and compound C was obtained by rapid column chromatography with PE and EtOAc. (3) Compound C, 5-bromo-2,1,3-benzothiadiazole, PdPPh4, and Na2CO3 were added to a dry two-necked flask, the air was replaced with N2, toluene was added and the reaction was refluxed; after the reaction was completed, the mixture was extracted with EtOAc and H2O, the extracted substances were combined, the organic phase was dried with anhydrous magnesium sulfate, and then concentrated by rotary evaporator. PE and EtOAc were used as eluents for column chromatography to obtain compound D; (4) Compound D and CoCl2 .6H2O and NaBH4 were added to a dry container, the air was replaced with N2, and a mixed solvent of EtOH and THF was added and the reaction was heated. After the reaction was complete, the resulting solution was poured into distilled water and extracted with dichloromethane. The resulting substances were combined, and the organic phase was dried with anhydrous magnesium sulfate and then concentrated by rotary evaporation to obtain the crude product. The crude product was purified by silica gel rapid column chromatography to obtain the desired fluorescent probe compound.

[0014] As a preferred embodiment of this application, in the method for preparing the fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, the specific operation of step (1) is as follows: 5 mmol of di(4-bromophenyl)yne, 10 mmol of AgOAc, and 10 mmol of diphenylphosphine oxide were added to a 100 mL dry two-necked flask and then dissolved in 30 mL of DMF. The reaction was carried out under N2 conditions at 100 ± 5 °C for 10 ± 0.5 h. After cooling to room temperature, the mixture was extracted with EtOAc and H2O. The combined organic phases were dried over anhydrous magnesium sulfate and then concentrated by rotary evaporation. Column chromatography was performed using PE and EtOAc as eluents to obtain compound B.

[0015] As a preferred embodiment of this application, in the preparation method of the fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, the specific operation of step (2) is as follows: Compound B, B2pin2, Pd(dppf)Cl2, and KOAc were added to a dry two-necked flask, the air was replaced with N2, 20 mL of 1,4-dioxane was added, and the mixture was refluxed at 120 °C ± 5 °C for 5 h. The reaction was detected by TLC using DCM and MeOH in a volume ratio of 50:1. After the reaction was completed, the organic phases were extracted with DCM and H2O and dried with anhydrous magnesium sulfate. The mixture was then concentrated by rotary evaporation and compound C was obtained by rapid column chromatography with PE and EtOAc. The molar ratio of compounds B, B2pin2, Pd(dppf)Cl2, and KOAc was 1:2-2.2:0.1-0.15:6-6.5 (more preferably 1:2.2:0.1:6).

[0016] As a preferred embodiment of this application, in the method for preparing the fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, the specific operation of step (3) is as follows: 1 mmol of compound C, 1.5 mmol of 5-bromo-2,1,3-benzothiadiazole, 6% mmol of PdPPh4, and 4 mmol of Na2CO3 were added to a dry two-necked flask. The air was replaced with N2, and 20 mL of toluene was added. The mixture was refluxed at 85±5 °C for 24 h. The reaction was monitored by TLC with a PE:EtOAc ratio of 1:1. After the reaction was completed, the mixture was extracted with EtOAc and H2O. The combined organic phases were dried over anhydrous magnesium sulfate and then concentrated by rotary evaporation. Column chromatography was performed using PE and EtOAc as eluents to obtain compound D.

[0017] As a preferred embodiment of this application, in the method for preparing the fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, the specific operation of step (4) is as follows: Compound D, CoCl2 . 6H₂O and NaBH₄ were added to a dry two-necked flask, the air was replaced with N₂, and a mixed solvent EtOH:THF = 2:1 was added. The reaction was carried out at 50 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, the resulting solution was poured into 100 mL of distilled water and extracted with 2 × 100 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and then concentrated by rotary evaporation. The crude product was purified by silica gel rapid column chromatography to obtain the desired fluorescent probe compound. Compound D reacted with CoCl₂ . The molar ratio of 6H2O to NaBH4 is 1:1-1.5:4-4.5 (more preferably 1:1:4).

[0018] This invention protects a fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, which contains a fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide as described above, or a fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide prepared by the method described above.

[0019] This invention protects the application of the fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide described above in the detection of sodium nitrite in meat food preservation testing, water quality testing, and waste pollution from heavy industry.

[0020] Furthermore, in the aforementioned application, when used to detect sodium nitrite, the probe emits a strong fluorescence signal at 505 nm, with a minimum detection limit of 5 nM. The probe also exhibits good specificity and selectivity, making it suitable for mixed solution systems containing numerous interfering ions. The probe has a short reaction time, achieving stable fluorescence intensity after contacting the analyte hydrogen sulfide for 10 ± 1 min.

[0021] The working principle of this invention is as follows: By using o-phenylenediamine (OPD) as NO2 A novel colorimetric and fluorescent probe, BPO-OPD, was developed by attaching the reaction site to a fluorescent scaffold of benzo[b]phosphazenecyclopentadiene-1-oxide. The free probe exhibits significant fluorescence quenching due to the efficient PET effect of the bimolecular o-phenylenediamine. (The text abruptly ends here, likely due to an incomplete translation or missing information.) A specific reaction occurs, generating a triazole structure that effectively inhibits the PET effect, emitting bright green fluorescence at a wavelength of 505 nm. Simultaneously, the visible color changes from yellow to colorless. The BPO-OPD probe was applied to water samples and food samples (spiced beef, sauerkraut, kimchi, dried radish) from different environments to detect NO2. The content was determined. A convenient test strip was successfully developed, allowing users to test NO2 using a smartphone. The content was monitored in real time, and the results were satisfactory.

[0022] Compared with existing technologies, the beneficial effects of the present invention are as follows: (i) This invention uses benzo[b]phosphacyclopentadiene-1-oxide as the basic framework, and then introduces o-phenylenediamine, which can recognize the sodium nitrite structure, to obtain the final structure. Due to the presence of the o-phenylenediamine structure, the fluorescence of this structure is quenched. When sodium nitrite is detected, a cyclization reaction occurs to generate a triazole structure, which triggers fluorescence recovery, and the fluorescence intensity is significantly enhanced at 505 nm.

[0023] (II) The characteristics of benzo[b]phosphacyclopentadiene-1-oxide in this invention are as follows: Before contact with sodium nitrite, its maximum UV absorption peak is 322 nm, and its excitation wavelength is also 322 nm. Due to the presence of the o-phenylenediamine structure in the probe structure, the probe is in a fluorescence-quenched state. After contact with sodium nitrite, its maximum UV absorption peak is 354 nm, and it produces strong fluorescence emission at 505 nm. In a 10% (volume percentage) PBS buffer solution of DMSO (pH=2.5), the addition of sodium nitrite enhances the fluorescence at 505 nm by at least a hundredfold.

[0024] (iii) In this invention, the benzo[b]phosphacyclopentadiene-1-oxide molecule is not disturbed in solution systems containing various interfering ions, such as inorganic salts, metal ions, and amino acids, and can still achieve highly selective recognition of hydrogen sulfide, which can meet the complex environment faced in actual detection processes. Attached Figure Description

[0025] Figure 1 This is a comparison of the UV absorption spectra of a fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide before and after contact with sodium nitrite in Example 4. Figure 2 This is a comparison of the fluorescence emission spectra of the fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide before and after contact with sodium nitrite in Example 4. Figure 3 This is a comparison of fluorescence emission and fluorescence intensity of the fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide in Example 5 under the presence of different interfering ions; Figure 4 The UV absorption and fluorescence emission spectra of the fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide after adding different concentrations of sodium nitrite are shown in Example 6. Figure 5 This is a time-titration result diagram of sodium nitrite after adding a fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide in Example 6; Figure 6 This is a graph showing the cytotoxicity results of a fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide in Example 7. Detailed Implementation

[0026] A fluorescent probe for the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, the structure of which is shown in formula (1):

[0027] Equation (1).

[0028] A novel fluorescent preparation method for the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, the synthetic route of which is as follows: .

[0029] As a preferred embodiment of this application, the preparation method of the novel fluorescent probe with the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide includes the following steps: The synthesis of compound B involved adding 1.67 g (5 mmol) of di(4-bromophenyl)yne, 1.67 g (10 mmol) of AgOAc, and 1.78 g (10 mmol) of diphenylphosphine oxide to a 100 mL dry two-necked flask, dissolving them in 30 mL of DMF, and reacting in N2 at 100 °C for 10 h. After cooling to room temperature, the mixture was extracted with EtOAc and H2O. The combined organic phases were dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, and then subjected to column chromatography using PE and EtOAc as eluents to obtain the product, compound B (1.6 g, 60% yield). Synthesis of compound C: B (1.06 g, 2 mmol), B2pin2 (1.1 g, 4.4 mmol), Pd(dppf)Cl2 (146 mg, 10% mmol), and KOAc (1.17 g, 12 mmol) were added to a dry two-necked flask. The air was replaced with N2, and 20 mL of 1,4-dioxane was added. The mixture was refluxed at 120 °C for 5 h. The reaction was monitored by TLC using DCM:MeOH-50:1. After the reaction was completed, the DCM and H2O were extracted, and the combined organic phases were dried over anhydrous magnesium sulfate. The mixture was then concentrated by rotary evaporation and the product (1.2 g, 95% yield) was obtained by rapid column chromatography using PE and EtOAc.

[0030] Synthesis of compound D: C (630 mg, 1 mmol), 5-bromo-2,1,3-benzothiadiazole (320 mg, 1.5 mmol), PdPPh4 (69 mg, 6% mmol), and NaCO3 (424 mg, 4 mmol) were added to a dry two-necked flask. The air was replaced with N2, and 20 mL of toluene was added. The mixture was refluxed at 85 °C for 24 h. The reaction was monitored by TLC with a PE:EtOAc ratio of 1:1. After the reaction was completed, the mixture was extracted with EtOAc / H2O. The combined organic phases were dried over anhydrous magnesium sulfate and then concentrated by rotary evaporation. Column chromatography was performed using PE and EtOAc as eluents to obtain product D (459 mg, 71% yield).

[0031] Synthesis of Probe BPO-OPD: D (646 mg, 1 mmol), CoCl2 . 238 mg of 6H₂O and 1 mmol of NaBH₄ were added to a dry two-necked flask. The air was replaced with N₂, and a mixed solvent of EtOH:THF = 2:1 was added. The mixture was reacted at 50 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, the resulting solution was poured into 100 mL of distilled water and extracted with dichloromethane (2 × 100 mL). The combined organic phases were dried over anhydrous magnesium sulfate and then concentrated by rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain the final product.

[0032] As a preferred embodiment of this application, in step 1) of the method for preparing a novel fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, the molar ratio of compound A, compound 1, and AgOAc is 1:2:2; the reaction solvent is DMF; the reflux temperature is 100±5℃ (more preferably 100℃); and the reflux time is 10±0.5h (more preferably 10h).

[0033] In a preferred embodiment of this application, in step 2) of the method for preparing the novel fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, the molar ratio of compound B, B2pin2, Pd(dppf)Cl2 and KOAc is 1:2-2.2:0.1-0.15:6-6.5 (more preferably 1:2.2:0.1:6); the stirring temperature is 120℃±5℃ (more preferably 120℃); and the stirring time of the mixed solution system at room temperature is 5h.

[0034] In a preferred embodiment of this application, in step 3) of the method for preparing the novel fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, the molar ratio of compound C to PdPPh4 and compound 2 is 1:0.06-0.65:1.5-1.8 (more preferably 1:0.06:1.5); the reflux temperature is 85℃±5℃ (more preferably 85℃); and the reaction time is 24 hours.

[0035] As a preferred embodiment of this application, in step 4) of the method for preparing a novel fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, compound D reacts with CoCl2. . The molar ratio of 6H2O to NaBH4 was 1:1-1.5:4-4.5 (more preferably 1:1:4); the reaction time was 2 hours. The final structure obtained was quenched due to the presence of the o-phenylenediamine structure. Upon detection of sodium nitrite, a cyclization reaction occurred to generate a triazole structure, triggering fluorescence recovery, with a significant increase in fluorescence intensity at 505 nm.

[0036] This invention also protects a novel fluorescent probe with a benzo[b]phosphacyclopentadiene-1-oxide molecular structure, wherein the fluorescent probe contains a novel fluorescent probe compound with a benzo[b]phosphacyclopentadiene-1-oxide molecular structure as described above, or a novel fluorescent probe compound with a benzo[b]phosphacyclopentadiene-1-oxide molecular structure prepared by the methods or steps described above.

[0037] This invention also protects the application of the novel fluorescent probe with the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide described above in the detection of sodium nitrite.

[0038] Furthermore, its specific recognition mechanism for sodium nitrite is shown in equation (3):

[0039] (3).

[0040] The probe synthesized in this invention exhibits high sensitivity and selectivity for recognizing sodium nitrite. Upon contact with the analyte sodium nitrite, the probe emits strong fluorescence at 505 nm. Furthermore, the probe solution gradually changes from yellow to colorless upon contact, allowing for accurate determination of the presence of sodium nitrite through this color change. This invention can be applied to the detection of meat product preservation and water quality.

[0041] Furthermore, when used to detect sodium nitrite, the probe emits a strong fluorescence signal at 505 nm when in contact with hydrogen sulfide; and the probe also has an extremely low detection limit of 5 nM.

[0042] Furthermore, when the aforementioned fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide is used to detect sodium nitrite, the probe exhibits good specificity and selectivity, making it suitable for mixed solution systems containing numerous interfering ions. In addition, the probe has a short reaction time, achieving a stable fluorescence intensity approximately 10 minutes after contact with the analyte sodium nitrite.

[0043] Based on the specific implementation methods, the technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0045] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this description, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0046] In this invention, some conventional operating equipment, devices and components have been omitted or only briefly described.

[0047] Unless otherwise specified in the examples, the conditions shall be performed according to the standard conditions or the conditions recommended by the manufacturer.

[0048] In this application, unless otherwise specified, % refers to volume percentage.

[0049] In this application, a novel fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, with the molecular formula C 38 H 31 N4OP has a molecular weight of 590.2235.

[0050] Example 1: Based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, the fluorescent probe compound shown has the following structural formula: .

[0051] Example 2: The preparation method of fluorescent probe compounds based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide is as follows: .

[0052] Example 3: The preparation method of fluorescent probe compounds based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide follows the same synthetic route as in Example 2, and includes the following steps: Preparation of compound B:

[0053] Di(4-bromophenyl)yne (1.67 g, 5 mmol), AgOAc (1.67 g, 10 mmol), and diphenylphosphine oxide (1.78 g, 10 mmol) were added to a 100 mL dry two-necked flask and dissolved in 30 mL of DMF. The mixture was reacted in N2 at 100 °C for 10 h. After cooling to room temperature, the mixture was extracted with EtOAc and H2O. The combined organic phases were dried over anhydrous magnesium sulfate and then concentrated by rotary evaporation. Column chromatography using PE and EtOAc as eluents was performed to obtain the product, compound B (1.6 g, 60% yield). 1 HNMR (600 MHz, DMSO- d 6) δ 7.70 (s, 5H), 7.62 – 7.52 (m, 2H), 7.47 (s, 3H), 7.44 – 7.27 (m, 4H), 7.16 (s, 1H), 7.11 (d, J = 7.3 Hz, 2H). 13 C NMR (151 MHz, DMSO- d6) δ 149.41, 149.27, 142.29, 133.45, 133.32, 132.69, 132.53, 132.43,132.18, 131.77, 131.71, 131.56, 131.44, 131.13, 130.62, 130.35, 129.81,129.74, 129.24, 129.16, 128.89, 124.04, 122.43, 121.62, 39.80. Synthesis of compound C:

[0054] Compound B (1.06 g, 2 mmol), B2pin2 (1.1 g, 4.4 mmol), Pd(dppf)Cl2 (146 mg, 10% mmol), and KOAc (1.17 g, 12 mmol) were added to a dry two-necked flask. The air was replaced with N2, and 20 mL of 1,4-dioxane was added. The mixture was refluxed at 120 °C for 5 h. The reaction was detected by TLC using DCM:MeOH at a volume ratio of 50:1. After the reaction was completed, DCM and H2O were extracted, and the combined organic phases were dried over anhydrous magnesium sulfate. The mixture was then concentrated by rotary evaporation and the product, compound C (1.2 g, 95% yield), was obtained by rapid column chromatography with PE and EtOAc.

[0055] Preparation of compound D:

[0056] Compound C (630 mg, 1 mmol), 5-bromo-2,1,3-benzothiadiazole (320 mg, 1.5 mmol), PdPPh4 (69 mg, 6% mmol), and NaCO3 (424 mg, 4 mmol) were added to a dry two-necked flask. The air was replaced with N2, and 20 mL of toluene was added. The mixture was refluxed at 85 °C for 24 h. The reaction was detected by TLC using a 1:1 volume ratio of PE and EtOAc. After the reaction was complete, the mixture was extracted with EtOAc and H2O. The combined organic phases were dried over anhydrous magnesium sulfate and then concentrated by rotary evaporation. The product, compound D (459 mg, 71% yield), was obtained by column chromatography using PE and EtOAc as eluents. 1 H NMR (600 MHz, Chloroform- d ) δ 8.24 (s, 1H), 8.07 (d, J= 9.1 Hz, 1H), 8.03 (s, 1H), 7.96 –7.91 (m, 2H), 7.86 – 7.81 (m, 4H), 7.77 – 7.73 (m, 2H), 7.56 – 7.46 (m, 6H),7.45 – 7.40 (m, 5H), 7.32 (dd, J = 7.6, 2.8 Hz, 1H) . 13 C NMR (151 MHz, Chloroform-d) δ 155.36, 155.31, 154.36, 154.23, 143.56, 141.53, 141.44,140.03, 138.86, 134.43, 134.33, 132.44, 131.07, 129.97, 129.76, 129.54,129.35, 129.10, 128.19, 124.18, 124.11, 121.79, 121.48, 118.82, 118.40,77.29. Preparation of compound BPO-OPD:

[0057] Compound D (646 mg, 1 mmol), CoCl2 . 238 mg of 6H₂O and 1 mmol of NaBH₄ were added to a dry two-necked flask. Air was replaced with N₂, and a mixed solvent of EtOH:THF = 2:1 (v / v) was added. The mixture was reacted at 50 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, the resulting solution was poured into 100 mL of distilled water and extracted with dichloromethane (2 × 100 mL). The combined organic phases were dried over anhydrous magnesium sulfate and then concentrated by rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane to methanol v / v = 10:1) to obtain product BPO-OPD, a fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide (385 mg, yield 65.3%). 1 H NMR (600MHz, DMSO- d 6) δ 7.74 (dd, J = 12.2, 7.2 Hz, 2H), 7.71 – 7.63 (m, 3H), 7.61 –7.54 (m, 2H), 7.51 – 7.47 (m, 2H), 7.45 (td, J= 7.3, 3.6 Hz, 1H), 7.41 (d, J =7.5 Hz, 2H), 7.31 – 7.24 (m, 3H), 7.22 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 2.1Hz, 1H), 6.85 (dd, J = 8.0, 2.1 Hz, 1H), 6.78 (d, J = 2.2 Hz, 1H), 6.67 (dd, J =8.1, 2.1 Hz, 1H), 6.61 (d, J = 8.0 Hz, 1H), 6.49 (d, J = 8.1 Hz, 1H), 4.62 (s, 8H). 13 C NMR (151 MHz, DMSO) δ 148.96, 148.81, 142.98, 141.52, 141.07, 140.59, 135.52, 135.43, 135.25, 135.04, 133.31, 132.43, 132.35, 132.34, 131.73, 130.92, 130.58, 130.51, 129.96, 129.30, 129.24, 129.16, 128.88, 128.84, 128.63, 127.94, 127.67, 125.82, 124.98, 123.85, 123.78, 115.88, 115.76, 114.70, 114.57, 112.51, 112.32, 106.36 Example 4: UV absorption and fluorescence emission experiments of the reaction between the probe (prepared using the fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide obtained in Example 3) and sodium nitrite: Control group (Probe-BPO-OPD): probe (20 uM), system was DMSO:PBS = 4:1 (volume ratio) buffer solution (5 mL), pH = 2.5; Experimental group (Probe-BPO-OPD+NaNO2): probe (20 uM), system was DMSO:PBS = 4:1 (volume ratio) buffer solution (5 mL), pH = 2.5, NaNO2 (0.1 mM). Both the control and experimental groups were brought to a final volume of 5 mL. After reacting at room temperature for 5 minutes, the UV absorption was measured to obtain the maximum absorption peak. The fluorescence emission spectrum was measured using the maximum absorption as the single-photon excitation wavelength. The spectrum is shown below. Figure 1 and Figure 2 , Figure 1 and Figure 2 The horizontal axis represents wavelength (nm). Figure 1 The vertical axis represents ultraviolet absorption intensity. Figure 2 The vertical axis represents fluorescence intensity. For example... Figure 1 As shown, the probe exhibits the strongest UV absorption at 322 nm. When the probe comes into contact with hydrogen sulfide, it produces a new UV absorption at 354 nm, as... Figure 2 As shown, when the excitation wavelength is 322 nm, the excitation slit is 5 nm, and the emission slit is 5 nm, the probe does not produce fluorescence emission when it is not in contact with hydrogen sulfide, but produces strong fluorescence emission at 505 nm when it is in contact with hydrogen sulfide.

[0058] Example 5: Anti-interference experiment. The following probes were prepared using fluorescent probe compounds based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide obtained in Example 3.

[0059] Normal group: probe (20 μM), system is DMSO:PBS=4:1 (volume ratio) buffer solution (5 mL), pH=2.5, NaNO2 (0.1 mM); Interference group: probe (20 μM), system is DMSO:PBS=4:1 (volume ratio) buffer solution (5 mL), pH=2.5, interfering ion (0.1 mM).

[0060] Interference ion determination: 200 μM NO3 was added to the buffer solution system of the probe (20 μM). - Cu 2+ Zn 2+ S2O3 2- , I - ,HS - HCO3 - , GSH, Cys, CH3COO - , and Al 3+ After 5 minutes of incubation, the fluorescence absorption was detected using 322 nm as the excitation wavelength. Figure 3 As shown, most interfering ions do not cause fluorescence enhancement of the probe. Subsequently, sodium nitrite was added to the mixture containing interfering ions, and after reacting at room temperature for five minutes, the probe still produced a strong fluorescence signal enhancement upon contact with sodium nitrite, even in the presence of interfering ions. Therefore, the probe exhibits good specificity for sodium nitrite and can meet the detection requirements in most practical situations.

[0061] Example 6: Probe Detection Dynamics Experiment The probes below were prepared using fluorescent probe compounds based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide obtained in Example 3.

[0062] Twelve 5 mL volumetric flasks were prepared, and 5 mL of 20 μM probe solution (DMSO to PBS volume ratio = 4:1, pH = 2.5) was added to each flask sequentially. Then, sodium nitrite solutions of different concentrations were added to each flask. After reacting at room temperature for 10 minutes, UV absorption and fluorescence emission spectra were measured. Figure 4 It can be seen that when NaNO2 reaches 55 μM, its ultraviolet and fluorescence spectra are basically stable and no longer fluctuate significantly. Then, the corresponding time of the probe was detected by adding 10 eq of NaNO2 solution to 5 mL of volumetric sample containing 20 μM probe solution. The change in the fluorescence emission spectrum of the probe after the addition of 10 eq of NaNO2 solution was recorded to determine the required detection time of the probe. When the reaction time reached 15 min, its fluorescence intensity tended to stabilize.

[0063] Example 7: Cytotoxicity assay of the probe To further investigate the applicability of the probe for in vivo imaging, well-grown 293T cells were digested and seeded into 96-well plates at a density of 6000 cells per well. When the cells reached approximately 80% confluence, the culture medium was replaced with fresh medium at concentrations of 10 μM, 30 μM, 50 μM, 60 μM, 80 μM, and 100 μM. A control group of normally cultured cells without the probe was used. After culturing for 24 h, the old medium was discarded, and fresh medium containing CCK8 solution (10 μL of CCK8 reagent per 100 μL of complete medium) was added to each well with gentle agitation. The cells were then incubated for 1.5 h, and the absorbance of each group was measured at 450 nm, and cell viability was calculated. The results showed that cell viability remained above 90%, indicating that the probe BPO-OPD exhibited low cytotoxicity in the 100 μM concentration range. Figure 6 As shown.

[0064] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

[0065] This background section is provided to generally present the context of the invention. The work of the currently named inventors, the work to the extent described in this background section, and aspects of this section that did not constitute prior art at the time of application are neither expressly nor impliedly acknowledged as prior art to the invention.

Claims

1. A fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, characterized in that: The structure of the compound is shown in formula (1): Equation (1).

2. A fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, characterized in that, The synthetic route for this compound is as follows: 。 3. The method for preparing the fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide according to claim 1 or 2, characterized in that... Includes the following steps: (1) Di(4-bromophenyl)yne, AgOAc and diphenylphosphine oxide were added to a container containing DMF and heated in N2 atmosphere. After the reaction was completed, the mixture was cooled to room temperature and extracted with EtOAc and H2O. The extracted substances were combined and the organic phase was dried with anhydrous magnesium sulfate. The mixture was then concentrated by rotary evaporator and column chromatography was performed using PE and EtOAc as eluents to obtain compound B. (2) Compound B, B2pin2, Pd(dppf)Cl2 and KOAc were added to a dry container, the air was replaced with N2, 1,4-dioxane was added and refluxed. After the reaction was completed, DCM and H2O were used for extraction. The extracted substances were combined and the organic phase was dried with anhydrous magnesium sulfate. Then it was concentrated by rotary evaporator and compound C was obtained by rapid column chromatography with PE and EtOAc. (3) Compound C, 5-bromo-2,1,3-benzothiadiazole, PdPPh4, and Na2CO3 were added to a dry two-necked flask, the air was replaced with N2, toluene was added and the reaction was refluxed; after the reaction was completed, the mixture was extracted with EtOAc and H2O, the extracted substances were combined, the organic phase was dried with anhydrous magnesium sulfate, and then concentrated by rotary evaporator. PE and EtOAc were used as eluents for column chromatography to obtain compound D; (4) Compound D and CoCl2 . 6H2O and NaBH4 were added to a dry container, the air was replaced with N2, and a mixed solvent of EtOH and THF was added and the reaction was heated. After the reaction was complete, the resulting solution was poured into distilled water and extracted with dichloromethane. The resulting substances were combined, and the organic phase was dried with anhydrous magnesium sulfate and then concentrated by rotary evaporation to obtain the crude product. The crude product was purified by silica gel rapid column chromatography to obtain the desired fluorescent probe compound.

4. The method for preparing the fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide according to claim 3, characterized in that, The specific operation of step (1) is as follows: 5 mmol of di(4-bromophenyl)yne, 10 mmol of AgOAc, and 10 mmol of diphenylphosphine oxide were added to a 100 mL dry two-necked flask and then dissolved in 30 mL of DMF. The reaction was carried out under N2 conditions at 100 ± 5 °C for 10 ± 0.5 h. After cooling to room temperature, the mixture was extracted with EtOAc and H2O. The combined organic phases were dried over anhydrous magnesium sulfate and then concentrated by rotary evaporation. Column chromatography was performed using PE and EtOAc as eluents to obtain compound B.

5. The method for preparing the fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide according to claim 3, characterized in that, The specific operation of step (2) is as follows: Compounds B, B2pin2, Pd(dppf)Cl2, and KOAc were added to a dry two-necked flask, the air was replaced with N2, 20 mL of 1,4-dioxane was added, and the mixture was refluxed at 120 °C ± 5 °C for 5 h. The reaction was detected by TLC using DCM and MeOH in a volume ratio of 50:

1. After the reaction was completed, the organic phases were extracted with DCM and H2O, dried with anhydrous magnesium sulfate, concentrated by rotary evaporation, and then subjected to rapid column chromatography with PE and EtOAc to obtain compound C. The molar ratio of compounds B, B2pin2, Pd(dppf)Cl2, and KOAc was 1:2-2.2:0.1-0.15:6-6.

5.

6. The method for preparing the fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide according to claim 3, characterized in that, The specific operation of step (3) is as follows: 1 mmol of compound C, 1.5 mmol of 5-bromo-2,1,3-benzothiadiazole, 6% mmol of PdPPh4, and 4 mmol of Na2CO3 were added to a dry two-necked flask. The air was replaced with N2, and 20 mL of toluene was added. The mixture was refluxed at 85℃±5℃ for 24 h. The reaction was detected by TLC with PE and EtOAc at a volume ratio of 1:

1. After the reaction was completed, the mixture was extracted with EtOAc and H2O. The combined organic phases were dried with anhydrous magnesium sulfate and then concentrated by rotary evaporation. Column chromatography was performed using PE and EtOAc as eluents to obtain compound D.

7. The method for preparing the fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide according to claim 3, characterized in that, The specific operation of step (4) is as follows: Compound D, CoCl2 . 6H₂O and NaBH₄ were added to a dry two-necked flask, the air was replaced with N₂, and a mixed solvent EtOH:THF = 2:1 was added. The reaction was carried out at 50 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, the resulting solution was poured into 100 mL of distilled water and extracted with 2 × 100 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and then concentrated by rotary evaporation. The crude product was purified by silica gel rapid column chromatography to obtain the desired fluorescent probe compound. Compound D reacted with CoCl₂ . The molar ratio of 6H2O to NaBH4 is 1:1-1.5:4-4.

5.

8. A fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide, characterized in that: The fluorescent probe contains a fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide as described in claim 1 or 2, or a fluorescent probe compound based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide prepared by any one of claims 3-7.

9. The application of the fluorescent probe based on the molecular structure of benzo[b]phosphacyclopentadiene-1-oxide as described in claim 8 in the detection of sodium nitrite.

10. The application as described in claim 9, characterized in that: When used to detect sodium nitrite, the probe emits a strong fluorescence signal at 505 nm, with a minimum detection limit of 5 nM. The probe also exhibits good specificity and selectivity, making it suitable for mixed solution systems containing numerous interfering ions. The probe has a short reaction time, achieving stable fluorescence intensity after contacting the analyte hydrogen sulfide for 10 ± 1 min.

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