Fluorescent chemical sensor for detecting biological sulfhydryl compound and preparation method of fluorescent chemical sensor
By designing specific molecular structures M1 and M2 of fluorescent chemical sensors and utilizing maleic acid to react with thiol compounds, the problems of high cost and complexity of existing detection methods are solved, and highly sensitive and simple detection of biological thiol compounds is achieved.
Patent Information
- Application Number
- CN202510792749.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-13
- Publication Date
- 2025-09-16
AI Technical Summary
Existing methods for detecting biothiol compounds require special instruments, professional operations, high costs, and complex sample pretreatment, which limits their scope of application.
A fluorescent chemical sensor is used. By designing specific molecular structures M1 and M2, maleic acid is used as a recognition site to undergo electrophilic addition with the thiol group in the thiol compound, inhibiting the photoinduced charge transfer effect, realizing fluorescence emission onset, and performing selective detection.
It achieves high sensitivity, simple operation, and on-site detection of biothiol compounds, has good selectivity and sensing performance, and reduces detection cost and complexity.
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Figure CN120647568A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of fluorescent chemical sensors, and in particular to a fluorescent chemical sensor for detecting biological thiol compounds and a preparation method thereof. Background Art
[0002] Sulfhydryl compounds in organisms primarily refer to amino acids containing a sulfhydryl group (-SH), common examples of which include cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). These amino acids are essential to the human body. Within organisms, the sulfhydryl groups of sulfhydryl compounds have a strong affinity for heavy metals, effectively chelating toxic heavy metal ions and excreting them from the body, thereby playing a detoxifying role. Furthermore, sulfhydryl groups possess strong nucleophilicity, readily reacting with electrophilic substances, particularly free radicals, to combat disease and prevent aging. In summary, biological sulfhydryl compounds are numerous and play important physiological roles in the body. They not only participate in maintaining redox homeostasis but also play a key role in resisting oxidative damage and protecting biomembrane systems. However, abnormal concentrations of sulfhydryl amino acids in organisms can also lead to a range of diseases, such as leukocyte loss, psoriasis, liver damage, and cancer. Therefore, monitoring the concentration of sulfhydryl amino acids in organisms is crucial. Currently, there are a variety of methods for detecting biothiols, including ion exchange chromatography, high-performance liquid chromatography, gas chromatography, electrophoresis, mass spectrometry, and electrochemical methods. However, these detection methods require specialized instruments, are costly, and require specialized personnel to operate, which limits their application to a certain extent. In addition, some methods require complex sample pretreatment processes, increasing the difficulty and cost of detection. Fluorescent chemical sensors have multiple advantages in detecting biothiols, such as high sensitivity, high selectivity, real-time performance, ease of operation, and visualization. These advantages make fluorescent chemical sensors widely applicable and promising in the detection and research of biothiols. Summary of the Invention
[0003] The present invention aims to solve the problems existing in the prior art and provides a fluorescent chemical sensor for detecting biothiol compounds and a preparation method thereof. The fluorescent chemical sensor prepared by the method exhibits good biothiol sensing performance and can efficiently detect biothiol compounds.
[0004] To achieve the above object, the present invention adopts the following technical solutions:
[0005] (1) The present invention provides a fluorescent chemical sensor for detecting biothiol compounds, wherein the molecular structure of the fluorescent chemical sensor is M1, which has the structural formula: In the M1, R1 is one of the following structures:
[0006] (II) The present invention also provides a method for preparing the fluorescent chemical sensor for detecting biothiol compounds described in the invention summary (I), the preparation process is as follows:
[0007]
[0008] Furthermore, the preparation method comprises the following steps:
[0009] A1, compound L1, 4-aminophenylboronic acid, tetrakis(triphenylphosphine)palladium, an inorganic base, and an organic solvent / water mixture are mixed and degassed, and then the reaction system is heat-treated and reacted. After the reaction is completed, the product is separated to obtain compound P1; the compound L1 is a monobromosubstituted product of R1;
[0010] B1. Dissolve compound P1 and maleic anhydride in a solvent and react. After the reaction, remove the solvent to obtain an intermediate. Mix the intermediate, acetic anhydride and anhydrous sodium acetate, heat and continue the reaction. After the reaction, separate the product to obtain compound M1.
[0011] Furthermore, in step A1, the molar ratio of compound L1, 4-aminophenylboronic acid, tetrakis(triphenylphosphine)palladium, and inorganic base is 1.0:1.3-1.5:0.04-0.06:2.5-3.0,
[0012] Furthermore, in step B1, the addition ratio of compound P1, maleic anhydride, acetic anhydride, and anhydrous sodium acetate is 1.0 mmol: 1.0-1.1 mmol: 50-100 mL: 1.0-1.1 mmol.
[0013] Furthermore, in the step A1, the heat treatment temperature is 90-100° C., and the reaction time is 2-3 days.
[0014] Furthermore, in the step B1, the first reaction temperature is 45-60° C., and the reaction time is 6-12 h; the second reaction temperature is 100-110° C., and the reaction time is 12-18 h.
[0015] Furthermore, the inorganic base is one of Na2CO3, K2CO3, and Cs2CO3.
[0016] Furthermore, in the organic solvent / water mixture, the ratio of organic solvent / water is 15 / 1 to 20 / 1, and the organic solvent is one of dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide.
[0017] Furthermore, the ratio of compound L1, organic solvent, and water is 5.0 mmol:60 mL:3 mL.
[0018] Furthermore, in step A1, the operation for isolating the product is as follows: after the reaction is completed, the reaction mixture is cooled to room temperature, poured into water, extracted with dichloromethane, and the organic phase is dried over anhydrous Na2CO3, concentrated using a rotary evaporator, and then purified to obtain compound P1.
[0019] Furthermore, in step B1, the operation of isolating the product is as follows: after the reaction is completed, the reactant is poured into ice water and stirred, then extracted with dichloromethane, the organic phase is dried over anhydrous Na2CO3, concentrated using a rotary evaporator, and then purified to obtain compound M1.
[0020] Furthermore, the degassing treatment is: subjecting the mixed system to at least one freezing-vacuuming-nitrogen filling-thawing operation.
[0021] (III) The present invention provides a fluorescent chemical sensor for detecting biothiol compounds, wherein the molecular structure of the fluorescent chemical sensor is M2, which has the structural formula: In the M2, R2 is one of the following structures:
[0022] (IV) The present invention also provides a method for preparing the fluorescent chemical sensor for detecting biothiol compounds described in the invention summary (III), the preparation process is as follows:
[0023]
[0024] Furthermore, the preparation method comprises the following steps:
[0025] A2, compound L2, 4-aminophenylboronic acid, tetrakis(triphenylphosphine)palladium, an inorganic base, and an organic solvent / water mixture are mixed and degassed, and then the reaction system is heat-treated and reacted. After the reaction is completed, the product is separated to obtain compound P2; the compound L2 is a dibromosubstituted product of R2;
[0026] B2. Dissolve compound P2 and maleic anhydride in a solvent and react. After the reaction, remove the solvent to obtain an intermediate. Mix the intermediate, acetic anhydride and anhydrous sodium acetate, heat and continue the reaction. After the reaction, separate the product to obtain compound M2.
[0027] Furthermore, in step A2, the molar ratio of compound L2, 4-aminophenylboronic acid, tetrakis(triphenylphosphine)palladium, and inorganic base is 1.0:2.5-3.0:0.08-0.12:5.0-7.5.
[0028] Furthermore, in step B2, the addition ratio of compound P2, maleic anhydride, acetic anhydride, and anhydrous sodium acetate is 1.0 mmol: 2.0-2.2 mmol: 50-100 mL: 2.0-2.2 mmol.
[0029] Furthermore, in step A2, the heat treatment temperature is 90-100° C., and the reaction time is 2-3 days.
[0030] Furthermore, in step B2, the first reaction temperature is 45-60° C., and the reaction time is 6-12 h; the second reaction temperature is 100-110° C., and the reaction time is 12-18 h.
[0031] Furthermore, the inorganic base is one of Na2CO3, K2CO3, and Cs2CO3.
[0032] Furthermore, in the organic solvent / water mixture, the ratio of organic solvent / water is 15 / 1 to 20 / 1, and the organic solvent is one of dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide.
[0033] Furthermore, the ratio of compound L2, organic solvent, and water is 5.0 mmol:80 mL:4 mL.
[0034] Furthermore, in step A2, the operation for isolating the product is as follows: after the reaction is completed, the reaction mixture is cooled to room temperature, poured into water, extracted with dichloromethane, and the organic phase is dried over anhydrous Na2SO4, concentrated using a rotary evaporator, and then purified to obtain compound P2.
[0035] Furthermore, in step B2, the operation of isolating the product is as follows: after the reaction is completed, the reactant is poured into ice water with stirring, then extracted with dichloromethane, the organic phase is dried over anhydrous Na2SO4, concentrated using a rotary evaporator, and then purified to obtain compound M2.
[0036] Furthermore, the degassing treatment is to subject the mixed system to at least one freezing-vacuuming-nitrogen filling-thawing operation. The beneficial effects of the present invention are:
[0037] (1) The present invention provides a fluorescent chemical sensor for detecting biothiol compounds and a preparation method thereof. The fluorescent chemical sensor prepared by this method can selectively sense and respond to biothiol compounds and can exhibit good biothiol sensing performance. The maleoyl group in the sensing molecule is an electron-deficient acceptor group, and the anthracene group is an electron-donating fluorophore. Due to the photoinduced charge transfer effect (PET effect), the fluorescence of the sensing molecule is quenched. When combined with a biothiol compound, the maleoyl group (maleic acid group) in the sensing molecule acts as a recognition site and undergoes electrophilic addition with the thiol group (-SH) in the thiol compound, resulting in the inhibition of the PET effect and the activation of fluorescence emission, thereby identifying and detecting the thiol compound.
[0038] (2) The scheme of the present invention is reasonably designed and has many advantages such as simple operation, high sensitivity, convenient preparation, and on-site detection, providing another option for the efficient detection of biothiol compounds. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Figure 1 The fluorescence emission spectra of sensor M1-1 in acetonitrile solvent in response to cysteine and 16 other amino acids;
[0040] Figure 2 The fluorescence emission spectra of sensor M2-1 in acetonitrile solvent in response to cysteine and 16 other amino acids;
[0041] Figure 3 The fluorescence emission intensity (418 nm) bar graph of the sensor M1-1 selectively responding to cysteine and other 16 amino acids in acetonitrile solvent;
[0042] Figure 4 The fluorescence emission intensity (422 nm) bar graph of the sensor M2-1 selectively responding to cysteine and other 16 amino acids in acetonitrile solvent;
[0043] Figure 5 is the fluorescence emission spectrum of sensor M1-1 in response to different concentrations of cysteine in acetonitrile solvent;
[0044] Figure 6 is the fluorescence emission spectrum of sensor M2-1 in response to different concentrations of cysteine in acetonitrile solvent;
[0045] Figure 7 is the fluorescence emission spectrum of sensor M1-1 in response to interference of other amino acids on cysteine in acetonitrile solvent;
[0046] Figure 8 Figure 5. Fluorescence emission spectrum of sensor M2-1 in acetonitrile solvent in response to interference of other amino acids on cysteine. DETAILED DESCRIPTION
[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.
[0048] Example 1
[0049] This example provides a fluorescent chemical sensor M1-1 for detecting biothiol compounds. The preparation route is as follows:
[0050]
[0051] Step A1, 9-bromoanthracene L1-1 (1.29g, 5.0mmol), 4-aminophenylboronic acid (1.03g, 7.5mmol), tetrakis(triphenylphosphine)palladium (0.29g, 0.25mmol), cesium carbonate (4.08g, 12.5mmol) were added to a 100mL sealed bottle, and then 60mL of 1,4-dioxane and 3mL of water were added. Ultrasonication for 10min was performed to uniformly disperse the reactants. After three cycles of freezing-vacuuming-filling with nitrogen-thawing, the tube was sealed and the reaction system was heated in a 90°C oil bath for 3 days. After the reaction was completed, it was cooled to room temperature, the reactant was poured into water, extracted with dichloromethane, the organic phase was dried over anhydrous Na2SO4, concentrated with a rotary evaporator, and purified by column chromatography to obtain compound P1-1.
[0052] Step B1: Compound P1-1 (1.35 g, 5 mmol) and maleic anhydride (0.49 g, 5 mmol) were dissolved in 100 mL of CHCl3 and reacted at 60°C for 6 h. After completion of the reaction, the solvent was removed by distillation under reduced pressure to obtain an intermediate, which was directly transferred to the next step without purification. The intermediate, acetic anhydride (250 mL) and anhydrous sodium acetate (0.41 mg, 5 mmol) were then added to a flask and the reaction was continued at 110°C for 12 h. After completion of the reaction, the reaction mixture was poured into 100 mL of ice water and stirred for 1 h, then extracted with dichloromethane. The organic phase was dried over anhydrous Na2SO4 and concentrated on a rotary evaporator to obtain a crude product, which was purified by column chromatography to obtain the target compound M1-1.
[0053] Example 2
[0054] This example provides a fluorescent chemical sensor M1-2 for detecting biothiol compounds. The preparation steps are the same as those in Example 1, except that compound L1-1 is replaced by compound L1-2, and compound P1-1 is replaced by compound P1-2.
[0055] The preparation route is as follows:
[0056]
[0057] Example 3
[0058] This example provides a fluorescent chemical sensor M1-3 for detecting biothiol compounds. The preparation steps are the same as those in Example 1, except that compound L1-1 is replaced by compound L1-3, and compound P1-1 is replaced by compound P1-3.
[0059] The preparation route is as follows:
[0060]
[0061] Example 4
[0062] This example provides a fluorescent chemical sensor M2-1 for detecting biothiol compounds. The preparation route is as follows:
[0063]
[0064] Step A2: 9,10-dibromoanthracene L2-1 (1.68 g, 5.0 mmol), 4-aminophenylboronic acid (2.05 g, 15.0 mmol), tetrakis(triphenylphosphine)palladium (0.578 g, 0.5 mmol), and cesium carbonate (8.15 g, 25.0 mmol) were added to a 150 mL sealed vial, followed by the addition of 80 mL of 1,4-dioxane and 4 mL of water. Ultrasonication was performed for 10 min to uniformly disperse the reactants. After three cycles of freezing, vacuuming, filling with nitrogen, and thawing, the vial was sealed. The reaction system was then heated in a 90°C oil bath for 3 days. After completion of the reaction, the reaction was cooled to room temperature, poured into water, extracted with dichloromethane, and the organic phase dried over anhydrous Na2SO4, concentrated using a rotary evaporator, and purified by column chromatography to obtain the target molecule P2-1.
[0065] Step B2: Compound P2-1 (1.80 g, 5 mmol) and maleic anhydride (0.98 g, 10 mmol) were dissolved in 100 mL of CHCl3 and reacted at 60°C for 6 h. After completion of the reaction, the solvent was removed by distillation under reduced pressure to obtain the intermediate, which was directly transferred to the next step without purification. The intermediate, acetic anhydride (250 mL) and anhydrous sodium acetate (0.82 mg, 10 mmol) were then added to a flask and the reaction was continued at 110°C for 12 h. The reaction mixture was then poured into 100 mL of ice water and stirred for 1 h, then extracted with dichloromethane. The organic phase was dried over anhydrous Na2SO4 and concentrated using a rotary evaporator. The crude product was purified by column chromatography to obtain the target molecule M2-1.
[0066] Example 5
[0067] This example provides a fluorescent chemical sensor M2-2 for detecting biothiol compounds. The preparation steps are the same as those in Example 4, except that compound L2-1 is replaced by compound L2-2, and compound P2-1 is replaced by compound P2-2.
[0068] The preparation route is as follows:
[0069]
[0070] Example 6
[0071] This example provides a fluorescent chemical sensor M2-3 for detecting biothiol compounds. The preparation steps are the same as those in Example 4, except that compound L2-1 is replaced by compound L2-3, and compound P2-1 is replaced by compound P2-3.
[0072] The preparation route is as follows:
[0073]
[0074] Example 7
[0075] This example provides a fluorescent chemical sensor M2-4 for detecting biothiol compounds. The preparation steps are the same as those in Example 4, except that compound L2-1 is replaced by compound L2-4, and compound P2-1 is replaced by compound P2-4.
[0076] The preparation route is as follows:
[0077]
[0078] Effect test:
[0079] H and C NMR spectra were measured using a Bruker AVANCE II NMR spectrometer, using tetramethylsilane as the internal standard and deuterated chloroform or deuterated dimethyl sulfoxide as the solvent. Fluorescence spectra were measured using a Shimadzu RF-5301PC fluorescence spectrophotometer.
[0080] From the fluorescence emission spectra of sensor M1-1 in response to 17 amino acids, we can see that ( Figure 1 ), after adding cysteine to the sensor solution, an obvious fluorescence emission peak appeared at 418nm, while the fluorescence emission intensity was only one tenth or even lower when the same equivalent of other amino acids were added. This can be seen from the bar graph of the selective response of sensor M1-1 to 17 amino acids ( Figure 3 ) can be observed more intuitively. Therefore, the M1-1 molecule exhibits excellent detection selectivity for cysteine, and the detection process can be directly observed by the naked eye (the fluorescence color changes from colorless to blue). Sensor M2-1 also exhibits similar fluorescence sensing performance, and the sensor solution responds selectively to cysteine ( Figure 4 ), producing a strong fluorescence emission peak at 427nm ( Figure 2 ), the solution color can still be observed to change from colorless to blue with the naked eye. This is because the maleoyl group (maleyl) in the sensor molecule acts as a recognition site, undergoing electrophilic addition with the sulfhydryl group (-SH) in the sulfhydryl compound, resulting in the suppression of the PET effect and the activation of fluorescence emission, thereby enabling the identification and detection of the sulfhydryl compound.
[0081] In order to explore the effective detection concentration range of cysteine by the fluorescence sensor, we adjusted the concentration of cysteine in the test solution (0.1eq~10.0eq). Figure 5 and Figure 6 ) As the cysteine concentration increases, the fluorescence intensity increases after adding the sensor molecule. This is because excess cysteine ensures complete recognition of the maleoyl groups in the sensor molecule, completely suppressing the PET effect and fully activating fluorescence emission. However, when the cysteine concentration is low, the fluorescence intensity is weaker after adding the sensor molecule. This is because some maleoyl groups are not recognized, the PET effect still exists, and the fluorophore does not fully activate fluorescence emission. Compared with M1-1, the fluorescence intensity of the sensor molecule M2-1 is weaker after interacting with cysteine. This is because M2-1 contains more maleoyl groups, requiring a higher concentration of cysteine to completely suppress the PET effect.
[0082] In order to explore the potential influence of other amino acids on cysteine determination, 16 mixed solutions containing different amino acids and cysteine were prepared and added to the sensor solution in sequence to test their fluorescence spectra. Figure 7 and Figure 8 As shown, the results indicate that the presence of the other 16 amino acids in the system has a weak effect on the detection of cysteine. Although the fluorescence emission intensity decreases, the obvious change of fluorescence color can still be observed intuitively.
[0083] In summary, the fluorescent chemical sensor provided by the present invention can selectively sense and respond to biothiol compounds. This technical route has many advantages, such as simple operation, high sensitivity, convenient preparation, and on-site detection, providing a new option for biothiol sensing detection.
[0084] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions based on the principles of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should be considered within the scope of protection of the present invention.
Claims
1. A fluorescent chemical sensor for detecting biothiol compounds, characterized in that: The molecular structural formula of the fluorescent chemical sensor is M1, In the M1, R1 is one of the following structures: R1： 2. The method for preparing a fluorescent chemical sensor for detecting biothiol compounds according to claim 1, wherein The following steps are involved: A1, compound L1, 4-aminophenylboronic acid, tetrakis(triphenylphosphine)palladium, an inorganic base, and an organic solvent / water mixture are mixed and degassed, and then the reaction system is heat-treated and reacted. After the reaction is completed, the product is separated to obtain compound P1; the compound L1 is a monobromosubstituted product of R1; B1. Dissolve compound P1 and maleic anhydride in a solvent and react. After the reaction, remove the solvent to obtain an intermediate. Mix the intermediate, acetic anhydride and anhydrous sodium acetate, heat and continue the reaction. After the reaction, separate the product to obtain compound M1.
3. The method for preparing a fluorescent chemical sensor for detecting biological thiol compounds according to claim 2, wherein In the step A1, the molar ratio of compound L1, 4-aminophenylboronic acid, tetrakis(triphenylphosphine)palladium, and inorganic base is 1.0:1.3-1.5:0.04-0.06:2.5-3.0; In the step B1, the addition ratio of compound P1, maleic anhydride, acetic anhydride, and anhydrous sodium acetate is 1.0 mmol: 1.0-1.1 mmol: 50-100 mL: 1.0-1.1 mmol.
4. The method for preparing a fluorescent chemical sensor for detecting biosulfhydryl compounds according to claim 2, wherein: In the step A1, the heat treatment temperature is 90-100°C and the reaction time is 2-3 days; In the step B1, the first reaction temperature is 45-60° C., and the reaction time is 6-12 h; the second reaction temperature is 100-110° C., and the reaction time is 12-18 h.
5. The method for preparing a fluorescent chemical sensor for detecting biosulfhydryl compounds according to claim 2, wherein: The inorganic base is one of Na2CO3, K2CO3, and Cs2CO3; In the organic solvent / water mixture, the ratio of organic solvent / water is 15 / 1 to 20 / 1, and the organic solvent is one of dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide.
6. A fluorescent chemical sensor for detecting biothiol compounds, characterized in that: The molecular structural formula of the fluorescent chemical sensor is M2, In the M2, R2 is one of the following structures: R2： 7. The method for preparing a fluorescent chemical sensor for detecting biothiol compounds according to claim 6, wherein: The following steps are involved: A2, compound L2, 4-aminophenylboronic acid, tetrakis(triphenylphosphine)palladium, an inorganic base, and an organic solvent / water mixture are mixed and degassed, and then the reaction system is heat-treated and reacted. After the reaction is completed, the product is separated to obtain compound P2; the compound L2 is a dibromosubstituted product of R2; B2. Dissolve compound P2 and maleic anhydride in a solvent and react. After the reaction, remove the solvent to obtain an intermediate. Mix the intermediate, acetic anhydride and anhydrous sodium acetate, heat and continue the reaction. After the reaction, separate the product to obtain compound M2.
8. The method for preparing a fluorescent chemical sensor for detecting biothiol compounds according to claim 7, wherein: In step A2, the molar ratio of compound L2, 4-aminophenylboronic acid, tetrakis(triphenylphosphine)palladium, and inorganic base is 1.0:2.5-3.0:0.08-0.12:5.0-7.5; In the step B2, the addition ratio of compound P2, maleic anhydride, acetic anhydride, and anhydrous sodium acetate is 1.0 mmol: 2.0-2.2 mmol: 50-100 mL: 2.0-2.2 mmol.
9. The method for preparing a fluorescent chemical sensor for detecting biothiol compounds according to claim 7, wherein: In step A2, the heat treatment temperature is 90-100° C., and the reaction time is 2-3 days; In step B2, the first reaction temperature is 45-60° C., and the reaction time is 6-12 h; the second reaction temperature is 100-110° C., and the reaction time is 12-18 h.
10. The method for preparing a fluorescent chemical sensor for detecting biothiol compounds according to claim 7, wherein: The inorganic base is one of Na2CO3, K2CO3, and Cs2CO3; In the organic solvent / water mixture, the ratio of organic solvent / water is 15 / 1 to 20 / 1, and the organic solvent is one of dioxane, N,N-dimethylformamide, and N,N-dimethylacetamide.