CN109608382B - Fluorescent probe for detecting cyanide ions and hypochlorous acid as well as preparation and application thereof - Google Patents

Fluorescent probe for detecting cyanide ions and hypochlorous acid as well as preparation and application thereof Download PDF

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CN109608382B
CN109608382B CN201910072311.1A CN201910072311A CN109608382B CN 109608382 B CN109608382 B CN 109608382B CN 201910072311 A CN201910072311 A CN 201910072311A CN 109608382 B CN109608382 B CN 109608382B
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刘益江
潘华
刘书智
欧志鹏
陈红飙
黎华明
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Abstract

Discloses a fluorescent probe for detecting cyanide ions and hypochlorous acid, and preparation and application thereof. The probe of the invention takes phenylhydrazine-4-sulfonic acid and methyl isopropyl ketone as raw materials, and synthesizes N-ethyl-2, 3, 3-trimethylindole-5-potassium sulfonate through Fischer reaction and the like; synthesizing a condensing agent through a Vilsmeier-Haack reaction; the condensing agent and N-ethyl-2, 3, 3-trimethylindole-5-sulfonic acid potassium are subjected to Knoevenagel Condensation reaction and then subjected to Suzuki coupling reaction with N-phenyl-3-carbazole boric acid to obtain the compound. The probe is a near-infrared probe, and has the characteristics of high selectivity, high sensitivity, quick response and the like for cyanide ions and hypochlorous acid.

Description

Fluorescent probe for detecting cyanide ions and hypochlorous acid as well as preparation and application thereof
Technical Field
The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a fluorescent probe for detecting cyanide ions and hypochlorous acid, and a preparation method and an application method thereof.
Background
Cyanide plays an important role in industrial production and is widely applied to the fields of gold ore mining, electroplating, resin and fiber synthesis and the like. However, cyanide is a virulent substance, and its lethal dose is 0.5-3.5 mg/kg. Cyanide can be taken into the body through the lungs, skin, contaminated food and contaminated drinking water. Cyanide ions inhibit cellular respiration in mammals and cause death by inhibiting the central nervous system. According to the World Health Organization (WHO), the highest allowable cyanide concentration in drinking water is 1.9. mu.M. Therefore, cyanide ion detection is of great significance.
Hypochlorous acid is widely used in daily life as a disinfectant, an antibacterial agent, and a bleaching agent. Meanwhile, hypochlorous acid is also an important Reactive Oxygen Species (ROS) and plays an important role in various physiological and pathological processes. However, excessive hypochlorous acid is harmful to the human body, and causes various diseases such as arthritis, neurodegenerative diseases, cardiovascular diseases, and cancer. Therefore, hypochlorous acid detection is of great significance.
To date, a number of methods have been developed to detect cyanide ions and hypochlorous acid. Including atomic absorption spectroscopy, electrochemical analysis, and the like. However, these methods have disadvantages such as high cost, complicated sample preparation, and long test time. The organic fluorescent probe is concerned by the majority of researchers because of the advantages of rapid detection, simple operation, high sensitivity and the like. In recent years, various fluorescent probes for detecting cyanide ions and hypochlorous acid have come into existence, and the probes are challenged by high selectivity, high sensitivity, quick response and good water solubility. Generally, a relatively large number of probes are available for detecting cyanide ions and hypochlorous acid, but a probe capable of detecting cyanide ions and hypochlorous acid simultaneously, rapidly and efficiently is lacking.
Disclosure of Invention
The invention aims to provide a chemical fluorescent probe which is easy to prepare, low in cost, rapid in detection and stable in performance, a synthetic method of the probe and a method for detecting cyanide ions and hypochlorous acid with high selectivity and high sensitivity.
The invention is characterized in that a classic near-infrared dye-heptamethine indocyanine derivative is used as a fluorescent group and a recognition group, a carbazole-modified novel heptamethine indocyanine derivative probe Cz-Cy7 is designed by utilizing the nucleophilicity of cyanide ions and the oxidability of hypochlorous acid, and the probe can simultaneously, rapidly and efficiently recognize the cyanide ions and the hypochlorous acid.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
according to a first embodiment of the present invention, there is provided a compound having the general formula (I):
Figure GDA0002369003490000021
according to a second embodiment of the present invention, there is provided a process for the preparation of the above compound, characterized in that the process comprises the steps of:
1) taking phenylhydrazine-4-sulfonic acid, methyl isopropyl ketone and ethyl bromide as raw materials, synthesizing N-ethyl-2, 3, 3-trimethylindole-5-potassium sulfonate with a general formula (II) through Fischer reaction and alkylation reaction, and marking the potassium sulfonate as an indole derivative; the reaction is as follows:
Figure GDA0002369003490000022
2) using cyclohexanone and phosphorus oxychloride as raw materials, dichloromethane as a solvent, N, N-Dimethylformamide (DMF) as a solvent and a reaction reagent, and synthesizing a condensing agent with the following general formula (III) through Vilsmeier-Haack formylation reaction:
Figure GDA0002369003490000023
3) subjecting the indole derivative having general formula (II) synthesized in step 1) to Knoevenagel Condensation reaction with the condensing agent having general formula (III) synthesized in step 2) to obtain a Condensation product (M1) having general formula (IV) below:
Figure GDA0002369003490000024
4) subjecting the condensation product (M1) having the general formula (IV) obtained in step 3) to a Suzuki coupling reaction with N-phenyl-3-carbazole boronic acid (B) to obtain the target probe compound having the general formula (I):
Figure GDA0002369003490000031
preferably, in the above production method, step 1) is carried out as follows:
adding acetic acid, phenylhydrazine-4-sulfonic acid and methyl isopropyl ketone into a reactor, and carrying out reflux reaction (preferably under the protection of inert gas for 3-15 hours); then adding an organic alcohol saturated solution of potassium hydroxide (e.g., an isopropyl alcohol saturated solution: a saturated potassium hydroxide isopropyl alcohol solution prepared by dissolving potassium hydroxide in isopropyl alcohol); then adding bromoethane and acetonitrile, and carrying out reflux reaction (preferably, carrying out reflux reaction for 5-48 hours under the protection of inert gas); after cooling to room temperature, suction filtration, washing (preferably with diethyl ether) and drying (preferably with vacuum) give potassium N-ethyl-2, 3, 3-trimethylindole-5-sulfonate of the general formula (II), labeled "indole derivative".
Preferably, the molar ratio of phenylhydrazine-4-sulfonic acid to methyl isopropyl ketone is 1:2 to 5, preferably 1:3 to 4, more preferably 1:3.5 to 3.8; .
Preferably, the molar ratio of phenylhydrazine-4-sulfonic acid to bromoethane is from 1:1 to 3, preferably from 1:1.2 to 2.5, more preferably from 1:1.5 to 2.
Preferably, in the above production method, step 1) is carried out as follows:
adding acetic acid, phenylhydrazine-4-sulfonic acid and methyl isopropyl ketone into a reactor, and refluxing under the protection of inert gas (for example, for 3 to 15 hours); the reaction mixture is cooled and then added dropwise to a poor solvent or a solvent for precipitation (e.g., ethyl acetate) to precipitate a pink solid; then, the reaction mixture is filtered by suction, and the solid is washed with a solvent (for example, diethyl ether) and dried; dissolving the dried pink powder with an organic solvent (e.g., methanol or ethanol) to obtain a solution of compound (a) (e.g., a methanol solution or an ethanol solution of compound (a)), then adding dropwise an organic alcohol-saturated solution of potassium hydroxide (e.g., an isopropanol-saturated solution: a saturated potassium hydroxide isopropanol solution prepared by dissolving potassium hydroxide in isopropanol) to the solution of compound (a), precipitating a yellow solid, centrifuging, washing with a solvent (e.g., diethyl ether), and drying to obtain the corresponding potassium salt; then adding the potassium salt, an organic solvent (such as acetonitrile) and ethyl bromide into another reactor, refluxing under the protection of inert gas (such as nitrogen) for 10-40 hours (such as 24 hours), and separating out a red solid; after cooling to room temperature, the reaction mixture is filtered with suction, the solid is washed with a solvent (e.g. diethyl ether) and dried to give the compound of the general formula (II).
More preferably, step 1) is carried out as follows: adding acetic acid, phenylhydrazine-4-sulfonic acid and methyl isopropyl ketone into a single-mouth bottle in sequence, and refluxing under the protection of inert gas (such as nitrogen gas) (for example, for 3-12 hours, such as 8 hours), wherein the solution turns dark red; after cooling to room temperature, slowly dropping the reaction solution into ethyl acetate to separate out pink solid in the solution; then, carrying out suction filtration, washing with diethyl ether for multiple times, and finally carrying out vacuum drying; dissolving the above dried pink powder with methanol, dissolving potassium hydroxide in isopropanol to obtain saturated solution, adding saturated solution of isopropanol of potassium hydroxide dropwise into methanol solution to obtain yellow solid, centrifuging, washing with diethyl ether (for example, 3-4 times), and vacuum drying; adding the yellow solid, acetonitrile and ethyl bromide into another single-neck bottle in sequence, and refluxing under the protection of inert gas (such as nitrogen) for 10-40 hr (such as 24 hr) to separate out red solid; after cooling to room temperature, suction filtration, washing with diethyl ether and vacuum drying, the compound of the general formula (II) is obtained.
Preferably, in the above preparation method, wherein the step 2) is performed as follows:
adding DMF into a reactor, adding phosphorus oxychloride and dichloromethane under cooling (for example, under the cooling of an ice bath at 0 ℃), reacting (preferably reacting at room temperature for 0.2-6 hours), adding cyclohexanone, and carrying out reflux reaction (preferably heating reflux reaction for 1-12 hours); after the reaction is cooled (preferably to room temperature), the reaction mixture is dropped into ice water, stirred (preferably for 6 to 48 hours), filtered with suction, and dried (preferably vacuum-dried) to obtain the condensing agent of the general formula (III).
Preferably, the molar ratio of cyclohexanone to phosphorus oxychloride to DMF is 1:1 to 5:2 to 8, preferably 1:1.5 to 4.5:2.5 to 7, more preferably 1:2 to 4:3 to 6.
Preferably, in the above preparation method, wherein the step 2) is performed as follows:
adding an organic solvent (such as DMF) into a reactor, slowly adding a mixed solution of phosphorus oxychloride and dichloromethane dropwise under cooling (such as cooling in an ice bath at 0 ℃), heating (such as raising the temperature to room temperature) the mixed solution, reacting (such as 0.5-3 hours, such as 1 or 2 hours), and slowly adding cyclohexanone dropwise into the mixed solution; after the addition of cyclohexanone is completed, heating and refluxing (for example, for 2 to 6 hours) are performed, at which time the mixed solution becomes orange red, the reaction mixed solution is cooled (for example, to room temperature), then the mixed solution is added dropwise to crushed ice, stirred (for example, stirred overnight), suction filtered, and dried (for example, vacuum dried), to obtain the condensing agent of the general formula (III).
Preferably, step 2) is performed as follows: adding DMF into a three-neck flask, and slowly dropwise adding a mixed solution of phosphorus oxychloride and dichloromethane at the temperature of 0 ℃ in an ice bath; removing ice bath, reacting the mixed solution at room temperature (for example, 0.5-3 hr, such as 1 or 2 hr), and slowly adding cyclohexanone dropwise into the mixed solution; after the addition is complete, the mixture is heated to reflux (e.g. to 40 ℃, e.g. 2-6 hours, such as 4 hours), at which time the solution becomes orange-red; after the reaction mixture was cooled to room temperature, the mixture was dropped into ice overnight, filtered with suction, and dried in vacuo (for example, for 24 hours) to obtain a condensing agent of the general formula (III).
Preferably, in the above preparation method, wherein the step 3) is performed as follows:
adding the indole derivative synthesized in the step 1) and having the general formula (II), the condensing agent synthesized in the step 2) and having the general formula (III), sodium acetate and acetic anhydride into a reactor, and performing reflux reaction (preferably under the protection of nitrogen for 6-24 hours); cooling (preferably to room temperature) and isolating (preferably by precipitation in diethyl ether and purification by column chromatography) to give the condensation product (M1) of general formula (IV) below.
Preferably, the molar ratio of the indole derivative having the general formula (II) synthesized in step 1) to the condensing agent having the general formula (III) synthesized in step 2) is 1 to 10:1, preferably 2 to 8:1, more preferably 3 to 5: 1.
Preferably, in the above preparation method, wherein the step 3) is performed as follows:
adding the compound of formula (II) obtained in step 1) potassium N-ethyl-2, 3, 3-trimethylindole-5-sulfonate, the condensing agent of formula (III) obtained in step 2), sodium acetate and acetic anhydride to a reactor, and refluxing under inert gas (e.g. heating to 80 ℃ under reflux, e.g. for 4-20 hours, e.g. 12 hours), at which time the solution appears green in color; cooling the reaction solution (for example, to room temperature), removing acetic anhydride under reduced pressure, dissolving the residue with a solvent (for example, methanol or ethanol), dropping the solution into a poor solvent or a solvent for precipitation (for example, diethyl ether), suction-filtering, washing with a solvent (for example, a large amount of diethyl ether), and drying; the resulting green crude product is purified (for example, by column chromatography) to give a green colored metallescent solid, i.e., the condensation product of formula (IV) (M1).
Preferably, the compound of formula (II) obtained in step 1), potassium N-ethyl-2, 3, 3-trimethylindole-5-sulfonate, the condensing agent of formula (III) obtained in step 2), sodium acetate and acetic anhydride are added sequentially to a single-neck flask and refluxed (for example heated to 80 ℃ for 4 to 20 hours, such as 12 hours) under the protection of an inert gas (for example nitrogen), at which time the solution appears green in color; cooling to room temperature, removing acetic anhydride under reduced pressure, dissolving with methanol, dripping into diethyl ether, filtering, washing with diethyl ether, and vacuum drying; the obtained green crude product is separated and purified by column chromatography, chloroform/methanol (such as 4:1, v/v) is used as eluent, a green color band is collected, and the solvent is evaporated to dryness to finally obtain a solid with green color band metallic luster, namely a condensation product (M1) of the general formula (IV).
Preferably, in the above preparation method, wherein the step 4) is performed as follows:
adding the condensation product (M1) of the general formula (IV) obtained in the step 3), N-phenyl-3-carbazole boric acid (B), a palladium catalyst, potassium carbonate and a solvent into a reactor, and carrying out reflux reaction (preferably at 78 ℃ for 12-48 hours, preferably under the protection of nitrogen); cooling (preferably to room temperature), removing the mixed solvent and purifying by column chromatography to obtain the target probe compound of the general formula (I).
Preferably, the condensation product (M1) of the general formula (IV) obtained in step 3) and the N-phenyl-3-carbazolboronic acid (B) are in a molar ratio of 1:0.8 to 5, preferably 1:1 to 4, more preferably 1:1.2 to 3.
Preferably, the solvent is a mixed solution of ethanol and water, and the volume ratio of ethanol to water is 2-10:1, preferably 3-8:1, and more preferably 4-6: 1.
Preferably, the palladium catalyst is Pd (PPh)3)4Or Pd (OAc)2Or PdCl2Or Pd (dba)2. The palladium catalyst is preferably Pd (PPh)3)4。
Preferably, the palladium catalyst is used in an amount of 0.1 to 2%, preferably 0.5 to 1.5%, more preferably 0.6 to 1.2% by mass of the condensation product M1.
Preferably, in the above preparation method, wherein the step 4) is performed as follows:
the condensation product (M1) of the general formula (IV) obtained in step 3), N-phenyl-3-carbazolboronic acid, Pd (PPh) are added to the reactor3)4Potassium carbonate and an ethanol/water mixed solvent (e.g., 5:1 ratio, v/v) under reflux (e.g., reflux temperature of 78 ℃ C., reflux for 10-30 hours, such as 20 hours) under protection of an inert gas (e.g., nitrogen); cooling the reaction product (e.g., to room temperature), removing the mixed solvent under reduced pressure, dissolving the reaction product in a solvent (e.g., methanol), dropping the resulting solution into a poor solvent or a solvent for precipitation (e.g., diethyl ether), suction-filtering the solution, washing the solution with a solvent (e.g., diethyl ether), and drying the solution; the resulting green crude product is purified (e.g., by column chromatography) to obtain a green colored metallic lustrous solid, i.e., the compound of formula (I).
Preferably, step 4) is performed as follows: adding the condensation product (M1) of the general formula (IV) obtained in the step 3), N-phenyl-3-carbazole boric acid and Pd (PPh) into a single-mouth bottle in sequence3)4Potassium carbonate and an ethanol/water mixed solvent (e.g., 5:1, v/v),refluxing under an inert gas (e.g., nitrogen) (e.g., at a reflux temperature of 78 deg.C for 10-30 hours, e.g., 20 hours); cooling the reaction product (e.g. to room temperature), removing the mixed solvent under reduced pressure, dissolving with methanol, dropping the obtained solution into diethyl ether, filtering, washing with diethyl ether, and vacuum drying; separating and purifying the obtained green crude product by column chromatography, collecting dark green color band with ethyl acetate/methanol (such as 5:1, v/v) as eluent, and evaporating the solvent to obtain a solid with green color band metallic luster, i.e. the compound of the general formula (I).
Preferably, the compounds of formula (I) prepared according to the present invention have the following characteristics:1H NMR(400MHz,DMSO-d6)δ(ppm):8.31-8.29(d,J＝8Hz,2H),8.20-8.19(d,J＝4.0Hz,2H),7.79(s,1H),7.77-7.62(t,2H),7.744-7.741(d,J＝1.2Hz,1H),7.57-7.54(t,2H),7.51-7.50(d,J＝4.0Hz,4H),7.497-7.495(d,J＝0.8Hz,1H),7.48-7.47(d,J＝4Hz,1H),7.29-7.28(t,1H),7.264-7.260(d,J＝1.6Hz,1H),7.21(s,1H),7.23(s,1H),6.24-6.21(d,J＝12Hz,CH),4.13-4.10(m,4H),2.75-2.74(t,4H),2.02-1.99(t,2H),1.25-1.15(t,6H),1.07-0.97(s,12H).13C NMR(100MHz,DMSO-d6)δ(ppm):171.34,163.03,148.09,145.36,142.16,141.36,140.42,137.24,132.08,130.93,128.50,128.07,127.4,127.2,126.55,123.41,122.81,122.29,121.27,120.86,120.23,110.16.100.43,48.63,27.28,24.81,21.43,12.42.MALDI-TOF MS(C52H51BrN3O6S2)m/z:calcd.958.01,found:878.93[Cz-Cy7-Br]+。
according to a third embodiment of the present invention, there is provided the use of the compound of the above general formula (I) or the compound produced by the above production method as a fluorescent probe for detecting cyanide ions and/or hypochlorous acid.
The compound is used as a probe to carry out qualitative detection and/or quantitative determination on cyanide ions and/or hypochlorous acid.
Preferably, the lower limit of detection of the concentration of cyanide ions by the probe is 0.09. mu.M.
Preferably, the lower limit of the detection of the hypochlorous acid concentration by the probe is 0.014. mu.M.
Preferably, when the detection is performed by ultraviolet absorption and fluorescence emission spectroscopy, the probe is dissolved in a solvent to detect cyanide ions and/or hypochlorous acid.
Preferably, in detecting cyanide ions, the probe is dissolved in a first solvent, and cyanide ions are detected by ultraviolet absorption and fluorescence emission spectroscopy. Preferably, the first solvent is a mixed solution of water and DMF at a volume ratio of 3:7, and more preferably, the first solvent is a mixed solution of water and DMF at a volume ratio of 3:7 with the addition of a pH buffer. The pH buffer is HEPES buffer solution. The pH of the first solvent is 9-11.
Preferably, the hypochlorous acid is detected by dissolving the probe in the second solvent and detecting the hypochlorous acid by ultraviolet absorption and fluorescence emission spectroscopy. Preferably, the second solvent is PBS buffer solution. The second solvent is more preferably a PBS buffer solution containing acetonitrile, the acetonitrile content being 40-60% by volume. The pH of the second solvent is 3-5.
Preferably, the detection of cyanide ions and/or hypochlorous acid by the probe is determined colorimetrically under daylight naked eye conditions, or the response of the probe to cyanide ions and hypochlorous acid is determined by ultraviolet absorption and fluorescence emission spectroscopy.
Preferably, cyanide ions are added to the solution containing the probe, and the solution changes from dark green to bright yellow in color and the fluorescence is reduced. Hypochlorous acid is added into the solution containing the probe, the color of the solution is changed from dark green to earthy yellow, and the fluorescence is weakened.
Preferably, when the probe has no fluorescence response to detection of cyanide ions, the molar ratio of cyanide ions to probe in the solution to be detected is 12: 1.
preferably, the probe has no longer an ultraviolet response to cyanide ion exposure for more than 5 minutes.
Preferably, when the probe no longer has a fluorescent response to the detection of hypochlorous acid, the molar ratio of hypochlorous acid to probe in the solution to be detected is 10:1
Preferably, the probe does not have an ultraviolet response to hypochlorous acid for more than 1 minute.
The above synthesis of the compound (I) of the present invention is as follows:
Figure GDA0002369003490000071
in the invention, the fluorescent probe with double functions of detecting cyanide ions and hypochlorous acid is a compound (I) based on a heptamethine indocyanine derivative, and is marked as a fluorescent probe Cz-Cy 7.
It is still another object of the present invention to provide an application method for detecting cyanide ions and hypochlorous acid using the fluorescent probe obtained by the above method, in which both the qualitative and quantitative determination of cyanide ions and hypochlorous acid is performed using the probe obtained by the above method.
Preferably, when the detection is carried out by ultraviolet absorption and fluorescence emission spectroscopy, the fluorescent probe with the component of the compound (I) is dissolved in a mixed solution of water and DMF (with the volume ratio of 3:7) to detect cyanide ions.
Preferably, after the cyanide ions are added into the probe solution, the color of the solution is rapidly changed from original green to bright yellow, and the color change can be clearly distinguished by naked eyes under a fluorescent lamp.
The fluorescent probe prepared by the invention is applied to cyanide ion detection. The probe was dissolved in a mixed solvent of water/DMF (volume ratio 3:7, preferably added with HEPES buffer, pH 10), preferably 5 μ M probe solution, then cyanide ion was added, and the response of the probe to cyanide ion was measured by uv absorption spectroscopy or the change in color of the solution was directly observed. The addition of cyanide ions changes the color of the probe solution from dark green to bright yellow, and the absorption intensity at 771nm is reduced.
Preferably, the quantitative detection performance of the probe on cyanide ions is researched by adopting a fluorescence spectroscopy, and the fluorescence emission intensity of the system at 806nm before and after the probe is added into the cyanide ions is collected; experiments show that after the cyanide ion solution is added into the probe solution for 5 minutes, the fluorescence intensity does not change any further along with the prolonging of time, and the probe can complete the response to the cyanide ions within 5 minutes. Meanwhile, the fluorescence intensity decreases with the increase of the concentration of cyanide ions, and when the concentration of the probe and the cyanide ions is 1: 12 hours, the fluorescence emission intensity of the system at 806nm reaches the lowest, which indicates that the probe and the cyanide ions have complete action, the change of the fluorescence intensity of the probe has a linear relationship with the concentration of the cyanide ions, and the lower detection limits are respectively 0.09 mu M and far lower than the 1.9 mu M safe drinking water standard determined by the world health organization.
Preferably, other ten common anions are selected for detecting the probe in the experiment, and the result shows that the detection of the probe is not obviously influenced, which shows that the probe has very excellent selectivity for detecting cyanide ions.
The fluorescent probe prepared by the invention is applied to hypochlorous acid detection. After dissolving the probe in a PBS buffer solution (pH 4, containing 50% acetonitrile) containing acetonitrile, preferably a probe solution having a concentration of 5 μ M, hypochlorous acid was added, and the response of the probe to hypochlorous acid was measured by ultraviolet absorption spectroscopy or the change in color of the solution was directly observed. The addition of hypochlorous acid changed the color of the probe solution from dark green to earthy yellow, and the absorption intensity at 762nm decreased.
Preferably, the quantitative detection performance of the probe on hypochlorous acid is researched by adopting a fluorescence spectroscopy method, and the fluorescence emission intensity of the system at the position of 795nm before and after the probe is added into the hypochlorous acid is collected; experiments show that after the hypochlorous acid solution is added into the probe solution for 1 minute, the fluorescence intensity does not change any further along with the prolonging of the time, and the probe can complete the response to the hypochlorous acid within 1 minute. Meanwhile, the fluorescence intensity decreases with the increase of the hypochlorous acid concentration, and when the concentration of the probe and the hypochlorous acid is 1: when 10, the fluorescence emission intensity of the system at 795nm reached the lowest, indicating that the probe acted completely with hypochlorous acid, and the change in the fluorescence intensity of the probe was linear to the hypochlorous acid concentration, with a lower limit of detection of 0.014. mu.M each.
The fluorescent probe prepared by the invention has high selectivity for detecting hypochlorous acid, and other common active oxygen species (ONOO-, HO-, H)2O2TBHP and. O2) Has no obvious influence on the detection of the probe.
The fluorescent probe prepared by the invention can be used for detecting cyanide ions and hypochlorous acid in a solid state, and if the probe is fixed on filter paper and the cyanide ions and the hypochlorous acid are dripped, the color change which is the same as that of a solution state can be observed by naked eyes under natural light.
In the present invention, hypochlorous acid is commonly used as hypochlorite ions.
The invention has the following beneficial effects:
1. the heptamethine indocyanine derivative-based fluorescent probe synthesized by the invention (i.e., the compound with the general formula (1)) can simultaneously detect cyanide ions and hypochlorous acid;
2. the invention provides a novel near-infrared probe, which can detect cyanide ions and hypochlorous acid in the presence of water and is expected to be applied to fluorescent tracing and imaging of cyanide ions and hypochlorous acid in biological living cells;
3. the probe has high response speed, and cyanide ion detection can be completed within 5 minutes; the hypochlorous acid detection can be completed within 1 minute, and can be applied to the on-site rapid detection and monitoring of cyanide ions and hypochlorous acid;
4. the heptamethine indocyanine derivative-based fluorescent probe synthesized by the invention has the characteristic of visual identification;
5. the detection lower limit of the probe to the concentration of the cyanide ions reaches 0.09 mu M, exceeds a plurality of other types of cyanide ion probes and is far lower than the concentration of the cyanide ions in safe drinking water permitted by the world health organization; the lower limit of detection of hypochlorous acid reaches 0.014 mu M, which is far lower than the hypochlorous acid probe based on indocyanine derivatives reported at present;
6. the probe has excellent selectivity and competitiveness, and other common anions and active oxygen species do not generate interference when detecting cyanide ions and hypochlorous acid.
Drawings
FIG. 1 is a general structural formula of the compound prepared by the invention.
FIG. 2 is a scheme showing the synthesis of the compound of formula (I) according to the present invention.
FIG. 3 is the ultraviolet-visible absorption spectrum response of the probe to cyanide ions, the linear relation curve and the photo thereof under natural light.
FIG. 4 is a curve showing the fluorescence spectral response and linear relationship between the probe and cyanide ions.
FIG. 5 is a graph showing the reaction kinetics of a probe with respect to cyanide ions.
FIG. 6 shows the high selectivity of the probe for detection of cyanide ions.
FIG. 7 is a graph showing the UV-VIS absorption spectrum response of a probe to hypochlorous acid, a linear relationship thereof, and a photograph thereof under natural light.
FIG. 8 is a graph showing the fluorescence spectral response of a probe to hypochlorous acid in a linear relationship.
FIG. 9 is a graph showing the relationship between the reaction kinetics of a probe and hypochlorous acid.
FIG. 10 shows the high selectivity of the probe for hypochlorous acid detection.
FIG. 11 is a photograph of a portable probe-based test paper (filter paper) for detecting cyanide ions and hypochlorous acid.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but the present invention is not limited to these examples.
Example 1: synthesis of target Probe Cz-Cy7
(1) Synthesis of potassium N-ethyl-2, 3, 3-trimethylindole-5-sulfonate (Compound (II))
To a 50mL single-neck flask, 10mL of acetic acid, phenylhydrazine-4-sulfonic acid (2.000g, 0.010mol), and 4.00mL (0.037mol) of methyl isopropyl ketone were added in this order, and the mixture was refluxed under nitrogen for 8 hours, whereupon the solution turned dark red. After cooling to room temperature, slowly dropping the reaction solution into ethyl acetate to separate out pink solid in the solution; filtering, washing with diethyl ether for several times, and vacuum drying; dissolving the dried pink powder with an appropriate amount of methanol, dissolving 0.561g of potassium hydroxide in isopropanol to prepare a saturated solution, then dropwise adding the isopropanol saturated solution of potassium hydroxide into the methanol solution to obtain a yellow solid, centrifuging, washing with diethyl ether for 3-4 times, and vacuum drying. Another 50mL single-neck flask was charged with the yellow solid, 20mL acetonitrile, and 1.49mL (0.020mol) bromoethane in that order, and refluxed under nitrogen for 24 hours to precipitate a red solid. After cooling to room temperature, suction filtration and washing with ether (3X 10mL) was carried out and drying in vacuo afforded compound (II).
(2) Synthesis of condensing agent (Compound (III))
A100 mL three-necked flask was charged with 10mL (0.130mol) of DMF, and a mixed solution of 10mL (0.107mol) of phosphorus oxychloride and 5mL of dichloromethane was slowly added dropwise under an ice bath at 0 ℃. The ice bath was removed, the reaction was carried out at room temperature for 1 hour, and 3.5mL (0.034mol) of cyclohexanone was slowly added dropwise to the mixed solution. After the addition was completed, the mixture was heated under reflux for 4 hours, at which time the solution became orange-red. After the reaction is cooled to room temperature, the mixed solution is dropped into ice, and the compound (III) is obtained after overnight, suction filtration and vacuum drying.
(3) Synthesis of Compound (IV), condensation product M1
To a 25mL single-neck flask, N-ethyl-2, 3, 3-trimethylindole-5-sulfonic acid potassium salt (0.843g, 2.2mmol), a condensing agent (compound (III) obtained in step 2, 0.188g, 1.1mmol), sodium acetate (0.180g, 2.2mmol) and 10mL of acetic anhydride were added in this order, and the mixture was refluxed under nitrogen for 12 hours, whereupon the solution appeared green in color. Cooling to room temperature, removing acetic anhydride under reduced pressure, dissolving with a small amount of methanol, dropping into diethyl ether, filtering, washing with a large amount of diethyl ether, and vacuum drying; the resulting green crude product was isolated and purified by column chromatography using chloroform/methanol (4:1, v/v) as eluent, collecting the green color band, and evaporating the solvent to dryness to obtain 0.664g of a green band metallic lustrous solid (M1) with a yield of 64.4%.
(4) Synthesis of aimed product Cz-Cy7 (Compound (I))
To a 100mL single-necked flask, the condensation product M1(1.220g, 2.00mmol) (Compound (IV) obtained in step 3), N-phenyl-3-carbazolboronic acid (1.040g, 3.60mmol), Pd (PPh) were added in this order3)4(0.230g, 0.20mmol), potassium carbonate 0.280g, 2.00mmol) and 50mL ethanol/water (5:1, v/v) were refluxed under nitrogen for 20 hours. After cooling to room temperature, the mixed solvent is removed under reduced pressure, then dissolved with a small amount of methanol, added dropwise to diethyl ether, filtered, washed with diethyl ether and dried in vacuum. The obtained green crude product is separated and purified by column chromatography, ethyl acetate/methanol (5:1, v/v) is used as eluent, a dark green color band is collected, the solvent is evaporated to dryness, and 1.197g of solid with green color band metallic luster is finally obtained.
And (3) probe characterization:1H NMR(400MHz,DMSO-d6)δ(ppm):8.31-8.29(d,J＝8Hz,2H),8.20-8.19(d,J＝4.0Hz,2H),7.79(s,1H),7.77-7.62(t,2H),7.744-7.741(d,J＝1.2Hz,1H),7.57-7.54(t,2H),7.51-7.50(d,J＝4.0Hz,4H),7.497-7.495(d,J＝0.8Hz,1H),7.48-7.47(d,J＝4Hz,1H),7.29-7.28(t,1H),7.264-7.260(d,J＝1.6Hz,1H),7.21(s,1H),7.23(s,1H),6.24-6.21(d,J＝12Hz,),4.13-4.10(m,4H),2.75-2.74(t,4H),2.02-1.99(t,2H),1.25-1.15(t,6H),1.07-0.97(s,12H).13CNMR(100MHz,DMSO-d6)δ(ppm):171.34,163.03,148.09,145.36,142.16,141.36,140.42,137.24,132.08,130.93,128.50,128.07,127.4,127.2,126.55,123.41,122.81,122.29,121.27,120.86,120.23,110.16.100.43,48.63,27.28,24.81,21.43,12.42.MALDI-TOF MS(C52H51BrN3O6S2)m/z:calcd.958.01,found:878.93[Cz-Cy7-Br]+。
example 2: titration experiment of cyanide ion pair probe
A probe solution having a concentration of 5. mu.M was prepared in a mixed solvent of water and DMF (in a volume ratio of 3:7, preferably with HEPES buffer solution, pH 10). Then, different concentrations of cyanide ions (0. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, 25. mu.M, 30. mu.M, 35. mu.M, 40. mu.M, 45. mu.M, 50. mu.M, 55. mu.M, 60. mu.M) were added dropwise, and after 5min, the absorption and fluorescence emission responses of the mixed system were tested by ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy.
As can be seen from FIG. 3, the absorption peak intensity at 771nm of the probe decreased with increasing concentration of cyanide ions, and the solution color changed from dark green to bright yellow.
As can be seen from FIG. 4, the emission peak intensity of the probe at 806nm decreases with the increase of the concentration of cyanide ions, the probe completely reacts with 12 equivalents of cyanide ions, and the emission peak intensity has a linear relationship with the concentration of cyanide ions, which indicates that the probe can quantitatively detect cyanide ions.
Example 3: reaction kinetics test of probe for cyanide ions
At room temperature, a 5 μ M probe solution (water/DMF in a volume ratio of 3:7, preferably HEPES buffer, pH 10) was prepared, followed by the addition of cyanide ion using a microsyringe and recording of the uv absorption intensity at 771nm for the samples at different times.
As can be seen from FIG. 5, the absorption intensity of the probe at 771nm decreases with time, and after 5 minutes, the ultraviolet absorption intensity of the system does not change any more with time, which indicates that the probe can complete the detection of cyanide ions within 5 minutes.
Example 4: selective test for detecting cyanide ions by probe
At room temperature, probe solutions (water/DMF in a volume ratio of 3:7, HEPES buffer, pH 10) and various anion solutions (Na) were prepared at a concentration of 5 μ M2CO3，KSCN，Na2SO4，KF，NaCl，NaBr，KI，NaNO2，NaHCO3NaOAc), adding various anions and cyanide ions into the probe solution respectively, shaking uniformly, standing for 5 minutes, and measuring the fluorescence emission intensity of the probe on a fluorescence spectrometer.
As can be seen from FIG. 6, common anions do not significantly interfere with the detection of the probe, indicating that the probe has good selectivity for detecting cyanide ions.
Example 5: titration experiment of hypochlorous acid on Probe
A probe solution was prepared at a concentration of 5. mu.M in PBS buffer (pH 4, 50% acetonitrile). Then, hypochlorous acid (0. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, 25. mu.M, 30. mu.M, 35. mu.M, 40. mu.M, 45. mu.M, 50. mu.M) was added dropwise at different concentrations, and after 1 minute, the absorption and fluorescence emission responses of the mixed system were measured by ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy.
As can be seen from FIG. 7, the absorption peak intensity at 762nm of the probe decreased with the increase in the hypochlorous acid concentration, and the color of the solution changed from greenish black to yellowish brown.
As is clear from FIG. 8, the emission peak intensity at 795nm of the probe decreased with the increase in the hypochlorous acid concentration, the probe reacted completely with 10 equivalents of hypochlorous acid, and the emission peak intensity linearly correlated with the hypochlorous acid concentration, indicating that the probe can quantitatively detect hypochlorous acid.
Example 6: probe reaction kinetics test for hypochlorous acid
Probe solutions were prepared at 5 μ M concentration (in PBS buffer, pH 4, 50% acetonitrile) at room temperature, then hypochlorous acid was added with a micro-syringe and the uv absorption intensity at 762nm was recorded for the samples at different times.
As can be seen from FIG. 9, the absorption peak intensity at 762nm of the probe decreased with time, and after 1 minute, the UV absorption intensity of the system did not change any more with time, indicating that the probe could complete the detection of hypochlorous acid within 1 minute.
Example 7: probe-based selectivity test for detecting hypochlorous acid
At room temperature, probe solutions (pH 4, 50% acetonitrile in PBS buffer) at 5. mu.M concentration and various active oxygen species (HO, H) were prepared2O2,ONOO-,·O2TBHP), various active oxygen species and hypochlorous acid were added to the probe solution, respectively, shaken and left for 1 minute, and then the fluorescence emission intensity of the probe was measured on a fluorescence spectrometer.
As can be seen from FIG. 10, the common active oxygen species do not significantly interfere with the detection of hypochlorous acid by the probe, indicating that the probe has good selectivity for the detection of hypochlorous acid.
Example 8: application of portable probe (probe fixed on filter paper) in detection of cyanide ions and hypochlorous acid
Preparing a probe solution with a certain concentration, immersing the cut filter paper in the probe solution, and performing vacuum drying at room temperature after the probe is fully adsorbed.
Subsequently:
(1) the filter paper is dripped with cyanide ion and hypochlorous acid solution with different concentrations, dried in vacuum at room temperature, and photographed by a digital camera (see figure 11 a). Wherein: the concentration of cyanide ions is 3eq, 6eq, 9eq and 12eq from left to right in sequence; the hypochlorite ion concentration was 2.5eq, 5eq, 7.5eq, and 10eq from left to right.
(2) Several representative anions (AcO) were added dropwise to the filter paper-,SO4 2-,CO3 2-,HCO3 -,NO2 -) And active oxygen (HO, H)2O2,ONOO-,TBHP,·O2) And dried in vacuum at room temperature and photographed by a digital camera (see fig. 11 b). Wherein: detection ofAcO is sequentially dripped from left to right of cyanide ions-、SO4 2-、CO3 2-、HCO3 -、NO2 -(ii) a HO and H are added from left to right in turn for detecting hypochlorous acid2O2、ONOO-、TBHP,·O2。
As can be seen from FIG. 11, the same color change occurred in the portable test paper as in the solution, i.e., the test paper gradually changed from dark green to bright yellow as the concentration of cyanide ions increased; with the increase of the hypochlorous acid concentration, the test paper gradually changes from dark green to earthy yellow; other interfering ions and active species do not interfere with the detection of cyanide ions and hypochlorous acid by the probe. The probe designed and synthesized by the invention can be used for detecting cyanide ions and hypochlorous acid in a portable, rapid and efficient manner.

Claims (23)

1. A compound having the general formula (I):
Figure FDA0002369003480000011
2. a process for the preparation of a compound according to claim 1, characterized in that it comprises the following steps:
1) taking phenylhydrazine-4-sulfonic acid, methyl isopropyl ketone and ethyl bromide as raw materials, synthesizing N-ethyl-2, 3, 3-trimethylindole-5-potassium sulfonate with a general formula (II) through Fischer reaction and alkylation reaction, and marking the potassium sulfonate as an indole derivative; the reaction is as follows:
Figure FDA0002369003480000012
2) using cyclohexanone and phosphorus oxychloride as raw materials, dichloromethane as a solvent, N, N-Dimethylformamide (DMF) as a solvent and a reaction reagent, and synthesizing a condensing agent with the following general formula (III) through Vilsmeier-Haack formylation reaction:
Figure FDA0002369003480000013
3) subjecting the indole derivative having general formula (II) synthesized in step 1) to Knoevenagel Condensation reaction with the condensing agent having general formula (III) synthesized in step 2) to obtain a Condensation product (M1) having general formula (IV) below:
Figure FDA0002369003480000014
4) subjecting the condensation product (M1) of general formula (IV) obtained in step 3) to a Suzuki coupling reaction with N-phenyl-3-carbazole boronic acid (B) to obtain the target probe compound having general formula (I):
Figure FDA0002369003480000021
3. the method of claim 2, wherein step 1) is performed by: adding acetic acid, phenylhydrazine-4-sulfonic acid and methyl isopropyl ketone into a reactor, and carrying out reflux reaction; then adding an organic alcohol saturated solution of potassium hydroxide; then adding bromoethane and acetonitrile for reflux reaction; cooling to room temperature, filtering, washing and drying to obtain the potassium N-ethyl-2, 3, 3-trimethylindole-5-sulfonate with the general formula (II) which is marked as the indole derivative.
4. The method of claim 3, wherein: the organic alcohol saturated solution of the potassium hydroxide comprises: dissolving potassium hydroxide in isopropanol to obtain saturated isopropanol solution of potassium hydroxide; washing with diethyl ether; the drying adopts vacuum drying; the molar ratio of the phenylhydrazine-4-sulfonic acid to the methyl isopropyl ketone is 1: 2-5; the molar ratio of the phenylhydrazine-4-sulfonic acid to the bromoethane is 1: 1-3.
5. The method of claim 4, wherein: the molar ratio of the phenylhydrazine-4-sulfonic acid to the methyl isopropyl ketone is 1: 3-4; the molar ratio of the phenylhydrazine-4-sulfonic acid to the bromoethane is 1: 1.2-2.5.
6. The method of claim 5, wherein: the molar ratio of the phenylhydrazine-4-sulfonic acid to the methyl isopropyl ketone is 1: 3.5-3.8; the molar ratio of the phenylhydrazine-4-sulfonic acid to the bromoethane is 1: 1.5-2.
7. The method of claim 2, wherein step 2) is performed by: adding DMF into a reactor, adding phosphorus oxychloride and dichloromethane under cooling, reacting, adding cyclohexanone, and performing reflux reaction; after the reaction is cooled, the reaction mixed liquid is dropped into ice water, stirred, filtered and dried to obtain the condensing agent with the general formula (III).
8. The method of claim 7, wherein: the cooling is carried out in an ice bath at 0 ℃; the reaction of the phosphorus oxychloride and the dichloromethane is carried out for 0.2 to 6 hours at room temperature; the reflux reaction is heating reflux reaction for 1 to 12 hours; the cooling is to cool to room temperature; the stirring is performed for 6 to 48 hours; the drying is vacuum drying; the molar ratio of cyclohexanone to phosphorus oxychloride to DMF is 1:1-5: 2-8.
9. The method of claim 8, wherein: the molar ratio of cyclohexanone to phosphorus oxychloride to DMF is 1:1.5-4.5: 2.5-7.
10. The method of claim 9, wherein: the molar ratio of cyclohexanone to phosphorus oxychloride to DMF is 1:2-4: 3-6.
11. The method of claim 2, wherein step 3) is performed by: adding the indole derivative synthesized in the step 1) and having the general formula (II), the condensing agent synthesized in the step 2) and having the general formula (III), sodium acetate and acetic anhydride into a reactor, and carrying out reflux reaction; cooling and separating to obtain the condensation product (M1) with the following general formula (IV).
12. The method of claim 11, wherein: the reflux reaction is carried out for 6 to 24 hours under the protection of nitrogen; the cooling is to cool to room temperature; the separation adopts sedimentation in ether and column chromatography separation and purification; the molar ratio of the indole derivative with the general formula (II) synthesized in the step 1) to the condensing agent with the general formula (III) synthesized in the step 2) is 1-10: 1.
13. The method of claim 12, wherein: the molar ratio of the indole derivative with the general formula (II) synthesized in the step 1) to the condensing agent with the general formula (III) synthesized in the step 2) is 2-8: 1.
14. The method of claim 13, wherein: the molar ratio of the indole derivative with the general formula (II) synthesized in the step 1) to the condensing agent with the general formula (III) synthesized in the step 2) is 3-5: 1.
15. The method of claim 2, wherein step 4) is performed by: adding the condensation product (M1) of the general formula (IV) obtained in the step 3), N-phenyl-3-carbazole boric acid (B), a palladium catalyst, potassium carbonate and a solvent into a reactor, and carrying out reflux reaction; and (3) cooling, removing the mixed solvent, and performing column chromatography separation and purification to obtain the target probe compound shown in the general formula (I).
16. The method of claim 15, wherein: the reflux reaction is carried out for 12 to 48 hours under the protection of nitrogen; the cooling is to cool to room temperature; the molar ratio of the condensation product (M1) of the general formula (IV) obtained in step 3) to the N-phenyl-3-carbazole boronic acid (B) is 1: 0.8-5; the solvent is a mixed solution of ethanol and water; the palladium catalyst is Pd (PPh)3)4Or Pd (OAc)2Or PdCl2Or Pd (dba)2(ii) a The amount of the palladium catalyst used is 0.1 to 2% by mass of the condensation product M1.
17. The method of claim 16, wherein: the molar ratio of the condensation product (M1) of the general formula (IV) obtained in step 3) to the N-phenyl-3-carbazole boronic acid (B) is 1: 1-4; the solvent is a mixed solution of ethanol and water, and the volume ratio of the ethanol to the water is 2-10: 1; the amount of the palladium catalyst used is 0.5 to 1.5% by mass of the condensation product M1.
18. The method of claim 16, wherein: the molar ratio of the condensation product (M1) of the general formula (IV) obtained in step 3) to the N-phenyl-3-carbazole boronic acid (B) is 1: 1.2-3; the solvent is a mixed solution of ethanol and water, and the volume ratio of the ethanol to the water is 3-8: 1; the palladium catalyst is Pd (PPh)3)4The amount of the palladium catalyst used is 0.6 to 1.2% by mass of the condensation product M1.
19. Use of a compound of general formula (I) or a compound prepared by the method of any one of claims 2 to 18 as a fluorescent probe for detecting cyanide ions and/or hypochlorous acid, wherein the compound is used as a probe for qualitative and/or quantitative detection of cyanide ions and/or hypochlorous acid.
20. Use according to claim 19, characterized in that: the lower limit of detection of the probe for the concentration of cyanide ions was 0.09. mu.M, and the lower limit of detection of the probe for the concentration of hypochlorous acid was 0.014. mu.M.
21. The use according to claim 19, wherein the probe is dissolved in a first solvent and cyanide ions are detected by uv absorption and fluorescence emission spectroscopy; the first solvent is a mixed solution of water and DMF in a volume ratio of 3: 7; and/or
Dissolving the probe in a second solvent, and detecting hypochlorous acid by adopting an ultraviolet absorption and fluorescence emission spectroscopy method; the second solvent is PBS buffer solution; and/or
The detection of the probe to the cyanide ions and/or the hypochlorous acid is carried out colorimetric judgment under the sunlight naked eye condition, or the response of the probe to the cyanide ions and the hypochlorous acid is measured through ultraviolet absorption and fluorescence emission spectrums.
22. Use according to claim 21, characterized in that: the first solvent is a mixed solution of water added with a pH buffering agent and DMF in a volume ratio of 3: 7; the pH buffer is HEPES buffer solution, and the pH of the first solvent is 9-11; and/or
The second solvent is PBS buffer solution containing acetonitrile, the volume content of the acetonitrile is 40-60%, and the pH value of the second solvent is 3-5; and/or
Adding cyanide ions into the solution containing the probe, wherein the color of the solution is changed from dark green to bright yellow, and the fluorescence is weakened; hypochlorous acid is added into the solution containing the probe, the color of the solution is changed from dark green to earthy yellow, and the fluorescence is weakened.
23. Use according to claim 21 or 22, characterized in that, when the probe no longer has a fluorescent response to detection of cyanide ions, the molar ratio of cyanide ions to probe in the solution to be detected is 12:1, and the time when the probe no longer has an ultraviolet response to the action of cyanide ions does not exceed 5 minutes; and/or
When the probe does not have fluorescence response to the hypochlorous acid detection, the mole ratio of the hypochlorous acid to the probe in the solution to be detected is 10:1, and the time when the probe does not have ultraviolet response to the hypochlorous acid is not more than 1 minute.
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