A fluorescent probe for copper ion suitable for strong alkaline conditions, its preparation method and application

Abstract

The application belongs to the technical field of analytical chemistry, and particularly relates to a copper ion fluorescence probe suitable for strong alkaline conditions and a preparation method and application thereof. The copper ion fluorescence probe contains a naphthalimide structure, and a structural formula is shown in the following formula. The fluorescence probe can detect Cu 2+ under strong alkaline conditions with pH greater than or equal to 10; the detection sensitivity is high, and the response speed is fast.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of analytical chemistry, and particularly relates to a copper ion fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] Copper is a metal that people often contact in daily life, which makes people often exposed to the environment of copper ions (Cu 2 + ). Appropriate intake of Cu 2+ has important significance for maintaining the health of living beings, and Cu 2+ in the body can directly participate in the construction of various types of functional proteins and regulate many life activities of the body. However, excessive Cu 2+ exposure will seriously threaten the health of the body, so effective identification and monitoring of Cu 2+ in the environment has important significance for avoiding the harm of Cu 2+ and maintaining the health of life. In daily life, the most common Cu 2+ wastewater includes dyeing and finishing wastewater and electroplating wastewater, and the typical feature of this type of wastewater is strong alkalinity. Under this strong alkaline condition, the conventional Cu 2+ detection method is difficult to use normally, and adjusting the pH of the system will interfere with the Cu 2+ concentration in the solution and affect the detection results. Therefore, it is urgent to develop a Cu 2+ identification probe suitable for application under strong alkaline conditions. SUMMARY

[0003] The purpose of the application is to provide a Cu 2+ fluorescent probe based on naphthalimide structure with high sensitivity and fast response speed, which is suitable for application under strong alkaline conditions, and a preparation method and application thereof.

[0004] The Cu 2+ fluorescent probe suitable for strong alkaline conditions provided by the application has a naphthalimide structure, and its structural formula is as follows:

[0005]

[0006] The above-mentioned fluorescent probe provided by the application is obtained by the following synthesis steps, and the synthesis route is as follows:

[0007]

[0008] The specific steps of synthesis are as follows:

[0009] (1) In a reaction flask (a tomato flask), the starting material 1 (4-bromonaphthalene anhydride) (1.0 eq.), N-(2-aminoethyl) morpholine (1-1.2 eq.) were dissolved in ethanol; the reaction was carried out under nitrogen protection and was heated to 70-90 °C for 2-12 hours; the reaction was monitored by TLC; after the reaction was completed, the ethanol was evaporated, and a small amount of deionized water was added; the mixture was separated by suction filtration with a Buchner funnel, and the filter cake was washed with a mixture of methanol and water three times; vacuum drying was performed to obtain intermediate compound 2 with a yield of 70-95%;

[0010] (2) Intermediate compound 2 (1 eq.), triethylamine (1-3 eq.) and tert-butyl 2-(methylamino)ethylcarbamate (1-1.5 eq.) were dissolved in N,N-dimethylformamide; the reaction was stirred under nitrogen protection and was heated to 90-110 °C for 2-12 hours; the reaction was monitored by TLC; after the reaction was completed, N,N-dimethylformamide was removed by distillation under reduced pressure, and a mixture of water and methanol was added; yellow solid was precipitated after ice bath cooling; the mixture was separated by suction filtration with a Buchner funnel, and the filter cake was washed with a small amount of ice methanol; vacuum drying was performed to obtain yellow solid intermediate compound 3 with a yield of 80-95%;

[0011] (3) Intermediate compound 3 was mixed with dichloromethane, and concentrated hydrochloric acid (1 g of intermediate 3 was added to 1-2 mL of hydrochloric acid) was slowly dropped into the mixture under ice bath cooling; the reaction was carried out for 1-12 hours; after the reaction was completed, the reaction flask was added with an appropriate amount of saturated sodium carbonate solution, and the pH of the aqueous phase was adjusted until the pH was greater than or equal to 7.5; the solution was poured into a separatory funnel, and dichloromethane was used for extraction three times; the organic phase was combined and dried with anhydrous sodium sulfate; after dichloromethane was removed by rotary evaporation, intermediate compound 4 was obtained with a yield of more than 90%;

[0012] (4) Intermediate compound 4 (1 eq.), 4-dimethylpyridine (0.1-1 eq.) and triethylamine (1-3 eq.) were dissolved in DCM and were stirred; p-nitrophenyl chloroformate (1-3 eq.) was weighed, dissolved in DCM, and slowly added to the reaction flask under ice water bath cooling and nitrogen protection; the reaction was carried out for 1-12 hours; after the reaction was completed, the reaction solution was extracted with water and DCM, the organic phase was combined and concentrated, and column chromatography (methanol:dichloromethane = 1:40) was performed to obtain intermediate yellow solid product 5 with a yield of 75-90%;

[0013] (5) Intermediate compound 5 (1 eq.) was dissolved in dichloromethane; hydrazine hydrate (3-6 eq.) was slowly dropped into the reaction flask at a constant rate through a constant pressure dropping funnel under ice water bath cooling and nitrogen protection; the reaction was carried out at room temperature for 1-12 hours; after the reaction was completed, the reaction solution was extracted with water and dichloromethane, the organic phase was combined and dried, and rotary evaporation was performed after concentration; column chromatography on alumina was performed to obtain yellow solid target product.

[0014] The present application also relates to the use of the above fluorescent probe for monitoring Cu 2+ under strong alkaline conditions (pH≥10); the specific operation is as follows:

[0015] The mother liquor of the compound is prepared using an organic solvent (DMSO, DMF, etc.), the specific concentration is determined according to the test system, and the commonly used concentration is 1 mM-10 mM, which is diluted into the responding test system (the specific concentration is determined according to the test system, and the commonly used test concentration is 5 μM or 10 μM), the excitation wavelength is 440 nm, the collection wavelength range is 460-700 nm, and the response time is more than 2 hours.

[0016] The present application relates to the Cu 2+ fluorescent probe has a significant difference from the existing Cu 2+ fluorescent probe, that is, it can realize the recognition of Cu 2+ under strong alkaline conditions. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of compound 6.

[0018] Figure 2 is the high-resolution mass spectrum of compound 6.

[0019] Figure 3 is the fluorescence intensity graph of 10 μM compound 6 in 10 mM PBS solution with pH equal to 6-10 and 50 μM copper and iron ions after 3 hours of response at 556 nm (the excitation wavelength is 440 nm). DETAILED DESCRIPTION

[0020] The present application will be further described below through specific examples.

[0021] The examples include the synthesis of related intermediates and target compounds and the detection examples of Cu 2+ under strong alkaline solution.

[0022] Example, preparation of Cu 2+ fluorescent probe:

[0023] (1) Preparation of intermediate 2

[0024] In a 100 mL vial, add 1 (2.77 g, 10 mmol, 1 eq.), N-(2-aminoethyl)morpholine (1.367 g, 10.5 mmol, 1.05 eq.) and 30 mL of ethanol. Heat to 85 °C under nitrogen protection. After about 8 h, the reaction is complete according to TCL. Remove about 15 mL of ethanol under reduced pressure. Cool the vial in an ice bath and add 4 mL of deionized water while stirring. Separate the mixture by suction filtration using a Buchner funnel and wash the filter cake three times with a mixture of methanol / water. Dry under vacuum to obtain compound 2, which weighs 3.63 g (93.2% yield).

[0025] (2) Preparation of intermediate 3

[0026] In a 100 mL vial, add compound 2 (3.11 g, 8 mmol, 1 eq.), triethylamine (1675 μL, 12 mmol, 1.5 eq.) and 20 mL of N,N-dimethylformamide. Transfer tert-butyl 2-(methylamino)ethylcarbamate (1.53 g, 8.8 mmol, 1.1 eq.) diluted with a small amount of N,N-dimethylformamide to the vial. Heat to 110 °C under nitrogen protection. The reaction is complete after about 10 h. Cool the system to about 95 °C, remove about 14 mL of N,N-dimethylformamide under reduced pressure and add an appropriate amount of a water / methanol mixture. Cool in an ice bath and precipitate a yellow solid. Separate the mixture by suction filtration using a Buchner funnel and wash the filter cake with a small amount of ice methanol. Dry under vacuum to obtain yellow solid compound 3, which weighs 3.25 g (84.2% yield).

[0027] (3) Preparation of intermediate 4

[0028] In a 100 mL vial, add compound 3 (2.90 g, 6 mmol) and 15 mL of dichloromethane and slowly add 2 mL of concentrated hydrochloric acid dropwise under ice bath. After the addition is complete, protect the reaction under nitrogen for about 4 h. The dichloromethane phase is colorless and clear, and the aqueous phase is transparent and orange-red. Add an appropriate amount of saturated sodium carbonate solution to the vial to adjust the pH of the aqueous phase until the pH is ≥ 7.5. Pour the solution into a separatory funnel and extract three times with 180 mL of dichloromethane. Dry the combined organic phases with anhydrous sodium sulfate. Remove the dichloromethane by rotary evaporation to obtain compound 4 as an orange-yellow solid. Weigh 2.20 g (96% yield).

[0029] (4) Preparation of intermediate 5

[0030] Compound 4 (382 mg, 1 mmol, 1 eq), 4-dimethylpyridine (0.012 g, 0.1 mmol, 0.1 eq.) and triethylamine (280 μL, 2 mmol, 2 eq.) were placed in a 100 mL jar, 8 mL DCM was added and stirred, the solid was dissolved. p-nitrophenyl chloroformate (0.3 g, 1.5 mmol, 1.5 eq) was weighed in a constant pressure dropping funnel and dissolved in 2 mL DCM. The solution was added slowly into the jar under ice water bath and nitrogen protection. TLC was used to monitor the reaction, which was stopped after about 12 h. The solution was extracted with water and DCM, the organic phase was combined and concentrated, and then separated by column (methanol: dichloromethane = 1 :40) to obtain a yellow solid 0.458 g (yield 83.7%).

[0031] (5) Preparation of the target product

[0032] A 50 mL jar was added with a magnetic stirrer and compound 5 (274 mg, 0.5 mmol, 1 eq). About 6 mL of dichloromethane was used for dissolution, and hydrazine hydrate (85%, 150 μL, 2.5 mmol, 5 eq.) was added into the jar through a constant pressure dropping funnel at a constant speed under ice water bath and nitrogen protection. After about 8 h of reaction at room temperature, the solution was extracted with water and dichloromethane, the organic phase was combined, dried and concentrated by rotary evaporation. The yellow solid product 6 (79.73 mg, yield 36.2%) was obtained by column chromatography on alumina. The nuclear magnetic resonance hydrogen spectrum and high resolution mass spectrum of compound 6 are shown in Figures Figure 1 and Figures Figure 2 .

[0033] (6) Cu 2+ detection

[0034] After 10 μM compound 6 was reacted with 50 μM copper, iron ions in 10 mM PBS solution with pH equal to 6-10 for 3 h, the fluorescence at 556 nm was collected with 440 nm as excitation. The results are shown in Figure Figure 3 , which clearly shows that probe 6 can respond to Cu 2+ at pH = 10, but not to Fe 3+ .

Claims

1. A Cu suitable for strong alkaline conditions 2+ A fluorescent probe, characterized in that The structural formula is shown below: ； The strong alkaline condition is pH ≥10.

2. Cu as claimed in claim 1 2+ A method for synthesizing a fluorescent probe, characterized in that: The synthetic route is: ； The specific steps of synthesis are: (1) In a reaction flask, 1.0 eq of the starting material 1 and 1-1.2 eq. of N-(2-aminoethyl)morpholine were dissolved in ethanol. The reaction was heated to 70-90 °C under nitrogen protection for 2-12 hours. After the reaction was completed, the ethanol was evaporated, the mixture was cooled, and a small amount of deionized water was added. The mixture was separated by suction filtration, and the filter cake was washed three times with a methanol / water mixture. The mixture was dried under vacuum to obtain the intermediate compound 2 with a yield of 70-95%. (2) 1 eq. of intermediate compound 2, 1-3 eq. of triethylamine, and 1-1.5 eq. of tert-butyl 2-(methylamino)ethylcarbamate were dissolved in N,N-dimethylformamide; the temperature was raised to 90-110°C under nitrogen protection, and the reaction was stirred for 1-12 hours; after the reaction was completed, N,N-dimethylformamide was removed by vacuum distillation, a water / methanol mixed solution was added, and the mixture was cooled in an ice bath to precipitate a yellow solid; the mixture was separated by filtration, and the filter cake was washed with a small amount of ice methanol; and vacuum dried to obtain yellow solid intermediate compound 3 with a yield of between 80% and 95%; (3) The intermediate compound 3 was mixed with dichloromethane, and concentrated hydrochloric acid was slowly added dropwise under ice bath for 1-12 hours. After the reaction was completed, an appropriate amount of saturated sodium carbonate solution was added to the reaction flask to adjust the pH of the aqueous phase until the pH was ≥7.

5. The solution was poured into a separatory funnel and extracted with dichloromethane three times. The organic phases were combined and dried over anhydrous sodium sulfate. After removing the dichloromethane by rotary evaporation, the intermediate compound 4 was obtained with a yield of more than 90%. (4) 1 eq of the intermediate compound, 0.1-1 eq. of 4-lutidine and 1-3 eq. of triethylamine were dissolved in DCM and stirred; 1-3 eq. of p-nitrophenyl chloroformate was weighed and dissolved in DCM, and slowly added dropwise to the reaction flask under an ice-water bath and nitrogen protection, and the reaction was carried out for 1-12 hours; after the reaction was completed, the solution was extracted with water and DCM, and the organic phase was combined and concentrated, and passed through a column, wherein methanol:dichloromethane = 1:40, to obtain the intermediate yellow solid product 5, with a yield between 75% and 90%; (5) Dissolve 1 eq of intermediate compound 5 in dichloromethane; add 3-6 eq. of hydrazine hydrate to the reaction flask at a constant speed through a constant pressure dropping funnel in an ice-water bath under nitrogen protection; react at room temperature for 1-12 hours. After the reaction is completed, extract with water and dichloromethane, combine the organic phases, dry them, and then concentrate them by rotary evaporation; obtain the target product as a yellow solid by chromatography on an alumina column.

3. The fluorescent probe according to claim 1 is prepared under strong alkaline conditions of pH ≥ 10 for Cu 2+ Used in reagents for monitoring.

4. The application according to claim 3, wherein the specific operations are: The fluorescent probe mother solution is prepared using an organic solvent with a concentration of 1 mM-10 mM; the mother solution is diluted into the corresponding test system with a test concentration of 5 µM-10 µM, an excitation wavelength of 440 nm, a collection wavelength range of 460-700 nm, and a response time of more than 2 hours.

5. The use according to claim 4, wherein the organic solvent is DMSO or DMF.

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