Preparation and application of rhodamine derivative modified beta-cyclodextrin
β-cyclodextrin and rhodamine B derivatives were prepared by acetonitrile method, and the aminerhodamine-cyclodextrin fluorescent probe was synthesized, which solved the toxicity and preparation methods of traditional fluorescence temperature probes, and achieved high stability and environmental protection Fluorescence temperature sensing effect.
Patent Information
- Application Number
- CN202311270493.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-28
- Publication Date
- 2024-12-06
AI Technical Summary
Traditional fluorescence temperature probes have problems of poor toxicity and light stability, which limits their use in some applications, and the existing preparation methods of β-cyclodextrin have problems of toxicity, high cost and difficulty in operation.
The active intermediate mono-(6-p-toluenesulfonyl-6-deoxy)-β-cyclodextrin was prepared by acetonitrile method, and the reaction was carried out with rhodamine B lactone by 1,3-propanediamine. The structured aminodrodamine-cyclodextrin fluorescent probe adopts green, non-toxic and simple operation method to obtain high-stability products.
The prepared fluorescent probe has good environmental protection, good water solubility and ambient temperature sensing properties, and is simple in process and industrially controllable, reducing toxicity and cost.
Smart Images
Figure CN119080969A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of fluorescent probe materials, and in particular to a preparation method and application of a fluorescent probe having a temperature-sensing rhodamine B derivative modified with beta-cyclodextrin. Background Art
[0002] Temperature detection has a wide range of applications in various fields, including biomedical research, food processing, material research, etc. Traditional temperature detection methods include thermocouples, thermistors, infrared temperature measurement, etc., but they may be limited by interference. Fluorescence temperature probe is a temperature measurement method based on fluorescence properties, which is highly selective, sensitive and non-invasive. Traditional probes may have shortcomings, such as toxicity and poor photostability, which limit their use in some applications. The fluorescent probe prepared by the present invention has lower toxicity and better biocompatibility.
[0003] Fluorescent probes can be divided into two categories: organic molecular probes and biomolecular probes. Organic molecular probes are usually organic dyes or molecular markers, such as fluorescent dyes, rhodamine, fluorescent proteins, etc. Biomolecular probes include DNA probes, RNA probes, fluorescent antibodies, etc. Among them, the organic dye rhodamine B has bright fluorescence characteristics. After being excited by light of a specific wavelength of 527nm, it will emit bright red fluorescence, but it has potential toxicity and is highly polluting to the environment.
[0004] Cyclodextrin has a cavity structure with hydrophobic interior and hydrophilic exterior, so it can provide one or more hydrophobic binding sites like an enzyme. Because of the special structure of cyclodextrin, it has an inclusion effect to increase drug solubility and bioavailability, a sustained release effect to stabilize volatile drugs, a stabilizing effect to improve the preservation of active ingredients, and a dissolving effect to promote the dissolution of substances that are poorly soluble in water. At present, the main methods for preparing mono-(6-toluenesulfonyl-6-deoxy)-β-cyclodextrin are: 1. The pyridine method, which has a high yield, but pyridine is toxic and harmful; 2. The acetonitrile method, which is relatively mild, green and low-toxic, but the yield is not high; 3. The p-toluenesulfonic anhydride method, which has expensive consumables and difficult to control the homemade conditions.
[0005] Based on this, the present invention adopts the acetonitrile method to prepare the active intermediate mono-(6-toluenesulfonyl-6-deoxy)-β-cyclodextrin. Using 1,3-propylenediamine as a linker, 1,3-propylenediamine-rhodamine B lactone is synthesized and reacted with mono-(6-toluenesulfonyl)-β-cyclodextrin to prepare an amino rhodamine-cyclodextrin fluorescent probe with a self-inclusion structure. The probe has good environmental protection and good water solubility. As the ambient temperature increases, the ultraviolet absorption and fluorescence intensity gradually increase. Summary of the invention
[0006] The present invention aims at the above technical analysis and existing problems, and provides a preparation method and application of a rhodamine derivative modified β-cyclodextrin temperature sensor. The green, non-toxic and simple-to-operate method can obtain a highly stable amino rhodamine β-cyclodextrin derivative product, and has industrial controllability.
[0007] To realize the above technical solution: a method for preparing an ambient temperature sensor of rhodamine derivative-modified β-cyclodextrin comprises:
[0008] Step 1, weigh β-cyclodextrin solid and disperse it in deionized water in a beaker, then add NaOH aqueous solution, stirring continuously during the addition process, and keep for 1 hour;
[0009] Step 2: Weigh p-toluenesulfonyl chloride and dissolve it in acetonitrile solution until it is completely dissolved;
[0010] Step 3: slowly add the above solution into the β-cyclodextrin solution and stir continuously at room temperature for 3 hours;
[0011] Step 4: Filter the reaction solution, add 3 mol / L hydrochloric acid to the filtrate to adjust the pH of the solution to 7-8, and then place it in a refrigerator at 2°C overnight;
[0012] Step 5: Filter and collect the precipitate, recrystallize it with water three times, and dry it in vacuum at 50°C for 10 hours to obtain a loose white solid, which is mono-(6-toluenesulfonyl)-β-cyclodextrin;
[0013] Step 6: Weigh the solid Rhodamine B hydrochloride and dissolve it in anhydrous ethanol in a round-bottom flask, then quickly add 2 mL of 1,3-propylenediamine solution, stirring continuously during the addition process;
[0014] Step 7: Install a condensation reflux device, and stir the mixture solution in a constant temperature water bath at 80°C for 24 hours;
[0015] Step 8, the reaction solution was cooled to room temperature, concentrated by rotary evaporation to half volume, and cooled to crystallize; water was added to the crystalline product, and extracted twice with CH2Cl2; the organic phases were combined, washed twice with water, and dried over anhydrous Na2SO4;
[0016] Step 9, filter the filtrate, remove the solvent by rotary evaporation and dry in vacuum at 50° C. for 12 h to obtain a pink solid, which is 1,3-propylenediamino-rhodamine B lactone;
[0017] Step 10: Under a nitrogen atmosphere, the prepared mono-(6-toluenesulfonyl)-β-cyclodextrin and 1,3-propylenediamino-rhodamine B lactone solid powders are dispersed in N,N-dimethylformamide in proportion;
[0018] Step 11: After obtaining a uniformly dispersed solution of the two, install a condensation reflux device, add Na2CO3, and gradually heat to 80°C and stir under reflux for 12 hours;
[0019] Step 12: Cool to room temperature, remove a large amount of solvent N,N-dimethylformamide by rotary evaporation under reduced pressure, add dropwise into 100 mL of water for preliminary purification, collect the filtrate by suction, remove a large amount of water by rotary evaporation, drop the crude product into 200 mL of acetone and stir for 6 hours, collect the precipitate by filtration, and repeat this process three times; the crude product is separated and purified by silica gel column to obtain 1,3-propylenediamino-rhodamine B lactone-β-cyclodextrin. Description of the drawings:
[0020] Figure 1 It is the infrared spectra of 1,3-propylenediamino-rhodamine B lactone, mono-(6-p-toluenesulfonyl)-β-cyclodextrin and 1,3-propylenediamino-rhodamine B lactone-β-cyclodextrin.
[0021] Figure 2 This is the nuclear magnetic resonance spectrum of 1,3-propylenediamino-rhodamine B lactone-β-cyclodextrin.
[0022] Figure 3 This is a graph of the ultraviolet absorption intensity of the 1,3-propylenediamine-rhodamine B lactone-β-cyclodextrin ambient temperature sensor at different temperatures.
[0023] Figure 4 This is a fluorescence intensity diagram of the 1,3-propylenediamine-rhodamine B lactone-β-cyclodextrin ambient temperature sensor at different temperatures. DETAILED DESCRIPTION The following are specific embodiments of the present invention, which are used to describe the present invention rather than to limit the present invention. Example 1 1) Add 1500mL of deionized water to a 2L beaker, add 300g of pre-dried β-cyclodextrin in portions to obtain a white emulsion; add 90mL of 4mol / L NaOH solution until the solution becomes clear, continue stirring for 1h, and rotate at 200rpm; add 130mL of 1.6mol / L p-toluenesulfonyl chloride-acetonitrile solution dropwise, control the dripping rate to 1.5mL / min, generate a large amount of white precipitate, continue stirring for 3h, and rotate at 100rpm; reduce the pressure to -0.1MPa and filter to remove insoluble matter, take the light yellow-green filtrate, adjust the solution pH to 7.1 with 3mol / L HCl solution, and place it at 2°C for 12h to precipitate a large amount of white precipitate; collect the precipitate by suction, recrystallize it with water 3 times, and vacuum dry it at 50°C for 10h to obtain a loose white solid, which is mono-(6-p-toluenesulfonyl)-β-cyclodextrin; 2) Install a condensation reflux device, in a 100 mL single-necked round-bottom flask, fully dissolve 1.2 g of rhodamine B hydrochloride in 30 mL of anhydrous ethanol; quickly add 2 mL of 1,3-propylenediamine solution to the above solution and heat it to 80°C, keep the temperature of the magnetic stirring water bath at 80°C, the speed at 200 rpm, and stir and reflux for 20 hours until the red color of the solution disappears. The reaction solution was concentrated by rotary evaporation under reduced pressure, with a rotary evaporation pressure of -0.1MPa, a rotation speed of 40rpm, and a temperature of 40°C; the reaction solution was concentrated by rotary evaporation under reduced pressure to 15mL, and cooled to precipitate crystals; the crude product was filtered to obtain a crude product, 60mL of water was added to the product, and it was extracted twice with 80mL of CH2Cl2; a total of 160mL of organic phases were combined, washed twice with 40mL of deionized water, and dried over anhydrous Na2SO4; the filtrate was filtered to obtain the filtrate, and the solvent CH2Cl2 was removed by rotary evaporation at room temperature and pressure, with a rotary evaporation speed of 30rpm; the solid product was dried in a vacuum at 50°C for 12h to obtain a pink solid, which was 1,3-propylenediamino-rhodamine B lactone; 3) Take 0.80g of mono-(6-toluenesulfonyl)-β-cyclodextrin and 0.50g of 1,3-diamino-rhodamine B lactone in a 100mL two-necked round-bottom flask, add 30mL of N,N-dimethylformamide, and stir the mixed solution under nitrogen for 30min to completely dissolve it; add 150mg Na2CO3, and gradually heat to 80℃, keep the temperature of the magnetic stirring water bath at 80℃, the speed is 200rpm, stir and reflux for 12h, and cool to room temperature; filter to remove insoluble solids, take the filtrate and remove a large amount of solvent N, N-dimethylformamide by vacuum rotary evaporation, the rotary evaporation pressure is -0.1MPa, the speed is 60rpm, the temperature is 80℃, the remaining solution is dripped into 100mL water and the speed is maintained at 200rpm, and stirring is continued for 4h; the filtrate is collected by suction filtration, and a large amount of water is removed by vacuum rotary evaporation, the rotary evaporation pressure is -0.1MPa, the speed is 50rpm, and the temperature is 50℃; the collected solution is dripped into 200mL acetone and the speed is maintained at 100rpm, and stirring is continued for 6h, and the precipitate is collected by filtration, and this process is repeated three times; the crude product is separated and purified by silica gel column (methanol: water: isopropanol = 10:4:3) to obtain 1,3-propylenediamino-rhodamine B lactone-β-cyclodextrin; 4) Take the above fluorescent probe 1,3-propylenediamine-rhodamine B lactone-β-cyclodextrin solid, use deionized water as solvent, prepare it into a 1 mmol / L solution, take 5 mL of the solution and place it in an oven at 20°C, 40°C, 60°C, 80°C, and 100°C for 10 min, and measure the spectral changes of the probe at different temperatures using an ultraviolet spectrophotometer and a fluorescence photometer. Example 2 1) Add 1200mL of deionized water to a 2L beaker, add 240g of pre-dried β-cyclodextrin in portions to obtain a white emulsion; add 60mL of 4mol / L NaOH solution until the solution becomes clear, continue stirring for 1h at 200rpm; add 100mL of 1.6mol / L p-toluenesulfonyl chloride-acetonitrile solution dropwise at a drip rate of 2mL / min to generate a large amount of white precipitate, continue stirring for 3h at 100rpm; reduce the pressure to -0.1MPa and filter to remove insoluble matter, take the light yellow-green filtrate, adjust the solution pH to 8.0 with 3mol / L HCl solution, place at 2°C for 12h, and precipitate a large amount of white precipitate; collect the precipitate by suction, recrystallize it with water 3 times, and vacuum dry it at 50°C for 10h to obtain a loose white solid, which is mono-(6-p-toluenesulfonyl)-β-cyclodextrin; 2) Install a condensation reflux device, in a 100mL single-mouth round-bottom flask, fully dissolve 1.2g of rhodamine B hydrochloride in 30mL of anhydrous ethanol; add 2mL of ethylenediamine solution to the above solution and heat it to 80°C, keep the temperature of the magnetic stirring water bath at 80°C, the speed is 200rpm, stir and reflux for 24h until the red color of the solution disappears; vacuum rotary evaporation concentrate the solution after the reaction, the rotary evaporation pressure is -0.1MPa, the speed is 40rpm, and the temperature is 40°C; vacuum rotary evaporation concentrate the reaction solution to 15mL, cool it to precipitate crystals; filter to obtain a crude product, add 60mL of water to the product, and use 80mL Extract twice with CH2Cl2; combine the organic phases (160 mL in total), wash twice with 40 mL of deionized water, and dry over anhydrous Na2SO4; filter the filtrate, remove the solvent CH2Cl2 by rotary evaporation at room temperature and pressure, with a rotary evaporation speed of 30 rpm; dry the solid product in a vacuum at 50°C for 12 h to obtain a pink solid, which is ethylenediamine-rhodamine B lactone; 3) Take 0.80g of mono-(6-toluenesulfonyl)-β-cyclodextrin and 0.48g of ethylenediamine-rhodamine B lactone in a 100mL two-necked round-bottom flask, add 30mL of N,N-dimethylformamide, and stir the mixed solution under nitrogen for 30min to completely dissolve it; add 150mg Na2CO3, and gradually heat to 80°C, maintain the temperature of the magnetic stirring water bath at 80°C, the speed is 200rpm, stir and reflux for 12h, and cool to room temperature; filter to remove insoluble solids, take the filtrate and remove a large amount of solvent N, N-dimethylformamide by vacuum rotary evaporation, the rotary evaporation pressure is -0.1MPa, the speed is 60rpm, the temperature is 80°C, the remaining solution is dripped into 100mL water and the speed is maintained at 200rpm, and stirring is continued for 4h; the filtrate is collected by suction filtration, and a large amount of water is removed by vacuum rotary evaporation, the rotary evaporation pressure is -0.1MPa, the speed is 50rpm, and the temperature is 50°C; the collected solution is dripped into 200mL acetone and the speed is maintained at 100rpm, stirring is continued for 6h, and the precipitate is collected by filtration, and this process is repeated three times; the crude product is separated and purified by silica gel column (methanol: water: isopropanol = 10:4:3) to obtain ethylenediamine-rhodamine B lactone-β-cyclodextrin.
Claims
1. A method for preparing rhodamine derivative-modified β-cyclodextrin, which is mainly characterized by The following steps are involved: 1) Preparation of mono-(6-p-toluenesulfonyl)-β-cyclodextrin (6-OTs-β-CD): disperse β-cyclodextrin in a deionized water solution, add a sodium hydroxide solution until the solution becomes clear and pale yellow-green, and continue stirring for 1 hour; slowly add a p-toluenesulfonyl chloride-acetonitrile solution dropwise to the alkaline solution to produce a large amount of white precipitate, and continue stirring at a constant temperature for 3 hours; remove insoluble matter by suction filtration, and adjust the pH of the filtrate with a hydrochloric acid solution; The mixture was placed at 2°C overnight to precipitate a large amount of white precipitate, which was collected by filtration, recrystallized three times with water, and dried under vacuum at 50°C for 10 h to obtain a loose white solid, which was mono-(6-toluenesulfonyl)-β-cyclodextrin. 2) Preparation of 1,3-propylenediamine-rhodamine B lactone (1,3-PDA-RhoB): install a condensation reflux device, dissolve rhodamine B hydrochloride in 30 mL of anhydrous ethanol; quickly drop 1,3-propylenediamine into the solution and reflux at 80°C until the red color of the solution disappears; concentrate the reaction solution by vacuum rotary evaporation to half the volume, and cool for crystallization; The crude product was filtered, 60 mL of water was added to the product, and it was extracted twice with 80 mL of CH2Cl2; the organic phases were combined, washed twice with 40 mL of water, and dried over anhydrous Na2SO4; the filtrate was filtered, the solvent was removed by rotary evaporation at normal pressure, and it was dried in a vacuum at 50°C for 12 h to obtain a pink solid, which was 1,3-diaminopropane-rhodamine B lactone; 3) Preparation of 1,3-propylenediamino-rhodamine B lactone-β-cyclodextrin (1,3-PDA-RhoB-β-CD): 0.80 g of mono-(6-toluenesulfonyl)-β-cyclodextrin prepared in 1) and 0.50 g of 1,3-propylenediamino-rhodamine B lactone prepared in 2) were completely dissolved in 30 mL of N,N-dimethylformamide solution under nitrogen, Na2CO3 was added thereto, and the temperature was gradually raised to 80°C, stirred and refluxed for reaction for 12 h, and then cooled to room temperature; a large amount of solvent N,N-dimethylformamide was removed by rotary evaporation under reduced pressure, and the remaining reaction solution was dripped into 100 mL of water; the filtrate was collected by suction filtration, a large amount of water was removed by rotary evaporation, the crude product was dripped into 200 mL of acetone and stirred for 6 h, and the precipitate was collected by filtration, and this process was repeated three times; the crude product was separated and purified by silica gel column to obtain 1,3-propylenediamino-rhodamine B lactone-β-cyclodextrin.
2. The method for preparing a rhodamine derivative modified β-cyclodextrin according to claim 1, characterized in that: In the step 1), the mass ratio of β-cyclodextrin to deionized water is 1:5, the concentration of the sodium hydroxide solution is 4 mol / L, the dropping speed of the p-toluenesulfonyl chloride-acetonitrile solution is controlled at 1.5-2 mL / min, and the pH of the filtrate is adjusted to 7-8 with 3 mol / L hydrochloric acid solution.
3. The method for preparing a rhodamine derivative modified β-cyclodextrin according to claim 1, characterized in that: In the step 2), the molar ratio of rhodamine B hydrochloride to 1,3-propylenediamine is 1:1.5, and the stirring reflux reaction time is 20 to 24 hours.
4. The method for preparing a rhodamine derivative modified β-cyclodextrin according to claim 1, characterized in that: In the step 3), 150 mg of Na2CO3 is added to neutralize the p-toluenesulfonic acid generated during the reaction and promote the forward reaction. The developing agent ratio is methanol: water: isopropanol = 10: 4: 3.