CN115073443B - Visual fluorescence enhancement type copper ion fluorescence detection probe and preparation method thereof - Google Patents

Abstract

The invention discloses a visualized fluorescence enhancement type copper ion fluorescence detection probe and a preparation method thereof. Coumarin structure with longer emission wavelength and high fluorescence quantum yield is selected as a fluorophore, thiadiazole structure with larger electron density and stronger coordination binding capacity is used as a recognition group, and the molecular plane rigidity, the conjugated system size and the coordination recognition capacity are comprehensively considered to design and synthesize the imine fluorescent probe. The purposes of enhancing conjugation and improving fluorescence effect are achieved, and visual simplicity and easy identification are achieved while a fluorescence enhancement response mode is achieved. The structure of the fluorescence detection probe is as follows:

Description

Visual fluorescence enhancement type copper ion fluorescence detection probe and preparation method thereof

Technical Field

The invention relates to an organic small molecule fluorescent probe and a preparation method thereof, in particular to a visualized fluorescence enhanced copper ion fluorescent detection probe and a preparation method thereof.

Background

With continuous innovation of science and technology, subjects such as molecular biology, chemistry, biomedicine and the like develop rapidly, and technical methods for detecting ions and small molecules gradually developAnd (3) diversification. The fluorescence analysis method has the advantages of high analysis speed, simple testing instrument, high sensitivity, good selectivity and the like, and the fluorescence probe continuously improves the sensitivity and the accuracy, expands the application range and simultaneously develops towards targets such as trace, automation, visualization, convenient application and the like. Copper ion (Cu) ²⁺ ) As one of the most important trace metal elements in the human body, it plays an irreplaceable role in various biological processes such as cell energy generation, oxygen transfer and activation, signal transduction, etc. The lack of Cu in human body can cause anemia, hypopigmentation, coronary heart disease, brain dysfunction and various nervous system diseases, conversely, if excessive Cu exists in human body ²⁺ Can disturb the original stable state of cells and induce Alzheimer's disease, schizophrenia, mentha's disease and other diseases. Thus, development can be used to detect Cu in real environment samples and cellular environments ²⁺ Is important.

The organic small molecule fluorescent probe generally consists of three parts of a recognition group, a connecting group and a fluorescent signal group: the recognition group is a part which has specific action with the object to be detected, and affects the selectivity, the anti-interference capability and the sensitivity to a certain extent; the connecting group is a bridge for connecting the fluorescent group and the recognition group, and plays a role in transmitting signals; the fluorescent signal group is the part with the signal changed, so that the aim of effectively identifying the detected object is fulfilled, and the fluorescent signal group directly or indirectly determines the spectral characteristics and the performance of the probe.

In 2018, mukherjee et al entitled "Sensing phenomena, extraction and recovery of Cu ²⁺ followed by smart phone application using a luminescent pyrene based chemosensor "based on pyrene and 2-hydrazinobenzoic acid, a reversible, highly selective hydrazone-based ratio-type fluorescent probe was synthesized (Journal of Luminescence,2018, 204:145-153). The probe shows weak fluorescence in DMSO solution, and Cu is added ²⁺ The charge transfer transition in the rear ligand causes 44 times of emission intensity increase, and Cu can be conveniently monitored by taking an RGB collector in the smart phone as an analysis tool ²⁺ Concentration. In 2019, xu et al have a human hair designation of "A dual-function chemosensor based on coumarin for fluorescent turn-on recognition of Hg ²⁺ and colorimetric detection of Cu ²⁺ in aquos media ", a Schiff base bifunctional fluorescent probe based on coumarin units was synthesized (Journal of the Chinese Chemical Society,2019,67 (2): 298-305). Probe pair Hg ²⁺ Shows fluorescence enhanced response, better selectivity and sensitivity, and is characterized by CH ₃ CN/H ₂ Cu can be identified by naked eye colorimetry in O solution ²⁺ However, there is no intensive study and explanation on fluorescence response and detection mechanism. In 2020, jiang et al issued the title "A highly selective and sensitive" turn-on "fluorescent probe for rapid recognition and detection of Cu ²⁺ in aqueous solution and in living cells "(Journal of Molecular Structure,2020, 1219:128573-128582), cu based on diaminomaleonitrile and 5-formyl-2-hydroxybenzoic acid was designed and synthesized ²⁺ Fluorescent probe, fluorescence intensity of probe and Cu ²⁺ The concentration is in good linear relation, and Cu in a water sample can be accurately detected ²⁺ Shows high selectivity and sensitivity. In addition, the probe has excellent cell permeability and can be applied to recognizing Cu in HepG2 cells ²⁺ .2021, ma et al published under the title "A pyrene-containing Schiff base fluorescent ratiometric probe for the detection of Cu ²⁺ in aqueous solutions and in cells "(Journal of Photochemistry and Photobiology A: chemistry,2021,408: 113086-113092), synthesizing hydrazone analog fluorescent probes based on pyrene and p-toluenesulfonyl hydrazide groups, the probes responding to Cu with a fluorescence intensity ratio between themselves and pyrenyl monomers ²⁺ Can be used for detecting Cu with high selectivity and sensitivity and specificity in acetonitrile-HEPES buffer solution ²⁺ . In addition, the probe shows good potential for visualization application and can be used for detecting Cu in GS cells of groupers ²⁺ 。

As can be seen from the results reported in the prior literature, the design and application research of the copper ion fluorescent probe is one of research hotspots in the field of fluorescent materials, but the development of the visualized fluorescence-enhanced copper ion fluorescent probe is still a problem to be solved in the field.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art and provides a visualized fluorescence enhanced copper ion fluorescence detection probe and a preparation method thereof

The technical scheme of the invention is a visual and fluorescence-enhanced copper ion fluorescence detection probe, and the structural formula is as follows:

the preparation method of the copper ion detection probe with visualization and fluorescence enhancement comprises the following steps in sequence:

step 1, adding choline chloride into a mixed solvent of ethanol and water, stirring and dissolving, adding substituted salicylaldehyde and 1.1 times of McUn acid, stirring at room temperature for 10-48 hours, and directly leaching by suction filtration to obtain a compound L1;

step 2, dissolving the compound L1 and the thiosemicarbazide in dioxane, and slowly dripping POCl under the ice bath condition while stirring ₃ Stirring is continued for 0.5h; heating to 102 ℃ for continuous reaction for 0.5h, cooling to room temperature, adding water, and continuously refluxing for 4h; pouring the solution into ice water while the solution is hot, regulating the pH to 8-9 by using concentrated ammonia water, sodium hydroxide or potassium hydroxide, and recrystallizing the crude product after suction filtration and drying by using ethanol to obtain solid L2;

step 3. Dissolving solid L2 and 4-substituted-salicylaldehyde in absolute ethanol, adding anhydrous MgSO ₄ And glacial acetic acid, heating and refluxing until the solid L2 is completely reacted, cooling to room temperature, filtering, drying, and recrystallizing with ethanol to obtain the final product.

The preparation steps are as follows:

according to the invention, a coumarin structure with longer emission wavelength and high fluorescence quantum yield is selected as a fluorophore, a thiadiazole structure with larger electron density and stronger coordination binding capacity is used as a recognition group, and the molecular plane rigidity, the conjugated system size and the coordination recognition capacity are comprehensively considered to design and synthesize the imine fluorescent probe. The purposes of enhancing conjugation and improving fluorescence effect are achieved, a fluorescence enhancement response mode is achieved, and meanwhile visual simplicity and easy identification can be achieved. Compared with the prior art, the method has the following advantages:

1. the synthesis route is simple, complex raw materials and expensive equipment are not needed, and the method can be used for experimental operation and large-scale production;

2. prepared fluorescent probe pair Cu ²⁺ Shows strong fluorescence enhancement response and remarkable visual recognition capability. The probe has a relatively stable fluorescence response in the pH range of 4-9, and shows excellent selectivity, anti-interference performance and relatively high sensitivity, and the detection limit is as low as 0.017 mu M; implementing visualization and selectively recognizing Cu ²⁺ Making Cu ²⁺ The detection of the method has the advantages of simple operation, obvious effect, low price and the like.

Drawings

FIG. 1 is a schematic illustration of a compound prepared in accordance with example 1 of the present invention ¹ H NMR spectrum.

FIG. 2 is a graph showing the changes in ultraviolet absorption and fluorescence spectra of the compound of example 1 of the present invention in various solvents.

FIG. 3 shows the addition of Cu to the compound prepared in example 1 of the present invention ²⁺ Front-back ultraviolet absorption spectrum and fluorescence spectrum change diagrams.

FIG. 4 shows the addition of Cu to the compound prepared in example 1 of the present invention ²⁺ The front and back change patterns under the irradiation of visible light and ultraviolet light.

FIG. 5 is a graph showing fluorescence spectra of the compound of example 1 of the present invention as a fluorescent probe interacting with different anions and cations.

FIG. 6 is a diagram of a compound prepared in example 2 of the present invention ¹ H NMR spectrum.

FIG. 7 is a graph showing changes in ultraviolet absorption and fluorescence spectra of the compound of example 2 of the present invention in various solvents.

Detailed Description

Example 1:

the visual fluorescence enhancement type copper ion fluorescence detection probe is prepared by the following steps in sequence:

step 1. Catalyst-Choline chloride (0.0560 g,0.4 mmol) was added to 4mL of the mixed solvent (H ₂ O: etOH=3:1), adding 4- (diethylamino) salicylaldehyde (0.3944 g,2.0 mmol) and McO (0.3172 g,2.2 mmol) after stirring and dissolving, and stirring at room temperature for 10-48h to obtain a pure product (m.p. = 223.8-226.3 ℃), namely a compound L1;

step 2, dissolving the compound L1 and thiocarbamide (0.1637 g,1.8 mmol) in dioxane, stirring under ice bath condition, slowly dropwise adding 0.8mL phosphorus oxychloride, and continuing stirring for 0.5h; heating to 102 ℃ to react for 0.5h, cooling to room temperature, adding 1.5mL of water, and refluxing the mixture for 4h; pouring the solution into ice water while the solution is hot after the reaction is finished, regulating the pH to 8-9, and recrystallizing the crude product after suction filtration and drying by ethanol to obtain an orange-red solid (m.p. = 230.5-231.7 ℃), namely a compound L2;

step 3. Compound L2 and 4-chlorosalicylaldehyde (2.0 mmol,2 eq.) are dissolved in absolute ethanol, 0.0180g of anhydrous MgSO4,4 drops of glacial acetic acid as catalyst are added, heated to reflux and monitored by TLC until complete reaction; cooling to room temperature, suction filtering, drying and recrystallizing with proper solvent to obtain the final product, red solid and m.p.>300 ℃. The end product prepared ¹ The H NMR spectrum is shown in FIG. 1, and the structural formula is as follows:

example 2:

step 1: catalyst-Choline chloride (0.0560 g,0.4 mmol) was added to 4mL of the mixed solvent (H ₂ O: etOH=3:1), adding 4- (diethylamino) salicylaldehyde (0.3944 g,2.0 mmol) and McO (0.3172 g,2.2 mmol) after stirring and dissolving, and stirring at room temperature for 10-48h to obtain pure product, namely compound L1;

step 2, dissolving the compound L1 and thiocarbamide (0.1637 g,1.8 mmol) in dioxane, stirring under ice bath condition, slowly dropwise adding 0.8mL phosphorus oxychloride, and continuing stirring for 0.5h; heating to 102 ℃ to react for 0.5h, cooling to room temperature, adding 1.5mL of water, and refluxing the mixture for 4h; pouring the solution into ice water while the solution is hot after the reaction is finished, adjusting the pH to 8-9, and recrystallizing the crude product after suction filtration and drying by ethanol to obtain an orange-red solid, namely a compound L2;

step 3. Compound L2 and 4-methoxysalicylaldehyde (2.0 mmol,2 eq.) are dissolved in absolute ethanol, 0.0180g of anhydrous MgSO4,4 drops of glacial acetic acid are added as catalyst, heated to reflux and monitored by TLC until complete reaction, cooled to room temperature, filtered off with suction, dried and the appropriate solvent recrystallized to give the final product as an orange solid m.p.>300 ℃. The end product prepared ¹ The H NMR spectrum is shown in FIG. 6, and the structural formula is as follows:

experiment 1: fluorescent Property experiment of the Compound prepared in example 1

Considering the test natural environment and the type of ions in the cells, the ultraviolet-visible absorption spectrum (a) and fluorescence spectrum (b) of the final compound of example 1 were measured in 6 solvents capable of water-mixing with methanol, ethanol, acetonitrile, DMF, DMSO and acetone, as shown in fig. 2.

The results show that: the excitation wavelength of the compound in different solvents is similar, but the fluorescence intensity is greatly different, the emission in methanol and acetonitrile solvents is the best, and the fluorescence intensity in DMSO and DMF is the worst, thus showing the potential of fluorescence enhancement probes. Comprehensively considering factors such as solubility, fluorescence emission intensity, cytotoxicity of solvent and the like of the compound, and further researching Cu in DMSO ²⁺ Detection capability.

Experiment 2: compound prepared in example 1 vs. Cu ²⁺ Spectral characteristics of (2)

Compounds in DMSO/H ₂ O mixed solution (DMSO: H) ₂ O=3:1) was prepared in 10 μm of test solution. Specifically, 2mL of the probe solution was added to a quartz cell having a path width of 1cm, and different metal ion stock solutions were added to the quartz cell by a pipette for testing, the voltage was set at 500V,the excitation and emission slit widths were 5nm.

The ultraviolet absorption spectrum characteristics are shown in FIG. 3a, and the absorbance is lower at 285nm and 470 nm. Addition of Cu to Probe solution ²⁺ After that, the absorption peak intensity at 285nm gradually decreases, and the maximum absorption peak is observed to be enhanced at 470nm and a red shift trend occurs. Fluorescence spectrum along with Cu ²⁺ The added change is shown in FIG. 3b, with a significant enhancement of the emission peak at 530 nm. Probe pair Cu ²⁺ The visual detection effect of (4) is remarkable, and Cu is added ²⁺ The probe solution turned from pale pink to bright yellow under sunlight, and a change in fluorescence from colorless to bright green was observed under a 365nm ultraviolet lamp.

Experiment 3: test of the Selectivity and anti-interference Capacity of the Compounds prepared in example 1

By adding Cu to the probe solution ²⁺ Cu and Cu ⁺ 、Ag ⁺ 、K ⁺ 、Ca ²⁺ 、Zn ²⁺ 、Fe ²⁺ 、Pb ²⁺ 、Fe ³⁺ 、Cl ^- 、ClO ^- The plasma assesses the selectivity of the compounds. As a result, as shown in FIG. 5, the probe was used for Cu alone ²⁺ Shows an increase in fluorescence and none of the other analytes responded.

Experiment 4: fluorescent Property experiment of the Compound prepared in example 2

The ultraviolet-visible absorption spectrum (a) and the fluorescence spectrum (b) of the compound prepared in example 2 were measured in 6 solvents capable of water-mixing with methanol, ethanol, acetonitrile, DMF, DMSO, and acetone, in consideration of the test natural environment and the type of ions in cells. As shown in FIG. 7, the fluorescence emission spectra show the potential of fluorescence enhancement probes with little difference in emission wavelengths of the compounds in different solvents, but with a large difference in fluorescence intensity.

Claims (2)

1. A visual fluorescence enhancement type copper ion fluorescence detection probe is characterized by comprising the following structural formula:

2. a method for preparing the visualized fluorescence enhanced copper ion fluorescence detection probe according to claim 1, which is characterized by comprising the following steps in sequence:

step 1, adding choline chloride into a mixed solvent of ethanol and water, stirring and dissolving, adding substituted salicylaldehyde and 1.1 times of McUtility acid, stirring at room temperature for 10-48h, and directly leaching by suction filtration to obtain a compound L1, wherein the structural formula of the compound L1 is

Step 2, dissolving the compound L1 and the thiosemicarbazide in dioxane, and slowly dripping POCl under the ice bath condition while stirring ₃ Stirring is continued for 0.5h; heating to 102 ℃ for continuous reaction for 0.5h, cooling to room temperature, adding water, and continuously refluxing for 4h; pouring the solution into ice water while it is hot, regulating pH to 8-9 with concentrated ammonia water, sodium hydroxide or potassium hydroxide, vacuum filtering, and recrystallizing the dried crude product with ethanol to obtain solid L2, wherein the compound L2 has the structural formula of

Step 3. Dissolving solid L2 and 4-substituted-salicylaldehyde in absolute ethanol, adding anhydrous MgSO ₄ And glacial acetic acid, heating and refluxing until the solid L2 is completely reacted, cooling to room temperature, filtering, drying, and recrystallizing with ethanol to obtain the final product.

Images