阅读说明：本技术 一种选择性识别氟离子的荧光探针及其制备方法 (Fluorescent probe for selectively identifying fluorine ions and preparation method thereof ) 是由 赵宝华 夏艳 赵爽 柳翱 魏境宣 李东风 于 2021-07-01 设计创作，主要内容包括：本发明公开了一种选择性识别氟离子的荧光探针及其制备方法。它以香豆素作为荧光基团,进一步拓展了其作为荧光基团的功能性。在香豆素荧光基团上修饰了含不同取代的苯并咪唑基团,咪唑环上的H会与香豆素的羰基氧之间形成分子内氢键来保持分子的平面性。利用氟离子的强电负性、电荷密度高的特点,氟通过与咪唑环上的氢通过形成氢键作用,破环了原本的分子内氢键,导致分子内结构产生变化。合成的化合物10作为荧光探针对氟离子有较好的选择性,较快的响应速度,检出限为3.50μmol/L。(The invention discloses a fluorescent probe for selectively identifying fluorine ions and a preparation method thereof. The coumarin is used as a fluorescent group, and the functionality of the coumarin as the fluorescent group is further expanded. The fluorescent group of coumarin is modified with benzimidazole group with different substitution, and H on imidazole ring and carbonyl oxygen of coumarin form intramolecular hydrogen bond to maintain the planarity of molecule. By utilizing the characteristics of strong electronegativity and high charge density of fluorine ions, fluorine and hydrogen on an imidazole ring form hydrogen bond action, the original intramolecular hydrogen bond is broken, and the intramolecular structure is changed. The synthesized compound 10 has better selectivity for fluorine ions as a fluorescent probe, has higher response speed and has a detection limit of 3.50 mu mol/L.)

1. A coumarin-containing fluorescent probe compound is characterized in that the structural formula is as follows:

。

2. the coumarin-containing fluorescent probe compound of claim 1,

the 3- (1H-benzo [ d ] imidazole-2-yl) -7- (diethylamino) coumarin series compound is prepared by the following method:

(1) preparation of 7- (diethylamino) coumarin

Adding 0.01mol of 4- (diethylamino) salicylaldehyde, 0.02mol of diethyl malonate and 0.1mL of piperidine at normal temperature, and dissolving in 30mL of absolute ethyl alcohol; refluxing for 11h under the protection of nitrogen at 85 ℃, evaporating, adding 10mL of AcOH/HCl, heating to 100 ℃, and continuing to reflux for 10h, wherein the pH value is neutral; suction filtration, drying, and eluting by normal phase chromatography, wherein the eluent is dichloromethane: petroleum ether =2: 1; the volume ratio of AcOH to HCl is 1: 1;

(2) preparation of Compound 5

Measuring DMF and POCl₃Stirring 5mL of each liquid under the protection of nitrogen at normal temperature for 0.5h, heating to 60 ℃, and slowly dropwise adding 7- (diethylamino) coumarin (1.08g, 0.005mol) dissolved in 15mL of DMF into the reaction liquid; after reacting for 12h, adding ice water to quench the reaction solution, adjusting the pH to be neutral, performing suction filtration and drying, and performing normal phase chromatography elution, wherein an eluent is dichloromethane: ethyl acetate =10: 1; the structural formula of the compound 5 is as follows:

；

(3) preparation of fluorescent probe 3- (1H-benzo [ d ] imidazole-2-yl) -7- (diethylamino) coumarin compound 8

Dissolving 0.25g of compound 5 and 0.17g of 2-nitroaniline in 20mL of absolute ethanol, adding 0.5g of sodium thiosulfate solid, heating and refluxing for 2h under the protection of nitrogen, concentrating the naturally cooled reaction solution, extracting with dichloromethane and water, drying, evaporating to obtain a crude product, and recrystallizing with ethyl acetate;

the 3- (6-methoxy-1H-benzo [ d ] imidazole-2-yl) -7- (diethylamino) coumarin compound 9 is prepared by the following method:

dissolving 0.25g of compound 5 and 0.20g of 4-methoxy-2-nitroaniline in 20mL of absolute ethyl alcohol, adding 0.5g of sodium thiosulfate solid, then, under the protection of nitrogen, heating and refluxing for 2h, concentrating the naturally cooled reaction solution, extracting with dichloromethane and water, drying, evaporating to obtain a crude product, and recrystallizing with ethyl acetate;

the 3- (6-cyano-1H-benzo [ d ] imidazole-2-yl) -7- (diethylamino) coumarin compound 10 is prepared by the following method:

dissolving 0.25g of compound 5 and 0.19g of 3-amino-4-nitrobenzonitrile in 20mL of absolute ethyl alcohol, adding 0.5g of sodium thiosulfate solid, heating and refluxing for reaction for 2h under the protection of nitrogen, concentrating the naturally cooled reaction solution, extracting with dichloromethane and water, drying, evaporating to obtain a crude product, and recrystallizing with ethyl acetate.

3. An application of a fluorescent probe compound containing coumarin in the detection of fluorine ions.

Technical Field

The invention relates to the technical field of chemical analysis and detection, in particular to a fluorescent probe for selectively identifying fluorine ions and a preparation method thereof.

Background

Fluoride anions are widely used as additives for toothpastes and are also an important ingredient in the pharmaceutical industry and it is well known that excessive exposure to or intake of fluoride can lead to urinary tract stones and stomach and kidney disease.

Among a plurality of detection and analysis methods, the traditional detection methods such as a flame photometry method, an atomic absorption spectrometry method and the like are relatively high in cost, often require more samples, are complicated in preparation steps, cannot track dynamic changes of the samples, are long in detection time, and cannot meet the application requirements in actual detection work. Compared with the method, the method based on the fluorescent probe detection has obvious advantages in the aspects of sensitivity, selectivity, response time, local observation (such as fluorescence imaging spectrum) and the like. Nowadays, the application of fluorescent probes has made a great progress, and has been widely applied to various life and production fields because of more and more attention paid by people.

The fluorescent probes currently used for detecting fluorine ions are mostly based on chemical reactions in which fluorine breaks silicon-oxygen bonds, or the deprotonation effect of probe molecules caused by the strong electronegativity of fluorine ions. The fluorescent probes are mostly rearranged after the two action processes, the molecular structure is greatly changed, and the fluorine ions are difficult to continuously detect. Therefore, it is important to develop a method for detecting fluoride in biological systems with high efficiency, sensitivity and accuracy.

Disclosure of Invention

A coumarin-containing fluorescent probe compound is characterized in that the structural formula is as follows:

；

a method for preparing a coumarin-containing fluorescent probe compound 8 is characterized by comprising the following steps:

(1) preparation of 7- (diethylamino) coumarin

Adding 0.01mol of 4- (diethylamino) salicylaldehyde, 0.02mol of diethyl malonate and 0.1mL of piperidine at normal temperature, and dissolving in 30mL of absolute ethyl alcohol; refluxing for 11h under the protection of nitrogen at 85 ℃, evaporating, adding 10mL of AcOH/HCl, heating to 100 ℃, and continuing to reflux for 10h, wherein the pH value is neutral; suction filtration, drying, and eluting by normal phase chromatography, wherein the eluent is dichloromethane: petroleum ether =2: 1; the volume ratio of AcOH to HCl is 1: 1;

(2) preparation of Compound 5

Measuring DMF and POCl₃After stirring 5mL each of the liquids under nitrogen protection at room temperature for 0.5h, the temperature was raised to 60 ℃ and 7- (diethylamino) coumarin (1.08g, 0.005mol) dissolved in 15mL of DMF was slowly added dropwise to the reaction mixture. After 12h of reaction, the reaction solution was quenched with ice water and the pH was adjusted to neutral. Suction filtration, drying, and eluting by normal phase chromatography, wherein the eluent is dichloromethane: ethyl acetate =10: 1; the structural formula of the compound is as follows:

；

(3) preparation of fluorescent Probe Compound 8

Dissolving 0.25g of compound 5 and 0.17g of 2-nitroaniline in 20mL of absolute ethanol, adding 0.5g of sodium thiosulfate solid, heating and refluxing for 2h under the protection of nitrogen, concentrating the naturally cooled reaction solution, extracting with dichloromethane and water, drying, evaporating to obtain a crude product, and recrystallizing with ethyl acetate;

a preparation method of a coumarin-containing fluorescent probe compound 9 comprises the following steps:

dissolving 0.25g of compound 5 and 0.20g of 4-methoxy-2-nitroaniline in 20mL of absolute ethyl alcohol, adding 0.5g of sodium thiosulfate solid, then, under the protection of nitrogen, heating and refluxing for 2h, concentrating the naturally cooled reaction solution, extracting with dichloromethane and water, drying, evaporating to obtain a crude product, and recrystallizing with ethyl acetate;

a preparation method of a coumarin-containing fluorescent probe compound 10 comprises the following steps:

dissolving 0.25g of compound 5 and 0.19g of 3-amino-4-nitrobenzonitrile in 20mL of absolute ethyl alcohol, adding 0.5g of sodium thiosulfate solid, heating and refluxing for reaction for 2h under the protection of nitrogen, concentrating the naturally cooled reaction solution, extracting with dichloromethane and water, drying, evaporating to obtain a crude product, and recrystallizing with ethyl acetate;

an application of a fluorescent probe compound containing coumarin in the detection of fluorine ions.

The invention provides a fluorescent probe for selectively identifying fluorine ions and a preparation method thereof. The coumarin is used as a fluorescent group, and the functionality of the coumarin as the fluorescent group is further expanded. The fluorescent group of coumarin is modified with benzimidazole group with different substitution, and H on imidazole ring and carbonyl oxygen of coumarin form intramolecular hydrogen bond to maintain the planarity of molecule. By utilizing the characteristics of strong electronegativity and high charge density of fluorine ions, fluorine and hydrogen on an imidazole ring form hydrogen bond action, the original intramolecular hydrogen bond is broken, and the intramolecular structure is changed. The synthesized compound 10 has better selectivity for fluorine ions as a fluorescent probe, has higher response speed and has a detection limit of 3.50 mu mol/L.

Drawings

FIG. 1 is a scheme for the synthesis of 3- (1H-benzo [ d ] imidazol-2-yl) -7- (diethylamino) coumarins;

FIG. 2 shows the changes of absorption spectra of compounds 8,9,10 (a, b, c) affected by different anions;

FIG. 3 shows the change of emission spectra of compound 10 affected by different anions;

FIG. 4 concentration of Compound 10 is 10^-5The change condition of the absorption spectrum of the solution of mol/L along with the addition of the fluorinion;

FIG. 5 emission spectra of Compound 10 with F^-The concentration of the ions is increased (0-25 times F)^-Ion equivalent);

FIG. 6 shows the change in intensity of the emission peak at 510nm with the increase in the ratio of the equivalent weight of fluoride ion to that of Compound 10;

FIG. 7 fitting curve of the change in maximum fluorescence emission intensity of Compound 10 with fluoride ion addition;

FIG. 8 changes in fluorescence emission intensity at 510nm with time after addition of 100 equivalents of fluoride ion to Compound 10;

FIG. 9 shows the possible interactions of the series of compounds with fluoride ions;

FIG. 10 variation of emission spectra of Compound 10 in different solvents.

Detailed Description

EXAMPLE 1 preparation of fluorescent Probe

The preparation route of the fluorescent probe of the invention is shown in figure 1.

(1) Preparation of 7- (diethylamino) coumarin

4- (diethylamino) salicylaldehyde (1.93g, 0.01mol), diethyl malonate (2.4g, 0.02mol), 0.1mL piperidine were added at room temperature, and dissolved in 30mL absolute ethanol. Refluxing reaction for 11h under the protection of nitrogen at 85 deg.C, evaporating excess solvent, adding 10mL of AcOH/HCl (1:1), heating to 100 deg.C, and refluxing reaction for 10 h. After the reaction is completed, the pH is adjusted to be neutral. Filtering, collecting and drying a filter cake to obtain a crude product, and eluting by using normal phase chromatography, wherein an eluent is dichloromethane: petroleum ether =2: 1. 2.05g of a dark yellow oil solid was obtained in a yield of 94.4%.

(2) Preparation of intermediate Compound 5

Measuring DMF and POCl₃After 5mL of each liquid was stirred for 0.5h under nitrogen at room temperature, the temperature was raised to 60 ℃. 7- (diethylamino) coumarin (1.08g, 0.005mol) dissolved in 15ml DMF was slowly added dropwise to the reaction solution. After 12h of reaction, the reaction solution was quenched with ice water and the pH was adjusted to neutral. Filtering, collecting and drying a filter cake to obtain a crude product, and eluting by using normal phase chromatography, wherein an eluent is dichloromethane: ethyl acetate =10: 1. 0.98g of orange-red solid is obtained, and the yield is 80.4%.

(3) Preparation of fluorescent Probe Compound 8

Compound 5(0.25g, 1.0mmol) and 2-nitroaniline (0.17g, 1.2mmol) were dissolved in 20mL of anhydrous ethanol, and after adding 0.5g of sodium thiosulfate solid, the mixture was heated under reflux for 2 hours under nitrogen protection. After the reaction is finished, concentrating the naturally cooled reaction solution, extracting with dichloromethane and water, drying, and evaporating the redundant solvent to obtain a crude product. Then recrystallized by ethyl acetate to obtain orange-red solid 0.19g, and the yield is 57.6%.

Fluorescent probe compound 8:¹H NMR (CDCl₃ ,400MHz): δ=11.25(s, 1H), 8.91(s, 1H), 7.76(d, J=7.2 Hz, 1H), 7.49(d, J=6.4 Hz, 1H), 7.45(d, J=7.2Hz, 1H), 7.29-7.23(m, 2H), 6.67(dd, J=8.8, 2.4Hz, 1H), 6.65(d, J=2.0Hz, 1H), 3.45(q, J=7.2Hz, 4H), 1.24(t, J=7.2Hz, 6H); ¹³C NMR(CDCl₃ ,100MHz): δ=164.1, 148.4, 142.1, 138.9, 128.9, 124.7, 123.9, 123.2, 117.2, 115.2, 47.5, 28.9, 20.1, 13.8; FTIR (cm^-1):3350, 2970, 2927, 2830, 2696, 1698, 1618, 1594, 1529, 1439, 1402, 1358, 1258, 1186, 1131, 806, 773, 735, 648, 493; MALDI-TOF-MS (m/z): 333.38 (100%, M+-1, calcd. 333.39)。

(4) preparation of fluorescent Probe Compound 9

Compound 5(0.25g, 1.0mmol) and 4-methoxy-2-nitroaniline (0.20g, 1.2mmol) were dissolved in 20mL of anhydrous ethanol, and after adding 0.5g of sodium thiosulfate solid, the mixture was refluxed for 2 hours under nitrogen. After the reaction is finished, concentrating the naturally cooled reaction solution, extracting with dichloromethane and water, drying, and evaporating the redundant solvent to obtain a crude product. Then recrystallized by ethyl acetate to obtain orange-red solid 0.23g, and the yield is 63.8%.

Fluorescent probe compound 9:¹H NMR (CDCl₃ ,400MHz): δ=11.16(s, 1H), 8.87(d, J=10.8 Hz, 1H), 7.64(d, J=8.8 Hz, 1H), 7.46(dd, J=8.8, 2.8 Hz, 1H), 7.37(d, J=8.8 Hz, 1H), 6.96-6.87(m, 1H), 6.65(d, J=8.8, 1H), 6.53(s, 1H), 3.85(s, 3H) 3.45(q, J=7.2Hz, 4H), 1.22(t, J=7.2Hz, 6H); ¹³C NMR(CDCl₃ ,100MHz): δ=161.6, 154.4, 149.8, 134.8, 132.7, 128.4, 123.6, 117.3, 97.3, 70.5, 44.7, 35.4, 12.3; FTIR (cm^-1):3338, 3046, 2972, 2705, 1691, 1621, 1593, 1529, 1402, 1356, 1311, 1276, 1254, 1190, 1135, 1079, 973, 820, 778, 740, 658, 634, 473; MALDI-TOF-MS (m/z): 364.19 (100%, M+-1, calcd. 363.42)。

(5) preparation of fluorescent Probe Compound 10

Compound 5(0.25g, 1.0mmol) and 3-amino-4-nitrobenzonitrile (0.19g, 1.2mmol) were dissolved in 20mL of absolute ethanol, and after addition of 0.5g of sodium thiosulfate solid, the mixture was refluxed for 2 hours under nitrogen. After the reaction is finished, concentrating the naturally cooled reaction solution, extracting with dichloromethane and water, drying, and evaporating the redundant solvent to obtain a crude product. Then recrystallized by ethyl acetate to obtain orange-red solid 0.25g, and the yield is 69.8%.

Fluorescent probe compound 10:¹H NMR (CDCl₃ ,400MHz): δ=11.53(s, 1H), 8.92(d, J=5.2Hz, 1H), 8.04(s, 1H), 7.84-7.76(m, 1H), 7.56-7.46(m, 2H), 6.70(dd, J=9.2, 2.0 Hz, 1H), 6.56(s, 1H), 3.48(q, J=7.2Hz, 4H), 1.26(t, J=7.2Hz, 6H); ¹³C NMR(CDCl₃ ,100MHz): δ=161.77, 157.56, 150.59, 138.98 , 128.99, 121.66, 108.89, 108.27, 96.95, 114.92, 50.32, 44.81, 38.93, 12.44; FTIR (cm^-1):3338, 3046, 2972, 2929, 2704, 1694, 1621, 1593, 1529, 1403, 1356, 1275, 1254, 1190, 1135, 1079, 820, 778, 740, 634, 472; MALDI-TOF-MS (m/z): 358.39 (100%, M+-1, calcd. 358.40)。

example 2 fluorescent Probe pairs F^-Detection of the ability to recognize

To evaluate the fluoride ion-recognizing effect of compounds 8,9,10, a test in the UV-visible absorption spectrum was performed on a 10. mu. mol/L standard solution of the target compounds 8,9, 10. All three compounds have wider absorption peaks at 430-460 nm, and then a small amount of 1mmol/L ion solution to be detected is added to the compounds 8,9 and 10 respectively to make the concentration of the ion solution to be detected 100 mu mol/L, as shown in FIG. 2, the absorption peak heights of the compounds are all found to be reduced a small amount, wherein the absorption peak height of the compound 10 is reduced most obviously when fluorine ions are added, the absorption peak height of the compound 10 is reduced secondly when the fluorine ions are added, the compound 9 is reduced at the minimum, and compared with the influence of other anions on the absorption spectra of the compounds 8,9 and 10, the compound 10 can be considered to have selective effect on the fluorine ions. And the fluorescence emission spectrum of compound 10, in which the absorption spectrum was most affected by fluoride ions, was tested with compound 10, in which the absorption peak was most significantly changed, as shown in fig. 3. The wavelength at the maximum absorbance in the absorption spectrum was taken as the excitation wavelength, and the results showed that Compound 10 had emission peaks at 480 nm and 510nm, and other anions (Cl) were added^-, Br^-, OH^-) After that, the emission spectrum was not significantly changed. After addition of the fluoride ion solution, the emission peak at 480 nm of the emission spectrum was enhanced, while the emission peak at 510nm was diminished. Demonstrates the selectivity of compound 10 for fluorescence enhancement of fluoride ionAnd (4) acting.

In order to study the spectral properties of the series of compounds, the compound 10 most sensitive to the fluoride ion concentration was selected as a representative, and the absorption spectrum of the compound was measured as a function of the fluoride ion concentration. As shown in FIG. 4, it can be seen that Compound 10 has a broad absorption peak at 460 nm prior to the addition of fluoride ion. With the addition of fluoride ions, the absorption peak of the absorption spectrum of the compound 10 is gradually reduced, the absorption peak is gradually blue-shifted to 420 nm from 460 nm, and the absorption spectrum is continuously increased after the fluoride ion concentration exceeds 20 times of the probe concentration and does not change. The reason for the change of the absorption spectrum is probably that the concentration of the fluorine ions is increased, the carbonyl oxygen of coumarin in the molecule competes with hydrogen on an imidazole ring to form a hydrogen bond, fluorine with stronger electronegativity is easier to form the hydrogen bond, the hydrogen bond in the original molecule is weakened, the hydrogen bond maintains the planarity and rigidity of the molecule to a certain extent, so that a conjugated system is reduced, the absorption peak is reduced, and the absorption peak at 420 nm is the absorption peak of the coumarin skeleton. The emission spectrum of compound 10 affected by the increase in fluoride ion concentration was then determined. As shown in FIG. 5, Compound 10 had stronger emission peaks at 480 nm and 510nm before the addition of fluoride ion. When the fluoride ion concentration exceeds the compound 10 concentration by a factor of 25, no significant change in the emission spectrum occurs. The emission peak at 480 nm gradually increased as the fluoride ion concentration increased, while the absorption peak at 510nm gradually decreased to disappear. The increase in the emission peak at 480 nm is due to the fluorescence emission possessed by the coumarin fluorophore itself. As described above, the decrease in coplanarity and rigidity of the molecule leads to a decrease in the conjugated system of the molecule, the free rotation between the coumarin and the imidazole group generates dihedral angles, the degree of twisting of the molecule increases, and the molecule dissipates a portion of the absorbed energy in the form of molecular vibrations. The absorption peak at 510nm is caused by the fact that the Stokes shift of the molecule is increased due to the ICT effect generated between an electron-rich coumarin group and an electron-deficient imidazole ring introduced into an electron-deficient group. With the addition of the fluorinion, the coplanarity of the molecules is destroyed, the ICT effect is blocked, and the emission peak disappears.

Sensitivity is an important index for evaluating fluorescent probes, and a jobs's spot experiment was performed in order to measure the sensitivity of a target compound. The change in intensity of the emission peak at 510nm of the fluorescence emission spectrum with the addition of low concentration of fluoride ion using the formulated standard solution of compound 10. The working curve is shown in fig. 6, and a calibration curve is drawn by selecting a portion of the working curve with a better linear relationship at low concentration, as shown in fig. 7. The detection limit for compound 10 was calculated by the slope of the calibration curve to be 3.50 μmol/L (90% confidence), below the national standard for fluorine content in drinking water (about 53.63 μmol/L).

Example 3 response time of fluorescent Probe Compound 10

After the compound 10 and 100 times equivalent of fluorine ions are mixed, the time required for the fluorescence emission intensity to be stable is considered, as shown in fig. 8, the fluorescence intensity shows a gradually increasing trend along with the time, reaches the maximum value in 7 min and then can be stabilized at the same level for a long time, and the change of the fluorescence emission intensity in the first few minutes shows that the target compound 10 can rapidly identify the fluorine ions within 5 min, can meet the response time requirement of real-time monitoring of the fluorine ions in an actual sample, and has higher sensitivity and selectivity.

Example 4 fluorescent Probe Compounds 8,9,10 recognize the F-mechanism

The fluorine ions compete for hydrogen bonds preferentially formed on the imidazole ring, disrupting the molecular configuration resulting in changes in its spectral properties, as shown in fig. 9. The fluorescence emission spectra of compound 10 were measured in solvents of different polarity. As shown in fig. 10, the emission peak of compound 10, which has a longer emission wavelength, has a lower emission intensity and a shorter emission wavelength in a less polar solvent, and the emission peak has a greater emission intensity and a slightly red shift than the peak of compound 10 in a less polar solvent in a more polar solvent. It is presumed that the longer peak of the emission wavelength is due to the ICT effect. Because the introduction of the electron-withdrawing cyano group enhances this effect, a distinct red-shifted emission peak appears on the emission spectrum of compound 10. It is therefore speculated that the change in spectral properties is the disruption of intramolecular hydrogen bonds, resulting in a loss of planarity of the molecule that blocks the ICT process and hence the disappearance of the red-shifted emission peak. Since electron withdrawing groups enhance the ICT effect. The stronger the absorption peak that produces the red shift, the higher the sensitivity to fluoride ions.