Preparation method and application of a fluorescein probe with specific selectivity

Abstract

The application discloses a preparation method of a fluorescein probe with specific selectivity and application thereof, and relates to the fluorescein probe field. The fluorescein probe with specific selectivity is prepared through the following steps: mixing a compound of formula (III) with 1,6-naphthylpyridine-2-methyl aldehyde in a solvent, adding triethylamine to obtain a compound of formula (II); mixing the compound of formula (II) with 1-pyrene carboxylic acid in a solvent, and adding dicyclohexyl carbodiimide and 4-dimethylaminopyridine to obtain the fluorescein probe with specific selectivity. The fluorescein probe with specific selectivity prepared by the application has excellent fluorescence performance, specific selectivity to copper ions, and extremely low detection limit, and has important application significance in copper ion detection.

Description

Technical Field

[0001] This invention relates to the field of fluorophore probes, and more specifically to a method for preparing a fluorophore probe with specific selectivity and its application. Background Technology

[0002] Fluorescein probes, due to their advantages such as high sensitivity, ease of operation, and visual detection, have been widely used in environmental monitoring, biomedicine, food safety, and other fields. Among these, the specific detection of metal ions is one of the hot topics in fluorescein probe research. Copper ions (Cu...) 2+ Cu, as an essential trace element for the human body, participates in various physiological processes (such as redox reactions, enzyme catalysis, and electron transfer), but excessive Cu... 2+ This can lead to the accumulation of reactive oxygen species, triggering neurodegenerative diseases (such as Alzheimer's disease) and environmental toxicity (such as heavy metal pollution in water). Therefore, the development of Cu... 2+ Fluorescent probes with high selectivity and low detection limits are of great significance. Currently reported Cu... 2+ Fluorescent probes are mostly based on recognition units such as Schiff bases, polyamines, and crown ethers, combined with fluorophores such as fluorescein, coumarin, and naphthalimide. They achieve detection through photoinduced electron transfer, intramolecular charge transfer, or chelation to enhance fluorescence. However, existing probes still have problems such as insufficient selectivity, poor fluorescence performance, and lack of chiral recognition, which limit the application of probes.

[0003] Chinese invention patent CN109575004A discloses a method for preparing a novel pyrazolylcoumarin Schiff base copper ion fluorescent probe. The novel pyrazolylcoumarin Schiff base prepared by this invention is a rapid, highly sensitive, and stable Cu ion fluorescent probe. 2+ Fluorescent probes exhibit strong resistance to interference from other common metal ions and can be used for qualitative and quantitative detection of Cu in the environment. 2+ It can overcome the limitations of existing methods for detecting Cu 2+ The fluorescent probe has shortcomings such as slow response, high cytotoxicity of ligands, small Stokes transfer, inability to uniquely recognize a single cation, and low fluorescence quantum yield in aqueous media. It is easy to implement and promote and has great application value, but its fluorescence emission wavelength is relatively short. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing a fluorophore probe with specific selectivity and its application.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A fluorescent probe with specific selectivity has the structural formula shown in formula (I):

[0007] (I).

[0008] A method for preparing a fluorescein probe with specific selectivity, the process route is as follows:

[0009] S1: Compound of formula (III) (English name: Tert-butyl((S)-1-(((S)-1-((S)-7-hydroxy-3-(((R)-1,23,4-tetrahydronaphthalen-1-yl)carbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-3,3-dimethyl-1-0xobutan-2-yl)amino)-1-0xopropan-2-) yl)(methyl)carbamate; Chinese name is (S)-tert-butyl((S)-1-(((S)-1-((S)-7-hydroxy-3-(((R)-1,2,3,4-tetrahydronaphthyl-1-yl)carbamoyl)-1,2,3,4-tetrahydroisoquinoline-2(1H)-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-1-oxopropane-2-yl)(methyl)carbamate) and 1,6-naphthylpyridine-2-carboxaldehyde were mixed in a solvent, and then triethylamine was added to react to give compound (II);

[0010] S2: Compound of formula (II) is mixed with 1-pyrenic acid in a solvent, and then dicyclohexylcarbodiimide and 4-dimethylaminopyridine are added to react and a fluorescein probe with specific selectivity is obtained.

[0011] The reaction equation is as follows:

[0012]

[0013] In step S1, the molar ratio of the compound of formula (III) to 1,6-naphthylpyridine-2-carboxaldehyde is 1:(1.05-1.2).

[0014] In step S1, the solvent is anhydrous ethanol.

[0015] In step S1, the temperature during the reaction is 30-40℃.

[0016] In step S1, the reaction time is controlled to be 5-7 hours.

[0017] In step S2, the molar ratio of the compound of formula (II) to 1-pyrenic acid is 1:(1.05-1.1).

[0018] In step S2, the solvent is DMF.

[0019] In step S2, the reaction time is controlled to be 10-14 hours.

[0020] Application of a fluorescein probe with specific selectivity in the detection of copper ions.

[0021] Due to the adoption of the above technical solutions, the beneficial effects of the present invention include:

[0022] The fluorophore probe prepared by this invention has specific selectivity and can specifically detect copper ions with an extremely low detection limit. Moreover, the prepared fluorophore probe has excellent long-wavelength fluorescence emission, which can reduce the interference of autofluorescence in biological organisms, and has important application significance in copper ion detection. Attached Figure Description

[0023] Figure 1 The image shows the 1H NMR spectrum of the fluorescein probe prepared in Example 1.

[0024] Figure 2 This is a high-resolution mass spectrum of the fluorescein probe prepared in Example 1.

[0025] Figure 3 The image shows the fluorescence intensity of the fluorophore probe prepared in Example 1 against different metal ions. Detailed Implementation

[0026] The following description, in conjunction with specific embodiments, provides further details, but the present invention is not limited to these embodiments.

[0027] Example 1 Preparation of compound (I):

[0028] S1: Under nitrogen protection, 800 ml of anhydrous ethanol was mixed with 0.2 mol of compound (III) and 0.21 mol of 1,6-naphthylpyridine-2-carboxaldehyde, and 0.2 mol of triethylamine was added. The mixture was reacted at 30 °C for 7 h. TLC detection (methanol / dichloromethane = 1 / 10 (V / V), product Rf = 0.6) showed complete conversion of the starting material. The reaction solution was adjusted to pH 5 with 1M hydrochloric acid, quenched with 2.0 L of water, and extracted with ethyl acetate (3 × 400 ml). The organic phases were combined, washed with saturated brine (3 × 300 ml), dried with 30 g of anhydrous sodium sulfate for 1 h, filtered, concentrated under reduced pressure at 50 °C for 2 h, and the crude product was purified by silica gel column chromatography (methanol / dichloromethane V / V = 1:10). The product was then distilled under reduced pressure at 40 °C for 3 h to obtain compound (II) (yield 72.3%, HPLC purity 99.8%). Its 1H NMR data are as follows: 1 H NMR (400MHz, DMSO- d6) δ 9.10 (d, J = 0.6 Hz, 1H), 8.59 (s, 1H), 8.56 (s, 1H), 8.28 (d, J = 0.5 Hz, 1H), 8.26 (s, 1H), 8.13 (d, J = 0.6 Hz, 1H), 7.68 (d, J = 0.5Hz, 1H), 7.64 (d, J = 0.6 Hz, 1H), 7.24-7.12 (m, 4H), 6.98 (dt, J = 7.5, 1.0Hz, 1H), 6.58 (dd, J = 7.5, 2.0 Hz, 1H), 6.48 (dt, J = 2.0, 1.0 Hz, 1H), 4.98(t, J = 0.7 Hz, 1H), 4.90 (d, J = 1.0 Hz, 1H), 4.70 (s, 1H), 4.66 (s, 1H),4.59 (d, J = 1.0 Hz, 1H), 3.37-3.00 (m, 2H), 2.83-2.78 (m, 2H), 1.95 (s, 1H),1.84 (d, J = 0.6 Hz, 2H), 1.79 (s, 1H), 0.98 (s, 9H); HRMS (m / z):576.2891[M+H] + .

[0029] S2: Mix 500 ml DMF with 0.1 mol of compound (II) and 0.105 mol of 1-pyrene carboxylic acid, add 0.1 mol of dicyclohexylcarbodiimide and 0.4 g of 4-dimethylaminopyridine, and stir at room temperature for 10 h; TLC detection (petroleum ether / ethyl acetate = 2 / 1 (V / V), product Rf = 0.5) showed that the starting material was completely converted. The reaction solution was quenched with 1.0 L of water and extracted with ethyl acetate (3 × 300 ml). The organic phases were combined, washed with saturated brine (3 × 300 ml), dried over 35 g of anhydrous sodium sulfate for 1 h, filtered, concentrated under reduced pressure at 40 °C for 3 h, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate V / V = 2:5), distilled under reduced pressure at 40 °C for 3 h, and dried under vacuum at 45 °C for 12 h to obtain compound (I) (yield 73.4%, HPLC purity 99.9%), which is a fluorescein probe with specific selectivity; its proton NMR spectrum is shown below. Figure 1 As shown, the proton NMR data are as follows: 1 H NMR (400 MHz, Acetone- d6) δ9.09 (d, J = 0.6 Hz, 1H), 8.56 (s, 1H), 8.29-8.21 (m, 2H), 8.12 (dd, J = 7.6,0.5 Hz, 2H), 8.03-7.95 (m, 3H), 7.76 (d, J = 0.5 Hz, 1H), 7.73-7.66 (m, 2H), 7.63 (d, J = 1.3 Hz, 2H), 7.60-7.54 (m, 2H), 7.22-7.15 (m, 4H), 7.15-7.04 (m, 2H), 6.86 (dt, J = 2.0, 1.0 Hz, 1H), 5.02 (d, J = 0.6 Hz, 1H), 4.91 (d, J = 1.0 Hz, 1H), 4.68 (d, J = 17.6 Hz, 2H), 4.59 (d, J = 0.9 Hz, 1H), 3.24 (dd, J = 119.7, 1.0 Hz, 2H), 2.81 (d, J = 3.4 Hz, 2H), 1.97 (s, 1H), 1.83 (d, J = 3.1 Hz, 2H), 1.79 (s, 1H), 1.01 (s, 9H); its high-resolution mass spectrum is as follows. Figure 2 As shown, HRMS (m / z): 804.3342 [M+H] + .

[0030] Example 2 Preparation of compound (I):

[0031] S1: Under nitrogen protection, 800 ml of anhydrous ethanol was mixed with 0.2 mol of compound (III) and 0.22 mol of 1,6-naphthylpyridine-2-carboxaldehyde, and 0.2 mol of triethylamine was added. The mixture was reacted at 35 °C for 6 h. TLC detection (methanol / dichloromethane = 1 / 10 (V / V), product Rf = 0.6) showed complete conversion of the starting material. The reaction solution was adjusted to pH 5 with 1M hydrochloric acid, quenched with 2.0 L of water, and extracted with ethyl acetate (3 × 400 ml). The organic phases were combined, washed with saturated brine (3 × 300 ml), dried with 30 g of anhydrous sodium sulfate for 1 h, filtered, concentrated under reduced pressure at 50 °C for 2 h, and the crude product was purified by silica gel column chromatography (methanol / dichloromethane V / V = 1:10). The product was then distilled under reduced pressure at 40 °C for 3 h to obtain compound (II) (yield 73.8%, HPLC purity 99.9%).

[0032] S2: Mix 500 ml DMF with 0.1 mol of compound (II) and 0.108 mol of 1-pyrene carboxylic acid, add 0.1 mol of dicyclohexylcarbodiimide and 0.4 g of 4-dimethylaminopyridine, and stir at room temperature for 12 h; TLC detection (petroleum ether / ethyl acetate = 2 / 1 (V / V), product Rf = 0.5) showed complete conversion of the starting material. Quench the reaction solution with 1.0 L of water and extract with ethyl acetate (3 × 300 ml), combine the organic phases, wash with saturated brine (3 × 300 ml), dry with 35 g of anhydrous sodium sulfate for 1 h, filter, concentrate under reduced pressure at 40 °C for 3 h, purify the crude product by silica gel column chromatography (petroleum ether / ethyl acetate V / V = 2:5), distill under reduced pressure at 40 °C for 3 h, and dry under vacuum at 45 °C for 12 h to obtain compound (I) (yield 74.7%, liquid phase purity 99.8%), which is the fluorophore probe with specific selectivity.

[0033] Example 3 Preparation of compound (I):

[0034] S1: Under nitrogen protection, 800 ml of anhydrous ethanol was mixed with 0.2 mol of compound (III) and 0.24 mol of 1,6-naphthylpyridine-2-carboxaldehyde, and 0.2 mol of triethylamine was added. The mixture was reacted at 40 °C for 5 h. TLC detection (methanol / dichloromethane = 1 / 10 (V / V), product Rf = 0.6) showed complete conversion of the starting material. The reaction solution was adjusted to pH 5 with 1M hydrochloric acid, quenched with 2.0 L of water, and extracted with ethyl acetate (3 × 400 ml). The organic phases were combined, washed with saturated brine (3 × 300 ml), dried with 30 g of anhydrous sodium sulfate for 1 h, filtered, concentrated under reduced pressure at 50 °C for 2 h, and the crude product was purified by silica gel column chromatography (methanol / dichloromethane V / V = 1:10). The product was then distilled under reduced pressure at 40 °C for 3 h to obtain compound (II) (yield 74.5%, HPLC purity 99.8%).

[0035] S2: Mix 500 ml DMF with 0.1 mol of compound (II) and 0.11 mol of 1-pyrene carboxylic acid, add 0.1 mol of dicyclohexylcarbodiimide and 0.4 g of 4-dimethylaminopyridine, and stir at room temperature for 14 h; TLC detection (petroleum ether / ethyl acetate = 2 / 1 (V / V), product Rf = 0.5) showed complete conversion of the starting material. Quench the reaction solution with 1.0 L of water and extract with ethyl acetate (3 × 300 ml), combine the organic phases, wash with saturated brine (3 × 300 ml), dry with 35 g of anhydrous sodium sulfate for 1 h, filter, concentrate under reduced pressure at 40 °C for 3 h, purify the crude product by silica gel column chromatography (petroleum ether / ethyl acetate V / V = 2:5), distill under reduced pressure at 40 °C for 3 h, and dry under vacuum at 45 °C for 12 h to obtain compound (I) (yield 75.6%, liquid phase purity 99.7%), which is the fluorescein probe with specific selectivity.

[0036] Comparative Example 1

[0037] The preparation method of the fluorescein probe with specific selectivity is basically the same as that in Example 2, except that 1,6-naphthylpyridine-2-carboxaldehyde in step S1 is replaced with an equimolar amount of isoquinoline-6-carboxaldehyde.

[0038] Comparative Example 2

[0039] Fluorescein probes with specific selectivity are prepared by the following method:

[0040] S1: Under nitrogen protection, 800 ml of anhydrous ethanol was mixed with 0.2 mol of compound (III) and 0.21 mol of 1,6-naphthidine-2-carboxylic acid. At 0 °C, a mixed solution of 1.2 mol of N-methylimidazole and 0.16 mol of N,N,N',N'-tetramethylchloromethanesulfonate hexafluorophosphate was added in 5 equal batches (15 min apart). The mixture was stirred at room temperature for 12 h. TLC detection (methanol / dichloromethane = 1 / 10 (V / V), product Rf = 0.4) showed that the starting material was completely converted. The reaction solution was quenched with 2.0 L of water and extracted with ethyl acetate (3 × 400 ml). The organic phases were combined, washed with saturated brine (3 × 300 ml), dried over 40 g of anhydrous sodium sulfate for 1 h, filtered, concentrated under reduced pressure at 40 °C for 2 h, and the crude product was purified by silica gel column chromatography (methanol / dichloromethane V / V = 1:10). The crude product was then distilled under reduced pressure at 40 °C for 2 h to give compound (II-1) (yield 73.9%, HPLC purity 99.8%).

[0041] S2: 500 ml of DMF was mixed with 0.1 mol of compound (II-1) and 0.108 mol of 1-pyrene carboxylic acid. 0.1 mol of dicyclohexylcarbodiimide and 0.4 g of 4-dimethylaminopyridine were added, and the mixture was stirred at room temperature for 12 h. TLC analysis (petroleum ether / ethyl acetate = 2 / 1 (V / V), product Rf = 0.5) showed complete conversion of the starting material. The reaction mixture was quenched with 1.0 L of water and extracted with ethyl acetate (3 × 300 ml). The organic phases were combined, washed with saturated brine (3 × 300 ml), dried over 35 g of anhydrous sodium sulfate for 1 h, filtered, concentrated under reduced pressure at 40 °C for 3 h, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate V / V = 2:5), distilled under reduced pressure at 40 °C for 3 h, and dried under vacuum at 45 °C for 12 h to obtain compound (I-1) (yield 74.1%, HPLC purity 99.8%). The reaction equation is as follows:

[0042]

[0043] Comparative Example 3

[0044] The preparation method of the fluorescein probe with specific selectivity is basically the same as that in Example 2, except that 1-pyrene carboxylic acid in step S2 is replaced with an equimolar amount of 3-phenanthrolic acid.

[0045] The fluorescence performance and specificity of the fluorophore probes prepared in Example 1 and Comparative Examples 1-3 were tested for copper ion detection. The test results are shown in Table 1.

[0046] Weigh out 10 mg of the fluorescein probe prepared in Example 1 and Comparative Examples 1-3 respectively, mix thoroughly with 2 ml of dichloromethane, pour into a fluorescence cuvette, and measure its fluorescence spectrum using an LF-1804004 fluorescence spectrophotometer (fluorescence spectral scanning conditions: excitation wavelength 365 nm, slit width 5.0 nm, scanning range 380 nm-1000 nm), and record the fluorescence emission wavelength;

[0047] Cu with a concentration of 0.01 mol / L was prepared separately. 2+ Zn 2+ and Mg 2+ The solutions were prepared by taking 2 ml of the fluorescein probe solution and 20 μL of 0.01 mol / L Cu solution. 2+ Zn 2+ Mg 2+ The solution was mixed thoroughly and poured into a fluorescence cuvette. Its fluorescence spectrum was measured using an LF-1804004 fluorescence spectrophotometer (fluorescence spectral scanning conditions: excitation wavelength 365 nm, slit width 5.0 nm, scanning range 380 nm-1000 nm), and the fluorescence intensity was recorded.

[0048] Take 2 ml of the prepared fluorescein probe solution and 1 ml of Cu at concentrations of 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, and 0.20 mol / L, respectively. 2+ After thoroughly mixing the solution, pour it into a fluorescence cuvette and measure its fluorescence spectrum using an LF-1804004 fluorescence spectrophotometer (fluorescence spectral scanning conditions: excitation wavelength 365 nm, slit width 5.0 nm, scanning range 380 nm-1000 nm). Record the fluorescence intensity. 2+ The fluorescein probe solution was used as a blank control. The LOD was calculated based on the formula LOD = 3σ / K (where σ represents the standard deviation obtained from ten measurements of the blank sample, and k represents the slope of the calibration curve). 2+ The detection limit.

[0049] Table 1

[0050]

[0051] As shown in Table 1, the fluorophore probe with specific selectivity prepared in this application exhibits excellent fluorescence performance and can specifically detect Cu. 2+ It has a low detection limit. Figure 3 The fluorescence intensity diagrams of the fluorophore probe prepared in Example 1 for different metal ions show that the fluorophore probe is effective against Cu. 2+ It has specific selectivity.

[0052] The fluorophore probe prepared in this application exhibits a longer fluorescence emission wavelength, primarily due to the presence of a large π-conjugated system within it. This reduces the energy difference between the highest occupied orbital and the lowest unoccupied orbital, enabling long-wavelength luminescence. Longer-wavelength fluorescence emission allows for deeper tissue penetration and reduces interference from autofluorescence within the organism, thereby improving detection sensitivity. In contrast, the fluorophore probe prepared in Comparative Example 3 has a smaller conjugated system than the examples, resulting in a smaller π-electron delocalization range and a larger energy difference between the highest occupied and lowest unoccupied orbitals. This leads to a shorter fluorescence emission wavelength, less variation in fluorescence intensity, and a relatively higher detection limit compared to the examples.

[0053] The fluorescein probe prepared in this application can specifically detect Cu. 2+ The low detection limit is mainly due to the presence of a Schiff base and a pyridine ring structure in the prepared fluorescein probe. The nitrogen atom of the Schiff base has a lone pair of electrons, readily coordinating with metal ions. The pyridine nitrogen atom and the Schiff base nitrogen atom work together to provide coordination sites, forming a stable four-coordinate environment, thus enabling the detection of Cu. 2+ Specific detection of ions. Furthermore, the presence of chiral structures and amide bonds in the fluorescein probe molecule imparts conformational rigidity, reducing non-specific binding and enhancing targeting of Cu through spatial configuration matching. 2+ The coordination selectivity was observed. In Comparative Example 1, the luciferin probe prepared using isoquinoline-6-carboxaldehyde lacked a pyridine ring structure, and in Comparative Example 2, the luciferin probe prepared using 1,6-naphthyl-2-carboxylic acid lacked a Schiff base structure. Neither a single Schiff base nor a pyridine ring structure in the luciferin molecule could bind with Cu. 2+ The formation of a stable four-coordination environment leads to an inability to specifically select Cu. 2+ .

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. However, any modifications, alterations, and variations made by those skilled in the art without departing from the scope of the present invention based on the disclosed technical content are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. A fluorescent probe with specific selectivity, characterized in that, It has the structural formula shown in equation (I): （I）。 2. A method for preparing a fluorophore probe with specific selectivity, characterized in that, Includes the following steps: S1: Compound (III) was mixed with 1,6-naphthylpyridine-2-carboxaldehyde in a solvent, and then reacted with triethylamine to give compound (II); S2: Compound of formula (II) is mixed with 1-pyrenic acid in a solvent, and then dicyclohexylcarbodiimide and 4-dimethylaminopyridine are added to react and a fluorescein probe with specific selectivity is obtained. The reaction equation is as follows: 。 3. The method for preparing a fluorophore probe with specific selectivity according to claim 2, characterized in that, In step S1, the molar ratio of the compound of formula (III) to 1,6-naphthylpyridine-2-carboxaldehyde is 1:(1.05-1.2).

4. The method for preparing a fluorophore probe with specific selectivity according to claim 2, characterized in that, In step S1, the solvent is anhydrous ethanol.

5. The method for preparing a fluorophore probe with specific selectivity according to claim 2, characterized in that, In step S1, the temperature during the reaction is 30-40℃.

6. The method for preparing a fluorophore probe with specific selectivity according to claim 2, characterized in that, In step S1, the reaction time is controlled to be 5-7 hours.

7. The method for preparing a fluorophore probe with specific selectivity according to claim 2, characterized in that, In step S2, the molar ratio of the compound of formula (II) to 1-pyrenic acid is 1:(1.05-1.1).

8. The method for preparing a fluorophore probe with specific selectivity according to claim 2, characterized in that, In step S2, the solvent is DMF.

9. The method for preparing a fluorophore probe with specific selectivity according to claim 2, characterized in that, In step S2, the reaction time is controlled to be 10-14 hours.

10. The application of the fluorescent probe with specific selectivity as described in claim 1 in the detection of copper ions.

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