Fluorescent probe molecule based on perylene bisimide-thiophene boron ester structure as well as preparation method and application of fluorescent probe molecule

Abstract

The invention relates to the technical field of fluorescence sensing, and particularly discloses a fluorescent probe based on a perylene bisimide-thiophene boron ester structure and a preparation method and application thereof.The fluorescent probe takes 1, 7-dibromo perylene bisimide as a light-emitting main body, boron ester modified perylene bisimide derivatives with a large conjugate surface are prepared through a series of reactions, and the boron ester modified perylene bisimide derivatives with the large conjugate surface are prepared into the fluorescent probe. The fluorescent probe molecule is obtained. Ultraviolet, fluorescence and ion interference test experiments prove that the molecule has rapid photoresponse and good ion interference resistance to hydrogen peroxide, and the fluorescent probe molecule has good H2O2 sensing selectivity, shows high selectivity to hypochlorous acid and other interference ions, and can be used for detecting hydrogen peroxide in the field of hydrogen peroxide detection. The rapid spectral response and the low detection limit value provide possibility for real-time microscopic imaging of cell biology.

Description

Technical Field

[0001] This invention relates to the field of fluorescence sensing technology, specifically to a fluorescent probe molecule based on a perylene imide-thiophene boron ester structure, its preparation method, and its application. Background Technology

[0002] Fluorescent probe molecular imaging technology has promising applications in surgery and biomedicine. Its advantages include convenience, non-invasiveness, real-time in-situ dynamic monitoring, high sensitivity, and high spatiotemporal resolution, making it an important tool in the field of biological fluorescent probe research.

[0003] Ratio-modulation fluorescence assay is an analytical method that determines a target analyte by measuring the fluorescence intensity at two different wavelengths and using the ratio of these intensities as a signal parameter. This method provides built-in correction by simultaneously measuring two different emission signals, offering more accurate data analysis compared to single-channel detection. It better avoids systematic and human errors, exhibiting better reproducibility and stability. It reduces or eliminates data distortion caused by factors such as substrate concentration, external environment, and instrument conditions. Furthermore, changes in the intensity of the two emitted light wavelengths cause changes in the color of the detection system, making the detection process more reliable.

[0004] Hydrogen peroxide is a product and bridge of various enzyme-catalyzed reactions, and many biological indicators can be indirectly obtained through measurement. Fluorescent sensing technology has seen widespread application in hydrogen peroxide detection. Currently, hydrogen peroxide-based fluorescent probes mainly focus on single-molecule unit site recognition, while fluorescent probes based on dual recognition sites are rarely reported compared to single-detection fluorescent probes. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention uses 1,7-dibromoperyleneimide as the luminescent host and prepares a boron ester-modified peryleneimide derivative with a large conjugated surface through a series of reactions. Based on this structure, the molecule exhibits a unique dual-site, dual-emission variation. Ultraviolet, fluorescence, and ion interference tests demonstrate that the molecular probe designed in this invention achieves dual-channel fluorescence response at 560 nm and 600 nm, respectively.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A fluorescent probe molecule based on the peryleneimide-thiophene boron ester structure has the following structural formula: .

[0007] The second objective of this invention is to provide a method for preparing a fluorescent probe molecule based on a peryleneimide-thiophene boron ester structure, comprising the following steps: Step 1: The compound dibromoperylene tetracarboxylic anhydride and 2-aminooctane were heated under reflux in anhydrous ethanol for 12 hours to obtain the compound dibromoperylene imide derivative; Step 2: Dibromoperylene imide derivatives and 2-aldehyde thiophene boron ester compounds are coupled via palladium-catalyzed reaction to prepare 2-aldehyde thiophene-modified perylene imide compounds; Step 3: Then, the 2-aldehyde thiophene-modified perylene imide compound is condensed with p-boron ester benzyltriphenylphosphine bromide to obtain a boron ester-modified perylene imide derivative with a large conjugated surface.

[0008] Furthermore, after the reflux reaction in step one, the mixture is filtered, washed with water, dried, and recrystallized from dichloromethane and methanol.

[0009] Furthermore, the specific steps of step two are as follows: Under nitrogen protection, dibromoperylimide derivative, 2-aldehyde thiophene boron ester compound, and potassium carbonate were dissolved in 1,4-dioxane. After bubbling with nitrogen for 30 minutes, tetra-triphenylphosphine palladium was added to the reaction solution as a catalyst. The mixture was heated under reflux for 24 hours. The reaction solution was then poured into ice water to precipitate the solid, which was dried under vacuum and then purified by dichloromethane / methanol column chromatography.

[0010] Furthermore, the specific steps of step three are as follows: Under nitrogen protection, a 2-aldehyde thiophene-modified perylene imide compound and a bromotriphenylphosphine boron ester compound were dissolved in a mixed solution of anhydrous ethanol / DMF. Potassium tert-butoxide was added in portions at 0°C, and the mixture was stirred at room temperature for 30 minutes. Then, the temperature was raised to 80°C and heated for 12 hours. An aqueous solution of ammonium chloride was added dropwise to the reaction solution, and a large amount of red precipitate was produced. After filtration, washing, drying, and methanol / dichloromethane column chromatography, the probe molecule was obtained.

[0011] The third objective of this invention is to provide an application of the peryleneimide-thiophene phenylborone ester-based fluorescent probe as described above in hydrogen peroxide fluorescence imaging.

[0012] This invention uses 1,7-dibromoperyleneimide as the luminescent host. First, a dibromoperylenetetracarboxylic anhydride and 2-aminooctane are heated under reflux in anhydrous ethanol for 12 hours to obtain a dibromoperyleneimide derivative. Then, the dibromoperyleneimide derivative is coupled with a 2-aldehydethiophene boron ester compound via a palladium-catalyzed coupling reaction to prepare a 2-aldehydethiophene-modified peryleneimide compound. Finally, this 2-aldehydethiophene-modified peryleneimide compound is condensed with p-boron ester benzyltriphenylphosphine bromide to obtain a boron ester-modified peryleneimide derivative with a large conjugated surface.

[0013] The beneficial effects of this invention are: the recognition process of H2O2 by the perylene imide-based fluorescent probe molecule provided by this invention is accompanied by obvious color change; the fluorescent probe molecule has good sensitivity to H2O2 and exhibits high selectivity to hypochlorous acid and other interfering ions. Its rapid spectral response and low detection limit make it possible for real-time imaging in cell biology microscopy. Attached Figure Description

[0014] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0015] Figure 1 The image shows the H NMR spectrum of the probe molecule PDI-SCO.

[0016] Figure 2 The image shows the H NMR spectrum of the probe molecule PDI-SBZ.

[0017] Figure 3 This is a diagram illustrating the mechanism of action of the probe molecule PDI-SBZ on H2O2.

[0018] Figure 4 The fluorescence spectrum of the probe molecule PDI-SBZ in response to the concentration of H2O2 is shown (C=10μm).

[0019] Figure 5 The graph shows the linear fitting curve of the fluorescence response of the probe molecule to H2O2.

[0020] Figure 6 Cell imaging images of the probe molecule PDI-SBZ (left: interaction with the probe; right: imaging image after hydrogen peroxide treatment).

[0021] Figure 7 The probe molecule PDI-SBZ is used to probe various molecules and ions (1-14 correspond to Br). − ,Cl − SO4 2+ CO3 2- ,F − ,PO4 3- H2O2,Fe 3+ Ag + Co 2+ CN - Ni 2+ Cu 2+ Zn 2+The fluorescence (at 560 nm) bar chart after the reaction with H2O2. Detailed Implementation

[0022] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Example

[0024] Step 1: The reaction equation is as follows Under nitrogen protection, 1,7-dibromo-3,4,9,10-perylenetetracarboxylic dianhydride PDI-O (12 g, 22 mmol) and 2-aminooctane (6.0 g, 46 mmol) were dissolved in 100 mL of anhydrous ethanol and heated to reflux for 12 hours. A large amount of red precipitate was formed. The precipitate was filtered, washed with water, dried, and recrystallized from dichloromethane and methanol to give 15.2 g of the dibromoperylene imide derivative, denoted as PDI-Br, with a yield of 90%.

[0025] 1 H NMR (300 MHz, CDCl3): δ 8.24 (s, 2H), 8.10-8.08 (d, J = 6.0Hz, 4H), 3.54(m, 2H), 1.72-1.66(m, 12H), 1.54-1.48(m, 8H), 1.29-0.88 (m, 12H).

[0026] Step 2: The reaction equation is as follows Under nitrogen protection, compound PDI-Br (9.0 g, 12 mmol), 2-aldehyde thiophene boron ester compound (6.2 g, 26 mmol), and potassium carbonate (4.0 g, 40 mmol) were dissolved in 100 mL of 1,4-dioxane. After bubbling with nitrogen for 30 minutes, 0.5 g of tetraphenylphosphine palladium was added to the reaction solution. The mixture was heated under reflux for 24 hours. When the reaction solution was poured into ice water, a large amount of red solid precipitated. After vacuum drying, the solid was purified by dichloromethane / methanol column chromatography to obtain a 2-aldehyde thiophene-modified perylene imide compound, designated as PDI-SCO8 g, with a yield of 82%.

[0027] 1 H NMR (300 MHz, CDCl3): δ 9.88 (s, 2H), 8.22 (s, 2H), 8.06-8.04 (d, J= 6.0Hz, 4H), 7.82-7.78 (m, 4H), 3.52(m, 2H),1.68-1.62(m, 12H), 1.52-1.46(m,8H), 1.28-0.88 (m, 12H)( Figure 1 ).

[0028] Step 3: The reaction equation is as follows Under nitrogen protection, compounds PDI-SCO (4.0 g, 4.8 mmol) and Br-PPh (6.0 g, 10.5 mmol) were dissolved in a mixture of anhydrous ethanol / DMF. Potassium tert-butoxide (1.7 g, 15 mmol) was added in portions at 0 °C, and the mixture was stirred at room temperature for 30 minutes. Then, the temperature was raised to 80 °C and heated for 12 hours. An aqueous solution of ammonium chloride was added dropwise to the reaction solution, and a large amount of red precipitate was formed. After drying, the compound was purified by methanol / dichloromethane column chromatography to obtain the boron ester-modified perylene imide derivative, designated as PDI-SBZ, 4.2 g, yield: 72%.

[0029] 1 H NMR (300 MHz, CDCl3): δ 8.42 (s, 2H), 7.82-7.80 (d, J = 6.0Hz, 4H), 7.76-7.74 (m, 4H), 7.45 -7.38(m, 8H), 6.86-6.82(d, 4H, J = 6.0Hz), 3.48(m,2H), 1.66-1.58(m, 12H), 1.52-1.46(m, 8H), 1.25(s, 24H), 1.18-0.88 (m, 12H)( Figure 2 ).

[0030] Fluorescence and ultraviolet spectroscopy were performed on the PDI-SBZ prepared in the examples, and the fluorescence emission intensity of the materials with different THF / H2O2 ratios (0 μM~50 μM) was measured. Figure 4 ), combined Figure 1-3 It is known that the present invention constructs an intramolecular symmetrical conjugated structure with a long-range conjugated structure. The NMR data proves that the probe structure is correct. Based on this structure, the molecule has a dual-site recognition function. After reacting with H2O2, the boron ester group on the molecule becomes a hydroxyl group, which leads to a change in the entire conjugated structure of the molecule. The direct result is that the emission spectrum changes from red light around 600nm to yellow light around 560nm.

[0031] linear fitting curve of fluorescence response ( Figure 5 The fluorescence quantum yield was calculated, and the spectral data of the corresponding molecules are shown in Table 1 below. The detection limit 3σ / slope = 0.2µm was calculated, indicating that the probe molecule has high sensitivity to the detection of H2O2 and that the fluorescent probe molecule has high quantum yield and high fluorescence lifetime.

[0032] Table 1 Imaging experiments follow general cell imaging methods, such as... Figure 6 and 7 Further fluorescence cell imaging and interference experiments demonstrated that the fluorescent probe molecules of the present invention have good cell penetration and high selectivity for hypochlorous acid and other interfering ions, and have potential application value in cell fluorescence imaging.

[0033] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A fluorescent probe molecule based on a peryleneimide-thiophene boron ester structure, characterized in that, It has the following structural formula: 。 2. A method for preparing a fluorescent probe molecule based on a peryleneimide-thiophene boron ester structure as described in claim 1, characterized in that, Includes the following steps: Step 1: The compound dibromoperylene tetracarboxylic anhydride and 2-aminooctane were heated under reflux in anhydrous ethanol for 12 hours to obtain the compound dibromoperylene imide derivative; Step 2: Dibromoperylene imide derivatives and 2-aldehyde thiophene boron ester compounds are coupled via palladium-catalyzed reaction to prepare 2-aldehyde thiophene-modified perylene imide compounds; Step 3: Then, the 2-aldehyde thiophene-modified perylene imide compound is condensed with p-boron ester benzyltriphenylphosphine bromide to obtain a boron ester-modified perylene imide derivative with a large conjugated surface.

3. The method for preparing a peryleneimide-thiophene phenylborone ester-based fluorescent probe according to claim 2, characterized in that, After the reflux reaction in step one, the mixture is filtered, washed with water, dried, and then recrystallized from dichloromethane and methanol.

4. The method for preparing a peryleneimide-thiophene phenylborone ester-based fluorescent probe according to claim 2, characterized in that, The specific steps of step two are as follows: Under nitrogen protection, dibromoperylimide derivative, 2-aldehyde thiophene boron ester compound, and potassium carbonate were dissolved in 1,4-dioxane. After bubbling with nitrogen for 30 minutes, tetra-triphenylphosphine palladium was added to the reaction solution as a catalyst. The mixture was heated under reflux for 24 hours. The reaction solution was then poured into ice water to precipitate the solid, which was dried under vacuum and then purified by dichloromethane / methanol column chromatography.

5. The method for preparing a peryleneimide-thiophene phenylborone ester-based fluorescent probe according to claim 2, characterized in that, The specific steps of step three are as follows: Under nitrogen protection, a 2-aldehyde thiophene-modified perylene imide compound and a bromotriphenylphosphine boron ester compound were dissolved in a mixed solution of anhydrous ethanol / DMF. Potassium tert-butoxide was added in portions at 0°C, and the mixture was stirred at room temperature for 30 minutes. Then, the temperature was raised to 80°C and heated for 12 hours. An aqueous solution of ammonium chloride was added dropwise to the reaction solution, and a large amount of red precipitate was produced. After filtration, washing, drying, and methanol / dichloromethane column chromatography, the probe molecule was obtained.

6. An application of the peryleneimide-thiophene phenylborone ester-based fluorescent probe as described in claim 1 in hydrogen peroxide fluorescence imaging.

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