A fluorescein-fused nitric oxide donor, its preparation method and application

Abstract

This invention relates to the field of nitric oxide donor technology, and particularly to a fluorescein-fused nitric oxide donor, its preparation method, and its application. The method includes the following steps: adding N,N-dimethylformamide to a mixture of an aromatic compound containing a para-active halogenated side chain and potassium carbonate; stirring; adding 3-trifluoromethyl-4-nitroaniline; quenching the reaction with saturated brine; drying and vacuum concentration; and purifying. Then, mixing fluorescein, potassium carbonate, and dimethyl sulfoxide and stirring; placing the first mixture in an ice-water bath; adding a second mixture; quenching the reaction with saturated brine; drying and vacuum concentration; and purifying to obtain the fluorescein-fused nitric oxide donor. This constructs a functional molecule that combines NO release function and fluorescence response characteristics, achieving an integrated release-response-imaging function. This solves the problem that existing NO donors are difficult to monitor in real time, making it impossible to intuitively and in real-time trace NO release behavior, thus limiting the practical application of nitric oxide donors.

Description

Technical Field

[0001] This invention relates to the field of nitric oxide donor technology, and particularly to a fluorescein-fused nitric oxide donor, its preparation method, and its application. Background Technology

[0002] Nitric oxide (NO) is an important endogenous gaseous signaling molecule that plays a crucial role in antibacterial, anti-inflammatory, angiogenesis-promoting, and wound-healing processes. Nitric oxide donors are an important pathway for providing exogenous NO, enabling the controlled release of nitric oxide molecules under physiological conditions or external stimuli.

[0003] Fluorescein compounds exhibit fluorescence upon photoexcitation, possessing characteristics such as high fluorescence quantum yield, good photostability, and excellent biocompatibility. Furthermore, these compounds also possess excellent optical properties, chemical stability, water solubility, and pH responsiveness, making them widely used in the design and construction of various fluorescent probes and indicators.

[0004] Existing common NO donor systems, such as nitrobenzene derivatives, nitrosothiols, N-nitrosamines, and diazoxide glycol salts, can release NO, but the NO release process is difficult to monitor in real time and lacks release indication function, making it impossible to intuitively and in real time trace the NO release behavior, thus limiting the practical application of nitric oxide donors. Summary of the Invention

[0005] The purpose of this invention is to propose a method for preparing a fluorescein-fused nitric oxide donor, which constructs a functional molecule that combines NO release function and fluorescence response characteristics. This fluorescein-fused nitric oxide donor retains the excellent optical properties of the fluorescent group and releases NO under specific illumination conditions, demonstrating the ability to controllably release NO. It achieves an integrated function of release-response-imaging, solving the problems of existing NO donors, such as difficulty in real-time monitoring of the NO release process and lack of release indication function, which prevents intuitive and real-time tracking of NO release behavior and thus limits the practical application of nitric oxide donors.

[0006] Another objective of this invention is to propose a fluorescein-fused nitric oxide donor prepared using the above-described method for preparing a fluorescein-fused nitric oxide donor, which retains the excellent optical properties of the fluorescent group and releases NO under specific illumination conditions, thus possessing the ability to release NO in a controllable manner and achieving an integrated function of release-response-imaging.

[0007] Another object of the present invention is to propose the application of the above-mentioned fluorescein-fused nitric oxide donor in wound repair, infection control and antimicrobial biomedical materials.

[0008] To achieve the above objectives, the present invention proposes a method for preparing a fluorescein-fused nitric oxide donor, comprising the following steps: Step S1, Synthesis of NO donor intermediate: Under an inert gas atmosphere, N,N-dimethylformamide was added to a mixture of an aromatic compound containing a para-active halogenated side chain and potassium carbonate. After stirring, 3-trifluoromethyl-4-nitroaniline was added and the reaction was heated. After the reaction was completed, the mixture was cooled to room temperature, and the reaction was quenched by adding saturated brine. Ethyl acetate was added and mixed, and the mixture was washed with saturated brine. The organic phase was dried and concentrated under vacuum to obtain the first crude product. The first crude product was purified by column chromatography to obtain the NO donor intermediate. Step S2, Synthesis of fluorescein-fused nitric oxide donor: Fluorescein, potassium carbonate, and dimethyl sulfoxide are mixed and stirred to obtain a first mixture; the NO donor intermediate is dissolved in an organic solvent to obtain a second mixture; the first mixture is placed in an ice-water bath, the second mixture is added, and the mixture is heated to react. After the reaction is completed, the mixture is cooled to room temperature, and the reaction is quenched by adding saturated brine. Ethyl acetate is added, and the mixture is washed with saturated brine. The organic phase is dried and concentrated under vacuum to obtain a second crude product. The second crude product is purified by column chromatography to obtain the fluorescein-fused nitric oxide donor.

[0009] Optionally, the aromatic compound containing a para-active halogenated side chain is selected from any one of 1,4-di(bromomethyl)benzene, 1,4-p-dichlorobenzene, or 1,4-bis(iodomethyl)benzene.

[0010] Optionally, the molar ratio of the aromatic compound containing the para-active halogenated side chain to the 3-trifluoromethyl-4-nitroaniline is 1.2:1.

[0011] Optionally, in step S1, the N,N-dimethylformamide is measured under an inert gas atmosphere; The inert gas is nitrogen; The stirring conditions are: stirring at room temperature for 0.5 hours; the heating reaction temperature is 80°C and the time is 12 hours.

[0012] Optionally, in step S2, the molar ratio of the fluorescein to the NO donor intermediate is 1:1.

[0013] Optionally, in step S2, fluorescein, potassium carbonate, and dimethyl sulfoxide are mixed and stirred at room temperature for 0.5 h to obtain a first mixture; the NO donor intermediate is dissolved in dimethyl sulfoxide to obtain a second mixture; the first mixture is placed in an ice-water bath at 0°C and the second mixture is added to carry out a heating reaction at 80°C for 10 h.

[0014] Optionally, in step S1, after adding ethyl acetate and mixing, the mixture is washed three times with saturated saline solution; in step S2, after adding ethyl acetate and mixing, the mixture is washed three times with saturated saline solution. In step S1, anhydrous sodium sulfate is used for drying; in step S2, anhydrous sodium sulfate is used for drying.

[0015] Optionally, in step S1, the column chromatography uses ethyl acetate and petroleum ether in a volume ratio of 5:1 as the eluent. In step S2, the column chromatography uses dichloromethane and petroleum ether as eluents, and performs gradient elution at volume ratios of 10:1 and 5:1.

[0016] The present invention also proposes a fluorescein-fused nitric oxide donor, which is prepared using any of the above-described methods for preparing fluorescein-fused nitric oxide donors.

[0017] This invention also proposes the application of the above-mentioned fluorescein-fused nitric oxide donor in wound repair, infection control, and antimicrobial biomedical materials.

[0018] Compared with the prior art, the embodiments of the present invention have the following beneficial effects: 1. This invention uses an aromatic compound containing a para-active halogenated side chain, which has less steric hindrance in its para-substituted structure, thus facilitating the preparation of a fluorescein-fused nitric oxide donor with regular configuration and uniform substitution. By reacting the aromatic compound containing the para-active halogenated side chain with 3-trifluoromethyl-4-nitroaniline, the prepared NO donor intermediate not only has a NO donor group but also a reaction site that can be linked to fluorescein. This facilitates the stable connection between the subsequent NO donor intermediate and fluorescein, further ensuring the stable connection of the NO donor group to the fluorescein core, thereby guaranteeing the stability and controllable release performance of the fluorescein-fused nitric oxide donor molecule. This invention establishes an intrinsic correlation between NO release and optical response through changes in fluorescence signal, enabling visualized monitoring of the NO release process.

[0019] 2. This invention fuses the fluorophore structure with the NO donor structure at the molecular level. The fluorescent parent structure and the NO donor intermediate are covalently integrated into the same molecular backbone, constructing a functional molecule that combines NO release and fluorescence response characteristics. This fluorophore-fused nitric oxide donor retains the excellent optical properties of the fluorescent group and releases NO under specific illumination conditions, demonstrating controllable NO release and achieving an integrated release-response-imaging function. This invention not only achieves real-time fluorescence indication of the NO release process but also allows for quantitative or semi-quantitative monitoring of the release behavior through changes in optical signals. The prepared fluorophore-fused nitric oxide donor features high controllability and visualization capabilities. Attached Figure Description

[0020] Figure 1This is a schematic diagram illustrating the principle of NO release from a fluorescein-fused nitric oxide donor (DPL-NO) under 500nm wavelength illumination, according to an embodiment of the present invention. Figure 2 The hydrogen nuclear magnetic resonance spectrum of the NO donor intermediate (DPL-Br) of Example 1 of the present invention; Figure 3 The carbon NMR spectrum of the NO donor intermediate (DPL-Br) of Example 1 of the present invention; Figure 4 The hydrogen nuclear magnetic resonance spectrum of the fluorescein-fused nitric oxide donor (DPL-NO) of Example 1 of the present invention; Figure 5 The carbon NMR spectrum of the fluorescein-fused nitric oxide donor (DPL-NO) of Example 1 of the present invention is shown. Figure 6 The nuclear magnetic resonance fluorine spectrum of the fluorescein-fused nitric oxide donor (DPL-NO) of Example 1 of the present invention; Figure 7 The image shows the UV-Vis spectrum of the photolysis process of the fluorescein-fused nitric oxide donor (DPL-NO) in a mixed solution of phosphate buffer and DMSO according to Example 1 of the present invention. The inset is a fitting graph of the first-order reaction rate equation of absorbance versus time at a wavelength of 500 nm. Figure 8 The figure shows the absorbance-concentration standard curves of fluorescein fused with nitric oxide donor (DPL-NO) at different concentrations in Example 1 of the present invention, with concentrations of 50µM, 25µM, 12.5µM, 6.25µM, 3.125µM and 1.5625µM. The inset is a linear fitting graph of absorbance and concentration at a wavelength of 500nm. Figure 9 The fluorescein-fused nitric oxide donor (DPL-NO) (50.0 μM, λ) of Example 1 of the present invention ex Fluorescence emission spectrum of 405 nm (irradiated by 500 nm light source, 25 °C); Figure 10 This is a graph showing the detection of reactive oxygen species release according to the present invention, wherein, Figure 10 In the figure, 'a' represents the UV-Vis spectrum of ABDA in dimethyl sulfoxide at 25°C as a function of time. Figure 10 In the figure, b is the UV-Vis spectrum of ABDA containing DPL-NO in dimethyl sulfoxide as a function of time at 25℃; Figure 11 This is a schematic diagram showing the NO release results of the fluorescein-fused nitric oxide donor (DPL-NO) in Example 1 of the present invention under light and dark conditions; Figure 12This is a schematic diagram of the femtosecond transient absorption spectrum and half-life detection results of the fluorescein-fused nitric oxide donor (DPL-NO) in Example 1 of the present invention. Figure 12 a, b, and c in the figure are femtosecond transient absorption spectra of fluorescein-fused nitric oxide donor (DPL-NO) at different delay times in a mixed solution of phosphate buffer and dimethyl sulfoxide. Figure 12 In the diagram, d represents the half-life detection results of fluorescein fused with nitric oxide donor (DPL-NO); Figure 13 These are confocal laser scanning microscope images and fluorescence images of the fluorescein-fused nitric oxide donor (DPL-NO) according to Example 1 of the present invention, wherein... Figure 13 In the image, a and b are confocal laser scanning microscope images of DPL-NO under different fields of view (excitation wavelength λ). ex =405nm), Figure 13 c in the image is a fluorescence imaging image of intracellular nitric oxide production induced by DPL-NO at different time points monitored by confocal laser scanning microscopy. Figure 14 This figure shows the results of cytotoxicity studies on different cell lines using the fluorescein-fused nitric oxide donor (DPL-NO) from Example 1 of this invention. Figure 14 Figures a, b, c, and d in the figure show the results of a cytotoxicity study of luciferin-fused nitric oxide donor (DPL-NO) on B16 cells, HepG2 cells, 4T1 cells, and HeLa cells, respectively. Figure 15 The images show colony diagrams of *Escherichia coli* and *Staphylococcus aureus* treated with 5% OHA-CMCS-DPL-NO injectable hydrogel and 6% OHA-CMCS injectable hydrogel, respectively, according to this invention. Figure 15 In the image, 'a' represents colony images of non-illuminated OHA-CMCS, non-illuminated OHA-CMCS-DPL-NO, illuminated OHA-CMCS, and illuminated OHA-CMCS-DPL-NO grown on E. coli culture plates. Figure 15 In the figure, b represents the colony diagrams of non-illuminated OHA-CMCS, non-illuminated OHA-CMCS-DPL-NO, illuminated OHA-CMCS, and illuminated OHA-CMCS-DPL-NO grown on Staphylococcus aureus agar plates. Figure 16 This diagram illustrates the effects of 6% OHA-CMCS injectable hydrogel and 5% OHA-CMCS-DPL-NO injectable hydrogel on the cell viability of *Escherichia coli* and *Staphylococcus aureus*, respectively. Figure 16In the figure, 'a' represents the effects of non-illuminated OHA-CMCS (-laser-OHA-CMCS), non-illuminated 5% OHA-CMCS-DPL-NO (-laser-OHA-CMCS-DPL-NO-5%), illuminated OHA-CMCS (+laser-OHA-CMCS), and illuminated 5% OHA-CMCS-DPL-NO (+laser-OHA-CMCS-DPL-NO-5%) on Escherichia coli cell viability (n=3, *p≤0.05, **p≤0.01, ***p≤0.001), and the statistical significance analysis. Figure 16 Figure b shows the effects of non-illuminated OHA-CMCS (-laser-OHA-CMCS), non-illuminated 5% OHA-CMCS-DPL-NO (-laser-OHA-CMCS-DPL-NO-5%), illuminated OHA-CMCS (+laser-OHA-CMCS), and illuminated 5% OHA-CMCS-DPL-NO (+laser-OHA-CMCS-DPL-NO-5%) on Staphylococcus aureus cell viability (n=3, *p≤0.05, **p≤0.01, ***p≤0.001). Figure 17 This is a time-scan analysis of the changes in G′ and G′′ over time for the 6% OHA-CMCS injectable hydrogel and the 6% OHA-CMCS-DPL-NO injectable hydrogel in this invention. Figure 16 In the figure, 'a' represents the time-scan analysis of the changes in G′ and G′′ over time for 6% OHA-CMCS injectable hydrogel. Figure 16 b in the figure represents the time-scan analysis of the changes of G′ and G′′ over time in 6% OHA-CMCS-DPL-NO injectable hydrogel. Figure 18 The graphs show the changes of G′ and G′′ as a function of shear strain for the 5%OHA-CMCS injectable hydrogel, 6%OHA-CMCS injectable hydrogel, and 6%OHA-CMCS-DPL-NO injectable hydrogel in this invention. Figure 19 The graphs show the changes of G′ and G′′ with angular frequency for the 5%OHA-CMCS injectable hydrogel, 6%OHA-CMCS injectable hydrogel, and 6%OHA-CMCS-DPL-NO injectable hydrogel in this invention. Figure 20 The images show scanning electron microscope (SEM) images of the 6% OHA-CMCS injectable hydrogel and the 6% OHA-CMCS-DPL-NO injectable hydrogel used in this invention, as well as an image showing the elemental distribution in the 6% OHA-CMCS-DPL-NO injectable hydrogel. Figure 20 Scanning electron microscope image of 6% OHA-CMCS injectable hydrogel (a). Figure 20 b is a scanning electron microscope image of 6% OHA-CMCS-DPL-NO injectable hydrogel. Figure 20 c, d, e, and f in the image represent the C, N, O, and F element distribution images in 6% OHA-CMCS-DPL-NO injectable hydrogel, respectively. Figure 21 The Fourier transform infrared spectra of the 6%OHA-CMCS injectable hydrogel and the 6%OHA-CMCS-DPL-NO injectable hydrogel in this invention, along with HA, OHA, and CMCS. Detailed Implementation

[0021] This invention proposes a method for preparing a fluorescein-fused nitric oxide donor.

[0022] In this embodiment of the invention, the method for preparing the fluorescein-fused nitric oxide donor includes the following steps: Step S1, Synthesis of NO donor intermediate: Under an inert gas atmosphere, N,N-dimethylformamide (DMF) was added to a mixture of an aromatic compound containing a para-active halogenated side chain and potassium carbonate. After stirring, 3-trifluoromethyl-4-nitroaniline was added and the reaction was heated. After the reaction was completed, the mixture was cooled to room temperature, and the reaction was quenched by adding saturated brine. Ethyl acetate (EtoAc) was added and mixed, and the mixture was washed with saturated brine. The organic phase was dried and concentrated under vacuum to obtain the first crude product. The first crude product was purified by column chromatography to obtain the NO donor intermediate (named DPL-X). Step S2, Synthesis of fluorescein-fused nitric oxide donor: Fluorescein, potassium carbonate, and dimethyl sulfoxide (DMSO) were mixed and stirred to obtain a first mixture; the NO donor intermediate was dissolved in an organic solvent to obtain a second mixture; the first mixture was placed in an ice-water bath, the second mixture was added, and the mixture was heated to react. After the reaction was completed, the mixture was cooled to room temperature, and the reaction was quenched by adding saturated brine. Ethyl acetate (EtoAc) was added, and the mixture was washed with saturated brine. The organic phase was dried and concentrated under vacuum to obtain a second crude product. The second crude product was purified by column chromatography to obtain the fluorescein-fused nitric oxide donor (named DPL-NO).

[0023] This invention utilizes an aromatic compound containing a para-active halogenated side chain, whose para-substituted structure has less steric hindrance, which is beneficial for obtaining a fluorescein-fused nitric oxide donor with regular configuration and uniform substitution. By reacting the aromatic compound containing the para-active halogenated side chain with 3-trifluoromethyl-4-nitroaniline, the prepared NO donor intermediate not only has a NO donor group but also a reaction site that can be linked to fluorescein. This facilitates the stable connection between the subsequent NO donor intermediate and fluorescein, further ensuring that the NO donor group is stably linked to the fluorescein core, thereby guaranteeing the stability of the fluorescein-fused nitric oxide donor molecule and its controllable release performance. This invention establishes an intrinsic correlation between NO release and optical response through changes in fluorescence signal, enabling visualized monitoring of the NO release process.

[0024] This invention fuses the fluorophore structure with the NO donor structure at the molecular level. The parent fluorescent structure and the NO donor intermediate are covalently integrated into the same molecular backbone, constructing a functional molecule that combines NO release and fluorescence response. This fluorophore fused with a nitric oxide donor retains the excellent optical properties of the fluorescent group and releases NO (such as NO) under specific illumination conditions. Figure 1 The diagram illustrates the principle of NO release from a fluorescein-fused nitric oxide donor (DPL-NO) under 500nm wavelength illumination. This invention demonstrates the ability to controllably release NO, achieving an integrated release-response-imaging function. It not only enables real-time fluorescence indication of the NO release process but also allows for quantitative or semi-quantitative monitoring of the release behavior through changes in optical signals. The prepared fluorescein-fused nitric oxide donor exhibits high controllability and visualization capabilities.

[0025] Furthermore, the fluorescein-fused nitric oxide donor prepared by this invention maintains structural stability under normal storage and physiological conditions. It is structurally stable under light-protected conditions and can release NO under specific light conditions. The released byproducts have low toxicity, and the NO release process is accompanied by changes in fluorescence signal. Biocompatible fragments are introduced into the molecular structure, avoiding the introduction of highly toxic groups.

[0026] Furthermore, fluorescein-type structures exhibit high quantum yield and good photostability; their excitation and emission wavelengths are located in the visible light region, making them suitable for biological systems; the functional groups such as phenolic hydroxyl and carboxyl groups in these structures facilitate further chemical modification; and their fluorescence intensity is highly sensitive to changes in molecular structure, which is beneficial for constructing responsive systems.

[0027] Furthermore, the fluorescein-fused nitric oxide donor prepared by this invention can be loaded into hydrogels, nanomaterials, or other biomedical carriers to construct a smart material system with antibacterial, healing-promoting, and visualization functions, which has broad application prospects in wound repair, infection control, biomedical imaging, and precision treatment.

[0028] This invention solves the problem that the NO release process of existing NO donors is difficult to monitor in real time and lacks release indication function, making it impossible to intuitively and in real time track NO release behavior, thus limiting the practical application of nitric oxide donors.

[0029] To further clarify, the aromatic compound containing the para-active halogenated side chain is selected from any one of 1,4-di(bromomethyl)benzene, 1,4-p-dichlorobenzene, or 1,4-bis(iodomethyl)benzene.

[0030] When the aromatic compound containing the para-active halogenated side chain is selected from any one of 1,4-di(bromomethyl)benzene, 1,4-p-dichlorobenzyl, or 1,4-bis(iodomethyl)benzene, its para-substituted structure has less steric hindrance, which is beneficial for obtaining a fluorescein-fused nitric oxide donor with regular configuration and uniform substitution. Moreover, the benzylic halogen atom is a leaving group with moderate reactivity, which can undergo efficient nucleophilic substitution reaction under mild conditions to achieve stable grafting of NO donor fragments, while avoiding side reactions and system instability caused by excessive reactivity.

[0031] Preferably, the aromatic compound containing the para-active halogenated side chain is selected from 1,4-di(bromomethyl)benzene. If other bromine-containing substances, such as aliphatic bromoalkanes, are used as the aromatic compound containing the para-active halogenated side chain, although the reactivity is high, the structure is highly flexible and lacks stability, easily initiating cross-linking or side reactions; direct bromine attachment to the aromatic ring (such as bromobenzenes) results in low reactivity, requiring a coupling catalytic system, increasing process complexity and cost. Therefore, this invention, by using 1,4-di(bromomethyl)benzene, can effectively balance reaction selectivity, structural controllability, and process feasibility.

[0032] In another embodiment of the invention, the aromatic compound containing a para-active halogenated side chain is selected from p-bromoethylbenzene or p-chloroethylbenzene.

[0033] To further illustrate, when the aromatic compound containing the para-active halogenated side chain is selected from 1,4-di(bromomethyl)benzene, the preparation reaction formula for the NO donor intermediate (named DPL-Br) is as follows: ; The reaction formula for the preparation of fluorescein fused with a nitric oxide donor (DPL-NO) is as follows: .

[0034] To further illustrate, the molar ratio of the aromatic compound containing the para-active halogenated side chain to 3-trifluoromethyl-4-nitroaniline is 1.2:1.

[0035] To further explain, in step S1, N,N-dimethylformamide is measured under an inert gas atmosphere; The inert gas is nitrogen; The stirring conditions were: stirring at room temperature for 0.5 hours; and heating the reaction at 80°C for 12 hours.

[0036] To further clarify, in step S2, the molar ratio of fluorescein to NO donor intermediate is 1:1.

[0037] To further explain, in step S2, fluorescein, potassium carbonate and dimethyl sulfoxide (DMSO) are mixed and stirred at room temperature for 0.5 h to obtain a first mixture; the NO donor intermediate is dissolved in dimethyl sulfoxide to obtain a second mixture; the first mixture is placed in an ice-water bath at 0 °C and the second mixture is added, and the reaction is carried out by heating at 80 °C for 10 h.

[0038] To further explain, in step S2, a syringe is used to add the second mixture to the first mixture.

[0039] To further explain, in step S1, after adding ethyl acetate (EtoAc) and mixing, the mixture is washed three times with saturated saline solution; in step S2, after adding ethyl acetate (EtoAc) and mixing, the mixture is washed three times with saturated saline solution. In step S1, anhydrous sodium sulfate is used for drying; in step S2, anhydrous sodium sulfate is used for drying.

[0040] In one embodiment of the present invention, both steps S1 and S2 are performed using a Schlenk reaction tube for heating reaction.

[0041] To further explain, in step S1, column chromatography uses ethyl acetate and petroleum ether in a volume ratio of 5:1 as the eluent. In step S2, column chromatography uses dichloromethane and petroleum ether as eluents, and performs gradient elution at volume ratios of 10:1 and 5:1.

[0042] The present invention also proposes a fluorescein-fused nitric oxide donor, which is prepared using the above-described method for preparing a fluorescein-fused nitric oxide donor.

[0043] The prepared fluorescein-fused nitric oxide donor retains the excellent optical properties of the fluorescent group and releases NO under specific illumination conditions, demonstrating the ability to controllably release NO and achieve an integrated release-response-imaging function.

[0044] This invention also proposes the application of the above-mentioned fluorescein-fused nitric oxide donor in wound repair, infection control, and antimicrobial biomedical materials.

[0045] To facilitate understanding of the present invention, a more complete description is provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0046] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0047] The raw materials used in the following examples are all commercially available, wherein: Fluorescein: 90% purity, catalog number F100007, Aladdin Reagent (Shanghai) Co., Ltd. Hyaluronic acid: molecular weight 1.0 MDa~1.8 MDa, Shanghai Maclean Biochemical Technology Co., Ltd. Sodium periodate: 99% purity, Beijing Mairuida Technology Co., Ltd. Carboxymethyl chitosan: BioReagent, Aladdin Reagent (Shanghai) Co., Ltd.

[0048] Example 1 A method for preparing a fluorescein-fused nitric oxide donor includes the following steps: Step S1, Synthesis of NO donor intermediate: 1.81 g (6.83 mmol, 1.2 eq) of an aromatic compound containing a para-active halogenated side chain (the aromatic compound containing a para-active halogenated side chain is selected from 1,4-di(bromomethyl)benzene) and 944.1 mg (1.2 eq, 6.83 mmol) of potassium carbonate were weighed and added sequentially to a Schlenk reaction tube; Under an inert gas (nitrogen) atmosphere, 57 mL of N,N-dimethylformamide (DMF) (measured under nitrogen) was added to a mixture of an aromatic compound containing a para-active halogenated side chain and potassium carbonate to obtain a 0.1 M solution. The solution was stirred at room temperature for 0.5 h. After stirring, 1.17 g (5.693 mmol) of 3-trifluoromethyl-4-nitroaniline was added to the reaction tube, and the reaction was heated (80 °C for 12 h). The reaction result... After cooling to room temperature, the reaction was quenched with saturated brine. 100 mL of ethyl acetate (EtoAc) was added and mixed. The mixture was washed three times with saturated brine (3 × 100 mL). The organic phase was dried (using anhydrous sodium sulfate) and concentrated under vacuum to obtain the first crude product. The first crude product was purified by column chromatography (using ethyl acetate and petroleum ether in a volume ratio of 5:1 as eluent) to obtain 1.1 g of NO donor intermediate (DPL-Br) (yellow-green solid, yield 45%). The molar ratio of the aromatic compound containing the para-active halogenated side chain to 3-trifluoromethyl-4-nitroaniline is 1.2:1. In step S2, 204.9 mg (0.62 mmol, 1.0 eq) of fluorescein and 85.7 mg (0.62 mmol, 1.0 eq) of potassium carbonate were weighed and added to the Schlenk reaction tube in sequence, and 6 mL of dimethyl sulfoxide was measured and added to the Schlenk reaction tube. Fluorescein, potassium carbonate, and dimethyl sulfoxide (DMSO) were mixed and stirred at room temperature for 0.5 h to obtain a first mixture. 241.3 mg (0.62 mmol, 1.0 eq) of NO donor intermediate (DPL-Br) was weighed and dissolved in 3 mL of DMSO to obtain a second mixture with a concentration of 0.2 M. The first mixture was placed in an ice-water bath at 0 °C. The second mixture was slowly added to the reaction tube using a syringe, followed by heating to 80 °C for [time missing]. After 10 hours of reaction, the mixture was cooled to room temperature, and saturated brine was added to quench the reaction. 100 mL of ethyl acetate (EtoAc) was added and mixed, and the mixture was washed three times with saturated brine (3 × 100 mL). The organic phase was dried (using anhydrous sodium sulfate) and concentrated under vacuum to obtain the second crude product. The second crude product was purified by column chromatography (using dichloromethane and petroleum ether as eluents, with gradient elution at volume ratios of 10:1 and 5:1) to obtain 50 mg of fluorescein-fused nitric oxide donor (DPL-NO) (orange solid, yield 13%). The molar ratio of fluorescein to NO donor intermediate is 1:1.

[0049] Characterization of the NO donor intermediate (DPL-Br) of Example 1 using 1H NMR and 1C NMR spectra: Figure 2 The proton NMR spectrum of the NO donor intermediate (DPL-Br) is shown in the following data: 1 H NMR (400MHz, CDCl3) δ8.02 (d, J =8Hz, 1H), δ7.44(d, J =8Hz, 2H), δ7.34(d, J =8Hz, 2H), δ6.98(d, J =4Hz, 1H), δ6.7 (dd, J 1 = 8Hz J 2=8Hz, 1H), δ5.02(s, 1H), δ4.52(s, 2H), δ4.47(d, J =8Hz, 2H).

[0050] Figure 3The carbon NMR spectrum of the NO donor intermediate (DPL-Br) is shown in the following data: 13 C NMR (100MHz, CDCl3) δ151.27, 137.80, 136.94, 129.84, 129.16, 127.82, 112.94, 111.67, 47.19, 32.65.

[0051] By analyzing the molecular formula of DPL-Br (C 15 H 12 Based on the analysis of the structure (BrF3N2O2), the DPL-Br molecule contains a total of 12 hydrogen atoms. The 1,4-di(bromomethyl)benzene ring contains 4 H atoms, the benzyl C atom is connected to 2 H atoms, the amino group contains 1 H atom, and the aniline molecule contains a total of 3 H atoms. This information was used to further analyze the properties of DPL-Br. 1 ¹H NMR analysis revealed that the four atoms in the 1,4-di(bromomethyl)benzene ring appeared at chemical shifts of 7.44 and 7.34, exhibiting two doublets (d), each with an integral of 2. The four H atoms bonded to the benzyl C atom appeared at chemical shifts of 4.52 and 4.47, exhibiting one singlet (s) and one doublet (d), each with an integral of 2. The hydrogen atom in the amino group appeared at chemical shift 5.02, exhibiting one singlet (s), with an integral of 1. The three H atoms in the aniline molecule appeared at chemical shifts of 8.02, 6.98, and 6.7, exhibiting two doublets (d) and one doublet (dd), each with an integral of 1.

[0052] The fluorescein-fused nitric oxide donor (DPL-NO) from Example 1 was characterized and detected as follows: (1) Characterization of fluorescein fused with nitric oxide donor (DPL-NO) using 1H NMR, 1C NMR, and 1N NMR spectra. Figure 4 The 1H NMR spectrum of fluorescein fused with a nitric oxide donor (DPL-NO) is shown below. 1 H NMR (400MHz, CDCl3) δ8.27 (d, J =8Hz, 1H), δ7.91(d, J =8Hz, 1H), δ7.71(m, 2H), δ7.24(d, J =8Hz, 1H), δ7.09(d, J =8Hz, 2H), δ7.03(s, 1H), δ6.92(d, J =8Hz, 2H), δ6.79(d, J =8Hz, 2H), δ6.69 (m, 5H), δ4.84 (s, 2H), δ4.41 (s, 2H).

[0053] Figure 5 The carbon NMR spectrum of fluorescein fused with a nitric oxide donor (DPL-NO) is shown below. 13 C NMR (100MHZ, CDCl3) δ175.48, 165.45, 157.72, 156.68, 152.49, 137.75, 136.03, 133.48, 132.80, 131.53, 130.97, 130.48, 130.23, 130.12 ,129.43,129.32,127.55,122.03,114.96,112.36,103.49,67.28,46 .91, 33.96, 32.06, 31.63, 29.83, 29.49, 25.63, 25.01, 22.83, 14.27.

[0054] Figure 6 The carbon NMR spectrum of fluorescein fused with a nitric oxide donor (DPL-NO) is shown below. 19 F NMR (100MHz, CDCl3) δ-60.13.

[0055] By analyzing the molecular formula of DPL-NO (C 35 H 23 Based on the analysis of F3N2O7 and its structural formula, the DPL-NO molecule contains a total of 23 hydrogen atoms. Specifically, the 1,4-di(bromomethyl)benzene ring contains 4 H atoms, the benzyl C atom is connected to 2 H atoms, the amino group contains 1 H atom, the aniline molecule contains 3 H atoms, the fluorescein benzene ring contains 10 H atoms, and the hydroxyl group contains 1 H atom. This information was used to further analyze the properties of DPL-NO. 1 ¹H NMR analysis revealed that the four atoms in the 1,4-di(bromomethyl)benzene ring appeared at chemical shifts of 7.09 and 6.92, exhibiting two doublets (d) with integrals of 2 for each. The four hydrogen atoms attached to the benzyl C atom appeared at chemical shifts of 4.84 and 4.41, exhibiting two singlets (s) with integrals of 2 for each. No hydrogen atoms in the amino group showed peaks. The three H atoms in the aniline molecule appeared at chemical shifts of 8.27, 7.91, and 7.03, exhibiting two doublets (d) and one singlet (s) with integrals of 1 for each. The ten H atoms in the fluorescein benzene ring appeared at chemical shifts of 7.74–7.68, 7.24, 6.79, and 6.71–6.67, exhibiting two multiplets and one doublet with integrals of 2, 1, 2, and 5, respectively.

[0056] (2) UV-Vis spectral characterization of fluorescein fused with nitric oxide donor (DPL-NO) The degradation of DPL-NO (50 μM) under 500 nm green light excitation in a mixed solution of phosphate buffer (pH 7.40, 0.10 M) and dimethyl sulfoxide (DMSO) (phosphate buffer: DMSO = 95:5, v / v) was monitored by a UV-Vis spectrophotometer at 25 °C.

[0057] Figure 7 The corresponding spectral changes were shown. The results indicate that the characteristic absorption peak of DPL-NO at 387 nm decreased significantly and continuously after irradiation, while the characteristic absorption peaks at 475 nm and 500 nm increased significantly and continuously after irradiation. The rate constant k was obtained using a first-order equation based on the change in absorbance over time during photolysis at 500 nm. obs = (7.90 ± 0.30) × 10 -2 min -1 (t) 1 / 2 =8.77min) The fitting graph of the first-order reaction rate equation of absorbance versus time at a wavelength of 500nm is shown below. Figure 7 As shown in the illustration.

[0058] (3) Standard curve of fluorescein fused with nitric oxide donor (DPL-NO) DPL-NO was analyzed using a UV-Vis spectrophotometer, and its standard curve was determined. In the experiment, DPL-NO solutions of different concentrations (50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.125 µM, and 1.5625 µM) were measured in a 5:95 mixture of DMSO and buffer (the buffer was phosphate buffer solution). The correlation between DPL-NO concentration and absorbance at 500 nm was obtained. Based on these data, a standard curve for DPL-NO was successfully plotted (e.g., ...). Figure 8 As shown in the figure, the linear fitting graph of absorbance versus concentration at a wavelength of 500 nm is as follows. Figure 8 As shown in the illustration, the formula is: Y = 0.01636X + 0.00328, R 2 =0.99958.

[0059] (4) Fluorescence spectral characterization of fluorescein fused with nitric oxide donor (DPL-NO) The decomposition of DPL-NO (50.0 µM) in a mixed solution of phosphate buffer (pH 7.40, 0.10 mol / L) and dimethyl sulfoxide (phosphate buffer: dimethyl sulfoxide = 95:5, v / v) at 25°C was monitored using fluorescence spectroscopy.

[0060] Initially, the fluorescence intensity of DPL-NO was extremely low under 405 nm light excitation. Subsequently, when irradiated with 500 nm green light, the fluorescence emission gradually increased in the wavelength range of 520-550 nm as photolysis continued. Figure 9 As shown, there is a peak at a wavelength of 528 nm. After complete decomposition of DPL-NO, the fluorescence intensity at 528 nm is 33 times stronger than that of DPL-NO. Therefore, the fluorescence signal generated by the photolysis of DPL-NO can be used as a probe for the quantification and localization of NO. The site and dose of NO release can be precisely controlled by illumination.

[0061] (5) Detection of reactive oxygen species and NO release by fluorescein fused with nitric oxide donor (DPL-NO) Singlet oxygen was detected using 9,10-anthratridimyl-bis(methylene)dicarboxylic acid (ABDA). 1 To generate O2, DPL-NO and ABDA were prepared into 5µM and 50µM aqueous solutions and mixed in a cuvette (DMSO / PBS = 1 / 9). The absorption spectrum was recorded every 1 minute under 500nm green light irradiation. ABDA was used as a control group under its own illumination.

[0062] Using ABDA alone as a control, its absorbance changes negligibly over time under the same illumination conditions (e.g., Figure 10 As shown in a), this indicates that no significant photochemical reaction occurred in the system. However, in the presence of DPL-NO, the characteristic absorption peak of ABDA in the 360-400 nm band continuously decayed with irradiation time (as shown in a figure). Figure 10 As shown in b), this indicates that singlet oxygen was generated in the system. 1 O2).

[0063] The NO release from DPL-NO under both light and dark conditions was monitored in real time using a NOA (ThermoScientific, USA) analyzer.

[0064] Test results are as follows Figure 11 As shown, under illumination, the NO concentration in the system gradually increased from the baseline value of 0.00 ppb / mg to 8.90 ppb / mg; when the light source was turned off, the NO signal quickly dropped back to the baseline level. Further comparative analysis of the data from eight sets of cyclic illumination experiments showed that DPL-NO can stably and repeatedly release NO under photoexcitation conditions, and the amount of NO released in each cycle was basically consistent.

[0065] (6) Femtosecond transient absorption spectroscopy characterization of fluorescein fused with nitric oxide donor (DPL-NO) The photolysis kinetics of DPL-NO was systematically studied using a femtosecond transient absorption spectrometer in a mixed solution of phosphate buffer (0.10 M, pH 7.40) and dimethyl sulfoxide (phosphate buffer: dimethyl sulfoxide = 95:5, v / v) with an excitation wavelength of 450 nm.

[0066] like Figure 12 As shown in Figures a, b, and c, the evolution of its transient absorption spectrum over time can be divided into three typical time windows for analysis: early (0.71–2.45 ps), middle (2.45–22.3 ps), and late (655.8 ps–4.3 ns). In the early phase, the transient absorption signal at 500 nm rapidly decreases, while the absorption near 475 nm slightly increases. This phenomenon can be attributed to the excited-state molecule transitioning from a highly excited singlet state (S0). n The rapid internal transition process from the lowest excited singlet state (S1) to the lowest excited singlet state. After entering the mid-stage, the transient absorption signals at 470 nm and 580 nm decay significantly over time, and the corresponding kinetic fitting yields a characteristic half-life of 60.33 ps (e.g., ...). Figure 12 (as shown in d).

[0067] (7) Cell imaging of fluorescein fused with nitric oxide donor (DPL-NO) To verify the real-time reporting capability of DPL-NO in the photolysis process during bioimaging, 4T1 cells were treated with DPL-NO (50 µM) and then analyzed using confocal microscopy. Figure 13 (a) Using a 500nm light source (~100mW / cm²) 2 Irradiation was performed in photobleaching mode to create a 25µm demarcation zone. Figure 13 (b) The PBS control group showed no fluorescence signal, while DPL-NO-treated cells showed only weak initial emission. After 10 irradiation cycles (15 seconds each, totaling 150 seconds), significant fluorescence enhancement was observed only in DPL-NO-treated cells. Parallel experiments using NO-sensitive fluorescent probes (excitation / emission wavelengths: 488 / 610 nm) confirmed intracellular NO production (… Figure 13 (c). After 18 radiation pulses, enhanced green fluorescence indicated that DPL-NO successfully decomposed and released nitric oxide, verifying the system's dual functionality in space-controlled nitric oxide delivery and synchronous optical reporting.

[0068] (8) Cytotoxicity of luciferin fused with nitric oxide donor (DPL-NO) Nitric oxide exhibits a concentration-dependent dual role in cellular processes: at nanomolar concentrations, it promotes cell proliferation and immune regulation, while at micromolar concentrations, it induces apoptosis through oxidative damage to biomolecules by reacting with superoxide anions to form peroxynitrite. To quantify the photocytotoxic effect, this study assessed the viability of DPL-NO-treated B16, HepG2, 4T1, and HeLa cells (e.g., ...). Figure 14 (As shown in a, b, c, and d). All cell lines maintained high viability under dark conditions but exhibited significant light-dependent cytotoxicity under light conditions. Cell viability decreased continuously with increasing DPL-NO concentration, dropping to approximately 20% at a concentration of 80 μM under light, confirming that this donor system possesses dose-responsive and spatiotemporally controllable NO release capabilities.

[0069] The fluorescein-fused nitric oxide donor (DPL-NO) of Example 1 was used to prepare the OHA-CMCS-DPL-NO injectable hydrogel, and the OHA-CMCS injectable hydrogel was prepared as a blank control.

[0070] The preparation method of 5% OHA-CMCS injectable hydrogel is as follows: Weigh 125 mg of OHA and 125 mg of CMCS into two separate sample vials, and dissolve them in 2.5 ml of deionized water in each vial. Mix the two vials thoroughly in a syringe and then add the mixture into the sample vials.

[0071] The preparation method of 6% OHA-CMCS injectable hydrogel is as follows: Weigh 150 mg of OHA and 150 mg of CMCS into two separate sample vials, and dissolve each in 2.5 ml of deionized water. Mix the two vials thoroughly in a syringe and then add the mixture into the sample vials.

[0072] The preparation method of 5% OHA-CMCS-DPL-NO injectable hydrogel is as follows: Weigh 150 mg of OHA and 150 mg of CMCS into two separate sample vials, and dissolve them in 2.5 ml of deionized water in each. Mix the two vials thoroughly in a syringe and then add the mixture to the sample vials. Next, weigh 50 mg of DPL-NO and add it to the sample vials.

[0073] The preparation method of 6%OHA-CMCS-DPL-NO injectable hydrogel is as follows: Weigh 150 mg of OHA and 150 mg of CMCS into two separate sample vials, and dissolve them in 2.5 ml of deionized water in each. Mix the two vials thoroughly in a syringe and then add the mixture to the sample vials. Next, weigh 60 mg of DPL-NO and add it to the sample vials.

[0074] The prepared OHA-CMCS-DPL-NO injectable hydrogel and OHA-CMCS injectable hydrogel were subjected to the following detection and characterization: (1) Detection of the antibacterial properties of OHA-CMCS-DPL-NO injectable hydrogel To evaluate the antibacterial properties of the OHA-CMCS-DPL-NO injectable hydrogel, this invention used 5% OHA-CMCS-DPL-NO injectable hydrogel and 6% OHA-CMCS injectable hydrogel, respectively, to investigate the antibacterial activity against *Escherichia coli* (e.g., *Escherichia coli*) under light and dark conditions. Figure 15 (as shown in a) and Staphylococcus aureus (such as Figure 15 The bacteria were treated as shown in b), and their antibacterial effect was quantitatively analyzed by plate colony counting.

[0075] Experimental results showed that under 500 nm green light irradiation, the hydrogel system could effectively trigger the release of NO from DPL-NO and significantly inhibit bacterial growth. Compared with the dark control group, the survival rate of E. coli after light treatment decreased by 2.1 orders of magnitude, and the inhibition rate reached 97.15%. Figure 16 (a); Staphylococcus aureus survival rate decreased by 2.0 orders of magnitude, corresponding to an inhibition rate of 98.96% ( Figure 16 (b).

[0076] (2) Detection of the rheological properties of OHA-CMCS-DPL-NO injectable hydrogel The gelation process of two injectable hydrogels was monitored in real time using rheological analysis.

[0077] like Figure 17 As shown in Figure a, the 6% OHA-CMCS hydrogel solution rapidly underwent a sol-gel transition after mixing, with the storage modulus (G′) increasing rapidly over time. Its growth rate was significantly higher than that of the loss modulus (G″), and it exceeded G″ within approximately 1 minute, marking a shift from viscosity-dominated to elastic-dominated systems, indicating the formation of a three-dimensional cross-linked network. The introduction of DPL-NO further accelerated this process. Figure 17 (b) The gelation time of the 6% OHA-CMCS injectable hydrogel is approximately 25 s, while that of the DPL-NO-loaded hydrogel is shortened to approximately 20 s.

[0078] Figure 18The strain-dependent rheological behavior of the hydrogel was demonstrated. Taking the 5% OHA-CMCS system as an example, as the shear strain increases, G′ gradually decreases while G″ gradually increases. The intersection of these two points corresponds to a shift in the network structure from elastic dominance to viscous dominance, indicating that the three-dimensional network structure inside the hydrogel is disrupted. Increasing the polymer concentration can significantly enhance the structural stability of the material under large deformation. The system maintains similar mechanical stability after loading DPL-NO, indicating that the introduction of functional molecules does not significantly weaken the network integrity. Frequency scan results ( Figure 19 The results showed that the 5% OHA-CMCS hydrogel exhibited a high and stable G′ value in the low-frequency region, indicating good mechanical strength under slow deformation conditions; when the frequency exceeded the critical value, the network structure began to relax and disintegrate. With the increase of OHA and CMCS concentrations, the storage modulus of the system increased significantly, and G′ reached 1189 Pa when the concentrations of OHA and CMCS were both 6%, demonstrating higher crosslinking density and stronger network structure strength.

[0079] (3) Micromorphological characterization of OHA-CMCS-DPL-NO injectable hydrogel The microstructure of the two hydrogels was characterized by scanning electron microscopy (SEM), and the results are as follows: Figure 20 As shown. Blank 6% OHA-CMCS injectable hydrogel ( Figure 20 a) The surface morphology is relatively rough, with numerous irregular protrusions and dense areas observed. In contrast, the 6% OHA-CMCS-DPL-NO injectable hydrogel doped with DPL-NO ( Figure 20 (b) While maintaining a continuous network structure overall, a relatively loose structure appears in local areas, with further increases in surface roughness and more irregular protrusions. This morphological change may stem from the interference of DPL-NO molecules into the network, affecting the original polymer chain arrangement and hydrogen bonding, leading to a decrease in local crosslinking density or microstructure reconstruction. The appearance of additional surface protrusions also indicates, to some extent, that DPL-NO has been successfully loaded into the hydrogel matrix.

[0080] Further elemental mapping analysis was performed using energy-dispersive X-ray spectroscopy (EDS). Figure 20 The results (c–f) showed a significant fluorine signal in the modified hydrogel, while the element was not observed in the blank hydrogel. The uniform distribution of fluorine, due to the characteristic fluorine-containing groups in the DPL-NO molecular structure, directly proves its successful embedding in the hydrogel network structure.

[0081] (4) Fourier transform infrared spectroscopy characterization of OHA-CMCS-DPL-NO injectable hydrogel In this embodiment, the preparation method of oxidized hyaluronic acid is as follows: 5g of hyaluronic acid is dissolved in 100ml of distilled water. After the hyaluronic acid is completely dissolved, 2.5g of NaIO4 is added. The mixture is then stirred in a dark environment at 25°C for 14 hours. Excess ethylene glycol is then added to quench any unreacted NaIO4. The reaction is stirred for another hour, and then the solution is dialyzed with distilled water for 3 days. The water is changed every 2 hours on the first day and every 12 hours on the following two days.

[0082] Figure 21 The FT-IR spectral characteristics of natural hyaluronic acid (HA), oxidized hyaluronic acid (OHA), carboxymethyl chitosan (CMCS), 6% OHA-CMCS injectable hydrogel, and 6% OHA-CMCS-DPL-NO injectable hydrogel are shown. Natural hyaluronic acid (HA) exhibits FT-IR spectra at approximately 3410 cm⁻¹. -1 A broad and strong absorption band is observed at 1605cm. -1 The characteristic peak at 1730 cm⁻¹ corresponds to the asymmetric stretching vibration of the carboxylate group (-COO-). After oxidation with sodium periodate, the infrared spectrum of OHA is at 1730 cm⁻¹. -1 A new characteristic absorption peak appears at 3415 cm⁻¹, which is attributed to the stretching vibration of the aldehyde C=O group. The CMCS sample shows a peak at 3415 cm⁻¹. -1 The peak at 1600 cm⁻¹ exhibits the characteristic peak of OH / NH stretching vibration. -1 The location is the amide I band (C=O stretching vibration), 1410 cm. -1 The corresponding -COO- symmetric stretching vibration indicates that the molecule contains both amide groups and carboxymethyl substituents.

[0083] In 6% OHA-CMCS injectable hydrogel, 1730 cm -1 The characteristic peaks of the aldehyde group basically disappeared, while the peaks at 1645 cm⁻¹ disappeared. -1 A new absorption peak appears at 1510 cm⁻¹, which can be attributed to the stretching vibration of the imine bond (C=N). This indicates that the aldehyde group in the OHA molecule undergoes a Schiff base reaction with the amino group in the CMCS molecule, forming a dynamic covalent cross-linked structure, thereby constructing a stable three-dimensional network structure. A new absorption peak appears at 1510 cm⁻¹ in the FT-IR spectrum of the 6% OHA-CMCS-DPL-NO injectable hydrogel. -1 With 1345cm -1 Two new characteristic absorption peaks correspond to the asymmetric stretching vibration and symmetric stretching vibration of the -NO2 group in the nitroaniline unit, respectively, indicating that the DPL-NO molecule has been successfully introduced into the hydrogel system.

[0084] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a fluorescein-fused nitric oxide donor, characterized in that, Includes the following steps: Step S1, Synthesis of NO donor intermediate: Under an inert gas atmosphere, N,N-dimethylformamide was added to a mixture of an aromatic compound containing a para-active halogenated side chain and potassium carbonate. After stirring, 3-trifluoromethyl-4-nitroaniline was added and the reaction was heated. After the reaction was completed, the mixture was cooled to room temperature, and the reaction was quenched by adding saturated brine. Ethyl acetate was added and mixed, and the mixture was washed with saturated brine. The organic phase was dried and concentrated under vacuum to obtain the first crude product. The first crude product was purified by column chromatography to obtain the NO donor intermediate. Step S2, Synthesis of fluorescein-fused nitric oxide donor: Fluorescein, potassium carbonate, and dimethyl sulfoxide are mixed and stirred to obtain a first mixture; the NO donor intermediate is dissolved in an organic solvent to obtain a second mixture; the first mixture is placed in an ice-water bath, the second mixture is added, and the mixture is heated to react. After the reaction is completed, the mixture is cooled to room temperature, and the reaction is quenched by adding saturated brine. Ethyl acetate is added, and the mixture is washed with saturated brine. The organic phase is dried and concentrated under vacuum to obtain a second crude product. The second crude product is purified by column chromatography to obtain the fluorescein-fused nitric oxide donor.

2. The method for preparing a fluorescein-fused nitric oxide donor according to claim 1, characterized in that, The aromatic compound containing a para-active halogenated side chain is selected from any one of 1,4-di(bromomethyl)benzene, 1,4-p-dichlorobenzene, or 1,4-bis(iodomethyl)benzene.

3. The method for preparing a fluorescein-fused nitric oxide donor according to claim 2, characterized in that, The molar ratio of the aromatic compound containing the para-active halogenated side chain to the 3-trifluoromethyl-4-nitroaniline is 1.2:

1.

4. The method for preparing a fluorescein-fused nitric oxide donor according to claim 1, characterized in that, In step S1, the N,N-dimethylformamide is measured under an inert gas atmosphere; The inert gas is nitrogen; The stirring conditions are: stirring at room temperature for 0.5 hours; the heating reaction temperature is 80°C and the time is 12 hours.

5. The method for preparing a fluorescein-fused nitric oxide donor according to claim 1, characterized in that, In step S2, the molar ratio of the fluorescein to the NO donor intermediate is 1:

1.

6. The method for preparing a fluorescein-fused nitric oxide donor according to claim 1, characterized in that, In step S2, fluorescein, potassium carbonate, and dimethyl sulfoxide are mixed and stirred at room temperature for 0.5 h to obtain a first mixture; the NO donor intermediate is dissolved in dimethyl sulfoxide to obtain a second mixture; the first mixture is placed in an ice-water bath at 0°C and the second mixture is added to carry out a heating reaction at 80°C for 10 h.

7. The method for preparing a fluorescein-fused nitric oxide donor according to claim 1, characterized in that, In step S1, ethyl acetate is added and mixed, then washed three times with saturated saline solution; in step S2, ethyl acetate is added and mixed, then washed three times with saturated saline solution. In step S1, anhydrous sodium sulfate is used for drying; in step S2, anhydrous sodium sulfate is used for drying.

8. The method for preparing a fluorescein-fused nitric oxide donor according to claim 1, characterized in that, In step S1, the column chromatography uses ethyl acetate and petroleum ether in a volume ratio of 5:1 as the eluent. In step S2, the column chromatography uses dichloromethane and petroleum ether as eluents, and performs gradient elution at volume ratios of 10:1 and 5:

1.

9. A fluorescein-fused nitric oxide donor, characterized in that, It was prepared using the preparation method of fluorescein fused with nitric oxide donor as described in any one of claims 1-8.

10. The application of the fluorescein-fused nitric oxide donor as described in claim 9 in wound repair, infection control, and antimicrobial biomedical materials.

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