A light-controlled nitric oxide donor with fluorescent self-reporting function and preparation method and application thereof

Abstract

The application discloses a kind of photonic nitric oxide donor with fluorescent self-reporting function and its preparation method and application, belong to the field of biomedical materials.The nitric oxide donor has the structure as shown in formula 1.The donor itself weak fluorescence, but in the presence of reduced glutathione (GSH), after irradiation by ultraviolet light (such as 405 nm), it can efficiently and controllably release nitric oxide (NO), and it is accompanied by strong fluorescence enhancement signal (fluorescence intensity can be increased by about 100 times), so as to realize the real-time, high-resolution fluorescence monitoring of NO release process and site.The donor has good light stability and biocompatibility.Experiments show that it can realize the light-controlled release and imaging of NO in living cells, and has application potential in the field of biomedical research and related drug development.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and medicinal chemistry, specifically to a novel nitric oxide donor, particularly a compound that can release nitric oxide in the presence of reduced glutathione via light control accompanied by fluorescence activation (self-reporting), its preparation method, and its application in bioimaging and medical research. Background Technology

[0002] Nitric oxide is synthesized endogenously by isomers of nitric oxide synthase and participates in a wide variety of physiological functions, including vasodilation, neurotransmission, and immune regulation. Its biological activity is highly environment-dependent, primarily depending on local concentration and exposure time. In the low concentration range (approximately picomolar to nanomolar levels), nitric oxide mainly plays a regulatory and cytoprotective role, such as promoting vasodilation and tissue repair. Although nitric oxide has therapeutic potential, its inherent physicochemical properties, including its gaseous form, high reactivity, and short half-life, pose significant challenges to its in vivo application. Therefore, exogenous nitric oxide donors are frequently used as a source of nitric oxide in numerous biomedical studies.

[0003] However, existing light-controlled NO donors still face some challenges. For example, the release process of many donors is difficult to monitor in real time and intuitively, making it impossible to accurately obtain information on release sites, flux, and kinetics. Furthermore, the microenvironment of diseased tissues such as tumors often exhibits high reducing activity, one characteristic of which is a significantly elevated level of reduced glutathione (GSH). Developing donors that can specifically respond to such microenvironments and achieve controlled NO release with real-time feedback has significant scientific and practical value.

[0004] This invention aims to overcome the above-mentioned shortcomings and provide a novel photocontrolled NO donor. This donor not only enables photocontrolled NO release in the presence of GSH, but also generates significant fluorescence signal enhancement during the release process, i.e., it possesses a "self-reporting" function, thus providing a tool with high spatiotemporal resolution for the biological research of NO. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a novel light-controlled nitric oxide donor that can respond to light in a reducing microenvironment (such as high GSH) and simultaneously realize the controllable release of nitric oxide and the fluorescence visualization monitoring of the release process.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: First, this invention provides a light-controlled nitric oxide donor with fluorescence self-reporting function, which is a compound having the structure shown in Formula 1 or a salt thereof. The donor molecule is designed to bind a NO-releasing unit to a rhodamine derivative fluorophore via a specific linking group. In the absence of GSH or without illumination, the donor fluorescence is quenched; in the presence of GSH, illumination triggers the breakage of the linking group, releasing NO on one hand and restoring strong fluorescence emission from the fluorophore on the other.

[0007] Secondly, this invention provides a method for preparing the aforementioned photocontrolled nitric oxide donor. This method uses commercially available rhodamine 6G as a starting material, and proceeds sequentially through base-catalyzed ring-opening, Friedel-Crafts acylation, and nitrosation. The steps are simple, the yield is good, and the target product can be prepared efficiently.

[0008] Furthermore, this invention provides applications of the aforementioned photocontrolled nitric oxide donor. This donor can be used to prepare reagents for photocontrolled NO release, and is particularly suitable for studying the spatiotemporal controlled release and real-time imaging of NO in reducing biological microenvironments (such as tumors). In addition, this donor can also serve as a tool reagent for studying NO-related physiological and pathological processes or for the development of related drugs.

[0009] Compared with existing technologies, this invention has the following advantages: It achieves, for the first time, the synchronized photocontrolled release and fluorescence activation of NO in the presence of GSH, enabling real-time and intuitive monitoring of the kinetics and localization of NO release. The NO release efficiency of the donor depends on GSH, making it potentially targeted to disease microenvironments with high GSH levels. The donor exhibits a large Stokes shift, good photostability and pH stability, low background fluorescence, and a high signal-to-noise ratio. Cell imaging experiments show that the donor has low toxicity and can be successfully used for photocontrolled NO release and imaging at the in vivo level. The preparation method uses readily available raw materials, has a clear route, and is easy to operate and scale up. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0011] Figure 1 This is the hydrogen spectrum of donor formula 1.

[0012] Figure 2 This is the carbon spectrum of donor formula 1.

[0013] Figure 3 This is a high-resolution mass spectrum of donor formula 1.

[0014] Figure 4 This is a diagram illustrating the mechanism of nitric oxide release by donor type 1 under light irradiation.

[0015] Figure 5 This is the EPR diagram of nitric oxide release from donor type 1 under light irradiation.

[0016] Figure 6 This is the fluorescence emission spectrum of nitric oxide released by the photolysis of donor type 1.

[0017] Figure 7 This is the ultraviolet absorption spectrum of nitric oxide released by the photodecomposition of donor 1.

[0018] Figure 8 This is a graph showing the change in fluorescence intensity over time when the light donor solution (Formula 1) is exposed to interfering substances.

[0019] Figure 9 This is a photosensitivity test diagram of donor type 1.

[0020] Figure 10 This is a photostogram showing the photostability of donor 1 and fluorophore PIK-O after irradiation with a 500 nm xenon lamp.

[0021] Figure 11 This is a diagram showing the light-controlled release of nitric oxide in A549 cells by donor formula 1. Detailed Implementation

[0022] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the following embodiments.

[0023] Example 1: This example provides a novel method for preparing a light-controlled nitric oxide donor. The specific steps are as follows: (1) Synthesis of intermediate PIK: In a 250 mL round-bottom flask, add rhodamine 6G (4.4386 g, 10 mmol), sodium hydroxide (20 g, 0.5 mol), 30 mL of anhydrous ethanol, and 70 mL of distilled water. Heat the mixture to 180 °C and reflux for 72 hours. After the reaction is complete, cool to room temperature and adjust the pH to 2-3 with concentrated hydrochloric acid. Filter and wash the filter cake with a small amount of methanol. Combine the filtrate and washings and extract with dichloromethane (3 × 50 mL). Combine the organic phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent, yielding pink solid PIK.

[0024]

[0025] (2) Synthesis of intermediate PIK-O: Acetophenone (0.23 mL, 2 mmol) and concentrated sulfuric acid (2 mL) were added to a 25 mL round-bottom flask under ice-water bath cooling and stirred for 10 minutes. Then, PIK (0.3 g, about 1 mmol) obtained in the above step and an additional 3 mL of concentrated sulfuric acid were added. The ice bath was removed, and the mixture was heated to 100 °C and stirred for 3 hours. After the reaction was completed, the mixture was cooled to room temperature and the reaction solution was slowly poured into 10 mL of ice water. Perchloric acid was added dropwise while stirring until a large amount of precipitate was formed. The precipitate was filtered and washed with distilled water to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10:1, v / v), and the red band was collected and concentrated under reduced pressure to obtain red solid PIK-O.

[0026]

[0027] (3) Synthesis of the target donor (Formula 1): Under ice-water bath cooling and stirring, PIK-O (0.1 g, 0.26 mmol) and 5 mL of glacial acetic acid were added to a 25 mL round-bottom flask. A solution of sodium nitrite (0.1 g, 1.5 mmol) dissolved in 2 mL of water was slowly added dropwise using a constant-pressure dropping funnel. After the addition was complete, the reaction was stirred at 0 °C for 1.5 hours. After the reaction was completed, the reaction solution was poured into 20 mL of water and extracted with dichloromethane (3 × 20 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v), and the target component was collected and concentrated under reduced pressure to obtain a white solid, namely the target donor Formula 1, with a yield of 50%.

[0028]

[0029] Example 2: Photocontrolled NO release and fluorescence self-reporting performance of the donor This embodiment verifies the performance of the donor in the presence of GSH, which releases NO upon illumination and is accompanied by fluorescence activation.

[0030] Prepare a neutral phosphate buffer solution (PBS, pH 7.4) containing 10 μM donor formula 1 and 1 mM GSH. Irradiate the solution with a 405 nm UV lamp at 25 °C and measure its fluorescence emission spectrum (excitation wavelength λex = 500 nm) and UV-Vis absorption spectrum at regular intervals.

[0031] Figure 4 This is a diagram illustrating the mechanism of nitric oxide release from donor type 1 under light irradiation. Figure 5This is the EPR diagram of nitric oxide release from donor 1 under illumination. With increasing illumination time, the fluorescence emission intensity of the solution at approximately 600 nm gradually increases, reaching equilibrium after about 60 minutes, with the fluorescence intensity increasing approximately 100-fold. Figure 6 Meanwhile, the absorbance of the ultraviolet absorption spectrum at approximately 495 nm also gradually increases. Figure 7 The changes in fluorescence enhancement and absorption spectrum are consistent with the process of the donor molecule releasing NO under illumination and the fluorophore structure recovering, demonstrating its "self-report" function.

[0032] Example 3: Donor selectivity and interference resistance This embodiment examines the selectivity of the donor to the NO release triggering condition.

[0033] Under identical conditions (10 μM donor, PBS, pH 7.4), high concentrations of various potential interfering substances, including amino acids (Glu, Gly, Ser, Asp, Trp, Pro, Tyr, Cys, 500 μM each), other reducing agents (ascorbic acid CA, β-nicotinamide adenine dinucleotide NADH, 1 mM each), and physiological concentrations of GSH (1 mM), were added to the system. The system was then irradiated with a 405 nm light source, and the change in fluorescence intensity at 600 nm over time was monitored.

[0034] The results are as follows Figure 8 As shown, only in the presence of GSH can illumination induce a rapid and significant increase in fluorescence intensity. In the presence of other amino acids or reducing agents, the rate and magnitude of fluorescence enhancement are much lower than in the GSH group, indicating that this donor has good selectivity for GSH, its NO release process is specifically promoted by GSH, and it has strong anti-interference ability.

[0035] Example 4: Photosensitivity and Reversibility of the Donor This embodiment tests the donor's ability to respond quickly to external light sources.

[0036] The donor solution (10 μM in PBS) was placed under a 365 nm light source for 5 minutes, then the light source was turned off, and the solution was placed in the dark for 5 minutes. This constituted one cycle. The fluorescence intensity at 600 nm was recorded at the beginning and end of each illumination cycle.

[0037] The results are as follows Figure 9 As shown, the fluorescence intensity increased rapidly after each illumination and remained stable without a significant decrease after the light source was turned off. This indicates that the donor is highly responsive to light stimulation, and that the light-triggered NO release / fluorescence activation process is irreversible, consistent with design expectations.

[0038] Example 5: Photostability of the Donor This embodiment evaluates the stability of the donor and its fluorescent nucleus under long-term illumination.

[0039] PBS solutions containing 10 μM donor formula 1 and an equimolar concentration of fluorophore PIK-O were prepared. The solutions were continuously irradiated with a xenon lamp at a wavelength of 500 nm, and the fluorescence emission spectra were measured every 5 minutes.

[0040] The results are as follows Figure 10 As shown, under continuous irradiation for up to 60 minutes, the fluorescence intensity of both donor formula 1 and PIK-O did not decrease significantly, indicating that both have good photostability and are suitable for experiments requiring long-term illumination or observation.

[0041] Example 6: Photocontrolled release and imaging of NO in living cells This example demonstrates the application of donors in biological systems.

[0042] A549 cells were seeded in confocal culture dishes and cultured at 37°C and 5% CO2 until adherent. Culture medium containing 10 μM donor formula 1 was added and incubated at 37°C for 10 minutes. Cells were gently washed three times with PBS to remove any donors that had not entered the cells. Cells were divided into two groups: a control group (no illumination) and an experimental group. The experimental group was irradiated locally or globally with a 405 nm laser for different durations (0, 8, 15, 20 minutes), followed immediately by fluorescence confocal microscopy (excitation wavelength: 488 nm, emission / collection channel: 570-620 nm).

[0043] The results are as follows Figure 11 As shown, the control group cells, which were not exposed to light, showed only very weak background fluorescence. After illumination, the intracellular fluorescence signal in the experimental group cells significantly increased over time, and the fluorescence intensity was positively correlated with the illumination time. This indicates that the donor successfully entered the cells, and in the presence of intracellular GSH, photocontrolled release of NO and real-time fluorescence imaging were achieved.

[0044] in conclusion This invention successfully designed and synthesized a novel photocontrolled nitric oxide donor with fluorescence self-report function. This donor innovatively achieves synchronization of photocontrolled NO release and fluorescence self-report function under the triggering of a reducing microenvironment (GSH). Its preparation method is simple, the donor itself exhibits excellent performance, and its good biocompatibility and application effectiveness have been demonstrated in living cells. This donor provides a powerful tool for studying the spatiotemporal dynamics of NO in physiological and pathological processes, and has broad application prospects in basic biomedical research and related drug development.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A light-controlled nitric oxide donor with fluorescence self-reporting function, characterized in that, It is a compound having the structure shown in Formula 1: 。 2. A method for preparing the light-controlled nitric oxide donor as described in claim 1, characterized in that, Includes the following steps: (1) Synthesis of intermediate PIK: Rhodamine 6G and sodium hydroxide were dissolved in a mixed solvent of anhydrous ethanol and water and reacted under reflux conditions. After the reaction was completed, the reaction solution was cooled, the pH was adjusted to acidic with acid, filtered, and the filtrate was extracted with an organic solvent. The organic phases were combined, dried, and concentrated to obtain the solid product PIK. ； (2) Synthesis of intermediate PIK-O: Acetophenone was dissolved in concentrated sulfuric acid under ice bath cooling and stirring, and then PIK obtained in step (1) was added. The temperature was raised and the reaction was carried out under heating conditions. After the reaction was completed, the reaction solution was poured into ice water and perchloric acid was added until precipitation was complete. The solid was collected by filtration and purified by column chromatography to obtain the red solid product PIK-O. ； (3) Synthesis of the target donor (Formula 1): The PIK-O obtained in step (2) was dissolved in glacial acetic acid. Under ice bath cooling and stirring, an aqueous solution of sodium nitrite was slowly added dropwise to maintain the low temperature reaction. After the reaction was completed, the reaction mixture was extracted with dichloromethane, the organic phases were combined, dried and concentrated to obtain the crude product, and then purified by column chromatography to obtain the compound shown in Formula 1. 。 3. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of Rhodamine 6G to sodium hydroxide is 1: (40-60); the temperature of the reflux reaction is 170-190℃, and the reaction time is 65-80 hours; the acid used to adjust the pH is hydrochloric acid or sulfuric acid; the organic solvent used for extraction is dichloromethane or ethyl acetate. In step (2), the molar ratio of acetophenone to PIK is (1.5-2.5):1; the heating reaction temperature is 95-105℃, and the reaction time is 2-4 hours; the eluent used for column chromatography purification is a mixed solvent of dichloromethane and methanol, with a volume ratio of (8:1)-(12:1); In step (3), the molar ratio of PIK-O to sodium nitrite is 1: (5-7); the reaction temperature is 0-5℃ and the reaction time is 1-2 hours; the eluent used for column chromatography purification is a mixed solvent of dichloromethane and petroleum ether with a volume ratio of 1:1 to 1:

2.

4. The use of the photocontrolled nitric oxide donor as described in claim 1 in the preparation of a reagent for photocontrolled release of nitric oxide.

5. The application according to claim 4, characterized in that, The photocontrolled release is carried out in the presence of reduced glutathione.

6. The use of the light-controlled nitric oxide donor as described in claim 1 in the preparation of tool reagents or drugs for studying nitric oxide-related physiological and pathological processes.

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