A fluorescent probe for detecting peroxynitrite ions and its preparation method and application

Abstract

The present invention belongs to the technical field of fluorescent probes, and specifically relates to a fluorescent probe for detecting peroxynitrite ions, a preparation method thereof, and an application thereof. The fluorescent probe has the following general structural formula: wherein n is an integer between 1 and 4; R1, R2, and R3 are each independently selected from hydrogen, halogen, nitro, cyano, C 2‑6 The carboxylate group, C 1‑6 Alkyl, C 1‑6 Alkoxy, 1-3 halogen-substituted C 1‑6 Alkyl and C substituted by 1-3 halogen 1‑6 The fluorescent probe provided by the present invention can be used for the detection of peroxynitrite ions, and has the advantages of a wide linear range, fast response speed, high sensitivity, good selectivity, etc., and has good biocompatibility. It can be used to detect peroxynitrite ions in cells and organisms, and has high practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a fluorescent probe for detecting peroxynitrite ions, and a preparation method and application thereof. Background Art

[0002] Peroxynitrite (ONOO−) is an endogenous reactive oxygen / nitrogen species (ROS / RNS) in cells. It is a highly reactive product generated in situ by the reaction of nitric oxide (NO) and superoxide (O2•−) in biological systems. It exhibits strong oxidizing and nucleophilic properties. Excessive concentrations of ONOO− in vivo can oxidize and damage the structures of macromolecules such as DNA, proteins, and esters, causing cell damage and ultimately leading to cell death. Furthermore, ONOO− can nitrate small molecules such as cysteine, tyrosine, and methionine, thereby affecting normal cell growth. As research on ONOO− continues to deepen, it has been found to be closely associated with physiological diseases such as inflammation, cancer, diabetes, cardiovascular disease, Alzheimer's disease, and neurodegenerative disorders. Therefore, monitoring peroxynitrite in vivo has attracted widespread attention. Elucidating the physiological functions and pathological mechanisms of peroxynitrite has important implications for the diagnosis of diseases caused by it.

[0003] In recent years, fluorescent probe detection has become an indispensable tool for studying bioactive molecules due to its high sensitivity, good selectivity, non-invasiveness, and excellent spatiotemporal resolution. Several fluorescent probes have been reported for the detection of ONOO− in cells and organisms. However, due to the extremely short half-life of ONOO− (approximately 10 ms) and the numerous reaction pathways involving this ion in biological systems, the development of probes capable of detecting ONOO− with the desired sensitivity, selectivity, and time response remains a major challenge.

[0004] Therefore, it is necessary to develop an efficient ONOO− fluorescence detection method. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned problems existing in the conventional technology and provide a fluorescent probe for detecting peroxynitrite ions and a preparation method and application thereof.

[0006] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0007] The present invention provides a fluorescent probe for detecting peroxynitrite ions, which has the following general structural formula:

[0008]

[0009] wherein n is an integer between 1 and 4; R1, R2 and R3 are each independently selected from hydrogen, halogen, nitro, cyano, C 2-6 The carboxylate group, C 1-6 Alkyl, C 1-6 Alkoxy, 1-3 halogen-substituted C 1-6 Alkyl and C substituted by 1-3 halogen 1-6 One of the alkoxy groups.

[0010] Further, n = 3; R1 = H; R2 = H; R3 = -CH2CH3.

[0011] The present invention also provides a method for preparing the above-mentioned fluorescent probe, characterized in that 7-nitro-N-(pyrrolidin-3-yl)benzo[c][1,2,5]oxadiazole-4-amine and substituted benzoyl chloride are dissolved in a dichloromethane solvent, and then a certain amount of triethylamine is added, and the reaction is stirred to obtain the target probe.

[0012] Furthermore, the molar ratio of 7-nitro-N-(pyrrolidin-3-yl)benzo[c][1,2,5]oxadiazol-4-amine to the substituted benzoyl chloride is 1:1.1.

[0013] Furthermore, the molar ratio of triethylamine to 7-nitro-N-(pyrrolidin-3-yl)benzo[c][1,2,5]oxadiazol-4-amine is 3:1.

[0014] Furthermore, when the target probe is generated by the reaction, the reaction is stirred at 25° C. for 3-6 h.

[0015] Furthermore, after the target probe is generated by the reaction, the product is separated and purified; the separation and purification steps include: distilling the solvent off the reaction solution under reduced pressure, and subjecting the dried solid to silica gel column chromatography, with the eluent being a mixture of dichloromethane and methanol in a volume ratio of 100 to 75:1.

[0016] The synthetic route of the present invention is:

[0017] .

[0018] The present invention also provides application of the fluorescent probe in detecting peroxynitrite ions in cells and organisms.

[0019] The beneficial effects of the present invention are:

[0020] 1. The molecular fluorescent probe of the present invention has excellent water solubility. A 1 mM probe stock solution prepared with DMSO (dimethyl sulfoxide) as the solvent is soluble in PBS buffer solution. The probe test solution (5 μM) prepared with a DMSO to PBS buffer solvent ratio of 1:199 can sensitively detect ONOO−, thus having excellent biocompatibility.

[0021] 2. The molecular fluorescent probe of the present invention can detect ONOO− at the nanomolar level, and therefore has high sensitivity.

[0022] 3. The molecular fluorescent probe of the present invention has good selectivity and specifically recognizes ONOO−. Other substances such as reactive oxygen, reactive nitrogen, reactive sulfur, metal ions and anions have no interfering effect on it.

[0023] 4. The molecular fluorescent probe of the present invention can detect ONOO− in cells and organisms and can be used for biological imaging.

[0024] 5. The preparation method of the molecular fluorescent probe of the present invention is simple, the raw materials are easily available, and the yield is high.

[0025] Of course, any product implementing the present invention does not necessarily need to achieve all of the above advantages at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 The fluorescence intensity response diagram of NBD-ONOO fluorescent probe to different concentrations of ONOO−;

[0028] Figure 2 is the time response diagram of NBD-ONOO fluorescent probe to ONOO−;

[0029] Figure 3 This is the fluorescence response diagram of the NBD-ONOO fluorescent probe to interferents such as reactive oxygen, reactive nitrogen, and reactive sulfur;

[0030] Figure 4 This is the fluorescence imaging of endogenous peroxynitrite ions in HepG2 cells by NBD-ONOO fluorescent probe;

[0031] Figure 5 This is the fluorescence imaging of endogenous peroxynitrite ions in zebrafish using the NBD-ONOO fluorescent probe. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0033] The success of numerous ONOO− fluorescent probes is primarily due to the cleavage of carbon-nitrogen, carbon-carbon, and carbon-oxygen double bonds within their structures by ONOO−, resulting in fluorescence changes. In this study, the inventors designed and synthesized a new fluorescent probe based on the novel reaction mode of ONOO−, which oxidizes and breaks the C-N bond, for in vitro and in vivo sensing and imaging of ONOO−. Photophysical characterization revealed that NBD-ONOO exhibits high sensitivity (LOD = 11 nM), high water solubility, excellent selectivity, and a rapid response (~3 s) toward ONOO−. Cell imaging results demonstrated that NBD-ONOO can visualize ONOO− in living cells, exhibiting good cell membrane permeability and low cytotoxicity. Furthermore, NBD-ONOO was used for in vivo imaging of endogenous ONOO− in a rotenone-induced PD model. These results clearly demonstrate that NBD-ONOO may serve as a potential biological tool for further understanding the unique physiological and pathological significance of ONOO−.

[0034] The relevant specific embodiments of the present invention are:

[0035] Figure 1 The fluorescence intensity response of the NBD-ONOO fluorescent probe to different concentrations of ONOO− was plotted. The probe concentration was 5 μM, the peroxynitrite ion concentration ranged from 0 to 38 μM, and the detection system consisted of a mixture of DMSO and PBS buffer (volume ratio 1:199). The excitation wavelength was 474 nm. Using the detection limit calculation formula 3σ / k, the detection limit of the NBD-ONOO fluorescent probe was calculated to be 11 nM. Note: F0 is the fluorescence intensity of the reaction system before the addition of analyte, and F is the fluorescence intensity of the reaction system after the addition of analyte.

[0036] Figure 2 This is the time response diagram of the NBD-ONOO fluorescent probe to ONOO−. The probe concentration is 5 μM, the peroxynitrite ion concentration is 25 μM, the detection system is a mixed solution of DMSO and PBS buffer (volume ratio of 1:199), and the excitation wavelength is 474 nm.

[0037] Figure 3This figure shows the fluorescence response of the NBD-ONOO fluorescent probe to interfering substances such as reactive oxygen species, reactive nitrogen species, and reactive sulfur species. The probe concentration was 5 μM, the peroxynitrite concentration was 25 μM, and the interfering ion concentration was 100 μM. The detection system consisted of a mixture of DMSO and PBS buffer (1:199 by volume), with an excitation wavelength of 474 nm.

[0038] Figure 4 Fluorescence imaging of endogenous peroxynitrite ions in HepG2 cells using the NBD-ONOO fluorescent probe. The probe concentration was 10 μM, the detection system was a mixed solution of DMSO and PBS buffer (volume ratio of 1:199), and the excitation wavelength of the confocal fluorescence microscope was 488 nm.

[0039] Figure 5 Fluorescence imaging of endogenous peroxynitrite ions in zebrafish using the NBD-ONOO fluorescent probe. The probe concentration was 10 μM, and the green filter of an inverted fluorescence microscope was used.

[0040] Example 1 Synthesis of NBD-ONOO fluorescent probe

[0041] First, 7-nitro-N-(pyrrolidin-3-yl)benzo[c][1,2,5]oxadiazol-4-amine (265.3 mg, 1 mmol), 4-ethylbenzoyl chloride (185.5 mg, 1.1 mmol), and triethylamine (303.6 mg, 3 mmol) were added to 30 mL of dichloromethane and stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was washed twice with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The target probe NBD-ONOO was purified by silica gel column chromatography (DCM:CH3OH = 100:1 to 75:1, v / v) to obtain the target probe NBD-ONOO (78%).

[0042] 1H NMR (400 MHz, DMSO- d 6) δ 9.60 (d, J = 25.1 Hz, 1H), 8.50 (dd, J =28.1, 8.9 Hz, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.26 (dd, J = 13.8, 7.7 Hz, 2H),6.48 (dd, J = 29.2, 9.0 Hz, 1H), 4.51 (s, 1H), 3.91 (dd, J= 13.0, 6.3 Hz, 1H),3.73 – 3.59 (m, 2H), 3.56 (d, J = 21.7 Hz, 1H), 2.62 (p, J = 7.8 Hz, 2H), 2.36 –2.05 (m, 2H), 1.16 (t, J = 8.5 Hz, 3H). HRMS (ESI) m / z Calcd for C 19 H 20 N5O4+ [M+H]+: 382.1510; found: 382.1503.

[0043] Example 2 Fluorescence intensity of NBD-ONOO probe solution under different volumes of ONOO−

[0044] Different volumes of ONOO− solution were pipetted into the probe solution (5 μM) using a 200 μL pipette. After 2 min of reaction, fluorescence detection was performed (λex = 474 nm, λem = 544 nm). The fluorescence intensity in each system was detected and a curve was drawn using the relative fluorescence intensity versus ONOO− concentration. Figure 1 As shown in the figure, the final detection limit of the probe for ONOO− was 11 nM.

[0045] Example 3 Response time of NBD-ONOO probe to ONOO−

[0046] After adding peroxynitrite ions (25 μM) to the NBD-ONOO (5 μM) probe solution, the fluorescence intensity was measured and recorded every 1 s, and a line graph was drawn based on the fluorescence intensity-time of the system, as shown in the figure. Figure 2 As shown in the figure, the response time of the NBD-ONOO probe to peroxynitrite ions is within 3 s.

[0047] Example 4 Selectivity of NBD-ONOO fluorescent probe

[0048] Other substances such as reactive oxygen, reactive nitrogen, and reactive sulfur produced in the body may interfere with the detection of ONOO−. Therefore, it is necessary to determine whether the synthesized ONOO− fluorescent probe has good selectivity in identifying ONOO−. Prepare 10mM ONOO−, Na+, Al3+, K+, Fe2+, Fe3+, Mg2+, Ca2+, GSH, Cys, Hcy, S2-, HS-, SO32-, HSO32-, CO32-, and NO2- stock solutions respectively. Take 10 μL of the analyte stock solution and add it to 2 mL of the probe solution (5 μM) test system. After reacting for 2 minutes, record the fluorescence intensity of different reaction systems. Figure 3 It can be seen that the probe solution only responds to ONOO−, and the fluorescence intensity decreases by nearly 78 times, while the fluorescence intensity of other reaction systems does not change significantly, which shows that the molecular fluorescent probe is highly selective for ONOO−.

[0049] Example 5 Fluorescence imaging of endogenous ONOO− in human liver cancer cells (HepG2) using NBD-ONOO fluorescent probe

[0050] HepG2 cells were cultured in a medium containing 10% (v / v) fetal bovine serum (FBS) and 100 μg / ml penicillin-streptomycin at 37°C and 5% CO2. (1) 5 μM probe was added to the culture medium, incubated for 30 min, washed three times with phosphate buffer, and then subjected to laser confocal fluorescence imaging. (2) HepG2 cells were pre-stimulated with 1 μg / mL lipopolysaccharide (LPS) for 2 h, then 5 μM probe was added and incubated for 30 min, washed three times with phosphate buffer, and finally subjected to laser confocal fluorescence imaging. (3) HepG2 cells were pre-stimulated with 1 μg / mL lipopolysaccharide (LPS) and 500 μM free radical scavenger 2,2,6,6-tetramethylpiperidinyl oxide (TEMPO) for 2 h, then 5 μM probe was added and incubated for 30 min, washed three times with phosphate buffer, and finally subjected to laser confocal fluorescence imaging. Figure 4 As shown, when HepG2 cells were not pre-stimulated with LPS and TEMPO, HepG2 cells had strong fluorescence ( Figure 4 A); When HepG2 cells were only pre-stimulated with LPS, HepG2 cells had weak fluorescence ( Figure 4 B); When HepG2 cells were pre-stimulated with LPS and TEMPO, HepG2 cells had stronger fluorescence ( Figure 4 C). These results indicate that the probe can be used to detect endogenous ONOO− in cells.

[0051] Example 6 Fluorescence imaging of ONOO− changes in Parkinson's disease (PD) models using NBD-ONOO fluorescent probe

[0052] Given the critical role of ONOO− in the pathogenesis of Parkinson's disease (PD), we further investigated whether the probe NBD-ONOO could be used to visualize ONOO− levels in PD models. Studies have shown that rotenone can induce Parkinson's disease symptoms in zebrafish by inhibiting the mitochondrial electron transport chain, leading to oxidative stress and dopaminergic neuron loss. Furthermore, H2S may act as a neuroprotective agent through multiple mechanisms, including anti-oxidative stress and anti-inflammatory effects, protecting against neurotoxin-induced neurodegeneration. Therefore, we sought to investigate the effect of H2S on ONOO− overexpression during rotenone induction. Control group: Zebrafish were treated with 10 μM NBD-ONOO for 30 minutes. Rotenone group: Zebrafish were pretreated with rotenone (1 μM) for 1 hour and then treated with 10 μM NBD-ONOO for 30 minutes. H2S+rotenone group: Zebrafish were first pretreated with rotenone (1 μM) and H2S (20 μM) for 1 h, and then incubated with NBD-ONOO (10 μM) for 30 min. Figure 5 As shown, the control group produced a significant green fluorescence signal ( Figure 5 A), indicating that the ONOO− level in zebrafish is low and the probe fluorescence is not quenched; the rotenone-treated group alone emits a weaker green fluorescence signal, indicating that the ONOO− level in zebrafish is high ( Figure 5 B). The H2S and rotenone co-treatment group produced a stronger green fluorescence signal ( Figure 5 C), indicating that H2S has a certain neuroprotective effect. Therefore, the developed fluorescent probe NBD-ONOO can be used to detect fluctuations in ONOO− in PD models and evaluate the role of H2S in suppressing endogenous ONOO− overexpression during rotenone induction.

[0053] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to specific embodiments. Obviously, many modifications and variations are possible based on the contents of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A fluorescent probe for detecting peroxynitrite ions, characterized in that It has the following general structural formula: wherein n is an integer between 1 and 4; R1, R2 and R3 are each independently selected from hydrogen, halogen, nitro, cyano, C 2-6 The carboxylate group, C 1-6 Alkyl, C 1-6 Alkoxy, 1-3 halogen-substituted C 1-6 Alkyl and C substituted by 1-3 halogen 1-6 One of the alkoxy groups.

2. The fluorescent probe according to claim 1, wherein n = 3; R1= H; R2= H; R3= -CH2CH3.

3. The method for preparing a fluorescent probe according to claim 2, wherein: 7-nitro-N-(pyrrolidin-3-yl)benzo[c][1,2,5]oxadiazole-4-amine and -CH2CH3-substituted benzoyl chloride are dissolved in dichloromethane solvent, and triethylamine is added and stirred to react to obtain the target probe.

4. The preparation method according to claim 3, characterized in that The molar ratio of 7-nitro-N-(pyrrolidin-3-yl)benzo[c][1,2,5]oxadiazol-4-amine and -CH2CH3 substituted benzoyl chloride is 1:1.

1.

5. The preparation method according to claim 3, characterized in that The molar ratio of triethylamine to 7-nitro-N-(pyrrolidin-3-yl)benzo[c][1,2,5]oxadiazol-4-amine is 3:

1.

6. The preparation method according to claim 3, characterized in that When the target probe is generated by the reaction, the reaction is stirred at 25°C for 3-6 hours.

7. The preparation method according to claim 3, characterized in that After the target probe is generated by the reaction, the product is separated and purified; the separation and purification steps include: distilling the solvent off the reaction solution under reduced pressure, and subjecting the dried solid to silica gel column chromatography, with the eluent being a mixture of dichloromethane and methanol in a volume ratio of 100 to 75:1.

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