An endoplasmic reticulum-targeted long-wavelength fluorescent probe for detecting hydroxyl radicals, and its preparation method and application

Abstract

An endoplasmic reticulum-targeted long-wavelength fluorescent probe for detecting hydroxyl free radicals, and its preparation method and application. The structure of the fluorescent probe is shown in Formula I. The present invention also provides a method for preparing the fluorescent probe and its application in hydroxyl free radical detection. The fluorescent probe provided by the present invention can sensitively and specifically detect hydroxyl free radicals in the long analytical wavelength region of 640 nm. It also has the advantages of selectively targeting the endoplasmic reticulum, low biological background fluorescence signal, minimal photodamage to biological samples, good biocompatibility, and easy preparation and use. It has great application prospects in the fields of chemistry, biology, medicine, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical analysis, and in particular to an endoplasmic reticulum-targeted long-wavelength fluorescent probe for detecting hydroxyl radicals, and a preparation method and application thereof. Background Art

[0002] The endoplasmic reticulum (ER) is a crucial organelle within eukaryotic cells, playing a crucial role in the synthesis, modification, and transport of important biomacromolecules such as proteins, carbohydrates, and lipids. Furthermore, the ER is involved in the regulation of cell death processes such as apoptosis and ferroptosis. For example, due to its large lipid membrane structure, studies have shown that the ER is the earliest and primary site of lipid peroxidation (the execution step of ferroptosis) during ferroptosis. Lipid peroxidation refers to a series of lipid oxidation reactions of polyunsaturated fatty acids (PUFAs) within cells under the action of reactive oxygen species (ROS) and lipoxygenases. The hydroxyl radical (·OH) is one of the most destructive ROS in organisms. Its strong oxidizing and dehydrogenating properties make it one of the most important initiators of lipid peroxidation. Therefore, the development of real-time, in situ methods for detecting ·OH in the ER is of vital importance for the study of related physiological and pathological processes. However, due to the high reactivity, short lifetime (nanoseconds), and low concentration (nanomolar levels) of ·OH, accurate, efficient, real-time, and in situ analysis and detection of ·OH remains difficult.

[0003] Fluorescent probe imaging analysis technology holds great promise due to its high sensitivity, low cost, ease of use, noninvasive imaging, real-time imaging, and ultra-high spatiotemporal resolution. It has been widely used for fluorescence imaging analysis of ·OH. However, currently reported fluorescent probes for ·OH detection, including commercially available ·OH fluorescent probes, still suffer from shortcomings such as short analytical wavelengths and susceptibility to interference from other oxidizing species. Furthermore, there are currently no long-wavelength fluorescent probes that can target the endoplasmic reticulum to avoid interference from biological background fluorescence signals while improving the optical signal's ability to penetrate biological tissues and reduce photodamage to biological samples. Summary of the Invention

[0004] The purpose of the present invention is to provide an endoplasmic reticulum-targeted long-wavelength fluorescent probe for detecting ·OH and its preparation method and application. To this end, in a first aspect, the present invention provides an endoplasmic reticulum-targeted long-wavelength fluorescent probe for detecting ·OH, the structure of which is shown in Formula I,

[0005]

[0006] In a second aspect, the present invention provides a method for preparing a fluorescent probe of Formula I, comprising the following reaction steps: (1) reacting N-Boc piperazine and 4-fluoro-2-hydroxybenzaldehyde in an organic solvent via a base catalysis to obtain compound 1; (2) reacting compound 1 with ethyl acetoacetate in an organic solvent via a base catalysis to obtain compound 2; (3) removing the Boc protecting group from compound 2 in an organic solvent via an acid catalysis to obtain compound 3; (4) reacting compound 3 with p-toluenesulfonyl chloride in an organic solvent via a base catalysis to obtain compound 4; and (5) reacting compound 4 with 3-formyl-1-methylquinolinium in a water / organic mixed solvent via a base catalysis to obtain an electrophilic addition reaction to obtain the fluorescent probe of Formula I; see the following reaction scheme,

[0007]

[0008] Furthermore, the organic solvent in step (1) is at least one of dimethyl sulfoxide, toluene and N,N-dimethylformamide, the reaction temperature is 80-130°C, and the reaction time is 1-24h; the organic solvent in step (2) is at least one of ethanol, acetic acid and methanol, the base is at least one of piperidine, pyridine and potassium carbonate, the reaction temperature is 30-120°C, and the reaction time is 1-24h; the organic solvent in step (3) is at least one of dichloromethane and dioxane, and the acid is at least one of trifluoroacetic acid, hydrochloric acid, formic acid and p-toluenesulfonic acid. at least one of the above, the reaction temperature is 10-100° C., and the reaction time is 10 min-10 h; in step (4), the organic solvent is at least one of acetonitrile, dichloromethane, dimethyl sulfoxide and N,N-dimethylformamide, the base is at least one of potassium carbonate, cesium carbonate and sodium hydroxide, the reaction temperature is 20-100° C., and the reaction time is 1-20 h; in step (5), the organic solvent is at least one of ethanol and methanol, the base is at least one of sodium hydroxide and potassium hydroxide, the reaction temperature is 20-80° C., and the reaction time is 1-20 h.

[0009] In a third aspect, the present invention provides the use of the fluorescent probe described in Formula I in the detection of ·OH.

[0010] Preferably, ·OH is determined by detecting the fluorescence response intensity of the fluorescent probe at a wavelength of 640 nm.

[0011] The fluorescent probe provided by the present invention has the following features and advantages:

[0012] 1) After the probe reacts with ·OH, the conjugated system of the probe molecule expands, causing the fluorescence emission wavelength to red-shift to the long-wavelength region of 640 nm. The large spectral shift is beneficial to reducing the detection background fluorescence signal, improving the biological tissue penetration ability of the light signal, and reducing light damage to biological samples.

[0013] 2) The probe can selectively target the endoplasmic reticulum (ER), enabling in situ non-destructive fluorescence imaging analysis of ·OH in the ER.

[0014] 3) The probe has good selectivity for ·OH analysis and is not interfered with by other reactive oxygen species or physiologically active species;

[0015] 4) Good biocompatibility and easy to use;

[0016] 5) The preparation method is simple, can be produced in large quantities, and is easy to store. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Various other advantages and benefits will become apparent to those skilled in the art by reading the detailed description of the preferred embodiment below. The accompanying drawings are only for the purpose of illustrating the preferred embodiment and are not to be considered as limiting the present invention. In the accompanying drawings:

[0018] Figure 1 Graph showing the fluorescence response of the fluorescent probe shown in Formula I to TCBQ / H2O2 reagents at different concentrations.

[0019] Figure 2 Graph showing the fluorescence response of the fluorescent probe shown in Formula I to different ROS and physiologically active substances.

[0020] Figure 3 The fluorescent probe shown in formula I detects exogenous ·OH produced by Fenton's reagent in cells.

[0021] Figure 4 The fluorescent probe shown in formula I is used to detect ·OH produced during cell ferroptosis.

[0022] Figure 5 This is a co-localization fluorescence imaging diagram of the fluorescent probe represented by Formula I and the commercial endoplasmic reticulum fluorescent dye ER-tracker Green. DETAILED DESCRIPTION

[0023] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0024] Example 1 Preparation of the fluorescent probe represented by formula I

[0025] This embodiment provides a method for preparing the fluorescent probe represented by Formula I, and its synthetic route is as follows:

[0026]

[0027] The specific steps are as follows:

[0028] N-Boc piperazine (1.80 g, 12 mmol) and 4-fluoro-2-hydroxybenzaldehyde (1.40 g, 10 mmol) were weighed into a round-bottom flask. 25 mL of dimethyl sulfoxide (DMSO) was added to the flask to dissolve the solid. The flask was then placed in an oil bath at 100°C and allowed to react for 22 hours. 50 mL of deionized water was added to the reaction mixture, followed by extraction with ethyl acetate (EA) (20 mL × 3). The organic phase was washed with saturated aqueous sodium chloride solution and dried over anhydrous magnesium sulfate (Mg2SO4). The organic phase was removed under reduced pressure to obtain a solid residue, which was then purified by silica gel column chromatography (PE:EA = 2:1) to obtain Compound 1 (2.30 g, 75% yield) as a yellow solid.

[0029] Compound 1 (3.06 g, 10 mmol) and ethyl acetoacetate (1.40 g, 13 mmol) were weighed into a round-bottom flask, and 200 mL of anhydrous ethanol and 2 mL of piperidine were added successively. The round-bottom flask was then placed in an 80°C oil bath and refluxed for 4 hours. The flask was then transferred to ice water to produce a large amount of orange-yellow needle-like precipitates. After filtration and drying, an orange-yellow needle-like solid compound 2 (2.95 g, yield 79.4%) was obtained.

[0030] Compound 2 (1.86 g, 10 mmol) was weighed into a two-necked flask. 30 mL of 30% trifluoroacetic acid (containing 70 mL of CH₂Cl₂ and 30 mL of trifluoroacetic acid) was added to the flask at room temperature. After stirring for 30 minutes, the trifluoroacetic acid was removed with nitrogen for 1 hour, and the remaining trifluoroacetic acid was removed under reduced pressure. Extraction was performed with 50 mL of CH₂Cl₂, and the organic phase was washed three times with saturated sodium chloride solution. Finally, the organic solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (CH₂Cl₂:MeOH = 10:1) to obtain compound 3 as a yellow solid (2.14 g, 78.2% yield).

[0031] Compound 3 (2.72 g, 10 mmol), p-toluenesulfonyl chloride (2.86 g, 15 mmol), and potassium carbonate (2.76 g, 20 mmol) were weighed into a round-bottom flask, and 40 mL of acetonitrile was added. The flask was then placed in a 40°C oil bath and reacted for 8 hours. After cooling to room temperature, the organic phase was removed under reduced pressure to obtain a solid residue, which was then purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 4 (2.03 g, 47.5% yield) as a yellow solid.

[0032] Compound 4 (2.13 g, 5 mmol), 3-formyl-1-methylquinolinium (1.02 g, 6 mmol), and sodium hydroxide (0.40 mg, 10 mmol) were weighed into a round-bottom flask. 40 mL of a mixture of anhydrous ethanol and water (v / v = 1:1) was added. The mixture was reacted at 32°C in the dark for 24 hours. The solvent was removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography (DCM:MeOH = 100:1) to obtain a yellow powder, the fluorescent probe represented by Formula I. 1 H NMR (400MHz, Chloroform-d): δ9.10(s,1H),8.35(s,1H),7.64(d,J=8.1Hz,2H),7.46(d,J=6.0Hz,1H),7.39( d,J=8.9Hz,1H),7.33(d,J=8.0Hz,2H),7.17(t,J=6.9Hz,1H),7.05(t,J=7.4Hz,1H),6.98(s,1H),6.86(d,J= 8.2Hz,1H),6.71(dd,J=8.9,2.4Hz,1H),6.55(d,J=2.4Hz,1H),4.70(t,J=6.1Hz,1H),3.59(dd,J=15.3,5.7H z,1H),3.49(t,J=5.1Hz,4H),3.40(s,3H),3.12(d,J=5.0Hz,4H),3.00(dd,J=15.3,6.6Hz,1H),2.41(s,3H). 13 C NMR (101MHz, Chloroform-d): δ195.3,187.5,160.0,157.8,154.3,151.8,147.8,144.2,138.1,133.3,132.0,131.6,129. 99,129.9,127.8,126.9,124.6,119.0,114.8,112.8,111.9,110.5,100.0,52.1,46.7,44.9,39.4,30.8,21.6.HR-MS:m / z calcd for C33H31N3NaO6S([M+Na] + ),620.1826;found620.1830.

[0033] Example 2 Detection of hydroxyl radicals by the fluorescent probe represented by formula I

[0034] The probe was dissolved in DMF to prepare a 1 mM stock solution. 3 mL of 20 mM phosphate buffer was added to a test tube, followed by 30 μL of the probe stock solution to a final probe concentration of 10 μM. Finally, various concentrations of TCBQ / H₂O₂ solutions (to generate ·OH) were added. After 30 minutes, fluorescence spectra were measured at an excitation wavelength of 510 nm. All experiments were performed in triplicate.

[0035] Figure 1 The fluorescence response diagram of the fluorescent probe shown in Formula I to different concentrations of TCBQ / H2O2 reagents. Figure 1 It can be seen that with the increase of TCBQ / H2O2 reagent concentration (i.e., the increase of ·OH level), the fluorescence intensity of the probe at 640nm also increased significantly; and in the TCBQ / H2O2 reagent concentration range of 0 to 10μM, the fluorescence intensity of the probe showed a good linear relationship with the concentration, and the linear equation was F = 23.6×C (μM) + 34.7 (R 2 =0.989), and the detection limit was calculated to be 125 nM TCBQ / H2O2 reagent.

[0036] Example 3 Analytical selectivity of the fluorescent probe represented by Formula I

[0037] As in Example 2, 3 mL of phosphate buffer containing 10 μM of the fluorescent probe was prepared, and different common reactive oxygen species (including NO, OCl - 、H2O2、 1 O2, O2 ·- 、ONOO - and TBHP), different amino acids, common metal ions, glucose, ascorbic acid and BSA, etc. The test solutions were placed at room temperature for 30 min before fluorescence testing.

[0038] As attached Figure 2 As shown, the fluorescent probe represented by Formula I does not produce a significant fluorescence signal even after interaction with high concentrations of other ROS (10 times that of ·OH) except ·OH. Other physiologically active substances, such as amino acids, metal ions, glucose, ascorbic acid, and BSA, also do not cause changes in the probe's fluorescence signal. These results demonstrate that the fluorescent probe represented by Formula I has good analytical selectivity for ·OH.

[0039] Example 4: Detection of exogenous ·OH produced by Fenton's reagent in cells by the fluorescent probe shown in Formula I

[0040] Human fibrosarcoma cells (HT-1080) were cultured for 24 hours in glass-bottomed culture dishes designed for laser confocal microscopy in RPMI 1640 medium supplemented with 10% (v / v) newborn calf serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C, 5% CO2, 95% air, and 96% humidity. After 24 hours of growth, appropriate volumes of Fenton's reagent were added to final concentrations of 100 μM and 200 μM, respectively. Then, 10 μL of a 1 mM stock solution of the fluorescent probe (Formula I) was added to a final concentration of 10 μM. Fluorescence imaging was performed after 30 minutes of incubation. In a separate dish, cells were incubated with 200 μM tempol for 5 minutes, followed by 200 μM Fenton's reagent and 10 μM of the fluorescent probe (Formula I). ​​Fluorescence imaging was performed after 30 minutes of incubation. The excitation wavelength for fluorescence imaging was 510 nm, and the fluorescence signal was collected in the range of 600 nm to 700 nm.

[0041] The experimental results are as follows Figure 3 When cells were incubated with only the probe, only weak fluorescence was observed. However, after incubation with 100 and 200 μM Fenton's reagent, the fluorescence intensity significantly increased with increasing Fenton's reagent concentration. Furthermore, when the ·OH scavenger tempol was simultaneously added to the cells, the fluorescence signal decreased significantly. These results demonstrate that the fluorescent probe represented by Formula I can be used to detect exogenous ·OH in living cells.

[0042] Example 5: Detection of ·OH Produced During Cell Ferropoptosis Using the Fluorescent Probe Shown in Formula I. HT-1080 was cultured for 24 hours as described in Example 4. 10 μM of an ferroptosis inducer was added and the cells were incubated for 2, 4, and 6 hours, respectively. The old culture medium was then removed, serum-free culture medium was added, and 10 μM of the fluorescent probe shown in Formula I was added. Fluorescence imaging was performed after incubation for 30 minutes. Three more dishes of cells were taken. 10 μM of Erastin was added, along with three ferroptosis inhibitors: 200 μM DFO, 10 μM Fer-1, and 10 μM Lip-1. The cells were incubated for 6 hours and then treated as above for fluorescence imaging. The excitation wavelength for fluorescence imaging was 510 nm, and the fluorescence signal was collected in the range of 600 nm to 700 nm.

[0043] Erastin is a commonly used ferroptosis inducer that can trigger ferroptosis in HT-1080 cells and generate a large amount of ·OH in the cells. Figure 4 The fluorescence imaging diagram of the fluorescent probe shown in formula I on HT-1080 cells induced by Erastin. Figure 5As shown, the fluorescence signal of the ·OH channel gradually increased with the extension of erastin incubation time, indicating that erastin-induced ferroptosis is accompanied by an increase in ·OH levels. No significant fluorescence signal was observed in cells treated with a ferroptosis inhibitor. These results further demonstrate that the fluorescent probe represented by Formula I can be used for in situ non-destructive fluorescence imaging of intracellular ·OH.

[0044] Example 6 Endoplasmic Reticulum Targeting Ability of the Fluorescent Probe Represented by Formula I

[0045] HT-1080 cells were cultured for 24 hours according to the method of Example 4. 100 μM Fenton's reagent was then added, followed by 10 μM of the fluorescent probe of Formula I and 50 nM of the commercial endoplasmic reticulum fluorescent dye ER-tracker Green. The cells were incubated for 30 minutes, then washed with PBS, and 1 mL of serum-free medium was added. Finally, fluorescence imaging was performed using a laser confocal microscope. Red fluorescence channel (fluorescent probe of Formula I): λ ex =510nm,λ em =600–700 nm; Green fluorescence channel (ER-trackerGreen): λ ex =488nm,λ em =500–560nm.

[0046] Figure 5 This is a co-localized fluorescence imaging image of the fluorescent probe represented by Formula I and the commercial endoplasmic reticulum fluorescent dye ER-tracker Green. It can be seen that the fluorescence of the red fluorescent channel (fluorescent probe represented by Formula I) and the green fluorescent channel (ER-tracker Green) have a good overlap effect, and the Pearson coefficient is 0.86, indicating that the fluorescent probe represented by Formula I has good targeting ability to the endoplasmic reticulum.

[0047] Finally, it should be noted that the above examples only illustrate the preparation method and some applications of one fluorescent probe represented by Formula I, and the remaining preparation methods, analysis and testing, and imaging application conditions are not listed one by one.

[0048] The foregoing description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be readily conceived by a person skilled in the art within the technical scope disclosed herein are intended to be encompassed within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. An endoplasmic reticulum-targeted long-wavelength fluorescent probe for detecting hydroxyl radicals, characterized in that: The structure of the fluorescent probe is shown in Formula I, 。 2. The method for preparing a fluorescent probe according to claim 1, wherein: The method comprises the following reaction steps: (1) reacting N-Boc piperazine and 4-fluoro-2-hydroxybenzaldehyde in an organic solvent via base catalysis to obtain compound 1; (2) reacting compound 1 with ethyl acetoacetate in an organic solvent via base catalysis to produce a condensation reaction to obtain compound 2; (3) removing the Boc protecting group from compound 2 via acid catalysis in an organic solvent to obtain compound 3; (4) reacting compound 3 with p-toluenesulfonyl chloride in an organic solvent via base catalysis to produce a nucleophilic substitution reaction to obtain compound 4; (5) reacting compound 4 with 3-formyl-1-methylquinolinium in a water / organic mixed solvent via base catalysis to produce an electrophilic addition reaction to obtain a fluorescent probe represented by formula I; See the following reaction scheme, 。 3. The preparation method according to claim 2, wherein The organic solvent in step (1) is at least one of dimethyl sulfoxide, toluene and N,N-dimethylformamide, the reaction temperature is 80-130 ° C, and the reaction time is 1-24 h; the organic solvent in step (2) is at least one of ethanol, acetic acid and methanol, the base is at least one of piperidine, pyridine and potassium carbonate, the reaction temperature is 30-120 ° C, and the reaction time is 1-24 h; the organic solvent in step (3) is at least one of dichloromethane and dioxane, the acid is at least one of trifluoroacetic acid, hydrochloric acid, formic acid and p-toluenesulfonic acid, the reaction temperature is 10-100 ° C, and the reaction time is 10 min ~ 10 h; the organic solvent in step (4) is at least one of acetonitrile, dichloromethane, dimethyl sulfoxide and N,N-dimethylformamide, the base is at least one of potassium carbonate, cesium carbonate and sodium hydroxide, the reaction temperature is 20-100 ° C, and the reaction time is 1-20 h; the organic solvent in step (5) is at least one of ethanol and methanol, the base is at least one of sodium hydroxide and potassium hydroxide, the reaction temperature is 20~80 °C, and the reaction time is 1~20 h.

4. Use of the fluorescent probe as claimed in claim 1 in preparing a hydroxyl radical detection probe.

5. The use according to claim 4, characterized in that: The hydroxyl radical detection probe detects hydroxyl radicals by monitoring the fluorescence signal intensity at 640 nm.

6. The use according to claim 4, characterized in that: Hydroxyl radical detection probes are used in fluorescence imaging detection applications.

7. The use according to claim 4, characterized in that: Hydroxyl radical detection probes are used in cell imaging detection applications.

8. The use according to claim 4, characterized in that: Hydroxyl radical detection probes are used in endoplasmic reticulum imaging and detection applications.

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