High-sensitivity fluorescent probe for monitoring mitochondrial autophagy as well as preparation method and application of high-sensitivity fluorescent probe

Abstract

The invention discloses a fluorescent probe for high-sensitivity monitoring of cell mitochondrial autophagy as well as a preparation method and application thereof. The structure of the fluorescent probe is as shown in formula I in the specification. According to the fluorescent probe provided by the invention, a phenol group is used as an electron donor and a pH response unit, hydroxyl tricyanopyrrole is used as an electron acceptor, triphenylphosphine is used as a mitochondrial targeting group, fluorine atoms are introduced to a benzene ring to improve the response rate and the fluorescence intensity, and meanwhile, a thiophene ring is introduced to further expand the fluorescence emission wavelength. The probe can rapidly target mitochondria and emit corresponding fluorescence signals based on pH differences of different autophagy stages, so that real-time, high-sensitivity and high-selectivity monitoring of the mitochondria autophagy process is realized. In addition, the fluorescent probe is simple in molecular structure, small in molecular weight and easy to synthesize, purify and subsequently modify. Cell experiment results show that the probe can accurately trace the mitochondrial autophagy process in cells, and has good application potential in the field of biological imaging and detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochemical analysis, and particularly relates to a high-sensitivity monitoring mitochondrial autophagy fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] Mitochondrial autophagy is a core self-maintenance mechanism of cells to selectively remove dysfunctional mitochondria. This process is mainly cooperatively regulated by the PINK1-Parkin pathway and various mitochondrial receptor proteins (such as BNIP3, FUNDC1), and plays a core role in maintaining cellular energy metabolism homeostasis, inhibiting oxidative stress and apoptosis. Its dysfunction has been widely proven to be one of the key causes of major pathological processes such as neurodegenerative diseases, metabolic diseases, tumors and aging.

[0003] The process of mitochondrial autophagy is accompanied by significant changes in its intracellular environment: from the weakly basic environment (pH ~ 8.0) of normal mitochondria in the initial stage, to autophagosome formation, and finally to fusion with acidic lysosomes (pH ~ 4.5-5.5) in the degradation stage. Therefore, a tool capable of real-time, in-situ monitoring of this pH dynamic change (range about 5.0-8.0) is extremely important for analyzing the regulation mechanism of mitochondrial autophagy.

[0004] At present, fluorescence imaging technology is the mainstream method to achieve this goal. Although some pH-responsive fluorescent probes have been developed for such monitoring, most of the probes have fluorescence emission wavelengths in the visible light region (400-600 nm), which has weak tissue penetration ability and is easily disturbed by the spontaneous fluorescence of biological tissues, so the application effect is poor in deep tissue imaging and high signal-to-noise ratio dynamic monitoring of living cells. In addition, they usually cannot achieve high sensitivity and linear fluorescence response in the pH dynamic range (about 5.0-8.0) related to autophagy, so they cannot accurately distinguish different stages of autophagy, limiting their ability to perform high-fidelity real-time tracking of the mitochondrial autophagy process in living cells. Therefore, developing a new type of molecular probe with high sensitivity, good linearity and high signal-to-noise ratio for mitochondrial autophagy in this pH range has important research value for in-depth understanding of the mechanism of related diseases. SUMMARY

[0005] The purpose of the present application is to provide a fluorescent probe preparation method for high-sensitivity monitoring of mitochondrial autophagy and its application.

[0006] To this end, in a first aspect, the present application provides a fluorescent probe, the structure of which is shown as formula I,

[0007]

[0008] In a second aspect, the application provides a preparation method of the high-sensitivity fluorescent probe for monitoring mitochondrial autophagy, comprising: preparing the fluorescent probe of Formula I from the compounds of Formula II and Formula III in an organic solvent through copper catalysis,

[0009]

[0010] Further, the compound of Formula II is a compound of Formula IV, which is prepared into the fluorescent probe precursor of Formula II through an alkali catalytic reaction with Formula V,

[0011]

[0012] In a third aspect, the application provides application of the fluorescent probe in monitoring mitochondrial autophagy.

[0013] Specifically, the stage of mitochondrial autophagy is monitored by detecting the fluorescence intensity of the probe at 650 nm under different pH values.

[0014] The high-sensitivity fluorescent probe for monitoring mitochondrial autophagy provided by the application has the following characteristics and advantages:

[0015] 1) The fluorescent probe has a good pH response effect, and has a good linearity in the range of 5.0 to 8.0, so that the process of mitochondrial autophagy can be accurately monitored. The maximum fluorescence emission wavelength of the probe is 650 nm, which is located in the near-infrared region, can eliminate the interference of biological autofluorescence, and can be better used for in vivo animal imaging.

[0016] 2) The fluorescent probe can well target the mitochondria of cells, and can accurately monitor the mitochondrial autophagy process of various cells.

[0017] 3) The fluorescent probe has good biological safety, has no cytotoxicity to cells within the working concentration, and can avoid the interference of the probe to the mitochondrial autophagy of cells.

[0018] 4) The fluorescent probe has a simple molecular structure, a definite chemical structure, is easy to prepare and purify, has low production cost, and is highly practical. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The synthesis route of the high-sensitivity fluorescent probe for monitoring mitochondrial autophagy of Formula I.

[0020] Figure 2 The ultraviolet-visible absorption spectrum of the high-sensitivity fluorescent probe for monitoring mitochondrial autophagy of Formula I in PBS buffer with different pH values.

[0021] Figure 3Fluorescence spectra of the high-sensitivity monitoring mitochondrial autophagy fluorescent probe shown as formula I in PBS buffer with different pH values.

[0022] Figure 4 Nonlinear fitting of the fluorescence intensity of the high-sensitivity monitoring mitochondrial autophagy fluorescent probe shown as formula I in PBS buffer with different pH values and pH values.

[0023] Figure 5 Linear fitting of the fluorescence intensity of the high-sensitivity monitoring mitochondrial autophagy fluorescent probe shown as formula I in PBS buffer with pH values of 5.0-7.6 and pH values.

[0024] Figure 6 Time stability of the high-sensitivity monitoring mitochondrial autophagy fluorescent probe shown as formula I in different pH values.

[0025] Figure 7 Cyclic response and reversibility of the high-sensitivity monitoring mitochondrial autophagy fluorescent probe shown as formula I between pH 5.0 and pH 8.0.

[0026] Figure 8 Selectivity of the high-sensitivity monitoring mitochondrial autophagy fluorescent probe shown as formula I, where a in the abscissa is the high-sensitivity monitoring mitochondrial autophagy fluorescent probe, b: K + , c: Mg 2+ , d: Ca 2+ , e: Cu 2+ , f: Zn 2+ , g: Na + , h: NO 2- , i: NO 3- , j: Cl - , k: SO4 2- , l: Glu, m: Vc, n: Cys, o: Gln, p: Trp, q: Thr, r: H2O2, s: GSH, and the ordinate is the fluorescence emission intensity (a.u.).

[0027] Figure 9 Biological activity of the high-sensitivity monitoring mitochondrial autophagy fluorescent probe shown as formula I in three cells.

[0028] Figure 10 Co-localization of the high-sensitivity monitoring mitochondrial autophagy fluorescent probe shown as formula I in 4T1 cells.

[0029] Figure 11 pH correction test of the high-sensitivity monitoring mitochondrial autophagy fluorescent probe shown as formula I in 4T1 cells. DETAILED DESCRIPTION

[0030] Exemplary embodiments of the present disclosure will be described in greater detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it is understood that the present disclosure can be embodied in various forms without being limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure will be thoroughly and completely comprehended, and so that the scope of the present disclosure will be completely conveyed to those skilled in the art.

[0031] Example 1

[0032] Synthesis of the high-sensitivity fluorescent probe for monitoring mitophagy (Formula I) according to the present disclosure.

[0033] The synthesis route of the high-sensitivity fluorescent probe for monitoring mitophagy (Formula I) according to the present disclosure is shown in Figure 1 The synthesis method is as follows:

[0034] (i) 5-Formyl-2-thiopheneboronic acid (468 mg, 3.0 mmol), 2-fluoro-4-iodophenol (476 mg, 2.0 mmol), potassium carbonate (617 mg, 4.5 mmol) and palladium acetate (32 mg, 0.14 mmol) were dissolved in 8 mL of EtOH / H2O (1:1, v / v) and reacted at 80°C for 5 h. The reaction progress was monitored by TLC, after the reaction was completed, a small amount of water was added to quench and the reaction solution was extracted with ethyl acetate, the organic layer was dried over anhydrous sodium sulfate, then the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 1:3, v / v) to obtain compound a as a yellow powder. 1 H NMR (400 MHz, Deuterated dimethyl sulfoxide) δ 10.55 (s, 1H), 9.85 (s, 1H), 7.97 (s, 1H), 7.67-7.60 (m, 2H), 7.46-7.41 (d, J = 8.1 Hz, 1H), 7.05-6.99 (t, J = 8.4 Hz, 1H). 13 C NMR (101 MHz, Deuterated dimethyl sulfoxide) δ 184.4, 152.6, 150.4, 146.9, 141.5, 139.9, 124.9, 123.5, 118.8, 114.6.

[0035] (ii) Compound a (222 mg, 1.0 mmol), compound b (125 mg, 0.5 mmol) and ammonium acetate (77 mg, 1.0 mmol) were placed in a vial, tetrahydrofuran 15 mL was added, and the reaction was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol, 50:1, v / v) to obtain compound c as a red powder. 1 H NMR (400 MHz, Deuterated Methanol) δ 3.45 - 3.34 (m, 1H), 3.09 (p, J = 5.8 Hz, 1H), 2.42 (q, J = 5.5, 4.4 Hz, 2H), 2.31 (s, 3H), 2.27 (s, 1H), 1.65 - 1.55 (s, 3H). 13 C NMR (101 MHz, Deuterated Methanol) δ 175.9, 161.2, 113.5, 112.7, 109.9, 107.2, 97.6, 80.5, 69.5, 62.1, 21.7, 19.1, 11.9.

[0036] (iii) Compound a (222 mg, 1.0 mmol), compound b (125 mg, 0.5 mmol) and ammonium acetate (77 mg, 1.0 mmol) were placed in a vial, tetrahydrofuran 15 mL was added, and the reaction was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol, 50:1, v / v) to obtain compound c as a red powder. 1 H NMR (400 MHz, Deuterated Methanol) δ 3.45 - 3.34 (m, 1H), 3.09 (p, J = 5.8 Hz, 1H), 2.42 (q, J = 5.5, 4.4 Hz, 2H), 2.31 (s, 3H), 2.27 (s, 1H), 1.65 - 1.55 (s, 3H). 13C NMR(101MHz,DMSO-d6)δ166.6,161.3,152.5,150.1,149.4,146.4,138.7,138.1,137.0,125.3,124 .5,123.0,118.5,115.3,114.1,113.8,111.7,101.1,97.3,81.8,72.4,62.1,25.7,19.2.HR-MS:m / z C 25 H 17 FN4NaO2S + Theoretical value: 479.0948; Experimental value: [M+Na] + ,479.0954.

[0037] (iv) (3-aminopropyl)triphenylphosphine (150 mg, 0.5 mmol), 1H-imidazolium-1-sulfonyl azidohydrochloride (300 mg, 1.43 mmol), potassium carbonate (198 mg, 1.43 mmol), and copper sulfate pentahydrate (12 mg, 0.048 mmol) were dissolved in 10 mL of methanol and reacted at room temperature for 12 h. After the reaction was complete, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol, 50:1, v / v) to give compound d as a transparent oily liquid, which was directly used for the next reaction.

[0038] (v) Compound c (110 mg, 0.24 mmol), compound d (97 mg, 0.28 mmol), copper sulfate pentahydrate (70 mg, 0.28 mmol), and sodium ascorbate (55 mg, 0.28 mmol) were dissolved in 10 mL of THF / H2O (1:1, v / v) and reacted at room temperature for 3 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was extracted with dichloromethane. The organic layer was dried with anhydrous sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, 15:1, v / v) to obtain the red powder compound NSP (yield: 52%), which is the fluorescent probe shown in Formula I. 1¹H NMR (400 MHz, deuterated dimethyl sulfoxide) δ 10.47 (s, 1H), 10.34 (s, 1H), 7.93–7.85 (m, 4H), 7.83–7.71 (m, 12H), 7.64 (s, 2H), 7.57 (d, J = 3.9 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.03 (t, J = 8.8 Hz) ,1H),6.72(d,J=15.8Hz,1H),4.39(q,J=6.8Hz,2H),3.60(s,1H),3.52(d,J=7.7Hz, 2H),3.29–3.14(m,2H),2.86(t,J=6.8Hz,2H),2.02(d,J=14.0Hz,2H),1.73(s,3H). 13 C10 NMR (101 MHz, deuterated dimethyl sulfoxide) δ 166.6, 161.2, 152.4, 150.0, 149.3, 146.4, 138.6, 137.9, 137.1, 135.1, 133.6, 133.5, 130.4, 130.2, 125.1, 124.3, 122.9, 118.5, 117.6, 115.1, 111.6, 100.8, 97.1, 65.7, 62.8, 60.3, 49.1, 46.0, 25.6, 23.0. HR-MS: m / z C10 46 H 38 FN7O2PS + Theoretical value: 802.2524; Experimental value: [M] + ,802.2526.

[0039] Example 2

[0040] The present invention describes a high-sensitivity fluorescent probe (NSP) for monitoring mitochondrial autophagy, used in chemical analysis experiments.

[0041] UV-Vis absorption spectra of NSP in response to different pH values: The NSP prepared in Example 1 was dissolved in chromatographically pure dimethyl sulfoxide to prepare a 1 mM stock solution. The NSP stock solution was then added to PBS buffer (10 mM, pH 4.0-9.0) to obtain 10 μM NSP probe solutions. These solutions were then placed in a UV-Vis spectrophotometer (Shimadzu) and the UV-Vis absorption spectra were recorded. The results are shown below. Figure 5 The absorption spectrum of the NSP fluorescent probe showed a relatively stable trend, with a prominent peak at about 490 nm. As the pH increased from 4.0 to 9.0, the absorption spectrum red-shifted slightly.

[0042] The fluorescence spectra of NSP in response to different pH values ​​were obtained by adding NSP stock solution to PBS buffer (10 mM, pH 4.0-9.0) to obtain 10 μM NSP probe solutions, which were then placed in a fluorescence spectrophotometer (Agilent) and the fluorescence spectra were recorded. The results are shown below. Figure 6 It can be seen that the fluorescence intensity of the probe increases significantly as the pH decreases, and the fluorescence intensity increases significantly by 55 times between pH 4.0 and pH 9.0.

[0043] The pKa value of the NSP fluorescent probe was determined by the Henderson-Hasselbalch equation (log[(Fmax-F) / (F-Fmin)]=pH-pKa), which showed a value of 6.29±0.1.

[0044] Plotting standard curves of NSP fluorescence response at different pH values: NSP stock solution was added to PBS buffer (10 mM, pH 5.0–7.6) to obtain a 10 μM NSP probe solution. The solution was placed in a fluorescence spectrophotometer (Agilent), and the fluorescence intensity at 650 nm was recorded. The standard curves were obtained by plotting different pH values ​​against fluorescence intensity and fitting the plots. Figure 8 As shown, pH values ​​exhibit a negative linear correlation within the range of 5.0–7.6 (y = -299.56x + 2273.61, R0). 2 =0.984).

[0045] Photostability of NSP at different pH values: NSP stock solution was added to PBS buffer (10 mM, pH 6.0, 7.0, 8.0) to obtain a 10 μM NSP probe solution. The solution was then placed in a fluorescence spectrophotometer (Agilent) and the fluorescence intensity at 650 nm was recorded from 0 to 120 min. Results are as follows: Figure 9 As shown, the probe exhibited stable fluorescence for up to 2 hours at different pH levels, indicating that it has excellent photostability.

[0046] Cyclic response and reversibility of NSP between pH 5.0 and pH 8.0: NSP stock solution was added to PBS buffer (10 mM, pH 5.0) to obtain a 10 μM NSP probe solution. This solution was placed in a fluorescence spectrophotometer (Agilent) and the fluorescence intensity at 650 nm was recorded. The pH was then adjusted to 8.0, and the fluorescence intensity at 650 nm was recorded again. This procedure was repeated 10 times. Results are as follows: Figure 10 As shown, the fluorescence intensity disappeared when the pH increased from 5.0 to 8.0, and recovered when the pH returned to 5.0, indicating that the NSP probe responds well to H+. +The probe exhibits a rapid response. Furthermore, it can switch fluorescence between "on" and "off" states for at least 10 cycles with minimal intensity loss, demonstrating excellent reversibility and enabling rapid and sensitive monitoring of mitophagy.

[0047] NSP selectivity assessment: NSP stock solution was added to PBS buffer (10 mM, pH 6.0, 7.0, 8.0) to obtain a 10 μM NSP probe solution. This solution was then placed in a quartz cuvette, and K+ was added to each solution. + Mg 2+ Ca 2+ Cu 2+ Zn 2+ Na + NO 2- NO 3- ,Cl - SO4 2- Glu, VC, Cys, Gln, Trp, Thr, H2O2, and GSH (final concentrations all 10 μM) were added. The sample was placed in a fluorescence spectrophotometer (Agilent), and the fluorescence intensity at 650 nm was recorded, with three parallel studies. Results are shown below. Figure 11 As shown, the fluorescence response of NSP is not affected by common ions, amino acids, or reactive oxygen species.

[0048] Example 3

[0049] The high-sensitivity fluorescent probe (NSP) for monitoring mitochondrial autophagy described in this invention is used in cellular-level experiments.

[0050] Biocompatibility study of NSP: NSP prepared in Example 1 was dissolved in cell-grade dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution. HeLa cells were seeded in 96-well plates and cultured for 24 h to ensure adequate cell attachment. Subsequently, different concentrations of NSP stock solution (0, 2.5, 5, 10, 20, 40 μM) were added and the cells were incubated for 24 h. After incubation, the culture medium was removed, and 100 μL of MTT (0.5 mg / mL) solution was added for further incubation for 4 h. After gently removing the MTT solution, DMSO was added, and the cells were shaken for 10 min to fully dissolve the formazan within the cells. The absorbance was measured at 490 nm using a microplate reader. Cell viability was expressed as V =

[0051] The absorbance was calculated using the formula A / A0×100%, where A represents the absorbance of the experimental group and A0 represents the absorbance of the control group. The cell viability of the control group was defined as 100%, and six groups were treated in parallel. After treatment with 0–40 μM NSP, the cell viability remained above 80%, indicating that the probe had no significant cytotoxicity. The procedure for 4T1 and PC12 cells was the same as described above.

[0052] Cellular colocalization imaging of NSP: NSP prepared in Example 1 was dissolved in cell-grade dimethyl sulfoxide to prepare a 1 mM stock solution. 4T1 cells were pre-cultured in confocal dishes for 24 h. After removing the original culture medium, the cells were washed three times with serum-free medium. 1 mL of medium containing 10 μM NSP, 5 μM Hoechst 33342, and 100 nM MitoTracker Green (or LysoTracker Green) was added and the cells were incubated for 30 min. After incubation, the cells were washed twice with PBS solution, and then colocalization imaging was performed using a laser confocal scanning microscope. During fluorescence imaging, MitoTracker Green (or LysoTracker Green) was excited by a 488 nm light source, and fluorescence signals were collected in the range of 500 nm to 540 nm; Hoechst 33342 was excited by a 405 nm light source, and fluorescence signals were collected in the range of 421 nm to 470 nm; NSP was excited by a 540 nm light source, and fluorescence signals were collected in the range of 600 nm to 800 nm. Finally, the fluorescence images were overlaid and analyzed using ImageJ software, and the Pearson coefficients for each channel were calculated. This demonstrates that the probe can effectively target mitochondria and has the potential to monitor the process of mitophagy.

[0053] Intracellular pH regulation imaging of NSP: NSP prepared in Example 1 was dissolved in cell-grade dimethyl sulfoxide to prepare a 1 mM stock solution. 4T1 cells were pre-cultured in confocal dishes for 24 h. After removing the original culture medium, the cells were washed three times with serum-free medium. 1 mL of medium containing 10 μM NSP was added and incubated for 30 min, followed by washing with PBS (10 mM, pH 10).

[0054] 7.4) Wash cells twice. Then, add high-kJ electrolytes at different pH values ​​(5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0). + Buffer solution, add 10

[0055] Cells were incubated with μM Nigerian cytosine for 20 min, followed by imaging using a laser confocal scanning microscope. Results were processed using ImageJ software. The three groups of cells at pH 5.0–6.0 showed significant fluorescence, indicating successful intracellular pH regulation. Furthermore, the fluorescence intensity gradually decreased with increasing pH, indicating a linear correlation between fluorescence intensity and pH in 4T1 cells within the pH range of 5.0–9.0 (y = -4.23x + 38.01, R0). 2 =0.891), which is consistent with the in vitro analysis results. This demonstrates the practicality of NSP for flexible monitoring of mitophagy in living cells.

[0056] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A highly sensitive fluorescent probe for monitoring cellular mitochondrial autophagy, characterized in that: The chemical structure of the fluorescent probe is shown in Formula I.

2. The method for preparing the high-sensitivity fluorescent probe for monitoring mitochondrial autophagy as described in claim 1, comprising: The fluorescent probe of formula I was prepared by reacting the compounds shown in formulas II and III in an organic solvent with copper catalysis.

3. The preparation method according to claim 2, characterized in that: The compound shown in Formula II is the same as the compound shown in Formula IV. It is prepared by reacting Formula V with Formula V via a base-catalyzed reaction to yield the fluorescent probe precursor shown in Formula II.

4. The preparation method according to claim 3, characterized in that: The organic solvent is at least one of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, and dichloromethane.

5. The preparation method according to claim 3, characterized in that: The base is at least one of sodium acetate, ammonium acetate, potassium carbonate, and pyridine.

6. The preparation method according to claim 3, characterized in that: The organic solvent is tetrahydrofuran, and the base is ammonium acetate.

7. The preparation method according to claim 3, characterized in that: The condensation reaction is carried out at a temperature of 20-60℃ for 4-16 hours.

8. The application of the fluorescent probe as described in claim 1 in mitophagy.

9. The application as described in claim 8, characterized in that: Mitochondrial autophagy was monitored by observing changes in the fluorescence signal of a fluorescent probe at 650 nm in response to different pH values.

10. The application as described in claim 8, characterized in that: The application is a detection application in biological imaging, cell imaging, or mitochondrial autophagy imaging.

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