CN115246854B - Fluorescent probe for detecting mitochondrial hydrogen peroxide and preparation method and application thereof - Google Patents

Fluorescent probe for detecting mitochondrial hydrogen peroxide and preparation method and application thereof Download PDF

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CN115246854B
CN115246854B CN202211034998.8A CN202211034998A CN115246854B CN 115246854 B CN115246854 B CN 115246854B CN 202211034998 A CN202211034998 A CN 202211034998A CN 115246854 B CN115246854 B CN 115246854B
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probe
reaction
mito
dye
target product
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CN115246854A (en
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柏桦
王莉敏
李林
方斌
秦绪军
陈宏莉
彭勃
黄维
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Northwestern Polytechnical University
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    • G01N21/64—Fluorescence; Phosphorescence
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Abstract

The invention discloses a fluorescent probe for detecting mitochondrial hydrogen peroxide, a preparation method and application thereof, and belongs to the technical field of organic micromolecular fluorescent probe analysis and detection; adding 4-bromomethyl phenylboronic acid pinacol ester and introducing N 2 Reflux reaction; after the reaction is completed, the mixture is cooled to room temperature, and the solid is separated out by recrystallization and purified to obtain the target product. The probe has excellent photophysical property, good biocompatibility and inherent mitochondrial targeting capability, and can realize the targeting of mitochondrial H 2 O 2 Is selected from the group consisting of a plurality of detection probes. The synthesis raw material cost of the probe is low, the synthesis route is simple, the reaction condition is mild, the treatment is simple, the yield is high, the fluorescence background of the probe is low in sensitivity and low in detection limit, and the probe is matched with H 2 O 2 After the reaction, the obvious fluorescence of Turn on is realized, so that the interference of autofluorescence of biological tissues is avoided, and the method is suitable for mitochondria H in biological tissue samples 2 O 2 And (3) detecting the level.

Description

Fluorescent probe for detecting mitochondrial hydrogen peroxide and preparation method and application thereof
Technical Field
The invention belongs to the technical field of organic micromolecular fluorescent probe analysis and detection, and particularly relates to a fluorescent probe for detecting mitochondrial hydrogen peroxide, and a preparation method and application thereof.
Background
Mitochondria are organelles with semi-autonomy in eukaryotic cells, and are short rods or spheres, generally about 0.5-1.0 μm in diameter. Mitochondria produce Adenosine Triphosphate (ATP) through the aerobic respiration process, providing energy for the vital activity. Reactive Oxygen Species (ROS) are unavoidable byproducts of mitochondrial oxidative phosphorylation, and more than 80% of ROS in the cell are derived from mitochondria under physiological conditions. ROS include superoxide anions (O) 2 -hydroxyl radical (OH), hydrogen peroxide (H) 2 O 2 ) Etc. in various forms, wherein H 2 O 2 The most important active oxygen in the cell has the characteristics of low oxidation-reduction potential (weak reactivity), long half-life period (stable), small molecular polarity (capable of penetrating biological membranes), long diffusion distance and the like, and has the dual functions of causing cell damage and intracellular messengers. At sub-toxic concentrations, it will modulate the functional state of its target in a reversible binding or modification manner, acting by affecting the activity of a variety of signaling pathway proteins. In eukaryotic cells, H 2 O 2 The concentration is tightly controlled. During evolution, the organism divides H by compartmentalization against the oxidase system 2 O 2 Is limited to a specific subcellular range, while mitochondria are the only ones with intact H 2 O 2 Organelles of the production/removal system. Thus, mitochondrial H 2 O 2 The change in concentration is a key signal that it modulates specific target molecules, affecting the biological behavior of cells. However, due to technical limitations and complexity of the biological environment, mitochondrial H is currently not available in living cells or in vivo 2 O 2 And (3) accurately detecting the level.
In recent years, H in living body 2 O 2 The detection method of the level comprises electrochemical analysis, chemiluminescence, spectrophotometry, enzyme-linked immunosorbent assay, mass spectrum and the like. However, these methods require complex sample preparation, are time consuming and expensive, and destroy cell/tissue structures, and cannot meet the requirements of real-time non-invasive detection of living cells or living bodies. Fluorescent probe technology has sensitivityThe small molecular dye can realize target molecular marking on molecular, cell and living body levels, and is widely applied to real-time detection and imaging of a life system. Since the organism contains autofluorescent molecules (such as reduced Nicotinamide Adenine Dinucleotide (NADH), folic acid, etc.), background signals are generated to interfere with detection of target molecules, and H 2 O 2 Can freely diffuse in/between cells, and causes generation of pseudo signals outside mitochondria, so that development of a mitochondrial H with low background and high specificity is highly demanded 2 O 2 Visual detection tool for realizing mitochondrial H 2 O 2 Efficient, specific and spatiotemporal dynamic detection of mitochondrial H from multiple levels of molecules, cells and individuals 2 O 2 Signal conditioning and mechanism of (a) are provided.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a fluorescent probe for detecting mitochondrial hydrogen peroxide, a preparation method and application thereof, and designs a targeted mitochondrial H through a intramolecular twist charge transfer (TICT) mechanism 2 O 2 Low background, specific fluorescent probes. The probe has low fluorescence background and high sensitivity, and is matched with H 2 O 2 After the reaction, significant fluorescence "turnon" is achieved, thereby avoiding interference of autofluorescence of biological tissues.
In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
the invention provides a fluorescent probe for detecting mitochondrial hydrogen peroxide, which has the structural formula:
the invention provides a preparation method of a fluorescent probe for detecting mitochondrial hydrogen peroxide, which comprises the following steps:
s1: adding dye and acetonitrile, heating to a preset temperature, and stirring until the dye and the acetonitrile are completely dissolved;
s2: adding 4-bromomethyl phenylboronic acid pinacol ester and introducing N 2 Reflux reaction;
s3: after the reaction is completed, standing and cooling to room temperature, recrystallizing to separate out solid, and purifying to obtain a target product;
when the dye isWhen the target product is
When the dye isWhen the target product is
When the dye isWhen the target product is
The invention further provides that in S1, the preparation process is carried out in a 50mL Schlenk reaction bottle; the dosage of the acetonitrile is 5ml; the preset temperature is 50 ℃; in the step S2, the reaction time is 12h.
In the invention, in the step S3, the reaction is completely confirmed by adopting TLC (thin layer chromatography) plate tracking; the purification process is to carry out suction filtration washing by adopting EA.
The invention provides a dye of a fluorescent probe for detecting mitochondrial hydrogen peroxide, which has the following structure:
the invention provides a preparation method of a fluorescent probe dye for detecting mitochondrial hydrogen peroxide, which comprises the following steps:
s01: dissolving t-BuOK with anhydrous DMF under stirring, dissolving in N 2 Slowly injecting picoline under the atmosphere, and stirring the solution from colorless to brownish black;
s02: injecting na-CHO DMF solution, stirring at a certain temperature and reacting for a certain time;
s03: cooling to room temperature after the reaction is finished, adding distilled water, extracting by EA until an organic phase is colorless, washing, drying, removing a solvent, and purifying a crude product by column chromatography to obtain a target product;
when the picoline is 4-picoline, the target product is
Or (b)
When the picoline is 3-picoline, the target product is
Or (b)
When the picoline is 2-picoline, the target product is
In the invention, in the step S01, the stirring temperature is 40 ℃, and the stirring time is 1h.
In the step S02, TLC is adopted to monitor the reaction progress in the reaction process; the certain temperature is 50 ℃, and the certain time is 4 hours.
In the step S03, washing is performed by adopting potassium hydroxide solution; the drying is carried out by adopting anhydrous sodium sulfate; the chromatographic purification adopts petroleum ether and ethyl acetate for chromatographic purification; the volume ratio of petroleum ether to ethyl acetate is 2:1.
The fluorescent probe for detecting mitochondrial hydrogen peroxide is adopted to detect mitochondrial H in living cells 2 O 2 The specific detection and fluorescence microscopic imaging analysis.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a fluorescent probe for detecting mitochondrial hydrogen peroxide, which has excellent photophysical properties and is used for detecting mitochondrial H 2 O 2 The detection of the method has the characteristics of good selectivity, high sensitivity, low detection limit and the like; the synthesis raw material of the probe has low cost, simple synthesis route, mild reaction condition, simple treatment and higher yield, and has the advantage of commercialization; the probe synthesized by the invention has good biocompatibility and inherent mitochondrial targeting capability, so that the probe is suitable for living cell mitochondrial H 2 O 2 Specific detection and fluorescence microscopic imaging analysis of (2); the probe has low fluorescence background and high sensitivity, and is matched with H 2 O 2 After reaction, the obvious fluorescence 'Turn on' is realized, so that the interference of autofluorescence of biological tissues is avoided, and the method can be used for mitochondrial level H of living bodies and clinical serum samples 2 O 2 The detection has commercial development application value.
Drawings
FIG. 1, panels (a-d) are HPLC-MS monitoring probes and H 2 O 2 A process of gradually converting the dye after the reaction;
FIG. 2, panels (A-B), shows probe and dye absorption/emission spectroscopy tests;
FIG. 3 shows graphs (A-F) of probes Mito-W1 and H 2 O 2 Testing responsiveness;
FIG. 4 shows graphs (A-F) of probes Mito-W2 and H 2 O 2 Testing responsiveness;
FIG. 5 (A-F) shows probes Mito-W3 and H 2 O 2 ResponsivenessTesting;
FIG. 6, panels (A-B), shows probe biocompatibility and zeta potential tests;
FIG. 7 (A-C) shows the probe Mito-W2 for 0-10. Mu.M exogenous H in four different cell lines HepG2, SH-SY5Y, U87 and HUVED 2 O 2 Imaging, detection applications of (a);
FIG. 8 is a diagram (A-F) of endogenous H of probe Mito-W2 in both HepG2 and SH-SY5Y cells 2 O 2 Imaging, detection applications of (a);
FIG. 9 (A-F) shows the use of probe Mito-W2 in animal and clinical serum samples;
FIG. 10 shows the synthesis route of dye (pre-Mito-1/2/3);
FIG. 11, panels (A-C), shows the synthetic route of the probe (Mito-W1/W2/W3).
Detailed Description
The invention provides a fluorescent probe for detecting mitochondrial hydrogen peroxide, a preparation method and application thereof. A preparation method of a fluorescent probe for detecting mitochondrial hydrogen peroxide comprises the following steps:
s1: adding dye and acetonitrile, heating to a preset temperature, and stirring until the dye and the acetonitrile are completely dissolved;
s2: adding 4-bromomethyl phenylboronic acid pinacol ester and introducing N 2 Reflux reaction;
s3: after the reaction is completed, standing and cooling to room temperature, recrystallizing to separate out solid, and purifying to obtain a target product; when the dye isWhen the target product is->
When the dye isWhen the target product is
When the dye isWhen the target product is
A preparation method of a fluorescent probe dye for detecting mitochondrial hydrogen peroxide comprises the following steps:
s01: dissolving t-BuOK with anhydrous DMF under stirring, dissolving in N 2 Slowly injecting picoline under the atmosphere, and changing the stirring solution from colorless to brown-black;
s02: injecting na-CHO DMF solution, stirring at a certain temperature and reacting for a certain time;
s03: cooling to room temperature after the reaction is finished, adding distilled water, extracting by EA until an organic phase is colorless, washing, drying, removing a solvent, and purifying a crude product by column chromatography to obtain a target product;
when the picoline is 4-picoline, the target product is
When the picoline is 3-picoline, the target product is
When the picoline is 2-picoline, the target product is
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
1. Dye (pre-Mito-1/2/3) synthetic route
Example 1
Synthesis of dye pre-Mito-1: in a 50mL two-necked flask, t-BuOK (437 mg,3.9 mmol) and 10mL anhydrous DMF were added and dissolved with stirring. At N 2 4-methylpyridine (284 mg,5.2 mmol) was slowly injected under an atmosphere, stirred at 40℃for 1h, the solution turned from colorless to brownish black, then na-CHO (595 g,2.6 mmol) in DMF was injected and stirred at 50℃for 4h, and the progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled to room temperature, 100mL of distilled water was added, the organic phase was extracted to colorless with EA, and the organic phase was washed with a potassium hydroxide (1M) solution for 1 time and dried over anhydrous sodium sulfate. The solvent was removed and the crude product was purified by column chromatography (PE/ea=1/2) to give the yellow solid product pre-Mito-1 (514 mg, 65%).
1 H NMR(500MHz,DMSO-d 6 )δ(ppm):8.53(d,J＝4.5Hz,2H),7.87(d,J＝12.0Hz,1H),7.79–7.50(m,6H),7.22(d,2H),6.92(s,1H),4.73(d,J＝4.5Hz,1H),3.58(dd,J＝38.6,5.4Hz,4H),3.06(s,3H). 13 C NMR(125MHz,DMSO-d 6 )δ(ppm):150.44,148.30,145.21,135.58,134.07,129.65,129.47,128.09,126.84,126.06,124.27,124.08,121.06,116.65,105.33,58.81,54.70,40.51.HR-MS(ESI):calcd.for[C 20 H 20 N 2 O]m/z 304.1576,found[M+H] + 305.1633.
Synthesis route of probe (Mito-W1)
Synthesis of probe Mito-W1: in a 50mL Schlenk flask were added pre-Mito-1 (24 mg,0.08 mmol) and 5mL acetonitrile was heated to 50deg.C and stirred until completely dissolved, and 4-bromomethylphenylboronic acid pinacol ester (28 mg,0.096 mmol) was added thereto, followed by N-pass 2 The reaction was refluxed for 12h and the TLC plate was followed until the starting material was completely reacted. And (3) standing and cooling to room temperature, recrystallizing to precipitate a solid, and purifying, namely, carrying out suction filtration and washing by adopting EA to obtain a black-red crystalline product Mito-W1 (46 mg, 96%).
1 H NMR(500MHz,DMSO-d 6 )δ(ppm):8.98(d,J＝6.8Hz,2H),8.21(d,J＝6.8Hz,2H),8.11(d,J＝16.1Hz,1H),7.99(s,1H),7.75(ddd,J＝34.3,17.7,7.0Hz,5H),7.54–7.45(m,3H),7.26(dd,J＝9.2,2.4Hz,1H),6.95(d,J＝2.1Hz,1H),5.76(s,2H),4.75(t,J＝5.3Hz,1H),3.67–3.52(m,4H),3.09(s,3H),1.29(s,12H). 13 C NMR(125MHz,DMSO-d 6 )δ(ppm):154.28,149.21,144.54,142.90,138.24,136.71,135.64,131.00,130.20,128.45,127.14,125.69,124.40,123.96,120.92,116.80,105.27,84.36,62.39,58.81,54.55,40.51,25.10.HR-MS(ESI):calcd.for[C 33 H 38 BN 2 O 3 + ]m/z 521.2970,found[M] + 521.2996.
Example 2
Synthesis of dye pre-Mito-2: similar to the pre-Mito-1 synthesis, except for the difference in starting material (3-methylpyridine), the product pre-Mito-2 (399mg, 50%) was obtained as a yellow solid.
1 H NMR(500MHz,DMSO-d 6 )δ(ppm):8.78(s,1H),8.44(s,1H),8.05(d,J＝6.7Hz,1H),7.82(s,1H),7.75–7.62(m,3H),7.49–7.36(m,2H),7.24(dd,J＝24.8,12.6Hz,2H),6.91(s,1H),4.72(s,1H),3.57(d,J＝40.3Hz,4H),3.06(s,3H). 13 C NMR(125MHz,DMSO-d 6 )δ(ppm):148.54,148.41,148.09,135.26,133.72,132.79,131.48,130.22,129.30,127.24,126.78,126.16,124.24,124.16,123.15,116.64,105.38,58.81,54.74,40.59.HR-MS(ESI):calcd.for[C 20 H 20 N 2 O]m/z 304.1576,found[M+H] + 305.1629.
Synthetic route to the probe (Mito-W2):
synthesis of probe Mito-W2: similar to the Mito-W1 synthesis, except for the difference in starting material (pre-Mito-2), an orange-colored crystalline product Mito-W2 (41 mg, 86%) was obtained.
1 H NMR(500MHz,DMSO-d 6 )δ(ppm):9.38(s,1H),8.96(d,J＝6.0Hz,1H),8.79(d,J＝8.2Hz,1H),8.14(dd,J＝8.2,6.0Hz,1H),7.85(s,1H),7.83–7.65(m,6H),7.56(d,J＝8.1Hz,2H),7.36(d,J＝16.4Hz,1H),7.23(dd,J＝9.2,2.5Hz,1H),6.91(d,J＝2.2Hz,1H),5.86(s,2H),4.72(t,J＝5.3Hz,1H),3.59(t,J＝5.6Hz,2H),3.53(t,J＝5.9Hz,2H),3.05(s,3H),1.28(s,12H). 13 C NMR(125MHz,DMSO-d 6 )δ(ppm):148.63,142.62,142.34,141.53,139.05,137.73,136.84,135.95,135.65,129.71,128.95,128.85,128.70,127.07,125.89,123.98,119.27,116.77,105.29,84.38,63.91,58.79,54.63,40.60,25.10.HR-MS(ESI):calcd.for[C 33 H 38 BN 2 O 3 + ]m/z 521.2970,found[M] + 521.2917.
Example 3
Synthesis of dye pre-Mito-3: similar to the pre-Mito-1 synthesis, except for the difference in starting material (2-methylpyridine), the product pre-Mito-3 (356 mg, 45%) was obtained as a yellow solid.
1 H NMR(500MHz,DMSO-d 6 )δ(ppm):8.57(d,J＝3.8Hz,1H),7.86(s,1H),7.81–7.71(m,4H),7.65(d,J＝8.6Hz,1H),7.53(d,J＝7.9Hz,1H),7.29(d,J＝16.0Hz,1H),7.26–7.20(m,2H),6.91(d,J＝2.3Hz,1H),4.72(t,J＝5.3Hz,1H),3.67–3.49(m,4H),3.06(s,3H). 13 C NMR(125MHz,DMSO-d 6 )δ(ppm):155.93,149.93,148.17,137.21,135.43,133.11,129.89,129.40,127.88,126.80,126.36,126.18,124.40,122.52,122.36,116.59,105.36,58.81,54.73,40.60.HR-MS(ESI):calcd.for[C 20 H 20 N 2 O]m/z 304.1576,found[M+H] + 305.1625.
Synthesis route of probe (Mito-W3)
Synthesis of probe Mito-W3: similar to the Mito-W1 synthesis procedure, except for the difference in starting material (pre-Mito-3), a dark red crystalline product Mito-W3 (38 mg, 81%) was obtained.
1 H NMR(500MHz,DMSO-d 6 )δ(ppm):9.06(d,J＝6.2Hz,1H),8.63–8.49(m,3H),7.96(dd,J＝20.9,11.4Hz,3H),7.75(d,J＝9.2Hz,2H),7.68(dd,J＝8.4,4.4Hz,2H),7.52(d,J＝15.7Hz,1H),7.35(d,J＝7.9Hz,2H),7.25(dd,J＝9.2,2.4Hz,1H),6.93(d,J＝2.1Hz,1H),6.15(s,2H),4.74(t,J＝5.3Hz,1H),3.67–3.49(m,4H),3.08(s,3H),1.23(s,12H). 13 C NMR(125MHz,DMSO-d 6 )δ(ppm):153.16,149.32,146.28,145.06,144.69,138.02,136.82,135.54,131.57,130.28,128.17,127.29,127.12,125.72,125.53,125.14,124.39,116.82,114.64,105.25,84.31,60.43,58.80,54.55,40.60,25.09.HR-MS(ESI):calcd.for[C 33 H 38 BN 2 O 3 + ]m/z 521.2970,found[M] + 521.2922.
HPLC-MS test method and conditions
HPLC-MS mobile phase is methanol-water (0.1% HCOOH), flow rate 1mL/min (gradient setup: equilibration time 3min 40% CH 3 OH/60％H 2 O；0-5min:40％CH 3 OH/60％H 2 O-50％CH 3 OH/50％H 2 O；5-10min:50％CH 3 OH/50％H 2 O-70％CH 3 OH/30％H 2 O；10-15min:70％CH 3 OH/30％H 2 O-90％CH 3 OH/10％H 2 O；15-20min:90％CH 3 OH/10％H 2 O-100％CH 3 OH/0％H 2 O), uv detector (wavelength 254 nm) distinguishes chromatographic peaks, mass spectrum detector (ISQ-EC) recognizes molecular weights of different components. The probes Mito-W1/Mito-W2/Mito-W3 (50. Mu.M) in CH 3 OH (80%) and H 2 O (20%) mixed solution is respectively mixed with H 2 O 2 (5 mM) HPLC-MS test was performed after various times of reaction at 37 ℃. Intermediate captured by MS to speculate that the pinacol ester of phenylboronic acid compound was reacted with H 2 O oxidation reaction mechanism.
3. Absorption/emission spectrum test method and conditions
A certain amount of dye (pre-Mito-1/pre-Mito-2/pre-Mito-3) and probe (Mito-W1/Mito-W2/Mito-W3) solids were accurately weighed and prepared into a mother solution with a concentration of 1mM by using biological-grade DMSO. The solvent used for the spectroscopic test was PBS buffer (ph=7.42, 10mM,0.02%Triton X-100) with a sample concentration of 10 μm.
3.1 calculation of relative fluorescence Quantum yield
Using the reference method, the fluorescence quantum yield of the dye was based on fluorescein as reference substance (Φ in 0.1M NaOH solution r =0.92), the fluorescence quantum yield of the probe was referenced to rhodamine B (Φ in absolute ethanol r =0.89). The relative fluorescence quantum yields of the dye and probe in PBS buffer (ph=7.42, 10mM,0.02%Triton X-100) were calculated according to formula (1), respectively.
Wherein, the phi fluorescence quantum yield, A is absorbance, F is fluorescence integration area, and n is refractive index of the solution. r and s represent the standard substance and the substance to be measured, respectively.
3.2 reaction kinetic constant test
1. Mu.M probe (Mito-W1/Mito-W2/Mito-W3) was incubated with 10mM H in PBS buffer (pH=7.42, 10mM,0.02%Triton X-100), respectively 2 O 2 Mixing well, and recording fluorescence emission spectrum (lambda) at 400-650nm with fluorescence spectrometer ex =359 nm/358nm/378 nm), the relationship between the time and the fluorescence intensity was calculated and linear fitting was performed, and the first order reaction kinetic constant K was measured according to formula (2).
Wherein F is final As the final fluorescence intensity value, F t The fluorescence intensity values at different time points are shown, and t is time.
3.3 detection Limit test
10. Mu.M probe @ in PBS (pH=7.42, 10mM,0.02%Triton X-100) -acetone (V: V=1:1) solutionMito-W1/Mito-W2/Mito-W3) is added with 0-10eq of H respectively 2 O 2 (0-100. Mu.M), after incubation at 37℃for 2h, spectroscopic testing (lambda.) was performed using a fluorescence spectrometer ex =359 nm/358nm/378 nm), calculate H 2 O 2 The relationship between the concentration (0-100. Mu.M) and the fluorescence intensity of the probe (10. Mu.M) was subjected to linear fitting, and the detection limit LOD was measured according to the formula (2).
Where σ is the standard deviation of 11 blank samples tested, k is H 2 O 2 Slope of the linear relationship of concentration and fluorescence intensity.
3.4 anti-tamper test
The preparation of 10mM interference factor stock solution comprises: f1. F - ；2.Cl - ；3.Br - ；4.I - ；5.NO 3 - ；6.HS - ；7.SCN - ；8.HSO 3 - ；9.H 2 PO 4 - ；10.HCO 3 - ；11.SO 4 2- ；12.S 2 - ；13.CO 3 2- ；14.HPO 4 2- ；15.Na + ；16.K + ；17.Mg 2+ ；18.Zn 2+ ；19.Cu 2+ ；20.Fe 2+ ；21.Mn 2+ ；22.Ba 2+ ；23.Ni 2+ ；24.Ca 2+ ；25.Cd 2+ ；26.Fe 3+ ；27.Al 3+ ；28.Cr 3+ ；29.Gly；30.Ala；31.Val；32.Ile；33.Phe；34.Trp；35.Met；36.Pro；37.Ser；38.Thr；39.Cys；40.Tyr；41.His；42.Lys；43.Arg；44.Asp；45.Glu；46.H 2 O 2 ；47.·OH；48.ONOO - ；49.ROO·.50. 1 O 2 ；51.ClO - . 10. Mu.M probe and 100. Mu.M different interference factors were added to PBS buffer (pH=7.42, 10mM,0.02%Triton X-100), and after incubation at 37℃for 2h, fluorescence intensities of the probe at the dual excitation wavelengths (probe optimal absorption wavelength and dye optimal absorption wavelength) were recorded using a fluorescence spectrometer, respectively.
4. Biocompatibility Log P test
Octanol-water partition coefficient (log P) is a membrane permeability in vitro simulated test of a compound. In a 5mL EP tube, n-octanol (2 mL) and water (2 mL) were added, along with a 10. Mu.M sample, the mixture was centrifuged at 5000rpm for 3min after vortexing, the n-octanol layer was separated from the water layer, and the absorbance of the two phases was measured using UV-visible absorption spectroscopy. The Log P value is measured according to equation (4).
Wherein A is o And A w Absorbance of the sample in n-octanol and water phase, respectively.
5. Cell experiment
5.1 cell culture
Human HepG2 (hepatoma cell) and SH-SY5Y (neuroblastoma cell), U87 (glioma cell), HUVED (umbilical vein endothelial cell) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1% streptomycin and penicillin at 37℃under 5% CO 2 Is a cell culture box.
5.2 cell imaging experiments
Cells were inoculated in confocal dishes, confluence was 60% after adherence overnight, phenol red-free serum-free medium containing Mito-W2 and Mitotracker Deep Red at 1 μΜ was added, incubated with cells for 30min, washed 2 times with PBS, and laser confocal imaging was performed in phenol red-free serum-free medium. Imaging conditions: mito-W2 excitation light lambda ex Emission light λ=405 nm em Receiving wave band: 450-550nm; mitotracker Deep Red excitation λex=650 nm, emission λem reception band: 600-700nm.
5.3 flow cytometer detection
Cells were seeded in 6-well plates, incubated with phenol red-free serum-free medium cells containing 1 μm Mito-W2 and Mitotracker Deep Red for 30min, collected by digestion centrifugation (n=3), washed one to two times with PBS, detected by flow cytometry, excitation wavelength 405nm, detection wavelength 525nm.
5.4 Amplex Red detection
Cell mitochondria were isolated by differential centrifugation and mitochondrial H was detected by fluorescence according to the kit protocol (Invitrogen A22188) 2 O 2 Is a level of (c).
6. Animal experiment
Adult male C57 mice were selected from the experimental animals. In the first part, 200 mu L Mito-W2 with the concentration of 2 mu M/L is injected into tail vein after different time points (1 d/3d/5 d) after 70% partial hepatectomy, the liver is extracted after 2h injection, and a small animal living body imager is used for organ imaging, and a control group is a sham operation group; the second part, based on mCAT adeno-associated virus treated mice, was subjected to 70% hepatectomy, and after 1d tail vein injection Mito-W2, and after 2h, liver was extracted for organ imaging. Meanwhile, H in isolated mitochondria of liver tissue was detected using a commercial Amplex Red hydrogen peroxide detection kit (Invitrogen A22188) 2 O 2 Horizontal.
7. Clinical serum sample experiment
Serum samples were from Tang Dou hospital, beijing power release army Hospital, beijing rocket army Hospital. The disease group is myocardial infarction patient (Stroke) or Parkinson's Disease (PD), and the normal control group is selected from healthy people matched with the age range of the disease group. Firstly, adding an equal volume of acetone into serum, uniformly mixing, centrifuging at 12000rcf/min for 10min at 4 ℃, extracting supernatant and preserving at 0 ℃. The purpose is as follows: proteins in serum are removed, and autofluorescence of the proteins is avoided to interfere with detection results. Mito-W2 (1. Mu.M) was then added to the supernatant, and immediately fluorescence test (fluorescence intensity: F1) was performed, and after incubation for 3 hours, fluorescence test (fluorescence intensity: F2) was performed again. Finally, the change in fluorescence intensity before and after the reaction (F2-F1) was subjected to statistical analysis (t-test).
The invention provides a fluorescent probe for detecting mitochondrial hydrogen peroxide, which is designed to target mitochondrial H 2 O 2 Low background, specific fluorescent probes. The design of the fluorescent probe is based on an intramolecular distorted charge transfer (tic) mechanism. Wherein the electron donating group (Donor, D) is naphthylamine, and hydroxyethyl is introduced on amino nitrogen atom to increase molecular water solubilityThe method comprises the steps of carrying out a first treatment on the surface of the The electron withdrawing group (A) moiety contains a group which can be substituted with H 2 O 2 Specifically oxidizing the pyridinium salt of the identified pinacol ester of p-methylphenylboronic acid; the D and A parts are connected through pi bridge to construct D-pi-A type molecule, thus facilitating electron delocalization and increasing charge transmission performance. The overall intrinsic positive charge of the probe molecule effectively targets mitochondria (mitochondrial membrane potential-180 mV) by electrostatic attraction. Experimental results show that, with H 2 O 2 Prior to reaction, the probe Mito-W2 was extremely low in Brightness (Brightness (Mito-W2) =15), and H 2 O 2 After the reaction, the dye was converted to a dye pre-Mito-2 Brightness (Brightness (pre-Mito-2) =2555), is 1113 times the pre-reaction probe fluorescence intensity, achieving significant fluorescence "Turn-on". The selective test result shows that the probe pair H 2 O 2 Can specifically recognize and is not interfered by other molecules, and the detection Limit (LOD) is as low as 14.05nM. The above results indicate that Mito-W2 can be used as a low background specificity detection for mitochondrial H 2 O 2 Is provided.
In addition, we have systematically studied the pyridinium salts H at different pyridinium nitrogen positions (para/meta/ortho) linked to pi bonds 2 O 2 The photophysical properties of the probe (Mito-W1/Mito-W2/Mito-W3) verify that the probe Mito-W2 designed according to the invention has a low background for detecting mitochondrial H 2 O 2 Is a characteristic of (a). The invention provides two other (para and ortho) different pyridinium salts H 2 O 2 The probes (Mito-W1 and Mito-W3) were compared in nature and the three probe structures were as follows:
1. first, as shown in the diagrams (a-d) of FIG. 1, a probe (Mito-W1/W2/W3) was combined with H by high performance liquid chromatography-mass spectrometry (HPLC-MS) 2 O 2 The position of the chromatographic peak after the reaction and the molecular weight of the corresponding component are qualitatively characterized by the gradual conversion of the monitoring probe from the intermediate to the dye (pre-Mito-1/2/3).
2. Next, as shown in the graph (A-B) of FIG. 2,the photophysical properties of the three probes and dyes in PBS buffer (ph=7.42, 10mM,0.02%Triton X-100) were studied by an ultraviolet-visible spectrophotometer (UV-Vis spectrophotometer) and fluorescence spectrometer (Fluorescence spectrophotometer) system, including optimal absorption emission wavelength, stokes shift, relative fluorescence quantum yield, molar extinction coefficient, brightness (as shown in table 1). The optimal absorption/emission wavelengths of the probe Mito-W1 and the dye pre-Mito-1 are 470/647nm,359/521nm, respectively, and the Brightness (Brightness) is 514 and 543; the optimal absorption/emission wavelengths of Mito-W2 and pre-Mito-2 are 395/600nm,358/493nm and Brightness of 15 and 2555 respectively; the optimal absorption/emission wavelengths for Mito-W3 and pre-Mito-3 are 477/625nm,378/502nm, respectively, and Brightness is 402 and 2476. Indicating probe and H 2 O 2 After the reaction, the dye is converted into a dye with high brightness, and fluorescence of Turn-on is realized.
Table 1 shows the basic photophysical data of the dyes and probes.
3. Next, probe pair H was evaluated by fluorescence spectroscopy testing 2 O 2 Is a response (sensitivity and selectivity) capability of (a) a target. As shown in FIG. 3, panels (A-F), H was added to probe Mito-W1 (1. Mu.M) at 359nm as excitation wavelength 2 O 2 After (1 mM), the fluorescence intensity of the probe was significantly increased with time, and the reaction was completed at 110min, and the apparent rate constant K of the first order reaction was measured obs (Mito-W1)＝0.0118min -1 The method comprises the steps of carrying out a first treatment on the surface of the Mito-W1 (10. Mu.M) was added with different concentrations of H 2 O 2 Then, 359nm is used as excitation wavelength, and the fluorescence intensity of the probe is along with H 2 O 2 The concentration is increased and enhanced, and the linear relation is better in the range of 0-40 mu M, so that the detection limit LOD (Mito-W1) =53.15 nM is calculated; mito-W1 (10. Mu.M) and common anions and cations, amino acids and ROS (100. Mu.M) in organisms were incubated at 37deg.C for 2H, respectively, with 470/359nm as excitation wavelength, fluorescence test showed that H was removed 2 O 2 In addition, mito-W1 has no fluorescence intensity in the presence of other biomoleculesObvious change, indicating that the probe is H 2 O 2 Specific response, fluorescence was 93-fold enhanced.
As shown in FIG. 4, panels (A-F), probe Mito-W2 (1. Mu.M) was excited at 358nm, and H was added 2 O 2 After (1 mM), the fluorescence intensity of the probe was significantly increased with time, and the reaction was completed at 110min, and the apparent rate constant K of the first order reaction was measured obs (Mito-W2)＝0.0151min -1 The method comprises the steps of carrying out a first treatment on the surface of the Mito-W2 (10. Mu.M) was added at various concentrations of H 2 O 2 Then, the detection limit LOD (Mito-W2) =14.05 nm is calculated with a better linear relation in the range of 0-100 mu M; different biomolecular experiments showed Mito-W2 (10. Mu.M) vs. H 2 O 2 Specific response, fluorescence was significantly enhanced 1113-fold, achieving significant fluorescence "turnon".
As shown in FIG. 5, panels (A-F), probe Mito-W3 (1. Mu.M) was excited at 378nm, and H was added 2 O 2 After (1 mM), the fluorescence intensity of the probe was significantly enhanced with time, and the reaction was completed at 70min, and the apparent rate constant K of the first order reaction was measured obs (Mito-W3)＝0.0476min -1 The method comprises the steps of carrying out a first treatment on the surface of the Mito-W3 (10. Mu.M) was added at various concentrations of H 2 O 2 (0-100 μm), has a good linear relationship, and the detection limit LOD (Mito-W3) =44.79 nm; different biomolecular experiments showed Mito-W3 (10. Mu.M) vs. H 2 O 2 The specific response fluorescence was increased 407-fold.
4. The biological properties of the probes and dyes were further studied by oil/water separation coefficient (Log P) and Zeta potential tests. As shown in figure 6, panel a, the probe and dye (Log P < 0.7) have good lipophilic/oleophilic properties, and as shown in figure 6, panel B, zeta potential characterization results demonstrate that the probe is positively charged (> 10 mV) in aqueous solution, enabling targeting of mitochondria with negative membrane potential by electrostatic adsorption.
As a result of comparison of the above experimental results, mito-W2 is a P H 2 O 2 The probe with high selectivity and high sensitivity response has lower fluorescence before detection, and the fluorescence intensity after detection is obviously enhanced, and can be used as a low background H 2 O 2 Fluorescent probes were used in biological studies, thus also validating our original setupAnd (5) counting.
5. Laser confocal fluorescence imaging (Confocol), flow cytometry and commercial kit research probe Mito-W2 for mitochondrial H in cell and animal models 2 O 2 Horizontal detectability.
Exogenous H 2 O 2 And (3) detection:
as shown in FIG. 7, panel A, different concentrations of H were added to each of the four different types of cells HepG2, SH-SY5Y, U, HUVED, respectively 2 O 2 Confocal fluorescence imaging was performed after (0-10. Mu.M) co-incubation with Mito-W2 (1. Mu.M) and commercial dye Mitotracker Deep Red (1. Mu.M). With H 2 O 2 The increase of the dosage, the fluorescence signal of the cells is gradually enhanced and better in co-localization with Mitotracker Deep Red, and the mitochondrial targeting ability of Mito-W2 in the cells is verified. Mito-W2 (1. Mu.M) labelled cells were then detected by flow cytometry at different concentrations of H 2 O 2 (0-10. Mu.M) change in fluorescence intensity after treatment, consistent with confocal imaging results, as shown in FIG. 7, panel B. Finally, mitochondria in cells were extracted, and fluorescence intensity in mitochondria was detected with a commercial kit (duplex red kit), consistent with Mito-W2 confocal imaging and flow cytometry detection results, as shown in FIG. 7, panel C. The results in summary show that: mito-W2 has good compliance and high sensitivity in common fluorescent signal detection platforms such as microscopic imaging and flow.
Endogenous H 2 O 2 And (3) detection:
(1) Directly or indirectly regulating and controlling endogenous H of HepG2 and SH-SY5Y cells through gene editing means 2 O 2 Horizontal. First at 10 mu M H 2 O 2 The cells are transfected by using a viral vector on the basis of the treated cells to enable the cells to specifically overexpress mitochondrial catalase mCAT (control), thereby directly reducing H in mitochondria 2 O 2 After incubation with Mito-W2 for 30min, the fluorescence signal was detected by confocal microscopy (Mitotracker Deep Red co-localization) and flow cytometry. Addition of exogenous H to HepG2 control cells 2 O 2 Post fluorescence is significantly enhanced, while H 2 O 2 Treatment of mCAT expressing cellsNo significant enhancement of the fluorescence signal was seen, as shown in fig. 8, panel a. In the case where the processing conditions were unchanged, the flow cytometer (shown in fig. 8, panel B) and the kit detection result (shown in fig. 8, panel C) were consistent with the confocal. SH-SY5Y cells were similar to HepG2 cells in experimental results. The results show that: intracellular mCAT overexpression can significantly inhibit exogenous H 2 O 2 Mito-W2 specifically detectable mCAT overexpression induced mitochondrial H 2 O 2 And (3) lowering.
(2) Monoamine oxidase (MAO) of different subtypes is overexpressed by HepG2 and SH-SY5Y, respectively (MAO-B is overexpressed by HepG2, MAO-A is overexpressed by SH-SY 5Y), thereby causing mitochondrial H 2 O 2 The elevated levels were followed by mitochondrial antioxidant Mito Q treatment (control) and then incubation with Mito-W2 for 30min, after which fluorescence signals were detected using confocal microscopy (Mitotracker Deep Red co-localization) and flow cytometry. Confocal imaging results show that: mito-W2 showed a significant increase in fluorescence signal in MAO-overexpressing cells, whereas after Mito Q treatment, the signal was significantly reduced as shown in panel D of FIG. 8. SH-SY5Y cells were consistent with HepG2 cell experiments. The flow cytometer detection results (as shown in fig. 8, panel E) and the kit detection results (as shown in fig. 8, panel F) were consistent with confocal under unchanged processing conditions. The results show that: MAO overexpression can significantly increase endogenous H of cells 2 O 2 At the level, mito Q can effectively remove H 2 O 2 . Mito-W2 specifically detects MAOB over-expression induced mitochondrial H 2 O 2 Elevation and exogenous mitochondrial antioxidant enzyme induced H 2 O 2 And (3) lowering.
Mouse liver resection model:
Mito-W2 sensitively reflects intrahepatic H at various time points of hepatectomy using a 70% post hepatectomy regeneration model in mice as shown in FIG. 9, panel A 2 O 2 Level change of (2); as shown in FIG. 9, panel C, treatment of mice with adeno-associated virus to overexpress mCAT significantly inhibited liver tissue H1 d after hepatectomy 2 O 2 The level, as shown in FIG. 9, panel B or panel D, was consistent with the results of the kit liver tissue homogenate assay. Results tableBright: mito-W2 can realize endogenous H at tissue and organ level 2 O 2 Sensitive detection of changes.
6. Finally, the application performance of the probe Mito-W2 in clinical samples is studied by a fluorescence spectrometry. On the one hand, fluorescence detection was performed after incubation with Mito-W2 (1. Mu.M) added to the serum of the myocardial infarction patient (n=13) and the serum of the normal person of the same age (n=13), and as a result, as shown in FIG. 9, the difference in fluorescence intensity of the myocardial infarction group was significantly higher before and after the change than that of the normal control group (P=0.0058). On the other hand, fluorescence detection was performed after incubation with Mito-W2 (1 μm) added to PD patients (n=26) and normal human serum of the same age (n=26), respectively, and as a result, as shown in fig. 9, panel F showed that the difference in fluorescence intensity before and after PD group was significantly higher than that in the normal control group (P < 0.0001). The results show that: mito-W2 can be used for detecting clinical samples of mitochondrial oxidative stress related diseases.
The probe synthesized by the invention has excellent photophysical property and is suitable for mitochondria H 2 O 2 The detection of the method has the characteristics of good selectivity, high sensitivity, low detection limit and the like; the probe has the advantages of low cost of synthetic raw materials, simple synthetic route, mild reaction conditions, simple treatment, higher yield and commercialization;
the probe synthesized by the invention has good biocompatibility and inherent mitochondrial targeting capability, so that the probe is suitable for living cell mitochondrial H 2 O 2 Specific detection and fluorescence microscopic imaging analysis of (2); the probe has low fluorescence background and high sensitivity, and is matched with H 2 O 2 After reaction, the obvious fluorescence 'Turn on' is realized, so that the interference of autofluorescence of biological tissues is avoided, and the method can be used for mitochondrial level H of living bodies and clinical serum samples 2 O 2 The detection has commercial development application value.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims (7)

1. A method for preparing a fluorescent probe for detecting mitochondrial hydrogen peroxide, which is characterized by comprising the following steps:
s1: adding dye and acetonitrile, heating to a preset temperature, and stirring until the dye and the acetonitrile are completely dissolved;
s2: adding 4-bromomethyl phenylboronic acid pinacol ester and introducing N 2 Reflux reaction;
s3: after the reaction is completed, standing and cooling to room temperature, recrystallizing to separate out solid, and purifying to obtain a target product;
when the dye isWhen the target product is
When the dye isWhen the target product is
When the dye isWhen the target product is
The structural formula of the fluorescent probe is as follows:
the preparation method of the dye is characterized by comprising the following steps:
s01: dissolving t-BuOK with anhydrous DMF under stirring, dissolving in N 2 Slowly injecting picoline under the atmosphere, and stirring the solution from colorless to brownish black;
s02: injecting na-CHO DMF solution, stirring at a certain temperature and reacting for a certain time;
s03: cooling to room temperature after the reaction is finished, adding distilled water, extracting by EA until an organic phase is colorless, washing, drying, removing a solvent, and purifying a crude product by column chromatography to obtain a target product;
when the picoline is 4-picoline, the target product is
Or (b)
When the picoline is 3-picoline, the target product is
Or (b)
When the picoline is 2-picoline, the target product is
The na-CHO has the following structural formula:
2. the method of claim 1, wherein in S1, the preparation process is performed in a 50mL Schlenk flask; the dosage of the acetonitrile is 5ml; the preset temperature is 50 ℃; in the step S2, the reaction time is 12h.
3. The method of claim 1, wherein in S3, the reaction is completely confirmed by TLC plate tracking; the purification process is to carry out suction filtration washing by adopting EA.
4. The method according to claim 1, wherein in S01, the stirring temperature is 40 ℃ and the stirring time is 1h.
5. The method according to claim 1, wherein in S02, TLC is used to monitor the progress of the reaction during the reaction; the certain temperature is 50 ℃, and the certain time is 4 hours.
6. The method according to claim 1, wherein in S03, the washing is performed with a potassium hydroxide solution; the drying is carried out by adopting anhydrous sodium sulfate; the chromatographic purification adopts petroleum ether and ethyl acetate for chromatographic purification; the volume ratio of petroleum ether to ethyl acetate is 2:1.
7. A fluorescent probe prepared by the preparation method of claim 1 for living cell mitochondria H 2 O 2 The application of the detection reagent for the specific detection and fluorescence microscopic imaging analysis is characterized in that the structural formula of the fluorescent probe is as follows:
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Citations (1)

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Publication number Priority date Publication date Assignee Title
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Publication number Priority date Publication date Assignee Title
CN106281310A (en) * 2016-08-09 2017-01-04 济南大学 A kind of Mitochondrially targeted hydrogen peroxide molecule fluorescent probe and its preparation method and application

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Title
Fluororeactands for the Detection of Saccharides Based on Hemicyanine Dyes with a Boronic Acid Receptor;Trupp, Sabine et al.;Microchimica Acta;第153卷;127-131 *

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