CN116514706A - A position-shifting two-color fluorescent probe, preparation method and application thereof - Google Patents

Abstract

The invention relates to a position migration type double-color fluorescent probe, a preparation method and application thereof, and belongs to the technical field of fluorescent probes. The structural formula of the fluorescent probe is The fluorescent probe dyes lipid drops when living cells are in a normal physiological state, emits blue fluorescence, and when cells undergo autophagy, part of probe molecules migrate from the lipid drops to lysosomes to emit red fluorescence, so that the application of double-color and dyeing position migration observation of the autophagy process of the cells is realized. The fluorescent probe has the characteristics of low price, quick dyeing time, no washing and good biocompatibility.

Description

A position-shifting two-color fluorescent probe, its preparation method and its application

technical field

The invention relates to a position-transfer type two-color fluorescent probe, a preparation method and an application thereof, and belongs to the technical field of fluorescent probes.

Background technique

Autophagy is a process in which cells engulf their own organelles or proteins to maintain their own metabolism or renew organelles. When autophagy occurs, the substances to be removed in the cytoplasm are first coated by vesicles called "isolation membranes", which eventually form a double-membrane structure called an autophagosome. Autophagosomes gradually fuse with lysosomes to form autolysosomes. Substances to be removed in the isolated cytoplasm can be degraded by hydrolytic enzymes in lysosomes into free nucleotides, amino acids or fatty acids and other small biomolecules, and these nascent biomolecules can be reused by cells. Therefore, the nutrient cycle of autophagy can effectively promote the survival of cells. Studies have shown that autophagy plays an important role in the occurrence and development of cancer and the response of tumor cells to anti-cancer treatment. In addition, the reduction of autophagy activity is closely related to aging, neurodegenerative diseases, cardiovascular diseases, autoimmune diseases, etc. relevant.

At present, the methods for studying autophagy mainly include Western Blot detection, transmission electron microscope observation and fluorescent protein observation. When autophagy is formed, the cytoplasmic LC3 protein will enzymatically hydrolyze a small section of polypeptide to form LC3-I, and LC3-I will combine with phosphatidylethanolamine to form LC3-II, so the change of LC3-II/I ratio can be detected by Western Blot Evaluation of autophagy formation. The microstructure of phagocytic vesicles, autophagosomes, and autolysosomes can be observed by transmission electron microscopy. Both of the above two methods are "destructive" analysis methods, and both need to fix the active cell samples first to make them lose their activity. They are not suitable for in situ, real-time, dynamic observation and analysis of the autophagy process of living cells. The fluorescent protein observation method can connect green fluorescent protein (GFP) to the LC3 protein to observe the location of GFP-LC3 aggregation in living cells. When autophagy occurs, GFP-LC3 translocates to the autophagosome membrane. Multiple bright green spots are formed below to observe the autophagy process. However, the operation steps of fluorescent protein technology are complicated and cumbersome, and the fluorescent protein is too large to interfere with the normal function of the target protein.

Compared with the above methods, the fluorescent imaging method based on molecular fluorescent probes can color living cells by simply incubating molecular fluorescent probes, and image and observe targets in a nearly non-destructive way under a fluorescent microscope. This method can not only realize in situ and real-time imaging of targets in living cells, but also observe target dynamics and detect changes in target concentration and distribution under various physiological and pathological conditions. At present, the molecular fluorescent probes used to observe the autophagy process are mainly based on lysosome probes, such as commercial lysosome tracking series probes LysoTrakcer Red, LysoTracker DeepRed, etc. When autophagy occurs, the number of lysosomes increases , the fluorescent signal in the cell will increase. However, it is difficult for such probes to give information about the whole process of autophagy, and the increase or decrease of fluorescence intensity is used as a response signal, which is easily affected by external factors such as probe distribution concentration, polarity, temperature, excitation light source of the instrument itself, detection Sensitivity, etc. Therefore, designing a fluorescent probe capable of real-time, in situ, and intuitive observation of the autophagy process in living cells remains a great challenge.

Contents of the invention

In view of this, the object of the present invention is to provide a position shifting type two-color fluorescent probe, a preparation method and an application thereof. The fluorescent probe stains lipid droplets and emits blue fluorescence when cells are in a normal physiological state. When cells undergo autophagy, some probe molecules migrate from lipid droplets to lysosomes and emit red fluorescence. The change of staining position can be used to visually observe the autophagy process of living cells.

To achieve the above object, the technical scheme of the present invention is as follows:

A position shift type two-color fluorescent probe, the structural formula of the fluorescent probe is as follows:

R for /> That is, the structure of the fluorescent probe is

Wherein, the fluorescent probe emits blue fluorescence in a lipophilic environment, and emits red fluorescence in an acidic environment; and the fluorescent probe can migrate from a lipophilic environment to an acidic environment.

A method for preparing a position-shifting two-color fluorescent probe of the present invention, the method steps comprising:

4-Bromotriphenylamine and 4-vinylpyridine generate (E)-N,N-diphenyl-4-(2-pyridine)vinyl)aniline (referred to as TPAP) through coupling reaction (Heck reaction), namely R is Position-shifting dual-color fluorescent probes;

4-Dianilinobenzaldehyde and 4-methylquinoline are obtained by the Knoevenagel reaction (Knoevenagel reaction) to obtain (E)-N,N-diphenyl-4-(2-quinoline)vinyl)aniline (record is TPAQ), that is, R is A position-shifting two-color fluorescent probe.

An application of the position-shifting dual-color fluorescent probe of the present invention, the fluorescent probe is used to observe the process of cell autophagy.

Preferably, when the fluorescent probe is used, when the living cell is in a normal physiological state, the lipid droplet is stained and emits blue fluorescence; when the cell undergoes autophagy, some fluorescent probe molecules migrate from the lipid droplet to the lysosome to emit Red fluorescence, so as to realize two-color, dyeing position migration to observe the process of cell autophagy.

Beneficial effect

The invention provides a position shifting type two-color fluorescent probe, which can visually observe the autophagy process of living cells through the change of fluorescent color and staining position in living cells. The mechanism of its fluorescent color and dyeing position change is shown in Figure 1: both TPAQ and TPAP are lipophilic fluorophores with a weak electron-donating-π-electron-withdrawing (D-π-A) structure. Lipophilicity and lower polarity, TPAQ and TPAP stained normal living cells first stained lipid droplets and emitted blue fluorescence. When autophagy occurs in cells, the pH of lysosomes in the cells decreases. Since TPAQ and TPAP are weakly alkaline, some molecules will migrate from lipid droplets to lysosomes. In the acidic environment of lysosomes, TPAQ and TPAP It will be protonated into TPAQ-H and TPAP-H, and its chemical structure will change from weak D-π-A structure to strong D-π-A structure, and the fluorescence will be significantly red-shifted. Therefore, the fluorescent probe can visually observe the autophagy process of living cells through the change of fluorescent color and staining position in living cells.

The invention provides a preparation method of a position-transfer type two-color fluorescent probe. The probe is prepared through a Heck reaction or a Brainwenger reaction, and the method is simple to operate.

The present invention provides the application of a position-shifting two-color fluorescent probe, which can first stain lipid droplets in living cells and emit blue fluorescence. Migrate to lysosomes and emit red fluorescence, so the autophagy process in living cells can be observed more intuitively through changes in color and staining position. The fluorescent probe of the invention has the characteristics of low price, fast staining time, no washing and good biocompatibility. It is especially suitable for real-time and in situ tracking of the autophagy process of living cells under confocal fluorescence microscopy and universal fluorescence microscopy, and has broad application prospects in the fields of fluorescent biomarkers and important biological process tracking.

Description of drawings

Fig. 1 is a mechanism diagram of the change of fluorescent color and staining position of the fluorescent probe of the present invention in the process of autophagy.

Fig. 2 is the pH titration experiment result of TPAQ (A) and TPAP (B) described in embodiment 3. Probe concentration was 10 μM.

FIG. 3 is a confocal fluorescent image of cancer cells (A549) stained in a normal physiological state and a starvation state in Example 4. FIG. The excitation wavelength of the blue light channel is 405nm, and the collection band is 410-480nm; the excitation wavelength of the red light channel is 488nm, and the collection band is 600-700nm. Scale bar is 20 μm.

Fig. 4 is a confocal fluorescence picture of the co-localization experiment with commercial lipid droplet probe Nile Red and lysosome probe LysoTrackerDeep Red (LTDR) described in Example 5. (A) The excitation wavelength of TPAQ and TPAP is 405nm, and the collection band is 410-480nm; the excitation wavelength of Nile Red is 561nm, and the collection band is 580-650nm; (B) The excitation wavelength of TPAQ and TPAP is 488nm, and the collection band is 600nm -700nm; the excitation wavelength of LTDR is 640nm, and the collection band is 650-700nm. Scale bar is 20 μm.

FIG. 5 is a confocal fluorescence picture of adding the autophagy inhibitor chloroquine as described in Example 6. The excitation wavelength of the blue light channel is 405nm, and the collection band is 410-480nm; the excitation wavelength of the red light channel is 488nm, and the collection band is 600-700nm. Scale bar is 20 μm.

Detailed ways

The present invention will be described in further detail below in conjunction with specific examples.

Example 1 Preparation of two-color, position-shifting fluorescent probes for observing autophagy

4-Dianilinobenzaldehyde (0.54g, 2mmol) was dissolved in dry DMF solvent, then 4-methylquinoline (0.29g, 2mmol) and potassium tert-butoxide (0.22g, 2mmol) were added, and the mixture was The reaction was stirred at room temperature for 24h. Then it was purified by silica gel column chromatography to finally obtain an orange-yellow solid with a yield of 8%.

Dissolve 4-bromotriphenylamine (0.97g, 3mmol) and 4-vinylpyridine (0.63g, 6mmol) in dry N-methylpyrrolidone solvent, add palladium acetate (0.067g, 0.3mmol), tri-orthomethyl phenylphosphine (0.27g, 0.9mmol), potassium carbonate (2.90g, 21mmol), and the resulting mixture was heated under reflux for 48h under the protection of nitrogen. After the reaction, the mixture was poured into water and extracted with dichloromethane. Then it was purified by silica gel column chromatography to finally obtain a yellow solid with a yield of 70%.

The preparation reaction formula is as follows:

The H NMR spectrum and carbon spectrum results of the orange-yellow solid are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ), δ(ppm): 8.86(d, J=4.80Hz, 1H), 8.49(d, J=8.40Hz, 1H), 8.02(d, J=8.40Hz, 1H), 7.95(d, J=16.00Hz, 1H), 7.83(d, J=4.80Hz, 1H), 7.79(d, J=6.80Hz, 1H), 7.74(d, J=8.40Hz, 2H) ,7.64(t,J=7.40Hz,1H),7.55(d,J=16.40Hz,1H),7.35(t,J=7.80Hz,4H),7.07-7.12(m,6H),6.99(d, J＝8.40Hz, 2H).

¹³ C NMR (100MHz, DMSO-d ₆ ), δ (ppm): 149.95, 148.13, 147.64, 146.59, 142.25, 134.39, 130.18, 129.72, 129.52, 129.39, 129.29, 129.20, 128.73, 128. 60, 128.04, 126.27, 125.58 , 124.36, 124.14, 123.89, 123.50, 122.09, 120.03, 116.05. The H NMR and C NMR results of the yellow solid are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ), δ (ppm): 8.54-8.46 (m, 2H), 7.58-7.45 (m, 5H), 7.38-7.27 (m, 4H), 7.11 (d, J= 7.1Hz, 2H), 7.08-7.02(m, 5H), 6.96(d, J=8.7Hz, 2H).

¹³ C NMR (100MHz, CDCl ₃ ), δ(ppm): 150.01, 148.50, 147.28, 145.13, 132.80, 129.78, 129.41, 129.35, 127.99, 124.92, 124.85, 123.86, 123.52, 122.82 ,120.66.

The results show that the orange-yellow solid is (E)-N,N-diphenyl-4-(2-quinoline)vinyl)aniline (TPAQ); the yellow solid is (E)-N,N- Diphenyl-4-(2-pyridine)vinyl)aniline (TPAP).

Embodiment 2 cancer cell (A549) culture

Cell lines were cultured at 37°C in a saturated humidity incubator with 5% CO ₂ . The A549 cell line was cultured adherently in H-DMEM medium containing 10% fetal bovine serum (containing 1% double antibody). When the cells grow to the logarithmic phase, wash the cells in the 100mL cell flask three times with PBS, digest with 1mL 0.25% trypsin for 3-5min, pour out the medium carefully, add a small amount of fresh medium and pipette evenly, and count the cells , leave the cells with a suitable density, add the medium to the required volume (control the final concentration of cells to be 1×10 ⁴ ), inoculate them into confocal glass-bottom culture dishes, and culture them in a CO ₂ incubator to make the cells grow in the glass Bottom-adherent growth.

The pH titration experiment of embodiment 3TPAQ and TPAP

Buffer solutions with pHs of 2.650, 2.923, 3.195, 3.332, 3.627, 3.939, 4.380, 4.765, 5.123, 5.421, 5.902, 6.224, 6.667, 7.091, and 7.569 were prepared respectively, and TPAQ and TPAP molecules were added at a molecular concentration of 10 μM. Test the UV absorption spectrum and fluorescence emission spectrum of TPAQ and TPAP in the above buffer.

The results are shown in Figure 2, with the decrease of pH, the absorption peak of TPAQ at 500nm is enhanced while the absorption peak at 400nm is decreased, the fluorescence peak at 560nm is decreased and the fluorescence peak at 670nm is enhanced. TPAP is similar to TPAQ. With the decrease of pH, the absorption peak of TPAP at 450nm increases and the absorption peak at 380nm decreases, and the fluorescence peak at 535nm decreases while the fluorescence peak at 620nm increases. According to the absorbance of TPAQ at 500nm and the absorbance of TPAP at 450nm, the calculated pKa value of TPAQ was 3.51, and the pKa value of TPAP was 4.01.

Example 4 staining experiments on cancer cells (A549) in normal physiological state and starvation state

Divide the confocal glass-bottom culture dishes inoculated with A549 cells into two groups. One group was directly stained with 2μMTPAQ and TPAP for 15min. The other group starved the cells with PBS buffer solution for 30 minutes to make the cells undergo autophagy, and then stained with 2 μM TPAQ and TPAP for 15 minutes.

After staining, laser scanning confocal microscopy was used to observe the stained parts of the cells. As shown in Figure 3, TPAQ and TPAP stained lipid droplets in A549 cells under normal physiological conditions and emitted blue fluorescence; Stain lipid droplets and lysosomes in A549 cells during phagocytosis, and blue and red fluorescence can be observed simultaneously.

Example 5 Colocalization experiment with commercial lipid droplet probe Nile Red and lysosome probe LysoTracker Deep Red (LTDR)

Divide the confocal glass-bottom culture dishes inoculated with A549 cells into two groups. One group was directly stained with 2 μM TPAQ and TPAP for 15 min, and then stained with 1 μM Nile Red for 15 min; the other group was starved with PBS buffer solution for 30 min to allow the cells to undergo autophagy, then stained with 2 μM TPAQ and TPAP for 15 min, and then stained with 0.2 μM LTDR staining for 15min.

After staining, laser scanning confocal microscopy was used to observe the stained parts of the cells. The results are shown in Figure 4. In cancer cells in a normal physiological state, the fluorescence signals of TPAQ, TPAP and Nile Red have a good overlap, indicating that TPAQ and TPAP Staining lipid droplets; in cancer cells in autophagy state, the fluorescent signals of TPAQ, TPAP and LTDR have good overlap, indicating that TPAQ and TPAP stain lysosomes when cells undergo autophagy.

Example 6 Adding the imaging experiment of autophagy inhibitor chloroquine

Divide the confocal glass-bottom culture dishes inoculated with A549 cells into two groups. One group first starved the cells with PBS buffer solution for 30 minutes to make the cells undergo autophagy, and then stained with 2 μM TPAQ and TPAP for 15 minutes; the other group first treated the cells with 50 μM chloroquine for 4 hours, then stained with 2 μM TPAQ and TPAP for 15 minutes, and then stained with PBS The buffer solution starved the cells for 30 min to allow the cells to undergo autophagy.

After staining, laser scanning confocal microscopy was used to observe the stained parts of the cells. The results are shown in Figure 5. When the cells undergo autophagy, the red fluorescence at the lysosomes is strong, and when the autophagy inhibitor chloroquine is added to make the lysosomes After the body was alkalized, the red fluorescence in the lysosome almost disappeared, indicating that TPAQ and TPAP would not change the fluorescent staining and staining position after autophagy was inhibited, and further indicated that TPAQ and TPAP could pass through the Changes in fluorescent staining and staining positions were used to observe the autophagy process.

In summary, the invention includes but is not limited to the above embodiments, and any equivalent replacement or partial improvement made under the spirit and principles of the present invention will be considered within the protection scope of the present invention.

Claims (4)

1. A position-shifting type double-color fluorescent probe, which is characterized in that: the structural formula of the fluorescent probe is as follows:

r is->

Wherein the fluorescent probe emits blue fluorescence in a lipophilic environment and emits red fluorescence in an acidic environment; and the fluorescent probe is capable of migrating from a lipophilic environment to an acidic environment.

2. A method for preparing the position-shifting type double-color fluorescent probe according to claim 1, wherein: the method comprises the following steps:

the 4-bromotriphenylamine and 4-vinyl pyridine are subjected to coupling reaction to generate (E) -N, N-diphenyl-4- (2-pyridine) vinyl) aniline, namely R isA position-shifting type double-color fluorescent probe;

reacting 4-diphenylaminobenzaldehyde with 4-methylquinoline to obtain (E) -N, N-diphenyl-4- (2-quinoline) vinyl) aniline by brain Wen Ge, namely R isIs a position-shifting type double-color fluorescent probe.

3. Use of a position-shifting double-colour fluorescent probe according to claim 1, characterized in that: the fluorescent probe is used to observe the autophagy process.

4. Use of a position-shifting bicolor fluorescent probe as claimed in claim 3, wherein: when the fluorescent probe is used, the lipid drops are stained when living cells are in a normal physiological state, and blue fluorescence is emitted; when autophagy occurs in cells, part of fluorescent probe molecules migrate from lipid droplets to lysosomes to emit red fluorescence, so that the double-color and staining site migration observation of the autophagy process of cells is realized.