Potential of hydrogen (pH) fluorescent probe based on sulfonium perchlorate and application of pH fluorescent probe

Abstract

The invention discloses a pH fluorescent probe based on sulfonium perchlorate. The structural formula of the pH fluorescent probe is as follows: # imgabs0 #. The invention also provides a preparation method of the pH fluorescent probe based on sulfonium perchlorate. The invention also provides an application of the pH fluorescent probe based on the sulfonium perchlorate in preparation of a positioning agent used as an intracellular lysosome. The invention further provides application of the pH fluorescent probe based on the sulfonium perchlorate in preparation of a reagent serving as a weakly acidic microenvironment in in-vivo visual tumor tissues. The probe provided by the invention has near-infrared emission fluorescence of 710 nm and large Stokes shift of 214 nm, has remarkable lysosome targeting ability, and shows excellent reversibility, selectivity and light stability at the same time. The pKa value of the probe is 7.5, and the probe can be used for visualization of a weakly acidic microenvironment of tumor tissues in a mouse body and has an application prospect of early tumor detection.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry and relates to a fluorescent probe, in particular to a pH fluorescent probe based on sulfonium perchlorate and an application thereof. Background Art

[0002] pH plays an important role in many physiological processes of life systems, including cell metabolism, growth, proliferation, apoptosis, signal transduction, and ion transport. Maintaining proper acid-base balance is essential for normal physiological functions and overall health. Studies have shown that abnormal intracellular pH can lead to enzyme activity disorders, increased free radical production, cell dysfunction, abnormal protein deposition, and other problems. These abnormal phenomena can easily lead to cancer, renal failure, lung disease, Alzheimer's disease and other diseases.

[0003] Taking tumors as an example, a notable feature of tumor tissue is its acidic microenvironment. The pH value of normal tissue is usually 7.2-7.4, while the pH value of tumor tissue is 6.2-6.9. The abnormal pH is caused by the active metabolism of tumor cells. Insufficient blood supply and hypoxia inside the tumor lead to the accumulation of lactic acid and other acidic metabolites. This acidic environment not only promotes the growth and spread of tumor cells, but may also affect the function of immune cells, thereby accelerating the development of tumors. Therefore, real-time monitoring of pH fluctuations in cells and in vivo systems is of great significance for a deep understanding of physiological and pathological processes related to pH.

[0004] Small molecule fluorescent probes developed based on fluorescence imaging technology have the advantages of non-invasiveness, fast response speed, and good real-time performance, and have become an indispensable tool in life sciences and medical diagnosis. Among them, near-infrared fluorescent probes with emission wavelengths in the range of 650-900nm (NIR-I) have attracted much attention due to their deep tissue penetration, low photodamage to biological samples, and weak autofluorescence of biological molecules. Given the unique advantages of near-infrared fluorescent probes in non-invasive optical imaging, the design of near-infrared fluorescent probes for real-time monitoring of pH fluctuations in cells and in vivo systems has shown good application prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a pH fluorescent probe based on sulfonium perchlorate and its application. The pH fluorescent probe based on sulfonium perchlorate and its application are intended to solve the technical problems of poor visibility and positioning of pH fluorescent probes in the prior art.

[0006] The present invention provides a pH fluorescent probe based on sulfonium perchlorate, and its structural formula is:

[0007]

[0008] The name of the probe is (E)-7-(dibutylamino)-4-(4-hydroxyphenylvinyl)-2-phenylsulfonium perchlorate perchlorate (SOH).

[0009] Its synthetic route is:

[0010]

[0011] Furthermore, the above-mentioned method for preparing a pH fluorescent probe based on sulfonium perchlorate comprises the following steps:

[0012] (1) adding polyphosphoric acid, 3-bromothiophenol and ethyl 3-oxo-3-phenylpropionate into a reaction container, mixing, reacting, and extracting after the reaction is complete to obtain compound 1;

[0013] (2) Compound 1, Pd 2 (dba) 3 , DavePhos (2-dicyclohexylphosphino-2'-(N,N-dimethylamino)-biphenyl) and Cs 2 CO 3 Add to a second reaction container, evacuate by filling with nitrogen, first add anhydrous dioxane, then add dibutylamine, and purify after the reaction is complete to obtain compound 2;

[0014] (3) In a nitrogen-filled third reaction vessel, compound 2 is dissolved in the first organic solvent, and then CH 3 MgBr, and stirred at room temperature for 1-4 hours, then the solution was poured into an aqueous perchloric acid solution with a mass percentage concentration of 8-12%, and purified to obtain compound 3;

[0015] (4) dissolving p-hydroxybenzaldehyde and compound 3 in a second organic solvent and reacting them under an argon atmosphere. After the reaction is completed, concentrating the reaction mixture by evaporation under reduced pressure and purifying it to obtain a pH fluorescent probe based on sulfonium perchlorate salt.

[0016] Furthermore, in step 1), the mass ratio of polyphosphoric acid, 3-bromothiophenol and ethyl 3-oxo-3-phenylpropionate is: (18-25): (1-3): (2-3).

[0017] Further, in step 2), compound 1, Pd 2 (dba) 3 , DavePhos (2-dicyclohexylphosphino-2'-(N,N-dimethylamino)-biphenyl) and Cs 2 CO 3, anhydrous dioxane, dibutylamine material ratio is (600-750 mg): (40-55 mg): (15-25 mg): (1.5-2 g): (8-20 mL): (1-3 mL). Further, in step 3), compound 2, CH 3 The material ratio of MgBr and a perchloric acid aqueous solution with a mass percentage concentration of 8-12% is (300-400 mg): (3-4 mmol): (8-15 mL).

[0018] Furthermore, in step 4), the material ratio of p-hydroxybenzaldehyde to compound 3 is: (50-70 mg): (200-300 mg).

[0019] The present invention also provides a use of a pH fluorescent probe based on sulfonium perchlorate in the preparation of a localization agent for intracellular lysosomes.

[0020] The present invention also provides a use of a pH fluorescent probe based on sulfonium perchlorate in preparing a reagent for visualizing a weakly acidic microenvironment in tumor tissue in vivo.

[0021] The probe SOH of the present invention can be used to visualize the weakly acidic microenvironment of tumor tissue in mice, and shows good application prospects in the early detection of tumors.

[0022] In the preparation process of the present invention, the preparation of compound 1 is simple, the product is obtained by extraction, and no further purification is required. The next step of reaction operation is directly carried out, which is suitable for large-scale preparation. The preparation of compound 2 is a typical halogenated hydrocarbon nucleophilic substitution reaction. The reaction requires the use of a catalyst DavePhos (2-dicyclohexylphosphino-2'-(N,N-dimethylamine)-biphenyl), and the reaction time is about 18 hours. Although the time is long, the yield is high. Compound 3 is prepared by methylation reaction of compound 2, and the reaction is fast and the yield is high. The target product SOH is obtained by dehydrating compound 3 and p-hydroxybenzaldehyde. Although the yield is slightly low, the synthesis steps are simple and have potential commercial application value.

[0023] Compared with the existing pH fluorescent probes, the probe SOH of the present invention has the following advantages: (1) The pKa of the probe SOH is 7.5, and the pH linear response range is 6.5-8.2. The probe can be used to visualize the weakly acidic microenvironment of tumor tissue in mice, showing good application prospects in the early detection of tumors. (2) The maximum fluorescence emission of the probe SOH is located at 710nm, which belongs to the near-infrared region, and can effectively reduce the light damage to cells and reduce the interference of the spontaneous fluorescence of biological samples. (3) The probe SOH has a large Stokes shift (214nm), which can effectively reduce the interference from the excitation light. (4) The probe SOH has high sensitivity and selectivity for pH response and is not interfered by common cations and anions. (5) The probe SOH has excellent lysosomal targeting ability and exhibits excellent reversibility, selectivity and photostability. (6) The synthesis steps of this probe are simple, the cost is low, and it has potential commercial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 .The UV-visible absorption spectrum of the probe SOH of the present invention changes with pH value.

[0025] Figure 2 .The fluorescence emission spectrum of the probe SOH of the present invention changes with pH value.

[0026] Figure 3 . The F of the probe SOH of the present invention 710 nm Boltzmann function plot as a function of pH, pK a The value is 7.5.

[0027] Figure 4 . The F of the probe SOH of the present invention 710 nm It is a linear function of pH value, and the linear range is pH 6.5-8.2.

[0028] Figure 5 The probe SOH of the present invention reacts with H in the presence of common cations and anions. + selectivity.

[0029] Figure 6 .Co-localization imaging of the probe SOH of the present invention and the commercially available lysosomal green fluorescent probe Lyso-Tracker Green in human liver cancer cells (HepG2 cells).

[0030] Figure 7 .Fluorescence imaging of the probe SOH of the present invention in a tumor mouse model. DETAILED DESCRIPTION

[0031] Example 1

[0032] Preparation of probe (E)-7-(dibutylamino)-4-(4-hydroxyphenylvinyl)-2-phenylsulfonium perchlorate (SOH):

[0033]

[0034] (1) Polyphosphoric acid (PPA, 22 g), 3-bromothiophenol (2 g, 10.6 mmol) and ethyl 3-oxo-3-phenylpropionate (2.24 g, 11.66 mmol) were mixed and stirred at 95°C for 2 hours. After cooling to room temperature, ice water was added to terminate the reaction, and DCM (dichloromethane) (100 mL×3) was used for extraction. The combined organic phase was washed with Na 2 SO 4 Drying, filtration and evaporation gave compound 1 without further purification.

[0035] (2) Compound 1 (694 mg, 2 mmol), Pd 2 (dba) 3 (45.8 mg, 0.05 mmol), DavePhos (2-dicyclohexylphosphino-2'-(N,N-dimethylamino)-biphenyl) (19.7 mg, 0.05 mmol) and Cs 2 CO 3 (1.63 g, 5 mmol) was added to a test tube. The test tube was sealed and filled with nitrogen evacuation. Anhydrous dioxane (10 mL) was added, followed by dibutylamine (1.7 mL, 10 mmol). The reaction was stirred at 100 ° C for 18 hours. After cooling to room temperature, the mixture was filtered and the filtrate was evaporated. The crude product was further purified by silica gel column chromatography (EA / DCM=1 / 10, v / v) to obtain compound 2 (592 mg, 81%). 1 H NMR (400 MHz, DMSO-d 6 )δ(ppm):8.34(d,J=8.0Hz,1H),7.67-7.69(m,2H),7.46-7.48(m,3H),7.10(s,1H),6.82-6.84(dd,J 1 =2.4Hz,J 2 =2.4Hz,1H),6.63(d,J=4.0Hz,1H),3.36(t,J=8.0Hz,4H),1.58-1.66(m,4H),1.37-1.42(m,4H),0.99(t,J=8.0Hz,6H).MS(MM-ES+APCI)m / zcalcd for C 23 H 28 NOS + [M]+ ,366.2;Found,366.1.

[0036] (3) Compound 2 (365 mg, 1 mmol) was dissolved in anhydrous THF (10 mL) in a nitrogen-filled dry flask. 1.0 M CH 3 MgBr (3.6 mL) was added and stirred at room temperature for 2 hours. The solution was poured into a 10% mass percent concentration of perchloric acid aqueous solution (10 mL) and extracted with DCM (25 mL × 3). The combined organic phase was washed with Na 2 SO 4 Dry, filter and evaporate. The crude product was further purified by flash column chromatography (MeOH / DCM=1 / 5, v / v) to give compound 3 (371 mg, 80%). 1 H NMR (400 MHz, CDCl 3 )δ(ppm):8.43(d,J=10.0Hz,1H),7.93(s,1H),7.88-7.90(m,2H),7.56-7.61(m,3H),7.48-7.51(dd,J 1 =2.4Hz,J 2 =2.4Hz,1H),7.39(d,J=4.0Hz,1H),3.59(t,J=8.0Hz,4H),2.99(s,3H),1.67 -1.73(m,4H),1.44-1.50(m,4H),1.01(t,J=8.0Hz,6H).MS(MM-ES+APCI)m / z calcd for C 24 H 30 NS + [M] + ,364.2;Found,364.2.

[0037] (4) p-Hydroxybenzaldehyde (61 mg, 0.5 mmol) and compound 3 (232 mg, 0.5 mmol) were dissolved in a mixed solution of toluene / n-butanol (3 mL / 3 mL). Under an argon atmosphere, the reaction mixture was stirred at 100° C. overnight. After the reaction was completed, the reaction mixture was concentrated by evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to obtain the target product SOH with a yield of 284 mg and a yield of 50%. 1 H NMR (400 MHz, DMSO-d 6)δ(ppm):10.43(s,1H),8.91(d,J=12.0Hz,1H),8.49(s,1H),8.37(d,J=16 .0Hz,1H),8.13(d,J=16.0Hz,1H),8.02(d,J=8.0Hz,2H),7.91(d,J=8.0Hz, 2H),7.60-7.66(m,4H),7.43-7.46(m,1H),6.89(d,J=8.0Hz,2H),3.60(t, J=8.0Hz,4H),1.54-1.62(m,4H),1.31-1.41(m,4H),0.91(t,J=8.0Hz,6H). 13 C NMR (126 MHz, DMSO-d 6 )δ161.9,156.7,153.9,152.1,147.0,145.9,135.7,132.8,132.6,130.3,128 .2,127.7,119.7,119.4,116.7,107.1,51.0,29.6,20.0,14.3.HRMS(ESI)m / z calcdfor C 31 H 34 NOS + [M] + ,468.2356;Found,468.2167.

[0038] Example 2

[0039] The probe SOH prepared in Example 1 was prepared with DMSO (dimethyl sulfoxide) to prepare a stock solution with a concentration of 1 mM. 2 The probe was diluted to a final concentration of 10 μM using an O / DMSO (V / V=5 / 1) system, and the pH value of the system was adjusted using a high-concentration, small-volume NaOH solution. The UV absorption spectrum was recorded ( Figure 1 ). As the pH value increases, the absorption peak at 500nm gradually decreases, the absorption peak at 700nm increases accordingly, and there is an isosbestic point at 620nm.

[0040] Example 3

[0041] The probe SOH was diluted to a final concentration of 10 μM, and the pH value of the system was adjusted with a high-concentration and small-volume NaOH solution. The excitation wavelength was fixed at 600 nm, and its fluorescence emission spectrum was recorded ( Figure 2 ). With the increase of pH value, the fluorescence intensity at 710nm gradually decreases. The Boltzmann function is used to fit F 710 nm Value and pH change curve, calculate pK aThe value is 7.5 ( Figure 3 ), the pH response linear range is 6.5-8.2, and the linear regression equation is F 710 nm =17784.69-1919.71×pH, correlation coefficient R 2 =0.9916( Figure 4 ).

[0042] Example 4

[0043] The concentration of the probe SOH prepared in Example 1 was kept at 10 μM, and the probe was investigated for H + Selectivity. Figure 5 As shown in Figure 2, the probe SOH had almost no response to the above substances, proving that the probe is sensitive to H + Highly selective. Figure 5 The order and concentration of the substances in the sample are as follows: 1, blank; 2, K + (150 μM); 3,Cl - (50μM); 4,I - (50 μM); 5,Br - (50 μM); 6, Na + (50μM); 7, NO 2 - (50 μM); 8, SO 3 2- (50 μM); 9, OAc - (50μM); 10,F - (50μM); 11,S 2 O 3 - (50μM); 12, Li + (50 μM); 13, NO 3 - (50 μM); 14, SO 4 2- (50 μM); 15, Mg 2+ (50 μM); 16, Cu 2+ (50μM); 17,Cd 2+ (50μM); 18,Fe 3+ (50 μM); 19, Zn 2+ (50μM); 20, Mn 2+ (50μM); 21,Co 2+ (50 μM); 22,Al 3+ (50 μM); 23, proline (50 μM).

[0044] Example 5

[0045] In order to confirm whether the probe SOH of the present invention has excellent lysosomal targeting properties, the probe SOH was co-localized with the commercially available lysosomal green fluorescent probe Lyso-Tracker Green. Adherent HepG2 cells were incubated with Lyso-Tracker Green (50 nM) at 37°C and 5% CO at pH 7.4. 2 Incubate in an incubator for 20 minutes, then add SOH (final concentration 10μM) and continue incubation for 15 minutes, then gently wash 3 times with phosphate buffer (pH 7.40) to remove excess probes, and observe the co-localization of the two under a laser confocal microscope. Lyso-Tracker Green has a fixed excitation wavelength of 488nm, collecting green emission in the range of 500-550nm; SOH has a fixed excitation wavelength of 633nm, collecting red emission in the range of 685-735nm. Figure 6 b shows that the red fluorescence of SOH is distributed in the cytoplasm, indicating that the probe has good cell membrane permeability. In addition, the red fluorescence of SOH is consistent with the green fluorescence of Lyso-Tracker Green ( Figure 6 a) can overlap well, and yellow fluorescence is obtained after software processing ( Figure 6 c), the colocalization coefficient of SOH and Lyso-Tracker Green is A=0.87 ( Figure 6 e), indicating that SOH and Lyso-Tracker Green have significant co-localization imaging, and the probe has excellent lysosomal targeting properties. Bright field imaging further confirmed the viability of cells after incubation with SOH ( Figure 6 d), indicating that SOH has low toxicity to cells.

[0046] Example 6

[0047] The probe SOH prepared in Example 1 was used for fluorescence imaging of weakly acidic environment in tumor tissue. HCT116 cells were implanted subcutaneously in the right leg of 5-week-old BALB / c nude mice until the tumor volume reached 500 mm 3 The near-infrared emission signal of SOH in nude mice was monitored by a small animal optical in vivo imaging system (PerkinElmer IVIS Spectrum), with the imaging settings of excitation wavelength 640 nm and emission wavelength 700 nm. Figure 7As shown in a, physiological saline (150 μL) was injected into the tumor area of ​​the right leg and the normal area of ​​the left leg. No photoluminescence was observed in the tumor area of ​​the right leg (red circle) and the normal area of ​​the left leg (green circle). 20 minutes after the same volume of SOH solution (150 μL, 200 μM) was subcutaneously injected into two different areas of nude mice, the fluorescence signal in the tumor area was significantly enhanced compared with the normal area ( Figure 7 b). Statistical data further showed that the SOH fluorescence intensity in the tumor area increased significantly ( Figure 7 c). These results clearly demonstrate that SOH can sensitively visualize the weakly acidic environment in tumor tissues in vivo.

Claims

1. A pH fluorescent probe based on sulfonium perchlorate, characterized in that: Its structural formula is as follows:

2. The method for preparing a pH fluorescent probe based on sulfonium perchlorate according to claim 1, characterized in that: The synthetic route is as follows:

3. The method for preparing a pH fluorescent probe based on sulfonium perchlorate according to claim 2, characterized in that The steps include: (1) adding polyphosphoric acid, 3-bromothiophenol and ethyl 3-oxo-3-phenylpropionate into a reaction container, mixing, reacting, and extracting after the reaction is complete to obtain compound 1; (2) Compound 1, Pd2(dba)3, 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)-biphenyl and Cs2CO3 are added to a second reaction vessel, the reaction is evacuated by nitrogen filling, anhydrous dioxane is first added, and then dibutylamine is added. After the reaction is complete, the compound 2 is purified; (3) In a third reaction vessel filled with nitrogen, compound 2 is dissolved in a first organic solvent, CH3MgBr is added, and the mixture is stirred at room temperature for 1-4 hours, and then the solution is poured into an aqueous perchloric acid solution having a mass percentage concentration of 8-12% to purify compound 3; (4) dissolving p-hydroxybenzaldehyde and compound 3 in a second organic solvent and reacting them under an argon atmosphere. After the reaction is completed, concentrating the reaction mixture by evaporation under reduced pressure and purifying it to obtain a pH fluorescent probe based on sulfonium perchlorate salt.

4. The method for preparing a pH fluorescent probe based on sulfonium perchlorate according to claim 3, characterized in that: In step 1), the mass ratio of polyphosphoric acid, 3-bromothiophenol and ethyl 3-oxo-3-phenylpropionate is: (18-25): (1-3): (2-3).

5. The method for preparing a pH fluorescent probe based on sulfonium perchlorate according to claim 3, characterized in that: In step 2), the material ratio of compound 1, Pd2(dba)3, 2-dicyclohexylphosphino-2'-(N,N-dimethylamine)-biphenyl and Cs2CO3, anhydrous dioxane and dibutylamine is (600-750 mg): (40~55mg): (15~25mg): (1.5~2g): (8~20mL): (1~3mL).

6. The method for preparing a pH fluorescent probe based on sulfonium perchlorate according to claim 2, characterized in that: In step 3), the material ratio of compound 2, CH3MgBr, and 8-12% perchloric acid aqueous solution is (300-400 mg): (3-4 mmol): (8-15 mL).

7. The method for preparing a pH fluorescent probe based on sulfonium perchlorate according to claim 3, characterized in that: In step 4), the material ratio of p-hydroxybenzaldehyde to compound 3 is: (50-70 mg): (200-300 mg).

8. Use of the pH fluorescent probe based on sulfonium perchlorate according to claim 1 in the preparation of a localizing agent for intracellular lysosomes.

9. Use of a pH fluorescent probe based on sulfonium perchlorate according to claim 1 in the preparation of a reagent for visualizing the weakly acidic microenvironment in tumor tissue in vivo.

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