CN119306696A - A pH fluorescent probe based on sulfonium perchlorate and its application - Google Patents

Abstract

The invention discloses a pH fluorescent probe based on a sulfonium perchlorate, which has the following structural formula: The invention also provides a preparation method of the pH fluorescent probe based on the sulfonium perchlorate. The invention also provides the application of the pH fluorescent probe based on the sulfonium perchlorate in preparing a positioning agent serving as an intracellular lysosome. The invention also provides application of the pH fluorescent probe based on the sulfonium perchlorate in preparing a reagent for visualizing weak acid microenvironment in tumor tissues in vivo. The probe has near infrared fluorescence of 710nm and large stokes shift of 214nm, has remarkable lysosome targeting capability, and simultaneously has excellent reversibility, selectivity and photostability. The pK _a value of the probe is 7.5, can be used for visualization of weak acidic microenvironment of tumor tissues in mice, and has an application prospect of early detection of tumors.

Description

PH fluorescent probe based on sulfonium perchlorate and application thereof

Technical Field

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 application thereof.

Background

The pH plays an important role in many physiological processes of the life system, including metabolism, growth, proliferation, apoptosis, signal transduction, ion transport, etc., and maintaining proper acid-base balance is critical to normal physiological function and overall health. Studies have shown that intracellular pH abnormalities can lead to problems such as disturbance of enzyme activity, increased free radical production, cell dysfunction, abnormal protein deposition, etc., and these abnormal phenomena are liable to cause diseases such as cancer, renal failure, lung disease, alzheimer's disease, etc.

Taking a tumor as an example, one of the remarkable characteristics of tumor tissue is its acidic microenvironment, the pH of normal tissue is typically 7.2-7.4, and the pH of tumor tissue is 6.2-6.9. The pH abnormality is caused by active metabolism of tumor cells, and insufficient blood supply and hypoxia inside the tumor result in accumulation of lactic acid and other acidic metabolites. The acidic environment not only promotes the growth and diffusion of tumor cells, but also can influence the functions of immune cells, thereby accelerating the development of tumors. Therefore, monitoring pH fluctuations in intracellular and in vivo systems in real time is of great importance for a deep understanding of the physiological and pathological processes associated with pH.

The small molecular fluorescent probe developed based on the fluorescent imaging technology has the advantages of non-invasiveness, high response speed, good real-time performance and the like, and becomes an indispensable tool in life science and medical diagnosis. Among them, near infrared fluorescent probes having an emission wavelength in the range of 650-900nm (NIR-I) are attracting attention because of their deep tissue penetration, small photodamage to biological samples, and weak autofluorescence of biomolecules. In view of the unique advantages of the near infrared fluorescent probe in non-invasive optical imaging, the design of the near infrared fluorescent probe monitors pH fluctuation in cells and in-vivo systems in real time, and shows good application prospects.

Disclosure of Invention

The invention aims to provide a pH fluorescent probe based on a sulfonium perchlorate and application thereof, and the pH fluorescent probe based on the sulfonium perchlorate and application thereof aim to solve the technical problems of weak visibility and positioning of the pH fluorescent probe in the prior art.

The invention provides a pH fluorescent probe based on a sulfonium perchlorate, which has the structural formula:

The name of the probe is (E) -7- (dibutylamino) -4- (4-hydroxystyryl) -2-phenyl sulfonium perchlorate (SOH).

The synthetic route is as follows:

Further, the preparation method of the pH fluorescent probe based on the sulfonium perchlorate comprises the following steps:

(1) Adding polyphosphoric acid, 3-bromothiophenol and 3-oxo-3-phenylpropionic acid ethyl ester into a reaction vessel, mixing, reacting, and extracting after the reaction is completed to obtain a compound 1;

(2) Adding a compound 1, pd ₂(dba)₃, davePhos (2-dicyclohexylphosphino-2' - (N, N-dimethylamine) -biphenyl) and Cs ₂CO₃ into a second reaction vessel, adding anhydrous dioxane, then adding dibutylamine, and purifying after the reaction is completed to obtain a compound 2;

(3) In a third reaction container filled with nitrogen, dissolving the compound 2 in a first organic solvent, then adding CH ₃ MgBr, stirring for 1-4 hours at room temperature, then pouring the solution into a perchloric acid aqueous solution with the mass percent concentration of 8-12%, and purifying to obtain a compound 3;

(4) The p-hydroxybenzaldehyde and the compound 3 are dissolved in a second organic solvent to react under the argon atmosphere, and after the reaction is completed, the reaction mixture is concentrated by reduced pressure evaporation and purified to obtain the pH fluorescent probe based on the sulfonium perchlorate.

Further, in the step 1), the mass ratio of the polyphosphoric acid to the 3-bromothiophenol to the 3-oxo-3-phenylpropionic acid ethyl ester is (18-25): 1-3): 2-3.

Further, in the step 2), the material ratio of the compound 1, pd ₂(dba)₃, davePhos (2-dicyclohexylphosphino-2' - (N, N-dimethylamine) -biphenyl) to Cs ₂CO₃, anhydrous dioxane and dibutylamine is (600-750 mg) (40-55 mg) (15-25 mg) (1.5-2 g) (8-20 mL) (1-3 mL). Further, in the step 3), the material ratio of the compound 2, CH ₃ MgBr and 8-12% perchloric acid aqueous solution by mass percent is (300-400 mg): 3-4 mmol): 8-15 mL.

In the step 4), the material ratio of the parahydroxyben-zaldehyde to the compound 3 is (50-70 mg) (200-300 mg).

The invention also provides an application of the pH fluorescent probe based on the sulfonium perchlorate in preparing a positioning agent serving as an intracellular lysosome.

The invention also provides an application of the pH fluorescent probe based on the sulfonium perchlorate in preparing a reagent for visualizing weak acid microenvironment in tumor tissues in vivo.

The probe SOH can be used for visualization of weak acidic microenvironment of tumor tissues in mice, and has good application prospect in early detection of tumors.

In the preparation process of the invention, the preparation of the compound 1 has simple reaction, the product is obtained through extraction, the next reaction operation is directly carried out without further purification, and the method is suitable for mass preparation. The preparation of compound 2 is a typical nucleophilic substitution reaction of halogenated hydrocarbon, and the reaction requires the use of catalyst DavePhos (2-dicyclohexylphosphino-2' - (N, N-dimethylamine) -biphenyl), and the reaction time is about 18 hours, and the yield is high although the reaction time is long. Compound 3 is prepared from compound 2 by methylation reaction, and the reaction is rapid and the yield is high. The target product SOH is obtained by dehydrating the compound 3 and the p-hydroxybenzaldehyde, and has low yield, but simple synthesis steps and potential commercial application value.

Compared with the existing pH fluorescent probe, the probe SOH has the advantages that (1) the pKa of the probe SOH is 7.5, the linear pH response range is 6.5-8.2, the probe can be used for visualizing the weak acidic microenvironment of the tumor tissue in a mouse body, and the probe SOH has good application prospect in early detection of tumors. (2) The maximum fluorescence emission of the probe SOH is 710nm, belongs to the near infrared region, and can effectively reduce the photodamage to cells and reduce the interference of autofluorescence of biological samples. (3) The probe SOH has extremely large Stokes displacement (214 nm) and can effectively reduce interference from self-excited light. (4) The probe SOH has high sensitivity and selectivity to pH response and is not interfered by common cations, anions and other substances. (5) The probe SOH has excellent lysosome targeting ability, and simultaneously shows excellent reversibility, selectivity and photostability. (6) The probe has simple synthesis steps and low cost, and has potential commercial application value.

Drawings

FIG. 1. Ultraviolet-visible absorption spectra of the probe SOH of the present invention as a function of pH.

FIG. 2 fluorescence emission spectra of the probe SOH according to the present invention as a function of pH.

FIG. 3 shows Boltzmann function of F _{710 nm} of the probe SOH according to the invention as a function of pH, pK _a value is 7.5.

FIG. 4 shows the linear dependence of F _{710 nm} of the probe SOH according to the present invention on pH, in the range of pH 6.5-8.2.

FIG. 5 shows the selectivity of the probe SOH of the present invention for H ⁺ in the presence of common cations and anions.

FIG. 6 is a co-localization imaging of the probe SOH of the present invention with a commercially available lysosome green fluorescent probe Lyso-TRACKER GREEN in human hepatoma cells (HepG 2 cells).

FIG. 7 fluorescence imaging of the probe SOH of the invention in a mouse model of tumor.

Detailed Description

Example 1

Preparation of probe (E) -7- (dibutylamino) -4- (4-hydroxystyryl) -2-phenyl sulfonium perchlorate onium perchlorate (SOH):

(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℃for 2 hours. After cooling to room temperature, the reaction was quenched by addition of ice water and extracted with DCM (dichloromethane) (100 mL. Times.3). The combined organic phases were dried over Na ₂SO₄, filtered and evaporated to give compound 1 without further purification.

(2) Compound 1 (694 mg,2 mmol), pd ₂(dba)₃ (45.8 mg,0.05 mmol), davePhos (2-dicyclohexylphosphino-2' - (N, N-dimethylamine) -biphenyl) (19.7 mg,0.05 mmol) and Cs ₂CO₃ (1.63 g,5 mmol) were added to a test tube. The tube was sealed and filled by nitrogen and vacuum. Anhydrous dioxane (10 mL) was added followed by dibutylamine (1.7 mL,10 mmol). The reaction was stirred at 100 ℃ 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 give the compound 2(592mg,81％).¹H NMR(400MHz,DMSO-d₆)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,J₁＝2.4Hz,J₂＝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/z calcd for C₂₃H₂₈NOS⁺[M]⁺,366.2;Found,366.1.

(3) In a nitrogen-filled dry flask, compound 2 (365 mg,1 mmol) was dissolved in anhydrous THF (10 mL). To the solution was added dropwise 1.0M CH ₃ MgBr (3.6 mL) and stirred at room temperature for 2 hours. The solution was poured into 10% strength by mass aqueous perchloric acid (10 mL) and extracted with DCM (25 ml×3). The combined organic phases were dried over Na ₂SO₄, filtered and evaporated. The crude product was further purified by flash column chromatography (MeOH/dcm=1/5, v/v) to give the compound 3(371mg,80％).¹H NMR(400MHz,CDCl₃)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,J₁＝2.4Hz,J₂＝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 C₂₄H₃₀NS⁺[M]⁺,364.2;Found,364.2.

(4) Paramyxol (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). The reaction mixture was stirred at 100 ℃ overnight under argon atmosphere. After the reaction was completed, the reaction mixture was concentrated by evaporation under reduced pressure. Purifying the crude product by silica gel column chromatography to obtain target product SOH with yield of 284mg 50％.¹H NMR(400MHz,DMSO-d₆)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).¹³C NMR(126MHz,DMSO-d₆)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 calcd for C₃₁H₃₄NOS⁺[M]⁺,468.2356;Found,468.2167.

Example 2

The probe SOH prepared in example 1 was stored in a stock solution prepared in DMSO (dimethyl sulfoxide) at a concentration of 1 mM. In the experiment, the probe was diluted to a final concentration of 10. Mu.M with an H ₂ O/DMSO (V/V=5/1) system, the pH of which was adjusted with a high concentration of a small volume of NaOH solution, and the UV absorption spectrum was recorded (FIG. 1). As the pH increases, the absorption peak at 500nm gradually decreases, the absorption peak at 700nm correspondingly increases, and there is an isosbestic point at 620 nm.

Example 3

The probe SOH was diluted to a final concentration of 10. Mu.M, the pH of the system was adjusted with a high concentration small volume of NaOH solution, the excitation wavelength was fixed at 600nm, and the fluorescence emission spectrum was recorded (FIG. 2). As the pH value increases, the fluorescence intensity at 710nm gradually decreases. The pK _a value was calculated to be 7.5 (fig. 3), the pH response linear range was 6.5-8.2, the linear regression equation was F _{710 nm} = 17784.69-1919.71 ×ph, and the correlation coefficient R ² = 0.9916 (fig. 4) by Boltzmann function fitting the F _{710 nm} value to the pH change curve.

Example 4

The SOH concentration of the probe prepared in example 1 was maintained at 10. Mu.M, and the selectivity of the probe to H ⁺ in the presence of common cations and anions, respectively, was examined. As shown in FIG. 5, the probe SOH hardly responds to the above substances, and it was confirmed that the probe has high selectivity for H ⁺. The order and concentration of the substances in FIG. 5 is 1, blank ;2,K⁺(150μM);3,Cl^-(50μM);4,I^-(50μM);5,Br^-(50μM);6,Na⁺(50μM);7,NO₂ ^-(50μM);8,SO₃ ^2-(50μM);9,OAc^-(50μM);10,F^-(50μM);11,S₂O₃ ^-(50μM);12,Li⁺(50μM);13,NO₃ ^-(50μM);14,SO₄ ^2-(50μM);15,Mg²⁺(50μM);16,Cu²⁺(50μM);17,Cd²⁺(50μM);18,Fe³⁺(50μM);19,Zn²⁺(50μM);20,Mn²⁺(50μM);21,Co²⁺(50μM);22,Al³⁺(50μM);23, proline (50. Mu.M).

Example 5

To confirm whether the probe SOH of the present invention has excellent lysosome targeting properties, a co-localization experiment was performed with a commercially available lysosome green fluorescent probe Lyso-TRACKER GREEN. The attached HepG2 cells were incubated with Lyso-TRACKER GREEN (50 nM) at pH 7.4 for 20min in an incubator with 37 ℃ and 5% co ₂, followed by further incubation with SOH (final concentration 10 μm) for 15min, followed by gentle washing 3 times with phosphate buffer (pH 7.40) to remove excess probe, and co-localization of both was observed under a laser confocal microscope. Wherein, the fixed excitation wavelength of Lyso-TRACKER GREEN is 488nm, the emission range of collecting green is 500-550nm, the fixed excitation wavelength of SOH is 633nm, and the emission range of collecting red is 685-735nm. As can be seen from FIG. 6b, the red fluorescence of SOH was distributed in the cytoplasmic region, demonstrating good cell membrane permeability of the probe. In addition, the red fluorescence of SOH and the green fluorescence of Lyso-TRACKER GREEN (fig. 6 a) can be well overlapped, and the yellow fluorescence is obtained by software treatment (fig. 6 c), and the co-localization coefficient a=0.87 of SOH and Lyso-TRACKER GREEN (fig. 6 e) shows that SOH and Lyso-TRACKER GREEN have significant co-localization imaging, and the probe has excellent lysosome targeting property. Bright field imaging further confirmed the viability of the cells after SOH incubation (fig. 6 d), indicating that SOH has low toxicity to cells.

Example 6

The probe SOH prepared in example 1 was used for fluorescence imaging of weakly acidic environments in tumor tissue. 5-week-old BALB/c nude mice were subcutaneously implanted with HCT116 cells in the right leg until the tumor volume reached 500mm ³. The near infrared emission signal of SOH in nude mice was monitored by a small animal optical live imaging system (PERKINELMER IVIS spectra) with imaging set to excitation wavelength 640nm and emission wavelength 700nm. As shown in fig. 7a, physiological saline (150 μl) was injected into the tumor area of the right leg and the normal area of the left leg, with the result that no photoluminescence was observed in both the tumor area of the right leg (red circle) and the normal area of the left leg (green circle). After subcutaneous injection of the same volume of SOH solution (150 μl,200 μΜ) into two different areas of nude mice for 20 minutes, the fluorescent signal of the tumor area was significantly enhanced compared to the normal area (fig. 7 b). The statistics further show that SOH fluorescence intensity of tumor regions was significantly increased (fig. 7 c). These results clearly demonstrate that SOH can sensitively visualize the weakly acidic environment in tumor tissue in vivo.

Claims (9)

1. A pH fluorescent probe based on sulfonium perchlorate, which is characterized by the following structural formula:

2. The method for preparing a pH fluorescent probe based on a sulfonium perchlorate as claimed in claim 1, wherein the synthetic route is as follows:

3. The method for preparing a pH fluorescent probe based on sulfonium perchlorate according to claim 2, characterized by comprising the following steps:

(1) Adding polyphosphoric acid, 3-bromothiophenol and 3-oxo-3-phenylpropionic acid ethyl ester into a reaction vessel, mixing, reacting, and extracting after the reaction is completed to obtain a compound 1;

(2) Adding the compound 1, pd ₂(dba)₃, 2-dicyclohexylphosphino-2' - (N, N-dimethylamine) -biphenyl and Cs ₂CO₃ into a second reaction vessel, adding anhydrous dioxane, then adding dibutylamine, and purifying after the reaction is completed to obtain a compound 2;

(3) In a third reaction container filled with nitrogen, dissolving the compound 2 in a first organic solvent, then adding CH ₃ MgBr, stirring for 1-4 hours at room temperature, then pouring the solution into a perchloric acid aqueous solution with the mass percent concentration of 8-12%, and purifying to obtain a compound 3;

(4) The p-hydroxybenzaldehyde and the compound 3 are dissolved in a second organic solvent to react under the argon atmosphere, and after the reaction is completed, the reaction mixture is concentrated by reduced pressure evaporation and purified to obtain the pH fluorescent probe based on the sulfonium perchlorate.

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

5. The method for preparing a pH fluorescent probe based on a sulfonium perchlorate according to claim 3, wherein in the step 2), the material ratio of the compound 1, pd ₂(dba)₃, 2-dicyclohexyl phosphino-2' - (N, N-dimethylamine) -biphenyl to Cs ₂CO₃, anhydrous dioxane and dibutylamine is (600-750 mg):

(40~55mg):(15~25mg):(1.5~2g):(8~20mL):(1~3mL)。

6. The preparation method of the pH fluorescent probe based on the sulfonium perchlorate according to claim 2 is characterized in that in the step 3), the material ratio of the compound 2, CH ₃ MgBr and 8-12% perchloric acid aqueous solution by mass percent is (300-400 mg), 3-4 mmol and 8-15 mL.

7. The method for preparing the pH fluorescent probe based on the sulfonium perchlorate according to claim 3, wherein in the step 4), the material ratio of the parahydroxyben-zaldehyde to the compound 3 is (50-70 mg) (200-300 mg).

8. Use of a sulphur onium perchlorate-based pH fluorescent probe according to claim 1 for the preparation of a localization agent as an intracellular lysosome.

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