Synthesis and application of a near-infrared fluorescent probe with dual response to pH and viscosity

Abstract

This invention discloses a method for preparing a near-infrared fluorescent probe that is dual-responsive to both pH and viscosity, belonging to the field of chemical analysis and detection technology. The probe, PTOH, can simultaneously detect changes in pH and viscosity under acidic conditions. The probe, PTOH, rapidly responds to changes in system pH within seconds, emitting strong fluorescence and exhibiting excellent photostability and anti-interference properties. Furthermore, the probe PTOH can exhibit reversible switching between pH values ​​of 4 and 10 several times. Furthermore, the probe PTOH can sensitively detect viscosity under acidic conditions and exhibits excellent photostability, thermal stability, and anti-interference properties.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a method for preparing a near-infrared fluorescent probe with dual responses to pH and viscosity. Background Art

[0002] As an important indicator of acidity and alkalinity, pH is one of the most important parameters in the chemical industry, biotechnology, and environmental science. Similarly, cell survival depends critically on maintaining a balanced pH. pH regulates many metabolic pathways, such as signal transduction, defense, and apoptosis. Therefore, abnormal pH is often associated with organelle dysfunction and many diseases, including cancer. Numerous studies have shown that diseases such as Alzheimer's disease, myocardial ischemia, breast cancer, and colorectal cancer are closely related to changes in intracellular pH.

[0003] Viscosity is an important parameter in the human microenvironment. It plays a key role in various cellular activities, including the diffusion of metabolites and signal transduction. Abnormal changes in viscosity are associated with a variety of diseases, such as malignant tumors, hypertension, and diabetes. Furthermore, near-infrared fluorescent probes offer advantages such as minimal photodamage, deep penetration, and low background interference.

[0004] To date, many fluorescent probes for detecting single analytes, such as pH or viscosity, have been developed, but probes that can simultaneously detect both pH and viscosity still require further research. Therefore, there is an urgent need to develop a near-infrared fluorescent probe that is dual-responsive to both pH and viscosity. This would provide an effective method for monitoring intracellular pH fluctuations and viscosity changes in vivo, signaling abnormal conditions and enabling more sensitive and accurate detection of disease onset. Summary of the Invention

[0005] The present invention aims to provide a method for synthesizing a near-infrared fluorescent probe with dual responses to pH and viscosity, and to effectively detect pH and viscosity at physiological levels.

[0006] The molecular structure of the fluorescent probe in the present invention is as follows:

[0007]

[0008] The fluorescent probe in the present invention is prepared by the following reaction, and the synthesis process is as follows:

[0009]

[0010] The preparation steps of the fluorescent probe PTOH are as follows:

[0011] Step 1: Preparation of intermediate 1

[0012] 4-Bromotriphenylamine and 5-formyl-2-thiopheneboronic acid were placed in a round-bottom flask, and anhydrous tetrahydrofuran was added and ultrasonically dissolved. Aqueous potassium carbonate solution and tetrakis(triphenylphosphine)palladium were then added, and the mixed solution was stirred evenly and then heated to reflux under argon protection. The mixture was cooled to room temperature and extracted with dichloromethane and water. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and column chromatography was performed to obtain a yellow product 1 (249 mg, 35.0% yield).

[0013] Step 2: Preparation of intermediate 2

[0014] 1,1,2-Trimethyl-1H-benz[e]indole and 2-iodoethanol were added to a round-bottom flask. Toluene was then added to the solution, stirred and dissolved, and heated to reflux under argon. The mixture was cooled to room temperature, filtered, washed several times with toluene, and dried under vacuum to obtain a light blue powdery solid 2 (3 g, 34.0% yield).

[0015] Step 3: Preparation of fluorescent probe PTOH

[0016] Compound 1 and compound 2 were placed in a round-bottom flask, anhydrous ethanol was added, ultrasonically dissolved, and then heated to reflux. After the reaction was completed, the solvent was removed under reduced pressure and column chromatography was performed to obtain a dark green product PTOH (220 mg, yield 58.0%).

[0017] The recognition principle of the fluorescent probe of the present invention for pH and viscosity is as follows:

[0018]

[0019] When the probe is photoexcited, a certain degree of intramolecular charge transfer (ICT) occurs, with electrons transferred from the donor to the acceptor. However, these two moieties within the molecule can rotate freely in a low-viscosity environment, transforming the molecule into a twisted intramolecular charge transfer (TICT) state. Viscosity sensitivity is achieved by suppressing the TICT process at high viscosity. The high viscosity environment hinders the rotation of intramolecular single bonds to form a large conjugated open ring form, reducing the probability of the system's TICT excited state and forcing it to return to the ground state via a non-radiative transition, thereby enhancing the fluorescence intensity. In addition, the benzindole salt acts as a pH-sensitive site. When the probe is in an acidic environment, a proton-triggered ring-opening process of the probe occurs. With increasing alkalinity, the carbon-nitrogen double bond changes, the nucleophile attacks the hydroxyl group, and the probe undergoes ring closure. Compared to the open ring form, the conjugation of the probe in the cyclized state is reduced, resulting in a blue shift in the emission wavelength.

[0020] The fluorescent probe of the present invention has an excitation wavelength of 370 nanometers and an emission wavelength of approximately 470 nanometers, and responds regularly to changes in pH between 2 and 12. Fluorescence gradually increases with increasing pH. Furthermore, the fluorescence intensity of the probe PTOH at approximately 730 nanometers increases 47-fold with increasing glycerol content in the glycerol-PBS system.

[0021] The fluorescent probe of the present invention has excellent photostability.

[0022] The fluorescent probe of the present invention has a large Stokes shift.

[0023] The fluorescent probe of the present invention has good anti-interference performance for detecting pH and can specifically detect viscosity.

[0024] Figure 3 The fluorescence quantum yields of the fluorescent probe of the present invention in several common solvents were measured using coumarin 102 and rhodamine B as references. The maximum absorption wavelengths of the probe PTOH in different solvents are similar and insensitive to changes in solution polarity. Using 600 nanometers as the excitation wavelength, the fluorescence intensity of the probe PTOH in glycerol is significantly higher than in other solvents.

[0025] Figure 4 (A) is the probe PTOH (10 μmol / L, V PBS :V DMSO =7:3) at different pH values. Inset: Probe PTOH (10 μmol / L, V PBS :V DMSO =7:3) under natural light at pH = 2 and pH = 12. As the pH of the system increases, the absorption at 370nm gradually increases, and the absorption at 580nm gradually decreases. (B) is the fluorescent probe PTOH of the present invention (10μmol / L, V PBS :V DMSO =7:3) when the pH value of the system is changed. The illustration shows the fluorescence emission spectra of the probe PTOH (10 μmol / L, V PBS :V DMSO =7:3) at pH = 2 and pH = 12 under a 365nm UV lamp. It shows that as the pH of the system increases, the fluorescence at 470nm gradually increases. (C) is the fluorescent probe PTOH of the present invention (10μmol / L, V PBS :V DMSO =7:3) at different pH values ​​at 470nm. Inset: Linear plot of pH and fluorescence intensity in the range of pH = 4 to 10. This shows that the probe PTOH can respond quickly to pH and has a good linear relationship. (D) is the fluorescent probe PTOH of the present invention (10μmol / L, VPBS :V DMSO =7:3) The change of fluorescence intensity between pH = 4 and pH = 10. This shows that the probe PTOH exhibits several reversible switching between pH 4 and 10, which can be completed rapidly within seconds.

[0026] Figure 5 (A) is the UV-visible absorption spectra of the probe PTOH in glycerol-PBS (pH=5) with different ratios. Inset: Photographs taken under natural light at 0.89 cP and 438.4 cP of glycerol. (B) is the fluorescence emission spectra of the probe PTOH in glycerol-PBS (pH=5) with different ratios. Inset: Fluorescence intensity of the probe at 730 nm (log I 730 ) is a linear relationship diagram between the fluorescence intensity of the probe PTOH and the viscosity (logη). This shows that the probe PTOH is sensitive to viscosity and has good response ability. (C) is a fluorescence intensity diagram of the probe PTOH at 730nm in a glycerol-PBS system with different pH and different viscosity values. This shows that the fluorescence intensity of the probe PTOH increases with the viscosity of the system under acidic conditions. (D) is a graph showing the change in fluorescence intensity of the probe PTOH in PBS solution and 95% glycerol within 60 minutes at different temperatures. This shows that the probe PTOH has good photostability at different temperatures and different viscosities.

[0027] Figure 6 (A) is the probe PTOH (10 μmol / L, V PBS :V DMSO =7:3) after adding various bioactive species at 470nm. (1) Ca 2+ ; (2) Cu 2+ ; (3)Fe 2+ ; (4)Fe 3+ ;(5)K + ; (6) Mg 2+ ; (7) Mn 2+ ; (8) Na + ; (9) Zn 2+ ;(10)F - ; (11) ClO - ;(12)S 2- ;(13)CO3 2- ;(14)HSO3 - ;(15)Cys;(16)GSH;(17)Hcy;(18)H2O2. (B) is the probe PTOH (10μmol / L, V PBS :V DMSO =7:3) after adding various bioactive species to the fluorescence intensity change at 730nm. (1) Ca 2+ ; (2) Cu2+ ; (3)Fe 2+ ; (4)Fe 3+ ;(5)K + ; (6) Mg 2+ ; (7) Mn 2+ ; (8) Na + ; (9) Zn 2+ ;(10)F - ; (11) ClO - ;(12)S 2- ;(13)CO3 2- ;(14)HSO3 - ;(15)Cys;(16)GSH;(17)Hcy;(18)H2O2. This indicates that the probe PTOH has good anti-interference ability in the process of detecting pH and viscosity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the design idea and synthesis route of the fluorescent probe PTOH of the present invention.

[0029] Figure 2 This is the mechanism of the fluorescent probe PTOH of the present invention for detecting pH and viscosity.

[0030] Figure 3 The fluorescence quantum yield of the fluorescent probe PTOH of the present invention in several common solvents is tested with coumarin 102 and rhodamine B as references.

[0031] Figure 4 (A) is the UV-visible absorption spectrum of the probe PTOH at different pH values. Inset: Photographs of the probe PTOH under natural light at pH = 2 and pH = 12. (B) is the fluorescence emission spectrum of the fluorescent probe PTOH of the present invention when the pH value of the system is changed. Inset is a photo of the probe PTOH under a 365nm UV handlamp at pH = 2 and pH = 12. (C) is a graph showing the fluorescence changes of the fluorescent probe PTOH of the present invention at 470nm at different pH values. Inset: a linear graph of pH and fluorescence intensity in the range of pH = 4 to 10. (D) is a graph showing the changes in fluorescence intensity of the fluorescent probe PTOH of the present invention between pH = 4 and pH = 10.

[0032] Figure 5 (A) is the UV-visible absorption spectra of the probe PTOH in glycerol-PBS (pH=5) with different ratios. Inset: Photographs taken under natural light at 0.89 cP and 438.4 cP of glycerol. (B) is the fluorescence emission spectra of the probe PTOH in glycerol-PBS (pH=5) with different ratios. Inset: Fluorescence intensity of the probe at 730 nm (log I 730) versus viscosity (logη). (C) Fluorescence intensity of probe PTOH at 730 nm in glycerol-PBS systems at different pH values ​​and viscosities. (D) Fluorescence intensity of probe PTOH in PBS and 95% glycerol over 60 minutes at different temperatures.

[0033] Figure 6 (A) is the fluorescence intensity change at 470nm after adding various bioactive species to the probe PTOH. (1) Ca 2+ ; (2) Cu 2+ ; (3)Fe 2+ ; (4)Fe 3+ ;(5)K + ; (6) Mg 2+ ; (7) Mn 2+ ; (8) Na + ; (9) Zn 2+ ;(10)F - ; (11) ClO - ;(12)S 2- ;(13)CO3 2- ;(14)HSO3 - ; (15) Cys; (16) GSH; (17) Hcy; (18) H2O2. (B) Fluorescence intensity changes at 730 nm after adding various bioactive species to the probe PTOH. (1) Ca 2+ ; (2) Cu 2+ ; (3)Fe 2+ ; (4)Fe 3+ ;(5)K + ; (6) Mg 2+ ; (7) Mn 2+ ; (8) Na + ; (9) Zn 2+ ;(10)F - ; (11) ClO - ;(12)S 2- ;(13)CO3 2- ;(14)HSO3 - ; (15) Cys; (16) GSH; (17) Hcy; (18) H2O2.

[0034] Figure 7 This is the hydrogen nuclear magnetic resonance spectrum of the probe PTOH of the present invention after adding 10 μL H 2 SO 4 (1 mol / L).

[0035] Figure 8 This is the hydrogen nuclear magnetic resonance spectrum of the probe PTOH of the present invention after adding 10 μL NaOH (1 mol / L).

[0036] Figure 9 This is the carbon NMR spectrum of the probe PTOH of the present invention after adding 10 μL H 2 SO 4 (1 mol / L).

[0037] Figure 10 This is a high-resolution mass spectrum of the probe PTOH of the present invention.

[0038] Specific implementation examples

[0039] Example 1: Synthesis of Compound 1

[0040] 4-Bromotriphenylamine (778 mg, 2.4 mmol) and 5-formyl-2-thiopheneboronic acid (2 mmol, 311 mg) were placed in a 50 mL round-bottom flask, and 8 mL of anhydrous tetrahydrofuran was added for ultrasonic dissolution. 2 mol / L K2CO3 aqueous solution (3.2 mL) and Pd(PPh3)4 (116 mg, 0.1 mmol) were then added, and the mixed solution was stirred evenly, and then heated to reflux for 18 hours under argon protection. The mixture was cooled to room temperature and extracted with DCM and water, and the organic phase was dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure and purified by column chromatography (eluent: V 石油醚 :V 乙酸乙酯 =20:1) to give a yellow product 1 (249 mg, 35.0%).

[0041] Example 2: Synthesis of Compound 2

[0042] 1,1,2-Trimethyl-1H-benz[e]indole (4.9 g, 24 mmol) and 2-iodoethanol (1.867 mL, 24 mmol) were added to a 50 mL round-bottom flask. 2 mL of toluene solution was then added to the solution, stirred and dissolved, and heated to reflux under argon. The mixture was cooled to room temperature, filtered, washed several times with toluene, and dried in vacuo to obtain a light blue powdery solid 2 (3 g, 34.0% yield).

[0043] Example 3: Synthesis of probe PTOH

[0044] Compound 1 (221.3 mg, 0.614 mmol) and compound 2 (216.8 mg, 0.57 mmol) were placed in a round-bottom flask, 8 mL of anhydrous ethanol was added, ultrasonically dissolved, and then heated under reflux for 30 h. After the reaction was completed, the solvent was removed under reduced pressure and column chromatography was performed for purification (eluent: V 二氯甲烷 :V 无水乙醇 =60:1) to obtain a dark green product PTOH (220 mg, yield 58.0%). HRMS (ESI+) m / z: calculated for C 40 H 35 N2OS + [M+H+ ],591.2465; found,591.2474. 1 H NMR(600MHz,DMSO-d6)δ8.76(d,J=15.9Hz,1H),8.42(d,J=8.4Hz,1H),8.25(d,J=8.9Hz, 1H),8.20(t,J=6.1Hz,2H),8.08(d,J=9.0Hz,1H),7.81(t,J=7.6Hz,1H),7.77-7.68(m,3 H),7.39(t,J=7.8Hz,3H),7.34(d,J=15.9Hz,1H),7.17(t,J=7.4Hz,2H),7.13(d,J=7.9H z, 3H), 7.00 (d, J = 8.5Hz, 2H), 5.75 (s, 1H), 4.82 (s, 2H), 3.99-3.90 (m, 2H), 2.04 (s, 5H). 13 C NMR (151MHz, DMSO-d6) δ182.9,154.1,149.3,146.7,144.7,140.1,139.2,138.9,138.2,133.4,131.2,130.5,130.3,12 8.8,127.9,127.4,127.2,125.9,125.8,125.7,124.9,123.4,121.9,113.8,110.3,59.3,56.5,53.9,49.6,26.3,19.0.

[0045] Example 4: Probe PTOH monitors the pH and viscosity of a solution.

[0046] Figure 4 (A) is the UV-visible absorption spectrum of the probe PTOH at different pH values. Inset: Photographs of the probe PTOH under natural light at pH = 2 and pH = 12. (B) is the fluorescence emission spectrum of the fluorescent probe PTOH of the present invention when the pH value of the system is changed. Inset is a photo of the probe PTOH under a 365nm UV handlamp at pH = 2 and pH = 12. (C) is a graph showing the fluorescence changes of the fluorescent probe PTOH of the present invention at 470nm at different pH values. Inset: a linear graph of pH and fluorescence intensity in the range of pH = 4 to 10. (D) is a graph showing the changes in fluorescence intensity of the fluorescent probe PTOH of the present invention between pH = 4 and pH = 10. Figure 5(A) is the UV-visible absorption spectra of the probe PTOH in glycerol-PBS (pH=5) with different ratios. Inset: Photographs taken under natural light at 0.89 cP and 438.4 cP of glycerol. (B) is the fluorescence emission spectra of the probe PTOH in glycerol-PBS (pH=5) with different ratios. Inset: Fluorescence intensity of the probe at 730 nm (log I 730 ) versus viscosity (logη). (C) Fluorescence intensity of probe PTOH at 730 nm in glycerol-PBS systems at different pH values ​​and viscosities. (D) Fluorescence intensity of probe PTOH in solution and 95% glycerol over 60 minutes at different temperatures.

[0047] In summary, using a simple organic synthesis method, we have developed a near-infrared fluorescent probe, PTOH, that is dually responsive to pH and viscosity. The probe PTOH exhibits rapid responses to both pH and viscosity. At 470 nm, the fluorescence intensity increases with increasing pH. Under acidic conditions, the probe also exhibits viscosity-sensitive fluorescence at 730 nm, emitting strong fluorescence in highly viscous solutions, unaffected by polarity.

Claims

1. Synthesis and application of a near-infrared fluorescent probe with dual response to pH and viscosity (E)-2-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)-3-(2-hydroxyethyl)-1,1-dimethyl-1H-benz[e]indol-3-ium iodide, whose structural formula is:

2. The method for preparing the pH and viscosity dual-responsive near-infrared fluorescent probe according to claim 1, comprising the following steps: 4-Bromotriphenylamine and 5-formyl-2-thiopheneboronic acid were placed in a round-bottom flask, anhydrous tetrahydrofuran was added and dissolved by ultrasonication, and then an aqueous potassium carbonate solution and tetrakis(triphenylphosphine)palladium were added. The mixed solution was stirred evenly and then heated to reflux under argon protection. The mixture was cooled to room temperature and extracted with dichloromethane and water. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and purification by column chromatography was performed to obtain a yellow product 1. 1,1,2-Trimethyl-1H-benz[e]indole and 2-iodoethanol were added to a round-bottom flask. Toluene solution was then added to the solution, stirred and dissolved, and heated to reflux under argon protection. The mixture was cooled to room temperature, filtered, washed several times with toluene, and vacuum dried to obtain a light blue powdery solid 2. Compound 1 and compound 2 were placed in a round-bottom flask, and anhydrous ethanol was added for ultrasonic dissolution, followed by heating under reflux. After the reaction was completed, the solvent was removed under reduced pressure and column chromatography was performed for purification to obtain a dark green product, PTOH. The synthesis route is as follows:

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