CN107603603A - A kind of fluorescence probe for identifying hydrogen peroxide - Google Patents

Abstract

The invention relates to a preparation method and application of a fluorescent probe for detecting hydrogen peroxide (H ₂ O ₂ ), belonging to the technical field of chemical analysis and detection. Its molecular structure is as follows: The maximum absorption wavelength of the probe molecule is at 480nm. After the probe molecule interacts with hydrogen peroxide (H ₂ O ₂ ), the intensity of the fluorescence spectrum at 619nm grows from nothing and increases continuously, showing a large Stokes shift ( Stokes shifts) can reduce fluorescence self-absorption and improve detection sensitivity; the emission wavelength in the red light region can reduce background fluorescence and photodamage of living cells during probe detection, and enhance the ability of organisms to penetrate tissues. The probe molecule of the present invention has good linearity in a certain time and concentration range, has strong recognition ability to hydrogen peroxide (H ₂ O ₂ ), and has strong selectivity and anti-interference ability. important application value.

Description

A Fluorescent Probe for Recognizing Hydrogen Peroxide

technical field

The invention belongs to the technical field of chemical analysis and detection, and in particular relates to a preparation method of a novel red light-near-infrared fluorescent probe with a large Stokes shift for detecting hydrogen peroxide and its detection of hydrogen peroxide in vitro and inside living cells Applications.

Background technique

Hydrogen peroxide (H ₂ O ₂ ) is one of the most important reactive oxygen species (ROS), playing key roles in many physiological and pathological processes. Hydrogen peroxide ( _{H2O2 )} can be produced endogenously in cells by activation of the NADPH oxidase complex (NOX) during cellular stimulation by hormones, cytokines, neurotransmitters and peptide growth factors. Endogenous _hydrogen peroxide (H2O2 ₎ is not only a major oxidative metabolite serving as an indicator of oxidative stress in biological systems, but also an important signaling molecule regulating many biological processes such as wound healing, immunity, cell proliferation, differentiation and migration . However, abnormal levels of _hydrogen peroxide (H2O2 ₎ are strongly associated with many diseases, including neurodegenerative diseases, diabetes, cancer and aging. Due to the great influence of hydrogen peroxide (H ₂ O ₂ ), it is meaningful and necessary to design effective methods for the qualitative and quantitative detection of hydrogen peroxide (H ₂ O ₂ ). Currently, a variety of fluorescent probes for detecting hydrogen peroxide (H ₂ O ₂ ) with high sensitivity have been reported. However, most of these probes emit short wavelengths and have the disadvantage of small Stokes shifts. However, a longer emission wavelength can reduce photodamage to living cells during probe detection and enhance tissue penetration, and a large Stokes shift can also reduce self-absorption and improve detection sensitivity. At present, there are relatively few near-infrared fluorescent probes that have a large Stokes shift and are used to detect hydrogen peroxide.

Contents of the invention

One of object of the present invention is to provide a kind of easy and efficient fluorescent probe synthesis method; Another object of the present invention is to provide a kind of good selectivity, strong anti-interference ability, large Stokes shift (Stokes shifts), emission wavelength In the near infrared, a fluorescent probe capable of detecting hydrogen peroxide in vitro or inside living cells.

The technical scheme that the present invention solves the problem and takes is, a kind of fluorescent switch (off-on) method recognizes hydrogen peroxide novel fluorescent probe, and its molecular structural formula is as follows:

The synthetic route is as follows:

The specific synthesis method is as follows: (a) Compound 1 (234.0 mg, 1.0 mmol) and potassium carbonate (300.1 mg, 2.2 mmol) were dissolved in 8.0 mL of acetone and stirred at room temperature for 10 minutes. Then 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (356.2 mg, 1.2 mmol), reacted at 70°C for 3h. After cooling to room temperature, the solvent was removed from the reaction mixture under reduced pressure, and compound 2 (197.2 mg, 43.7%) was obtained as a yellow solid through column chromatography. (b) Compound 2 (90.3 mg, 0.2 mmol) and ethyl cyanoacetate (48.2 mg, 0.4 mmol) were dissolved in 5.0 mL of ethanol at room temperature, and piperidine (5.0 μL, 0.05 mmol) was added. After stirring at room temperature for 5 minutes, the resulting solution was refluxed for 1 hour. Then, the reaction solution was concentrated under reduced pressure, and compound 3 (46.0 mg, 39.4% yield) was isolated as a red solid by column chromatography.

The action mechanism of the fluorescent probe of the present invention is as follows, the fluorescence of the probe molecule itself is in a quenched state. Hydrogen peroxide can oxidize the aromatic borate in the probe molecule, hydrolyze to obtain the phenolic hydroxyl group, then undergo intramolecular proton transfer to leave compound 4 to obtain intermediate 5, and finally undergo intramolecular cyclization to obtain compound 6 through nucleophilic substitution reaction. Therefore, the purpose of specifically detecting hydrogen peroxide (H ₂ O ₂ ) is achieved. The response process of the probe molecule is as follows:

The fluorescent probe of the present invention emits in the near infrared, and has no fluorescence before reacting with hydrogen peroxide (H ₂ O ₂ ), and the fluorescent emission peak is at 619 nm after reacting.

The fluorescent probe of the present invention has a large Stokes shift (Stokes shifts), the maximum absorption is at 480nm, and the maximum emission is at 619nm after interacting with hydrogen peroxide (H ₂ O ₂ ), and the Stokes shifts (Stokes shifts) are 139nm.

The fluorescent probe of the invention has good selectivity. The test system of the probe molecule is a 10 mM PBS buffer solution containing 30% ethanol at a pH of 7.4, measured at room temperature. The probe molecule itself has no fluorescence, and after adding 40 times the equivalent of hydrogen peroxide (H ₂ O ₂ ), the fluorescence intensity at the maximum emission wavelength of 619 nm increases by 22 times. While adding other reactive oxygen species and some anions and cations (ROO ^. , NO ^. , TBHP, ClO ^- , KO ₂ , ONOO ^- , OH ^- , I ^- , F ^- , PO ₄ ^3- , NO2-, NO ₃ ^- , SO ₃ ^2- , S ₂ O ₃ ^2- , SCN ^- , CN ^- , CO ₃ ^2- , Fe ²⁺ , Fe ³⁺ ), there was only an almost negligible increase in fluorescence.

The fluorescent probe of the present invention has strong anti-interference ability, and other active oxygen species and some anions and cations (ROO ^. , NO ^. , TBHP, ClO ^- , KO ₂ , ONOO ^- , OH ^- , I ^- , F ^- , PO ₄ ^3- , NO2-, NO ₃ ^- , SO ₃ ^2- , S ₂ O ₃ ^2- , SCN ^- , CN ^- , CO ₃ ^2- , Fe ²⁺ , Fe ³⁺ (H ₂ O ₂ ) effect.

After adding 40 times equivalent of hydrogen peroxide (H ₂ O ₂ ) to the fluorescent probe of the present invention, the fluorescence increases rapidly and reaches the maximum value in 40 minutes, and obvious changes in fluorescence can be observed.

The fluorescent probe of the present invention has a wide application range, and the probe molecule can selectively recognize hydrogen peroxide (H ₂ O ₂ ) in the pH range of 7 to 11.

The emission wavelength of the probe molecule of the present invention is in the near infrared after responding to hydrogen peroxide (H ₂ O ₂ ), the Stokes shift is large, and it has good selectivity and anti-interference ability, and has good sensitivity, and has Wide application range, the fluorescent probe has practical application value in fields such as biology and chemistry.

Description of drawings

Figure 1 is the selectivity of the fluorescent probe of the present invention, the fluorescent probe (10.0×10 ^-6 mol/L) in PBS (10mM, pH=7.4)/EtOH30% solution, and hydrogen peroxide (H ₂ O ₂ ) The fluorescence spectrum after the action, the abscissa is the wavelength, and the ordinate is the fluorescence intensity.

Fig. 2 shows the anti-interference ability of the fluorescent probe of the present invention. When hydrogen peroxide (H ₂ O ₂ ) coexists with other oxides and anions and cations, it is mixed with the fluorescent probe (10.0×10 ^-6 mol/L) in PBS (10mM , pH=7.4)/EtOH30% and hydrogen peroxide (H ₂ O ₂ ) in the histogram of the fluorescence intensity ratio (I/I ₀ ).

Fig. 3 is the fluorescence spectrum of the fluorescent probe (10.0×10 ^-6 mol/L) of the present invention in PBS (10mM, pH=7.4)/EtOH30% after reacting with different concentrations of hydrogen peroxide (H ₂ O ₂ ) The abscissa is the wavelength, and the ordinate is the fluorescence intensity.

Figure 4 shows the linear relationship between the fluorescent probe (10.0×10 ^-6 mol/L) of the present invention and the concentration of hydrogen peroxide (H ₂ O ₂ ) in PBS (10 mM, pH=7.4)/EtOH30%, on the abscissa is the wavelength, and the ordinate is the fluorescence intensity.

Figure 5 shows the fluorescence intensity at 619nm of the fluorescent probe (10.0×10 ^-6 mol/L) of the present invention in PBS (10mM, pH=7.4)/EtOH30% in the process of interacting with hydrogen peroxide (H ₂ O ₂ ) The change with time, the abscissa is the time, and the ordinate is the fluorescence intensity.

Figure 6 shows the fluorescence intensity at 619nm of the fluorescent probe (10.0×10 ^-6 mol/L) of the present invention in PBS (10mM, pH=7.4)/EtOH30% in the process of interacting with hydrogen peroxide (H ₂ O ₂ ) The linear relationship with time, the abscissa is time, and the ordinate is fluorescence intensity.

Fig. 7 shows the fluorescence intensity of the fluorescent probe (10.0×10 ^-6 mol/L) of the present invention in buffer solutions with different pH values before and after reacting with hydrogen peroxide (H ₂ O ₂ ), the abscissa is pH, and the ordinate is pH is the fluorescence intensity.

Figure 8 is the application of the fluorescent probe of the present invention to detect the activity of hydrogen peroxide (H ₂ O ₂ ) in Hela cells, A1-A3 are probes (10.0×10 ^-6 mol/L) and incubated in the cells for 30 minutes at 37°C imaging effect. B1-B3 are the imaging effects of cells first incubated with hydrogen peroxide (0.4×10 ^-3 mol/L) for 30 minutes at 37°C, and then incubated with probe (10.0×10 ^-6 mol/L) for 30 minutes.

Specific implementation examples

Embodiment 1: the synthesis of intermediate product 2

Compound 1 (234.0 mg, 1.0 mmol) and potassium carbonate (300.1 mg, 2.2 mmol) were stirred in 8.0 mL of acetone at room temperature for 10 minutes. Then 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (356.2 mg, 1.2 mmol), the resulting mixture was reacted at 70°C for 3h. After the reaction was cooled to room temperature, the reaction mixture was concentrated under reduced pressure to obtain a crude product. Finally, column chromatography was performed using petroleum ether/dichloromethane (5/1, v/v) as eluent to obtain compound 3 (197.2 mg, 43.7%) as a yellow solid. ¹ H NMR (500MHz, CDCl ₃ ) δ _H 10.26(s, 1H), 7.83(d, J=7.7Hz, 2H), 7.45(d, J=7.7Hz, 2H), 7.01(s, 1H), 6.04 , 1.55(s, 2H), 1.36(s, 12H), 1.16(t, J=7.0Hz, 3H), 1.12(t, J=7.1Hz, 3H)

Embodiment 2: the synthesis of probe molecule

Dissolve compound 2 (90.3 mg, 0.2 mmol) and ethyl cyanoacetate (48.2 mg, 0.4 mmol) in 5.0 mL of ethanol at room temperature, add piperidine (5.0 μL, 0.05 mmol), and stir at room temperature for 5 minutes , and the resulting solution was refluxed for 1 hour. After the reaction, the reaction solution was concentrated under reduced pressure to obtain a crude product. Finally, column chromatography using petroleum ether/ethyl acetate (4/1, v/v) as eluent afforded Probe1 as a red solid (46.0 mg, 39.4% yield). HRMS (ESI) m/z: C ₂₂ H ₂₁ N ₆ O ₅ [M+1]+, 449.1573; ¹ H NMR (400 MHz, CDCl ₃ ) δ _H 8.72 (s, 1H), 7.82 (d, J=8.0 Hz, 2H), 7.73(s, 1H), 7.42(d, J=8.0Hz, 2H), 5.98(s, 1H), 5.15(s, 2H), 4.31(q, J=7.1Hz, 2H), 3.31 (m, 8H), 1.35 (t, J = 15.5, 3H), 1.34 (s, 12H), 1.21 (t, J = 7.0Hz, 3H), 1.07 (t, J = 7.1Hz, 3H). ¹³ CNMR (100MHz, CDC _{l 3} ) δ _C 165.2, 155.9, 147.1, 143.4, 140.0, 135.1, 128.8, 126.1, 118.8, 109.6, 94.4, 90.3, 83.8, 71.1, 61.3, 48.0, 45.8, 45.5, 44.8, , 14.3, 10.7, 9.8.

Embodiment 3: the application of fluorescent probe of the present invention

The probe molecules were dissolved in PBS (10mM, pH=7.4)/EtOH30% to prepare a 10.0×10 ^-6 mol/L solution, and various reactive oxygen species and anions and cations (ROO., NO., TBHP, ClO ^- , KO ₂ , ONOO ^- , OH ^- , I ^- , F ^- , PO ₄ ^3- , NO2-, NO ₃ ^- , SO ₃ ^2- , S ₂ O ₃ ^2- , SCN ^- , CN ^- , CO ₃ ^2- , Fe ²⁺ , Fe ³⁺ ) did not cause significant changes in fluorescence, when hydrogen peroxide (H ₂ O ₂ ) mixed with interfering substances (ROO ^. , NO ^. , TBHP, ClO ^- , KO ₂ , ONOO ^- , OH ^- , I ^- , F ^- , PO ₄ ^3- , NO2-, NO ₃ ^- , SO ₃ ^2- , S ₂ O ₃ ^2- , SCN ^- , CN ^- , CO ₃ ^2- , Fe ²⁺ , When Fe ³⁺ ) coexists, the probe is not affected by interference factors, showing a strong anti-interference ability. The probe molecule has a good linear relationship with the hydrogen peroxide (H ₂ O ₂ ) response within a certain time and concentration range. The probe molecule can selectively recognize hydrogen peroxide (H ₂ O ₂ ) in the pH range of 7 to 11, showing good biological adaptability.

Claims (1)

1. A fluorescent probe for recognizing hydrogen peroxide, its structure is: