CN115232098A

CN115232098A - Rhodol fluorescent probe for rapidly and sensitively detecting peroxynitrate and preparation method and application thereof

Info

Publication number: CN115232098A
Application number: CN202210844439.7A
Authority: CN
Inventors: 叶天晴; 汪剑波; 林燕飞
Original assignee: Jiaxing University
Current assignee: Jiaxing University
Priority date: 2022-07-18
Filing date: 2022-07-18
Publication date: 2022-10-25
Anticipated expiration: 2042-07-18
Also published as: CN115232098B

Abstract

The invention discloses a method for detecting peroxynitrite (ONOO) ^‑ ) The structure of the Rhodol fluorescent probe is shown as the formula (I). The probe has obvious fluorescence enhancement change on peroxynitrite in a PBS (20mM, pH 7.4) buffer solution, quick detection response (within 1 minute), low detection limit (57 nM) and excellent detection selectivity, and is applied to the fluorescence imaging detection of exogenous and endogenous peroxynitrite in living cells.

Description

Rhodol fluorescent probe for rapidly and sensitively detecting peroxynitrate and preparation method and application thereof

Technical Field

The invention belongs to the technical field of fluorescence sensing detection, and particularly relates to a fluorescence-enhanced detection fluorescent probe which is based on a Rhodol derivative and has high speed, sensitivity and selectivity on peroxynitrate.

Background

In recent years, the death rate of serious diseases such as malignant tumor, diabetes, cardiovascular and cerebrovascular diseases and the like is continuously increased, and the health and the life quality are seriously harmed. The search and development of early diagnosis methods for biomarkers of related diseases has become a research hotspot. Peroxynitrite (ONOO) ^- ) Is an important active nitrogen component in physiological systemPredominantly of peroxy anions (O) ₂ . ^- ) And Nitric Oxide (NO), and is mainly produced in mitochondria. Low concentration of ONOO ^- Participate in the signal transduction process in organisms and maintain normal physiological functions. However, ONOO ^- The product has strong oxidizing property, and when the concentration is too high, the structure and the function of biomacromolecules such as nucleic acid, protein, lipid and the like can be damaged, so that the product is closely related to the onset of many diseases, such as inflammation, cardiovascular and cerebrovascular diseases, alzheimer disease, cancer, diabetes, drug-induced liver injury and the like. However, by now, ONOO ^- The specific physiological and pathological processes that lead to the above-mentioned diseases are not fully understood, and thus, the specific physiological and pathological processes of ONOO are described in ^- The detection and biological imaging research of (2) is of great importance for the diagnosis and treatment of many diseases.

The detection method for peroxynitrite comprises electrochemical analysis, electron spin resonance, ultraviolet spectrophotometer method and the like, but peroxynitrite in organisms has the characteristics of high reaction activity, low concentration, short half-life period, environmental sensitivity and the like, and the methods are not suitable for detection and analysis at present. The existing fluorescent probe has slow response time and is easy to be subjected to H ₂ O ₂ The interference causes the problems of poor selectivity and the like, so that the design and development of a fast, sensitive and high-selectivity peroxynitrite fluorescent probe has important significance.

Disclosure of Invention

The invention aims at the problems in the peroxynitrite fluorescent probe and provides a preparation method of a Rhodol fluorescent probe, rapid, sensitive and high-selectivity detection of peroxynitrite and biological cell imaging application thereof.

A Rhodol fluorescent probe has the following structural formula:

the Rhodol fluorescent probe of the invention introduces 1, 1-dimethylhydrazine into aldehyde group of a Rhodol dye frame to obtain 1, 1-dimethylhydrazone which is easily destroyed by specific oxidation of peroxynitrite as a response identification group, and methylates carboxylic acid of a Rholol dye skeleton to avoid interference of pH change on a fluorescent signal, thereby obtaining the fluorescent probe. The probe can realize the rapid, sensitive and high-selectivity detection of the peroxynitrite through the unique oxidation bond-breaking reaction with the peroxynitrite. In a test system PBS (20mM, pH 7.4), the probe has almost no fluorescence signal, after the peroxynitrite is added, the fluorescence signal is rapidly enhanced, the response is finished within 1 minute, the detection limit of the probe is 57nM, and the method has high selectivity and rapid sensitive detection, and can also be used for naked eye qualitative identification and fluorescent quantitative detection.

The invention provides a preparation method of a Rhodol fluorescent probe for detecting peroxynitrate, which comprises the following steps:

heating and condensing 4-

diethylamino keto acid

1 and 2, 4-dihydroxybenzaldehyde in methanesulfonic acid to obtain Rhodol dye 2, carrying out esterification reaction on the Rhodol dye 2 and concentrated sulfuric acid in methanol to obtain a compound 3, finally condensing the

compound

3 and 1, 1-dimethylhydrazine hydrochloride in dichloromethane/triethylamine, and carrying out post-treatment after the reaction is finished to obtain the fluorescent probe.

The reaction equation is as follows:

the invention also provides application of the Rhodol fluorescent probe in peroxynitrate detection and cell imaging.

The Rhodol fluorescent probe of the present invention has almost no fluorescence signal after being excited by 530nm light in PBS buffer solution (20mM, pH 7.4), the fluorescence quantum efficiency is measured to be 0.02, 1-dimethylhydrazone in the probe is oxidized and broken in the presence of peroxynitrite to obtain aldehyde group Rhodol dye, and at this time, strong orange-red fluorescence with an emission peak of 571nm is generated by excitation at 530nm, such that peroxynitrite is qualitatively detected by fluorescence enhancement, and the detection reaction is shown in FIG. 1.

The fluorescence emission intensity of the fluorescent probe is gradually enhanced at 571nm along with the increase of the concentration of peroxynitrite under the excitation of light of 530nm, and the change of the fluorescence intensity is in a linear relation with the concentration of peroxynitrite within a certain range, so that the change of the fluorescence intensity can be determined to quantitatively detect the concentration of peroxynitrite, as shown in figures 2 and 3.

Adding the fluorescent probe into Hela living cells to form an experimental group, and adding 3-morpholine pyridine imine hydrochloride (SIN-1) for pretreatment (providing ONOO) before adding the probe ^- ) And additional uric acid (ONOO) ^- Scavenger) of two control groups; as shown in FIG. 6, the fluorescence channel of the cells in the experimental group was weak, the fluorescence of the cell fluorescence channel of the SIN-1 treated control group was strong, and the fluorescence signal of the control group was significantly reduced after the treatment with uric acid. The change of the cell fluorescence imaging is obvious, which indicates that the probe can be used as an exogenous ONOO-imaging detection tool of living cells.

Endogenous ONOO of cells ^- Fluorescence imaging: after the HepG2 cell is cultured by LPS + IFN-gamma, adding a probe for post-treatment imaging; and a comparison group, wherein after the cells are cultured by LPS + IFN-gamma, active nitrogen scavenger GSH and TEMPO are added for treatment, and finally, a probe is added for confocal cell imaging detection. As shown in FIG. 7, the fluorescent signal of the probe in the cell is very weak, after the cell is incubated with LPS + IFN-gamma, the fluorescent signal in the cell is very strong, and the fluorescent signal in the cell treated by the active nitrogen scavenger GSH or TEMPO is very weak and reduced. The results indicate that the probes can be used for the endogenous production of ONOO in cells ^- And (4) fluorescence detection imaging.

The beneficial effects of the invention are as follows: the fluorescent probe is simple to synthesize, simple to operate and low in synthesis cost; can detect peroxynitrite quickly, sensitively and with high selectivity. The fluorescence emission intensity of the probe is enhanced at 571nM along with the increase of the concentration of the peroxynitrite, the fluorescence intensity and the concentration of the peroxynitrite are in a linear relation in a certain range, the detection limit is 57nM, the response is within 1 minute, and the response is free from H ₂ O ₂ ,ClO ^- And other active oxygen interference effects. The fluorescent materialThe optical probe can not only quantitatively detect peroxynitrite in a solution system, but also can be used for imaging detection of peroxynitrite in living cells.

Drawings

FIG. 1 shows the fluorescent probe and ONOO of the present invention ^- The structure of the effect changes.

FIG. 2 shows the fluorescent probe of the present invention and different concentrations of ONOO ^- Graph of fluorescence emission change of effect.

FIG. 3 shows the intensity of the fluorescent probe of the present invention versus different concentrations of ONOO ^- Change in fluorescence intensity after action (F) _571nm ) Spectra.

FIG. 4 shows different concentrations of ONOO for the fluorescent probes of the present invention ^- (0, 25 and 50. Mu. Mol/L) change in fluorescence intensity at 571nm (F) _571nm ) Time-varying spectra.

FIG. 5 shows the change in fluorescence intensity of the fluorescent probe of the present invention after it has acted on various interferents (F) _571nm ) Spectra.

FIG. 6 is a graph showing the fluorescence imaging of the fluorogenic probe of the present invention on exogenous ONOO-in Hela cells.

FIG. 7 is a graph of the fluorescence image of the inventive fluorescent probe against endogenous ONOO-in cells.

FIG. 8 is the nuclear magnetic hydrogen spectrum of the pure fluorescent probe prepared in example 3 (400MHz, CDCl) ₃ )。

FIG. 9 shows the NMR spectrum of a pure fluorescent probe prepared in example 3 (100MHz, CDCl) ₃ )。

Detailed Description

Example 1

4-Diethylaminoketo acid 1 (3.7g, 10mmol) was weighed and dissolved in methanesulfonic acid (10 mL), and 2, 4-dihydroxy-benzaldehyde (1.4g, 10mmol) was added to an ice-water bath and the mixture was heated at 90 ℃ for 3 hours under nitrogen atmosphere and stirred. After completion of the reaction, the reaction mixture was poured into ice water, filtered to obtain a solid, and purified by silica gel column chromatography to obtain compound 2 (yield, 62%).

Example 2

To a solution of Compound 2 (80mg, 0.25mmol) in methanol (15 ml) was added concentrated H under ice-water bath ₂ SO ₄ (1 ml). The reaction is carried out in a nitrogen atmosphereReflux for 6h. After cooling, the solvent was spin-dried and NaHCO was added to the residue ₃ Extracting the solution with dichloromethane, collecting organic layer, drying with anhydrous sodium sulfate, concentrating, purifying with silica gel column chromatography to obtain compound 3 (67mg, 52%), ¹ H NMR(400MHz),CDCl ₃ ):δ10.42(s,1H),8.26(dd,J＝8.0Hz,1.2Hz,1H),7.66-7.75(m,2H),7.44(s,1H),7.44(s,1H),7.28(d,J＝5.2Hz,1H),6.80(d,J＝9.2Hz,1H),6.51-6.59(m,3H),3.67(s,3H),3.47-3.53(m,4H),1.27(t,J＝7.2Hz,6H)； ¹³ C NMR(100MH _Z ,CDCl ₃ ):δ192.1,183.2,165.4,159.4,157.7,156.4,153.9,134.4,132.8,131.2,3.9,130.5.,9,132.,113.4,111.8,110.9,107.0,96.6,52.2,45.4,12.6。

example 3

Compound 3 (80mg, 0.25mmol) was weighed out and dissolved in dichloromethane (15 ml). Et was added ₃ N (0.2 ml) and 1, 1-dimethylhydrazine hydrochloride (30mg, 0.5 mmol) and the mixture was stirred at room temperature for 6h. After completion of the reaction, the solvent was distilled off under reduced pressure to give an oil, and the residue was purified by silica gel column chromatography to obtain a probe (30mg, 46%). ¹ H NMR(400MHz,CDCl ₃ ):δ8.27(d,J＝8.0Hz,1H),7.75-7.79(m,1H),7.67-7.71(m,1H),7.49(s,1H),7.34(d,J＝7.2Hz,1H),7.21(s,1H),6.90(d,J＝9.2Hz,1H),6.68-6.74(m,3H),3.63(s,3H),3.52-3.57(m,4H),2.93(s,6H)),1.27(t,J＝7.2Hz,6H)； ¹³ CNMR(100MHz，CDCl ₃ )：δ165.7、156.0、155.1、153.4、134.4、132.8、131.2、130.7、130.0、130.0、129.7、115.3、112.8、105.0、104.6、104.6、96.5、96.5、52.4、52.4、45.6、45.6、42.7、12.7、12.7。

Example 4

Probes to different concentrations of ONOO ^- Fluorescence emission change spectrum: the probe was prepared as a 5. Mu. Mol/L PBS buffer solution (20mM, pH 7.4), and different concentrations of ONOO were added ^- The solution, after equilibration, is subjected to fluorescence emission spectroscopy, the result is shown in FIG. 2, which shows the fluorescence intensity I at 571 _571nm The variation is as in figure 3.

As can be seen from FIGS. 2 and 3, addition of ONOO ^- Then, the fluorescence emission of the probe is significantly enhanced, and the fluorescence emission intensity and ONOO at 571nm ^- Is linear in the range of 0-40 mu mol/LAccordingly, the probe may be referred to as ONOO ^- The fluorescence sensor quantitatively detects the fluorescent substance.

Example 5

Probe pair ONOO ^- Change in response time of fluorescence detection of (2): the probe was prepared as a 5. Mu. Mol/L PBS buffer solution (20mM, pH 7.4) without or with addition of ONOO ^- The fluorescence emission spectra of the solutions (25 and 50. Mu. Mol/L) were measured, and the change in fluorescence intensity at 571nm with time is shown in FIG. 4.

As can be seen from FIG. 4, the probe itself was relatively stable and was able to detect ONOO ^- The detection response of (2) is sensitive, and the response is complete within 1 minute.

Example 6

Fluorescence intensity I of probes after acting on different interferents _571nm Changing: PBS buffer (20mM, pH 7.4) was prepared at a concentration of 5. Mu. Mol/L probe, and after adding 100. Mu. Mol/L of various interferents, the fluorescence emission spectra were measured, and the results were shown in FIG. 5.

The results show that only ONOO ^- The fluorescence of the probe is obviously enhanced and changed, and other interferents have little influence, indicating that the probe is on ONOO ^- Has excellent detection selectivity.

Example 7

Exogenous ONOO ^- The cell fluorescence imaging test of (1): and transferring the three groups of Hela cells into a culture dish for confocal imaging, culturing for 24h, directly culturing the first group of Hela cells by using a probe (10 mu M) for 30 min, washing the first group of Hela cells by PBS for three times, then carrying out confocal cell imaging detection, pre-treating the second group of cells by SIN-1, culturing for 30 min, then adding the probe for culturing, washing the second group of cells by PBS for three times, carrying out confocal cell imaging, pre-treating the last group of cells by using uric acid, then adding the probe (10 mu M), culturing for 30 min, and washing the third group of cells by PBS for confocal cell imaging detection. The excitation wavelength used was 488nm and the fluorescence channel collection wavelength was 520-620nm, as shown in FIG. 6.

As can be seen from FIG. 6, the fluorescence signal of the probe alone was very weak, but passed through the ONOO ^- After the donor SIN-is cultured, the intracellular fluorescence signal is very strong, and finally the signal passes through ONOO ^- After pretreatment with scavenger (uric acid), the fluorescence signal decreased significantly. The results show that the probe canFor exogenous ONOO ^- And (4) carrying out fluorescence detection imaging on the cells.

Example 8

Endogenous ONOO ^- The cell fluorescence imaging test of (1): after the cells are cultured by LPS + IFN-gamma, adding a probe, and performing post-treatment imaging; and a comparison group, wherein after the cells are cultured by LPS + IFN-gamma, the cells are added with active nitrogen scavenger GSH and TEMPO for treatment, and then the cells are added with a probe for confocal cell imaging detection. The excitation wavelength used was 488nm and the green channel collection wavelength was 520-620nm, as shown in FIG. 7.

As can be seen from FIG. 7, the fluorescent signal of the probe in the cell was very weak, but after the incubation with LPS + IFN-. Gamma.the fluorescent signal in the cell was very strong, while the fluorescent signal in the cell treated with the active nitrogen scavenger GSH and TEMPO was very weak and decreased. The results indicate that the probes can be used for the endogenous production of ONOO in cells ^- And (4) fluorescence detection imaging.

Claims

1. A Rhodol fluorescent probe for rapidly and sensitively detecting peroxynitrate is characterized in that the structural formula of the fluorescent probe is as follows:

2. the method for preparing a Rhodol-type fluorescent probe according to claim 1, comprising the following steps:

(1) Heating 4-diethylamino keto acid 1 and 2, 4-dihydroxybenzaldehyde in methanesulfonic acid for condensation, and performing post-treatment after the reaction to obtain Rhodol dye 2;

(2) Performing esterification reaction on Rhodol dye 2 and concentrated sulfuric acid in methanol, and performing post-treatment after the reaction to obtain a compound 3;

(3) And (3) condensing the compound 3 and 1, 1-dimethylhydrazine hydrochloride in dichloromethane and triethylamine, and performing post-treatment after the reaction is finished to obtain the fluorescent probe.

3. The method for preparing a Rhodol-based fluorescent probe according to claim 2, wherein the feeding molar ratio of 4-diethylaminoketo acid 1 to 2, 4-dihydroxybenzaldehyde in step 1) is 1 to 1.5, and the reaction temperature is 70 to 110 ℃.

Step 2) sulfuric acid is a solvent and a catalyst, and methanol includes but is not limited to methanol, ethanol, isopropanol, butanol and the like.

Step 3) the feeding molar ratio of the compound 3 to 1, 1-dimethylhydrazine hydrochloride and triethylamine is (1).

4. Use of the Rhodol-type fluorescent probe according to claim 1 in peroxynitrate detection.

5. The application of Rhodol-type fluorescent probe in peroxynitrate detection according to claim 4, wherein the detection method is as follows: and adding the solution to be detected into the PBS buffer solution of the fluorescent probe, irradiating by adopting light of 530nm, observing the fluorescence change of the probe solution, and judging whether the peroxynitrate is detected according to the fluorescence change of the probe solution.

6. The application of Rhodol-type fluorescent probe in peroxynitrate detection according to claim 4, wherein the detection method is as follows: adding the solution to be detected into the PBS buffer solution of the fluorescent probe, then measuring the fluorescence emission spectrum to obtain the fluorescence change value before and after the solution to be detected is added, and then comparing the fluorescence change value with the standard curve to obtain the peroxynitrate concentration in the solution to be detected.

7. The use of Rhodol-type fluorescent probe for peroxynitrate according to claim 5 or 6, wherein the PBS buffer solution has a concentration of 20mM and a pH of 7.4.

8. The application of Rhodol-type fluorescent probe to peroxynitrate according to claim 1, which is used for fluorescence imaging detection of endogenous and exogenous peroxynitrate in living cells.