CN115232098B

CN115232098B - Rhodol fluorescent probe for rapidly and sensitively detecting peroxynitrate as well as preparation method and application thereof

Info

Publication number: CN115232098B
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: 2023-10-03
Anticipated expiration: 2042-07-18
Also published as: CN115232098A

Abstract

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

Description

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

Technical Field

The invention belongs to the technical field of fluorescence sensing detection, and particularly relates to a fluorescence probe for rapidly, sensitively and highly selectively fluorescence-enhanced detection of peroxynitrate based on a Rhodol derivative.

Background

In recent years, the mortality rate of serious diseases such as malignant tumor, diabetes mellitus, cardiovascular and cerebrovascular diseases and the like is continuously increased, and the body health and the quality of life are seriously endangered. The search and development of early diagnostic methods for biomarkers of related diseases has become a current research hotspot. Peroxynitrite (ONOO) ^- ) Is an important active nitrogen molecule in physiological system, mainly composed of peroxy anion (O ₂ . ^- ) And Nitric Oxide (NO) and is produced mainly in mitochondria. Low concentration ONOO ^- Is involved in the signal transduction process in the living body, and maintains normal physiological functions. However, ONOO ^- Has strong oxidizing property, and can destroy the structure and function of biomacromolecule such as nucleic acid, protein, lipid, etc. when the concentration is too high, thereby being closely related to the pathogenesis of a plurality of diseases, such as inflammation, cardiovascular and cerebrovascular diseasesDiseases, alzheimer's disease, cancer, diabetes, and drug-induced liver injury. However, to date, ONOO ^- The specific physiological and pathological processes responsible for the above-mentioned diseases are not completely understood, and thus, for ONOO ^- The detection and the biological imaging research of the formula (I) have important significance for diagnosis and treatment of a plurality of diseases.

The detection method of the peroxynitrite comprises electrochemical analysis, electron spin resonance, ultraviolet spectrophotometry and the like, but the peroxynitrite in the organism has the characteristics of high reactivity, low concentration, short half-life period, environmental sensitivity and the like, and the methods are not suitable for detection analysis at present. Existing fluorescent probes have slow response time and are easy to H ₂ O ₂ The interference causes the problems of poor selectivity and the like, so the design and development of the rapid, sensitive and high-selectivity peroxynitrite fluorescent probe have important significance.

Disclosure of Invention

Aiming at the problems in the peroxynitrite fluorescent probe, the invention provides a preparation method of a Rhodol fluorescent probe and application of the Rhodol fluorescent probe in quick, sensitive and high-selectivity detection and biological cell imaging on peroxynitrite.

A Rhodol fluorescent probe has the following structural formula:

according to the Rhodol fluorescent probe, 1-dimethylhydrazine is introduced into aldehyde groups of a Rhodol dye framework to obtain 1, 1-dimethylhydrazone which is easy to be damaged by specific oxidation of peroxynitrite as a response recognition group, and carboxylic acid of the Rhodol dye framework is subjected to methylation treatment to avoid interference of pH change on fluorescent signals, so that the fluorescent probe is obtained. The rapid, sensitive and high-selectivity detection of the probe on the peroxynitrite is realized through the unique oxidation bond breaking reaction with the peroxynitrite. In a PBS buffer solution (20 mM, pH 7.4) of the test system, the probe has almost no fluorescent signal, the fluorescent signal is rapidly enhanced after peroxynitrite is added, the response is finished within 1 minute, the detection limit of the probe is 57nM, and the probe 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:

and (3) heating and condensing the 4-diethylamino keto acid 1 and 2, 4-dihydroxybenzaldehyde in methanesulfonic acid to obtain a Rhodol dye 2, esterifying the Rhodol dye 2 with concentrated sulfuric acid in methanol to obtain a compound 3, condensing the compound 3 with 1, 1-dimethylhydrazine hydrochloride in dichloromethane/triethylamine, and performing aftertreatment 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 has the advantages that in PBS buffer solution (20 mM, pH 7.4), after being excited by 530nm light, a fluorescent signal hardly exists, the fluorescent quantum efficiency is measured to be 0.02, 1-dimethyl hydrazone in the probe is oxidized and broken in the presence of peroxynitrite to obtain aldehyde Rhodol dye, at the moment, the excitation at 530nm can generate strong orange red fluorescence with an emission peak of 571nm, so that the peroxynitrite is detected by fluorescence enhancement qualitative detection, and the detection reaction is shown in figure 1.

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

The book is put intoThe fluorescent probe of the invention is added into Hela living cells to be an experimental group, and 3-morpholinopyridine imine hydrochloride (SIN-1) is added to be pretreated (ONOO is provided) before the probe is added ^- ) And with the addition of uric acid (ONOO) ^- Scavenger) staining of cells of the two control groups; as shown in FIG. 6, the cell fluorescence channel in the experimental group had a weak signal, the cell fluorescence channel in the SIN-1 treated control group had a strong fluorescence, and in the other control group, the fluorescence signal was significantly reduced after the uric acid treatment was added. The change of cell fluorescence imaging is obvious, which indicates that the probe can be used as an exogenous ONOO-imaging detection tool for living cells.

Cellular endogenous ONOO ^- Fluorescence imaging: after the HepG2 cells are cultivated by LPS+IFN-gamma, probes are added for post-treatment imaging; and in the comparison group, after the cells are cultivated by LPS+IFN-gamma, adding active nitrogen scavenger GSH and TEMPO for treatment, and finally adding a probe for confocal cell imaging detection. As shown in FIG. 7, the fluorescent signal of the probe in the cell is very weak, and after LPS+IFN-gamma incubation, the fluorescent signal in the cell treated with active nitrogen scavenger GSH or TEMPO is very weak. The results indicate that the probe can be used for ONOO endogenously produced by cells ^- Fluorescence detection imaging.

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

Drawings

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

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

FIG. 3 shows the intensity of the fluorescent probe versus the concentration of ONOO according to the invention ^- Change in fluorescence intensity after action (F _571nm ) A spectrogram.

FIG. 4 shows the fluorescence probe of the present invention for ONOO of different concentrations ^- (0, 25 and 50. Mu. Mol/L) change in fluorescence intensity at 571nm after the action (F) _571nm ) A time-dependent spectrum.

FIG. 5 shows the change in fluorescence intensity of the fluorescent probe according to the present invention after the fluorescent probe acts on different interferents (F _571nm ) A spectrogram.

FIG. 6 is a fluorescent image of exogenous ONOO-by the fluorescent probe of the invention in HeLa cells.

FIG. 7 is a fluorescent image of endogenous ONOO-in a cell using a fluorescent probe according to the invention.

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

FIG. 9 is a nuclear magnetic resonance spectrum (100 MHz, CDCl) of a fluorescent probe prepared in example 3 ₃ )。

Detailed Description

Example 1

4-diethylamino-keto acid 1 (3.7 g,10 mmol) was weighed out and dissolved in methanesulfonic acid (10 mL), 2, 4-dihydroxy-benzaldehyde (1.4 g,10 mmol) was added to the ice-water bath, and the mixture was stirred under nitrogen atmosphere at 90℃for 3 hours. After the reaction was completed, the reaction mixture was poured into ice water, and a solid was obtained after filtration, followed by purification by silica gel column chromatography to obtain compound 2 (yield, 62%).

Example 2

To a solution of Compound 2 (80 mg,0.25 mmol) in methanol (15 ml) was added concentrated H under an ice-water bath ₂ SO ₄ (1 ml). The reaction was refluxed under nitrogen atmosphere for 6h. After cooling, the solvent was dried by spin-drying, and NaHCO was added to the residue ₃ Extracting with dichloromethane, collecting organic layer, drying with anhydrous sodium sulfate, concentrating, purifying with silica gel column chromatography to obtain compound 3 (67 mg, 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 (80 mg,0.25 mmol) was weighed and dissolved in dichloromethane (15 ml). Addition of Et ₃ N (0.2 ml) and 1, 1-dimethylhydrazine hydrochloride (30 mg,0.5 mmol) and the mixture was stirred at room temperature for 6h. After the 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 give a probe (30 mg, 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

ONOO of different concentrations for probe pairs ^- Fluorescence emission change profile: the probe was prepared as 5. Mu. Mol/L PBS buffer (20 mM, pH 7.4) and ONOO was added at various concentrations ^- After the solution is equilibrated, the fluorescence emission spectrum is measured, and the result is shown in FIG. 2, which shows the fluorescence intensity I at 571 _571nm The variation is shown in fig. 3.

As can be seen from fig. 2 and 3, ONOO is added ^- After that, the fluorescence emission of the probe is significantly enhanced, and the fluorescence emission intensity at 571nm is equal to that of ONOO ^- In a linear relationship in the range of 0-40. Mu. Mol/L, so that the probe can be used as ONOO ^- The fluorescent sensor quantitatively detects the fluorescent sensor.

Example 5

Probe pair ONOO ^- Fluorescence detection response time variation of (2): the probe was prepared as 5. Mu. Mol/L PBS buffer (20 mM, pH 7.4), with or without the 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 in the arrangement with time is shown in FIG. 4.

As can be seen from FIG. 4, the probe itself is relatively stable and is also for ONOO ^- Is sensitive and responds completely within 1 minute.

Example 6

Fluorescence intensity I after probe action on different interferents _571nm The change is: after preparing a 5. Mu. Mol/L probe in PBS buffer (20 mM, pH 7.4), and then adding 100. Mu. Mol/L of different interferents, fluorescence emission spectra were measured, and the result of the arrangement is shown in FIG. 5.

The results show that only ONOO ^- Has obvious enhanced change to the fluorescence of the probe, and other interferents have little influence, which indicates that the probe has no effect on ONOO ^- Has excellent detection selectivity.

Example 7

Exogenous ONOO ^- Is measured by a cell fluorescence imaging test of (a): after three groups of Hela cells were transferred to a petri dish for confocal imaging and incubated for 24 hours, the first group was incubated with the probe (10. Mu.M) directly for 30 minutes, and after three times of PBS washing, confocal cell imaging was performed, the second group was incubated for 30 minutes after pretreatment with SIN-1, and after three times of PBS washing, confocal cell imaging was performed, and the last group was incubated for 30 minutes after pretreatment with uric acid, and after three times of PBS washing, confocal cell imaging was performed. 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-only cells was weak, but passed through ONOO ^- After donor SIN-incubation, the intracellular fluorescence signal is strong, and finally goes through ONOO ^- After pretreatment with scavenger (uric acid), the fluorescence signal is significantly reduced. The results indicate that the probe can be used for exogenous ONOO ^- And (5) performing cell fluorescence detection imaging.

Example 8

Endogenous ONOO ^- Is measured by a cell fluorescence imaging test of (a): after the cells are incubated with LPS+IFN-gamma, probes are added, and the cells are subjected to post-treatment imaging; and in the comparison group, after the cells are cultivated by LPS+IFN-gamma, adding active nitrogen scavenger GSH and TEMPO for treatment, and then adding a probe for confocal cell imaging detection. Excitation usedThe green channel collection wavelength is 520-620nm with a wavelength of 488nm, as shown in FIG. 7.

As can be seen from FIG. 7, the fluorescence signal of the probe in the cells was very weak, but after LPS+IFN-. Gamma.incubation, the fluorescence signal in the cells treated with the active nitrogen scavengers GSH and TEMPO was very weak. The results indicate that the probe can be used for ONOO endogenously produced by cells ^- Fluorescence detection imaging.

Claims

1. A Rhodol fluorescent probe for rapidly and sensitively detecting peroxynitrate, which is characterized by having the following structural formula:

。

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

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

(2) Carrying out esterification reaction on Rhodol dye 2 and concentrated sulfuric acid in methanol, and after the reaction is finished, carrying out aftertreatment to obtain a compound 3;

(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 the Rhodol fluorescent probe according to claim 2, wherein the feeding molar ratio of 4-diethylamino keto acid 1 to 2, 4-dihydroxybenzaldehyde in the step 1) is 1:1-1:1.5, and the reaction temperature is 70-110 ℃;

step 2) concentrated sulfuric acid is used as a solvent and a catalyst;

the feeding molar ratio of the compound 3 to the 1, 1-dimethylhydrazine hydrochloride to the triethylamine in the step 3) is 1:1:5-1:3:5.

4. Use of a Rhodol fluorescent probe according to claim 1 for peroxynitrate detection.

5. The use of a Rhodol fluorescent probe according to claim 4 for detecting peroxynitrate, wherein the detection method is as follows: and adding a liquid to be detected into the PBS buffer solution of the fluorescent probe, irradiating with 530 and nm light, observing the fluorescence change of the probe solution, and judging whether the probe solution contains peroxynitrate for detection according to the fluorescence change of the probe solution.

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

7. Use of a Rhodol fluorescent probe according to claim 5 or 6 for peroxynitrate detection, wherein the concentration of the PBS buffer solution is 20mm and the ph is 7.4.

8. The use according to claim 4 for the fluorescence imaging detection of endogenous and exogenous peroxynitrate in living cells.