CN113429389B - Preparation and application of solid-state fluorescent probe for detecting carbon monoxide - Google Patents

Abstract

The invention relates to preparation and application of a solid-state fluorescent probe for detecting carbon monoxide (CO), wherein the structural formula of the fluorescent probe is as follows:

the invention provides a preparation method for synthesizing a solid fluorescent probe by using 3- (7-chlorine-4-oxygen-3, 4-dihydroquinazoline) -4-hydroxybenzaldehyde, 1-ethyl-2-methylquinoline, allyl chloroformate and the like as raw materials; firstly, the solid-state fluorescent probe has longer wavelength, emits strong fluorescence at 650nm, is insoluble in water and has the anti-diffusion capability; secondly, the fluorescent probe shows high sensitivity to CO, and the fluorescence is obviously enhanced after the probe reacts with the CO; in addition, the solid-state fluorescent probe has high selectivity on CO, and is not interfered by other active oxygen, active nitrogen, active sulfur, biological mercaptan and common ions; in addition, the solid-state fluorescent probe has rapid action with CO, and the response time is within 100 s; finally, the solid-state fluorescent probe is applied to detection of the content of carbon monoxide in living cells, and can perform fluorescence imaging on CO.

Description

Preparation and application of solid-state fluorescent probe for detecting carbon monoxide

Technical Field

The invention belongs to the technical field of fluorescent probes, and particularly relates to preparation and application of a solid-state fluorescent probe for detecting carbon monoxide.

Background

Carbon monoxide (CO) is a colorless, odorless gas that has traditionally been considered a toxic substance due to its strong affinity for hemoglobin (l.k.weaver, n.engl.j.med.,2009,360, 1217-. However, in recent years, scientific evidence has shown that CO is associated with Nitric Oxide (NO), hydrogen sulfide (H)₂S), which is an essential gas transmitter in human body, plays an important role in a variety of physiological and pathological processes (f.watts, r.favory, s.lancet, r.neviere, d.mathieu, ball.acad.natl.med., 2006,190, 1961-; l.y.wu, r.wang, pharmacol.rev.,2005,57, 585-. For example: CO is involved in vasodilation, anti-apoptosis, anti-inflammation and neurotransmission. In addition, abnormalities in CO metabolism are associated with a number of diseases, such as: alzheimer's disease, hypertension, inflammation, heart failure, etc. (D.R.Premkumar, M.A.Smith, P.L.Richey, R.B.Petersen, R.Castellani, R.K.Kutty, J.Neurochem.,1995,65,1399-,1581-1589). Because of the important physiological and pathological effects of carbon monoxide, it is necessary to develop a method suitable for detecting the CO content in cells.

Compared with other traditional detection methods, the fluorescent probe has the advantages of convenience, sensitivity, no wound and the like. At present, some fluorescent probes for detecting CO have been developed, but none of the fluorescent probes can accurately image in cells after reacting with CO. Since the fluorophores released by these probes after reaction with CO are soluble and thus diffuse away from the site of action quickly, the released fluorescent signals can even penetrate outside the cell, resulting in a fluorescence imaging range that is too large for accurate in situ imaging (X.Chen, Y.ZHou, X.Peng, chem.Soc.Rev.,2010,39, 2120. 2135; X.Li, X.Gao, W.Shi, H.Ma, chem.Rev.,2014,114, 590. sup. 659). Therefore, it remains a challenge to develop a probe that can provide CO fluorescence imaging in situ in cells as well as in vivo.

HPQ is a solid-state fluorophore which can emit strong solid-state fluorescence, has anti-diffusion property and can realize accurate in-situ imaging. Solid-state fluorescent probes based on HPQ synthesis have been successfully used for furan, hypochlorous acid (ClO)^-) Detection of alkaline phosphatase (ALP), hydrogen peroxide, gamma-glutamyltranspeptidase (GGT), aminopeptidase n (apn) (k.li, x.x.hu, h.w.liu, s.xu, s.y.huangan, j.b.li, t.g.deng, x.b.zhang, anal.chem.,2018,90, 11680-; l.zhou, d.q.lu, q.wang, s.hu, h.wang, h.sun, x.zhang, spectrochim.acta.a mol.biomol.spectrosc.,2016,166,129-134.h.w.liu, k.li, x.x.hu, angelw.chem.int.ed., 2017,56, 11788-; z.li, t.b.ren, x.x.zhang, s.xu, x.y.gong, y.yang, g.l.ke, l.yuan, x.b.zhang, anal.chem.,2021,93, 2235-; J.Ou-Yang, Y.F.Li, P.Wu, ACS sens, 2018,3, 1354-1361; liu, C.xu, H.W.Liu, L.Teng, S.Huang, L.Yuan, X.B.Zhang, anal.chem.,2021,93, 6463-containing 6471). However, the short wavelength of these solid-state fluorescent probes limits their application in living organisms. Therefore, it is important to develop a solid-state fluorescent probe having a longer wavelength.

Disclosure of Invention

In light of the demands made, the present inventors have conducted intensive studies to provide a solid-state fluorescent probe for detecting carbon monoxide after a great deal of creative work.

The technical scheme of the invention is that the solid-state fluorescent probe for detecting carbon monoxide has the following structural formula:

a method for preparing a solid-state fluorescent probe for detecting carbon monoxide. The synthesis steps are as follows:

1) dissolving 1 equivalent of 3- (6-chloro-3, 4-dihydro-4-oxo-2-quinazolinyl) -4-hydroxybenzaldehyde and 0.8-1.2 equivalent of 1-ethyl-2-methylquinoline in 10-20 mL of acetonitrile in a 100mL round-bottom flask, adding 0.1-0.5 mL of piperidine, reacting the reaction mixture at 60-90 ℃ for 12-18 h under the protection of nitrogen, stopping the reaction, removing the solvent by reduced pressure distillation to obtain a purple solid crude product, and dissolving the product in CH₂Cl₂Extracting with saturated salt water for three times, drying the organic phase with anhydrous sodium sulfate, and collecting the crude product with CH at a volume ratio of 100: 1-100: 10₂Cl₂/CH₃Performing column chromatography with OH eluent to obtain a reddish brown solid compound HPQ-MQ-OH.

2) Dissolving 1 equivalent of compound HPQ-MQ-OH in 10-20 mL of dry dichloromethane in a 100mL round-bottom flask at 0-5 ℃ under ice bath, adding 1.5-2.5 equivalents of triethylamine, slowly adding 9-12 equivalents of allyl chloroformate under the protection of nitrogen after 0-15 min, stirring for 0-5 h under ice bath for changing the color of the solution, then heating to room temperature, stirring for reaction for 10-15 h, stopping the reaction, removing the solvent through reduced pressure distillation, and performing column chromatography on the crude product by using dichloromethane to obtain a brown solid product, namely the solid fluorescent probe HPQ-MQ-CO.

The invention has the beneficial effect that the solid-state fluorescent probe for detecting carbon monoxide has excellent spectral performance. First, the fluorescence spectrum properties of the probe were investigated. The probe itself showed only weak fluorescence when Pd was added²⁺And a strong fluorescent signal at 650nm after CO.And the fluorescence signal intensity is continuously enhanced along with the increase of the CO concentration; the detection range of the probe is 0.1 mu M to 100 mu M, and the detection limit is 0.035 mu M. This indicates that the probe can detect CO with high sensitivity. Next, the ultraviolet-visible absorption spectrum of the probe was investigated. The probe itself showed a weak absorption peak when Pd was added²⁺And CO showed distinct absorption peaks at 410nm and 550 nm. Subsequently, the fluorescence emission intensity changes after the action of the probe and CO were investigated in DMSO/PBS buffer at different ratios. Adding Pd²⁺And CO, the content of water is increased along with the continuous reduction of the proportion of DMSO in the buffer solution, and the emission intensity of the fluorophore at 650nm is observed to be increased continuously, which indicates that the probe has solid-state luminescence property. Then, the selectivity of the probe was investigated. Probes and active nitrogen species (ONOO) were investigated^-,NO,NO₂ ^-,NO₃ ^-) Active oxygen species (H)₂O₂,·OH,ClO^-,O₂ ^-) Active sulfur species (H)₂S,HSO₃ ^-,SO₃ ^2-) Amino acids (Ala, Trp, Leu), biological thiols (Cys, Hcy, GSH) and common ions (Na)⁺,Ca²⁺,Mg²⁺,Zn²⁺,K⁺) The fluorescent response of (c). As a result, only CO can cause the fluorescence signal to be obviously enhanced, and other detection objects almost have no response to the probe and only emit weak fluorescence. These indicate that the probe has good selectivity. Finally, the effect of pH on the CO measurement by the fluorescent probe was investigated, and the CO measurement by the fluorescent probe was not affected when the pH was between 7.0 and 9.0. In addition, the fluorescent probe has rapid response, and the response time is within 100 s.

Application of a solid-state fluorescent probe for detecting carbon monoxide. Only the fluorescent probe was added to the cells and the red channel had little fluorescence. Adding fluorescent probe and Pd simultaneously into cells²⁺The red channel still has no significant fluorescence change, indicating a low CO content in the cells. The cells are treated with a CO releasing agent and then Pd is added²⁺And simultaneously, the probe is used for staining, and a red channel detects a remarkable red fluorescent signal. The cells are treated with Heme (Heme) to stimulate the production of intracellular CO, and then P is addedd²⁺And simultaneously, staining with a probe to detect that a strong red fluorescent signal appears in the cells. These results indicate that the fluorescent probe can monitor changes in intracellular CO content.

Drawings

FIG. 1 shows a synthetic route of a solid-state fluorescent probe.

FIG. 2 is a fluorescence spectrum of a solid-state fluorescent probe after the solid-state fluorescent probe is reacted with CO at different concentrations.

The abscissa is wavelength and the ordinate is fluorescence intensity. Fluorescent probe and Pd²⁺The concentrations of (A) and (B) were all 10. mu.M, and the CO concentrations were respectively: 0,0.1,0.5,1,5,10,20,30,40,50,60,70,80,90, 100. mu.M. The fluorescence excitation wavelength was 550 nm.

FIG. 3 is a graph of the linear response of solid-state fluorescent probes to different CO concentrations.

FIG. 4 is a graph of the UV-VIS absorption spectra of a solid-state fluorescent probe and a solid-state fluorophore.

FIG. 5 is a graph showing fluorescence spectra of solid-state fluorescent probes after being exposed to CO in DMSO/PBS buffer solutions at different ratios.

Solid-state fluorescent probe and Pd²⁺The concentrations of (A) and (B) were 10. mu.M, the CO concentration was 100. mu.M, and the DMSO/PBS buffers were: 1%, 10%, 20%, 40%, 60%, 80%.

FIG. 6 is a graph of selectivity for solid-state fluorescent probes.

Solid-state fluorescent probe and Pd²⁺Concentrations of (D) were all 10. mu.M, CO concentrations and other analytes 100. mu.M.

FIG. 7 is a graph showing the effect of pH on solid-state fluorescent probes.

FIG. 8 is a graph showing the relationship between the fluorescence intensity of a solid-state fluorescent probe and the change of the fluorescence intensity with time after CO reaction.

FIG. 9 is a cytotoxicity assay. The abscissa is the concentration of the solid-state fluorescent probe and the ordinate is the survival rate of the cells.

FIG. 10 is an image of a cell imaged with solid-state fluorescent probes and CO. (a) Cells were stained with probe for 0.5 h. (b) Simultaneous application of probe and Pd to cells²⁺And dyeing for 0.5 h. (c) The cells were treated with CO releasing agent for 0.5h, then with probe and Pd simultaneously²⁺And dyeing for 0.5 h. (d) Cells were treated with Heme for 5h, then with both probe and Pd²⁺And dyeing for 0.5 h.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but is not limited thereto.

Example 1:

synthesis of solid-state fluorescent probes

The synthetic route is shown in figure 1.

Method for the synthesis of the solid-state fluorophore HPQ-MQ-OH: 0.35g of 1-ethyl-2-methylquinoline (1mmol), 0.30g of 3- (6-chloro-3, 4-dihydro-4-oxo-2-quinazolinyl) -4-hydroxybenzaldehyde (1mmol) and 20.0mL of acetonitrile were successively charged into a 100mL round-bottom flask. Then, 0.2mL of piperidine was added by a micropipette under a nitrogen atmosphere, and after stirring and refluxing at 80 ℃ for 15 hours, the solvent was removed by distillation under reduced pressure. Dissolving the solid product with dichloromethane, repeatedly extracting with saturated saline solution for three times, drying the lower organic phase, filtering, evaporating, and dissolving the solid product with CH₂Cl₂/CH₃Column chromatography with OH 100:7 (vol) as eluent gave 0.22g of the product as a reddish brown solid (HPQ-MQ-OH) in 50% yield.

The synthesis method of the solid-state fluorescent probe HPQ-MQ-CO comprises the following steps: 0.25g of HPQ-MQ-OH (1mmol) are dissolved in 10mL of dry CH at 0 ℃ under ice bath and inert gas protection₂Cl₂Then, 0.20g Et was added₃N (2mmol), stirring for reaction for 10min, adding 1.2g chloromethyl allyl ester (10mmol) by a microsyringe, stirring for reaction for 3h, changing the color of the solution, heating to room temperature, stirring for reaction for 12h, stopping the reaction, removing the solvent by reduced pressure distillation, and carrying out column chromatography on the crude product by dichloromethane eluent to obtain 0.11g brown solid product, namely the solid fluorescent probe HPQ-MQ-CO, wherein the yield is 20%.¹H NMR(400MHz,DMSO,ppm):8.65(s,1H),8.47(d,J＝16.0Hz,1H),8.33(d,J＝8.0Hz,1H),8.26(d,J＝8.0Hz,1H),8.18(d,J＝8.0Hz,1H),8.08(d,J＝8.0Hz,1H),8.02(s,1H),7.95(d,J＝8.0Hz,1H),7.87(d,J＝8.0Hz,1H),7.78(s,1H),7.74-7.68(m,1H),7.65-7.59(m,1H),7.26(d,J＝16.0Hz,1H),6.79(d,J＝8.0Hz,2H),5.82-5.74(m,1H),5.35(d,J＝8.0Hz,2H),4.67-4.61(m,2H),4.19(d,J＝4.0Hz,2H),1.43(t,J＝7.2Hz,3H).¹³C NMR(100MHz,DMSO,ppm):δ162.3,160.8,159.1,157.5,156.2,155.1,152.4,146.1,144.2,141.5,138.6,136.3,134.1,130.2,128.8,127.7,126.8,124.6,123.0,121.0,117.4,115.5,113.8,111.6,109.1,107.6,105.9,104.6,68.3,52.8,14.4.MS(TOF):538.4.

Example 2:

preparation of solid-state fluorescent probe and CO solution

Preparation of probe solution: weighing a certain amount of probe, dissolving in dimethyl sulfoxide to prepare 1 × 10^-4M probe solution. Simultaneously, weighing a certain amount of PdCl₂Dissolving in secondary distilled water to obtain 1 × 10 solution^-4A solution of M. Preparing a CO solution: dissolving a certain amount of CORM-3 in redistilled water, transferring to a 500mL volumetric flask, adding water to the scale mark to obtain a concentration of 1.0 × 10^-3CORM-3 of M. Mixing 1.0X 10^-3Gradually diluting CORM-3 solution of M to obtain 1.0X 10^-6-1.0×10^-3mol·L^-1An aqueous solution of CORM-3. 1.0mL of probe solution, 1.0mL of PdCl₂The solution and 1.0mL of CORM-3 aqueous solution were added to a 10mL volumetric flask, and the volume was determined by buffer solution to obtain a concentration of 1.0X 10^-5Fluorescent probe and PdCl of M₂，1.0×10^-7-1.0×10^-4And mixing the CO of the M with the solution to be detected.

Example 3:

determination of fluorescence spectra of solid-state fluorescent probes with CO interaction

FIG. 2 shows fluorescence spectra of solid-state fluorescent probe and CO, solid-state fluorescent probe and Pd²⁺The concentrations of (A) and (B) were all 10. mu.M, and the CO concentrations were respectively: 0,0.1,0.5,1,5,10,20,30,40,50,60,70,80,90, 100. mu.M. The excitation wavelength is fixed to be 550nm, and the emission wavelength range is 620-670 nm. The slit width was 5.0nm/5.0nm, and the fluorescence measuring instrument used was a Hitachi F4600 fluorescence spectrophotometer. As can be seen from FIG. 2, the probe itself emitted only weak fluorescence due to the quenching effect of allyl formate. Adding Pd²⁺And CO, a distinct emission peak at 650 nm. This is because of Pd²⁺First reduced to Pd by CO⁰Subsequent mediation of the Tsuji-Trost reaction results in cleavage of the allyl formate, liberating the fluorophore, thereby generating fluorescenceA signal. And the fluorescence intensity of the probe molecules is continuously enhanced along with the increase of the CO concentration. FIG. 3 is a graph of the linear response of the probe to different CO concentrations. The fluorescence intensity is in linear relation with the concentration of CO, and the linear range is 1.0 multiplied by 10^-7～1.0×10^-4M, the limit of detection is 0.035. mu.M. This indicates that the probe can detect CO with high sensitivity.

Example 4:

measurement of UV-visible absorption spectra of solid-state fluorescent probes and solid-state fluorophores

FIG. 4 shows the UV-VIS absorption spectra of a solid-state fluorescence probe and a solid-state fluorophore, wherein the concentration of the fluorescence probe is 10 μ M and the amount of CO is 20 μ M. The instrument for measuring the ultraviolet visible absorption spectrum is an Agilent Cary60 ultraviolet visible spectrophotometer. As can be seen from FIG. 4, the probe itself showed a weak absorption peak when Pd was added²⁺And CO showed distinct absorption peaks at 410nm and 550 nm.

Example 5:

determination of fluorescence spectra of probes after their interaction with CO in DMSO/PBS buffers containing different ratios

FIG. 5 shows fluorescence emission spectra of solid-state fluorescent probes after reaction in DMSO/PBS buffer containing different ratios. Solid-state fluorescent probe and Pd²⁺The concentrations of (A) and (B) were 10. mu.M, the CO concentration was 100. mu.M, and the DMSO/PBS buffers were: 1%, 10%, 20%, 40%, 60%, 80%. FIG. 5 shows that the addition of CO and Pd²⁺Then, with the increasing water content in DMSO/PBS buffer, the emission intensity at 650nm is increased, which indicates that the probe has solid-state luminescence property.

Example 6:

selectivity of solid-state fluorescent probes for CO determination

FIG. 6 is a graph of selectivity of solid-state fluorescent probes for CO determination. Examination of fluorescent Probe and Pd at a concentration of 10. mu.M²⁺Adding CO (100 μ M) and active nitrogen species (ONOO) into the solution^-,NO,NO₂ ^-,NO₃ ^-) Active oxygen species (H)₂O₂,·OH,ClO^-,O₂ ^-) Active sulfur species (H)₂S,HSO₃ ^-,SO₃ ^2-) Amino acids (Ala, Trp, Leu), active biological thiols (Cys, Hcy, GSH) and common ions (Na)⁺,Ca²⁺,Mg²⁺,Zn²⁺,K⁺,F^-) (100. mu.M) fluorescence response. As can be seen in FIG. 6, only CO caused a change in the fluorescence spectrum, and the other analytes had no significant effect on the fluorescence spectrum of the probe. These results indicate that the fluorescent probe is better selective for CO.

Example 7:

influence of solution pH value on fluorescence property of solid-state fluorescent probe for CO determination

The effect of pH on the fluorescence spectrum of CO measured with the fluorescent probe was examined, and the results are shown in FIG. 7. The pH range of the study is 2.0-12.0, and the fluorescent probe and Pd²⁺The concentrations of (A) and (B) were all 10. mu.M, and the concentration of CO was 100. mu.M. As can be seen from the figure, the fluorescence intensity of the fluorescent probe is basically unchanged along with the change of pH, which shows that the pH has no great influence on the probe. However, after the addition of CO, the fluorescence intensity ratio is remarkably enhanced in the pH range of 7.0-9.0. In summary, when the pH value is between 7.0 and 9.0, the determination of CO by the fluorescent probe is not affected, and the pH value range is more suitable, which is very beneficial for the probe to be used for determining CO in actual samples.

Example 8:

determination of response time of solid-state fluorescent probe to CO action

The response time of the fluorescent probe to CO was investigated, and the results are shown in FIG. 8. As can be seen from the figure, the response time of the probe to CO is 100s, and the requirement of real-time monitoring in actual samples can be met. As can be seen from FIG. 8, after the fluorescence intensity reaches the maximum value, the fluorescence intensity does not change any more in the following time, indicating that the fluorescence probe has better light stability.

Example 9:

application of solid-state fluorescent probe in living cells

First, the toxicity of the probe against the cells was investigated, and the results are shown in FIG. 9. When 0-30 mu M CO probe is added, the survival rate of the cells is over 90 percent. It can be shown that the fluorescent probe is toxicThe kit is small in size and can be applied to detecting CO in living cells. Then, the application of fluorescent probe in living cells was studied, and liver cancer cell HepG2 was selected for confocal microscopy, and the results are shown in fig. 10. Only fluorescent probes were added to the cells and the red channel barely fluoresced (fig. 10 a); adding fluorescent probe and Pd simultaneously into cells²⁺The red channel still did not show significant fluorescence change (fig. 10b), indicating a lower CO content in the cells. The cells were treated with CO releasing agent for 0.5h, then Pd was added²⁺At the same time, staining with the probe was performed for 0.5h, and a distinct red fluorescent signal was detected in the red channel (FIG. 10 c). Heme (Heme) is reported in the literature to stimulate the production of CO in cells. The cells are pretreated with Heme (Heme) for 5h to stimulate the production of CO in the cells, and then Pd is added²⁺At the same time, the probe was used for 0.5h, and a strong red fluorescence signal was detected in the cells (FIG. 10 d). FIG. 10e is a quantitative histogram of the fluorescence emission intensity of cells in the red channel. These results indicate that the fluorescent probe can monitor the change of the content of CO in cells, which provides a reliable means for monitoring the pathological changes related to CO in human body.

Claims (3)

1. A solid-state fluorescent probe HPQ-MQ-CO for detecting carbon monoxide has the following structure:

2. the method for preparing the solid-state fluorescent probe for detecting carbon monoxide according to claim 1, which is characterized by comprising the following reaction steps:

1) dissolving 1 equivalent of 3- (6-chloro-3, 4-dihydro-4-oxo-2-quinazolinyl) -4-hydroxybenzaldehyde and 0.8-1.2 equivalent of 1-ethyl-2-methylquinoline in 10-20 mL of acetonitrile in a 100mL round-bottom flask, adding 0.1-0.5 mL of piperidine, reacting the reaction mixture at 60-90 ℃ for 12-18 h under the protection of nitrogen, stopping the reaction, removing the solvent by reduced pressure distillation to obtain a purple solid crude product, and dissolving the product in CH₂Cl₂Extracting with saturated salt water for three times, drying the organic phase with anhydrous sodium sulfate, and collecting the crude product with CH at a volume ratio of 100: 1-100: 10₂Cl₂/CH₃Performing column chromatography by using OH eluent to obtain a reddish brown solid compound HPQ-MQ-OH, wherein the structure of the compound is as follows:

2) dissolving 1 equivalent of compound HPQ-MQ-OH in 10-20 mL of dry dichloromethane in a 100mL round-bottom flask at 0-5 ℃ under ice bath, adding 1.5-2.5 equivalents of triethylamine, slowly adding 9-12 equivalents of allyl chloroformate under the protection of nitrogen after 0-15 min, stirring for 0-5 h under ice bath for changing the color of the solution, then heating to room temperature, stirring for reaction for 10-15 h, stopping the reaction, removing the solvent through reduced pressure distillation, and performing column chromatography on the crude product by using dichloromethane to obtain a brown solid product, namely the solid fluorescent probe HPQ-MQ-CO.

3. The use of the solid-state fluorescent probe for detecting carbon monoxide according to claim 1, wherein the fluorescent probe is used for detecting the content of carbon monoxide in living cells.

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