CN111518071A - Preparation and application of cysteine near-infrared fluorescent probe - Google Patents
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Abstract
The invention relates to preparation and application of a cysteine (Cys) near-infrared fluorescent probe, wherein the structural formula of the fluorescent probe is as follows:
the invention provides a preparation method for synthesizing the fluorescent probe by taking 6-methoxy-2, 3-dihydro-1H-xanthene-4-formaldehyde, 2- (3,5, 5-trimethylcyclohexene-2-vinyl) malononitrile, acryloyl chloride and the like as raw materials; the fluorescent probe is a near-infrared cysteine fluorescent probe; firstly, the fluorescent probe has a large Stokes shift of 180 nm; in addition, the fluorescent probe can detect Cys with high sensitivity, and the fluorescence intensity is obviously enhanced by 6 times after the fluorescent probe reacts with Cys; then, the fluorescent probe can detect Cys with high selectivity and is free from other CysInterference of biological thiols, amino acids, cations, active oxygen and active sulfur; secondly, the response time of the fluorescent probe to Cys is 10 min; and the fluorescent probe can monitor the change of Cys content in the living cell.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to preparation and application of a cysteine near-infrared fluorescent probe.
Background
Cysteine (Cys) is a small-molecule biological thiol that plays an important role in regulating physiological processes and maintaining biological system balance, and is closely related to biological self-detoxification, protein synthesis, and biological processes such as human metabolism (y.li, w.liu, p.zhang, h.zhang, j.wu, j.ge, p.wang, biosens.bioelectrtron., 2017,90, 117. fig. 124; m.li, n.kang, c.zhang, w.liang, g.zhang, j.jia, c.dong, spectrochim.acta.a.mol.biom.spectrum, 2019,222,117262; y.dai, y.zheng, t.xue, f.he, h.ji, z.oi, spectum.a.Acrome.bio mol.spectrum, y.84, yang.g, y.zu, y.84; y.g.x.j, g.j.j.h.h.ju, y.j, y.g.z.j, r.g.z.z.z.j, m.g.z.z.z.h.h.h.h.12, m.g.h.h.h.h.h.h.h.h.g. Recent studies have shown that abnormal levels of Cys can lead to liver damage, growth retardation, and skin damage. Furthermore, Cys levels are closely related to cardiovascular complications, neurotoxicity, Alzheimer's disease, Parkinson's disease (X.Lu, W.Wang, Q.Dong, X.Bao, X.Lin, W.Zhang, W.Zhao, chem.Commun, 2015,51, 1498-fila 1501, X.ren, H.Tian, L.Yang, L.He, Y.Geng, X.Liu, X.Song, Sens.Actuators.B.chem.,2018,273,1170-1178, C.F.Yang, L.Y.Zeze, B.K.Ning, J.Y.Wang, H.Zhang, Z.H.Zhang, Spectrochem.acta.A.mol.biol.ACS.2418, Spectro. 2020,225,117482, GUS.Yang, C.Yao, Li.201J.2013-J.O, Qio.3, Qio.J.3, J.O.J.O.J.O.J.O.O.D. 2, S.O.D. 2, S.S.A. 2, S.A. E.A. A. E.E.E.E.E.E.E.E.E. Therefore, there is an urgent need to develop a method for detecting Cys in a biological system accurately and efficiently.
In recent years, fluorescent probes have attracted much attention and application due to their high sensitivity, non-invasiveness and high spatial-temporal resolution. To date, a number of probes for Cys detection have been successfully developed. These probes mostly use naphthalimide, fluorescein, carbazole, etc. as fluorophores, and have short emission wavelengths (K.B.Li, W.B.Qu, Q.Shen, S.Zhang, W.Shi, L.Dong, D.M.Han, Dyes pigments, 2020,173,107918; H.Song, J.Zhang, X.Wang, Y.Zhou, C.Xu, Sensa activators B chem.,2018,259, 233-doped 240; X.Hou, Z.Li, B.Li, C.Liu, Z.Xu, Sens.activators B.chem.,2018,260, 295-doped 302; Y.Huang, Q.ren, S.Li, Y.Feng.Zhang, G.Fang, Sens.activators B.chem.,2019, 201255-doped 240). However, probes using the novel naphthofluorescein, indole-based dye as a fluorophore have a small Stokes shift although they have a near infrared emission wavelength (S.Xue, S.Ding, Q.ZHai, H.Zhang, G.Feng, biosens. Bioelectron, 2015, 68; 316-. Therefore, it is of great interest to design and synthesize a Cys fluorescent probe with near-infrared emission wavelength and large Stokes shift.
Binding of isophorone derivatives to chromophores can give the resulting fluorophores a large stokes shift. The fluorescent probe has the advantages of large Stokes shift, low background interference, small light damage to biological samples, strong sample penetrability, high detection sensitivity and the like. At present, a fluorescence probe synthesized by isophorone derivative and p-methoxybenzaldehyde and 6-hydroxy-2-naphthaldehyde and having a large Stokes shift is used for measuring Cys, but the emission wavelength is short and is not enough to meet the requirements of biological imaging (J.Hou, P.Cai, C.Wang, Y.Shen, tetrahedron.Lett.,2018,59, 2581-K2585; W.Zhang, J.Liu, Y.W.Yu, Q.R.Han, T.Cheng, J.Shen, B.X.Wang, Y.L.Jiang, Talanta,2018, 477-K482). Therefore, it is highly desirable to design and synthesize a long wavelength probe based on an isophorone derivative-xanthene dye for detecting Cys.
Disclosure of Invention
In accordance with the proposed requirements, the present inventors have conducted intensive studies to provide a cysteine near-infrared fluorescent probe having a large stokes shift after a great deal of creative work.
The technical scheme of the invention is that the cysteine near-infrared fluorescent probe has the following structural formula:
a method for preparing a cysteine near-infrared fluorescent probe. The method comprises the following steps:
1) in a 100mL round bottom flask, 1 equivalent of the solid compound 6-methoxy-2, 3-dihydro-1H-xanthene-4-carbaldehyde is dissolved in 6-10 mL of acetic anhydride, then 1 equivalent of the solid compound 2- (3,5, 5-trimethylcyclohexene-2-vinyl) malononitrile and 2 equivalents of potassium carbonate are added thereto, and the reaction mixture is stirred in a N2Stirring the mixture for 13-15 h at 80 ℃ under the atmosphere, removing the solvent by reduced pressure distillation, and carrying out column chromatography on the crude product by using dichloromethane/petroleum ether eluent with the volume ratio of 1:1 to obtain a solid compound (IX-OMe) (yield is 50%). 2) Dissolving 1 equivalent of solid compound IX-OMe in 20-30 mL of anhydrous dichloromethane in a 100mL round-bottom flask in N2Placing the mixture into an ice water mixture at 0 ℃ under the atmosphere, then slowly dropwise adding 20 equivalents of boron tribromide, stirring the reaction mixture at room temperature for 15-17 h, stopping the reaction, dropwise adding a saturated sodium bicarbonate solution at 0 ℃ to quench the reaction, extracting an aqueous layer by using dichloromethane to obtain an organic layer, drying, filtering, removing the solvent of the organic layer by reduced pressure distillation, and carrying out column chromatography on a crude product by using a dichloromethane eluent to obtain a solid compound (IX-OH) (the yield is 60%). 3) Dissolving 1 equivalent of solid compound IX-OH in 20-30 mL of anhydrous dichloromethane in a 100mL round bottom flask, adding 2 equivalents of triethylamine and 2 equivalents of acryloyl chloride, stirring the reaction mixture at room temperature for 15-20 minutes, stopping the reaction, removing the solvent through reduced pressure distillation, and performing column chromatography on the crude product by using dichloromethane eluent to obtain a solid product (IX) (the yield is 71%), namely the fluorescent probe.
The cysteine near-infrared fluorescent probe has the beneficial effect of good spectral response performance. First, the fluorescence spectrum of the fluorescent probe IX was investigated. The fluorescent probe per se, under the excitation of 590nm, shows a weak near-infrared emission peak of you at 743 nm; after addition of Cys, a new near-infrared emission peak appeared at 770 nm. And the near infrared fluorescence intensity of the fluorescent probe at 770nm is continuously enhanced along with the increase of the concentration of Cys. The Stokes shift is large, 180 nm. The fluorescence intensity of IX increased 6-fold at a Cys concentration of 70. mu.M. Then, the ultraviolet absorption spectrum of the fluorescent probe IX was recorded. The fluorescent probe IX itself exhibited an absorption band at 534 nm; after addition of 70. mu.M Cys, the peak shifted to 570 nm. Second, fluorescent probe IX was evaluated for selectivity to Cys. The probe was measured separately from cysteine, biological thiol (Hcy, GSH), amino acid (His, Ala, IIe, Leu, Met, Ser, Thr, Tyr, Asp, Phe, Pro, Trp, Val, Asn, Gln, Gly, Glu, Arg, Lys), cation (K)+,Na+,Mg2+,Ca2+) Active oxygen (H)2O2HClO), active sulfur (H)2S,S2-) The fluorescent response of (a). The fluorescent probe IX responded weakly to Hcy and GSH, and did not respond substantially to other competitors. The fluorescence intensity of probe IX was significantly enhanced only in the presence of Cys. Next, the influence of pH on the detection of Cys by IX was examined, and fluorescence was observedA suitable pH range for Cys determination with light probe IX is 7.0-10.0. Finally, the response time of the fluorescent probe IX to Cys was determined within 10 min.
An application of cysteine near-infrared fluorescent probe. Only the fluorescent probe is added into the cell, and the red channel has weak fluorescence, which indicates that the probe can detect the endogenous Cys of the cell. The cells were pretreated with N-ethylmaleimide (NEM) followed by probe incubation, and the red channel did not fluoresce because NEM cleared Cys endogenous to living cells. NEM is added into cells for pretreatment, Cys is added for cultivation, and finally a probe is added for cultivation, so that a red channel has strong fluorescence. Therefore, the fluorescent probe IX can detect Cys in cells and monitor the change of the concentration of Cys, and is expected to be an advantageous tool for monitoring the content of Cys and diagnosing Cys-related diseases.
Drawings
FIG. 1 shows the synthetic route of fluorescent probe IX.
FIG. 2 is a graph showing the fluorescence spectra of fluorescent probe IX after reaction with Cys at various concentrations.
The abscissa is wavelength and the ordinate is fluorescence intensity. The concentration of the fluorescent probe IX is 10 mu M, and the concentration of Cys is respectively as follows: 0,10,20,30,40,50,60,70 μ M. The fluorescence excitation wavelength was 590 nm.
FIG. 3 is a graph of the linear response of fluorescent probe IX to various concentrations of Cys.
FIG. 4 is a diagram of the UV-VIS absorption spectrum of the fluorescent probe IX after reaction with Cys.
FIG. 5 is a diagram showing the selectivity of the fluorescent probe IX.
The concentration of the fluorescent probe IX was 10. mu.M, the concentration of Cys was 70. mu.M, and the concentration of the other analytes was 400. mu.M.
FIG. 6 is a graph showing the effect of pH on the fluorescent probe IX.
FIG. 7 is a graph showing the time response of fluorescent probe IX after reaction with varying concentrations of Cys.
FIG. 8 is a cytotoxicity assay. The abscissa is the concentration of the fluorescent probe IX and the ordinate is the viability of the cells.
FIG. 9 is an image of a cell after reaction of fluorescent probe IX with Cys. (a) Culturing the cells and the probe for 1 h; (b) culturing cells with NEM for 30min, adding probe, and culturing for 1 h; (c) culturing cells with NEM for 30min, adding Cys for 30min, and adding probe for 1 h; (d) fluorescence intensity map of (a).
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 fluorescent Probe IX
The synthetic route is shown in figure 1. Synthesis of Compound IX-OMe: in a 100mL round-bottom flask, the solid compound 6-methoxy-2, 3-dihydro-1H-xanthene-4-carbaldehyde (0.24g, 1.0mmol) was dissolved in 10mL of acetic anhydride, to which was then added the solid compound 2- (3,5, 5-trimethylcyclohexene-2-vinyl) malononitrile (0.22g, 1.2mmol) and potassium carbonate (0.28g, 2.0mmol), and the reaction mixture was stirred in N2Stirring at 80 deg.C for 14h under atmosphere, distilling under reduced pressure to remove solvent, and subjecting the crude product to column chromatography with dichloromethane/petroleum ether eluent at volume ratio of 1:1 to obtain solid compound (0.21g, yield: 50%) as compound IX-OMe.
Synthesis of Compound IX-OH: in a 100mL round-bottom flask, the solid compound IX-OMe (0.21g, 0.5mmol) was dissolved in 30mL dry dichloromethane in N2Placing the mixture into an ice water mixture at 0 ℃ under the atmosphere, then slowly dropwise adding boron tribromide (0.95mL, 10mmol), stirring the reaction mixture at room temperature for 16h, stopping the reaction, dropwise adding a saturated sodium bicarbonate solution at 0 ℃ to quench the reaction, standing for layering, extracting a water layer by dichloromethane to obtain an organic layer, drying, filtering, removing the solvent of the organic layer by reduced pressure distillation, and carrying out column chromatography on a crude product by using a dichloromethane eluent to obtain a solid compound (0.12g, the yield: 60%) which is the compound IX-OH.
Synthesis of fluorescent Probe IX: in a 100mL round-bottom flask, the solid compound IX-OH (0.41g, 1.0mmol) was dissolved in 30mL of anhydrous dichloromethane, triethylamine (0.28mL, 2.0mmol) and acryloyl chloride (0.16mL, 2.0mmol) were added, the reaction mixture was stirred at room temperature for 15min to stop the reaction, the solvent was removed by distillation under reduced pressure, and the crude product was subjected to column chromatography with dichloromethane eluent to give a solid product (0.32g, yield; 71%) which was a fluorescent probe.1H NMR(400MHz,CDCl3,ppm):7.56(d,J＝16.0Hz,1H),7.09(d,J＝8.0Hz,1H),6.96(s,1H),6.81(d,J＝8.0Hz,1H),6.75(s,1H),6.66(d,J＝17.2Hz,1H),6.43-6.31(m,3H),6.08(d,J＝10.4Hz,1H),2.60-2.47(m,8H),1.83(t,J＝6.0Hz,2H),1.11(s,6H).13C NMR(100MHz,CDCl3,ppm):169.0,164.3,155.4,153.2,150.9,150.7,133.3,132.0,130.5,127.6,126.5,126.3,122.5,122.1,120.2,114.4,113.6,112.5,109.0,75.5,43.0,39.2,32.1,28.1,24.6,20.7.MS(TOF):450.2.
Example 2:
preparation of fluorescent probe and Cys solution
The probe solution is prepared by weighing a certain amount of fluorescent probe and dissolving in acetonitrile to obtain 2 × 10-4mol·L-1Weighing a certain amount of Cys, dissolving the Cys in secondary distilled water, transferring the solution into a 250mL volumetric flask, and fixing the volume with the secondary distilled water to prepare 1 × 10-3mol·L-10.5mL of the probe solution, 3.5mL of acetonitrile, and 0.4 to 3.6mL of the aqueous cysteine solution were put into a 10mL volumetric flask, and the volume was adjusted to 1.0 × 10 with PBS buffer solution-5mol·L-1Fluorescent Probe, 4.0 × 10-5mol·L-1-3.6×10-4mol·L-1Cys is mixed with the solution to be tested.
Example 3:
measurement of fluorescence Spectrum after reaction of fluorescent Probe with Cys
FIG. 2 shows the fluorescence spectra of the fluorescent probe IX reacted with Cys at different concentrations, the fluorescent probe concentration being 10. mu.M, and the Cys concentrations being: 0,10,20,30,40,50,60,70 μ M. The excitation wavelength is 590nm, and the emission wavelength is 680-880 nm. The slit width was 10.0nm/20.0nm, and measured by Hitachi F4600 spectrofluorometer. Only in the presence of the probe, there was a weak near-infrared emission peak at 743 nm. Cys was added, the emission peak red-shifted to 770nm, and the Stokes shift of the probe reached 180 nm. This is because Cys reacts with the probe, cleaving propenyl formate, releasing the fluorophore IX-OH, and the fluorescence intensity is positively correlated with Cys concentration. When Cys was added at 70. mu.M, the fluorescence intensity of the probe was increased 6-fold. FIG. 3 is a graph showing the linear response of the probe to Cys at various concentrations, and the fluorescence intensity of the probe has a good linear relationship with the concentration of Cys. From the above results, it can be seen that the fluorescent probe IX can detect Cys with high sensitivity.
Example 4:
determination of ultraviolet-visible absorption spectrum before and after reaction of fluorescent probe and Cys
FIG. 4 shows UV-VIS absorption spectra before and after reaction of fluorescent probe with Cys, the concentration of fluorescent probe was 10. mu.M, and the concentration of Cys was 70. mu.M. The measurement was carried out using an agilent Cary60 uv-vis spectrophotometer. In the case of only the probe, there is an absorption peak at 534 nm; after addition of Cys, the peak shifted to 570 nm.
Example 5:
selectivity of fluorescent probes for Cys
FIG. 5 shows the selectivity of fluorescent probe for Cys. The fluorescent probe (10. mu.M) was measured separately from Cys (70. mu.M), biological thiol (Hcy, GSH), amino acids (His, Ala, IIe, Leu, Met, Ser, Thr, Tyr, Asp, Phe, Pro, Trp, Val, Asn, Gln, Gly, Glu, Arg, Lys), and cation (K)+,Na+,Mg2+,Ca2+) Active oxygen (H)2O2HClO), active sulfur (H)2S,S2-) (400. mu.M) and it was found that strong fluorescence emission was generated only in the presence of Cys. Therefore, the fluorescent probe IX can detect Cys with high selectivity.
Example 6:
influence of pH value on Cys detection by fluorescent probe
FIG. 6 is a graph showing the effect of pH on fluorescent probes. Fluorescence emission spectra before and after reaction of the fluorescent probe (10. mu.M) and Cys (70. mu.M) under different pH conditions (2.0-12.0) were measured, respectively. The pH of the solution had substantially no effect on the fluorescence intensity of the probe itself. However, after addition of Cys, the probe generated intense fluorescence emission only when pH was 7.0-10.0. From the above conclusions, the probe can detect Cys in the pH range of 7.0-10.0. Thus, fluorescent probe IX can detect Cys in cells under physiological conditions.
Example 7:
time response of fluorescent probe to Cys reaction
FIG. 7 is a time response of a fluorescent probe to Cys. The response time of the fluorescent probe IX to Cys is within 10min, and after 10min, the fluorescence intensity of the probe is not increased any more. Thus, it is known that the fluorescent probe IX can detect Cys in real time.
Example 8:
application of fluorescent probe in living cell
First, we used the MTT method to assess the cytotoxicity of fluorescent probes. As can be seen from FIG. 8, when 0-30. mu.M Cys probe is added, the survival rate of the liver cancer cell HepG2 is higher than 90%, and the survival rate is not greatly influenced, which proves that the fluorescent probe IX has low cytotoxicity and can be used for detecting Cys in living cells. Next, we investigated the use of fluorescent probes in HepG2 cells. As can be seen in FIG. 9, when the cells were incubated with the probe for 1h, fluorescence was generated in the red channel (FIG. 9a), indicating that the probe can detect Cys endogenous to living cells. Cells were preincubated with N-ethylmaleimide (NEM) for 30min, then incubated for 1h with the addition of a fluorescent probe, and no fluorescence was observed in the red channel (FIG. 9b), since NEM cleared endogenous Cys from living cells. Cells were incubated with NEM for 30min, followed by Cys for 30min and finally probe for 1h, with strong fluorescence in the red channel (FIG. 9 c). Then, we analyzed the fluorescence intensity of the cells, and the relative fluorescence intensities of the a, b, c fluorescence imaging plots were 1, 0.1, 4.3, respectively (FIG. 9 d). In conclusion, fluorescent probe IX could monitor changes in Cys concentration in living cells and could potentially be a powerful tool for the diagnosis of Cys-related diseases.
Claims (3)
1. A cysteine near-infrared fluorescent probe has the following structure:
2. the method for preparing the cysteine near-infrared fluorescent probe according to claim 1, characterized in that the reaction steps are as follows:
1) in a 100mL round bottom flask, 1 equivalent of the solid compound 6-methoxy-2, 3-dihydro-1H-xanthene-4-carbaldehyde is dissolved in 6-10 mL of acetic anhydride, then 1 equivalent of the solid compound 2- (3,5, 5-trimethylcyclohexene-2-vinyl) malononitrile and 2 equivalents of potassium carbonate are added thereto, and the reaction mixture is stirred in a N2Stirring for 13-15 h at 80 ℃ in the atmosphere, removing the solvent by reduced pressure distillation, and carrying out column chromatography on the crude product by using dichloromethane/petroleum ether eluent with the volume ratio of 1:1 to obtain a solid compound IX-OMe, wherein the structure is as follows:
2) dissolving 1 equivalent of solid compound IX-OMe in 20-30 mL of anhydrous dichloromethane in a 100mL round-bottom flask in N2Placing the mixture into an ice water mixture at 0 ℃ under the atmosphere, then slowly dropwise adding 20 equivalents of boron tribromide, stirring the reaction mixture at room temperature for 15-17 h, stopping the reaction, dropwise adding a saturated sodium bicarbonate solution at 0 ℃, quenching the reaction, standing for layering, extracting a water layer by using dichloromethane to obtain an organic layer, drying, filtering, removing the solvent by reduced pressure distillation, and performing column chromatography on a crude product by using a dichloromethane eluent to obtain a solid compound IX-OH, wherein the structure of the solid compound IX-OH is as follows:
3) dissolving 1 equivalent of solid compound IX-OH in 20-30 mL of anhydrous dichloromethane in a 100mL round-bottom flask, adding 2 equivalents of triethylamine and 2 equivalents of acryloyl chloride, stirring the reaction mixture at room temperature for 15-20 minutes, stopping the reaction, removing the solvent through reduced pressure distillation, and performing column chromatography on the crude product by using dichloromethane eluent to obtain a solid product IX, namely the fluorescent probe.
3. The application of the cysteine near-infrared fluorescent probe as claimed in claim 1, wherein the fluorescent probe has a large Stokes shift and is applied to the detection of cysteine content in living cells.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112876392A (en) * | 2021-02-05 | 2021-06-01 | 新乡医学院 | Near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity and preparation method and application thereof |
| CN114605432A (en) * | 2022-03-22 | 2022-06-10 | 湘潭大学 | Preparation and application of cyanine dye-based targeted cysteine fluorescent probe |
| CN116410172A (en) * | 2023-04-13 | 2023-07-11 | 湘潭大学 | Preparation and application of viscosity fluorescent probe based on isophorone-xanthene |
| CN116410175A (en) * | 2023-04-11 | 2023-07-11 | 青岛科技大学 | Hypochlorite near infrared fluorescent probe with large Stokes shift and preparation method and application thereof |
| CN117586247A (en) * | 2023-11-16 | 2024-02-23 | 南华大学 | Mitochondrial targeting near infrared fluorescent probe with viscosity and cysteine specific response and preparation method and application thereof |
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