CN111518071B - Preparation and application of cysteine near infrared fluorescent probe - Google Patents
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Abstract
The invention relates to a cysteinePreparation and application of an amino acid (Cys) near infrared fluorescent probe, wherein the structural formula of the fluorescent probe is as follows:
the invention provides a preparation method for synthesizing a fluorescent probe by taking 6-methoxy-2, 3-dihydro-1H-xanthene-4-formaldehyde, 2- (3, 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 180nm; 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 not interfered by other biological thiols, amino acid, cations, active oxygen and active sulfur; secondly, the response time of the fluorescent probe to Cys is 10min; and, the fluorescent probe can monitor the change of Cys content in living cells.
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 which plays an important role in regulating physiological processes and maintaining the balance of biological systems, and is closely related to biological self-detoxification, protein synthesis, metabolism of the human body, and other life processes (Y.Li, W.Liu, P.Zhang, H.Zhang, J.Wu, J.Ge, P.Wang, biosens.Bioelectron.,2017,90,117-124; M.Li, N.Kang, C.Zhang, W.Liang, G.Zhang, J.Jia, C.Dong, spectrohem. Acta. A. Mol. Biomol. Spectrosc.,2019,222,117262; Y.Dai, Y.zheng, T.Xue, F.He, H.Ji, Z.Oi, spectrohem. Acta. A. Mol. Biomol. Spectrosc.,2020,225,117490; X.Song, Y.Yang, J.Ru, Y.Wang, F.Qiau, Y, feng, W.Liu, 2019). Recent studies have shown that abnormal levels of Cys can lead to liver damage, retarded growth, 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.,2015,51,1498-1501; X.ren, H.Tian, L.Yang, L.He, Y.Geng., X.Liu, X.Song, sens.Actators.B.chem., 2018,273,1170-1178; C.F.Yang, L.Y.Zeng, B.K.Ning, J.Y.Wang, H.Zhang, Z.H.Zhang, spectrochim.acta.A.mol. Biomol.Spectrosc.,2020,225,117482;S.Yang,C.Guo,Y.Li,J.Guo,J.Xiao,Z.Qing,ACS Sens, 2018,3,2415-2422). Therefore, there is an urgent need to develop a method for accurately and efficiently detecting Cys in biological systems.
In recent years, fluorescent probes have received a great deal of attention and application due to their high sensitivity, non-invasiveness, and high spatial-temporal resolution. To date, many probes for Cys detection have been successfully developed. Most of these probes use naphthalimide, fluorescein, carbazole, etc. as fluorophores and have a relatively short emission wavelength (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,Sens Actuators B Chem, 2018,259,233-240; X.Hou, Z.Li, B.Li, C.Liu, Z.xu, sens.Actors.B.chem., 2018,260,295-302; Y.Huang, Q.ren, S.Li, Y.Feng, W.zhang, G.fang, sens.Actors.B.chem., 2019,293,247-255.). However, probes using novel naphthalene fluorescein, indole dyes as fluorophores, although having near infrared emission wavelengths, have small Stokes shifts (S.Xue, S.Ding, Q.Zhai, H.Zhang, G.Feng, biosens.Bioelectron.,2015,68;316-321; S.J.Li, Y.J.Fu, C.Y.Li, Y.F.Li, L.H.Yi, J.ou-Yang, anal.Chim.acta.,2017,994,73-81). Therefore, it is very interesting to design and synthesize a Cys fluorescent probe with a near infrared emission wavelength, a large Stokes shift.
The isophorone derivative is combined with chromophore, so that the synthesized fluorophore has large Stokes shift. The fluorescent probe has the advantages of large Stokes displacement, low background interference, small optical damage to biological samples, strong sample penetrability, high detection sensitivity and the like. Currently, there are fluorescent probes working to synthesize isophorone derivatives with p-methoxybenzaldehyde, 6-hydroxy-2-naphthalene aldehyde with large Stokes shift for determining Cys, but with emission wavelengths short enough to meet the requirements of biological imaging (J.Hou, P.Cai, C.Wang, Y.Shen, tetrahedron.Lett.,2018,59,2581-2585; W.Zhang, J.Liu, Y.W.Yu, Q.R.Han, T.Cheng, J.Shan, B.X.Wang, Y.L.Jiang, talanta,2018,185, 477-482). Therefore, it is urgent to design and synthesize a long wavelength probe based on isophorone derivative-xanthene dye for detecting Cys.
Disclosure of Invention
In light of the requirements set forth, the inventors have conducted intensive studies on this, and after a lot of creative effort, provided a cysteine near infrared fluorescent probe having a large stokes shift.
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 was dissolved in 6 to 10mL of acetic anhydride, followed by 1 equivalent of the solid compound 2- (3, 5-trimethylcyclohexene-2-vinyl) malononitrile and 2 equivalents of potassium carbonate were added thereto, and the reaction mixture was reacted under N 2 Stirring at 80 ℃ for 13-15 h under atmosphere, removing solvent by reduced pressure distillation, and performing column chromatography on the crude product by using a volume ratio of 1:1 methylene chloride/petroleum ether eluent to obtain a solid compound (IX-OMe) (yield 50%). 2) In a 100mL round bottom flask, 1 equivalent of solid compound IX-OMe was dissolved in 20 to 30mL anhydrous dichloromethane in N 2 Placing in an ice-water mixture at 0 ℃ under atmosphere, slowly dropwise adding 20 equivalent of boron tribromide, stirring the reaction mixture at room temperature for 15-17 h, stopping the reaction, dropwise adding saturated sodium bicarbonate solution at 0 ℃ to quench the reaction, extracting an aqueous layer with 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 with a dichloromethane eluent to obtain a solid compound (IX-OH) (yield 60%). 3) In a 100mL round bottom flask, 1 equivalent of solid compound IX-OH was dissolved in 20 to 30mL of anhydrous dichloromethane, 2 equivalents of triethylamine and 2 equivalents of allyl chloride were added, the reaction mixture was stirred at room temperature for 15 to 20 minutes, the reaction was stopped, the solvent was removed by distillation under reduced pressure, and the crude product was subjected to column chromatography with a dichloromethane eluent to give solid product (IX) (yield 71%) which was the fluorescent probe.
The invention has the beneficial effects that the cysteine near infrared fluorescent probe has good spectral response performance. First, the fluorescence spectrum of the fluorescent probe IX was studied. Fluorescent probe itself, with excitation at 590nm, emits weak near infrared radiation at 743nmA peak; after addition of Cys, a new near infrared emission peak appears at 770 nm. And as the concentration of Cys increases, the near infrared fluorescence intensity of the fluorescent probe at 770nm is continuously enhanced. The Stokes shift is larger, 180nm. When the Cys concentration was 70. Mu.M, the fluorescence intensity of IX was enhanced 6-fold. Then, the ultraviolet absorption spectrum of the fluorescent probe IX was recorded. The fluorescence probe IX itself presents an absorption band at 534 nm; after addition of 70. Mu.M Cys, the absorption peak shifted red to 570nm. Next, the selectivity of fluorescent probe IX for Cys was evaluated. The probes were assayed for 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 + ,Mg 2+ ,Ca 2+ ) Active oxygen (H) 2 O 2 HClO), active sulfur (H) 2 S,S 2- ) Is a fluorescent response of (a). Fluorescent probe IX had a weak response to Hcy, GSH and was substantially unresponsive to other competitors. Only in the presence of Cys, the fluorescence intensity of probe IX was significantly enhanced. Next, the effect of pH on the Cys detection by IX was examined, and the pH range for Cys detection by fluorescent probe IX was suitably 7.0-10.0. Finally, the response time of fluorescent probe IX to Cys was determined to be within 10 min.
Use of a cysteine near infrared fluorescent probe. Only fluorescent probes are added into the cells, and the red channel has weak fluorescence, which indicates that the probes can detect Cys endogenous to the cells. The cells were pretreated with N-ethylmaleimide (NEM) followed by incubation with the probe, and the red channel was not fluorescent, as NEM cleared the endogenous Cys of the living cells. NEM pretreatment is added to the cells, cys incubation is added, and finally probes incubation is added, and the red channel has strong fluorescence. Thus, fluorescent probe IX is capable of detecting intracellular Cys and monitoring changes in Cys concentration, and is expected to be an advantageous tool for monitoring Cys content and diagnosing Cys-related diseases.
Drawings
FIG. 1 shows the synthetic route of fluorescent probe IX.
FIG. 2 is a graph showing fluorescence spectra of fluorescent probe IX after reaction with different concentrations of Cys.
The abscissa is wavelength and the ordinate is fluorescence intensity. The fluorescent probe IX concentration was 10. Mu.M and Cys concentration was respectively: 0,10,20,30,40,50,60,70. Mu.M. The fluorescence excitation wavelength was 590nm.
FIG. 3 is a graph showing the linear response of fluorescent probe IX with various concentrations of Cys.
FIG. 4 is a chart showing the UV-visible absorption spectrum of fluorescent probe IX after reaction with Cys.
FIG. 5 is a selective diagram of fluorescent probe IX.
The fluorescent probe IX was 10. Mu.M, cys was 70. Mu.M, and the other analytes were 400. Mu.M.
FIG. 6 is a graph showing the effect of pH on fluorescent probe IX.
FIG. 7 is a graph showing the time response of fluorescent probe IX after reaction with different concentrations of Cys.
FIG. 8 is a cytotoxicity test. The abscissa indicates the concentration of the fluorescent probe IX and the ordinate indicates the viability of the cells.
FIG. 9 is a cellular image of fluorescent probe IX after reaction with Cys. (a) incubating the cells with the probe for 1h; (b) incubating the cells with NEM for 30min, adding probe and incubating for 1h; (c) The cells are firstly incubated for 30min by NEM, then incubated for 30min by adding Cys, and finally incubated for 1h by adding a probe; (d) a fluorescence intensity profile of (a).
Detailed Description
The invention is described in detail below with reference to the drawings and the specific examples, but is not limited thereto.
Example 1:
synthesis of fluorescent Probe IX
The synthetic route is shown in FIG. 1. Compound IX-OMe synthesis: in a 100mL round bottom flask, the solid compound 6-methoxy-2, 3-dihydro-1H-xanthene-4-carbaldehyde (0.24 g,1.0 mmol) was dissolved in 10mL acetic anhydride, to which was subsequently added the solid compound 2- (3, 5-trimethylcyclohexene-2-vinyl) malononitrile (0.22 g,1.2 mmol) and potassium carbonate (0.28 g,2.0 mmol), the reaction mixture was taken up in N 2 The solvent was removed by distillation under reduced pressure under stirring at 80℃for 14 hours under an atmosphere, and the crude product was subjected to column chromatography using a volume ratio of 1:1 methylene chloride/petroleum ether as eluent to give a solid compound (0.21 g, yield: 50%) as compound IX-OMe.
Synthesis of Compound IX-OH:in a 100mL round bottom flask, solid compound IX-OMe (0.21 g,0.5 mmol) was dissolved in 30mL anhydrous dichloromethane under N 2 In an atmosphere, the mixture was placed in an ice-water mixture at 0℃followed by slowly dropping boron tribromide (0.95 mL,10 mmol), stirring the reaction mixture at room temperature for 16 hours, stopping the reaction, dropping a saturated sodium bicarbonate solution at 0℃to quench the reaction, standing for delamination, extracting the aqueous layer with methylene chloride to obtain an organic layer, drying, filtering, removing the solvent of the organic layer by distillation under reduced pressure, and subjecting the crude product to column chromatography with a methylene chloride eluent to obtain a solid compound (0.12 g, yield: 60%) which was compound IX-OH.
Synthesis of fluorescent probe IX: in a 100mL round bottom flask, solid compound IX-OH (0.41 g,1.0 mmol) was dissolved in 30mL of anhydrous dichloromethane, triethylamine (0.28 mL,2.0 mmol) and acryloyl chloride (0.16 mL,2.0 mmol) were added, the reaction mixture was stirred at room temperature for 15min, the reaction was stopped, 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 (0.32 g, yield; 71%) as a fluorescent probe. 1 H NMR(400MHz,CDCl 3 ,ppm):δ7.56(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). 13 C NMR(100MHz,CDCl 3 ,ppm):δ169.0,164.3,155.4,153.2,150.9,150.7,133.3,132.0,130.5,127.6,126.5,126.3,122.5,122.1,120.2,114.4,113.6,112.5,109.0,75.5,43.0,39.2,32.1,28.1,24.6,20.7.MS(TOF):450.2.
Example 2:
fluorescent probe and Cys solution preparation
Preparation of probe solution: weighing a certain amount of fluorescent probe, dissolving in acetonitrile, and preparing into 2×10 -4 mol·L -1 Is a probe solution of (a). Preparing Cys solution: weighing a certain amount of Cys, dissolving in secondary distilled water, transferring into 250mL volumetric flask, and fixing volume with secondary distilled water to obtain 1×10 -3 mol·L -1 Is a cysteine aqueous solution of (a). 0.5mL of the probe solution, 3.5mL of acetonitrile and 0.4-3.6mL of the cysteine aqueous solution were added to a 10mL volumetric flask, usingPBS buffer solution is fixed in volume and is configured to be 1.0X10 -5 mol·L -1 Fluorescent probe, 4.0X10 -5 mol·L -1 -3.6×10 -4 mol·L -1 Mixing Cys and testing solution.
Example 3:
determination of fluorescence Spectrum after reaction of fluorescent Probe with Cys
FIG. 2 shows fluorescence spectra of fluorescent probe IX after reaction with Cys at different concentrations of 10. Mu.M, cys concentrations: 0,10,20,30,40,50,60,70. Mu.M. The excitation wavelength is 590nm, and the emission wavelength is 680-880nm. The slit width was 10.0nm/20.0nm, measured using a Hitachi F4600 fluorescence spectrophotometer. Only in the presence of the probe, there was a weak near infrared emission peak at 743 nm. Cys was added and the emission peak red shifted to 770nm and the Stokes shift of the probe to 180nm. This is because Cys reacts with the probe, cleaving the propenyl formate, releasing the fluorophore IX-OH, and the fluorescence intensity is positively correlated with the Cys concentration. When Cys was added in an amount of 70. Mu.M, the fluorescence intensity of the probe was enhanced 6-fold. FIG. 3 is a graph showing the linear response of fluorescence of a 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, it was found that the fluorescent probe IX can detect Cys with high sensitivity.
Example 4:
determination of UV-visible absorption Spectrum before and after reaction of fluorescent Probe with Cys
FIG. 4 shows the UV-visible absorption spectra of a fluorescent probe at a concentration of 10. Mu.M and Cys at a concentration of 70. Mu.M before and after reaction with Cys. The measurements were made using an Agilent Cary60 UV-Vis spectrophotometer. Only in the presence of the probe, there is an absorption peak at 534 nm; after addition of Cys, the absorption peak shifted to 570nm.
Example 5:
selectivity of fluorescent probe for Cys
FIG. 5 shows the selectivity of fluorescent probe towards Cys. Determination of fluorescent Probe (10. Mu.M) and Cys (70. Mu.M), 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 + ,Mg 2+ ,Ca 2+ ) Active oxygen (H) 2 O 2 HClO), active sulfur (H) 2 S,S 2- ) (400. Mu.M) fluorescence emission spectrum, it was found that the probe produced intense fluorescence emission only in the presence of Cys. Thus, fluorescent probe IX can detect Cys with high selectivity.
Example 6:
effect of pH on fluorescence probe detection of Cys
FIG. 6 is a graph showing the effect of pH on fluorescent probes. Fluorescence emission spectra of the fluorescent probe (10. Mu.M) and Cys (70. Mu.M) before and after the reaction at different pH values (2.0-12.0) were measured, respectively. The pH of the solution has substantially no effect on the fluorescence intensity of the probe itself. However, after addition of Cys, the probe produced intense fluorescence emission only when ph=7.0-10.0. From the above, it can be seen that the probe can detect Cys in the ph=7.0-10.0 range. Thus, fluorescent probe IX can detect intracellular Cys under physiological conditions.
Example 7:
time response of fluorescent probe to Cys reaction
FIG. 7 shows the time response of fluorescent probe to Cys. The response time of the fluorescent probe IX to Cys is within 10min, and after 10min, the fluorescent intensity of the probe is not increased any more. Thus, it is known that fluorescent probe IX can detect Cys in real time.
Example 8:
application of fluorescent probe in living cells
First, we used the MTT method to evaluate the cytotoxicity of fluorescent probes. As can be seen from FIG. 8, when 0-30. Mu.M Cys probe was added, the survival rate of liver cancer cell HepG2 was higher than 90%, and was not greatly affected, which proves that fluorescent probe IX has less cytotoxicity and can be used for detection of Cys in living cells. Next, we studied 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, the red channel fluoresced (FIG. 9 a), indicating that the probe could detect Cys endogenous to the living cells. Cells were pre-incubated with N-ethylmaleimide (NEM) for 30min, followed by 1h incubation with fluorescent probes, and red channels did not see any fluorescence (FIG. 9 b) because NEM cleared endogenous Cys of living cells. Cells were incubated with NEM for 30min, followed by Cys addition for 30min, and finally probe addition for 1h, the red channel was strongly fluorescent (FIG. 9 c). Then we analyzed the fluorescence intensity of the cells, the relative fluorescence intensities of the a, b, c fluorescence imaging plots were 1, 0.1, 4.3, respectively (fig. 9 d). In summary, fluorescent probe IX can monitor changes in Cys concentration in living cells and has the potential to be a powerful tool for diagnosing Cys-related diseases.
Claims (3)
1. A cysteine near infrared fluorescent probe has the following structure:
2. the method for preparing a cysteine near infrared fluorescent probe according to claim 1, wherein 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 was dissolved in 6 to 10mL of acetic anhydride, followed by 1 equivalent of the solid compound 2- (3, 5-trimethylcyclohexene-2-vinyl) malononitrile and 2 equivalents of potassium carbonate were added thereto, and the reaction mixture was reacted under N 2 Stirring at 80 ℃ for 13-15 h under atmosphere, removing solvent by reduced pressure distillation, and performing column chromatography on the crude product by using a 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) In a 100mL round bottom flask, 1 equivalent of solid compound IX-OMe was dissolved in 20 to 30mL anhydrous dichloromethane in N 2 In the atmosphere, putting the mixture into an ice-water mixture at the temperature of 0 ℃, slowly dropwise adding 20 equivalents of boron tribromide, stirring the reaction mixture for 15-17 h at room temperature, stopping the reaction, and dropwise adding saturated carbon at the temperature of 0 DEG CThe reaction was quenched with sodium hydrogen carbonate solution, allowed to stand for delamination, the aqueous layer was extracted with methylene chloride to give an organic layer, dried, filtered, the solvent was removed by distillation under reduced pressure, and the crude product was subjected to column chromatography with methylene chloride eluent to give a solid compound IX-OH having the following structure:
3) In a 100mL round bottom flask, 1 equivalent of solid compound IX-OH is dissolved in 20-30 mL of anhydrous dichloromethane, 2 equivalents of triethylamine and 2 equivalents of acryloyl chloride are added, the reaction mixture is stirred for 15-20 minutes at room temperature, the reaction is stopped, the solvent is removed by reduced pressure distillation, and the crude product is subjected to column chromatography by using a dichloromethane eluent to obtain a solid product IX, namely the fluorescent probe.
3. The use of a near infrared fluorescent probe for cysteine according to claim 1, characterized in that the fluorescent probe has a large stokes shift and is used for the detection of the cysteine content in living cells for non-disease diagnosis and treatment purposes.
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| CN109897625B (en) * | 2017-12-08 | 2022-04-19 | 南京理工大学 | Fluorescent probe for selectively detecting cysteine, and synthetic method and application thereof |
| CN108129365B (en) * | 2018-01-03 | 2020-09-11 | 湖南韵邦生物医药有限公司 | Fluorescent probe for near-infrared detection of cysteine, and preparation method and application thereof |
| CN109320490A (en) * | 2018-10-26 | 2019-02-12 | 济南大学 | A kind of fluorescence probe of near-infrared specific detection cysteine |
| CN109369455B (en) * | 2018-11-23 | 2021-08-31 | 曲阜师范大学 | Two-photon near-infrared double-large Stokes shift fluorescent dye and synthetic method and application thereof |
| CN110563689B (en) * | 2019-08-27 | 2022-12-30 | 山西大学 | Long-wavelength emission fluorescent probe for specifically detecting cysteine in living cells and preparation method and application thereof |
| CN110698454B (en) * | 2019-09-12 | 2022-04-15 | 徐州医科大学 | Isophorone hydrogen sulfide fluorescent probe and preparation method and application thereof |
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