CN110028463B - Fluorescent probe with large Stokes displacement and synthetic method and application thereof - Google Patents
Fluorescent probe with large Stokes displacement and synthetic method and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent probe with large Stokes displacement and a synthesis method and application thereof. When cysteine was added to the system, cleavage of the acrylate moiety was achieved rapidly and specifically, which resulted in recovery of the ESIPT process and allowing the probe to exhibit the "on" fluorescence detection procedure for cysteine, showing strong fluorescence at 595 nm. According to the invention, the identification effect of the fluorescent probe with large Stokes shift on various biological thiols and sulfides is researched by ultraviolet-visible absorption spectrometry, fluorescence spectrometry and the like, and the result shows that the fluorescent probe can efficiently identify cysteine in a phosphate buffer solution and has high response sensitivity on cysteine.
Description
Technical Field
The invention belongs to the technical field of small molecular fluorescent probes, and relates to a method strategy for detecting cysteine. More particularly, the invention relates to a fluorescent probe with large Stokes shift, a synthetic method and an application thereof.
Background
Cysteine (Cys) is a thiol-containing important amino acid that is involved in cell growth, and the level of Cys is implicated in a number of diseases. Therefore, the development of fluorescent probes for Cys detection and imaging is important. To date, a number of fluorescent probes for Cys detection have been successfully constructed. However, most probes have difficulty distinguishing Cys from Hcy, GSH, or other active sulfur species (RSS), and the emission wavelength of the probe is ultraviolet/short wavelength, typically with a small stokes shift pattern (<100 nm).
Therefore, the development of a fluorescent probe with high selectivity and high sensitivity and large Stokes shift not only helps to better understand the biological functions of cysteine, but also has a far-reaching significance for monitoring endogenous cysteine in clinical diagnosis.
Disclosure of Invention
In view of the above, the present invention provides a fluorescent probe with high selectivity and high sensitivity and large stokes shift, which is directed to the problems in the prior art.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a fluorescent probe with large Stokes shift is characterized in that the structural formula of the fluorescent probe is as follows:
a disclosed fluorescent probe with a large Stokes shift uses a typical ESIPT dye (HBT) as a fluorescence signal reporter group, and an acrylate group as an ESIPT blocker and recognition unit. Wherein cleavage of the acrylate moiety can be achieved rapidly and specifically by Cys in aqueous buffer, resulting in recovery of the ESIPT process and allowing the probe to show an "on" fluorescence detection procedure for Cys, capable of addressing a variety of potential interferences (Hcy, GSH, Na) 2 S and NaHSO 3 ) Has excellent selectivity, and the existence of Cys is judged by monitoring and observing fluorescence through a laser confocal microscope.
Exemplarily, referring to the attached figures 1-3 of the specification, the invention performs structural characterization on the fluorescent probe with large Stokes shift through a nuclear magnetic resonance hydrogen spectrum, a nuclear magnetic resonance carbon spectrum, an ultraviolet-visible spectrum and a fluorescence spectrum.
Another object of the present invention is to provide a method for synthesizing the fluorescent probe with large Stokes shift.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for synthesizing a fluorescent probe with a large stokes shift, which comprises the following steps:
(1) dissolving 2-aminothiophenol, 5-methyl salicylaldehyde and iodine in methanol, stirring at normal temperature for reacting for 3-5 h, and performing suction filtration to obtain a compound 1;
(2) dissolving a compound 1 and hexamethylenetetramine in trifluoroacetic acid, heating and refluxing for 22-24 h at 105-115 ℃, adjusting the pH to be neutral, separating out a solid, and filtering to obtain a compound 2;
(2) dissolving the compound 2 in acetone, heating to 45-55 ℃, then slowly dropwise adding an alkali solution until the solution turns red, and continuously heating for 3-5 h to react to obtain a compound 3;
(4) dissolving the compound 3 in CH containing triethylamine 2 Cl 2 And stirring at a low temperature, slowly adding acryloyl chloride for reaction, transferring the reaction solution to the room temperature, stirring for 3-5 h, and then sequentially carrying out reduced pressure concentration and silica gel chromatography purification on the reaction solution to finally obtain the fluorescent probe with large Stokes shift.
By adopting the technical scheme, the invention has the following beneficial effects:
compared with the traditional fluorescent probe synthesis method, the synthesis method disclosed by the invention is simple to operate and convenient and rapid to purify.
Preferably, in the step (1), the molar ratio of the 2-aminothiophenol to the 5-methylsalicylaldehyde to the iodine is 2:2: 1.
Preferably, in the step (2), the molar ratio of the compound 2 to the hexamethylenetetramine is 1: 1.
Preferably, in the step (3), the volume of acetone used as a solvent is 15-25 mL, and the low-temperature condition is 0-5 ℃.
Preferably, in the step (4), the molar ratio of the compound 3, the acryloyl chloride and the triethylamine is 1: 1.2: and 3, the volume of the anhydrous dichloromethane is 25-35 mL.
Exemplary, the most preferred synthetic schemes of the present invention are:
(1) dissolving 2-aminothiophenol, 5-methyl salicylaldehyde and iodine in methanol, stirring for 4h at normal temperature, and performing suction filtration to obtain a compound 1;
(2) heating and refluxing the compound 1 and hexamethylenetetramine in trifluoroacetic acid at 110 ℃ for 24h, adjusting the pH to be neutral by using NaOH, separating out a solid, and filtering to obtain a compound 2;
(3) dissolving the compound 2 in acetone, heating to 50 ℃, then slowly dropwise adding a sodium hydroxide solution until the solution turns red, and continuously heating for 4 hours to react to obtain a compound 3;
(4) dissolving the compound 3 in CH containing triethylamine 2 Cl 2 And stirring at 0 ℃, simultaneously slowly adding acryloyl chloride for reaction, moving the reaction solution to room temperature, stirring for 4h, and then sequentially carrying out reduced pressure concentration and silica gel chromatography purification on the reaction solution to finally obtain the fluorescent probe with large Stokes shift.
In the step (1), the molar ratio of the 2-aminothiophenol, the 5-methyl salicylaldehyde and the iodine is 2:2: 1.
In the step (2), the molar ratio of 2- (2-hydroxyphenyl) -benzothiazole (HBT) to hexamethylenetetramine is 1: 1.
In the step (3), the solvent acetone is used in an amount of 20 mL.
In the step (4), the mol ratio of the compound 3 to the acryloyl chloride to the triethylamine is 1: 1.2: 3; the volume of anhydrous dichloromethane was 30 mL.
Exemplarily, referring to the attached figures 1-3 in the specification, the invention shows that the fluorescent probe with large Stokes shift is successfully synthesized through the representation of nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, ultraviolet-visible spectrum and fluorescence spectrum.
It is a further object of the present invention to provide the use of fluorescent probes with large Stokes' shifts for the selective recognition and quantitative detection of cysteine.
In some application scenes, the application of the fluorescent probe in detection of the endogenous cysteine and the synthetic performance of the endogenous cysteine is also included.
In some application scenarios, the application of the fluorescent probe in detection and imaging with cysteine as a marker is also included.
The cysteine is detected by the strategy, and the fluorescent probe with the large Stokes shift can efficiently and selectively identify the cysteine, has higher sensitivity to the cysteine, and can respond to the intracellular cysteine by a small amount of probes.
According to the technical scheme, compared with the prior art, the fluorescent probe with large Stokes displacement and the synthesis method and application thereof are provided.
First, the invention discloses a fluorescent probe with large Stokes shift, which uses a typical ESIPT dye (HBT) as a fluorescence signal reporter group and an acrylate group as an ESIPT blocker and a recognition unit to realize the detection of endogenous cysteine. When cysteine was added to the system, cleavage of the acrylate moiety was achieved rapidly and specifically, which resulted in recovery of the ESIPT process and allowing the probe to display the "on" fluorescence detection procedure for cysteine, showing strong fluorescence at 595nm to achieve detection of cysteine.
The invention further discloses a synthetic method of the fluorescent probe with the large Stokes displacement, and the synthetic method is simple to operate, convenient and quick to purify and has good industrial application potential.
Finally, the invention discloses the application of the fluorescent probe with large Stokes displacement, the fluorescent probe can efficiently and selectively identify cysteine, the fluorescent probe has low toxicity and high sensitivity, Cys can be efficiently and selectively identified in an aqueous buffer solution, a small amount of the probe can respond to Cys in a cervical cancer cell and image in the cervical cancer cell so as to monitor the change of the content of Cys in the cervical cancer cell;
in addition, the fluorescent probe with the large Stokes shift can be used for specifically detecting cells with cysteine as a marker, and has good application potential and research value in cell imaging and preparation of diagnostic reagents or medical devices for diagnosing, predicting and treating and monitoring diseases related to cysteine dysfunction.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 shows the NMR spectrum of the fluorescent probe of the present invention in DMSO.
FIG. 2 shows the NMR spectrum of the fluorescent probe of the present invention in DMSO.
FIG. 3 is a graph showing the UV-Vis spectrum and the fluorescence spectrum of the fluorescent probe of the present invention interacting with Cys in PBS buffer solution.
FIG. 4 is a graph comparing the reaction of the fluorescent probe of the present invention with other thiols and active thiols in PBS buffer.
FIG. 5 is a confocal time tracking of the imaging of the fluorescent probes of the present invention in cervical cancer cells.
FIG. 6 shows the detection of Cys concentration changes in cervical cancer cells by the fluorescent probe of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention discloses a fluorescent probe with high sensitivity and high selectivity and large Stokes shift for detecting cysteine, and a synthetic method and application thereof.
The present invention will be further specifically illustrated by the following examples for better understanding, but the present invention is not to be construed as being limited thereto, and certain insubstantial modifications and adaptations of the invention by those skilled in the art based on the foregoing disclosure are intended to be included within the scope of the invention.
The invention discloses a fluorescent probe with large Stokes displacement, which is characterized in that the structural formula of the fluorescent probe is as follows:
the invention also discloses a synthetic method of the fluorescent probe with large Stokes shift, which comprises the following specific steps:
(1) dissolving 2-aminothiophenol, 5-methyl salicylaldehyde and iodine in methanol, stirring at normal temperature for reacting for 3-5 h, and performing suction filtration to obtain a compound 1;
(2) dissolving a compound 1 and hexamethylenetetramine in trifluoroacetic acid, heating and refluxing for 22-24 h at 105-115 ℃, adjusting the pH to be neutral, separating out a solid, and filtering to obtain a compound 2;
(3) dissolving the compound 2 in acetone, heating to 45-55 ℃, then slowly dropwise adding an alkali solution until the solution turns red, and continuously heating for 3-5 h to react to obtain a compound 3;
(4) dissolving the compound 3 in CH containing triethylamine 2 Cl 2 And stirring at a low temperature, slowly adding acryloyl chloride for reaction, transferring the reaction solution to the room temperature, stirring for 3-5 h, and then sequentially carrying out reduced pressure concentration and silica gel chromatography purification on the reaction solution to finally obtain the fluorescent probe with large Stokes shift.
In order to further optimize the technical scheme, in the step (1), the molar ratio of the 2-aminothiophenol, the 5-methyl salicylaldehyde and the iodine is 2:2: 1.
In order to further optimize the technical scheme, in the step (2), the molar ratio of the compound 2 to the hexamethylenetetramine is 1: 1.
In order to further optimize the technical scheme, in the step (3), the volume of acetone used as a solvent is 15-25 mL, and the low-temperature condition is 0-5 ℃.
In order to further optimize the technical scheme, in the step (4), the molar ratio of the compound 3 to the acryloyl chloride to the triethylamine is 1: 1.2: and 3, the volume of the anhydrous dichloromethane is 25-35 mL.
The technical solution of the present invention will be further described with reference to the following specific examples.
Example 1
The synthesis method of the fluorescent probe with large Stokes shift comprises the following steps:
(1) synthesis of Compound 1:
2-aminothiophenol (10.0mmol), 5-methylsalicylaldehyde (10.0mmol) and iodine (5.0mmol), stirring at normal temperature, and filtering to obtain compound 1; the solvent of the reaction is methanol; the reaction time was 4 h.
(2) Synthesis of Compound 2:
0.432g (2.0mmol) of 2- (2-hydroxyphenyl) -benzothiazole (HBT) and 0.840g (6.0mmol) of hexamethylenetetramine are dissolved in 20mL of TFA and the contents are heated under reflux at 110 ℃ and stirred for 24 hours, after which the heating is stopped and allowed to cool to room temperature; the solution was neutralized with 1n naoh, and the resulting precipitate was filtered and washed with water; finally in a silica gel Column (CH) 2 Cl 2 And MeOH 100: 1, v/v) to give the pure product compound 2.
(3) Synthesis of Compound 3:
dissolving 0.400g (1.5mmol) of compound 2 in 20mL of acetone, heating to 50 ℃, then slowly dropping 2mL of 10% sodium hydroxide solution, heating the contents at 50 ℃ and stirring for 4h, then cooling to room temperature, neutralizing with HCl, filtering the precipitate and washing with water 3 times; finally in a silica gel Column (CH) 2 Cl 2 And MeOH 80: 1, v/v) to finally obtain a pure product compound 3;
(4) synthesis of fluorescent probe with large Stokes shift
Compound 3(0.155g, 0.5mmol) was dissolved inCH containing 100. mu.L TEA 2 Cl 2 (30mL) and stirred at 0 ℃ and acryloyl chloride (100. mu.L) was added slowly via syringe, and then the reaction solution was stirred at room temperature for 4 hours. Then, the solution was concentrated under reduced pressure and finally subjected to silica gel Column (CH) 2 Cl 2 MeOH, 100: 1, v/v) to finally obtain the fluorescent probe (ABT-MVK) with large Stokes shift.
Example 2
The synthesis method of the fluorescent probe with large Stokes shift comprises the following steps:
this embodiment differs from example 1 in that: the reaction time of 4h was replaced by 3h for step one in example 1, and the remaining reactants and experimental parameters were as in example 1.
Example 3
The synthesis method of the fluorescent probe with large Stokes shift comprises the following steps:
this embodiment differs from example 1 in that: the reaction time of 4h was replaced by 5h for step one in example 1, and the rest of the reactants and experimental parameters are seen in example 1.
Example 4
The synthesis method of the fluorescent probe with large Stokes shift comprises the following steps:
this embodiment differs from example 1 in that: the heating temperature of 110 ℃ in step two of example 1 was replaced by 105 ℃, and the stirring reaction time of 24h was replaced by 22h, with the rest of the reactants and experimental parameters being seen in example 1.
Example 5
The synthesis method of the fluorescent probe with large Stokes shift comprises the following steps:
this embodiment differs from example 1 in that: the heating temperature of 110 ℃ in step two of example 1 was replaced by 115 ℃ and the stirring reaction time of 24h was replaced by 23h, with the rest of the reactants and experimental parameters being seen in example 1.
Example 6
The synthesis method of the fluorescent probe with large Stokes shift comprises the following steps:
this embodiment differs from example 1 in that: the heating temperature of 50 ℃ in step three in example 1 was replaced by 45 ℃ and the heating and stirring time of 4h was replaced by 3h, and the rest of the reactants and experimental parameters were found in example 1.
Example 7
The synthesis method of the fluorescent probe with large Stokes shift comprises the following steps:
this embodiment differs from example 1 in that: the heating temperature of 50 ℃ in step three in example 1 was replaced by 55 ℃, and the heating and stirring time of 4h was replaced by 5h, and the rest of the reactants and experimental parameters were as in example 1.
Example 8
The synthesis method of the fluorescent probe with large Stokes shift comprises the following steps:
this embodiment differs from example 1 in that: the low temperature condition 0 of step four in example 1 was replaced by 3 ℃ and the room temperature stirring time 4h was replaced by 3h, the rest of the reactants and experimental parameters are seen in example 1.
Example 9
The synthesis method of the fluorescent probe with large Stokes shift comprises the following steps:
this embodiment differs from example 1 in that: the low temperature condition 0 of step four in example 1 was replaced by 5 ℃ and the room temperature stirring time 4h was replaced by 5h, the rest of the reactants and experimental parameters are seen in example 1.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The inventive content is not limited to the content of the above-mentioned embodiments, wherein combinations of one or several of the embodiments may also achieve the object of the invention.
To further verify the excellent effects of the present invention, the inventors also conducted the following experiments:
experiment 1: synthesis and structural characterization of probes
1. Synthesis of fluorescent probe with large Stokes shift
(1) Synthesis of Compound 1:
stirring 2-aminothiophenol (10.0mmol), 5-methyl salicylaldehyde (10.0mmol) and iodine (5.0mmol) at normal temperature, and performing suction filtration to obtain a product compound 1; the solvent of the reaction is methanol; the reaction time was 4 h.
(2) Synthesis of Compound 2:
0.432g (2.0mmol) of 2- (2-hydroxyphenyl) -benzothiazole (HBT) and 0.840g (6.0mmol) of hexamethylenetetramine are dissolved in 20mL of TFA. The contents were heated and stirred under reflux for 24 hours, then the heating was stopped and allowed to cool to room temperature. The solution was neutralized with 1N NaOH, and the resulting precipitate was filtered and washed with water. Then in a silica gel Column (CH) 2 Cl 2 And MeOH 100: 1, v/v) to give the pure product compound 2.
(3) Synthesis of Compound 3:
0.400g (1.5mmol) of Compound 2 was dissolved in 20mL of acetone, and the solution was heated at 50 ℃. Then 2mL of 10% sodium hydroxide solution was slowly added dropwise, and the contents were heated and stirred at 50 ℃ for another 4 hours, and then cooled to room temperature. Neutralized with HCl, filtered the precipitate and washed 3 times with water. Finally in a silica gel Column (CH) 2 Cl 2 And MeOH 80: 1, v/v) to obtain the pure product compound 3.
(4) Synthesis of fluorescent probe with large Stokes shift
Compound 3(0.155g, 0.5mmol) was dissolved in CH containing 100. mu.L TEA 2 Cl 2 (30mL) and stirred at 0 ℃. Acryloyl chloride (100 μ L) was added slowly via syringe. After that, the reaction solution was stirred at room temperature for 4 hours. Then, the solution was concentrated under reduced pressure. Chromatography on silica gel (CH) 2 Cl 2 MeOH, 100: 1, v/v) to finally obtain the compound ABT-MVK (fluorescent probe with large Stokes shift).
2. And (3) testing and analyzing:
FIG. 1 shows a probe 1 H NMR spectrum, specific spectrum peak value is:
1 H NMR(600MHz，DMSO-d 6 )δ(ppm)8.18(d,J＝7.7Hz,2H),8.05(d，J＝8.0Hz,1H),7.97(s，1H),7.60-7.57(m,1H),7.52-7.49(m，1H),7.47(d,J＝16.2Hz，1H),7.00(d,J＝16.2Hz,1H),6.72-6.68(m,1H),6.67-6.62(m,1H),6.35(dd,J＝10.0,1.5Hz,1H),2.49(s,3H),2.32(s,3H), which correspond to the probe group, can prove successful probe synthesis.
FIG. 2 shows a probe 13 C NMR spectrum, specific peak value:
13 C NMR(151MHz，DMSO-d 6 ) Delta (ppm)197.48,164.01,161.41,152.16,144.29,136.82,135.80,134.95,134.35,131.93,130.56,129.78,128.51,126.96,126.72,126.32,125.8,122.89,122.10,27.97, 20.23. The number of carbons and the peak positions correspond to the probe one by one, and the structure of the probe is further verified to be correct. To sum up from 1 HNMR and 13 c NMR proves the chemical structure of the probe and the fluorescent probe can be successfully synthesized by the technical scheme disclosed by the invention.
Experiment 2: test of ability of Probe to react with Cys in buffer solution
Dissolving a fluorescent probe in dimethyl sulfoxide to prepare a solution of 5.00mmol/L, taking 4 mu L of the solution in 2mL of PBS buffer solution (10mmol/L, pH 8.5), and detecting an ultraviolet visible absorption spectrum and a fluorescence spectrum of the solution; and adding 4 mu L of Cys (20mmol/L), incubating with the probe for 20 minutes, and detecting the ultraviolet visible absorption spectrum and the fluorescence spectrum of the Cys, wherein the specific test result is shown in figure 3.
FIG. 3 shows the UV-VIS absorption spectrum (FIG. 3a) and the fluorescence spectrum (FIG. 3b) before and after the reaction of the fluorescent probe with Cys. Wherein, line A represents the fluorescent probe, and line B represents the mixture of the fluorescent probe and Cys. It can be seen that the addition of Cys reduced the peak at 290nm with a slight blue shift and a new peak at 370 nm; meanwhile, the fluorescence intensity at 485nm and 595nm is enhanced, so that the cysteine reacts with the probe, and a new compound is obtained through the acrylic ester cyclization and shedding process. In addition, as can be seen from fig. 3, after the fluorescent probe reacts with Cys, a new absorption peak appears at 370nm in the absorption spectrum, the fluorescence emission wavelength is 595nm (in the red region), so the stokes shift is 225nm, and the stokes shift of the synthetic fluorescent probe disclosed by the invention is larger than that of the conventional fluorescent probe (<100 nm).
Experiment 3: selective recognition of cysteine by fluorescent probes with large Stokes shifts
Dissolving the fluorescent probe in dimethyl sulfoxide to prepare a solution of 5.00mmol/L, respectively taking 4 μ L of the fluorescent probe in 5 parts of 2mL PBS buffer solution (10mmol/L, pH 8.5) (marked as 1-5#), wherein 1# is that Cys (10mmol/L)4 μ L is taken in 2mL PBS buffer solution (20 μmol/L), and 2-5# is that other active sulfur and biological thiol (Hcy, GSH, Na) are respectively taken 2 S and NaHSO 3 ) (0.1mol/L) 2. mu.L in 2mL of PBS buffer (50. mu. mol/L) was incubated with the probe for 20 minutes, and then the fluorescence spectrum was detected. As can be seen from FIG. 4, only cysteine can cause the spectral change of the probe, and the probe has no signal response to other active sulfur and biological thiol, thereby confirming that the probe has higher selectivity to cysteine. And it is also known from fig. 4 that the fluorescent probe shows high sensitivity and specific selection even at low concentration of Cys.
Experiment 4: cys response of fluorescent probe with large Stokes shift in cervical cancer cell and detection of Cys content in cell
For intracellular Cys imaging, cells were incubated with 10 μ M ABT-MVK (with 0.2% DMSO, v/v) for 30 minutes at 37 ℃ and washed three times with PBS buffer for immediate imaging.
For NEM treated experiments, HeLa cells were pretreated with 1.0mM NEM for 30 min at 37 ℃, washed three times with PBS buffer and incubated with 10 μm ABT-MVK (or with 100 μm Cys, Hcy or GSH for 30 min), and cell imaging was performed after washing with PBS (fig. 5).
For H 2 O 2 Experiment on treatment, HeLa cells were used with 200. mu. M H 2 O 2 Pre-treatment at 37 ℃ for 30 minutes, washing with PBS buffer, and 10 u M ABT-MVK temperature with 30 minutes. Cell imaging was performed after washing the cells with PBS. Fluorescence imaging of HeLa cells was performed using a Leica SP8 point scanning confocal fluorescence microscope (FIG. 6), with an excitation wavelength fixed at 405nm and a fluorescence wavelength of 550-650 nm.
As can be seen from FIG. 5(A1), the fluorescent probe was able to detect Cys in cervical cancer cells, and showed significant fluorescence. FIG. 5(B1) shows no fluorescence when it was imaged after NEM treatment and incubated with the fluorescent probe. Fig. 5(C1), fig. 5(D1) and fig. 5(E1) are exogenous Cys, Hcy, GSH, respectively, with fig. 5(C1) having strong fluorescence, while fig. 5(D1) and fig. 5(E1) have almost no fluorescence.
From FIG. 6, it can be seen that 2 O 2 After treatment, this resulted in a decrease in intracellular Cys concentration (fig. 6(B1)), and thus weaker fluorescence than in fig. 6(a 1); whereas FIG. 6(C1) shows exogenously added Cys, with strong fluorescence.
In conclusion, the fluorescent probe can specifically detect Cys in cervical cancer cells, and other biological thiols cannot affect the detection; changes in intracellular Cys concentration may also be detected.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The method disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the description of the method part.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A fluorescent probe with large Stokes shift is characterized in that the structural formula of the fluorescent probe is as follows:
2. the method for synthesizing a fluorescent probe with a large Stokes shift according to claim 1, which comprises the following steps:
(1) dissolving 2-aminothiophenol, 5-methyl salicylaldehyde and iodine in methanol, stirring at normal temperature for reacting for 3-5 h, and performing suction filtration to obtain a compound 1;
(2) dissolving a compound 1 and hexamethylenetetramine in trifluoroacetic acid, heating and refluxing for 22-24 h at 105-115 ℃, adjusting the pH to be neutral, separating out a solid, and filtering to obtain a compound 2;
(3) dissolving the compound 2 in acetone, heating to 45-55 ℃, then slowly dropwise adding an alkali solution until the solution turns red, and continuously heating for 3-5 h to react to obtain a compound 3;
(4) dissolving the compound 3 in CH containing triethylamine 2 Cl 2 And stirring at a low temperature, slowly adding acryloyl chloride for reaction, moving the reaction solution to the room temperature, stirring for 3-5 h, and then sequentially carrying out reduced pressure concentration and silica gel chromatography purification on the reaction solution to finally obtain the fluorescent probe with the large Stokes shift.
3. The method for synthesizing a fluorescent probe with large Stokes shift according to claim 2, wherein in the step (1), the molar ratio of 2-aminothiophenol, 5-methylsalicylaldehyde and iodine is 2:2: 1.
4. The method for synthesizing a fluorescent probe with large Stokes shift according to claim 2, wherein in the step (2), the molar ratio of the compound 1 to the hexamethylenetetramine is 1: 1.
5. The method for synthesizing a fluorescent probe with large Stokes shift according to claim 2, wherein in the step (3), the volume of acetone used as a solvent is 15-25 mL.
6. The method for synthesizing a fluorescent probe with large Stokes shift according to claim 2, wherein in the step (4), the molar ratio of the compound 3 to the acryloyl chloride to the triethylamine is 1: 1.2: and 3, the volume of the anhydrous dichloromethane is 25-35 mL.
7. Use of a fluorescent probe with a large stokes shift according to claim 1 for non-diagnostic purposes in the selective recognition and quantitative detection of cysteine.
8. The use of a fluorescent probe with a large Stokes shift according to claim 7, further comprising the use of said fluorescent probe for non-diagnostic purposes in the detection and imaging of cysteine as a marker.
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