阅读说明：本技术 一种新型荧光探针的合成及在检测半胱氨酸中的应用 (Synthesis of novel fluorescent probe and application of novel fluorescent probe in cysteine detection ) 是由 朱海亮 张萌 王保忠 于 2018-09-13 设计创作，主要内容包括：一种新型荧光探针,它具有如下结构式。经实验证明,该新型荧光探针对半胱氨酸具有较好的选择性,可以定量检测生物体内外的半胱氨酸。本发明公开了该新型荧光探针的合成方法。<Image he="186" wi="700" file="DSA0000170610960000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(A novel fluorescent probe has the following structural formula. Experiments prove that the novel fluorescent probe has better selectivity on cysteine and can quantitatively detect cysteine in and out of organisms. The invention discloses a synthetic method of the novel fluorescent probe.)

1. A novel fluorescent probe, the Chinese name: trans-ethan-3- (7- (acryloyloxy) -2-oxo-2-hydrobenzopyran-3-yl) acrylate characterized by the following chemical structure:

2. the method for preparing a novel fluorescent probe as claimed in claim 1, which is characterized by comprising the following steps:

step 1 Compound 1(740mg, 5.36mmol), Compound 2(1.05g, 5.65mmol) and piperidine (44.64. mu.L, 0.57mmol) were weighed into a 50mL round-bottom flask and dissolved in 15mL absolute ethanol. Heating and refluxing for 12h at 85 ℃, and detecting the reaction by TLC until the raw materials are completely consumed. And concentrating the reaction solution by a rotary evaporator to obtain a crude product. Purifying by 200-mesh 300-mesh silica gel column chromatography with the elution ratio of ethyl acetate to petroleum ether being 1: 10 to finally obtain the compound 3. The product was a yellow solid in 76.15% yield.

Step 2 Compound 3(200mg, 0.77mM) and triethylamine (77mg, 0.77mM) were weighed into a 50mL round bottom flask and dissolved in 15mL dichloromethane. Acryloyl chloride (415.8. mu.L, 4.62mM) was added slowly at 0 ℃ and the reaction checked by TLC until the starting material was consumed. The reaction solution was concentrated by rotary evaporator to obtain a crude product. Purifying by 200-mesh 300-mesh silica gel column chromatography with the elution ratio of ethyl acetate to petroleum ether being 1: 20 to finally obtain the compound 4, namely the fluorescent probe. The product was a white solid in 88.54% yield.

3. The method for preparing the novel fluorescent probe according to claim 2, wherein the silica gel column used in the purification in step 1 and step 2 is 200-300 mesh, and the elution ratio is 1: 10 and 1: 20, respectively.

4. The preparation and use of a novel fluorescent probe according to claim 1 for the detection of cysteine.

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to a preparation method of a novel fluorescent probe, activity evaluation and application of the novel fluorescent probe in cysteine detection.

Background

Sulfur is one of important nutrients for plant growth and development, and cysteine (Cys) is the only amino acid containing sulfhydryl (-SH) in the amino acids that constitute proteins in the organism. Cysteine has important physiological functions, and because the functions of cysteine relate to a plurality of physiological regulation processes such as trace element metabolism, heavy metal detoxification, free radical elimination and the like, the dysfunction of cysteine metabolism in vivo can cause a series of immune system functional diseases. By monitoring cysteine concentrations in an organism, a prophase diagnosis of certain diseases can be achieved. Therefore, it is of great significance to develop a method for detecting cysteine with high speed, sensitivity and selectivity.

At present, methods for measuring Cys mainly include an electrochemical method, a high performance liquid chromatography, a resonance scattering spectrometry, a capillary zone electrophoresis method, a spectrophotometry, a fluorescence spectrometry and the like. The fluorescence spectroscopy is distinguished by the advantages of convenience, rapidness, rapid reaction, low cost and the like. And after the fluorescent probe and the Cys act, fluorescence changes, such as changes of fluorescence intensity or emission wavelength, and the like occur, so that the analysis and the determination of the fluorescent probe are realized.

Therefore, the invention designs and synthesizes a novel fluorescent probe, and the potential value of the fluorescent probe in detecting the activity of cysteine is researched by measuring the cysteine in vivo and in vitro.

Disclosure of Invention

The invention relates to synthesis of a novel fluorescent probe and application of the novel fluorescent probe in cysteine detection.

The technical scheme of the invention is as follows:

a novel fluorescent probe, the Chinese name: trans-ethan-3- (7- (acryloyloxy) -2-oxo-2-hydrobenzopyran-3-yl) acrylate. The specific chemical structure is as follows:

the synthesis method of the novel fluorescent probe is characterized by comprising the following steps:

step 1, weighing a proper amount of compounds 1 and 2, pouring the compounds into a reactor with a certain volume, dissolving the compounds in a certain volume of solvent, adding the compound 2 and piperidine, reacting at a specific reaction temperature until the raw materials are completely consumed, and treating to obtain a compound 3.

Step 2, weighing a proper amount of the compound 3 and triethylamine, pouring the mixture into a container containing a certain solvent, placing the container at a certain temperature, slowly adding a certain amount of acryloyl chloride, reacting until the raw materials are completely consumed, and processing to obtain a compound 4, namely the fluorescent probe.

The synthesized novel fluorescent probe has a good effect of measuring cysteine in vivo and in vitro, so that the fluorescent probe can quantitatively detect cysteine.

Drawings

FIG. 1 is an ultraviolet absorption spectrum of the fluorescent probe of the present invention

FIG. 2 is a fluorescence spectrum of the fluorescent probe of the present invention

FIG. 3 is a bar graph showing the selectivity of the fluorescent probe of the present invention for amino acids such as cysteine

FIG. 4 is a bar graph of interference of the fluorescent probe of the present invention with respect to biological thiol substances such as cysteine

FIG. 5 fluorescent imaging of the fluorescent probes of the present invention for intracellular cysteine detection

Detailed Description

The present invention is illustrated in detail by the following examples, but the scope of the present invention is not limited by any of the examples.

The first implementation example is as follows: synthesis and preparation of fluorescent probes

Step 1 Compound 1(740mg, 5.36mmol), Compound 2(1.05g, 5.65mmol) and piperidine (44.64. mu.L, 0.57mmol) were weighed into a 50mL round-bottom flask and dissolved in 15mL absolute ethanol. Heating and refluxing for 12h at 85 ℃, and detecting the reaction by TLC until the raw materials are completely consumed. And concentrating the reaction solution by a rotary evaporator to obtain a crude product. Purifying by 200-mesh 300-mesh silica gel column chromatography with the elution ratio of ethyl acetate to petroleum ether being 1: 10 to finally obtain the compound 3. The product was a yellow solid in 76.15%. 1H NMR (600MHz, DMSO-d6) δ 8.44(s, 1H), 7.59-7.47(m, 2H), 6.90-6.79(m, 2H), 6.74(d, J ═ 2.0Hz, 1H), 4.18(q, J ═ 7.1Hz, 2H), 1.25(t, J ═ 7.1Hz, 3H), 13C NMR (151MHz, DMSO-d6) δ 166.74, 163.69, 159.71, 155.89, 146.02, 139.44, 131.32, 120.05, 116.52, 114.63, 118.7102.44, 60.52, 14.65 yield.

Step 2 Compound 3(200mg, 0.77mM) and triethylamine (77mg, 0.77mM) were weighed into a 50mL round bottom flask and dissolved in 15mL dichloromethane. Acryloyl chloride (415.8. mu.L, 4.62mM) was added slowly at 0 ℃ and the reaction checked by TLC until the starting material was consumed. The reaction solution was concentrated by rotary evaporator to obtain a crude product. Purifying by 200-mesh 300-mesh silica gel column chromatography with the elution ratio of ethyl acetate to petroleum ether being 1: 20 to finally obtain the compound 4, namely the fluorescent probe. The product was a white solid in 88.54% yield. 1H NMR (600MHz, DMSO-d6) δ 8.59(s, 1H), 7.80(d, J ═ 8.5Hz, 1H), 7.56(d, J ═ 16.0Hz, 1H), 7.43(d, J ═ 2.1Hz, 1H), 7.28(dd, J ═ 8.5, 2.2Hz, 1H), 6.96(d, J ═ 16.0Hz, 1H), 6.59(dd, J ═ 17.3, 1.1Hz, 1H), 6.45(dd, J ═ 17.3, 10.4Hz, 1H), 6.22(dd, J ═ 10.4, 1.1Hz, 1H), 4.21(q, J ═ 7.1Hz, 2H), 1.27(t, J ═ 7.3, J ═ 10.4Hz, 1H), 6.21 (q, J ═ 7.1Hz, 2H), 1.27(t, J ═ 7.3, t ═ 7.3, J ═ 10.4Hz, 1H, 1H, 13.62, 17.35, 13, 35, 24, 26.

Example two was performed: ultraviolet absorption spectrum of fluorescent probe

Blank and control groups were set for this experiment. Blank group: 3 1.5mL centrifuge tubes were used, and 100. mu.L of PBS, 98. mu.L of water, and 2. mu.L of probe solution (DMSO, 1mM) were added to each tube. Control group: 3 of 1.5mL centrifuge tubes were taken and 100. mu.L of PBS, 96. mu.L of water, 2. mu.L of probe solution (DMSO, 1mM), 2. mu.L of Cys (water, 10mM) were added to each tube. The blank group and the control group are placed at 37 ℃ for reaction for 15min, and an absorption spectrum is measured by an ultraviolet spectrophotometer within the range of 200-800 nm.

Example three was performed: fluorescence spectrum of fluorescent probe

Blank and control groups were set for this experiment. Blank group: 3 1.5mL centrifuge tubes were used, and 100. mu.L of PBS, 98. mu.L of water, and 2. mu.L of probe solution (DMSO, 1mM) were added to each tube. Control group: 3 of 1.5mL centrifuge tubes were taken and 100. mu.L of PBS, 96. mu.L of water, 2. mu.L of probe solution (DMSO, 1mM), 2. mu.L of Cys (water, 10mM) were added to each tube. The blank group and the control group are placed at 37 ℃ for reaction for 15min, and a fluorescence spectrophotometer is utilized to set an excitation wavelength of 428nm, a slit of 5nm and a voltage of 400v, and an emission wavelength is collected in a range of 430-650 nm.

Example four was performed: bar chart of selectivity of fluorescent probe for amino acids such as cysteine

Blank and 20 control groups were set for this experiment. Blank group: 3 1.5mL centrifuge tubes were used, and 100. mu.L of PBS, 98. mu.L of water, and 2. mu.L of probe solution (DMSO, 1mM) were added to each tube. Control group: 20 control groups, 3 per 1.5mL centrifuge tube, 100. mu.L PBS, 96. mu.L water, 2. mu.L probe solution (DMSO, 1mM), 2. mu.L amino acid (water, 10mM) per centrifuge tube. The 20 control groups were in order: 1) cys, 2) Val 3) Lys, 4) Ala, 5) Alg, 6) Leu, 7) Ile, 8) Pro, 9) Phe, 10) Tyr, 11) Trp, 12) Thr, 13) Ser, 14) Met, 15) Asn, 16) Gln, 17) Asp, 18) Asn, 19) His, 20) Gly blank and control were reacted at 37 ℃ for 15min, and the excitation wavelength of 428nm, the slit of 5nm, the voltage of 400v were set by a fluorescence spectrophotometer, and the emission wavelength was collected in the range of 430 and 650 nm. The intensity of fluorescence at an emission wavelength of 505nm was plotted in a bar graph.

Example five of the implementation: bar chart of interference of fluorescent probe on biological sulfhydryl substances such as cysteine

Blank and 3 control groups were set for this experiment. Blank group: 3 of 1.5mL centrifuge tubes were taken and 100. mu.L of PBS, 96. mu.L of water, 2. mu.L of probe solution (DMSO, 1mM), 2. mu.L of Cys (water, 10mM) were added to each tube. Control group: 3 control groups, 3 per 1.5mL centrifuge tube, 100. mu.L PBS, 94. mu.L water, 2. mu.L probe solution (DMSO, 1mM), 2. mu.L Cys (water, 10mM), 2. mu.L biological thiol substrate (water, 10mM) were added to each centrifuge tube. The 3 control groups were in order: 1) hcy, 2) Na2S, 3) GSH. The blank group and the control group are placed at 37 ℃ for reaction for 15min, and a fluorescence spectrophotometer is utilized to set an excitation wavelength of 428nm, a slit of 5nm and a voltage of 400v, and an emission wavelength is collected in a range of 430-650 nm. The intensity of fluorescence at an emission wavelength of 505nm was plotted in a bar graph.

Example six: fluorescence imaging of fluorescent probes for intracellular detection of cysteine

Blank and 5 control groups were set for this experiment. Blank group: the hela cells were pretreated with NEM DMEM medium at a final concentration of 2mM for 30min at 37 ℃, then washed 3 times with PBS, and incubated with probe DMEM medium at a final concentration of 10. mu.M for 30 min. Control group: the hela cells were pretreated with NEM DMEM medium at a final concentration of 2mM for 30min at 37 ℃, then washed 3 times with PBS, and incubated with probe DMEM medium at a final concentration of 10. mu.M for 30 min. PBS was washed 3 times, and hela cells were incubated for 30min by sequentially adding final concentrations of 20. mu.M, 40. mu.M, 60. mu.M, 80. mu.M, and 100. mu.M Cys DMEM medium.

Through a single-photon confocal fluorescence microscope, an excitation channel is arranged at 405nm, and the change of the fluorescence intensity of each group is observed.