A near-infrared cysteine fluorescent probe and a preparation method and application thereof

Abstract

The application discloses a near-infrared cysteine fluorescent probe and a preparation method and application thereof. The near-infrared cysteine fluorescent probe is prepared by taking 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) as a fluorescent reporter group and modifying the fluorescent group by an acrylate linking group. The near-infrared cysteine fluorescent probe can be used for visual identification of cysteine, determination of intracellular cysteine in living cells and determination of cysteine in food, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of molecular probe technology, and in particular to a near-infrared cysteine ​​fluorescent probe, its preparation method, and its application. Background Technology

[0002] Cysteine ​​(Cys) is an essential amino acid in the human body, abundant in cells and involved in various physiological and pathological processes. As one of the thiol-containing amino acids, Cys has a significant impact on metabolism, protein synthesis, intracellular oxidative homeostasis regulation, and other physiological processes. However, both excess and deficiency of Cys are considered closely related to various diseases such as Parkinson's disease, cardiovascular disease, liver damage, myasthenia gravis, edema, skin lesions, growth retardation, and lethargy. On the other hand, Cys, as a low-cost sulfur-containing compound, is widely used in the industrial production of cosmetics, pharmaceuticals, fragrances, and perfumes. In addition, Cys can be used as an additive to enhance the elasticity of baked goods dough and as an effective antioxidant to prevent browning in vegetables and beverages. Cys is also often used as a food detoxifier to neutralize harmful substances such as formaldehyde, tetrodotoxin, acrylamide, and methylmercury.

[0003] Because Cys shares similar structural features and reactivity with homocysteine ​​(Hcy) and glutathione (GSH), effective differentiation between them is challenging. Therefore, developing a simple and rapid analytical method for real-time, highly sensitive detection of Cys in biological systems and food samples is crucial. Summary of the Invention

[0004] The main objective of this invention is to provide a near-infrared cysteine ​​fluorescent probe, its preparation method, and its application, aiming to solve at least one technical problem in the prior art.

[0005] To achieve the above objectives, the present invention provides a near-infrared cysteine ​​fluorescent probe, the structural formula of which is shown in Formula 1:

[0006] Formula 1 Where X is selected from any one of O, S, and N.

[0007] This invention also provides a method for synthesizing a near-infrared cysteine ​​fluorescent probe, comprising the following steps: (1) Using tetrahydrofuran and water as the reaction medium, 5-bromo-2-furan carbaldehyde and 4-hydroxyphenylboronic acid as reactants, tetra-triphenylphosphine palladium as the catalyst, and potassium carbonate as the reaction base, the reaction was carried out at 75°C. After the reaction was completed, the solvent was removed by vortexing, and ethyl acetate and water were added for extraction. The obtained organic phase was washed with saturated brine and dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated by vacuum distillation to obtain the crude product, which was washed with diethyl ether to obtain a yellow solid compound. (2) Using anhydrous ethanol as the reaction medium, malononitrile and 3-hydroxy-3-methyl-2-butanone as reactants, and sodium ethoxide as the reaction base, the reaction was carried out at room temperature. After the reaction was completed, the mixture was filtered and washed with ethanol and ether in sequence to obtain a gray solid compound. (3) Using tetrahydrofuran and anhydrous ethanol as the reaction solvent, the yellow solid compound obtained in step (1) and the gray solid compound obtained in step (2) as the reactants, and ammonium acetate as the reaction base, the reaction was carried out at room temperature. After the reaction was completed, distilled water and dichloromethane were added for extraction. The organic phase obtained was washed with saturated brine and dried with anhydrous sodium sulfate. The mixture was then filtered, and the filtrate was concentrated by vacuum distillation to obtain the crude product. The crude product was separated by silica gel column chromatography with dichloromethane / methanol as the eluent to obtain the black solid compound. (4) The black solid compound obtained in step (3) was dissolved in dichloromethane and triethylamine was added to adjust the alkaline environment. Acryloyl chloride was then added and reacted at room temperature. After the reaction was completed, dichloromethane and water were added for extraction. The resulting organic phase was washed with saturated brine and dried with anhydrous sodium sulfate. The phase was then filtered and the filtrate was concentrated by vacuum distillation to obtain the crude product. The crude product was separated by silica gel column chromatography with dichloromethane as the eluent to obtain a dark red solid, which is the near-infrared cysteine ​​fluorescent probe.

[0008] Further, in step (1), the molar ratio of 5-bromo-2-furan carbaldehyde, 4-hydroxyphenylboronic acid, tetratriphenylphosphine and palladium carbonate is 1:1.5~2:0.2~0.5:16~18.

[0009] Furthermore, in step (2), the molar ratio of sodium ethoxide, 3-hydroxy-3-methyl-2-butanone and malononitrile is 1:6-7:12-14.

[0010] Furthermore, in step (3), the molar ratio of the yellow solid compound, the gray solid compound and ammonium acetate is 1:1.1:1.

[0011] Furthermore, in step (4), the molar ratio of the black solid compound, acryloyl chloride and triethylamine is 1:1.5-2.5:3-5.

[0012] The present invention also provides an application of the above-mentioned near-infrared cysteine ​​fluorescent probe in the qualitative or quantitative detection of cysteine ​​in organisms and food.

[0013] The beneficial effects of this invention are reflected in: This invention relates to a near-infrared cysteine ​​fluorescent probe that uses 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) as a fluorescent reporter group and modifies the fluorescent group (TCF-OH) with an acrylate linker. It allows for visual identification of cysteine, and can also be used to measure intracellular cysteine ​​levels in living cells. Furthermore, it can be used to determine cysteine ​​levels in food, demonstrating promising application prospects.

[0014] The near-infrared cysteine ​​fluorescent probe of the present invention has the advantages of readily available raw materials, simple synthesis, good selectivity, high detection sensitivity, and strong anti-interference ability. Attached Figure Description

[0015] Figure 1 The image shows the 1H NMR spectrum of the intermediate black solid compound 7 obtained in Example 1.

[0016] Figure 2A -C represent the fluorescent probe TCF-C1 prepared in Example 1. 1 H, 13 CNMR and HRMS maps.

[0017] Figure 3A The fluorescence emission spectra of TCF-C1 (10 μM) after reaction with different analytes (100 μM) are shown. Figure 3B The fluorescence intensity at 664 nm is the result of the reaction of TCF-C1 (10 μM) with different analytes (100 μM). Figure 3C The color change of probe TCF-C1 (10 μM) against different analytes (100 μM).

[0018] Figure 4A The fluorescence emission spectra of TCF-C1 (10 μM) and Cys (100 μM) after reaction in the presence of different interfering analytes are shown. Figure 4B The fluorescence intensity at 664 nm is the result of the reaction of TCF-C1 (10 μM) and Cys (100 μM) in the presence of different interfering analytes.

[0019] Figure 5A The fluorescence spectra of TCF-C1 (10 μM) and different concentrations of Cys in EtOH / PBS buffer (8 / 2, v / v) are shown. Figure 5B The graph shows the linear relationship between fluorescence intensity at 664 nm and Cys concentration (0 ~ 28 μM).

[0020] Figure 6The curves show the response fluorescence intensity at 664 nm as a function of pH after the probe TCF-C1 (10 μM) and TCF-C1 (10 μM) react with Cys (100 μM).

[0021] Figure 7 The fluorescence spectra of probes TCF-C1 (10 μM) and Cys (100 μM) at different times in EtOH / PBS (8 / 2, v / v) buffer solution are shown.

[0022] Figure 8 Intracellular / exogenous Cys fluorescence imaging for MHCC97H cells.

[0023] Figure 9 The fluorescence emission spectra of TCF-C1 for measuring Cys content in different foods.

[0024] Figure 10 The mechanism of action of the probe TCF-C1 binding to Cys. Detailed Implementation

[0025] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0026] Unless otherwise specified, the raw materials, reagents or devices used in the following embodiments can be obtained from conventional commercial sources or by existing known methods; unless otherwise specified, the methods used in the embodiments of the present invention are methods mastered by those skilled in the art.

[0027] Example 1 Synthesis of the near-infrared cysteine ​​fluorescent probe TCF-C1 (i.e., the probe shown in Formula 1 when X is O). The synthesis route is as follows:

[0028] The synthesis steps are as follows: (1) Take a 100 mL single-necked round-bottom flask and add 5-bromo-2-furanaldehyde (1 g), 4-hydroxyphenylboronic acid (0.95 g) as reactants, tetra-triphenylphosphine palladium (330 mg) as catalyst, and potassium carbonate (2.36 g) as reaction base to the flask in sequence. Then add a mixture of tetrahydrofuran (25 mL) and distilled water (5 mL) as reaction medium to the flask, and simultaneously place a magnetic stir bar, remove the air from the flask through a three-way valve device, and fill with inert gas. Then raise the temperature to 70 °C using an oil bath heating device and stir the reaction for 12 hours. After the reaction is completed, cool the reaction mixture to room temperature, remove the solvent by vortexing, add distilled water and ethyl acetate for extraction, wash the obtained organic phase with saturated brine, dry with anhydrous sodium sulfate, filter, concentrate the filtrate by vacuum distillation to obtain crude product, and then wash three times with cold diethyl ether to obtain yellow solid compound 3 (0.8 g), with a yield of 75%.

[0029] Yellow solid compound 3 1 H NMR spectrum: 1 H NMR (400 MHz, DMSO- d 6) δ 9.52 (s, 1H),7.74 – 7.68 (m, 2H), 7.61 (d, J = 3.7 Hz, 1H), 7.07 (d, J = 3.8 Hz, 1H), 6.92 –6.86 (m, 2H). (2) Take a 100 mL single-necked round-bottom flask, add 5.0 g of 3-hydroxy-3-methyl-2-butanone and 8.0 g of malononitrile as reactants, 0.66 g of NaOEt as the base for the reaction, and 40 mL of anhydrous ethanol as the reaction medium. After stirring at room temperature for 1 hour, the temperature is raised to 85 °C and refluxed for 1.5 hours. After the reaction is completed, filter the reaction mixture and wash it three times each with cold ethanol and cold diethyl ether to obtain a gray solid compound 6 (5.0 g), with a yield of 51%.

[0030] gray solid compound 6 1 H NMR spectrum: 1 H NMR (400 MHz, DMSO- d 6) δ 2.37 (s, 3H), 1.59 (s, 6H). (3) Take a 100 mL single-necked round-bottom flask, add the yellow solid compound 3 (2.2 g) obtained in step (1) and the gray solid compound 6 (2.0 g) obtained in step (2) as reactants, ammonium acetate (0.780 g) as the base for the reaction, and a mixed solvent of 16 mL tetrahydrofuran and 4 mL ethanol as the reaction solvent. Stir the reaction mixture at room temperature for 1.5 hours. After the reaction is completed, cool the reaction mixture to room temperature, add distilled water and dichloromethane for extraction, wash the obtained organic phase with saturated brine, dry with anhydrous sodium sulfate, filter, concentrate the filtrate by vacuum distillation to obtain the crude product, and purify and separate it by silica gel column chromatography with dichloromethane / methanol (dichloromethane:methanol = 100:1) as the eluent to obtain black solid compound 7 (2.18 g), with a yield of 59%.

[0031] Ink-black solid compound 7 1 H NMR spectrum, see Figure 1 : 1 H NMR (400 MHz, DMSO- d 6) δ10.16 (s, 1H), 7.83 – 7.75 (m, 3H), 7.44 (d, J = 3.8 Hz, 1H), 7.18 (d, J = 3.8Hz, 1H), 6.95 – 6.83 (m, 3H), 1.78 (s, 6H). 13 C NMR (101 MHz, DMSO) δ 177.49,175.08, 160.75, 159.97, 150.51, 132.77, 127.60, 126.60, 120.14, 116.66,113.55, 112.76, 111.84, 110.20, 109.71, 99.07, 96.49, 53.18, 40.64, 40.59,40.38, 40.17, 39.96, 39.75, 39.55, 39.34, 25.91. (4) Take a 50 mL single-necked round-bottom flask, add the black solid compound 7 (0.184 g) obtained in step (3), triethylamine (0.25 mL), and 10 mL of dichloromethane. After dissolving, add acryloyl chloride (50 μL) dropwise under ice bath. React for 10 minutes, and then stir at room temperature for 12 hours. Subsequently, add 30 mL of water to quench the reaction, and then add distilled water and dichloromethane for extraction. The obtained organic phase is washed with saturated brine and dried with anhydrous sodium sulfate. After filtration, the filtrate is concentrated by vacuum distillation to obtain the crude product. It is purified and separated by silica gel column chromatography with pure dichloromethane as the eluent to obtain the dark reddish-black solid compound TCF-C1 (0.038 g), with a yield of 18%.

[0032] TCF-C1 1 H, 13 CNMR and HRMS spectra, see Figure 2A -C: 1 H NMR (400 MHz, DMSO- d 6) δ 8.00(d, J = 8.4 Hz, 2H), 7.83 (d, J = 16.1 Hz, 1H), 7.49 – 7.35 (m, 4H), 6.97 (d,J = 16.1 Hz, 1H), 6.64 – 6.54 (m, 1H), 6.50 – 6.39 (m, 1H), 6.19 (dd, J =10.4, 1.3 Hz, 1H), 1.79 (s, 6H). 13 C NMR (101 MHz, DMSO- d 6) δ 177.46, 174.99, 164.42, 158.44, 151.59, 151.36, 134.52, 132.80, 127.95, 126.75, 125.15, 123.25, 113.37, 112.58, 111.77, 111.52, 99.35, 25.79. Calculated m / z values ​​from high-resolution mass spectrometry (HRMS) (ESI) for [C25H17N3O4 + Na]. + 446.1111, measured value 446.1195.

[0033] Example 2 Performance testing of near-infrared cysteine ​​fluorescent probe TCF-C1 Using the fluorescent probe TCF-C1 prepared in Example 1 as the test object, the test items and specific operations are as follows: (1) Selectivity and anti-interference test of near-infrared cysteine ​​fluorescent probe TCF-C1: The fluorescent probe TCF-C1 was prepared into a 1 mM concentrated stock solution using DMSO, and various analytes were prepared into 10 mM concentrated stock solutions using deionized water.

[0034] Selective testing group: Add 10 μL of probe concentrate to each 1000 μL colorimetric tube, then add 10 μL of concentrate for each analyte, and bring the volume to 1000 μL with EtOH / PBS (8 / 2, v / v) buffer to achieve a final concentration of 10 μM for the fluorescent probe and 100 μM for the analytes. The analytes added to each group were Tyr. (tyrosine), His. (histidine), Ser. (serine), Ala. (alanine), Thr. (threonine), Gly. (glycine), Glu. (glutamate), GSH. (glutathione), Asn. (asparagine), Cys. (cysteine), Lys. (lysine), Gln. (glutamine), Arg. (arginine), Hcy. (homocysteine), and various ions ClO. - S 2- Na + Al 3+ Cu 2+ Co 2+ Ca 2+ Mn 2+ Mg 2+ Ni 2+ Cr 3+ Pb 2+ Fe 3+ Cd 2+ Zn 2+ Mix thoroughly, let stand for 10 minutes to allow the reaction to proceed, and then perform fluorescence emission spectroscopy testing at an excitation wavelength of 500 nm.

[0035] The test results are shown in Figure 3: Figure 3A The fluorescence emission spectra are those of TCF-C1 (10 μM) reacted with different analytes (100 μM); Figure 3B The fluorescence intensity at 664 nm is the result of TCF-C1 (10 μM) reacting with different analytes (100 μM). The numbers on the horizontal axis correspond to the numbers of the different analytes below. Figure 3C This shows the color change of the probe TCF-C1 (10 μM) in response to different analytes (100 μM). The numbers below correspond to the different analyte numbers. The analyte numbers are as follows: 1. TCF-C1; 2. His; 3. Ser; 4. Ala; 5. Thr; 6. Gly; 7. ClO -;8.Glu;9.GSH;10.Asn;11.S 2- ;12.Tyr;13.Cys;14.Lys;15.Gln;16.Arg;17.Na + ;18.Al 3+ ;19.Cu 2+ ;20.Co 2+ ;21.Ca 2+ ;22.Mn 2+ ;23.Mg 2+ ;24.Ni 2+ ; 25.Cr 3+ ;26.Pb 2+ ;27.Fe 3+ ;28.Cd 2+ ;29.Zn 2+ ;30.Hcy. λex = 500 nm, λem = 664 nm.

[0036] Interference substance test group: Add 10 μL of probe concentrate to each 1000 μL colorimetric tube. Then, set the first group as a blank control group and do not add any analyte. For the remaining groups, first add 10 μL of Cys concentrate, then add 10 μL of concentrate of different interference analytes. Finally, adjust the volume to 1000 μL with EtOH / PBS (8 / 2, v / v) buffer solution so that the final concentration of the fluorescent probe is 10 μM, the final concentration of Cys is 100 μM, and the final concentration of the interference analyte is 100 μM. The interfering analytes added to the probe test systems for the remaining groups were Tyr. (tyrosine), His. (histidine), Ser. (serine), Ala. (alanine), Thr. (threonine), Gly. (glycine), Glu. (glutamic acid), GSH. (glutathione), Asn. (asparagine), Lys. (lysine), Gln. (glutamine), Arg. (arginine), Hcy. (homocysteine), and various ions ClO. - S 2- Na + Al 3+ Cu 2+ Co 2+ Ca 2+ Mn 2+ Mg 2+ Ni 2+ Cr 3+ Pb 2+ Fe 3+ Cd 2+ Zn 2+Mix thoroughly, let stand for 10 minutes to allow the reaction to proceed, and then perform fluorescence emission spectroscopy testing at an excitation wavelength of 500 nm.

[0037] The test results are shown in Figure 4: Figure 4A The fluorescence emission spectra of TCF-C1 (10 μM) and Cys (100 μM) after reaction in the presence of different interfering analytes are shown. Figure 4B The fluorescence intensity at 664 nm is the result of the reaction between TCF-C1 (10 μM) and Cys (100 μM) in the presence of different interfering analytes. The numbers on the horizontal axis represent different interfering analytes, where: 1. TCF-C1; 2. His; 3. Ser; 4. Ala; 5. Thr; 6. Gly; 7. ClO - ;8.Glu;9.GSH;10.Asn;11.S 2- ;12.Tyr;13.Lys;14.Gln;15.Arg;16.Na + ;17.Al 3+ 18.Cu 2+ ;19.Co 2+ ;20.Ca 2+ ;21.Mn 2+ ;22.Mg 2+ ;23.Ni 2+ ;24.Cr 3+ ;25.Pb 2+ ;26.Fe 3+ ;27.Cd 2+ 28.Zn 2+ ;29.Hcy. λex = 500 nm, λem = 664 nm.

[0038] As can be seen, under 500 nM excitation, only Cys significantly increased the fluorescence intensity of probe TCF-C1 at 664 nM, while the changes caused by other analytes were negligible, indicating that TCF-C1 has excellent selectivity for Cys. Furthermore, in the presence of other interfering analytes, the fluorescence intensity of probe TCF-C1 at 664 nM remained almost unchanged after the addition of Cys, indicating that TCF-C1 has strong anti-interference ability for the recognition and detection of Cys. In addition, it can be observed with the naked eye that only Cys can change the color of the probe TCF-C1 solution from pink to purple, indicating that the probe can be used for the visual detection of Cys.

[0039] (2) Titration test of near-infrared cysteine ​​fluorescent probe TCF-C1: The fluorescent probe TCF-C1 was prepared into a 1 mM concentrated stock solution using DMSO, and Cys was prepared into a 10 mM concentrated stock solution using deionized water. 10 μL of the probe stock solution was added to each 1000 μL colorimetric tube, followed by different volumes of Cys stock solution. The solutions were then diluted with EtOH / PBS (8 / 2, v / v) buffer to a final probe concentration of 10 μM. The Cys concentrations added to each group were maintained at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 μM, respectively. After thorough mixing and standing for 10 min, fluorescence emission spectroscopy was performed at an excitation wavelength of 500 nm.

[0040] The test results are shown in Figure 5: Figure 5A The fluorescence spectra of TCF-C1 (10 μM) and different concentrations of Cys (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 μM) in EtOH / PBS buffer (8 / 2, v / v) were compared. Figure 5B The graph shows the linear relationship between fluorescence intensity at 664 nm and Cys concentration (0 ~ 28 μM).

[0041] It can be seen that the fluorescence intensity emitted by the probe TCF-C1 itself is negligible, while the fluorescence peak intensity at 664 nm gradually increases with increasing Cys concentration (0~100 μM). Furthermore, the fluorescence intensity shows a good linear relationship with the Cys concentration (2~28 μM) (R0). 2 = 0.9970, y = 1.092 × 10 5 The detection limit was 35.98 nM (3σ / k, where δ is the blank standard deviation and k is the slope of the standard curve). Fluorescence titration analysis showed that the probe TCF-C1 was highly sensitive to the Cys response.

[0042] (3) Determination of pH sensitivity: The fluorescent probe TCF-C1 was prepared into a 1 mM concentrated stock solution using DMSO, and Cys was prepared into a 10 mM concentrated stock solution using deionized water. Experimental group: 10 μL of the probe concentrated stock solution and 10 μL of the Cys concentrated stock solution were added to each 1000 μL colorimetric tube. Then, the volume was adjusted to 1000 μL using pre-prepared EtOH / PBS (8 / 2, v / v) buffers with different pH values ​​(pH 3 ~ 11), resulting in a final concentration of 10 μM for the fluorescent probe and 100 μM for Cys. Control group: The control group was identical to the experimental group except for the absence of Cys. After thorough mixing and standing for 10 min, fluorescence emission spectroscopy was performed at an excitation wavelength of 500 nm.

[0043] Test results are available Figure 6 : Figure 6 The curves show the response fluorescence intensity at 664 nm as a function of pH after the probe TCF-C1 (10 μM) and TCF-C1 (10 μM) react with Cys (100 μM).

[0044] It can be seen that when Cys is absent, the fluorescence intensity of TCF-C1 at 664 nm does not change significantly. However, when Cys is present, after TCF-C1 and Cys react under different pH conditions, the fluorescence intensity response is best in the pH range of 7.0 to 10.0, indicating that the probe can detect Cys under physiological conditions and can be further applied in biological systems and complex systems.

[0045] (4) Response time measurement: Prepare a 1 mM concentrated stock solution of the fluorescent probe TCF-C1 using DMSO, and a 10 mM concentrated stock solution of Cys using deionized water. Add 10 μL of the probe stock solution and 10 μL of the Cys stock solution to a 1000 μL colorimetric tube, and dilute with EtOH / PBS (8 / 2, v / v) buffer to a final probe concentration of 10 μM and a final Cys concentration of 100 μM. After thorough mixing, fluorescence emission spectroscopy can be performed at an excitation wavelength of 500 nm.

[0046] Test results are available Figure 7 : Figure 7 The fluorescence spectra of probes TCF-C1 (10 μM) and Cys (100 μM) at different times in EtOH / PBS (8 / 2, v / v) buffer solution are shown.

[0047] It can be seen that after adding 100 μM cysteine ​​(Cys), the fluorescence emission intensity at 664 nm increased rapidly and reached saturation within 10 minutes, indicating that the probe TCF-C1 can serve as a promising sensor for real-time monitoring of Cys.

[0048] The above tests show that the probe TCF-C1 of this invention has very good selectivity for cysteine, high detection sensitivity, and strong anti-interference ability. The detection mechanism of probe TCF-C1 is as follows: Figure 10 As shown, the thiol group in cysteine ​​(Cys) reacts with the electron-deficient double bond in TCF-C1 to form a thioether intermediate, which then undergoes a spontaneous intermolecular cyclization reaction to finally generate a fluorophore (compound 7).

[0049] Example 3 Qualitative and quantitative detection of intracellular cysteine ​​using the near-infrared fluorescent probe TCF-C1 The fluorescent probe TCF-C1 prepared in Example 1 was used to test the fluorescence imaging of cysteine ​​in liver cancer cells (MHCC97H). The specific operation is as follows: MHCC97H cells were seeded into confocal microarrays and incubated overnight at 37°C with 5% CO2. The culture medium was aspirated from the confocal microarrays, and the cells were washed once with serum-free medium. The following treatments were then applied to different cell groups: (a) Control group: no drugs added. (b) 1 mL of MB-Cys solution (10 μM) was added to the confocal microarray and incubated for 10 min. (c) 1 mL of NEM solution (100 μM) was added to the confocal microarray and incubated for 30 min. The culture medium was then discarded, and 1 mL of TCF-C1 solution (10 μM) was added and incubated for 10 min. (d) 1 mL of NEM solution (100 μM) was added and incubated for 30 min. The culture medium was then discarded, and 1 mL of Cys solution (100 μM) was added and incubated for 30 min. The culture medium was then discarded again, and 1 mL of TCF-C1 solution (10 μM) was added and incubated for 10 min. Finally, fluorescence imaging was performed on the different cell groups. The excitation wavelength was 647 nm, and the emission wavelength acquisition range was 645 ~ 700 nm.

[0050] Test results are available Figure 8 : Figure 8Imaging of intracellular / exogenous Cys fluorescence in MHCC97H cells. Control group: no treatment; MCF-C1 group: cells incubated with MCF-C1 (10 μM) for 10 min; NEM group: cells pretreated with NEM (100 μM) for 30 min, then incubated with MCF-C1 (10 μM) for 10 min; NEM + Cys group: cells pretreated with NEM (100 μM) for 30 min, then incubated with Cys (100 μM) for 30 min, and finally incubated with MCF-C1 (10 μM) for 10 min. λex = 647 nm, λem = 645 ~ 700 nm. Scale bar: 20 μM.

[0051] It can be seen that after the addition of probe TCF-C1, cells showed a strong fluorescence signal, while the fluorescence signal of cells with the addition of the thiol eliminator NEM (N-ethylmaleimide) was suppressed. In another control group, cells pre-incubated with NEM and then with exogenous Cys showed a significant enhancement of red fluorescence. These results indicate that probe TCF-C1 has the ability to detect both endogenous and exogenous Cys in cells, and probe TCF-C1 can also be applied to bioimaging.

[0052] Example 4 Qualitative and quantitative detection of cysteine ​​in food using the near-infrared fluorescent probe TCF-C1 The fluorescent probe TCF-C1 prepared in Example 1 was used to test spinach, green beans, carrots, and broccoli (purchased from a supermarket). The specific procedures are as follows: The fluorescent probe TCF-C1 was prepared into a 1 mM concentrated stock solution using DMSO. The food sample was ground and filtered, and the resulting filtrate was diluted 100-fold with EtOH / PBS (8 / 2, v / v) buffer. Subsequently, 990 μL of the diluted food buffer was added to 10 μL of the concentrated TCF-C1 stock solution, mixed thoroughly, and allowed to stand for 10 min before fluorescence emission spectroscopy was performed at an excitation wavelength of 500 nm to determine Cys in the food.

[0053] Test results are available Figure 9 : It can be seen that TCF-C1 can determine the Cys content in food, and spinach, green beans, and broccoli have high Cys content.

[0054] The above examples demonstrate the synthesis method, structural characterization, and detection effect verification of the near-infrared cysteine ​​fluorescent probe of Formula 1 when X is O. When preparing the near-infrared cysteine ​​fluorescent probe of Formula 1 when X is S or N, simply replace 5-bromo-2-furanaldehyde in step (1) of Example 1 with 5-bromo-2-thiaphenaldehyde or 5-bromo-2-pyrrolealdehyde. The corresponding probe obtained also has a good cysteine ​​detection effect.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A near-infrared cysteine ​​fluorescent probe, characterized in that, The structural formula of the fluorescent probe is shown in Formula 1: Formula 1 Where X is selected from any one of O, S, and N.

2. The method for synthesizing the near-infrared cysteine ​​fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) Using tetrahydrofuran and water as the reaction medium, 5-bromo-2-furan carbaldehyde and 4-hydroxyphenylboronic acid as reactants, tetra-triphenylphosphine palladium as the catalyst, and potassium carbonate as the reaction base, the reaction was carried out at 75°C. After the reaction was completed, the solvent was removed by vortexing, and ethyl acetate and water were added for extraction. The obtained organic phase was washed with saturated brine and dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated by vacuum distillation to obtain the crude product, which was washed with diethyl ether to obtain a yellow solid compound. (2) Using anhydrous ethanol as the reaction medium, malononitrile and 3-hydroxy-3-methyl-2-butanone as reactants, and sodium ethoxide as the reaction base, the reaction was carried out at room temperature. After the reaction was completed, the mixture was filtered and washed with ethanol and ether in sequence to obtain a gray solid compound. (3) Using tetrahydrofuran and anhydrous ethanol as the reaction solvent, the yellow solid compound obtained in step (1) and the gray solid compound obtained in step (2) as the reactants, and ammonium acetate as the reaction base, the reaction was carried out at room temperature. After the reaction was completed, distilled water and dichloromethane were added for extraction. The organic phase obtained was washed with saturated brine and dried with anhydrous sodium sulfate. The mixture was then filtered, and the filtrate was concentrated by vacuum distillation to obtain the crude product. The crude product was separated by silica gel column chromatography with dichloromethane / methanol as the eluent to obtain the black solid compound. (4) The black solid compound obtained in step (3) was dissolved in dichloromethane and triethylamine was added to adjust the alkaline environment. Acryloyl chloride was then added and reacted at room temperature. After the reaction was completed, dichloromethane and water were added for extraction. The resulting organic phase was washed with saturated brine and dried with anhydrous sodium sulfate. The phase was then filtered and the filtrate was concentrated by vacuum distillation to obtain the crude product. The crude product was separated by silica gel column chromatography with dichloromethane as the eluent to obtain a dark red solid, which is the near-infrared cysteine ​​fluorescent probe.

3. The method for synthesizing the near-infrared cysteine ​​fluorescent probe as described in claim 1, characterized in that, In step (1), the molar ratio of 5-bromo-2-furan carbaldehyde, 4-hydroxyphenylboronic acid, tetratriphenylphosphine and palladium carbonate is 1:1.5~2:0.2~0.5:16~18.

4. The method for synthesizing the near-infrared cysteine ​​fluorescent probe as described in claim 1, characterized in that, In step (2), the molar ratio of sodium ethoxide, 3-hydroxy-3-methyl-2-butanone and malononitrile is 1:6-7:12-14.

5. The method for synthesizing the near-infrared cysteine ​​fluorescent probe as described in claim 1, characterized in that, In step (3), the molar ratio of the yellow solid compound, the gray solid compound and ammonium acetate is 1:1.1:

1.

6. The method for synthesizing the near-infrared cysteine ​​fluorescent probe as described in claim 1, characterized in that, In step (4), the molar ratio of the black solid compound, acryloyl chloride and triethylamine is 1:1.5-2.5:3-5.

7. The application of the near-infrared cysteine ​​fluorescent probe as described in claim 1 in the qualitative or quantitative detection of cysteine ​​in organisms and food.

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