A persistent imaging cysteine fluorescent probe and a preparation method and application thereof

Abstract

This invention discloses a persistent imaging cysteine ​​fluorescent probe, its preparation method, and its applications. The probe structure is one of the following formulas 1-2. This probe has the function of detecting and persistently imaging cysteine ​​(Cys) levels in cells and organisms. The persistent imaging cysteine ​​fluorescent probe provided by this invention is a novel type of Cys small molecule fluorescent probe. Compared with previously reported Cys small molecule fluorescent probes, it has the ability to monitor Cys content in cells and in vivo for extended periods, and has broad application prospects in early clinical diagnosis of diseases and future long-term in vivo imaging based on small molecule probes.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a persistent imaging cysteine ​​fluorescent probe, its preparation method, and its application. Background Technology

[0002] Cysteine ​​(Cys), a type of biothiol, plays a crucial role in life processes, and changes in its concentration can trigger various diseases. Activated near-infrared (NIR) cysteine ​​fluorescent probes possess advantages such as high selectivity, strong sensitivity, and deep imaging depth, making them highly promising for early disease diagnosis and treatment intervention. However, the rapid diffusion and metabolic clearance of activated fluorophores in vivo significantly limits the application of small-molecule fluorescent probes. Considering that the observation time of fluorescence signals after probe application may vary, there is a strong need for Cys probes that can maintain fluorescence signals for a longer period.

[0003] Currently, most reported Cys probes can image Cys in cells and organisms, but during persistent imaging and fixation, fluorescent hydrolysis products leak out of the cells. Literature review reveals that while some fluorescent probes for persistent Cys imaging have been developed, these are all nanoprobes or solid-state fluorescent probes; there are no reports on the use of single-molecule fluorescent probes for persistent Cys imaging.

[0004] Therefore, the rational design of fluorescent probes for persistent imaging based on single-molecule systems is of great significance for the detection and persistent imaging of Cys levels in cells and organisms, and for the early diagnosis and treatment of diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a persistent imaging cysteine ​​fluorescent probe, its preparation method, and its application. This probe has the function of detecting and persistently imaging Cys levels in cells and organisms, and it has great advantages in the early diagnosis and treatment intervention of diseases.

[0006] The technical solution to achieve the objective of this invention is:

[0007] This invention provides a persistent imaging cysteine ​​fluorescent probe, the structure of which is one of the following formulas 1 and 2:

[0008]

[0009] The synthetic route of the persistent imaging cysteine ​​fluorescent probe of this invention is as follows:

[0010]

[0011] The method for preparing a persistent imaging cysteine ​​fluorescent probe of the present invention includes the following steps:

[0012] (1) Weigh anthocyanins, 2,4-dihydroxybenzyl alcohol and sodium hydride into a round-bottom flask, add N,N-dimethylformamide to dissolve, and after dissolving, place it in an oil bath to react for a period of time to obtain the crude product.

[0013] (2) Cool the crude product obtained in step (1), pour it into a beaker, add distilled water and perchloric acid in sequence, stir to precipitate solid, filter, dissolve in dichloromethane, concentrate with a rotary evaporator, elute with eluent, and then purify with a silica gel chromatography column to obtain the fluorescent dye with the structure shown in formula (I).

[0014] (3) Weigh the fluorescent dye with the structure shown in formula (Ⅰ) obtained in step (2), acryloyl chloride and triethylamine into a round bottom flask, add dichloromethane to dissolve, place in an oil bath and react for a period of time to obtain a crude product;

[0015] (4) Pour the crude product obtained in step (3) into a beaker containing water, add dichloromethane for extraction, concentrate it using a rotary evaporator, elute with eluent, and then purify it using a silica gel chromatography column to obtain a fluorescent probe with the structure shown in formula (II).

[0016] (5) Weigh the fluorescent probe with the structure shown in formula (II) obtained in step (4), add dichloromethane to dissolve it, add diethylaminotrifluoride (DAST) under ice bath conditions, and react at room temperature for a period of time.

[0017] (6) After the reaction was completed, methanol was added to quench DAST, then concentrated by rotary evaporator, eluted with eluent, and purified by silica gel chromatography column to obtain a persistent imaging cysteine ​​fluorescent probe with the structure shown in formula (Ⅲ) (same as formula 1).

[0018] Furthermore, in the preparation method, the equivalent ratio of anthocyanin to 2,4-dihydroxybenzyl alcohol in step (1) is 1:2, the equivalent ratio of anthocyanin to sodium hydride is 1:1, and the amount of N,N-dimethylformamide used is preferably sufficient to dissolve the raw materials participating in the reaction.

[0019] The oil bath temperature is 45-65℃, and the oil bath reaction lasts for 4-6 hours.

[0020] Furthermore, in the preparation method, the amount of distilled water and perchloric acid added in step (2) is 50 mL: 3-5 mL, and the amount of dichloromethane used is preferably enough to dissolve the raw materials participating in the reaction;

[0021] The crude product was eluted with dichloromethane and methanol at a volume ratio of 100:1 and purified using a 200-300 mesh silica gel chromatography column.

[0022] Furthermore, in the preparation method, the molar ratio of the fluorescent dye, acryloyl chloride and triethylamine in step (3) is 1:2:1, and the amount of dichloromethane used is preferably sufficient to dissolve the raw materials participating in the reaction.

[0023] The bath temperature is 45-65℃, and the oil bath reaction lasts for 4-6 hours.

[0024] Furthermore, in the preparation method, the amount of water and dichloromethane added in step (4) is 150 mL: 50 mL;

[0025] The eluent is dichloromethane and methanol in a volume ratio of 100:1, and the mixture is purified using a 200-300 mesh silica gel chromatography column.

[0026] Furthermore, in the preparation method, the molar ratio of the fluorescent probe with the structure shown in formula (II) in step (5) to DAST is 1:2, and the amount of dichloromethane used is preferably sufficient to dissolve the raw materials participating in the reaction.

[0027] The ice bath is subjected to a reaction at 0°C for 4-6 hours at room temperature.

[0028] Furthermore, in the preparation method, in step (6), methanol is added to quench DAST, and the amount of methanol used is preferably sufficient to quench DAST;

[0029] The eluent is dichloromethane and methanol in a volume ratio of 100:1, and the mixture is purified using a 200-300 mesh silica gel chromatography column.

[0030] In addition, this invention also applies the probe to persistent imaging of Cys in vivo, specifically for detecting changes in Cys content in cells and organisms, so as to realize the function of detecting and persistently imaging Cys levels in cells and organisms.

[0031] The specific application method of the cysteine ​​fluorescent probe for persistent imaging of this invention is as follows:

[0032] (1) Dissolve the fluorescent probe with the structure shown in formula (Ⅲ) in dimethyl sulfoxide (DMSO) to prepare a probe stock solution;

[0033] (2) Add the probe stock solution to the test solution and biological sample;

[0034] (3) Add Cys stock solution (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 μL) with a total concentration of 1 mmol, mix well for five minutes, and then observe the changes in the fluorescence spectrum of the test solution containing the Cys fluorescent probe using a UV and fluorescence spectrometer.

[0035] (4) Add the prepared calcium chloride (CaCl2), copper bromide (CuBr2), glutathione (GSH), homocysteine ​​(Hcy), hydrogen peroxide (H2O2), tyrosine (Tyr), hydrogen sulfide (H2S), sodium bisulfite (NaHSO3), sodium nitrite (NaNO2), sodium sulfate (Na2SO4), allyl thiourea (ATU), and cysteine ​​(Cys) respectively. After these analytes are diluted in the solution, the final concentration of each analyte is 120 μM. After the solution is mixed evenly for five minutes, observe the changes in the fluorescence spectra of the probes with different analytical interfering substances and Cys using ultraviolet and fluorescence spectrometers.

[0036] (5) The probe was applied to intracellular fluorescence imaging. The first group was incubated with probe (5.0 μM) for 10 minutes. The other three groups were incubated with NEM (10.0 μM) for 30 minutes, then Cys (100.0 μM), Hcy (100.0 μM) and GSH (100.0 μM) were added for 30 minutes respectively, and then probe (5.0 μM) was added. The fifth group was incubated with NEM (10.0 μM) for 30 minutes, and then probe (5.0 μM) was added. The images were taken and analyzed using a laser confocal microscope with an excitation wavelength of 633 nm.

[0037] (6) Apply the probe to intracellular Cys long-term fluorescence imaging. Add the probe (5.0 μM) and monitor the change in fluorescence intensity over 0-6 hours on a live cell workstation.

[0038] (7) The probe was applied to in vivo fluorescence imaging of zebrafish. The first group was incubated with probe Cys (5.0 μM) for 10 minutes. The other three groups were incubated with NEM (10.0 μM) for 30 minutes, then Cys (100.0 μM), Hcy (100.0 μM) and GSH (100.0 μM) were added for 30 minutes respectively, and then probe (5.0 mM) was added. The fifth group was incubated with NEM (10.0 μM) for 30 minutes, and then probe (5.0 mM) was added. The images were taken and analyzed using a laser confocal microscope with an excitation wavelength of 633 nm to observe the changes in fluorescence intensity.

[0039] (8) Apply the probe to long-term Cys fluorescence imaging in zebrafish. Add the probe (5.0 μM) and monitor the change in fluorescence intensity of the probe in zebrafish within 0-6 hours.

[0040] The change in fluorescence spectrum refers to the change in fluorescence intensity at 725 nm. Fluorescence spectra were observed using a fluorescence spectrometer with an excitation wavelength of 633 nm. The resulting fluorescence imaging indicates that the probe can specifically and persistently image Cys in cells and zebrafish.

[0041] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0042] (1) The cysteine ​​fluorescent probe provided by the present invention is a novel small molecule fluorescent probe with the function of detecting and persistently imaging Cys levels in cells and organisms. It has broad application prospects in the early diagnosis and treatment intervention of diseases.

[0043] (2) The fluorescent probe of the present invention has simple preparation steps, convenient purification, and high yield;

[0044] (3) This invention enables persistent Cys imaging in cells and zebrafish. Attached Figure Description

[0045] Figure 1 It is the Cy-Cys-F prepared in Example 1. 1 H NMR spectrum;

[0046] Figure 2 It is the Cy-Cys-F prepared in Example 1. 13 C NMR spectrum;

[0047] Figure 3 This is the mass spectrum of Cy-Cys-F prepared in Example 1;

[0048] Figure 4 This is a graph showing the changes in the UV and fluorescence spectra of Cy-Cys-F prepared in Example 1 during Cys titration;

[0049] Figure 5 This is a fluorescence ratio diagram at 724 nm of Cy-Cys-F prepared in Example 1 against different analytes.

[0050] Figure 6 This is a fluorescence image of Cy-Cys-F prepared in Example 1, specifically imaging Cys in cells;

[0051] Figure 7 This is a long-term fluorescence imaging image of Cys in cells prepared by Cy-Cys-F in Example 1;

[0052] Figure 8 This is a fluorescence image of Cy-Cys-F prepared in Example 1 specifically imaging Cys in zebrafish;

[0053] Figure 9 This is a long-term imaging fluorescence image of Cys in zebrafish prepared by Cy-Cys-F in Example 1. Detailed Implementation

[0054] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0055] Example 1

[0056] Preparation of a persistent imaging cysteine ​​fluorescent probe: A fluorescent probe was prepared using anthocyanins and named Cy-Cys-F;

[0057] Preparation of Cy-Cys-F:

[0058] (1) Weigh anthocyanin (100mg, 0.1mmol), 2,4-dihydroxybenzyl alcohol (90mg, 0.2mmol) and sodium hydride (25mg, 0.1mmol) into a round-bottom flask, add 10mL of N,N-dimethylformamide to dissolve, and place in an oil bath. Initially set the temperature to 45℃ and react for about half an hour. Then raise the temperature to 55℃ and monitor by TLC. Under nitrogen protection, it takes 5 hours to react to obtain the crude product.

[0059] (2) Cool the crude product obtained in step (1), pour it into a beaker, add 50 mL of distilled water and 3-5 mL of perchloric acid in sequence, stir and place in a refrigerator to stand. After a large amount of solid precipitates, filter it, dissolve it in 20 mL of dichloromethane, concentrate it in a rotary evaporator, elute with dichloromethane and methanol in a volume ratio of 100:1, and purify it with a 200-300 mesh silica gel chromatography column to obtain a fluorescent dye with the structure shown in formula (Ⅰ) (molecular formula: C 28 H 30 NO3 + );

[0060] (3) Weigh the fluorescent dye (22 mg, 0.04 mmol), acryloyl chloride (0.76 mg, 0.08 mmol), and triethylamine (4 mg, 0.04 mmol) with the structure shown in formula (Ⅰ) obtained in step (2) into a round-bottom flask, add 50 mL of dichloromethane to dissolve, place it in an oil bath, initially set the temperature to 45 °C, react for about half an hour, raise the temperature to 55 °C, monitor by TLC, and the crude product needs to be obtained after reacting for 5 hours under nitrogen protection.

[0061] (4) Pour the crude product obtained in step (3) into a beaker containing 50 mL of water, add 150 mL of dichloromethane for extraction, then concentrate it using a rotary evaporator, elute with dichloromethane and methanol in a volume ratio of 100:1, and then purify it using a 200-300 mesh silica gel chromatography column to obtain the fluorescent probe with the structure shown in formula (II).

[0062] (5) Weigh the fluorescent probe (15 mg, 0.025 mmol) with the structure shown in formula (II) obtained in step (4), add 15 mL of dichloromethane to dissolve it, add DAST (8 mg, 0.05 mmol) rapidly in an ice bath at 0 °C, and react at room temperature for 5 hours under nitrogen protection.

[0063] (6) After the reaction was completed, 2 mL of methanol was added to quench DAST, the mixture was concentrated by rotary evaporator, and eluted with dichloromethane and methanol in a volume ratio of 100:1. The mixture was then purified by silica gel chromatography column of 200-300 mesh to obtain a persistent imaging cysteine ​​fluorescent probe with the structure shown in formula (III).

[0064] The structural formula of the probe Cy-Cys-F prepared in Example 1 is as follows:

[0065]

[0066] Reference Figure 1 , 1 H NMR(500MHz,DMSO-d6)δ8.60(d,J=15.2Hz,1H),7.82–7.74(m,2H),7.59(d,J=8.1Hz,2H ),7.53(d,J=7.8Hz,2H),6.70(d,J=15.2Hz,1H),6.62(d,J=17.2Hz,1H),6.47(dd,J=17. 2,10.4Hz,1H),6.25(d,J=10.4Hz,1H),5.75(s,2H),5.67(s,1H),4.51(q,J=7.3Hz,2H), 2.75 (dt, J = 34.8, 6.1 Hz, 4H), 1.86 (p, J = 5.9 Hz, 2H), 1.76 (s, 6H), 1.40 (t, J = 7.1 Hz, 3H).

[0067] Reference Figure 2 , 13 C NMR (126MHz, DMSO-d6) δ178.83,164.24,158.66,153.41,151.90,145.95,143.20,141.33,135.03,134.24,131.01,129.96,129.51, 128.35,127.71,127.16,123.35,118.51,114.90,114.18,111.28,106.92,80.66,55.36,51.40,28.85,27.57,22.85,20.23,13.37.

[0068] Reference Figure 3 The theoretical molecular weight is 484.2282, but the actual high-resolution results show that the molecular weight is 484.2306.

[0069] Spectroscopic experiments of the probe Cy-Cys-F prepared in Example 1 on the titration of Cys concentration:

[0070] Prepare a DMSO stock solution of the fluorescent probe prepared in Example 1 with a concentration of 1 mM; then add 1970 μL of liquid (PBS:DMSO = 9:1) and 30 μL of probe stock solution to the UV and fluorescence dishes respectively, and add Cys to the dishes until the UV and absorption of the compound no longer change.

[0071] When the concentration of Cys added was (0-120 μm), the UV spectrum of Cy-Cys-F showed a red shift of 110 nm, and the fluorescence intensity increased 24-fold at 725 nm. This indicates that the probe can be used as a fluorescent probe for Cys, as shown in the results. Figure 4 As shown.

[0072] Experimental testing of Cys selectivity of probe Cy-Cys-F prepared in Example 1:

[0073] Prepare the fluorescent probe DMSO stock solution prepared in Example 1 with a concentration of 1 mM.

[0074] The probe's response to 16 intracellular analytes (amino acids, metal ions, reactive oxygen species (ROS), and reactive nitrogen species (RNS) was tested. A significant change in fluorescence intensity was observed in the probe-Cys mixture, with a fluorescence ratio of 10. Conversely, the fluorescence intensity of the solution showed almost no change after the addition of other analytes. This indicates that the probe has good selectivity for Cys and can specifically detect Cys under physiological conditions. The results are as follows... Figure 5 As shown.

[0075] Example 1: Fluorescence imaging experiments of endogenous and exogenous Cys cells using the probe Cy-Cys-F prepared in Example 1.

[0076] The DMSO stock solution of the fluorescent probe prepared in Example 1 was prepared at a concentration of 1 mM. After incubation with the probe (5 μM) for 15 minutes, fluorescence imaging was performed. N-ethylmaleimide (NEM, 10 μM) was added to the cells beforehand and incubated for 30 minutes to eliminate a certain amount of intracellular biothiols as a control. Cells in the group treated with NEM first, followed by the probe, showed almost no fluorescence. After NEM pretreatment, cells were incubated with Cys (100 μM), Hcy (100 μM), and GSH (100 μM), respectively, before the probe was added. Confocal fluorescence imaging results showed that only the cell group treated with Cys exhibited significant fluorescence enhancement, indicating that the probe specifically recognizes Cys in the cells. The results are as follows. Figure 6 As shown.

[0077] Example 1: Long-term imaging test of Cys in cells using the probe Cy-Cys-F prepared in Example 1.

[0078] Digested HeLa (human cervical cancer) cell suspension was prepared at a concentration of 1×10⁻⁶ cells per well. 5180 μL per cell -1 Cells were seeded at a density of 40-50% in 96-well plates and incubated for 24 hours. When the cell density reached 40-50%, Cy-Cys-F (5 μM) was added to each well, and the cells were incubated for different time periods (0-9 hours). Confocal microscopy results are shown below. Figure 7 As shown, the results indicate that the fluorescence intensity of the probe in cells gradually increases with the extension of incubation time from half an hour to nine hours, indicating that the probe has the ability to image Cys in cells for a long time.

[0079] Example 1: Fluorescence imaging experiments of endogenous and exogenous Cys in zebrafish using the probe Cy-Cys-F prepared in Example 1.

[0080] The fluorescent probe DMSO stock solution prepared in Example 1 was prepared at a concentration of 1 mM. After incubation of the probe (5 μM) for 3 hours, fluorescence imaging was performed. N-ethylmaleimide (NEM, 200 μM) was added to the zebrafish nutrient solution beforehand and incubated for 30 minutes to eliminate a certain amount of biothiols in the zebrafish as a control. Zebrafish treated with NEM first, followed by probe, showed almost no fluorescence. After NEM pretreatment, zebrafish were incubated with Cys (200 μM), Hcy (200 μM), and GSH (200 μM), respectively, before the probe was added. Confocal fluorescence imaging results showed that only the zebrafish group treated with Cys exhibited significant fluorescence enhancement, indicating that the probe can specifically recognize Cys in zebrafish. The results are as follows. Figure 8 As shown.

[0081] Example 1: Long-term Cys imaging test of the probe Cy-Cys-F prepared in zebrafish:

[0082] Prepare a 1 mM DMSO stock solution for the fluorescent probe prepared in Example 1. Incubate the probe (5 μM) for 3-24 hours and perform fluorescence imaging. The confocal experimental results are as follows: Figure 9 As shown, the results indicate that the fluorescence intensity of the probe in zebrafish gradually increases with the extension of incubation time from 3 to 24 hours, indicating that the probe has the ability to image Cys in zebrafish for a long time.

[0083] The fluorescent probe prepared in Example 1 has been shown in experiments to have Cys-specific detection and persistent imaging capabilities in cells and zebrafish, which is of great significance for the early diagnosis and treatment of diseases.

[0084] Finally, it should be noted that the above embodiments do not limit the present invention in any way. Those skilled in the art can make modifications and improvements based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A persistent imaging fluorescent probe for cysteine, the structure of which is shown in Formula 1 below: 1。 2. A method for preparing a persistent imaging cysteine ​​fluorescent probe, characterized in that, The synthesis route is as follows: The preparation method includes the following steps: (1) Weigh anthocyanins, 2,4-dihydroxybenzyl alcohol and sodium hydride into a round-bottom flask, add N,N-dimethylformamide to dissolve, and then place in an oil bath to react for a period of time to obtain the crude product; (2) Cool the crude product obtained in step (1), pour it into a beaker, add distilled water and perchloric acid in sequence, stir to precipitate solid, filter, dissolve in dichloromethane, concentrate with a rotary evaporator, and then purify with a silica gel chromatography column to obtain the fluorescent dye with the structure shown in formula (I). (3) Weigh the fluorescent dye with the structure shown in formula (Ⅰ) obtained in step (2), acryloyl chloride and triethylamine into a round bottom flask, add dichloromethane to dissolve, place in an oil bath and react for a period of time to obtain the crude product; (4) Pour the crude product obtained in step (3) into a beaker containing water, add dichloromethane for extraction, concentrate it using a rotary evaporator, and then purify it using a silica gel chromatography column to obtain a fluorescent probe with the structure shown in formula (II). (5) Weigh the fluorescent probe with the structure shown in formula (II) obtained in step (4), add dichloromethane to dissolve it, add DAST under ice bath conditions, and react at room temperature for a period of time. (6) After the reaction was completed, methanol was added to quench DAST, then concentrated by rotary evaporator, and then purified by silica gel chromatography column to obtain a persistent imaging cysteine ​​fluorescent probe with the structure shown in formula (III).

3. The method for preparing the cysteine ​​fluorescent probe according to claim 2, characterized in that: In step (1), the equivalent ratio of anthocyanin to 2,4-dihydroxybenzyl alcohol is 1:2, and the equivalent ratio of anthocyanin to sodium hydride is 1:

1. The amount of N,N-dimethylformamide used should be such that it can dissolve the raw materials participating in the reaction. The oil bath temperature is 45-65℃, and the oil bath reaction lasts for 4-6 hours.

4. The method for preparing the cysteine ​​fluorescent probe according to claim 2, characterized in that: In step (2), the amount of distilled water and perchloric acid added is 50 mL: 3-5 mL, and the amount of dichloromethane used is appropriate to dissolve the raw materials participating in the reaction. The silica gel chromatography column was used for purification, with dichloromethane and methanol as eluents in a volume ratio of 100:1, and 200-300 mesh silica gel as the eluent.

5. The method for preparing the cysteine ​​fluorescent probe according to claim 2, characterized in that: In step (3), the molar ratio of the fluorescent dye, acryloyl chloride and triethylamine is 1:2:1, and the amount of dichloromethane used is preferably sufficient to dissolve the raw materials participating in the reaction. The bath temperature is 45-65℃, and the oil bath reaction lasts for 4-6 hours.

6. The method for preparing the cysteine ​​fluorescent probe according to claim 2, characterized in that: In step (4), the ratio of water to dichloromethane is 150 mL: 50 mL. The silica gel chromatography column was used for purification, with dichloromethane and methanol in a volume ratio of 100:1 as the eluent, and 200-300 mesh silica gel as the elution medium.

7. The method for preparing the cysteine ​​fluorescent probe according to claim 2, characterized in that: In step (5), the molar ratio of the fluorescent probe with the structure shown in formula (II) to DAST is 1:2, and the amount of dichloromethane used is preferably sufficient to dissolve the raw materials participating in the reaction. The ice bath is subjected to a reaction at 0°C for 4-6 hours at room temperature.

8. The method for preparing the cysteine ​​fluorescent probe according to claim 2, characterized in that: In step (6), methanol is added to quench DAST. The amount of methanol used should be sufficient to quench DAST. The silica gel chromatography column was used for purification, with dichloromethane and methanol as eluents in a volume ratio of 100:1, and 200-300 mesh silica gel as the eluent.

9. The application of the persistent imaging cysteine ​​fluorescent probe according to claim 1, characterized in that: The probe is used to detect Cys content in cells and organisms for non-disease diagnostic purposes.

Images