A near-infrared fluorescent probe for detecting biological thiols with mitochondrial targeting and large stokes shift and preparation and application thereof

Abstract

This invention discloses a near-infrared fluorescent probe for the rapid and sensitive detection of biothiols (Cys / Hcy) with a large Stokes shift targeting mitochondria. The structural formula of the fluorescent probe is shown in Formula (I). The probe of this invention not only exhibits rapid response time, high sensitivity, and selectivity to cysteine / homocysteine, but also a near-infrared emission response with a large Stokes shift (approximately 200 nm). Furthermore, this probe possesses good cell permeability and mitochondrial targeting ability, making it highly suitable for imaging and detection of Cys / Hcy in live cells.

Description

Technical Field

[0001] This invention relates to a fluorescence sensing detection method and its application, specifically to a near-infrared fluorescent probe with mitochondrial targeting and large Stokes shift properties for rapid and sensitive detection of biothiols, its preparation and application, belonging to the field of organic fluorescence sensing technology. Background Technology

[0002] Cysteine ​​(Cys) and homocysteine ​​(Hcy) play important roles in physiological and pathological processes as biothiols. Cysteine, for example, participates in enzyme and peptide synthesis and is a sulfur ligand in the iron sulfide complex in organisms. Abnormal concentrations of these compounds in physiological processes are frequently associated with many major diseases. Abnormalities in cysteine ​​(Cys) can lead to numerous diseases, such as rheumatoid arthritis, growth retardation, liver damage, and others. More importantly, oxidative damage to mitochondria caused by reactive oxygen species leads to apoptosis, while Cys / Hcy acts as antioxidants to remove these damages and prevent this process. Therefore, detecting Cys / Hcy in mitochondria is crucial for understanding their role in physiological activities and early disease diagnosis.

[0003] Fluorescence imaging technology, due to its high selectivity, non-invasiveness, and real-time detection capabilities, has become an effective method for detecting and visualizing target analytes in live cells and organisms. To date, many fluorescent probes have been developed for Cys / Hcy monitoring and imaging, achieving excellent imaging detection results. However, many fluorescent probes detect fluorescence wavelengths in the visible light region; therefore, their in vivo application is limited by interference from in vivo autofluorescence and scattered light. Near-infrared (NIR) fluorescent probes have strong potential for in vivo imaging applications due to their low background biofluorescence signal, weak light loss, and high penetration into biological tissues. Currently, biothiol fluorescent probes are mainly based on nucleophilic substitution cyanine dyes and Michael addition-based hemicyanine dyes. However, the inefficient nucleophilic substitution mechanism of cyanine dye-based probes leads to higher cysteine ​​concentrations (50 times or more) and longer response times (60 minutes or longer). Furthermore, the small Stokes shift characteristics (<100 nm) of these probes make them more susceptible to interference from excitation sources in bioimaging. In addition, targeted detection of biothiols in mitochondria is of great significance for studying their physiological activities and related disease processes. Therefore, there is a need to develop near-infrared mitochondrial-targeting biothiol fluorescent probes with high sensitivity, fast response time, and large Stokes shift. Summary of the Invention

[0004] The main objective of this invention is to overcome the shortcomings of existing technologies and provide a fluorescent probe with mitochondrial targeting and large Stokes shift, as well as rapid and sensitive detection of biothiols in the near-infrared region.

[0005] This invention provides a near-infrared fluorescent probe for rapid and sensitive detection of biothiols with mitochondrial targeting and large Stokes shift, the molecular structure of which is shown in formula (I):

[0006]

[0007] The present invention also provides a method for preparing the near-infrared fluorescent probe, comprising the following steps:

[0008] (1) 9-chlorotetrahydroacridine 1 was reacted with iodomethane in sulfolane by heating. After the reaction was completed, diethyl ether was added to treat the solid salt compound 2.

[0009] (2) The product of step (1) is condensed with 3-aldehyde coumarin 3 in acetic anhydride. After the reaction is completed, the near-infrared fluorescent probe is obtained by post-treatment.

[0010] The preparation chemical reaction formula is as follows:

[0011]

[0012] The present invention also provides an application of the aforementioned near-infrared fluorescent probe in the detection and imaging of biothiols.

[0013] The fluorescent probe of this invention was used to perform fluorescence spectroscopy on biothiols in DMSO / PBS (v / v 1 / 1 pH 7.4) buffer. Under excitation at 480 nm, the probe solution showed a weak fluorescence signal. With increasing Cys concentration, a strong fluorescence peak appeared at 674 nm, and the fluorescence increased significantly and reached saturation at a Cys concentration of 30 μmol / L, with a 20-fold increase in fluorescence. Biothiols (Hcy) exhibited a similar spectral response, while the probe showed a weak response to GSH spectroscopy, thus enabling the probe to detect Cys / Hcy. Figure 1 and 2 As shown. Secondly, the probe detection has strong selectivity. Various interfering substances, including various amino acids, anions, metal ions, sulfur species, HClO and H2O2, do not cause any significant fluorescence changes in the probe. Only Cys / Hcy shows a significant fluorescence change.

[0014] Cellular imaging of Cys / Hcy was further performed by adding the near-infrared fluorescent probe described in this invention to A549 live cells. In the experimental group, the fluorescent probe was added to A549 live cells, and a fluorescent signal was observed, indicating that the probe can detect endogenous biothiols. Conversely, when cells were pretreated with NEM and then incubated with the probe, almost no fluorescent signal was observed in the red channel. Furthermore, when biothiols including Cys and GSH were added to NEM-pretreated cells and then incubated with the probe, a significant fluorescent signal was observed in Cys / Hcy. Almost no fluorescent signal was observed in the control group and the GSH group, indicating that the probe can also monitor exogenous Cys / Hcy in cells.

[0015] The beneficial effects of this invention are as follows: The probe is constructed based on a coumarin-hybridized tetrahydroacrylidine salt. The highly reactive chlorine atom in the probe facilitates the nucleophilic substitution reaction of biothiols, thereby giving the probe a rapid response time (responding to Cys within 3 minutes), high sensitivity, and high selectivity for Cys / Hcy detection. Furthermore, it can be distinguished and detected with a large Stokes shift (approximately 200 nm) in near-infrared fluorescence. In addition, the probe can specifically target mitochondria within cells, enabling imaging and detection of Cys / Hcy in living cells. Attached Figure Description

[0016] Figure 1 This diagram illustrates the reaction process between the near-infrared fluorescent probe prepared in this invention and Cys / Hcy.

[0017] Figure 2 The fluorescence emission spectra of the probe (10 μmol / L) of this invention reacting with different concentrations of Cys (left) and Hcy (right), with excitation at 480 nm.

[0018] Figure 3 The probe of this invention (10 μmol / L) at 674 nm (I 674 Linear relationship between fluorescence intensity (nm) and Cys (a) and Hcy (b) at low concentrations of 1–9 μmol / L.

[0019] Figure 4 The figures show the fluorescence spectra of the probe of this invention over time when 30 μmol / L Lys (a), Hcy (b), and GSH (c) were added to DMSO / PBS (pH 7.4, 20 mmol / L, v / v 1:1), and the fluorescence intensity of the probe at 674 nm over time (d).

[0020] Figure 5 This is the ESI-MS spectrum of the reaction product of the probe of this invention and Cys.

[0021] Figure 6 This is the ESI-MS spectrum of the reaction product of the probe of this invention and Hcy.

[0022] Figure 7 This is the ESI-MS spectrum of the reaction product of the probe of this invention and GSH.

[0023] Figure 8 The fluorescence spectra of the probe (10 μmol / L) of this invention against various interfering substances (50 μmol / L) in DMSO / PBS (pH 7.4, 20 mmol / L, v / v 1:1) are shown.

[0024] Figure 9 The fluorescence intensity changes of the probe (10 μmol / L) of this invention at 674 nm at different pH values, both naturally occurring and when Cys is added.

[0025] Figure 10 The image shows the fluorescence of the probe (10 μmol / L) and MitoTrackerGreen (5 μmol / L) in A549 cells under 488 nm excitation, according to the present invention.

[0026] Figure 11 This is a fluorescence imaging image of the fluorescent probe of the present invention in A549 cells. Detailed Implementation

[0027] Example 1

[0028] Iodomethane (1.4 g, 10 mmol) was added to a solution of compound 1 (0.44 g, 2 mmol) in sulfolane (5 mL). The reaction mixture was heated overnight at 60 °C under argon. A distinct solid was formed and obtained by filtration, followed by washing with diethyl ether to give compound 2 as a pale yellow solid (0.41 g, 57%). 1 H NMR(400MHz, CDCl3)δ:8.55–8.47(m,2H),8.16–8.14(m,1H),7.96 –7.92(m,1H),4.70(s,3H),3.73(t,J=6.0Hz,2H),3.19(t,J=6.0Hz,2H),2.18(m,2H),2.08–2.06(m 2H).

[0029] Example 2

[0030] Compound 2 (360 mg, 1 mmol) and 7-dialkylaminocoumarin-3-carboxaldehyde (compound 3, 250 mg, 1 mmol) were dissolved in acetic anhydride (10 mL). The reaction mixture was stirred at 60 °C for 6 hours. After completion, the mixture was evaporated under vacuum to remove the solvent. The residue was purified directly by flash column chromatography to give the probe as a red powder (150 mg, 26%). 1H NMR(400MHz,d6-DMSO) δ:8.56–8.52(m,2H),8.34(s,1H),8.29–8.25(m,1H),8.11–8.07(m,1H) ,7.67(d,J=9.2Hz,1H),7.17(s,1H),6.84(d,J=10Hz,1H),6.61(s,1H), 4.58(s,3H),3.53–3.48(m,4H),3.10–3.07(m,2H),3.01–2.98(m,2H),1.98–1.95(m,2H),1.16(t,J=6.8Hz,6H); 13 C NMR (100MHz, CDCl3) δ: 161.9, 161.3, 156.9, 152.1, 146.5, 146.0, 139.9, 138.5, 135.1, 134.7, 131.3, 131.2, 129.8, 126.6, 125.7, 119.7, 114.1, 110.0, 108.8, 105.0, 97.0, 46.7, 45.2, 29.3, 27.2, 20.9, 12.6. MS (ESI): Calculated C 28 H 28 ClN2O2[M-ClO4] + 459.1834, discovered 459.1853.

[0031] Example 3

[0032] Fluorescence spectroscopy of the probe against different biothiols: A 10 μmol / L test sample was obtained by adding the probe to a DMSO / PBS (pH 7.4, 20 mmol / L, v / v 1:1) aqueous solution. Different concentrations of Cys / Hcy aqueous solutions were then added. After equilibration, the fluorescence emission spectra were measured. The results are shown below. Figure 2 Its fluorescence intensity at 674 nm varies at low concentrations of 1-9 μmol / L Cys / Hcy, as shown in the figure. Figure 3 .

[0033] Depend on Figure 2 It was observed that with the gradual addition of Cys / Hcy, a new fluorescence peak appeared at 674 nm, which significantly increased and reached saturation at a Cys concentration of 30 μmol / L, with a 20-fold increase in fluorescence. Hcy exhibited a similar spectral response. This indicates that the probe can distinguish and detect Cys / Hcy in glutathione with a large Stokes shift (approximately 200 nm) in near-infrared fluorescence, and can also quantitatively detect it.

[0034] Example 4

[0035] Response time test of probe to Cys / Hcy: The probe was added to DMSO / PBS (pH 7.4, 20 mmol / L, v / v 1:1) to prepare a 10 μmol / L solution. Then, 30 μmol / L GSH, Hcy, and Cys were added, and the fluorescence spectra at different times were recorded. Figure 4 As shown.

[0036] Depend on Figure 4 The fluorescence intensity of the probe at 674 nm increased rapidly with time for both Cys and Hcy, reaching its maximum within 3 and 15 minutes, respectively. Conversely, the fluorescence response of the probe to GSH was very slight. These results indicate that the probe exhibits a rapid response time to Hcy / Cys.

[0037] Example 5

[0038] The probe was used to investigate the response mechanism of Cys / Hcy. The probe was added to a DMSO / PBS (pH 7.4, 20 mmol / L, v / v 1:1) test solution to prepare a 10 μmol / L solution. Then, 30 μmol / L Cys, Hcy, and GSH were added. After the response reached equilibrium, the mass spectrometry (ESI-MS) of the solution was measured. Figure 5 , 6 As shown in Figure 7.

[0039] Depend on Figure 5-7 As is known, the inferred response mechanism is as follows: Figure 1 As shown, the probe reacts with Cys / Hcy to yield an S-substituted product, but the relatively close spatial distance between the amine and thiol groups leads to further intramolecular substitution, generating an N-substituted product. Conversely, GSH only allows the probe to undergo thiol substitution, yielding an S-substituted product. Compared to chlorine atoms, the stronger electron-donating nature of the N-substituent results in a significant enhancement of fluorescence in the near-infrared region, while the S-substituent... - The substitution product provides a weak fluorescence signal enhancement, enabling the probe to distinguish Cys / Hcy and GSH in near-infrared fluorescence.

[0040] Example 6

[0041] Selectivity test of probe for Hcy / Cys: Various interfering substances, such as Cys, Hcy, GSH, Na2S, and Br2, were added to the probe (10 μmol / L) test solution in DMSO / PBS (pH 7.4, 20 mmol / L, v / v 1:1). - Cl - H2O2, ClO - I - NO3 - Mg 2+ SO32- S2O3 2- NO2 - Leu, Phe, Pro, Ser, Tgr, Thr, Trp, Arg, His, Ala, Gln, Glu, Gly, Asn, were tested for changes in fluorescence spectra.

[0042] Depend on Figure 8 It was found that many related analytes, including amino acids, anions, metal ions, sulfur species, HClO, and H2O2, did not cause any significant fluorescence changes; only Cys / Hcy showed a significant fluorescence change. This indicates that the probe has high detection selectivity for Cys / Hcy.

[0043] Example 7

[0044] The effect of system pH on probe detection: The changes in autofluorescence emission of the probe (10 μmol / L) in buffer solutions / DMSO test solutions at different pH values ​​and the changes in fluorescence emission after the addition of Cys were measured. Figure 9 As shown.

[0045] As shown in the figure, the probe exhibits almost no fluorescence within a pH range of 3.0 to 9.0. However, the addition of Cys / Hcy within a pH range of 6.0 to 8.0 results in a significant fluorescence response. Therefore, the probe can effectively monitor Cys / Hcy at physiological pH levels.

[0046] Example 8

[0047] Specific targeting of mitochondria by fluorescent probe: A549 cells were cultured with the probe (10 μmol / L) and MitoTracker Green FM (5 μmol / L) for 30 minutes, followed by three washes with PBS and confocal (Olympus FV3000) cell imaging. Under 488 nm excitation, the green channel signal at 500–580 nm and the red channel signal at 650–750 nm were collected, respectively.

[0048] Depend on Figure 10 It can be seen that due to the presence of endogenous Cys / Hcy in the cell, the red channel has obvious fluorescence signal and good overlap with the green channel signal. The Pearson colocalization coefficient is 0.91, indicating that the probe can specifically target the mitochondria in the cell.

[0049] Example 9

[0050] Fluorescence imaging assays of the probe in A549 cells: Cell imaging of the probe was performed using a confocal fluorescence microscope. A549 cells were seeded in 35 nm glass-bottomed culture dishes for 24 hours. To image endogenous Cys / Hcy, after removing the old cell culture medium, the cells were further incubated for 30 minutes in fresh medium containing the probe (10 μmol / L) without FBS. The cell culture dishes were then washed three times with PBS buffer. The red channel signal was collected from 650–750 nm under excitation at 488 nm. To remove intracellular biothiols, the cells were pretreated with N-ethylmaleic anhydride (NEM, 0.5 mmol / L) for 30 minutes and further cultured with the probe (10 μmol / L) for 30 minutes. To image exogenous biothiols, the cells were pre-impregnated with NEM (0.5 mmol / L) for 30 minutes, then incubated with the probe (10 μmol / L) for 30 minutes, and finally cultured with Cys / Hcy / GSH (100 μmol / L) for 30 minutes.

[0051] Depend on Figure 11 It can be seen that the probe has a weak fluorescent signal in the cell, which is some endogenous Cys / Hcy in the cell. After the cells are treated with the thiol scavenger NEM, the fluorescent signal is almost non-existent. However, when Cys / Hcy / GSH is added, only Cys and Hcy cells have obvious red fluorescent signals. These results indicate that the probe can detect endogenous and exogenous Cys / Hcy in living cells by near-infrared fluorescence imaging.

Claims

1. A near-infrared fluorescent probe with fast and sensitive detection of biological thiols with targeting mitochondria and large Stokes shift, characterized in that, The structural formula of the near-infrared fluorescent probe is as follows: 。 2. A method for preparing a near-infrared fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) 9-chlorotetrahydroacridine 1 was reacted with iodomethane in sulfolane and then post-treated to obtain salt compound 2; (2) Salt compound 2 and compound 3 were condensed in acetic anhydride, and after the reaction was completed, a near-infrared ratio fluorescent probe was obtained by post-treatment. The reaction formula is as follows: 。 3. The application of the near-infrared fluorescence probe as described in claim 1 in the preparation of reagents for the detection and imaging of biothiols, characterized in that, The biothiols mentioned are cysteine ​​(Cys) and homocysteine ​​(Hcy); The imaging reagent is used for targeted fluorescence imaging within mitochondria in cells, and the imaging reagent comprises the near-infrared fluorescent probe and the commercially available MitoTracker Green dye; The specific method is as follows: The cells to be tested are incubated with a near-infrared fluorescent probe and commercial MitoTracker Green dye, then washed three times with PBS, and confocal cell imaging is performed. During confocal cell imaging, the excitation wavelength is 488 nm, the green channel collection wavelength is 500-580 nm, and the red channel collection wavelength is 650-750 nm.

4. The application of the near-infrared fluorescent probe according to claim 3 in the preparation of reagents for the detection and imaging of biothiols, characterized in that, The near-infrared fluorescent probe is used for the qualitative detection of biothiols, and the specific method is as follows: The near-infrared fluorescent probe was prepared into a test solution, and then the sample to be tested was added. Under 480 nm excitation, the change in fluorescence emission intensity at 674 nm was measured. If the fluorescence intensity at 674 nm increased, it was determined that the sample to be tested contained cysteine ​​(Cys) and homocysteine ​​(Hcy).

5. The application of the near-infrared fluorescent probe according to claim 3 in the preparation of reagents for the detection and imaging of biothiols, characterized in that, The near-infrared fluorescent probe is used for the quantitative detection of biothiols. The specific method is as follows: the near-infrared fluorescent probe is prepared into a test solution, and then the sample to be tested is added. Under 488nm excitation, the fluorescence emission intensity at 674nm is measured to obtain the fluorescence change value before and after adding the test solution. Then, the fluorescence change value is compared with the standard curve to obtain the concentration of cysteine ​​(Cys) and homocysteine ​​(Hcy) in the test solution.

6. The application of the near-infrared fluorescent probe according to claim 5 in the preparation of reagents for the detection and imaging of biothiols, characterized in that, The test solution is prepared as follows: The fluorescent probe was added to a DMSO / PBS buffer solution to prepare a test solution with a concentration of 10 μmol / L. The buffer solution has a pH of 7.4, and the volume ratio of DMSO to PBS solution is 1:

1.

7. The application of the near-infrared fluorescent probe according to claim 3 or 4 in the preparation of reagents for the detection and imaging of biothiols, characterized in that, The imaging reagent contains the fluorescent probe, and the specific method is as follows: the cells to be tested are incubated with a solution containing the near-infrared fluorescent probe, then washed three times with PBS, and confocal cell imaging detection is performed; During confocal cell imaging, the excitation wavelength is 488 nm, and the collection wavelength for the red channel is 650-750 nm.

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