Near-infrared fluorescent probe for detecting nadh in cells and preparation and application thereof

Abstract

The application discloses a kind of for detecting NADH near-infrared fluorescent probe QB, its molecular structure is as shown in formula (I). Probe QB can be opened fluorescent signal in near-infrared (em:686nm), and significantly increase with the increase of NADH concentration. Its fluorescence intensity and NADH concentration are linear in a certain range, the QB has significant selectivity, fast response time (6min), high sensitivity (detection limit is 43nM) and the like superior performance. The QB probe has near-infrared fluorescent response, can quickly and sensitively detect NADH. The probe not only has lower biological toxicity and excellent mitochondrial targeting ability, highly selective monitoring intracellular endogenous and exogenous NADH, but also can distinguish normal cells and tumor cells. In addition, the probe can be used for imaging of diabetic cell model and alzheimer cell model, and can visualize the fluctuation of NADH level.

Description

Technical Field

[0001] This invention relates to the preparation and application of a novel fluorescent dye with near-infrared mitochondrial targeting, based on quinoline conjugated benzo[cd]indole double salt for rapid detection of NADH in cells. Specifically, it relates to the construction of a highly selective and highly sensitive fluorescent probe for detecting NADH in diabetic, Alzheimer's disease cells and tumor cells, and its bioimaging application, belonging to the field of organic fluorescence sensing technology. Background Technology

[0002] NADH is nicotinamide adenine dinucleotide (NAD) + NADH, in its reduced form, acts as a key reducing coenzyme in organisms. This important cofactor plays a multifaceted role in various redox processes, including energy metabolism, mitochondrial function, immune responses, calcium homeostasis, and apoptosis. Primarily located in mitochondria, NADH promotes energy transfer through oxidative phosphorylation, making it a key marker of mitochondrial energy production and often referred to as a mitochondrial hormone. Notably, diseases such as diabetes, Parkinson's disease, and cancer are significantly associated with mitochondrial NADH levels. Therefore, assessing NADH levels can enhance our understanding of disease progression and underlying pathological mechanisms.

[0003] Currently, fluorescent probes for NADH detection have attracted considerable attention due to their high sensitivity, excellent selectivity, and ease of visualization in cells or organisms. Many NADH fluorescent probes are designed based on four recognition groups: quinoline, pyridine, ralzazulin, and quinone. Given that NADH is primarily found in mitochondria, the development of mitochondrial-targeted fluorescent probes holds greater promise for bioimaging. Most reported mitochondrial NADH detection probes primarily utilize 1-methylquinoline salt and another electron acceptor to construct a receptor-π-receptor (A-π-A). The quinoline salt component readily reacts with NADH to form 1,4-dihydroquinoline. Therefore, the probe transforms into a donor-π-receptor (D-π-a) structure, initiating an intramolecular charge transfer (ICT) process and triggering fluorescence emission. Utilizing this mechanism, we summarized and analyzed related fluorescent probes. For example, the Fomin group (AcsSens. 2016.1. 702-709) successfully designed the probe Indicator1 for NADH detection, which exhibits a rapid response to NADH (5 min), but the maximum emission peak only emits a fluorescent signal in the visible region (E). max(Emax: 560 nm). Furthermore, Liang's group (Sens. Actuators B Chem. 2022. 373. 132694.) synthesized the probe Rh-QL, which, although responsive to NADH in the near-infrared (Emax: 750 nm) region, had a reaction time exceeding 30 min or longer. Therefore, developing a rapidly responsive (within 10 min) mitochondrial-targeting NIR probe for NADH remains a significant challenge. Summary of the Invention

[0004] This invention provides a novel fluorescent probe QB with a rapid and specific near-infrared response, based on quinoline conjugated benzo[cd]indole double salt for detecting NADH in cells, its preparation method, and its fluorescence detection technology for detecting NADH in cells.

[0005] The chemical structure of the novel fluorescent probe QB obtained in this invention is shown in formula (I):

[0006]

[0007] The novel fluorescent probe QB of this invention utilizes benzo[cd]indole as an electron acceptor in a 1-methylquinoline acceptor-π-acceptor (A-π-A) system, increasing the induction of quinoline salts by the electron acceptor and accelerating their response to NADH. Furthermore, due to the strong electron absorption capacity of benzo[cd]indole, it enhances electron conjugation density, enabling the activation of near-infrared fluorescence signals. This probe allows for highly selective and sensitive detection and differentiation of NADH. As described above, the probe is formed by an ethylene linker connecting the 1-methylquinoline moiety and benzo[cd]indole, creating an acceptor-π-acceptor (A-π-A) structure.

[0008] This invention also provides a method for preparing a novel fluorescent probe QB for detecting NADH in cells, which has a rapid and specific near-infrared mitochondrial targeting response and is based on quinoline conjugated benzo[cd]indole double salt, comprising the following steps:

[0009] (1) 1-Methylquinolinaldehyde (compound 1) and 1-ethyl-2-methylbenzo[cd]indole-1-iodide ammonium (compound 2) were condensed in acetic acid. After the reaction was completed, compound 3 was obtained by post-treatment.

[0010] (2) Compound 3 was methylated with methyl trifluoromethanesulfonate in DCM, and the probe QB was obtained after post-processing after the reaction.

[0011] The preparation chemical reaction formula is as follows:

[0012]

[0013] This invention also provides a novel fluorescent probe QB with an ultra-large Stokes shift, based on quinoline conjugated benzo[cd]indole double salt for detecting NADH in cells, and its application in spectral response to NADH and cell imaging.

[0014] The novel near-infrared fluorescent mitochondrial targeting probe QB based on quinoline conjugated benzo[cd]indole double salt for detecting NADH in cells, as described in this invention, produces an on-off fluorescence response upon the addition of NADH to PBS (20mM, v / v, pH 7.4). Figure 3 As shown, under 630 nm excitation, the addition of NADH resulted in strong red fluorescence at 686 nm, which increased with increasing NADH concentration, reaching saturation at 10 μM. Furthermore, as... Figure 4 As shown, emission intensity (F) 686nm The detection limit (LOD) showed a good linear relationship with (1-5 μM) NADH. According to the formula LOD = 3σ / k, the detection limit (LOD) was calculated to be 43 nM, indicating that the probe QB can sensitively detect NADH.

[0015] The novel near-infrared fluorescent probe QB described in this invention, such as Figure 5 As shown, under excitation at 630 nm, the fluorescence intensity at 686 nm gradually increased over time after the addition of 10 μM NADH, reaching a peak at about 6 minutes, indicating that the probe QB has a rapid response to NADH.

[0016] The novel fluorescent probe QB based on quinoline conjugated benzo[cd]indole double salt for detecting NADH in cells, as described in this invention, exhibits high selectivity and strong anti-interference properties. Different interfering substances (reducing agents such as H2S and SO3) were added to PBS (20 mM, v / v, pH 7.4). 2- HSO3 - ); anion (CO3) 2- SO4 2- CI - ,OH - ); Metal ions (Mg 2+ Cu 2+ ,Fe 3+ ,K + Cd 2+ The selectivity and interference of amino acids (Leu, Prs, Thr, Arg, Gly, Ser, Tyr, GSH, Hcy, Cys) were detected. Figure 6 As shown, NADH caused a significant increase in the fluorescence intensity of the probe, while other interfering substances did not cause significant changes in fluorescence intensity, indicating that QB has significant selectivity for NADH and strong anti-interference ability.

[0017] This invention relates to exogenous fluorescence imaging using a novel fluorescent probe, QB, based on quinoline conjugated benzo[cd]indole double salts for detecting NADH in cells. Cells were pretreated with the probe (QB) alone as a control group, followed by incubation of other cells with different concentrations of NADH. Figure 7 As shown, compared to the control group, cells exhibited a strong red near-infrared fluorescence signal in the presence of NADH. Notably, even low concentrations of NADH (10 μM) were detectable. Figure 10 (b) This demonstrates the sensitivity of QB to NADH in cells. These findings suggest that the probe QB can specifically detect exogenous NADH in vivo.

[0018] This invention relates to the intrinsic fluorescence imaging of QB, a novel fluorescent probe based on quinoline conjugated benzo[cd]indole double salt for detecting NADH in cells. For example... Figure 8 As shown, compared to the control group containing only the QB probe, a significantly enhanced fluorescence signal was observed in the red channel after incubation with the probe following glucose stimulation. This observation is consistent with the results observed after the addition of NADH, indicating that glucose induces cellular oxidative phosphorylation, leading to an increase in intracellular NADH levels. Pyruvate is known to inhibit the decrease in intracellular NADH levels; pretreatment of cells with pyruvate resulted in a significant decrease in fluorescence signal upon incubation with the probe. These data confirm that the QB probe can visualize the detection of endogenous NADH. Therefore, the QB probe can effectively visualize and detect fluctuations in intracellular NADH levels.

[0019] This invention relates to the fluorescence imaging of a diabetic cell model using a novel fluorescent probe, QB, based on quinoline conjugated benzo[cd]indole double salt to detect NADH in cells. Type II diabetes is associated with insufficient insulin secretion, and its insulin resistance is intrinsically linked to oxidative stress; excessive reactive oxygen species (ROS) can lead to mitochondrial damage. + The imbalance in NADH redox kinetics contributes to the accumulation of this ROS, providing a potential visualization method for monitoring diabetes progression.

[0020] DEX induced insulin resistance in A549 cells, which were then treated with a specific probe. For example... Figure 9 As shown in b and 9c, DEX-treated cells exhibited significantly enhanced fluorescence signals compared to the control group, indicating that the QB probe effectively detected NADH levels in diabetic cells. Furthermore, as Figure 10 As shown in Figure d, the fluorescence intensity in diabetic cells was significantly reduced after treatment with metformin (a commonly used drug for treating type 2 diabetes). These findings confirm that the probe QB provides a reliable method for monitoring changes in NADH levels within diabetic cells.

[0021] This invention relates to the novel fluorescent probe QB, based on quinoline conjugated benzo[cd]indole double salt, for detecting NADH in cells, and its use in fluorescence imaging of an Alzheimer's disease cell model. Metal ions (Fe...) 2+ Cu 2+ This causes the destruction of metal ions in the body, leading to oxidative stress, which is closely related to the occurrence of Alzheimer's disease (AD). Therefore, by adding different concentrations of Cu to PC-12 cells... 2+ To construct an AD cell model. For example... Figure 10 As shown in b-10d, the fluorescence intensity increases with Cu 2+ The concentration gradually increases with increasing Cu, indicating that NADH is present in Cu. 2+ This NADH is produced during induced oxidative stress. The addition of urea (UA) significantly reduced the red fluorescence signal in PC-12 cells, indicating an effective reduction in intracellular NADH levels. In conclusion, the QB probe can detect changes in NADH levels in an AD model.

[0022] The beneficial effects of this invention are as follows: a novel fluorescent probe for rapid detection of NADH in cells using near-infrared fluorescence. The probe is based on a quinoline conjugated benzo[cd]indole disalt, which reacts with NADH, achieving a near-infrared fluorescence response within 10 minutes, exhibiting high selectivity and a low detection limit (43 nM). The probe QB can target mitochondria and image both endogenous and exogenous NADH in cells. Furthermore, the probe QB has the ability to distinguish between normal cells and tumor cells. The probe can also be used for tracking and imaging NADH in diabetic cells and Alzheimer's disease cells. Attached Figure Description

[0023] Figure 1 This invention presents the synthetic route for a novel near-infrared fluorescent probe, QB, based on quinoline conjugated benzo[cd]indole double salt for detecting NADH in cells.

[0024] Figure 2 This invention relates to a novel fluorescent probe for detecting NADH in cells based on quinoline conjugated benzo[cd]indole double salts. It describes the response sensing mechanism of the novel near-infrared fluorescent probe QB to NADH in cells.

[0025] Figure 3 This invention presents the fluorescence emission changes of the novel near-infrared fluorescent probe QB in cells, based on quinoline conjugated benzo[cd]indole double salt, after being treated with different concentrations of NADH.

[0026] Figure 4 This is the LOD diagram of the novel near-infrared fluorescent probe QB in cells, based on the quinoline conjugated benzo[cd]indole double salt for detecting NADH in cells.

[0027] Figure 5 This invention presents a novel fluorescent probe based on quinoline conjugated benzo[cd]indole double salt for detecting NADH in cells. The novel near-infrared fluorescent probe QB in cells is shown as a time-dependent fluorescence emission spectrum after being treated with 10 μM NADH.

[0028] Figure 6 This invention presents the fluorescence spectrum of the novel near-infrared fluorescent probe QB for detecting NADH in cells based on quinoline conjugated benzo[cd]indole double salt, after interaction with different species.

[0029] Figure 7 This invention presents a novel near-infrared fluorescent probe, QB, based on quinoline conjugated benzo[cd]indole double salt, for detecting NADH in cells. The fluorescence image of exogenous NADH in HeLa cells is shown.

[0030] Figure 8 This invention presents a novel near-infrared fluorescent probe, QB, based on quinoline conjugated benzo[cd]indole double salt, for detecting NADH in cells. The fluorescence image of endogenous NADH in HeLa cells is shown.

[0031] Figure 9 This invention relates to a novel near-infrared fluorescent probe, QB, based on quinoline conjugated benzo[cd]indole double salt for detecting NADH in cells, and its fluorescence imaging of NADH in diabetic cells.

[0032] Figure 10 This invention relates to a novel near-infrared fluorescent probe (QB) for detecting NADH in cells based on quinoline conjugated benzo[cd]indole double salts, and to fluorescence imaging of NADH in Alzheimer's disease cells. Detailed Implementation

[0033] Example 1

[0034] 1-Methylquinolinaldehyde (compound 1) (200 mg, 1.27 mmol) and 1-ethyl-2-methylbenzo[cd]indole-1-iodide (compound 2) (200 mg, 1.27 mmol) were dissolved in 6 mL of CH3COOH. Triethylamine (0.6 mL) and acetic anhydride (0.6 mL) were added to the mixture. The mixture was stirred at 60 °C for 1 hour. After cooling the reaction mixture to room temperature, 10 mL of anhydrous ether was added dropwise. The mixture was filtered, the solid was collected, and washed with ethyl acetate to give compound 2 (320 mg, 91%). 1H NMR(400MHz,d6-DMSO),δ:9.69(s,1H),9.45(s,1H),9.21(s,1H),8.98(d,J =15.6Hz,1H),8.80(s,1H),8.51(s,1H),8.45(s,1H),8.33(d,J=15.6Hz,1H ),8.23(s,1H),8.12(d,J=8Hz,2H),8.02(s,1H),7.92(s,1H),7.75(t,J=7.2Hz,1H),4.97(d,J=4.8Hz,2H),1.59(t,J=6.8Hz,3H).MS(ESI):calculated for C 24 H 19 N2 + [M] + 335.1543, found 335.1538.

[0035] Compound 3 (120 mg, 0.26 mmol) and methyl trifluoromethanesulfonate (127 mg, 0.78 mmol) were dissolved in 10 mL of CH₂Cl₂. The reaction mixture was stirred at 25 °C for 48 hours. After the reaction was complete, the solvent was removed by filtration, and the solid was washed with petroleum ether, ether, and dichloromethane, respectively. The desired probe QB (150 mg, 92%) was obtained. 1 H NMR (400MHz, CD3OD), δ: 10.12 (s, 1H), 9.85 (s, 1H), 9.34 (d, J = 6.8Hz, 1H), 8.93-8.89(m,1H),8.79(d,J=8.8Hz,1H),8.61-8.55(m,2H),8.51-8.46(m ,2H),8.41-8.38(m,2H),8.25(t,J=7.2Hz,1H),8.16(t,J=6.8Hz,1H),8.0 5(t,J=8Hz,1H),5.01(t,J=7.2Hz,2H),4.84(s,3H),1.76(t,J=7.2Hz,3H). 13 C NMR(100MHz,CD3OD)exhibited peaks atδ:161.3,146.6,146.0,145.6,145.3,138.9,138.6,138.5,137.3,136.2,132.3,132.2,130.1,12 9.3,129.2,128.8,127.3,123.3,122.0,121.2,121.1,118.8,45.4,42.4,15.0.MS(ESI):calculated forC 25 H22 N 22 + [M] + 175.0886, found 175.0882.

[0036] Example 2

[0037] Spectral changes of probe QB with different concentrations of NADH: The probe was added to PBS (pH 7.4, 20 mM, v / v) test solution to prepare a 10 μmol / L solution. Then, different concentrations of NADH aqueous solutions were added. After equilibration, the fluorescence emission spectra were measured. The results are shown in the figure. Figure 3 .

[0038] from Figure 3 It was found that under 630 nm excitation, the addition of NADH resulted in strong red fluorescence at 686 nm, which stabilized with increasing NADH concentration, reaching saturation at 10 μM. This indicates that the probe has a significant response to NADH.

[0039] Example 3

[0040] The probe was added to PBS (pH 7.4, 20 mM, v / v) test solution to prepare a 10 μmol / L solution, and then 10 μmol / L NADH was added. Fluorescence spectra at different times were recorded, such as... Figure 5 As shown.

[0041] The result is Figure 5 It can be seen that after adding 10 μM NADH, under excitation at 630 nm, the fluorescence intensity at 686 nm gradually increases over time and reaches a peak at about 6 minutes, indicating that the probe QB responds rapidly to NADH.

[0042] Example 4

[0043] Selectivity of the novel quinoline conjugated benzo[cd]indole dual salt fluorescent probe QB was tested: The probe was added to PBS (pH 7.4, 20 mM, v / v) to prepare a 10 μmol / L solution, and then various interfering substances (reducing agents such as H2S and SO3) were added. 2- HSO 3- ); anion (CO3) 2- SO4 2- CI - ,OH - ); Metal ions (Mg 2+ Cu 2+ ,Fe 3 + ,K + Cd 2+The fluorescence intensity of amino acids (Leu, Prs, Thr, Arg, Gly, Ser, Tyr, GSH, Hcy, Cys) was measured under 630 nm excitation.

[0044] Depend on Figure 6 It can be seen that the fluorescence intensity of the probe only changes significantly with NADH, while the effect on other substances is very weak, indicating that the selectivity of the probe for NADH is higher than that for other substances.

[0045] Example 5

[0046] Intracellular fluorescence imaging assay: HeLa cells, A549 cells, HepG2 cells, and 3T3 cells were cultured in DMEM containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) and incubated at 37°C in a 5% CO2 atmosphere. First, cells were seeded in confocal culture dishes for 24 hours to allow cells to multiply at a density of 5 × 10⁶ cells per dish. 4 Cell density adhesion was then assessed. Probes were prepared by obtaining a DMSO solution, which was added to the adherent cells, ensuring a final probe concentration of 10 μM. A series of experiments were conducted to verify the imaging capability of the probe after reaction with NADH. In the exogenous experiments, different amounts of NADH were incubated for 30 minutes. Figure 7 As shown, the fluorescence intensity increased significantly with increasing NADH concentration, indicating that the probe can detect exogenous NADH. According to previous studies, glucose induces NADH production in cells, while pyruvate inhibits NADH production via lactate dehydrogenase. Therefore, HeLa cells were treated with 20 mM glucose or 5 mM pyruvate in endogenous form for 30 min, followed by incubation with probe QB (10 μM) for 30 min. Figure 8 As shown, consistent with predictions, glucose induces an increase in NADH in cells, while pyruvate decreases NADH concentration. These results further confirm the probe's superior ability to selectively detect both endogenous and exogenous NADH.

[0047] Example 6

[0048] Probe-based detection of a diabetic cell model: Insulin resistance was induced in A549 cells using different concentrations of DEX, followed by treatment with the probe QB for 30 min. Figure 9 As shown in b and 9c, DEX-treated cells exhibited significantly enhanced fluorescence signals compared to the control group, indicating that the QB probe effectively detected NADH levels in diabetic cells. Furthermore, as Figure 10As shown in Figure d, the fluorescence intensity in diabetic cells was significantly reduced after treatment with metformin (a commonly used drug for treating type 2 diabetes). These findings confirm that the QB probe provides a reliable means to monitor changes in NADH levels within diabetic cells.

[0049] Probe detection of Alzheimer's disease cell model test: using metal ions (Fe) 2+ Cu 2+ Cu ions cause the destruction of metal ions within PC-12 cells, leading to oxidative stress, which is closely related to the development of Alzheimer's disease. Adding different concentrations of Cu to PC-12 cells... 2+ To construct an AD cell model. For example... Figure 10 As shown in b-10d, the fluorescence intensity increases with Cu 2+ The concentration gradually increases with increasing Cu, indicating that NADH is present in Cu. 2+ This NADH is produced during induced oxidative stress. The addition of urea (UA) significantly reduced the red fluorescence signal in PC-12 cells, indicating an effective reduction in intracellular NADH levels. In conclusion, the QB probe can detect changes in NADH levels in an AD model.

Claims

1. A near-infrared fluorescent probe QB for detecting NADH, characterized in that, The structure of the fluorescent probe is as follows: 。 2. A method for preparing the near-infrared fluorescent probe QB according to claim 1, characterized by, The method comprises the following steps: (1) condensation reaction of 1-ethyl-2-methylbenzo[c, d] indole-1-iodide and 1-methylquinoline aldehyde in acetic acid, after the reaction is completed, post-treatment is performed to obtain compound 3; (2) substitution reaction of compound 3 obtained in step (1) and methyl triflate in DCM, after the reaction is completed, post-treatment is performed to obtain the near-infrared fluorescent probe QB; The reaction formula is as follows: 。 3. The application of the near-infrared fluorescent probe QB in the preparation of an NADH detection reagent.

4. Use according to claim 3, characterized in that, The NADH detection reagent is used for NADH spectral response and cell imaging.

5. Use according to claim 3, characterized in that, The NADH detection reagent is used for detecting NADH in tumor cells, type 2 diabetes cells and Alzheimer's disease cells.

6. Use according to claim 3, characterized in that, The NADH detection reagent is used for qualitative detection of NADH, and the specific method is as follows: The near-infrared fluorescent probe QB is configured into a test solution, then the sample to be detected is added, under excitation at 630 nm, the change in fluorescence emission intensity at 686 nm is detected, if the fluorescence at 686 nm is stronger, it is determined that the sample to be detected contains NADH.

7. Use according to claim 6, characterized in that, The preparation method of the test solution is as follows: (1) the near-infrared fluorescent probe QB is added to DMSO to configure a QB solution with a concentration of 2.5 mmol / L; (2) 10 μL of the QB solution is added to 2.5 ml of a PBS buffer solution to configure a test solution with a concentration of 10 μmol / L; The concentration of the PBS buffer solution is 20 mM, and the pH is 7.

4.

8. Use according to claim 3, characterized in that, The fluorescent probe is used for intracellular fluorescence imaging test, and the specific method is as follows: the cell to be detected is incubated with fresh DMEM-free medium containing the near-infrared fluorescent probe QB, then washed with PBS for three times, and confocal cell imaging detection is performed; During the confocal cell imaging detection, the red channel: excitation wavelength is 640 nm, and the collection wavelength is 650-750 nm.

Images