A ratio fluorescent probe for accurate diagnosis of liver fibrosis by dual detection of nitric oxide and viscosity

Abstract

The present application relates to a kind of double detection nitric oxide and viscosity accurate diagnosis of liver fibrosis ratio type fluorescent probe, belong to fluorescent probe field.Its molecular structure is as follows: the fluorescent probe molecule presents red fluorescence, and after complete response with NO, the red fluorescence at 715nm is weakened until disappear, emits intense orange fluorescence, the fluorescence intensity ratio (I 625nm / I 715nm ) of probe BDP with NO concentration has good linear relationship;High viscosity leads to the significant fluorescence enhancement of probe before and after response NO, realizes the effect of simultaneous response.The probe molecule described in the present application can not only qualitatively analyze NO and viscosity, but also can realize rapid, quantitative detection NO and viscosity, has important application value in the field of biochemistry molecular analysis.

Description

[0001] The present application relates to a ratiometric fluorescent probe BDP for dual detection of nitric oxide and viscosity for accurate diagnosis of liver fibrosis, comprising synthetic steps, fluorescence spectrum test and application in the field of biological imaging, and belongs to the field of fluorescent probes. BACKGROUND

[0002] Liver fibrosis (HF) is a stage in the progression of chronic liver disease to cirrhosis. In chronic liver disease, there is an imbalance between fibroplasia and catabolism in the liver parenchyma, resulting in fibrous connective tissue to treat liver damage. However, persistent chronic inflammation and necrosis of the liver parenchyma lead to excessive accumulation of fibrous connective tissue, resulting in liver fibrosis, and eventually leading to heart failure. Therefore, heart failure is an extremely complex dynamic process. Due to the lack of characteristic clinical manifestations, there is currently no specific tool for diagnosing HF except liver biopsy. Studies have shown that it is possible to prevent HF from developing into cirrhosis at an early stage. Therefore, accurate diagnosis of heart failure is crucial for controlling the progression of liver disease and treatment.

[0003] Studies have shown that the levels of NO and inducible nitric oxide synthase (iNOS) in liver tissue of HF patients are much higher than those in healthy liver tissue. In addition, a significant increase in viscosity is observed in fibrous hepatocytes, which is the cause of HF progression. Since NO overexpression or viscosity increase is found in other liver diseases, detecting either of them alone can lead to false diagnosis. Therefore, simultaneous detection of NO and viscosity in liver tissue helps to accurately diagnose HF at an early stage, and also helps to further understand the in vivo mechanism of HF.

[0004] Fluorescent probes have flourished in the past decade due to their high sensitivity, fast response, and non-invasive detection. Compared with turn-on fluorescent probes, ratiometric measurement probes are more desirable because of their high accuracy. In addition, fluorescent probes with long-wavelength spectra are preferred because long-wavelength light can penetrate biological tissues deeply. Notably, multi-factor responsive probes are more accurate and reliable than single-factor responsive probes because the former can avoid false signals generated by the expression of one factor in multiple diseases. In recent years, many fluorescent probes have been developed for detecting NO or viscosity, respectively. So far, only one fluorescent probe has been used to detect NO and viscosity, which shows turn-on fluorescent response of NO (green signal) and viscosity (blue signal) in cells. In fact, no matter how viscous the medium is, the probe only emits green fluorescence, and cannot monitor NO and viscosity simultaneously. Therefore, it is of great significance to develop a long-wavelength, quantitative fluorescent probe to effectively detect NO and viscosity for accurate diagnosis of HF.

[0005] In the present patent, the ratio fluorescent probe BDP is constructed to detect overexpressed NO and high viscosity for HF diagnosis. The electron-rich aromatic secondary amine in the molecule is regarded as the NO reaction group, which adjusts the push-pull system to perform ratio imaging by N-nitrosylation. It is assumed that the C=C bond between the N-methylaniline and the BODIPY moiety rotates freely under low viscosity, thus showing low fluorescence intensity of the probe. The high viscous environment of fibrotic liver restricts intramolecular rotation, thus enhancing the fluorescence signal. NO can react with the probe BDP, leading to a rapid transition from secondary amine to N-nitrosylation, accompanied by a change in fluorescence from red to orange. High viscosity leads to a significant fluorescence enhancement of the probe before and after the response to NO. By monitoring the changes in NO concentration and viscosity enhancement, it is expected that the probe BDP will be a promising tool for HF detection. SUMMARY

[0006] The present application aims to develop a ratio fluorescent probe for dual detection of nitric oxide and viscosity for accurate diagnosis of liver fibrosis, which solves the technical problem that the existing ratio fluorescent probe cannot simultaneously distinguish and detect active species NO and viscosity. The fluorescent probe BDP has the following structural formula:

[0007]

[0008] The synthesis route is as follows:

[0009] (a) Compound 1 and compound 2 are dissolved in anhydrous toluene, then acetic acid is added to the mixture, piperidine is used as a base catalyst, heated to reflux at 120℃ for 5h, then the mixture is cooled to room temperature, extracted with dichloromethane, dried with anhydrous sodium sulfate, and rotary evaporated under reduced pressure to obtain the crude product, which is purified by column chromatography to obtain compound BDP. The technical route is as follows:

[0010]

[0011] In step (a), the molar ratio of 5,5-difluoro-1,3,7,9-tetramethyl-10-phenyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine and 4-(methylamino)benzaldehyde is 1:1.625.

[0012] In step (a), the eluent used in column chromatography is (V petroleum ether:V ethyl acetate = 3 / 1).

[0013] The test method of the fluorescent probe of the present application is as follows: the probe molecule is dissolved in DMSO / PBS (3:7, v / v, 10mM, pH=7.40) and tested at room temperature. NO and viscosity can be qualitatively and quantitatively detected, and the specific implementation method is described in detail in the implementation examples.

[0014] The response mechanism of the fluorescent probe of the present application is as follows: the electron-rich aromatic secondary amine of the probe BDP is regarded as the NO reaction group, which regulates the push-pull electron system to perform ratio imaging by N-nitrosylation. It is assumed that the C=C bond between the N-methylaniline and the BODIPY moiety is free to rotate at low viscosity, thus showing low fluorescence intensity of the probe. The high viscous environment of the fibrotic liver restricts intramolecular rotation, thus enhancing the fluorescence signal. NO can react with the probe BDP, leading to a rapid transition from the secondary amine to N-nitrosylation, accompanied by a change in fluorescence from red to orange. High viscosity leads to a significant fluorescence enhancement before and after the probe responds to NO. Simultaneous detection of NO and viscosity can be achieved.

[0015] The fluorescent probe parent of the present application presents red fluorescence, and after complete response to NO, the red fluorescence at 715 nm is weakened until disappeared, and intense orange fluorescence is emitted, and the fluorescence intensity ratio (I 625nm / I 715nm ) of the probe BDP has a good linear relationship with the NO concentration; high viscosity leads to a significant fluorescence enhancement before and after the probe responds to NO, achieving the effect of simultaneous response.

[0016] The probe molecule described in the present application has a simple synthesis route, low cost, good biocompatibility, and can achieve high selectivity and sensitivity in detecting NO and viscosity. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe BDP of the present application in deuterated chloroform, the horizontal coordinate is chemical shift, and the vertical coordinate is intensity.

[0018] Figure 2 It is the nuclear magnetic resonance carbon spectrum of the fluorescent probe BDP of the present application in deuterated chloroform, the horizontal coordinate is chemical shift, and the vertical coordinate is intensity.

[0019] Figure 3 It is the change of the fluorescence intensity ratio (I 625nm / I 715nm ) of the fluorescent probe BDP (10 μM) of the present application in PBS buffer (0.01 M, pH = 7.4, 30% DMSO) before and after responding to 400 μM NO with time. The horizontal coordinate is wavelength, and the vertical coordinate is fluorescence intensity ratio.

[0020] Figure 4 It is the change of the ultraviolet absorption spectrum and fluorescence spectrum (absorption spectrum (solid line) and fluorescence spectrum (dashed line) λex=550 nm) of the fluorescent probe BDP (10 μM) of the present application in PBS buffer (0.01 M, pH = 7.4, 30% DMSO) before and after responding to 400 μM NO. The horizontal coordinate is wavelength, and the vertical coordinate is absorbance or fluorescence intensity.

[0021] Figure 5 The change of the fluorescence spectrum of the probe solution after the response of the fluorescent probe BDP (10 μM) of the application to related substances (ONOO - , H2O2, KO2, ROO·, Cys, Hcy, GSH, Cl - , S 2- , CO3 2- , K + , Mg 2+ , NO; the concentration is 400 μM) is shown in the figure, in which the abscissa represents wavelength and the ordinate represents fluorescence intensity.

[0022] Figure 6 The change of the fluorescence spectrum of the probe solution after the response of the fluorescent probe BDP (10 μM) of the application to related substances (ONOO

[0023] Figure 7 The fluorescence image of the fluorescent probe BDP (5 μM) in HeLa cells to exogenous NO.

[0024] Figure 8 The fluorescence image of the fluorescent probe BDP (5 μM) in HeLa cells to endogenous NO.

[0025] Figure 9 The fluorescence image of the fluorescent probe BDP (5 μM) in normal and high viscosity HeLa cells to NO.

[0026] Figure 10 The fluorescence image of the fluorescent probe BDP (100 μM, 100 μL) in normal mice and HF mice to NO.

[0027] Figure 11 The response process mechanism diagram of the fluorescent probe of the application.

[0028] Specific implementation examples

[0029] Example 1: Synthesis of compound BDP

[0030] Compound 1 and compound 2 were dissolved in anhydrous toluene, then acetic acid was added to the mixture, piperidine was added as a base catalyst, and the mixture was heated to reflux at 120°C for 5 h. After the mixture was cooled to room temperature, it was extracted with dichloromethane, dried over anhydrous sodium sulfate, and rotary evaporated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain compound BDP with a yield of 18.3%. The molecular structure characterization is as follows: 1H NMR (400 MHz, CDC13) δ 7.51 - 7.43 (m, 6H), 7.33 - 7.28 (m, 2H), 7.19 (d, J = 16.1 Hz, 1H), 6.69 (d, J = 6.2 Hz, 2H), 6.58 (s, 1H), 5.97 (s, 1H), 2.90 (s, 3H), 2.59 (s, 3H), and 1.56 (s, 6H). 13 C NMR (100 MHz, CDC13) δ 189.3, 153.3, 149.2, 136.9, 134.2, 131.3, 129.8, 128.4, 128.1, 128.0, 127.8, 127.3, 127.2, 127.1, 125.2, 124.5, 119.5, 116.7, 113.2, 112.0, 111.2, 110.4, 29.3, 28.9, 13.6, and 13.2. HRMS (ESI) m / z calcd for C 27 H 27 BF2N3 + [M+H] + The molecular weight of 442.2261, found at 442.2228 corresponding value.

[0031] Example 2: Probe BDP detection of the response study

[0032] The probe was dissolved in the test solution DMSO / PBS (3:7, v / v, 10 mM, pH = 7.40) to configure 1.0 x 10 - 3 The UV absorption spectrum and fluorescence spectrum of the test solution were tested over time. For the fluorescence spectrum, the fluorescence intensity at 625 nm was observed to rise rapidly after the addition of NO molecules, and the fluorescence intensity at 715 nm rose rapidly after the addition of viscosity. The detection time was short, and qualitative and quantitative detection of NO and viscosity could be achieved.

[0033] Example 3: Selectivity experiment of probe BDP

[0034] The probe was dissolved in the test solution DMSO / PBS (3:7, v / v, 10 mM, pH = 7.40) to configure 1.0 x 10 - 3 The probe was dissolved in the test solution DMSO / PBS (3:7, v / v, 10 mM, pH = 7.40) to configure 1.0 x 10 - , H202, KO2, ROO·, Cys, Hcy, GSH, Cl - , S 2- , CO3 2- , K + , Mg 2+Neither of them caused the change of fluorescence, but the addition of NO and the increase of viscosity caused the enhancement of the ratio fluorescence signal, Figures 5-6 It is shown that the single-wavelength excitation double-response type probe has high selectivity for NO and viscosity. The different pH response experiments show that the probe molecule has good detection effect under normal physiological pH conditions, so the fluorescent probe has good detection performance, which confirms the application of the probe.

Claims

1. A ratiometric fluorescent probe for the dual detection of nitric oxide and viscosity for the accurate diagnosis of liver fibrosis, having the structure: ###0001###

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