Near-infrared dual-lock fluorescent probe co-activated by CO and viscosity, and its synthesis method and application

Abstract

The present invention provides a near-infrared dual-lock fluorescent probe co-activated by CO and viscosity, and its structural formula is as follows: The near-infrared dual-lock fluorescent probe co-activated by CO and viscosity provided by the present invention is used to simultaneously detect CO and viscosity of liver tissue, solving the technical problem that existing fluorescent probes cannot be activated with dual biomarkers and accurately diagnose non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and particularly relates to a near-infrared dual-lock fluorescent probe co-activated by CO and viscosity, and a synthesis method and application thereof. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a clinical syndrome characterized by excessive fat deposition in hepatocytes. If not diagnosed and treated in time, NAFLD may deteriorate into cirrhosis or even liver cancer, and increase the risk of developing related diseases such as diabetes and thick blood lipids. Therefore, early and accurate diagnosis is of great help for the prevention and treatment of NAFLD. At present, the diagnosis of NAFLD in clinical practice mainly relies on liver biopsy. Liver biopsy is an invasive examination, which brings a lot of trauma and pain to patients, and at the same time has problems such as false positives and complex operations. Therefore, it is very meaningful to develop a non-invasive, non-toxic and effective diagnostic tool for NAFLD.

[0003] Molecular diagnosis refers to a method for quickly and accurately diagnosing diseases by detecting specific small molecules, DNA, RNA or proteins in human tissues based on modern molecular biology techniques. Molecular diagnosis includes two main elements: diagnostic tools and disease biomarkers. A key parameter of the cellular microenvironment is viscosity. It has been reported that excessive fat deposition in NAFLD hepatocytes increases the number of lipid droplets in hepatocytes, resulting in a high-viscosity environment. The abnormal viscosity of hepatocytes has been identified as a biomarker of NAFLD. In recent years, fluorescent probes have been widely used in disease diagnosis due to their advantages such as non-invasiveness, high sensitivity and high selectivity. It has been reported that several viscosity-sensitive probes have been reported for the diagnosis of NAFLD. However, an increase in intracellular environmental viscosity also exists in other diseases, and only detecting viscosity changes may lead to misdiagnosis.

[0004] For a long time, carbon monoxide (CO) has been associated with its biological toxicity. However, under certain physiological and pathological conditions, the intracellular CO content also increases. In addition, in mitochondria, it has been reported that CO plays a role in regulating the formation of reactive oxygen species (ROS), and viscosity will change significantly due to the production of ROS. Real-time monitoring of CO changes may provide diagnostic information for NAFLD. And the increase in viscosity during oxidative stress in NAFLD may be related to the production of CO.

[0005] Compared with probes activated by a single biomarker, probes activated by dual biomarkers show more accurate and real signals and are more suitable for disease diagnosis. Therefore, developing an effective fluorescent probe that can simultaneously detect CO and viscosity in liver tissue will be very helpful for the diagnosis of NAFLD. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a near-infrared dual-lock fluorescent probe co-activated by CO and viscosity, which is used to simultaneously detect CO and viscosity in liver tissue, and solves the technical problem that existing fluorescent probes cannot be activated by dual biomarkers and accurately diagnose non-alcoholic fatty liver disease.

[0007] The technical solution of the present invention is as follows:

[0008] A near-infrared dual-lock fluorescent probe co-activated by CO and viscosity, and its structural formula is as follows:

[0009]

[0010] A synthesis method of a near-infrared dual-lock fluorescent probe co-activated by CO and viscosity, comprising the following steps:

[0011] Step S1, dissolve Changsha near-infrared fluorescent dye CS-red, hydrazine hydrate and Carter condensing agent BOP in dry DCM solution, react at room temperature for 6-10 h, after the reaction is completed, spin-dry the reaction solution under reduced pressure, and purify the crude product by silica gel column chromatography to obtain compound CS-NH 2 , and its technical route is:

[0012]

[0013]

[0014] Step S2, dissolve compound CS-NH 2 and 5-methoxypyridine-2-carbaldehyde in anhydrous methanol solution, then react at room temperature for 6-10 h, after the reaction is completed, spin-dry the solvent, and purify the crude product by silica gel column chromatography to obtain the fluorescent probe product HCO-9, and its technical route is:

[0015]

[0016] Furthermore, in step S1, the molar ratio of CS-red, hydrazine hydrate and Carter condensing agent is 1:7.2:0.5.

[0017] Furthermore, in step S2, the molar ratio of CS-NH 2 and 5-methoxypyridine-2-carbaldehyde is 1:1.

[0018] Furthermore, in step S1, the eluent used for column chromatography is V 二氯甲烷 / V 乙酸乙酯 =8 / 1; in step S2, the eluent used for column chromatography is V 二氯甲烷 / V 乙酸乙酯 =10 / 1.

[0019] The present invention also provides an application of a CO and viscosity co-activated near-infrared dual-lock fluorescent probe in detecting CO and viscosity in liver tissue.

[0020] Compared with the prior art, the CO and viscosity co-activated near-infrared dual-lock fluorescent probe provided by the present invention, its synthesis method and application have the beneficial effects that:

[0021] First, the CO and viscosity co-activated near-infrared dual-lock fluorescent probe provided by the present invention is in the form of a closed-loop spirolactam and has no fluorescence. Under the activation of CO, the first "lock" is opened to keep the spirolactam in an open-ring state. However, since the C═C bond remains free to rotate, only weak near-infrared fluorescence is shown as a whole. However, when the probe HCO-9 is co-activated by CO and high viscosity, the two "locks" are opened simultaneously, and strong near-infrared fluorescence is rapidly released. Therefore, the fluorescent probe of the present invention can realize the simultaneous detection of CO and viscosity.

[0022] Second, the synthesis route of the CO and viscosity co-activated near-infrared dual-lock fluorescent probe provided by the present invention is simple, the cost is low, and the biocompatibility is good. It can realize the simultaneous detection of CO and viscosity and has been successfully applied to the detection of non-alcoholic fatty liver in a mouse model. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 1H NMR spectrum of the fluorescent probe of the present invention in deuterated chloroform, with the chemical shift on the abscissa and the intensity on the ordinate;

[0025] Figure 2 13C NMR spectrum of the fluorescent probe of the present invention in deuterated chloroform, with the chemical shift on the abscissa and the intensity on the ordinate;

[0026] Figure 3 Fluorescence titration diagram of the probe HCO-9 to CO and viscosity;

[0027] Figure 4 Response process mechanism diagram of the probe HCO-9 to CO and viscosity;

[0028] Figure 5 Response of the fluorescent probe HCO-9 (10 μM) of the present invention in (10.0 mM PBS, pH = 7.4, containing 80% glycerol) to related substances, including oxidizing substances (ClO - and H 2O 2 )), reducing substances (Cys, Hcy, GSH), other gas signal molecules (H 2 S and NO), and anions and cations (Cl - , F - , I - , NO 3 - , CO 3 2- , SO 4 2- , S 2 O 3 - , K + , Na + , Zn 2+ , Cu 2+ ), with a concentration of 100.0 μM, the abscissa is the wavelength, and the ordinate is the fluorescence intensity;

[0029] Figure 6 is the pathological analysis of probe HCO-9 in liver tissue sections of normal mice and non-alcoholic fatty liver tissue;

[0030] Figure 7 is the detection of non-alcoholic fatty liver and imaging of major organs by probe HCO-9 in a mouse model. Detailed implementation manners

[0031] To enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention and make the above objects, features, and advantages of the present invention more obvious and understandable, the following further describes the detailed implementation manners of the present invention.

[0032] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0033] Example 1: Synthesis of compound CS-NH 2

[0034] Dissolve compound CS-red (0.2 g, 0.36 mmol), hydrazine hydrate (0.13 mL, 2.6 mmol), and BOP (75.0 mg, 0.17 mmol) in 10.0 mL of dry DCM solution, and then react at room temperature for 8 hours. After the reaction is completed, spin-dry the reaction solution, and purify the crude product by silica gel column chromatography (silica gel 200 - 300 mesh, eluent: V 乙酸乙酯 / V​​二氯甲烷 = 8 / 1) to obtain 80.0 mg of compound CS-NH 2 with a yield of 31.7%.

[0035] Example 2: Synthesis of probe HCO-9

[0036] Dissolve compound CS-NH 2 (0.1 mmol) and 5-methoxypyridine-2-carbaldehyde (0.1 mmol) in 15.0 mL of anhydrous methanol solution, and then react at room temperature for 8 hours. After the reaction, the solvent was evaporated to dryness, and the crude product was separated by column chromatography (silica gel 200 - 300 mesh, eluent: V 乙酸乙酯 / V 二氯甲烷 = 10 / 1) to obtain 34 mg of the fluorescent probe product HCO-9; the yield was 49.3%.

[0037] Please refer to Figure 1 and Figure 2 , Figure 1 which is the proton nuclear magnetic resonance spectrum of the fluorescent probe of the present invention in deuterated chloroform, with the chemical shift on the abscissa and the intensity on the ordinate; Figure 2 which is the carbon nuclear magnetic resonance spectrum of the fluorescent probe of the present invention in deuterated chloroform, with the chemical shift on the abscissa and the intensity on the ordinate.

[0038] The characterization data of probe HCO-9 are as follows: 1 H NMR (400 MHz, CDCl 3 ) δ 8.58 (s, 1H), 8.19 (d, J = 2.7 Hz, 1H), 8.06 (d, J = 8.9 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 7.4 Hz, 2H), 7.44 (t, J = 7.4 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.18 (d, J = 6.9 Hz, 2H), 7.15 (d, J = 2.6 Hz, 1H), 6.85 (t, J = 7.3 Hz, 1H), 6.61 (d, J = 7.7 Hz, 1H), 6.50 (d, J = 8.8 Hz, 1H), 6.39 (s, 1H), 6.24 (d, J = 8.1 Hz, 1H), 5.37 (d, J = 12.7 Hz, 1H), 3.83 (s, 3H), 3.33 (dd, J = 13.8, 6.8 Hz, 4H), 3.14 (s, 3H), 2.49 (s, 2H), 1.76 (d, J = 11.1 Hz, 6H), 1.27 (d, J = 8.2 Hz, 4H), 1.16 (t, J = 6.9 Hz, 6H). 13 C NMR (100 MHz, CDCl 3)δ165.6,156.0,152.0,147.4,145.6,136.3,133.4,128.9,128.4,127.7,127.2,123.4,123.3,121.6,121.0,119.6,119.3,105.7,55.6,45.5,44.3,34.7,34.5,31.6,29.7,29.1,29.1,28.4,25.3,22.8,22.7,22.1,20.7,14.1,12.6,11.4.

[0039] Example 3: Fluorescence titration experiment of probe HCO-9

[0040] The test method of the fluorescence probe HCO-9 of the present invention is as follows: An appropriate amount of probe HCO-9 is dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1 mM, and it is stored in the refrigerator for later use. Under the condition of no viscosity, the spectral test experiment is carried out in a mixed buffer (10.0 mM PBS, pH = 7.4, containing 50% DMSO). Under the condition of having viscosity, the spectral test experiment is carried out in a mixed buffer (10.0 mM PBS, pH = 7.4, containing 80% glycerol). The excitation wavelength is 700 nm, and the slit widths of excitation and emission are set to 5.0 nm / 5.0 nm respectively.

[0041] The present invention studied the absorption and emission spectra of the response of probe HCO-9 to CO and viscosity ( Figure 3 a and 3b). The probe HCO-9 after reacting with CO has an obvious absorption peak at 715 nm, and adding glycerol has little effect on its spectral change. The probe HCO-9 has no fluorescence in both low-viscosity and high-viscosity environments. In contrast, after reacting with CO, weak fluorescence appears at 747 nm; after reacting with both CO and high viscosity, strong fluorescence is observed. The results show that the probe HCO-9 can detect CO in a viscous system. Please refer to Figure 4 , the response mechanism of the fluorescence probe of the present invention is as follows: The probe HCO-9 is in the form of a closed-loop helical lactam (non-fluorescent); under the activation of CO, the first "lock" is opened to keep the spiro-lactam in an open-loop state, but since the C═C bond remains free to rotate, the whole only shows weak near-infrared fluorescence. However, when the probe HCO-9 is jointly activated by CO and high viscosity, the two "locks" are opened simultaneously, and strong near-infrared fluorescence is rapidly released.

[0042] Then, the quantitative detection of the fluorescence spectrum of the probe HCO-9 to CO in a high-viscosity environment was studied. As Figure 3As shown in c, in glycerol / PBS buffer (V / V = 8 / 2), when CO (0 - 10.0 equivalents) was added to the probe HCO-9, a fluorescence peak appeared at 747 nm, and the fluorescence peak increased with the increase in the CO concentration. And there was a good linear relationship y = -3.309 + 209.976x (R 747 nm ) between the fluorescence intensity (I 2 ) at 747 nm and the CO concentration (0 - 6.0 equivalents). According to the signal-to-noise ratio of S / N = 3, the detection limit of CO was calculated to be 60 nM( Figure 3 d). The results showed that the HCO-9 probe was a highly sensitive probe for quantitatively detecting CO under high-viscosity conditions.

[0043] In addition, we also studied the changes in the fluorescence spectra of the probe HCO-9 after reacting with CO in solutions with different viscosities. As Figure 3 shown in e, the probe HCO-9 responded to CO in PBS buffer to PBS buffer containing 80% glycerol (η = 1.0 - 195.0 cp), and I 747 nm increased by 10 times. As Figure 3 shown in f, there was a linear relationship y = -24.786 + 142.612x (R 747 nm ) between I 2 and the viscosity coefficient, indicating that the HCO-9 probe could be used for viscosity detection in the presence of CO.

[0044] Example 4: Selectivity experiment of the probe HCO-9

[0045] To evaluate the selectivity of the probe HCO-9, its response to other related substances was studied in glycerol / PBS buffer (V / V = 8 / 2). For the detection of CO, potential interfering factors were considered, including oxidizing substances (ClO - and H 2 O 2 ), reducing substances (Cys, Hcy, GSH), other gas signaling molecules (H 2 S and NO), and anions and cations (Cl - , F - , I - , NO 3 - , CO 3 2- , SO 4 2- , K + , Na + , Mg 2+ , Ca 2+ ).

[0046] As Figure 5 shown, only CO can cause a significant increase in I 747 nm while other substances only cause slight fluorescence changes. This indicates that the probe HCO-9 has the ability to highly selectively detect CO in a viscosity system.

[0047] Example 5: Detection of non-alcoholic fatty liver by probe HCO-9 in a mouse model

[0048] In this invention, the imaging of abnormal viscosity and CO by the probe in a NAFLD mouse model was studied. Mice were fed a high-fat diet for 20 weeks to establish a NAFLD model. First, liver sections of the control group and high-fat-fed mice were prepared with a thickness of about 10 μm and stained with hematoxylin and eosin (H&E) and Oil Red O. As Figure 6 shown, obvious inflammatory damage, fat deposition, and fatty degeneration were observed in the experimental group, indicating the successful establishment of the NAFLD mouse model.

[0049] Then, after in-situ injection of the probe HCO-9 into normal mice and NAFLD mice, time-dependent imaging of the mice was observed. As Figure 7 shown in a, the normal group showed weak fluorescence in the red channel, indicating that the probe HCO-9 can sensitively detect low levels of CO in normal livers. A stronger red fluorescence signal was observed in the experimental group and increased with time, indicating higher levels of CO and viscosity in the livers of NAFLD mice.

[0050] In addition, after the imaging was completed, the main organs of the mice, including the liver, spleen, heart, kidneys, and lungs, were dissected for fluorescence imaging. As Figure 7 shown in b, only the liver had obvious fluorescence signals compared with other organs. The results indicate that the probe HCO-9 can well detect CO and viscosity in the NAFLD mouse model and can be used as a potential tool for the accurate diagnosis of NAFLD.

[0051] The fluorescent probe of the present invention itself has no fluorescence and emits weak near-infrared fluorescence after reacting with CO, with a maximum emission peak at 747 nm; it emits strong near-infrared fluorescence after reacting with both CO and viscosity, with a maximum emission peak at 747 nm. Therefore, the probe HCO-9 of the present invention can simultaneously detect CO and viscosity and can be used for the detection of non-alcoholic fatty liver.

[0052] The above has made a detailed description of the embodiments of the present invention, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations made to these embodiments without departing from the principles and spirit of the present invention still fall within the protection scope of the present invention.

Claims

1. A CO and viscosity co-activated near-infrared dual-lock fluorescent probe, characterized in that, its structural formula is as follows: 。 2. A synthesis method of a CO and viscosity co-activated near-infrared dual-lock fluorescent probe, characterized in that, it includes the following steps: Step S1, dissolve Changsha near-infrared fluorescent dye CS-red, hydrazine hydrate and Carter condensing agent BOP in dry DCM solution, react at room temperature for 6 - 10 h, after the reaction is completed, rotary evaporate the reaction solution under reduced pressure, and purify the crude product by silica gel column chromatography to obtain compound CS-NH 2 , and its technical route is as follows: ； Step S2, dissolve compound CS-NH 2 and 5-methoxypyridine-2-carbaldehyde in anhydrous methanol solution, then react at room temperature for 6 - 10 h. After the reaction is completed, spin-dry the solvent, and the crude product is purified by silica gel column chromatography to obtain the fluorescent probe product HCO-9. The technical route is as follows: 。 3. The synthesis method of the CO and viscosity co-activated near-infrared dual-lock fluorescent probe according to claim 2, characterized in that, in step S1, the molar ratio of CS-red, hydrazine hydrate and Carter condensing agent is 1:7.2:0.

5.

4. The synthesis method of the CO and viscosity co-activated near-infrared dual-lock fluorescent probe according to claim 2, characterized in that, In step S2, the molar ratio of CS-NH 2 to 5-methoxypyridine-2-carbaldehyde is 1:

1.

5. The synthesis method of the CO and viscosity co-activated near-infrared dual-lock fluorescent probe according to claim 2, characterized in that, The eluent used in column chromatography in step S1 is V 二氯甲烷 / V 乙酸乙酯 = 8 / 1; The eluent used in the column chromatography in step S2 is V 二氯甲烷 / V 乙酸乙酯 = 10 / 1.

6. An application of the CO and viscosity co-activated near-infrared dual-lock fluorescent probe according to claim 1 in the preparation of a reagent for detecting CO and viscosity in liver tissue.

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