A fluorescent probe based on rhodamine modified nucleoside and a preparation method and application thereof

Abstract

The application discloses a fluorescent probe based on rhodamine modified nucleoside and a preparation method and application thereof, and a structure formula of the fluorescent probe is as follows: the fluorescent probe is a novel fluorescent probe based on rhodamine modified nucleoside, is composed of rhodamine hydrazine-short-chain alkyne-2-deoxy cytidine, and has the advantages of high fluorescence intensity, good biological compatibility, good strong acid resistance, high sensitivity and good chemical stability. 3+ The fluorescent probe can be widely applied in the fields of in vivo and in vitro Fe detection, real-time monitoring of pH dynamics, preparation of pH test paper, cell imaging, construction of lysosome-targeting functional probes, design of in vivo contrast agents, treatment and diagnosis of gastrointestinal diseases and screening of related treatment drugs, and has great practical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fluorescent probe, in particular to a fluorescent probe based on rhodamine modified nucleoside and a preparation method and application thereof. BACKGROUND

[0002] In recent years, due to environmental pollution and the influence of many other natural processes, the types and contents of metal ions in daily life diet have increased. Iron ion, as the most important element in all life systems, plays a crucial role in various important cell functions such as hemoglobin formation, iron-containing enzymes and brain function. The excess and deficiency of Fe 3+ can destroy the intracellular homeostasis and cause various diseases, such as Parkinson's disease and Alzheimer's disease, which are related to the cytotoxicity caused by Fe 3+ ion. Therefore, the detection of iron ion is a key factor for early diagnosis of certain diseases and evaluation of human health. Therefore, it is urgent to develop a high-sensitivity and biocompatible fluorescent and visual probe for iron ion.

[0003] pH detection usually plays a key role in various environments, food and biological systems, such as industrial wastewater discharge detection, drinking water quality detection, food production auxiliary material detection, pH detection of algal culture environment, etc. Recently, various functional pH-responsive probes have been continuously reported, however, there are few reports on pH fluorescent probes with high sensitivity in strong acid (pH < 4). In view of the demand for strong acid application scenarios: such as industrial wastewater and gastric acid microenvironment, the use of strong acid sensitive sensors for detection is essential. It is well known that the acidity of gastric juice is an important indicator for the clinical diagnosis and treatment of gastrointestinal diseases. Under normal physiological conditions, the pH value is about 2.0, and when suffering from gastric ulcer, it is about 4. The commonly used method for clinically evaluating the pH value of gastric acid is mainly endoscopy or gastric juice extraction, but this method is invasive and has poor comfort, and is not suitable for long-term monitoring. Therefore, the pH fluorescent probe with in vivo gastrointestinal imaging function is the key to improve the clinical diagnosis means of gastric diseases and related drug development. The concentrated acid solution in the gastrointestinal tract can change the solubility, chemical stability and other physical / chemical properties or biological activity of the invading compounds, so it is still a challenging research topic to develop a chemiluminescent probe for in situ real-time monitoring of pH and non-invasive in vivo imaging.

[0004] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0005] In view of the above deficiencies of the prior art, the purpose of the present application is to provide a fluorescent probe based on rhodamine modified nucleoside and a preparation method and application thereof, aiming to solve the problems of poor biocompatibility of the existing Fe 3+ detection fluorescent probe and the scarcity of pH sensitive fluorescent probes for monitoring strong acid environment.

[0006] The technical scheme of the present application is as follows:

[0007] In a first aspect of the present application, a fluorescent probe based on rhodamine modified nucleoside (RBH-EdC) is provided, and the structural formula of the fluorescent probe is as follows:

[0008]

[0009] In a second aspect of the present application, a preparation method of the fluorescent probe based on rhodamine modified nucleoside is provided, and the preparation method comprises the following steps:

[0010] Compound 2 and compound 3 are provided, and Schiff base reaction is performed on the compound 2 and the compound 3 to obtain compound 4;

[0011] 5-iodo-2-deoxycytidine is provided, and coupling reaction is performed on the compound 4 and the 5-iodo-2-deoxycytidine to obtain the fluorescent probe;

[0012] The structural formula of the compound 2 is as follows: The structural formula of the compound 3 is as follows: The structural formula of the compound 4 is as follows:

[0013] Optionally, the preparation method of the compound 2 comprises the following steps:

[0014] 4-bromothiophene-2-carboxaldehyde and trimethylsilane acetylene are provided, and coupling reaction is performed on the 4-bromothiophene-2-carboxaldehyde and the trimethylsilane acetylene to obtain compound 1;

[0015] The compound 1 is reacted under the action of a base to obtain the compound 2;

[0016] The structural formula of the compound 1 is as follows:

[0017] Optionally, the preparation method of the compound 3 comprises the following step: rhodamine B and hydrazine hydrate are reacted to obtain the compound 3.

[0018] Optionally, the step of performing coupling reaction on the compound 4 and the 5-iodo-2-deoxycytidine to obtain the fluorescent probe comprises the following steps:

[0019] The compound 4, the 5-iodo-2-deoxycytidine, a catalyst, triethylamine and cuprous iodide are added into a first solvent to react under the protection of an inert gas, so as to obtain the fluorescent probe.

[0020] In a third aspect of the present application, the fluorescent probe is applied to the preparation of Fe 3+ detection probe.

[0021] The fourth aspect of the present application provides application of the fluorescent probe in the preparation of a solution pH monitoring probe.

[0022] The fifth aspect of the present application provides application of the fluorescent probe in cell imaging.

[0023] The sixth aspect of the present application provides application of the fluorescent probe in the preparation of a lysosome targeting probe.

[0024] The seventh aspect of the present application provides application of the fluorescent probe in the preparation of a gastric acid pH detection probe.

[0025] The eighth aspect of the present application provides application of the fluorescent probe in the preparation of a gastric ulcer diagnostic reagent.

[0026] Beneficial effects: the fluorescent probe of the present application is a new type of fluorescent probe based on rhodamine modified nucleosides, which is composed of rhodamine hydrazine-short chain alkyne-2-deoxy cytidine, and has the advantages of high fluorescence intensity, good biological compatibility, good strong acid resistance, high sensitivity and good chemical stability. The fluorescent probe described in the present application has at least the following six functions: first, it can sensitively and selectively detect and identify Fe 3+ and its concentration, with a detection limit as low as 0.03 μM; second, it can monitor the change of solution pH in a strong acid environment with a pH range of 1-4.5, and the fluorescence intensity change is as high as 20 times, which can be applied to the preparation of pH 2.5 test paper; third, it has high biological compatibility and can be applied to live cell fluorescence imaging; fourth, it can realize lysosome targeting function with a Pearson colocalization coefficient of 0.863; fifth, it has no toxicity to mice and can realize live fluorescence imaging; sixth, it can accurately feedback the dynamic change of gastric acid pH, and realize in-situ, non-invasive and real-time diagnosis of gastrointestinal diseases such as gastric ulcer. Therefore, the fluorescent probe can be widely applied in the fields of in vivo and in vitro Fe 3+ detection, real-time monitoring of pH dynamics, preparation of pH test paper, cell imaging, construction of lysosome targeting function probe, design of live contrast agent, diagnosis and treatment of gastrointestinal diseases and screening of related therapeutic drugs, etc., and has great practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a synthesis route map of RBH-EdC.

[0028] Figure 2 a is the hydrogen spectrum of RBH-EdC, and b is the mass spectrum of RBH-EdC.

[0029] Figure 3 It is a fluorescence spectrum of RBH-EdC mixed with different metal ions.

[0030] Figure 4 The fluorescence spectrum of RBH-EdC with different concentrations of Fe 3+ and the linear fitting of the fluorescence intensity of RBH-EdC with different concentrations of Fe 3+ (R 2 = 0.9979).

[0031] Figure 5 The fluorescence intensity comparison of RBH-EdC solution, RBH-EdC mixed with Fe 3+ , RBH-EdC mixed with other metal ions in the competitive experiment. 3+

[0032] Figure 6 a is the fluorescence intensity of RBH-EdC in different pH solutions, and b is the fluorescence intensity of RBH-EdC in the reversible experiment.

[0033] Figure 7 The survival rate of NIH-3T3 cells after 24 hours of co-incubation with different concentrations of RBH-EdC.

[0034] Figure 8 The cell fluorescence imaging of cells with different concentrations of Fe 3+ and RBH-EdC, where a is the cell fluorescence imaging under normal concentration of Fe 3+ , b is the cell fluorescence imaging under 50 μM concentration of Fe 3+ , and c is the cell fluorescence imaging under 100 μM concentration of Fe 3+ .

[0035] Figure 9 The cell fluorescence imaging and scatter plot of RBH-EdC lysosome co-localization, where a1 is the cell imaging of RBH-EdC in the red channel, a2 is the cell imaging of commercial lysosome targeting dye in the green channel, a3 is the superimposed image of a1 and a2, and a4 is the scatter plot of the red fluorescence sites of RBH-EdC and the green fluorescence sites of commercial lysosome targeting probe.

[0036] Figure 10 The toxicological tissue sections of control and experimental mice, where a is the stomach tissue section, and b is the liver tissue section.

[0037] Figure 11 The in vivo fluorescence imaging of control mice at different time points in acid suppression therapy.

[0038] Figure 12 The in vivo fluorescence imaging of experimental mice at different time points in acid suppression therapy. ​

[0039] Figure 13 The in vivo fluorescence imaging diagram of the experimental group and the control group of mice in the gastric ulcer experiment. DETAILED DESCRIPTION

[0040] The application provides a fluorescent probe based on rhodamine modified nucleosides and a preparation method and application thereof, in order to make the purpose, technical scheme and effect of the application more clear and definite, the application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the application and not to limit the application.

[0041] The application provides a fluorescent probe based on rhodamine modified nucleosides, wherein the structural formula of the fluorescent probe (RBH-EdC) is as follows:

[0042]

[0043] The fluorescent probe provided by the application is composed of rhodamine hydrazine-short-chain alkyne-2-deoxycytidine, has the advantages of high fluorescence intensity, good biological compatibility, good strong acid resistance, high sensitivity and good chemical stability, and can be widely applied to the fields of in vivo and in vitro Fe 3+ detection, real-time monitoring of pH dynamics, preparation of pH test paper, cell imaging, construction of lysosome targeting functional probes, design of in vivo contrast agents, treatment and diagnosis of gastrointestinal diseases and screening of related treatment drugs, and has great practical application value.

[0044] The application provides a preparation method of the fluorescent probe.

[0045] S1, providing compound 2 and compound 3, and subjecting the compound 2 and the compound 3 to Schiff base reaction to obtain compound 4;

[0046] S2, providing 5-iodo-2-deoxycytidine, and subjecting the compound 4 and the 5-iodo-2-deoxycytidine to coupling reaction to obtain the fluorescent probe;

[0047] The compound 2 has the structural formula as follows The compound 3 has the structural formula as follows The compound 4 has the structural formula as follows

[0048] The fluorescent probe provided by the application is obtained by subjecting the rhodamine hydrazine derivative (i.e., the compound 3) and the short-chain alkyne connected thiophene aldehyde (i.e., the compound 2) to Schiff base reaction to obtain an intermediate product (i.e., the compound 4), and then subjecting the intermediate product to sonagashira coupling reaction with 5-iodo-2-deoxycytidine. The preparation method provided by the application is simple, efficient, and raw materials are easy to obtain, and the preparation of the fluorescent probe can be realized.

[0049] In step S1, in an embodiment, the method for preparing the compound 2 comprises the following steps:

[0050] Providing 4-bromothiophene-2-carboxaldehyde and trimethylsilane acetylene, and coupling the 4-bromothiophene-2-carboxaldehyde and the trimethylsilane acetylene to obtain the compound 1;

[0051] Reacting the compound 1 under the action of a base to obtain the compound 2;

[0052] In an embodiment, the compound 1 has the following structural formula:

[0053] In an embodiment, the step of coupling the 4-bromothiophene-2-carboxaldehyde and the trimethylsilane acetylene to obtain the compound 1 specifically comprises:

[0054] Under the protection of an inert gas (such as nitrogen), 4-bromothiophene-2-carboxaldehyde, trimethylsilane acetylene, bis(triphenylphosphine)palladium(II) dichloride, anhydrous triethylamine (TEA), and cuprous iodide are added into anhydrous DMF solvent and uniformly mixed, and the compound 1 is obtained after stirring and reacting at 40°C for 24h. The specific chemical reaction formula is as shown below:

[0055]

[0056] In an embodiment, the base in the step of reacting the compound 1 under the action of a base to obtain the compound 2 is potassium carbonate, but is not limited thereto.

[0057] In an embodiment, the step of reacting the compound 1 under the action of a base to obtain the compound 2 specifically comprises:

[0058] In anhydrous methanol as a solvent, the compound 1 is reacted with potassium carbonate at 50°C overnight to obtain the compound 2. The specific chemical reaction formula is as shown below:

[0059]

[0060] In an embodiment, the method for preparing the compound 3 comprises the following step: reacting rhodamine B and hydrazine hydrate to obtain the compound 3.

[0061] In an embodiment, the step of reacting rhodamine B and hydrazine hydrate to obtain the compound 3 specifically comprises:

[0062] Rhodamine B and hydrazine hydrate are added into anhydrous methanol, and the compound 3 is obtained after reacting at 75°C for 6h. The specific chemical reaction formula is as shown below:

[0063]

[0064] In an embodiment, in order to improve the efficiency of the Schiff base reaction, an acid is added to the reaction system, preferably acetic acid, more preferably acetic acid with a concentration of 30%.

[0065] In an embodiment, the step of subjecting the compound 2 and the compound 3 to a Schiff base reaction to obtain the compound 4 specifically comprises:

[0066] The compound 2 and the compound 3 are added to a second solvent (such as ethanol, preferably anhydrous ethanol), and subjected to a Schiff base reaction at a temperature of 50-75°C (such as 50°C, 65°C or 75°C, etc.) for 12-24h (such as 12h, 20h or 24h, etc.) to obtain the compound 4. The specific chemical reaction formula is as follows:

[0067]

[0068] In an embodiment, the step of subjecting the compound 4 and the 5-iodo-2-deoxy cytidine to a coupling reaction to obtain the fluorescent probe specifically comprises:

[0069] The compound 4, the 5-iodo-2-deoxy cytidine, a catalyst, triethylamine and cuprous iodide are added to a first solvent under inert gas protection to obtain the fluorescent probe.

[0070] In an embodiment, the inert gas is nitrogen.

[0071] In an embodiment, the catalyst is a palladium catalyst, preferably the palladium catalyst is bis(triphenylphosphine)palladium(II) chloride, but is not limited thereto.

[0072] In an embodiment, the first solvent is N,N-dimethylformamide (DMF), but is not limited thereto.

[0073] In an embodiment, the compound 4 and the 5-iodo-2-deoxy cytidine are subjected to a sonagashira coupling reaction to obtain the fluorescent probe.

[0074] In an embodiment, the step of subjecting the compound 4 and the 5-iodo-2-deoxy cytidine to a coupling reaction to obtain the fluorescent probe specifically comprises:

[0075] The compound 4, the 5-iodo-2-deoxy cytidine, bis(triphenylphosphine)palladium(II) chloride, anhydrous triethylamine and cuprous iodide are added to anhydrous DMF solvent under nitrogen protection and mixed uniformly; after stirring at 40°C for 24h, the fluorescent probe (RBH-EdC) is obtained. The specific chemical reaction formula is as follows:

[0076]

[0077] This invention provides a fluorescent probe as described above for the preparation of Fe 3+ Applications in detection probes. On one hand, based on fluorescence spectroscopy experiments of the fluorescent probe detecting different metal cations in solution, it can be seen that the fluorescent probe reacts with Fe... 3+ When mixed, its fluorescence intensity increased nearly 20 times, and a significant color change occurred, with the solution turning from colorless to pink. Furthermore, Fe... 3+ The concentration of Fe2+ shows a clear linear relationship with fluorescence intensity, enabling the determination of Fe2+ in aqueous solution. 3+ Quantitative detection and visualization. On the other hand, based on intracellular Fe... 3+ Detection experiments showed that the fluorescent probe had almost no toxicity to cells, exhibited excellent biocompatibility, and effectively combined adherent cells with Fe... 3+ Maintaining intracellular Fe after co-incubation 3+ At a certain concentration, after incubation with the fluorescent probe, fluorescence imaging can clearly observe the effect of Fe... 3+ As the concentration increases, the fluorescence intensity gradually increases. Therefore, the fluorescent probe can effectively identify Fe in the detection solution or cells. 3+ And respond to Fe 3+ It provides a high concentration of Fe and presents visual feedback, making it an excellent Fe... 3+ Detection probe.

[0078] This invention provides an application of the fluorescent probe described above in solution pH monitoring. The fluorescent probe exhibits a gradual increase in fluorescence intensity in solutions with pH values ​​between 1 and 2.5, a gradual decrease in fluorescence intensity in solutions with pH values ​​between 2.5 and 4, and the strongest fluorescence intensity at pH 2.5. Furthermore, the color change of the solution is consistent with the fluorescence intensity; the stronger the fluorescence intensity, the redder the color. Therefore, this fluorescent probe can be well applied in solution pH monitoring, providing real-time feedback on changes in solution pH. It can serve as a pH detection probe in the pH range of 1-4.5 and can be used to prepare pH test strips for detecting pH 2.5.

[0079] This invention provides an application of the fluorescent probe described above in cell imaging. The fluorescent probe can effectively identify and detect intracellular Fe... 3+ And respond to Fe 3+ The concentration of the fluorescent probe is visualized, and the fluorescent probe can be used for cell imaging. Its fluorescence signal is stable within cells, and the intensity of the fluorescence signal increases with Fe. 3+ Increased concentration enhances imaging, resulting in a noticeable improvement in image quality.

[0080] The application provides application of the fluorescent probe in preparation of a lysosome-targeting probe.

[0081] The application provides application of the fluorescent probe in preparation of a gastric ulcer diagnosis reagent. After the probe is administered to a gastric ulcer model mouse by gavage, the fluorescence of the gastrointestinal part is quenched, and the normal mouse presents strong fluorescence. These results prove that the fluorescent probe can be used for in-situ non-destructive diagnosis of gastric ulcer.

[0082] The application provides application of the fluorescent probe in preparation of a gastric ulcer diagnosis reagent. After the probe is administered to a gastric ulcer model mouse by gavage, the fluorescence of the gastrointestinal part is quenched, and the normal mouse presents strong fluorescence. These results prove that the fluorescent probe can be used for in-situ non-destructive diagnosis of gastric ulcer.

[0083] The application is further described below through specific examples.

[0084] Example 1

[0085] Preparation of the fluorescent probe based on rhodamine-modified nucleosides (RBH-EdC) is as shown in the following synthesis route. Figure 1 The synthesis route is shown in the following.

[0086] 1. Synthesis of compound 1

[0087] In a two-neck flask, 950 mg of 4-bromothiophene-2-carboxaldehyde (5 mmol, 1 equivalent), 47.6 mg of cuprous iodide (0.25 mmol, 0.05 equivalent), 350 mg of Pd(PPh3)2Cl2 (0.5 mmol, 0.1 equivalent) and 3 mL of dry triethylamine (TEA) were weighed, dissolved in 12 mL of super-dry N,N-dimethylformamide (DMF), and stirred at a temperature of 40°C under nitrogen protection. After half an hour, 1 mL of trimethylsilane acetylene (10 mmol, 2 equivalents) was added to the reaction mixture and stirring was continued overnight. After the reaction was completed, ethyl acetate was added to quench the reaction, and the reaction mixture was filtered with diatomite using ethyl acetate as the eluent. The filtrate was washed with water three times. Finally, after water washing, the solution was column chromatography purified using petroleum ether and ethyl acetate (volume ratio of 30:1) as the eluent to obtain compound 1. The yield of the reaction was calculated to be 72%.

[0088] 2. Synthesis of compound 2

[0089] Compound 1 (680 mg, 3.28 mmol, 1 eq) and potassium carbonate (740 mg, 5.39 mmol, 1.5 eq) were dissolved in a single-neck flask containing 10 mL of anhydrous methanol (MeOH) and stirred at 50 °C overnight. When the reaction of compound 1 was monitored to be complete, the reaction was stopped. After the reaction mixture was cooled to room temperature, the part of insoluble potassium carbonate was filtered off with diatomite using dichloromethane as eluent. Finally, the filtrate was dried by rotary evaporation to obtain compound 2. The reaction yield was calculated to be 82%.

[0090] 3. Synthesis of compound 3 (rhodamine B hydrazide)

[0091] Compound 3 (rhodamine B hydrazide) was synthesized by dissolving rhodamine B (1270 mg, 2.8 mmol, 1 eq) and hydrazine hydrate (1 mL, 20.6 mmol, 7 eq) in a 100 mL dry flask and dissolving the powder raw materials in 20 mL of anhydrous methanol, and refluxing at 75 °C overnight. After the reaction was completed, the solvent was evaporated by a vacuum rotary evaporator, and then the reaction system was purified by column chromatography using ethyl acetate and petroleum ether (1:1, by volume) as eluent to obtain compound 3 as a pink powder with a yield of 46%.

[0092] 4. Synthesis of compound 4

[0093] Compound 3 (672 mg, 1.47 mmol, 1 eq) and compound 2 (200 mg, 1.47 mmol, 1 eq) were dissolved in 10 mL of anhydrous ethanol and refluxed at 60 °C. To improve the efficiency of the Schiff base reaction, 0.1 mL of 30% acetic acid was added to the reaction system. When the reaction of compound 2 was monitored to be complete, the heating was stopped and the reaction was completed. After the reaction mixture was cooled to room temperature, the reaction system was filtered with diatomite to obtain a filtrate, which was then extracted and washed with distilled water and ethyl acetate for 3 times, respectively, to obtain an organic layer solution. Finally, compound 4 was obtained as a light yellow solid powder by column chromatography using petroleum ether and ethyl acetate (4:1, by volume) as eluent, and the reaction yield was calculated to be 76.3%.

[0094] 5. Synthesis of rhodamine-modified nucleoside-based fluorescent probe (RBH-EdC)

[0095] In a two-necked round bottom flask, 178 mg of 5-iodo-2-deoxycytidine (0.5 mmol, 1 eq), 200 mg of compound 4 (0.336 mmol, 1 eq), 11.8 mg of Pd(PPh3)2Cl2(0.0168 mmol, 0.05 eq), 6.4 mg of cuprous iodide (0.0336 mmol, 0.1 eq) and 0.23 mL of anhydrous triethylamine were dissolved in 15 mL of super dry N,N-dimethylformamide (DMF) under nitrogen protection, and stirred at 40 °C overnight. After the reaction of compound 4 was monitored to be completed, the heating was stopped. After the reaction mixture was cooled to room temperature, the filtrate was obtained by filtering with diatomite. Then the filtrate was extracted and washed with ethyl acetate and cold water for 3 times respectively, and the organic layer solution was collected. Finally, the column chromatography purification was performed with dichloromethane and methanol (volume ratio of 12:1) as eluent to obtain the fluorescent probe based on rhodamine modified nucleoside (RBH-EdC) as dark red solid powder, and the reaction yield was calculated to be 56%. Figure 2 Figure 3a is the hydrogen spectrum of RBH-EdC. 1 Figure 3b is the mass spectrum of RBH-EdC. Figure 2 Figure 3b is the mass spectrum of RBH-EdC.

[0096] 1 H NMR (400 MHz, DMSO-d6): δ ppm 8.91-8.95 (s, 1H), 8.21-8.25 (s, 1H), 7.86-7.91 (m, 1H), 7.78-7.82 (s, 1H), 7.53-7.65 (m, 2H), 7.06-7.11 (m, 1H), 6.96-7.01 (s, 1H), 6.38-6.44 (m, 4H), 6.30-6.36 (m, 2H), 6.08-6.13 (m, 1H), 5.19-5.22 (d, 1H), 5.05-5.09 (t, 1H), 4.18-4.23 (m, 1H), 3.77-3.81 (m, 1H), 3.52-3.66 (m, 2H), 3.30-3.32 (m, 8H), 2.12-2.19 (m, 1H), 1.96-2.04 (m, 1H), 1.03-1.11 (t, 12H).

[0097] ESI-MS (m / z): 799.95, molecular formula: C 44 H 45 N7O6S, the molecular weight of theoretical calculation is 800.

[0098] Example 2

[0099] Application of fluorescent probe RBH-EdC (synthesized in Example 1) in Fe 3+ detection.

[0100] Aqueous solutions of different metal ions (Ag + , Al 3+ , Li + , Na + , K + , Cd 2+ , Co 2+ , Cr 2+ , Cr 3+ , Cu 2+ , Fe 2 + , Fe 3+ , Hg 2+ , Li + , Mg 2+ , Ni 2+ and Mn 2+ ) were prepared with double distilled water, all at a concentration of 1000 μM, and then diluted to different concentrations of 100 μM. Meanwhile, RBH-EdC solutions at a concentration of 25 μM were prepared with DMSO by diluting the 1000 μM RBH-EdC stock solution. The fluorescence spectra of each mixture were measured at an excitation wavelength of 515 nm after mixing the 25 μM RBH-EdC solution with different metal ion solutions (all at 100 μM). Figure 3 Figure 2 shows the fluorescence spectra of RBH-EdC mixed with different metal ions. The results show that the fluorescence intensity of RBH-EdC mixed with Fe 3+ increased nearly 20-fold, and the solution changed color from colorless to pink, which can be observed by the naked eye. Figure 4 Figure 3 shows the fluorescence spectra of RBH-EdC mixed with different concentrations of Fe 3+ and the linear fitting of the Fe 3+ concentration and fluorescence intensity (R 2 = 0.9979). The results show that the fluorescence intensity increased and the solution gradually changed from colorless to pink as the Fe 3+ concentration increased (1 μM-100 μM). The fluorescence intensity of the RBH-EdC mixed solution with different concentrations of Fe 3+ (1-100 μM) was calculated, and the results show that the fluorescence intensity and concentration were linearly related, and the detection limit was as low as 0.03 μM.

[0101] In addition, RBH-EdC still showed high selectivity for Fe 3+ in a complex solution containing Fe 3+ and another metal cation. Figure 5 Figure 4 shows the fluorescence spectra of RBH-EdC, RBH-EdC mixed with Fe 3+ and RBH-EdC mixed with Fe 3+ in a competitive experiment.A comparative analysis of fluorescence intensity in a mixture with other metal cations (where "others" in each column 3 corresponds to the metal cations on the horizontal axis) shows that, compared to the fluorescence intensity of RBH-EdC itself, the fluorescence intensity in the mixture of RBH-EdC and Fe... 3+ Mixture, RBH-EdC and Fe 3+ The fluorescence intensity was significantly enhanced in the mixture with another metal cation, demonstrating that RBH-EdC exhibits enhanced fluorescence intensity in Fe. 3+ It can still maintain its resistance to Fe in a mixture with another metal cation. 3+ High selectivity. These experiments strongly demonstrate that RBH-EdC can serve as a visually sensitive Fe... 3+ Detection probe.

[0102] Example 3

[0103] Application of fluorescent probe RBH-EdC (synthesized in Example 1) in solution pH monitoring.

[0104] Figure 6 Figure 'a' shows the fluorescence intensity statistics of double-distilled aqueous solutions of RBH-EdC at different pH values. The results show that the solution color changed significantly when RBH-EdC was added to solutions at different pH values. When the pH value was between 1 and 2.5, the solution changed from colorless to pink, and the color gradually deepened with increasing pH. When the pH value was between 2.5 and 4.5, the red color of the solution continuously weakened, and the higher the pH, the lighter the color. When the pH value was greater than 4.5, the solution returned to colorless, and the color remained unchanged until pH 12. Furthermore, the change in fluorescence intensity was consistent with the change in solution color. When the pH value was between 1 and 2.5, the fluorescence intensity continuously increased with increasing pH, reaching a peak at pH 2.5 with a fluorescence intensity exceeding 4000, nearly 40 times higher than at pH 1. When the pH value was between 2.5 and 4.5, the fluorescence intensity decreased sequentially, also showing a strong change of up to 40 times. When the pH value is greater than 4.5, the fluorescence intensity is very weak, dropping to its lowest level, and remains at its lowest level even at a pH value of 12. These experimental data show that RBH-EdC can sensitively reflect changes in the pH value of a solution in the pH range of 1-4.5, fully meeting the basic requirements of a pH probe, and can be used as a pH test strip for sensitive detection of pH value 2.5.

[0105] Furthermore, after adding RBH-EdC to a solution with a pH of 2.6, the pH was repeatedly adjusted eight times using hydrochloric acid and sodium hydroxide. Even after four acid-base cycles, RBH-EdC accurately responded with the corresponding fluorescence intensity, maintaining its sensitive pH detection. Figure 6 a), Figure 6Figure b shows the fluorescence intensity statistics of the fluorescent probe in the reversibility experiment. This demonstrates that RBH-EdC also exhibits excellent reversibility and can accurately reflect changes in solution pH in real time.

[0106] Example 4

[0107] The fluorescent probe RBH-EdC (synthesized in Example 1) was used in intracellular Fe 3+ Applications in detection and cell imaging.

[0108] Before conducting cell experiments, the cytotoxicity of RBH-EdC to NIH-3T3 cells was detected using the MTT assay. NIH-3T3 cells were co-incubated with different concentrations of RBH-EdC ranging from 0 to 400 μM for 24 hours, and cell viability at different concentrations was calculated. Figure 7 The survival rate of NIH-3T3 cells after co-incubation with different concentrations of RBH-EdC for 24 hours was measured. The results showed that the cell survival rate was greater than 85% at concentrations of 12.5-400 μM. Even at a probe concentration as high as 400 μM, it still showed almost no toxicity to the cells, demonstrating that RBH-EdC has excellent biocompatibility.

[0109] Intracellular Fe 3+ During the assay, appropriately adherent cells were first incubated with 50 μM and 100 μM Fe2+. 3+ Co-incubation for 1 hour allows intracellular Fe 3+ The concentration was kept constant. Then, after co-incubating the cells with 12.5 μM RBH-EdC for 4 hours, live-cell fluorescence imaging was performed. Figure 8 For cells with different concentrations of Fe 3+ Cellular fluorescence imaging of RBH-EdC under certain conditions, where a represents Fe. 3+ Cell fluorescence imaging at normal concentrations; b represents Fe 3+ Cell fluorescence imaging at a concentration of 50 μM; c represents Fe 3+ Cell fluorescence imaging at a concentration of 100 μM showed that, without altering intracellular Fe... 3+ At normal concentrations, RBH-EdC exhibits very weak fluorescence, making it difficult to even observe the cell outlines. When Fe... 3+ With increasing concentrations (50 μM and 100 μM), the fluorescence intensity of RBH-EdC significantly increased, allowing for clear observation of cell shape. Simultaneously, fluorescence analysis revealed specific locations of Fe... 3+ The concentration situation, such as Figure 8 In cell c, the cell in the lower left corner clearly shows stronger fluorescence intensity, indicating that the cell has Fe2+ fluorescence. 3+ Higher concentrations. These experimental results demonstrate that RBH-EdC can effectively detect intracellular Fe.3+ and in response to Fe 3+ concentration, and presents direct visual feedback. The fluorescent probe can also be used for cell imaging, which has stable fluorescence signal in cells, and the intensity of fluorescence signal is enhanced with the increase of Fe 3+ concentration, and the imaging effect is obvious.

[0110] Example 5

[0111] Application of fluorescent probe RBH-EdC (synthesized in Example 1) in lysosome targeting.

[0112] The green commercial lysosome targeting probe (concentration of 0.05 μM) and RBH-EdC (concentration of 25 μM) were sequentially stained on NIH-3T3 cells, and then cell fluorescence imaging was performed, and the green channel (commercial lysosome targeting probe) and red channel (RBH-EdC) of the cell fluorescence imaging image were compared using Image J software. Figure 9 The cell fluorescence imaging image and scatter plot of the lysosome co-localization of the probe, wherein a1 is the cell imaging image of RBH-EdC in the red channel, a2 is the cell imaging image of the commercial lysosome targeting probe in the green channel, a3 is the superimposed image of a1 and a2, and a4 is the scatter plot of the red fluorescence sites of RBH-EdC and the green fluorescence sites of the commercial lysosome targeting probe. From the scatter plot in a4, it can be seen that the scatter points tend to coincide into a straight line, that is, the degree of overlap of the fluorescence point sites of RBH-EdC and the commercial lysosome targeting probe is high, and the Pearson co-localization coefficient is 0.863. These results show that RBH-EdC has strong potential for lysosome targeting and can be applied to the improvement of lysosome-targeted drugs and related diagnostic means.

[0113] Example 6

[0114] Application of fluorescent probe RBH-EdC (synthesized in Example 1) in the detection of gastric acid pH in acid suppression therapy.

[0115] Before the verification experiment using mice, the toxicity of RBH-EdC to mice was evaluated by histological analysis (H&E). Ten 4-week-old, 10-15 g male BALB / c mice with similar gastric acid pH and digestive function were taken, and after adaptive feeding for one week, they were grouped, and the control group and the experimental group were set up, each with five mice. The control group of mice and the experimental group of mice were respectively given 200 μL of PBS solution and 2 mg of RBH-EdC, and then mouse toxicology tissue sections were taken. Figure 10Toxicological tissue slice images of the control group and experimental group mice, wherein a is a stomach tissue slice image, and b is a liver tissue slice image. The results show that the liver and stomach tissue edges of the two groups of mice are clear, and the tissue cells are full and have no obvious pathological changes. It is shown that RBH-EdC has good biocompatibility and can be used for in vivo imaging.

[0116] Subsequently, acid suppression experiments were performed on the mice. Ten 4-week-old male BALB / c mice with similar stomach acid pH values and digestive functions, weighing 10-15 g, were taken and adaptively fed for one week before being grouped. The control group and the experimental group each had five mice. The experimental group was orally administered 5.5 mg of an acid suppressant (sodium bicarbonate) and 2 mg of RBH-EdC, and the control group was orally administered 2 mg of RBH-EdC. Subsequently, in vivo imaging and stomach juice pH monitoring (using acid precision pH test paper) were performed on the mice at 5, 15, 30, 60, 90, and 120 minutes after administration. Figure 11 In vivo fluorescence imaging images of the control group mice at each time point during acid suppression treatment, Figure 12 In vivo fluorescence imaging images of the experimental group mice at each time point during acid suppression treatment, the results show that since the control group did not take the acid suppressant, its stomach acid did not fluctuate greatly, and the acidity remained at about pH 2. Therefore, the fluorescence of the mouse stomach remained at a certain brightness within 2 hours. After the experimental group mice took the acid suppressant, the pH of the stomach acid increased, and with the in vivo metabolism and self-regulation ability, the acidity of the mouse stomach acid gradually returned to normal. From the analysis of the in vivo fluorescence imaging images, Figure 12 ), within 5-30 minutes, the pH value of the mouse stomach juice was still high, and the fluorescence of the gastrointestinal tract was quenched. Around 60 minutes, the stomach fluorescence began to recover and was in a strong state, from which it can be concluded that the mouse stomach juice acidity basically returned to normal. After about 2 hours of administration, with the metabolism of the mouse body, the content of RBH-EdC in the body gradually decreased, and the fluorescence began to decline. These experimental results confirm that RBH-EdC is a high-sensitivity pH probe that can overcome a strong acid environment and can be used to track the changes in stomach acid pH in real time during acid suppression treatment, and is expected to be a powerful tool for gastrointestinal treatment and diagnosis and related drug design.

[0117] Example 7

[0118] Application of the fluorescence probe RBH-EdC (synthesized in Example 1) in the diagnosis of gastric ulcers.

[0119] Using the principle that excessive aspirin prevents the synthesis of endogenous prostaglandins to cause gastric ulcers, gastric ulcer modeling was achieved on the experimental group mice. The control group was normal mice, and the experimental group was gastric ulcer model mice. After all the mice were orally administered 2 mg of RBH-EdC, in vivo fluorescence imaging was performed, and the pH of the stomach juice was monitored using precision pH test paper. Figure 13The in vivo fluorescence imaging diagram of the experimental group and the control group mice in the gastric ulcer experiment shows that the control group mice present bright fluorescence in the gastrointestinal part, and the fluorescence of the experimental group is all quenched. From the gastric juice pH value, the gastric juice pH of the control group mice remains normal, about 2.5. The experimental group mice are in the state of gastric ulcer, and the gastric acid pH is between 3.5-4.2. These results confirm that the RBH-EdC can accurately feedback the pH value of gastric acid through fluorescence intensity, and it is shown that the probe can realize real-time, in-situ and non-invasive diagnosis of gastric ulcer disease, and has great application value in the diagnosis of gastric ulcer.

[0120] In summary, the present application provides a kind of fluorescence probe based on rhodamine modified nucleoside. The fluorescence probe is composed of rhodamine hydrazine-short chain alkyne-2-deoxy cytidine, has high fluorescence intensity, good biological compatibility, good strong acid resistance, high sensitivity and good chemical stability and the like. The preparation method of the fluorescence probe is simple, efficient, and raw materials are easy to obtain. The fluorescence probe can be widely applied to in vivo / Fe 3+ Detection, real-time monitoring of pH dynamics, preparation of pH test paper, cell imaging, construction of lysosome targeting functional probe, design of in vivo contrast agent, diagnosis and treatment of gastrointestinal diseases and screening of related therapeutic drugs, and has great practical application value.

[0121] It should be understood that the application of the present application is not limited to the above examples, and those skilled in the art can improve or change according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.

Claims

1. A fluorescent probe based on rhodamine-modified nucleoside, characterized by, The structural formula of the fluorescent probe is as follows: 。 2. A method for preparing the fluorescent probe based on rhodamine-modified nucleoside according to claim 1, characterized by, The method comprises the steps of: Providing compound 2 and compound 3, and subjecting the compound 2 and the compound 3 to Schiff base reaction to obtain compound 4; Under the protection of inert gas, the compound 4, 5-iodo-2-deoxy cytidine, a palladium catalyst, triethylamine and cuprous iodide are added into a first solvent to react, so as to obtain the fluorescent probe; wherein the compound 2 has a structural formula of , the compound 3 has a structural formula of , and the compound 4 has a structural formula of .

3. The method for preparing a rhodamine-modified nucleoside according to claim 2, wherein, The preparation method of the compound 2 comprises the steps of: Providing 4-bromothiophene-2-carboxaldehyde and trimethylsilane acetylene, and subjecting the 4-bromothiophene-2-carboxaldehyde and the trimethylsilane acetylene to coupling reaction to obtain compound 1; Subjecting the compound 1 to reaction under the action of a base to obtain the compound 2; The preparation method of the compound 3 comprises the steps of: subjecting rhodamine B and hydrazine hydrate to reaction to obtain the compound 3; wherein the structural formula of the compound 1 is: .

4. Use of a fluorescent probe based on rhodamine-modified nucleoside according to claim 1 for the preparation of Fe 3+ detection probes.

5. Application of the rhodamine-modified nucleoside-based fluorescent probe in claim 1 in the preparation of a solution pH monitoring probe.

6. Application of the rhodamine-modified nucleoside-based fluorescent probe in claim 1 in cell imaging for non-disease diagnosis or treatment purposes.

7. Application of the rhodamine-modified nucleoside-based fluorescent probe in claim 1 in the preparation of a lysosome-targeting probe.

8. Application of the rhodamine-modified nucleoside-based fluorescent probe in claim 1 in the preparation of a gastric acid pH detection probe.

9. Application of the rhodamine-modified nucleoside-based fluorescent probe in claim 1 in the preparation of a gastric ulcer diagnosis reagent.

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