Fluorescent probe compound based on hydrogen peroxide response group protection mechanism and synthesis and application thereof

Abstract

The invention discloses a lysosome targeting fluorescent probe compound based on a hydrogen peroxide response group protection mechanism. The lysosome targeting fluorescent probe compound comprises a naphthalimide fluorophore, a morpholine group and an N-methyl iminodiacetic acid borate (MIDA borate) protection structure. The morpholine group endows the lysosome with targeting ability, the MIDA borate improves the blood circulation stability, and the naphthalimide fluorophore generates a fluorescence signal after responding to H2O2. The synthesis method comprises the steps of synthesis of a naphthalimide precursor, introduction of a morpholine group and protection of MIDA borate. The probe can specifically recognize H2O2, the lowest detection limit reaches 0.25 mu M, the probe can target HeLa cell lysosome and realize high-sensitivity detection, and the cytotoxicity is low. Response group protection and targeting functions are creatively combined, the problems that an existing probe is poor in blood circulation stability and insufficient in detection specificity are solved, and the probe has important practical value in the fields of disease mechanism research, early diagnosis, medicine research and development and the like.

Description

Technical Field

[0001] This invention relates to a fluorescent probe compound with a hydrogen peroxide-responsive group protection mechanism, its synthesis method, and its application, belonging to the fields of chemical synthesis and biomaterials. Background Technology

[0002] Hydrogen peroxide (H2O2), as a key reactive oxygen species, plays a significant role in physiological and pathological processes. Under normal conditions, it maintains a low-concentration homeostasis within cells and participates in signal transduction; however, its level rises abnormally in diseases such as inflammation and tumors. Especially in the tumor microenvironment, the high metabolism of tumor cells and the infiltration of immune cells lead to H2O2 concentrations far exceeding those of normal tissues. This concentration change is closely related to disease development; therefore, accurate detection of its dynamic changes is of great significance for disease research, diagnosis, and efficacy evaluation. Fluorescent probe technology, with its advantages of high sensitivity, strong selectivity, rapid detection, and real-time in-situ observation, has become an important means of detecting H2O2. An ideal H2O2 fluorescent probe must meet three core conditions: specific response to H2O2 to avoid interference, good biocompatibility to ensure application safety, and stability in blood circulation to effectively reach the target site. However, existing H2O2 fluorescent probes suffer from insufficient stability in blood circulation. The blood environment is complex, containing abundant enzymes, proteins, and diverse physicochemical factors, which can easily lead to degradation, inactivation, or non-specific reactions of traditional probes. For example, some probes based on common organic molecules may have their response groups prematurely hydrolyzed by enzymes in the bloodstream, causing the probe to lose its responsiveness before reaching the target site, which seriously affects the reliability of the detection.

[0003] To address this issue, introducing a protective structure with a responsive group is an effective solution. N-Methyliminodiacetic acid borate (MIDA borate), as a special borate derivative, exhibits excellent chemical stability and resists interference from enzymatic degradation and other chemical reactions in the biological environment. Using it in fluorescent probe design can temporarily protect the responsive group, avoiding unnecessary reactions in the bloodstream. When the probe reaches a high-concentration H2O2 target site, H2O2 specifically reacts with MIDA borate, releasing the protection and restoring the probe's responsiveness and fluorescence signal, providing a feasible approach to improving stability. Meanwhile, lysosomes, as important organelles, are acidic (pH 4.5-5.5) and contain various hydrolases; their function and internal H2O2 levels are significantly altered in disease states. Specific detection of H2O2 within lysosomes is crucial for related research. Due to its unique structure and basicity, the morpholine group can interact with the acidic environment of lysosomes, exhibiting clear targeting. Introducing it into probes allows for probe enrichment within lysosomes, enabling precise detection.

[0004] While both strategies have their advantages, fluorescent probes that simultaneously possess the three properties of blood circulation stability, H2O2 responsiveness, and lysosomal targeting are still relatively rare. Existing probes struggle to achieve these key properties simultaneously, limiting their application in the biomedical field. Therefore, developing structurally innovative H2O2 fluorescent probes that can simultaneously address the issues of stability, responsiveness, and targeting is both an urgent practical need and of significant scientific importance. Summary of the Invention

[0005] The purpose of this invention is to provide a fluorescent probe compound based on the protection mechanism of hydrogen peroxide responsive groups and its synthesis method; Another object of the present invention is to provide the application of the probe compound in the detection of H2O2.

[0006] I. Fluorescent probe compounds with hydrogen peroxide-responsive group protection mechanism and their synthesis The probe compound (MIDA borate ester-naphthalimide derivative) provided by this invention has the following structural formula: The synthesis method provided by this invention includes the following steps: (1) Dissolve N-(2-aminoethyl)morpholine and 4-bromo-1,8-naphthalenedicarboxylic anhydride in ethanol solution, reflux at 75~80℃ for 4~6h, cool to room temperature, wash, and filter to obtain compound A (4-bromo-1,8-naphthalenedicarboxylic anhydride based on morpholine group). The structural formula of compound A is: ; The molar ratio of 4-bromo-1,8-naphthoic anhydride to morpholine derivative is 1:1.3 to 1:3.5.

[0007] (2) Compound A was dissolved in methanol solution. Copper sulfate was used as catalyst. Sodium methoxide was added and reacted at 60-70 °C for 40-50 min. HI was added and reacted at 40-55 °C for 2-3 h. The solvent was evaporated to obtain a milky yellow solid. After drying, it was dissolved in TFA and hexamethylenetetramine was added. After reacting at 50-75 °C for 3-6 h, saturated brine was added. The mixture was extracted with CH2Cl2. The organic phase was evaporated to dryness and separated by column chromatography to obtain compound B (4-hydroxy-1,8-naphthalenedicarboximide based on the morpholine group). The structural formula of compound B is: ; The molar ratio of compound A to sodium methoxide is 1:1.5 to 1:3; the molar ratio of compound A to copper sulfate is 1:0.1 to 1:0.5; the molar ratio of compound A to HI is 1:1.2 to 1:2.5; and the molar ratio of compound A to hexamethylenetetramine is 1:0.2 to 1:0.5.

[0008] (3) Compound B, N-methylimino diacetate borate and K2CO3 were dissolved in acetonitrile solution and stirred at room temperature for 9-14 h under nitrogen protection. After the reaction was complete, the mixture was filtered and NaBH4 was added to the filtrate and the reaction was continued at room temperature for 18-22 h. The probe compound (MIDA-substituted-1,8-naphthalenediamine based on morpholine group) was obtained by column chromatography and labeled as LGM.

[0009] The molar ratio of compound B, N-methylimino diacetate borate and K2CO3 is 1:1:1.5 to 1:1.5:2; the molar ratio of compound B to NaBH4 is 1:1 to 1:2.

[0010] II. Application of compound LGM in H2O2 detection 1. Fluorescence properties and H2O2 response of compound LGM Fluorescence was measured in a DMSO / PBS solution (DMSO:PBS = 1:9, c = 3 µM) of compound LGM. The study found that compound LGM exhibited no fluorescence emission due to the blocking effect of MIDA borate ester, and the fluorescent probe compound was quenched (e.g., ...). Figure 7 When 45 eq H2O2 was added, the fluorescence intensity reached its maximum at an emission wavelength of 525 nm, indicating that the compound LGM has a good responsiveness to H2O2 (e.g., Figure 7 ).

[0011] 2. Effect of different pH values ​​on the response of compound LGM to H2O2 Figure 8 The figure shows the fluorescence response curves of the compound LGM at different pH values. As can be seen from the figure, in the pH range of 1.0 to 8.0, without the participation of H2O2, the fluorescence of the compound is quenched, meaning its fluorescence intensity does not change significantly. However, when 45 eq H2O2 is added to the system, the fluorescence intensity of LGM increases with increasing pH. The fluorescence intensity of compound LGM reaches its maximum value in the pH range of 5 to 6. This indicates that compound LGM exhibits excellent optical properties under weakly acidic conditions, suggesting its potential for biological applications.

[0012] 3. Single-selective detection of H2O2 by compound LGM In a DMSO / PBS solution of compound LGM (DMSO:PBS = 1:9, c = 3 µM), 45 eq (relative to compound LGM) of reactive oxygen species H₂O₂, TBHP, HClO, KO₂, and NO were added, respectively. Only the addition of H₂O₂ caused the DMSO / PBS solution of LGM to produce strong fluorescence at 525 nm. The reactive oxygen species TBHP, HClO, KO₂, and NO did not significantly change the fluorescence intensity of the DMSO / PBS solution of compound LGM (e.g., ...). Figure 9 Therefore, LGM is able to selectively identify H2O2.

[0013] Fluorescent titration experiments were performed by adding 0–90 eq H2O2 to a DMSO / PBS solution of compound LGM (DMSO:PBS = 1:9, c = 3 µM). Figure 10 The results showed that under acidic conditions (pH=6.0), the fluorescence intensity of compound LGM significantly increased with increasing hydrogen peroxide concentration until saturation.

[0014] Based on fluorescence titration experiments, a linear regression equation (K=20.740, R0.05) was obtained for H2O2 concentration within the range of 0–45 eq. 2 =0.984), such as Figure 11 As shown in the figure. Calculations using the equation revealed that the limit of detection for H2O2 by compound LGM is 0.25 µM, indicating that compound LGM has very high sensitivity and that weakly acidic conditions are more conducive to improving the sensitivity of compound LGM.

[0015] III. Application of LGM in H2O2 Detection in Cells Studies have confirmed that fluorescent probes protected with hydrogen peroxide responsive groups exhibit both high sensitivity and high selectivity when detecting H2O2 in physiological environments. To investigate the intracellular response of this probe, HeLa cells were co-incubated with 1.0 µM of the derivative LGM at 37°C for 2 hours. Fluorescence imaging (Figure 12) revealed clear and bright green fluorescence. Due to LGM's pH-regulating properties, to verify its targeting ability to lysosomes, a co-localization experiment was conducted using 1.0 µM of commercially available LysoTracker Red (excitation 579 nm, emission 599 nm) as a control. After co-incubation with HeLa cells at 37°C for 30 minutes, a distinct yellow superimposed area appeared in the confocal imaging (Figure 12(c)). This area highly coincided with the LysoTracker Red staining area (Figure 12(b)), confirming that LGM can target lysosomes and effectively diffuse into the cytoplasm, specifically accumulating within lysosomes. Therefore, lysosomal fluorescence signals can be used to monitor endogenous H2O2 activity within cells. To evaluate the intracellular localization effectiveness, toxicity, and effects on apoptosis and proliferation of LGM, researchers conducted a cytotoxicity experiment (Figure 13): HeLa cells were co-incubated with different concentrations (1–35 μM) of LGM. The results showed high cell viability, with a survival rate greater than 79% even at a high concentration of 35 μM. This indicates that LGM has low toxicity and will not affect its effectiveness in detecting H2O2 within cells, making it suitable for rapid and sensitive detection of H2O2 via lysosomes in the cellular microenvironment. The probe compound of this invention comprises a naphthimide fluorophore, a morpholine group, and a responsive group protecting structure, N-methyliminodiacetate borate (MIDA borate). The morpholine group endows the probe with lysosomal targeting capability, enabling precise localization to intracellular lysosomes. The responsive group protecting structure (MIDA borate) significantly improves the stability of the probe in blood circulation, preventing premature activation or degradation. The naphthimide fluorophore, acting as a signal reporter unit, exhibits a significant change in fluorescence properties upon specific response to hydrogen peroxide. The synthesis method of this fluorescent probe compound includes the synthesis of a naphthimide fluorophore precursor, the introduction of the morpholine group, and the attachment of the responsive group protecting structure. In application, after incubating biological samples with the probe, highly sensitive and selective detection of hydrogen peroxide within lysosomes can be achieved using a fluorescence detection device.

[0016] The core advantage of this probe compound lies in the synergistic effect of the MIDA borate ester response protection mechanism and the lysosomal targeting of the morpholine group: MIDA borate ester can remain stable in the bloodstream and complex cellular environments, preventing premature activation or degradation of the response group. Upon contact with high concentrations of H2O2, it can specifically deprotect and generate a strong fluorescent signal. Compared with existing technologies, this probe solves the problems of poor cyclic stability, weak targeting, and low specificity of traditional probes, providing a reliable detection tool for disease-related oxidative stress research such as early tumor diagnosis and exploration of inflammation mechanisms, demonstrating significant practical value.

[0017] The beneficial effects of this invention are as follows: 1. Innovative Structural Design: This invention organically combines the MIDA borate ester responsive group protection structure, the naphthimide fluorophore, and the morpholine lysosome targeting group to construct a novel H2O2 fluorescent probe. This design enables the probe to possess high stability, high targeting, and high response specificity.

[0018] 2. Excellent blood circulation stability: The introduced MIDA borate ester protective structure can significantly improve the stability of the probe in blood circulation, effectively preventing the response group from being activated prematurely or the probe from being degraded, ensuring that the probe can effectively reach the target site.

[0019] 3. Precise lysosomal targeting capability: The morpholine group in the probe molecule can specifically target lysosomes in cells, enabling precise detection of H2O2 in this specific subcellular structure, providing a powerful tool for studying the relationship between lysosomal dysfunction and disease.

[0020] 4. High sensitivity and high selectivity: After the naphthalimide fluorophore in the probe specifically recognizes H2O2 and removes MIDA protection, its fluorescence properties change significantly (e.g., fluorescence enhancement), achieving highly sensitive detection of H2O2. Experiments have shown that this probe has good selectivity for H2O2 and is not affected by other common reactive oxygen species (such as TBHP, HClO, KO2, and NO).

[0021] 5. Good biocompatibility: Cytotoxicity experiments show that the probe has low cytotoxicity and is suitable for live cell imaging and detection.

[0022] 6. Suitable pH response range: The probe exhibits optimal fluorescence response under weakly acidic conditions of pH 5-6 (matching the internal pH environment of lysosomes), further ensuring its high sensitivity detection in lysosomes. Attached Figure Description

[0023] Figure 1 This is the mass spectrum of compound A of the present invention; Figure 2 This is the proton NMR spectrum of compound A of the present invention; Figure 3 This is the mass spectrum of compound B of the present invention; Figure 4 The hydrogen spectrum of compound B of this invention is shown below. Figure 5 This is the mass spectrum of the compound LGM of this invention; Figure 6 This is the proton NMR spectrum of the compound LGM of this invention; Figure 7 This is the fluorescence spectrum of the LGM of this invention; Figure 8 The fluorescence spectra of the LGM of this invention at different pH values ​​are shown below. Figure 9 This invention provides the selectivity of LGM for different interfering substances; Figure 10 The following are fluorescence spectra of the LGM of this invention for different concentrations of H2O2; Figure 11 This invention provides a linear fitting relationship between the LGM of the present invention at an excitation wavelength of 525 nm and different concentrations of H2O2 (0~45 eq); Figure 12 This invention provides fluorescence imaging of LGM in HeLa cells. Figure 13 This is a cytotoxicity experiment of LGM in this invention. Detailed Implementation

[0024] The preparation and application of the LGM of the present invention will be further illustrated below through specific embodiments.

[0025] Example 1: Preparation of LGM (1) N-(2-aminoethyl)morpholine (1.5 mmol, 0.195 g) and 4-bromo-1,8-naphthalenedicarboxylic anhydride (1 mmol, 0.28 g) were dissolved in 20 ml of ethanol solution, refluxed at 75 °C for 5 h, cooled to room temperature, washed, and filtered to obtain compound A (a 4-bromo-1,8-naphthalenedicarboxylic anhydride based on the morpholine group), with a yield of 66.2%; the mass spectrum and proton spectrum of compound A are shown below. Figure 1 and Figure 2 .

[0026] (2) Compound A (1.0 mmol, 0.38 g) was dissolved in 20 mL of methanol solution. Using copper sulfate (0.4 mmol, 0.064 g) as a catalyst, sodium methoxide (2.0 mmol, 0.12 g) was added and reacted at 65 °C for 45 min. Then, 1.5 mL of HI was added and reacted at 50 °C for 2 h. The solvent was evaporated to obtain a milky yellow solid. After drying, the solid was dissolved in 10 mL of TFA, and hexamethylenetetramine (0.3 mmol, 0.04 g) was added. The reaction was carried out at 60 °C for 4 h. Saturated brine was added, and the mixture was extracted with CH2Cl2. The organic phase was evaporated to dryness, and column chromatography was used to obtain compound B (a 4-hydroxy-1,8-naphthalenediamine compound based on the morpholine group), with a yield of 54.5%. The mass spectrum and proton NMR spectrum of compound B are shown below. Figure 3 and Figure 4 .

[0027] (3) Compound B (1.0 mmol, 0.33 g), N-methyliminodiacetate borate (1.2 mmol, 0.2 g), and 0.21 g K₂CO₃ were dissolved in 20 mL of acetonitrile solution. The mixture was stirred at room temperature for 10 h under nitrogen protection. After the reaction was complete, the mixture was filtered. NaBH₄ (1.2 mmol, 0.2 g) was then added to the filtrate, and the reaction was continued at room temperature for another 20 h. Column chromatography yielded MIDA-substituted-1,8-naphthalenediamine compounds based on the morpholine group, labeled LGM. The yield was 58.1%. The mass spectrum and proton NMR spectrum of LGM are shown below. Figure 5 and Figure 6 .

[0028] The LGM crafting route is as follows: .

[0029] Example 2: Detection of H2O2 by LGM In a DMSO / PBS solution of compound LGM (DMSO:PBS = 1:9, c = 3 µM), 45 eq (relative to compound LGM) of reactive oxygen species H2O2, TBHP, HClO, KO2, and NO were added respectively. If the DMSO / PBS solution of compound LGM produced strong fluorescence at 525 nm, then H2O2 was added; if the fluorescence intensity of the DMSO / PBS solution of LGM did not change significantly, then other reactive oxygen species were added.

Claims

1. A fluorescent probe compound based on the protection mechanism of hydrogen peroxide responsive groups, the structural formula of which is as follows: 。 2. The method for synthesizing the fluorescent probe compound as described in claim 1, comprising the following steps: (1) Dissolve N-(2-aminoethyl)morpholine and 4-bromo-1,8-naphthalenedicarboxylic anhydride in ethanol solution, reflux at 75~80℃ for 4~6h, cool to room temperature, wash, and filter to obtain compound A; The structural formula of compound A is: ； (2) Dissolve compound A in methanol solution, use copper sulfate as catalyst, add sodium methoxide and react at 60~70 ℃ for 40~50 min, add HI and react at 40~55 ℃ for 2~3 h, evaporate the solvent to obtain a milky yellow solid, dry it and dissolve it in TFA, add hexamethylenetetramine, react at 50~75 ℃ for 3~6 h, add saturated brine, extract with CH2Cl2, evaporate the organic phase, and separate by column chromatography to obtain compound B; The structural formula of compound B is: ； (3) Compound B, N-methylimino diacetate borate and K2CO3 were dissolved in acetonitrile solution and stirred at room temperature for 9-14 h under nitrogen protection. After the reaction was complete, the mixture was filtered and NaBH4 was added to the filtrate and the reaction was continued at room temperature for 18-22 h. The probe compound was obtained by column chromatography.

3. The method for synthesizing the fluorescent probe compound as described in claim 2, characterized in that: In step (1), the molar ratio of 4-bromo-1,8-naphthoic anhydride and morpholine derivative is 1:1.3 to 1:3.

5.

4. The method for synthesizing the fluorescent probe compound as described in claim 2, characterized in that: In step (2), the molar ratio of compound A to sodium methoxide is 1:1.5 to 1:3; the molar ratio of compound A to copper sulfate is 1:0.1 to 1:0.5; the molar ratio of compound A to HI is 1:1.2 to 1:2.5; and the molar ratio of compound A to hexamethylenetetramine is 1:0.2 to 1:0.

5.

5. The method for synthesizing the fluorescent probe compound as described in claim 2, characterized in that: In step (3), the molar ratio of compound B, N-methylimino diacetate borate and K2CO3 is 1:1:1.5 to 1:1.5:2; the molar ratio of compound B to NaBH4 is 1:1 to 1:

2.

6. The use of the fluorescent probe compound as described in claim 1 in the detection of hydrogen peroxide for purposes other than disease diagnosis or treatment.

7. The application as described in claim 6, characterized in that, The detection refers to the detection of hydrogen peroxide in an aquatic environment or in biological samples.

8. The application as described in claim 7, characterized in that, The biological sample includes cells, and the fluorescent probe compound is capable of targeting lysosomes within the cells.

9. The application as described in claim 6, characterized in that: In the DMSO / PBS solution of the fluorescent probe compound, reactive oxygen species H2O2, TBHP, HClO, KO2, and NO were added respectively. Only the addition of H2O2 caused the DMSO / PBS solution of the fluorescent probe compound to produce strong fluorescence at 525 nm. The other reactive oxygen species did not cause a significant change in the fluorescence intensity of the DMSO / PBS solution of the fluorescent probe compound. In the DMSO / PBS solution, the volume ratio of DMSO to PBS was 1:8 to 1:

15.

10. The use of the fluorescent probe compound as described in claim 1 in the preparation of fluorescent imaging detection reagents.

Images