Naphthalimide fluorescent probe for detecting hclo and preparation method thereof

Abstract

This invention discloses a naphthimide-based fluorescent probe for detecting HClO and its preparation method. The fluorescent molecular probe uses naphthimide as the fluorophore and C=N as the specific reaction site for HClO. Due to the presence of the naphthimide fluorophore, the probe molecule exhibits two-photon properties, enabling deep tissue penetration, reducing background noise, and facilitating fluorescence imaging of biological systems. Furthermore, the introduced morpholine group allows for lysosomal targeting, enabling the monitoring of hypochlorous acid concentration levels in lysosomes, making it a valuable tool for early disease diagnosis. In addition, this type of probe molecule is simple to synthesize, operates under mild reaction conditions, exhibits stable optical properties, and has a high synthesis yield. It can detect a wide range of HClO, making it practically valuable in fields such as biochemistry and environmental science.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, specifically to a naphthalimide fluorescent probe for targeted lysosomal detection of HClO and its preparation method. Background Technology

[0002] Reactive oxygen species (ROS) include the superoxide anion (O2), a one-electron reduction product. - The two-electron reduction products are H₂O₂ and three-electron reduction products, including hydroxyl radicals (·OH), hypochlorous acid (HClO), and peroxynitrite ions (ONOO). - ), singlet oxygen, and nitric oxide (NO), where HClO is formed by the reaction of H2O2 with Cl under the catalysis of myeloperoxidase (MPO). - The product of the reaction is an unstable weak acid (pka = 7.53), which undergoes partial dissociation under physiological conditions to yield its conjugate base—hypochlorite (ClO₂). - HClO / ClO is commonly used in daily life as a bleaching agent and disinfectant. - Reactive oxygen species (ROS) are a class of important reactive oxygen species widely distributed in organisms, participating in various biological processes and playing an irreplaceable role in human immune defense. They are mainly distributed in the acidic organelles lysosomes of macrophages. The normal concentration range of HClO / ClO... – It can kill harmful bacteria, HClO / ClO - Abnormal HClO / ClO concentration levels can cause oxidative stress, oxidizing or chlorinating other biomolecules, leading to a range of diseases such as neurodegeneration, cardiovascular disease, lung injury, rheumatoid arthritis, and even some cancers. Therefore, maintaining a proper HClO / ClO concentration is crucial. - Maintaining HClO / ClO concentrations within the normal range is crucial. Furthermore, abnormal HOCl concentrations can lead to redox imbalances in lysosomes, further causing lysosomal rupture and inducing apoptosis. Therefore, monitoring the HClO / ClO ratio in vivo is essential. - Abnormal concentration levels are crucial for the early diagnosis of diseases.

[0003] Currently, the detection of HClO / ClO - There are many methods for detecting HClO / ClO, mainly including chemiluminescence, potentiometry, and colorimetry. However, while these traditional methods can achieve high-sensitivity detection, they still have certain drawbacks, such as complex sample preparation, expensive instruments, and unsuitability for biological cells. Therefore, a series of methods for detecting HClO / ClO were designed and synthesized. - Fluorescent probes are of great significance. Summary of the Invention

[0004] One objective of this invention is to provide a method for synthesizing fluorescent probes that is simple to synthesize, has mild reaction conditions, and is inexpensive; another objective is to provide a series of lysosomal-targeting fluorescent probes that can detect HClO with high sensitivity and selectivity.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The structure of the naphthalimide fluorescent probe used for detecting HClO is as follows:

[0007]

[0008] Both probe molecules use 1,8-naphthalimide as the fluorophore and introduce morpholine groups as lysosomal targeting groups. In the synthesized compounds, C=N is the reaction site of HClO.

[0009] The preparation method of the novel molecular probe for detecting HClO specifically adopts the following process route:

[0010]

[0011]

[0012] The method for preparing the probe molecule for detecting HClO specifically includes the following steps:

[0013] (1) Dissolve 4-bromo-1,8-naphthalene anhydride and N-(2-aminoethyl)morpholine in EtOH, heat under reflux under inert gas protection, after the reaction is complete, add deionized water to quench the reaction, extract with dichloromethane, dry the obtained organic phase, remove the solvent by vacuum distillation and purify to obtain a white solid, which is compound 1.

[0014] (2) Compound 1 and p-formylphenylboronic acid were dissolved in an EtOH / H2O system, and Pd(PPh3)4 and K3PO4 were added. The mixture was heated under reflux and stirred in an inert atmosphere. After the reaction was completed, deionized water was added to quench the reaction, and the mixture was extracted with dichloromethane. The resulting organic phase was dried, and the solvent was removed by vacuum distillation to obtain a white solid compound 2. Preferably, the equivalence ratio of compound 1 to p-formylphenylboronic acid was 1:1.2-1:1.5.

[0015] (3) Dissolve the above compound 2 and 2,4-dinitrophenylhydrazine in EtOH, heat to reflux, after the reaction is completed, add deionized water to quench the reaction, extract with dichloromethane, dry the obtained organic phase, remove the solvent by vacuum distillation, and purify to obtain an orange solid, which is probe I. Preferably, the equivalent ratio of compound 2 to 2,4-dinitrophenylhydrazine is 1:1.2-1:1.5, or compound 2 and 2,3-diaminomaleonitrile are dissolved in MeOH, heated to reflux, and the reaction is monitored by TLC to be completed. After purification, a yellow solid is obtained, which is probe II. Preferably, the equivalent ratio of compound 2 to 2,3-diaminomaleonitrile is 1:1.2-1:1.5.

[0016] There are no particular limitations on the method of using the fluorescent molecular probes of this invention. Typically, the probe molecules can be dissolved in a suitable organic solvent, such as tetrahydrofuran (THF) or 1,4-dioxane, and the test can be performed at room temperature.

[0017] The detection principle of the fluorescent molecular probe for HClO in this invention is shown in the following formula:

[0018]

[0019] These probe molecules all possess C=N, and the isomerization of C=N lowers the energy of the excited-state compound, effectively promoting nonradiative transitions and resulting in relatively weak autofluorescence of the probe molecules. When HClO / ClO is added to the probe system... - After that, HClO / ClO - The strong oxidizing properties of the probe cause partial oxidative cleavage, which inhibits its isomerization and releases the fluorescence of the fluorophore, i.e., fluorescence enhancement. Attached Figure Description

[0020] Figure 1 The image shows the 1H NMR spectrum of compound 1 involved in Example 1.

[0021] Figure 2 The image shows the 1H NMR spectrum of compound 2 involved in Example 1.

[0022] Figure 3 The 1H NMR spectrum of probe I involved in Example 2 ( 1 H NMR).

[0023] Figure 4 The 1H NMR spectrum of probe II involved in Example 3 ( 1 H NMR).

[0024] Figure 5 Different concentrations (0-500 μM) of HClO / ClO were added to the PBS buffer system for probe I involved in Example 2. - The fluorescence intensity changes and linear relationship.

[0025] Figure 6 Different concentrations (0-100 μM) of HClO / ClO were added to the PBS buffer system for probe II involved in Example 3. - The fluorescence intensity changes and linear relationship.

[0026] Figure 7 This is a time response diagram of probe I involved in Example 2. Detailed Implementation

[0027] The present invention will be described below with reference to the accompanying drawings and specific embodiments. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0028] Example 1: Preparation of Compound 2

[0029] (1) Dissolve 4-bromo-1,8-naphthalene anhydride and N-(2-aminoethyl)morpholine in EtOH, heat under nitrogen protection and reflux. After the reaction is completed by TLC monitoring, add deionized water to quench the reaction, extract with dichloromethane, dry the obtained organic phase, remove the solvent by vacuum distillation and purify to obtain a white solid, which is compound 1 (yield: 95%).

[0030] (2) The above compound 1 and p-formylphenylboronic acid were dissolved in an EtOH / H2O system with an equivalent ratio of 1:1.2. Pd(PPh3)4 and K3PO4 were added and the mixture was refluxed and stirred under a nitrogen atmosphere. The reaction was monitored by TLC until it ended. The reaction was quenched with deionized water and extracted with dichloromethane. The resulting organic phase was dried and the solvent was removed by vacuum distillation. The mixture was purified to obtain a white solid compound 2 (yield: 89%).

[0031] Example 2: Preparation of Fluorescent Probe I

[0032] Compound 2 prepared in Example 1 and 2,4-dinitrophenylhydrazine were dissolved in EA / EtOH with an equivalent ratio of 1:1.2. The mixture was heated to reflux, and after the reaction was monitored by TLC, deionized water was added to quench the reaction. The mixture was then extracted with dichloromethane, and the resulting organic phase was dried. The solvent was removed by vacuum distillation, and the purified orange solid was obtained as probe I (yield: 86%).

[0033] Example 3: Preparation of Fluorescent Probe II

[0034] Compound 2 prepared in Example 1 was dissolved in methanol with 2,3-diaminomaleonitrile in an equivalent ratio of 1:1.3. The mixture was heated to reflux and the reaction was monitored by TLC until it ended. The resulting yellow solid was purified and was probe II (yield: 82%).

[0035] Example 4: Probe molecule detection of HClO / ClO - Application

[0036] The fluorescent probe molecules are used to buffer HClO / ClO in the system. - The specific methods for determining the content are as follows:

[0037] The probe molecules were added to the organic solvent DMF to obtain a probe molecule stock solution, and then different HClO / ClO ratios were prepared. - Concentration (10) -4 10 -3 10 -2 10 -1 A standard solution of M) was added. Different concentrations of HClO / ClO were added to a DMF:PBS = 1:9 (pH = 7.4) mixture. - After standing for 1 minute, the fluorescence intensity of the system at different concentrations was tested.

[0038] from Figure 5 , Figure 6 It can be seen that both probe I and probe II are effective against HClO / ClO - It has a wide detection range and can detect HClO / ClO - To achieve quantitative detection.

[0039] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A naphthalimide-based molecular probe for detecting HCIO, characterized in that, The structural formula is 2. A method for preparing the naphthalimide-based molecular probe for detecting HCIO according to claim 1, characterized in that, The preparation method adopts the following process route:

3. The method for preparing the naphthalimide-based molecular probe for detecting HClO according to claim 2, wherein, Specifically includes the following steps: (1) 4-bromo-1,8-naphthalene anhydride, N-(2-aminoethyl) morpholine is dissolved in EtOH, heated under reflux in inert gas protection, after the reaction is completed, deionized water is added to quench the reaction, extracted with dichloromethane, the obtained organic phase is dried, the solvent is removed by reduced pressure distillation and purified to obtain white solid, which is compound 1; (2) Compound 1 and p-formylphenylboronic acid are dissolved in EtOH / H2O system, Pd(PPh3)4 and K3PO4 are added, heated under reflux and stirred in inert gas atmosphere; after the reaction is completed, deionized water is added to quench the reaction, extracted with dichloromethane, the obtained organic phase is dried, the solvent is removed by reduced pressure distillation, and purified to obtain white solid compound 2; (3) Compound 2 and 2,3-diaminomaleonitrile are dissolved in MeOH, heated under reflux, and the reaction is completed by TLC monitoring, and purified to obtain yellow solid, which is probe II.

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