Nitric oxide fluorescent probe based on amine recognition site, preparation method and application

Abstract

This invention discloses nitric oxide fluorescent probes based on amino group recognition sites, their preparation methods, and applications. The nitric oxide fluorescent probes are IPAN-PPN and IPAN-PPNP. The preparation method involves synthesizing IPAN-Ph-Br from 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonadionitrile and p-bromobenzaldehyde; synthesizing IPAN-PPN from IPAN-Ph-Br and pinacol ester of 4-aminophenylboronic acid; and synthesizing IPAN-PPNP from IPAN-PPN and benzaldehyde. The probes are used for detection in nitric oxide. The aromatic amino group recognition of NO in this invention exhibits better specificity, selectivity, and stronger anti-interference ability.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe detection technology. Specifically, it relates to nitric oxide fluorescent probes based on amino group recognition sites, their preparation methods, and applications. Background Technology

[0002] Nitric oxide (NO) is a key signaling molecule in physiological systems, endogenously produced from L-arginine via nitric oxide synthases (NOS), including endothelial nitric oxide synthases, neuronal nitric oxide synthases, and inducible nitric oxide synthases (iNOS). NO plays a crucial role in various physiological and pathological processes, including those in the respiratory, immune, cardiovascular, and nervous systems. For example, it maintains vascular homeostasis, regulates cardiovascular function, and preserves the functional integrity of neurovascular units. Furthermore, abnormal NO secretion is highly correlated with the progression of various diseases, such as atherosclerosis, cancer progression, various neurodegenerative diseases, and the pathophysiology of major depressive disorder.

[0003] To date, several traditional methods have been applied to detect NO, such as electrochemical sensors, electron paramagnetic resonance, spectrophotometry, membrane inlet mass spectrometry, chemiluminescence, and fluorescent probes. Although these techniques are widely used, they also have limitations, such as low sensitivity, high invasiveness, time consumption in sample preparation, and high instrument costs.

[0004] Fluorescence spectroscopy is the most widely used method for NO determination due to its simplicity, low cost, selectivity, and excellent detection limit. Probe recognition mechanisms include: cyclization of o-phenylenediamine derivatives; trapping mechanisms of metal-ligand complexes; ring-opening reactions of rhodamine derivatives; reduction and deamination of aromatic primary amines; formation of the diazonium ring; aromatization processes associated with Hantzsch esters; and nitrosation of secondary amines.

[0005] In recent years, aromatic secondary amines have emerged as novel NO recognition groups, used to construct N-nitrosylated fluorescent probes with high sensitivity and excellent selectivity. These amine compounds react with NO to form N-nitroso products while effectively suppressing photoinduced electron transfer (PET) or intramolecular charge transfer (ICT) processes, accompanied by significant changes in fluorescence intensity. Aromatic primary amines exhibit faster reaction rates, more stable products, and are easier to modify, making them one of the most widely used and mature recognition sites among current NO fluorescent probes.

[0006] Both primary and secondary aromatic amines possess strong nucleophilic groups, exhibiting highly nonspecific chemical properties. They readily react with numerous substances in biological systems, are easily oxidized, and generate false positive signals, thus weakening selectivity, specificity, and resistance to interference. Furthermore, during preparation, primary aromatic amines are easily oxidized, requiring complete light and oxygen isolation during synthesis; their high polarity makes crystallization difficult, resulting in poor yield and purity. Secondary amine alkylation reactions are difficult to control, easily leading to over-alkylation and the formation of tertiary amines, resulting in low yields and numerous isomers. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to provide a nitric oxide fluorescent probe based on an amino group recognition site with better specificity, selectivity and stronger anti-interference ability, as well as its preparation method and application.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] A nitric oxide fluorescent probe based on an amino group recognition site, wherein the nitric oxide fluorescent probe is IPAN-PPN, and the molecular formula of IPAN-PPN is: .

[0010] A nitric oxide fluorescent probe based on an amino group recognition site, wherein the nitric oxide fluorescent probe is IPAN-PPNP; the molecular formula of IPAN-PPNP is: .

[0011] Two fluorescent probes, IPAN-PPN and IPAN-PPNP, were designed using IPAN as the fluorophore because: 1. The rigid, highly conjugated structure of IPAN stabilizes the electron transfer process, achieving a sensitive "off-on" fluorescence response and reducing non-specific signals; 2. The electron-withdrawing conjugation effect of IPAN can effectively regulate the electron cloud density and nucleophilic activity of the amine recognition site, making it specifically respond to NO without responding to other interfering substances such as reactive oxygen species and reactive nitrogen species; 3. The steric hindrance and long-wavelength emission characteristics of the IPAN framework can further reduce the influence of environmental interference and biological autofluorescence.

[0012] The preparation method of the above-mentioned nitric oxide fluorescent probe based on amino group recognition sites includes the following steps:

[0013] (1) IPAN-Ph-Br was synthesized using 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile and p-bromobenzaldehyde as raw materials, 1,4-dioxane as solvent, piperidine as catalyst, and acetic acid as the acid-base condition of the system.

[0014] (2) Using IPAN-Ph-Br and 4-aminophenylboronic acid pinacol ester as raw materials and Pd(PPh3)4 as catalyst, the Suzuki-Miyaura reaction was carried out under alkaline conditions to synthesize IPAN-PPN.

[0015] The above-mentioned method for preparing nitric oxide fluorescent probes based on amino group recognition sites includes the following steps in step (1):

[0016] (1-1) Weigh 0.745 g of 2-(3,5,5-trimethylcyclohex-2-en-1-yl)malononitrile and 0.740 g of p-bromobenzaldehyde and place them in a 50 mL three-necked flask;

[0017] (1-2) Under a nitrogen atmosphere, 30 mL of 1,4-dioxane and 4 mL of piperidine were added to a three-necked flask. After observing the color change of the system, 4 mL of acetic acid was added. The three-necked flask was then placed in a constant temperature oil bath and heated to 110 °C for 48 h.

[0018] The synergistic effect of the "piperidine and acetic acid forming a buffered acid-base catalytic system" is evident in the acetic acid-piperidine-acetic acid buffer system. Piperidine, as a nucleophilic base catalyst, can abstract the proton from the active methylene group of malononitrile, generating a carbanion, initiating the Knoevenagel condensation reaction, and promoting double bond formation. It provides an alkaline environment, accelerating the condensation and dehydration of the aldehyde with the active methylene compound, acting as the initiator of the reaction. Acetic acid can neutralize excess piperidine, avoiding side reactions caused by strong alkalinity (such as substrate decomposition and isomerization), while providing a weakly acidic environment, promoting the dehydration step (the rate-determining step of the condensation reaction). The acetic acid and piperidine form an acid-base buffer system, stabilizing the reaction pH and preventing localized over-alkalinity / over-acidity, making the reaction more mild and controllable. The core advantage of adding piperidine and acetic acid sequentially is: adding piperidine first: initiating the condensation at low / room temperature allows the active methylene group to fully add to the aldehyde, forming a stable aldol intermediate, avoiding substrate decomposition or polymerization caused by direct high temperature. Add acetic acid later: After the system changes color (indicating that the addition intermediate has been formed), add acetic acid to promote dehydration, avoid the premature acidic environment inhibiting the formation of carbanions, and ensure the orderly process of addition followed by dehydration.

[0019] (1-3) After the reaction is complete, the reaction solution is allowed to cool naturally to room temperature; it is extracted three times with dichloromethane / water at a volume ratio of 1:1, the lower organic phase is collected, concentrated and purified by column chromatography to obtain orange-yellow solid IPAN-Ph-Br, with dichloromethane / petroleum ether as the eluent and a volume ratio of DCM:PE=1:1.

[0020] In the preparation of IPAN-Ph-Br, 1,4-dioxane was chosen as the solvent, with a boiling point of 101.3℃. To ensure rapid and efficient reaction, this application selected a reaction temperature of 110℃. If the temperature is below 110℃, the solvent cannot reach a gentle boiling state, resulting in insufficient dissolution and uneven mixing of the reactants. This can lead to incomplete local reactions, generating more byproducts, reducing product purity, affecting the reaction rate, potentially prolonging the reaction cycle, or even causing reaction stagnation and incomplete conversion of the reactants. If the temperature is above 110℃, the solvent (1,4-dioxane) will boil, causing the reaction system to boil violently and surge, posing safety hazards. Furthermore, excessive solvent evaporation can lead to abnormal system concentrations and trigger side reactions; the catalyst (piperidine) may decompose and lose its catalytic effect; and high temperatures can accelerate the oxidation of amines in the system, affecting reaction stability.

[0021] The above-mentioned method for preparing nitric oxide fluorescent probes based on amino group recognition sites includes the following steps in step (2):

[0022] (2-1) Weigh 0.5 g IPAN-Ph-Br, 0.241 g 4-aminophenylboronic acid pinacol ester, 0.53 g Pd(PPh3)4 and 0.6897 g potassium carbonate into a 50 mL three-necked flask, and reflux at 100 °C for 10 h under a nitrogen atmosphere using 10 mL toluene, 2 mL anhydrous ethanol and 2.5 mL water as solvents.

[0023] (2-2) The reaction system was cooled to room temperature, and the reaction solution was concentrated and purified by column chromatography to obtain wine-red solid IPAN-PPN. The eluent was dichloromethane (DCM).

[0024] The above-mentioned method for preparing nitric oxide fluorescent probes based on amino group recognition sites uses IPAN-PPN and benzaldehyde as raw materials, dichloromethane as solvent, and sodium cyanoborohydride as reducing agent to synthesize IPAN-PPNP.

[0025] The preparation method of the above-mentioned nitric oxide fluorescent probe based on amino group recognition sites includes the following steps:

[0026] (3-1) Weigh 0.3216g IPAN-PPN, 0.093g benzaldehyde and 0.5mL acetic acid and dissolve them in 30mL dichloromethane. Stir at room temperature for 30min.

[0027] (3-2) Add 124 mg of sodium cyanoborohydride, react at 25 °C for 1 h, and then quench the reaction with water;

[0028] (3-2) Extract with chloroform and collect the lower organic phase; wash the organic phase with saturated sodium chloride solution, dry with anhydrous sodium sulfate, concentrate the solvent, and purify by column chromatography to obtain bright red solid IPAN-PPNP; the eluent is dichloromethane / petroleum ether, with a volume ratio of DCM:PE=1:1.

[0029] The above-mentioned nitric oxide fluorescent probe based on amino group recognition sites is used in the detection of nitric oxide.

[0030] The above-mentioned fluorescent nitric oxide probes are used in the detection and imaging of nitric oxide in solutions and living cells.

[0031] The above-mentioned nitric oxide fluorescent probes based on amino group recognition sites are used in the preparation of nitric oxide kits or test strips.

[0032] The technical solution of the present invention achieves the following beneficial technical effects:

[0033] 1. This invention uses 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile (IPAN) as the fluorophore and aromatic primary and secondary amines as recognition groups to design and synthesize IPAN-PPN and IPAN-PPNP fluorescent probes for the detection of nitric oxide. Compared with the traditional o-phenylenediamine group, the aromatic amine group of this invention has better specificity, selectivity and stronger anti-interference ability in recognizing NO.

[0034] Choosing IPAN as the fluorophore allows for synergistic effects with primary and secondary aromatic amines, enhancing specificity and selectivity. This is because: 1. The IPAN framework possesses a strong electron-withdrawing conjugated group, which moderately reduces the electron cloud density of the nitrogen atom in primary aromatic amines, weakening their excessive nucleophilic activity. This overcomes the non-specificity of traditional aromatic amine groups, ensuring specific recognition only with the target analyte and eliminating non-target substances, thus preventing non-specific binding at its source. 2. The IPAN framework has a large-volume, three-dimensional rigid structure, creating a spatial barrier for the aromatic amine recognition site. This effectively prevents interfering substances in the biological matrix (such as reactive oxygen species, reactive nitrogen species, common amino acids, and metal ions) from approaching the recognition site, avoiding side reactions between interfering molecules and amine groups, and significantly improving anti-interference performance. 3. The excellent structural stability of IPAN reduces false positive responses. 4. The IPAN conjugated framework further protects primary aromatic amines from oxidation and degradation, preventing false positive signals caused by the deterioration of traditional primary and secondary aromatic amine probes, further ensuring the specificity and accuracy of the detection results. (Traditional aromatic primary and secondary amine probes can usually only tolerate 8 to 12 simple interfering substances. They are prone to significant interference when reactive oxygen species, metal ions, and biothiols coexist, making it impossible to achieve accurate detection in complex systems.)

[0035] 2. This application uses isophorone fluorescent precursors, which possess long wavelengths, high stability, and low biofluorescence. The reaction route is simple and efficient, utilizing a simple, continuous two-step reaction to synthesize two target products, IPAN-PPN and IPAN-PPNP, and their optical properties and cell imaging effects are studied. After the probes react with nitric oxide, the fluorescence is significantly enhanced, exhibiting high selectivity and sensitivity. Two NO fluorescent sensors have been successfully constructed, providing an efficient tool for the visual monitoring of NO in biological systems.

[0036] 3. In the preparation of IPAN-Ph-Br, 1,4-dioxane was chosen as the solvent because it can completely dissolve both raw materials and is compatible with the piperidine and acetic acid catalytic system, ensuring uniform mixing of reactants and a more complete reaction. When heated to 110℃, the system is in a gentle boiling state, providing the necessary heat for the reaction without violent boiling that could lead to solvent evaporation or raw material spillage, thus ensuring a stable, isothermal reaction. 1,4-dioxane is chemically stable: it is an inert solvent, does not participate in the condensation reaction, and will not undergo side reactions with the raw materials, catalyst, or product, avoiding impurities and ensuring product purity. Simultaneously, 1,4-dioxane can synergistically work with the piperidine + acetic acid catalytic system without disrupting the acid-base balance, allowing the catalyst to function fully, accelerating the reaction rate, and increasing product yield. Existing catalysts are unsuitable as solvents for this application: Alcohols (methanol, ethanol): are too polar, easily reacting with the raw materials; their boiling points are too low, failing to reach the reaction temperature of 110°C, resulting in incomplete reactions and extremely low yields. Toluene: is too weakly polar, unable to dissolve the raw materials, leading to reactant stratification and localized reactions. Xylene: has an excessively high boiling point, easily causing thermal decomposition and carbonization of the products. Tetrahydrofuran: has a boiling point of only 66°C, far below the reaction temperature, easily resulting in violent boiling upon heating, leading to slow reactions and safety hazards.

[0037] Piperidine was chosen as the secondary amine organic base catalyst due to its excellent compatibility and outstanding advantages: moderate catalytic activity and controllable reaction; the formation of a buffered acid-base catalytic system between piperidine and acetic acid, resulting in synergistic enhancement and higher catalytic efficiency; and excellent solubility, making it suitable for the reaction system. Other inorganic base catalysts in the prior art are unsuitable as solvents for this application: such as inorganic base catalysts (sodium hydroxide, potassium carbonate, sodium carbonate): excessively alkaline, easily leading to side reactions such as hydrolysis, polymerization, and decyanation of the raw materials, damaging the raw material structure; and inorganic bases have poor solubility in organic solvents, belonging to heterogeneous catalysis, resulting in extremely slow reaction rates, extremely low yields, and numerous and difficult-to-separate impurities in the product. For example, triethylamine, a tertiary amine catalyst: triethylamine has weak basicity, insufficient ability to activate carbonyl groups and active hydrogens, making the reaction difficult to initiate, and even extending the reaction time cannot completely convert the raw materials. For example, pyridine: strong aromaticity, low catalytic activity, pungent odor, easy residue after post-processing, and difficulty in ensuring product purity.

[0038] 4. During the preparation of IPAN-PPN, 4-aminophenylboronic acid pinacol ester is prone to deboronization hydrolysis and self-coupling side reactions under heating and alkaline conditions, resulting in a reduction in the effective substrate actually participating in the coupling. Therefore, a slight excess of IPAN-Ph-Br can promote the reaction forward, maximize the conversion of borate ester raw materials, and avoid wasting valuable substrates. Furthermore, due to the significant polarity difference between IPAN-Ph-Br and the target product, even a small excess can be easily removed by subsequent column chromatography and recrystallization, without leaving any residue that would affect product purity.

[0039] Using 4-aminophenylboronic acid pinacol ester to provide the amino group: directly introducing the amino recognition site, eliminating the need for subsequent protection and deprotection, and simplifying the reaction.

[0040] Toluene, anhydrous ethanol, and water were chosen as the solvents: toluene is a nonpolar solvent that can fully dissolve the highly hydrophobic IPAN-Ph-Br and Pd(PPh3)4 catalysts; ethanol is a polar protic solvent that improves the miscibility between the organic and aqueous phases; and water can dissolve inorganic bases such as potassium carbonate, achieving a homogeneous reaction system that allows the substrate, catalyst, and base to come into full contact. The synergistic effect of these three solvents greatly improves the catalytic efficiency, ensuring complete dissolution and full contact of the raw materials, substrate, and catalyst, thus guaranteeing the complete reaction and increasing the yield.

[0041] The reaction condition of reflux at 100℃ for 10 hours was chosen because 100℃ provides the activation energy required for Suzuki coupling, accelerates the catalytic cycle rate, and the 10-hour duration ensures complete substrate conversion, balancing high yield and experimental efficiency.

[0042] Excessive temperature: Palladium catalysts rapidly decompose, aggregate, and deactivate, resulting in a sharp drop in catalytic efficiency; borate ester substrates undergo accelerated deboronization and self-coupling, leading to a surge in byproducts. Excessive temperature: Insufficient activation energy, slow catalytic cycling, and significantly prolonged reaction time; incomplete substrate conversion, resulting in substantial feedstock residue and a significantly reduced yield of the target product. Shortened reaction time: Leads to incomplete reaction between IPAN-Ph-Br and borate esters, resulting in substantial feedstock residue and low yield; prolonged reaction time and excessive heating cause product oxidation and decomposition, leading to a decrease in yield instead of an increase; accumulation of side reactions increases impurity content and makes purification more difficult.

[0043] 5. In the preparation of IPAN-PPNP, sodium cyanoborohydride is used as a reducing agent. It is a mild and weak reducing agent with extremely high selectivity. It does not over-reduce, but only directionally reduces the imine intermediate in the system. It will not damage the cyano group, aromatic ring and core recognition site amino group on the probe backbone, thus eliminating the problems of product structure damage and fluorescence performance failure. Attached Figure Description

[0044] Figure 1Synthesis route diagram of the probes IPAN-PPN and IPAN-PPNP of this invention;

[0045] Figure 2 The present invention relates to the sensing mechanism of the probes IPAN-PPN and IPAN-PPNP with NO;

[0046] Figure 3 The proton NMR spectrum of IPAN-Ph-Br;

[0047] Figure 4 Carbon NMR spectrum of IPAN-Ph-Br;

[0048] Figure 5 The proton NMR spectrum of IPAN-PPN;

[0049] Figure 6 Carbon NMR spectrum of IPAN-PPN;

[0050] Figure 7 The proton NMR spectrum of IPAN-PPNP;

[0051] Figure 8 Carbon NMR spectrum of IPAN-PPNP;

[0052] Figure 9 Fluorescence spectra of IPAN-PPNP probes in aqueous solutions with different PBS contents;

[0053] Figure 10. Fluorescence spectra of IPAN-PPN (10 μM) reacted with different concentrations of NO (0-50 μM);

[0054] Figure 10B Standard curves of IPAN-PPN (10 μM) reacting with different concentrations of NO (0-50 μM);

[0055] Figure 11A Fluorescence spectra of PAN-PPNP (10 μM) reacted with different concentrations of NO (0-50 μM);

[0056] Figure 11B Standard curves of PAN-PPNP (10 μM) reacting with different concentrations of NO (0-50 μM);

[0057] Figure 12A Fluorescence spectra of IPAN-PPN (10 μM) in MeOH in the presence of 3 equivalents of NO (curve 26) and interfering substances. (Curves 1 to 26 represent H2O2, ONOO, etc.) - , 1 O, ClO - NO2 -NO3 - ,MGO,DHA,Lys,Try,Glu,Cys,NH 4+ Fe 2+ Fe 3+ (30 μM; 3 eq), Na + (Cl) - ), Ca 2+ K + Mg 2+ F - ,Br - SO4 2- SO3 2- HCO3 - CO3 2- (100 μM; 10 eq);

[0058] Figure 12B Bar chart of IPAN-PPN response to NO and other substances (F-F0) / F0;

[0059] Figure 13A Fluorescence spectra of IPAN-PPNP (10 μM) in 10% PBS and 90% EtOH in the presence of 3 equivalents of NO (1) and interfering substances. (1 to 26 represent NO, H2O2, and O2O2, respectively.) - , 1 O, ClO - NO2 - NO3 - ,MGO,DHA,Lys,Try,Glu,Cys,NH 4+ Fe 2+ Fe 3+ Na + (Cl) - ), Ca 2+ K + Mg 2+ F - ,Br - SO4 2- SO3 2- HCO3 - CO3 2- (100 μM; 10 eq), NO (30 μM; 3 eq);

[0060] Figure 13B Bar chart of IPAN-PPNP response to NO and other substances (F-F0) / F0;

[0061] Figure 14A Cellular imaging of IPAN-PPN;

[0062] Figure 14B Cellular imaging of IPAN-PPNP. Detailed Implementation

[0063] Example 1

[0064] This invention designs and synthesizes IPAN-PPN and IPAN-PPNP fluorescent probes using 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonadionitrile (IPAN) as the fluorophore and aromatic primary and secondary amines as recognition groups, and uses them to detect nitric oxide.

[0065] The molecular formula of IPAN-PPN is: .

[0066] The molecular formula of IPAN-PPNP is: .

[0067] Example 2, Preparation Method

[0068] The synthetic routes of IPAN-PPN and IPAN-PPNP fluorescent probes are as follows: Figure 1 As shown.

[0069] Experimental materials and main equipment:

[0070] All chemical reagents and solvents were purchased from Shanghai Energy Chemical Co., Ltd. and Aladdin Reagent (Shanghai) Co., Ltd.

[0071] The interfering ions used were derived from their respective salts. The nitric oxide (NO) source was prepared from 2-(N,N-diethylamino)-diazepine-2-oxodiethylammonium salt (EDTA NONOates) provided by Shanghai Maclean Biochemical Technology Co., Ltd. The specific preparation method was as follows: 1 mg of 2-(N,N-diethylamino)-diazepine-2-oxodiethylammonium salt solid was dissolved in 0.1024 mL of high-purity water to obtain a 60 mM NO donor salt solution. Other low-concentration donor salt solutions were obtained by serial dilution from this concentration.

[0072] 1 H NMR, 13 C10 NMR spectra were acquired using a Bruker Ascend 400 MHz NMR spectrometer (Switzerland) with CDCl3 as solvent. Chemical shifts are expressed as parts per million (ppm) relative to tetramethylsilane (TMS), and solvent resonance peaks were used as internal standards. UV-Vis absorption and fluorescence emission spectra were acquired using a Shimadzu UV-2700i spectrophotometer and a Hitachi F-7000 fluorescence spectrometer (Japan), respectively.

[0073] Specific synthesis method:

[0074] (1) Synthesis of IPAN-Ph-Br

[0075] Weigh 0.745 g (4 mmol) of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonadionitrile and 0.740 g (4 mmol) of p-bromobenzaldehyde into a 50 mL three-necked flask. Under a nitrogen atmosphere, add 30 mL of 1,4-dioxane and 4 mL of piperidine to the reaction system. After observing a color change, add 4 mL of acetic acid. Then, place the reaction system in a constant temperature oil bath and heat to 110 °C for 48 h.

[0076] After the reaction was complete, the reaction solution was allowed to cool naturally to room temperature. It was extracted three times with dichloromethane / water (volume ratio 1:1), and the lower organic phase was collected, concentrated, and purified by column chromatography (eluent: DCM:PE = 1:1, volume ratio) to obtain 0.67 g of an orange-yellow solid (yield 47.42%). IPAN-Ph-Br 1 H NMR (400 MHz, CDCl3): δ 7.52 (d, J = 8.6Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 6.98 (s, 2H), 6.85 (s, 1H), 2.61 (s, 2H), 2.47 (s, 2H), 1.08 (s, 6H). Figure 3 and Figure 4 The images show the proton and carbon NMR spectra of IPAN-Ph-Br, respectively.

[0077] (2) Synthesis of IPAN-PPN

[0078] 0.5 g (1.41 mmol) of IPAN-Ph-Br, 0.241 g (1.24 mmol) of 4-aminophenylboronic acid pinacol ester, 0.53 g of Pd(PPh3)4, and 0.6897 g (5 mmol) of potassium carbonate were weighed into a 50 mL three-necked flask, using toluene (10 mL), anhydrous ethanol (2 mL), and water (2.5 mL) as solvents. The reaction mixture was refluxed at 100 °C for 10 h under a nitrogen atmosphere. After cooling to room temperature, the reaction mixture was concentrated and purified by column chromatography (eluent: DCM) to give 0.101 g of a wine-red solid (yield: 55.27%).

[0079] IPAN-PPN 1H NMR (400 MHz, CDCl3): δ 7.56 (q, J = 8.6 Hz, 4H), 7.46 (d, J = 8.5 Hz, 2H), 7.08 (d, J = 16.0 Hz, 1H), 7.00 (d, J = 16.3 Hz, 1H), 6.84 (s, 1H), 6.77 (d, J = 8.5 Hz, 2H), 3.81 (s, 2H), 2.61 (s, 2H), 2.49 (s,2H), 1.09 (s, 6H). Figure 5 and Figure 6 The images show the proton and carbon NMR spectra of IPAN-PPN, respectively.

[0080] (3) Synthesis of IPAN-PPNP

[0081] IPAN-PPN (0.3216 g, 0.88 mmol), benzaldehyde (0.093 g, 0.88 mmol), and acetic acid (0.5 mL) were dissolved in 30 mL of dichloromethane and stirred at room temperature for 30 min. Sodium cyanoborohydride (124 mg, 1.97 mmol) was added, and the reaction was quenched with water after 1 h, followed by extraction with chloroform. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was concentrated. The solution was then purified by column chromatography (DCM:PE = 1:1) to give 0.1526 g of bright red solid (yield = 38.15%).

[0082] IPAN-PPNP 1 H NMR (400 MHz, CDCl3) δ 7.61 – 7.51 (q, 4H), 7.49 (d, J =8.8 Hz, 2H), 7.38 (q, J = 7.0 Hz, 5H), 7.31 (d, 1H), 7.04 (q, J = 16.1 Hz, 2H), 6.84 (s, 1H), 6.72 (d, J = 8.6 Hz, 2H), 4.40 (s, 2H), 2.61 (s, 2H), 2.49 (s, 2H), 1.09 (s, 6H). Figure 7 and Figure 8 The images show the proton and carbon NMR spectra of IPAN-PPNP, respectively.

[0083] Example 3: The interaction between probes IPAN-PPN and IPAN-PPNP and NO

[0084] 1. For example Figure 2The diagram illustrates the sensing mechanism of probes IPAN-PPN and IPAN-PPNP with NO. From... Figure 2 It can be seen that IPAN-PPN is an aromatic primary amine, which detects NO using the principle of reductive deamination. IPAN-PPNP is an aromatic secondary amine, which detects NO using the nitrosation reaction of secondary amines.

[0085] 2. Solution preparation

[0086] The concentration to be prepared is 10×10 -3 mol / L PBS buffer solution (pH=7.4).

[0087] Prepare a concentration of 3.0 × 10⁻⁶ -3 mol / L IPAN-PPN and IPAN-PPNP solutions.

[0088] The prepared concentration is 60.0 × 10⁻⁶. -3 mol / L NO donor salt solution (EDTA NONOates).

[0089] Prepare 100 μM solutions of H2O2 and ONOO. - , 1 O, ClO - NO2 - NO3 - ,MGO,DHA,Lys,Try,Glu,Cys,NH4 + Fe 2+ Fe 3+ Na + (Cl) - ), Ca 2+ K + Mg 2+ F - ,Br - SO4 2- SO3 2- HCO 3- CO3 2- The aqueous solution is prepared for use.

[0090] 3. The interaction between probes IPAN-PPN and IPAN-PPNP and NO.

[0091] 3.1 After preliminary solvent screening, IPAN-PPN selected methanol, which has moderate polarity and high chemical stability, as the solvent.

[0092] IPAN-PPNP was initially selected using anhydrous ethanol, which has low toxicity and is readily miscible with water, as the solvent. After immobilizing IPAN-PPNP with anhydrous ethanol as the solvent, the probe was obtained by measuring different water contents (f... wAfter analyzing the fluorescence spectra of a mixed solution of anhydrous ethanol (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), it is noteworthy that by adding water (40%-100% PBS) to the solution system, the fluorescence signal wavelength of the original IPAN-PPNP probe at around 550 nm in pure ethanol shifted to around 620 nm. This involves an aggregation-induced emission mechanism, and the aggregation of fluorescence can be observed in subsequent cell imaging.

[0093] However, since the On / Off ratio of IPAN-PPNP after reaction with NO was optimal in the 90% EtOH + 10% PBS solution system, the 90% EtOH + 10% PBS solution was ultimately chosen as the solution system for subsequent tests. 2.7 mL of anhydrous ethanol and 0.3 mL of PBS buffer solution were transferred to a cuvette, and then 10 μL of a 3.0 × 10⁻⁶ solution was added. -3 The UV and fluorescence spectra were measured using a mol / L probe solution. Then, 1.5 μL of a 60.0 × 10⁻⁶ mol / L solution was added. -3 Prepare a mol / L NO donor salt solution, then measure the ultraviolet and fluorescence spectra and observe the changes in the curves.

[0094] 3.2 Fluorescence spectra of the interaction between IPAN-PPN and IPAN-PPNP and NO.

[0095] Through testing under different water content conditions, the probe IPAN-PPNP exhibited aggregation-induced emission (AIE) characteristics. Figure 9 In ethanol solution, when water (PBS content of 40%-100%) is added to the system, the fluorescence emission band of IPAN-PPNP redshifts from 550 nm to 620 nm. This result provides experimental evidence for subsequent mechanism studies.

[0096] When IPAN-PPN reacts with NO, it undergoes deamination, and its fluorescence emission peak gradually red-shifts from 560 nm as the NO concentration increases; when IPAN-PPNP reacts with NO to form nitroso compounds, its fluorescence emission peak gradually red-shifts from 550 nm.

[0097] In systems with gradually increasing NO concentrations (0.01–20 μM), the fluorescence intensity of the IPAN-PPN deamination product continuously increased near 580 nm, and the fluorescence intensity showed a good linear relationship with NO concentration within this range (R² = 0.995). Figure 10A and Figure 10B As shown.

[0098] Under the same conditions, the fluorescence intensity of the IPAN-PPNP nitrosation product at around 561 nm also continuously increased, and the fluorescence intensity showed good linearity with NO concentration within the same range (R²=0.989). Figure 11A and Figure 11B As shown.

[0099] 3.3 Selectivity of probes IPAN-PPN and IPAN-PPNP

[0100] To investigate the selectivity of fluorescent probes, the responses of IPAN-PPN and IPAN-PPNP fluorescent probes to potential interfering substances were studied. For example... Figure 12A and Figure 13A As shown, other substances, including NO, H2O2, and ONOO, were added in amounts equivalent to several times the probe concentration. - , 1 O, ClO - NO2 - NO3 - ,MGO,DHA,Lys,Try,Glu,Cys,NH4 + Fe 2+ Fe 3+ Na + (Cl) - ), Ca 2 + K + Mg 2+ F - ,Br - SO4 2- SO3 2- HCO 3- CO3 2- Besides showing a strong fluorescence response to NO, the two probes did not show a significant response to the addition of other interfering substances, indicating that both probes have good selectivity and can specifically detect NO.

[0101] Figure 12B and Figure 13B In this study, (F-F0) / F0 was used to evaluate the selectivity of the probes, where F is the fluorescence intensity after the system is stabilized with the addition of other substances, and F0 is the fluorescence intensity of the probe itself. This also indicates that the probes IPAN-PPN and IPAN-PPNP have good selectivity and can specifically detect NO.

[0102] 3.4 Imaging of probes IPAN-PPN and IPAN-PPNP in cells

[0103] To investigate the bioapplicability and biocompatibility of the probe, its imaging performance in cells was studied. For example... Figure 14AAs shown, group a cells had normal morphology and, as a control group, showed no red fluorescence signal in the fluorescence channel. In group b, after the addition of the IPAN-PPN probe, weak red fluorescent spots appeared in the fluorescence channel, and the overlay image showed that the fluorescence signal was located in the cytoplasm. Compared with group b, group c showed a significantly enhanced red fluorescence signal, and the overlay image clearly showed that the fluorescence signal was widely distributed in the cytoplasm.

[0104] Similarly, as Figure 14B As shown, group d cells had normal morphology and, as a control group, showed no red fluorescence signal in the fluorescence channel. In group e, after the addition of the IPAN-PPNP probe, weak red fluorescent spots appeared in the fluorescence channel, and the overlay image showed that the fluorescence signal was located in the cytoplasm. Compared with group e, group f showed a significant enhancement of the red fluorescence signal, and the overlay image clearly showed that the fluorescence signal was widely distributed in the cytoplasm.

[0105] Taking advantage of the specificity of IPAN-PPN and IPAN-PPNP probes for NO detection and based on their good selectivity, nitric oxide fluorescent probes IPAN-PPN and IPAN-PPNP can be used to prepare nitric oxide kits or test strips for convenient and rapid detection.

[0106] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A nitric oxide fluorescent probe based on an amino group recognition site, characterized in that, The nitric oxide fluorescent probe is IPAN-PPN, and the molecular formula of IPAN-PPN is: .

2. A nitric oxide fluorescent probe based on an amino group recognition site, characterized in that, The nitric oxide fluorescent probe is IPAN-PPNP; the molecular formula of IPAN-PPNP is: .

3. The method for preparing the nitric oxide fluorescent probe based on the amino group recognition site according to claim 1, characterized in that, Includes the following steps: (1) IPAN-Ph-Br was synthesized from 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile and p-bromobenzaldehyde as raw materials, 1,4-dioxane as solvent, and piperidine and acetic acid as catalysts. (2) Using IPAN-Ph-Br and 4-aminophenylboronic acid pinacol ester as raw materials and Pd(PPh3)4 as catalyst, the Suzuki-Miyaura reaction was carried out under alkaline conditions to synthesize IPAN-PPN.

4. The method for preparing a nitric oxide fluorescent probe based on an amino group recognition site according to claim 3, characterized in that, Step (1) includes the following steps: (1-1) Weigh 0.745 g of 2-(3,5,5-trimethylcyclohex-2-en-1-yl)malononitrile and 0.740 g of p-bromobenzaldehyde and place them in a 50 mL three-necked flask; (1-2) Under a nitrogen atmosphere, 30 mL of 1,4-dioxane and 4 mL of piperidine were added to a three-necked flask. After observing the color change of the system, 4 mL of acetic acid was added. The three-necked flask was then placed in a constant temperature oil bath and heated to 110 °C for 48 h. (1-3) After the reaction is completed, the reaction solution is allowed to cool naturally to room temperature. It is then extracted three times with dichloromethane / water at a volume ratio of 1:

1. The lower organic phase is collected, concentrated, and purified by column chromatography to obtain an orange-yellow solid IPAN-Ph-Br. The eluent is dichloromethane / petroleum ether at a volume ratio of DCM:PE = 1:

1.

5. The method for preparing a nitric oxide fluorescent probe based on an amino group recognition site according to claim 3, characterized in that, Step (2) includes the following steps: (2-1) Weigh 0.5 g IPAN-Ph-Br, 0.241 g 4-aminophenylboronic acid pinacol ester, 0.53 g Pd(PPh3)4 and 0.6897 g potassium carbonate into a 50 mL three-necked flask, and reflux at 100 °C for 10 h under a nitrogen atmosphere using 10 mL toluene, 2 mL anhydrous ethanol and 2.5 mL water as solvents. (2-2) The reaction system was cooled to room temperature, and the reaction solution was concentrated and purified by column chromatography to obtain wine-red solid IPAN-PPN. The eluent was dichloromethane (DCM).

6. The method for preparing a nitric oxide fluorescent probe based on an amino group recognition site according to claim 2, characterized in that, IPAN-PPNP was synthesized using IPAN-PPN and benzaldehyde as raw materials, dichloromethane as solvent, and sodium cyanoborohydride as reducing agent.

7. The method for preparing a nitric oxide fluorescent probe based on an amino group recognition site according to claim 6, characterized in that, Includes the following steps: (3-1) Weigh 0.3216g IPAN-PPN, 0.093g benzaldehyde and 0.5mL acetic acid and dissolve them in 30mL dichloromethane. Stir at room temperature for 30min. (3-2) Add 124 mg of sodium cyanoborohydride, react at 25 °C for 1 h, and then quench the reaction with water; (3-2) Extract with chloroform and collect the lower organic phase; wash the organic phase with saturated sodium chloride solution, dry with anhydrous sodium sulfate, concentrate the solvent, and purify by column chromatography to obtain bright red solid IPAN-PPNP; the eluent is dichloromethane / petroleum ether, with a volume ratio of DCM:PE=1:

1.

8. The application of the nitric oxide fluorescent probe based on the amino group recognition site according to claim 1 or 2 in the detection of nitric oxide.

9. The application of the nitric oxide fluorescent probe based on the amino group recognition site according to claim 1 or 2 in the detection and imaging of nitric oxide in solution and living cells.

10. The application of the nitric oxide fluorescent probe based on the amino group recognition site according to claim 1 in the preparation of nitric oxide kits or test strips.

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