High-sensitivity hypochlorous acid fluorescent probe based on donor-receptor porphyrin molecular engineering and synthetic method thereof

Abstract

The invention discloses a donor-receptor porphyrin molecular engineering-based high-sensitivity hypochlorous acid fluorescent probe and a synthesis method thereof, the structural formula of the probe is as follows: # imgabs0 #, the probe can detect hypochlorous acid (HClO) with high sensitivity, phenothiazine groups are introduced at two meso positions of porphyrin, and a donor-receptor (D-A) system is formed in molecules. The probe has rapid response performance to hypochlorous acid, sulfur atoms in phenothiazine are oxidized through hypochlorous acid, phenothiazine electrons are prevented from being transferred to porphyrin under light excitation, and therefore red fluorescence is shown, and selective detection of hypochlorous acid is achieved. After the probe reacts with hypochlorous acid, 667 nm red fluorescence is emitted under the excitation wavelength of 450 nm, the fluorescence appears obviously, and the Stokes shift is 217 nm. The reaction product has the advantages of good water solubility, high fluorescence quantum yield, large Stokes shift and the like. The probe can detect hypochlorous acid with high sensitivity, and has a huge application prospect in the technical fields of analytical chemistry, life science, environment detection, biomedical treatment and the like.

Description

Technical Field

[0001] The present invention belongs to the cross - field of organic fluorescent sensing materials and bioanalytical chemistry, and specifically relates to a porphyrin compound based on a novel donor - acceptor (D - A) structure, its preparation method, and its application in highly sensitive fluorescence detection of hypochlorous acid (HClO). The technology covers the molecular design of functionalized porphyrins, efficient synthesis strategies, and a fluorescence sensing mechanism regulated by the electron - pushing and - pulling effect, and is applicable to the specific recognition and quantitative analysis of HClO in environmental water quality monitoring (such as the detection of free chlorine in drinking water) and biomedical fields (such as real - time imaging of inflammation - related reactive oxygen species), solving the technical bottlenecks of insufficient sensitivity, poor anti - interference ability, and complex synthesis steps in traditional detection methods. Background Art

[0002] Hypochlorous acid (HClO), as an important member of the reactive oxygen species family, plays a dual role both in vivo and in vitro: it is not only a key effector molecule for neutrophils to kill pathogens in the immune defense system but also a potential inducer of inflammatory diseases, tissue damage, and cancer development ( Nat. Chem. Biol. 2008, 4 , 278 - 286.). In the environmental field, as the core reagent for drinking water disinfection, its residual concentration directly affects public health safety Sensor. Actuat. B-chem. 2023, 394 , 134357.). However, the high reactivity of HClO makes its accurate detection face great challenges ( Analyst 2022, 147 , 987 - 1005.). Traditional detection methods ( Anal. Chem. 2020, 92 , 15079 - 15086.), such as chromatography, electrochemistry, and colorimetry, although having certain applications, generally have defects such as cumbersome operation, strong equipment dependence, and poor real - time performance, and are particularly difficult to meet the requirements in in - vivo imaging and on - site rapid screening scenarios. In recent years, fluorescence sensing technology has become a research hotspot due to its advantages of high sensitivity, non - destructive analysis, and spatio - temporal resolution ( Sensor. Actuat. B-chem. 2024, 407 , 135453.). However, most fluorescence sensing mainly utilizes the strong oxidizing property of HClO to destroy the conjugated structure, block the intramolecular charge transfer (ICT), and restore fluorescence emission. Therefore, searching for and designing fluorescence emission structures with HClO response is the focus of research. Currently, most fluorescence emission structures with HClO response are mainly based on phenothiazines, spirothiolactones, thiophene hydrazides, etc. ( Sensor. Actuat. B-chem. 2017, 240 , 18 - 36.). Although these structures have good fluorescence emission quantum yields, most reported detection limits for HClO are higher than 30 nM ( Talanta 2024,274 , 126063.), it is difficult to capture the trace HClO signal in physiology or the environment. At the same time, its anti-interference ability is weak. In particular, coexisting reactive oxygen species such as H2S, H2O2, and ONOO⁻ are prone to cause cross-response, resulting in false positive results ( Spectrochim. Acta. A 2024, 316 , 124312.). Therefore, finding new hypochlorous acid fluorescent probes is the focus of research. Among them, porphyrin-based probes have attracted much attention due to their large conjugated structure, tunable optical properties, and biocompatibility. However, most of the existing porphyrin-based probes detect hypochlorous acid based on porphyrin polymers, and their synthesis routes are complex ( Analytical Chemistry ,2024, 52 , 1132), with relatively low repeatability, low yield, and difficulty in large-scale preparation. Summary of the Invention

[0003] In view of the above situation and overcoming some deficiencies of the prior art, the purpose of the present invention is to provide the synthesis of a novel porphyrin-based hypochlorous acid fluorescent probe, which can rapidly detect hypochlorous acid by fluorescence under specific detection conditions.

[0004] The purpose of synthesizing porphyrin derivatives in the present invention is also to provide the synthesis and application of a probe with a simple preparation method, high sensitivity, low detection limit, and low cost.

[0005] The specific technical solution adopted by the present invention to solve the problem is to innovatively design a donor-acceptor (D-A) type porphyrin structure through a molecular engineering strategy, introduce a bisphenothiazine group at the meso site to construct an intramolecular electron transfer system, use the specific oxidation of sulfur atoms by HClO to block the electron transfer path, achieve a significant enhancement of red fluorescence, and cooperate with the "one-pot" condensation-oxidation synthesis process to improve the yield. Finally, a hypochlorous acid probe with a low detection limit and a high Stokes shift is obtained, providing a breakthrough solution for in vivo inflammation imaging and trace environmental detection.

[0006] The chemical structural formula of the probe of the present invention is as follows:

[0008] A highly sensitive hypochlorous acid fluorescent probe based on donor-acceptor porphyrin molecular engineering and its synthesis method are characterized in that the preparation method of the fluorescent molecular probe includes the following steps:

[0009] Under nitrogen protection and in the dark, 1 equivalent of methyl 4-formylbenzoate and 25 equivalents of pyrrole monomer were successively added to a degassed two-necked flask. After three degassing-nitrogen replacement cycles, 0.1 equivalent of trifluoroacetic acid catalyst was injected through a syringe. After sealing, it was wrapped with tin foil to avoid light, and the reaction was stirred at reflux temperature. After 1 hour of reaction, 1 equivalent of solid sodium hydroxide was added to neutralize the system, and stirring was continued for 15 minutes to terminate the reaction; the reaction solution was vacuum filtered in the dark, and the filtrate was transferred to a round-bottom flask. Excess pyrrole was removed by vacuum distillation. The distillation residue was cooled to room temperature, dispersed by ultrasonic treatment with absolute ethanol, filtered under light protection and washed with absolute ethanol to obtain a white solid (methyl 4-(bis(1H-pyrrol-2-yl)methyl)benzoate);

[0010] (1)Under nitrogen protection, DMF (10 mL) was successively added to a degassed round-bottom flask. 2.5 equivalents of NaH were slowly added under an ice-water bath, and then 1.1 equivalents of methyl iodide and 1 equivalent of phenothiazine were successively added. The reaction flask was transferred to room temperature and stirred for 2 h. After the reaction was complete, water was added to quench the reaction. The mixture was extracted with dichloromethane, and the combined organic phases were dried over anhydrous magnesium sulfate and concentrated by rotary evaporation under reduced pressure. The product was separated by silica gel column chromatography (gradient of petroleum ether / ethyl acetate) to obtain a white solid (N-methylphenothiazine); (2)Under nitrogen protection, an appropriate amount of dry and redistilled N,N-dimethylformamide (DMF) was slowly added to an equal volume of phosphorus oxychloride (POCl3). The mixture was stirred at 20 - 50 °C for 30 - 60 minutes to obtain a yellow solution. N-Methylphenothiazine was dissolved in an appropriate amount of DMF and added dropwise to the yellow mixed solution. The mixture was continuously stirred and reacted at 60 °C for 12 h under nitrogen protection; after the reaction was complete, the reaction solution was poured into an appropriate amount of ice water to precipitate a solid, which was filtered by suction and washed with cold water. The solid was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 9:1) to obtain a yellow solid (10-methyl-10H-phenothiazine-3-carbaldehyde).

[0011] Under an inert atmosphere in the dark, methyl 4-(bis(1H-pyrrol-2-yl)methyl)benzoate and 10-methyl-10H-phenothiazine-3-carbaldehyde in equimolar ratio were dissolved in dichloromethane. After degassing, 1 - 3% equivalents of trifluoroacetic acid were injected. After stirring at room temperature for 1 h, a methanol solution containing 3 equivalents of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) was injected into the reaction solution, and stirring was continued in the dark for 6 h; after the reaction was completed, purification was carried out by silica gel column chromatography with 200 - 300 mesh silica gel, using dichloromethane as the eluent, and the main red band was collected by monitoring with an ultraviolet lamp. The solution was concentrated by rotary evaporation to obtain a purple-red solid; the solid was dissolved in methanol and dispersed by ultrasonic treatment, and then filtered under reduced pressure and washed with methanol to obtain a high-purity purple-red probe product. Description of the Drawings

[0012] Figure 11H NMR spectrum of a highly sensitive hypochlorous acid fluorescent probe based on donor-acceptor porphyrin molecular engineering according to the present invention.

[0013] Figure 2 UV-visible absorption spectrum response graph of the highly sensitive hypochlorous acid fluorescent probe based on donor-acceptor porphyrin molecular engineering according to the present invention and hypochlorous acid concentration.

[0014] Figure 3 The highly sensitive hypochlorous acid fluorescent probe based on donor-acceptor porphyrin molecular engineering according to the present invention exhibits a characteristic absorption peak at 424 nm in the UV-visible absorption spectrum, and its absorbance shows a linear response relationship with the hypochlorous acid concentration in the range of 0 - 1.5 μM. This linear standard curve (R² = 0.992) provides a reliable basis for establishing a quantitative analysis model for hypochlorous acid.

[0015] Figure 4 Response graph of the fluorescence intensity of the highly sensitive hypochlorous acid fluorescent probe based on donor-acceptor porphyrin molecular engineering according to the present invention and hypochlorous acid concentration at an excitation wavelength of 395 nm.

[0016] Figure 5 The highly sensitive hypochlorous acid fluorescent probe based on donor-acceptor porphyrin molecular engineering according to the present invention exhibits a characteristic absorption peak at 667 nm in its fluorescence emission spectrum at an excitation wavelength of 395 nm, and its fluorescence emission intensity shows a linear response relationship with the hypochlorous acid concentration in the range of 0 - 1.5 μM. This linear standard curve (R² = 0.992) provides a reliable basis for establishing hypochlorous acid fluorescence sensing. Detailed implementation manners

[0017] The present invention will be further described below in conjunction with the synthesis route. The synthesis route of the highly sensitive hypochlorous acid fluorescent probe based on donor-acceptor porphyrin molecular engineering according to the present invention is as follows:

[0018] Under nitrogen protection and in the dark, methyl 4-formylbenzoate (13 g, 79.3 mmol) and pyrrole monomer (136 ml, 1.98 mol) were successively added to a degassed two-necked flask. After three degassing-nitrogen replacement cycles, trifluoroacetic acid (0.6 ml, 7.93 mmol) catalyst was injected through a syringe. After sealing, it was wrapped with foil to avoid light, and the reaction was stirred at the reflux temperature; after 1 hour of reaction, solid sodium hydroxide (3.172 g, 79.3 mmol) was added to neutralize the system, and stirring was continued for 15 minutes to terminate the reaction; the reaction solution was vacuum filtered in the dark, and the filtrate was transferred to a round-bottom flask. Excess pyrrole was removed by distillation under reduced pressure; the distillation residue was cooled to room temperature, anhydrous ethanol was added for ultrasonic dispersion, filtered in the dark and washed with anhydrous ethanol to obtain a white solid (27.76 g, yield 98%).

[0019] Under nitrogen protection, DMF (10 mL) was successively added to a degassed round-bottom flask. Under an ice-water bath, NaH (1.00 g, 25.12 mmol, 60% purity) was slowly added. Subsequently, methyl iodide (1.57 g, 11.05 mmol) and phenothiazine (2.0 g, 10.05 mmol) were successively added. The reaction flask was transferred to room temperature and stirred for 2 h; after the reaction was complete, the reaction was quenched with water, extracted with dichloromethane, the combined organic phases were dried over anhydrous magnesium sulfate, dried under reduced pressure by rotary evaporation, and separated by silica gel column chromatography (petroleum ether / ethyl acetate gradient) to obtain a white solid (N-methylphenothiazine) (1.93 g, yield 90%); Under nitrogen protection, an appropriate amount of dry and redistilled N,N-dimethylformamide (DMF, 30 ml) was slowly added to an equal volume of phosphorus oxychloride (POCl3, 30 ml), and the mixture was stirred at 20 - 50 °C for 30 - 60 minutes to obtain a yellow solution; N-methylphenothiazine (1.5 g, 7.04 mmol) was dissolved in 15 ml of DMF and added dropwise to the yellow mixed solution. The mixture was continuously stirred and reacted at 60 °C under nitrogen protection for 12 hours; after the reaction was complete, the reaction solution was poured into 150 ml of ice water, and a solid was precipitated, filtered, and washed with cold water; the solid was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 9:1) to obtain a yellow solid (10-methyl-10H-phenothiazine-3-carbaldehyde, 1.39 g, yield 82%).

[0020] Under a light - avoiding inert atmosphere, methyl 4-(bis(1H - pyrrol - 2 - yl)methyl)benzoate (0.42 g, 1.5 mmol) and 10 - methyl - 10H - phenothiazine - 3 - carbaldehyde (0.36 g, 1.5 mmol) were dissolved in 150 ml of dichloromethane. After degassing, 0.15 ml of trifluoroacetic acid was added by syringe. After stirring at room temperature for 1 hour, a methanol (2 ml) solution of 2,3 - dichloro - 5,6 - dicyanobenzoquinone (DDQ, 1.02 g, 4.5 mmol) was injected into the reaction solution, and stirring was continued under light avoidance for 6 hours. After the reaction was completed, purification was carried out by silica gel column chromatography with 200 - 300 mesh silica gel, using dichloromethane as the eluent. The main red band was collected under ultraviolet lamp monitoring, and the solution was concentrated by rotary evaporation to obtain a purple - red solid. The solid was dissolved in methanol and ultrasonically dispersed, and then obtained a high - purity purple - red probe product (22.5 mg, yield 3%) through vacuum filtration and methanol washing.

[0021] The present invention relates to a highly sensitive hypochlorous acid fluorescent probe based on donor - acceptor porphyrin molecular engineering and its synthesis method. The probe constructs an intramolecular electron transfer channel through the precise coupling of the phenothiazine group and the porphyrin core, and realizes specific fluorescence response under the trigger of hypochlorous acid. Its mechanism of action is as follows: HClO selectively oxidizes the sulfur atom in the phenothiazine unit to generate a sulfoxide structure, resulting in the blockage of the intramolecular donor - acceptor (D - A) electron transfer channel, thereby activating the red fluorescence emission of the porphyrin large π - conjugate system. At an excitation wavelength of 450 nm, after the probe reacts with hypochlorous acid, a significant fluorescence enhancement occurs at 667 nm, and the Stokes shift reaches 217 nm, effectively overcoming the signal - to - noise ratio limitation caused by spectral overlap of traditional probes. This probe is synthesized by a one - pot condensation - oxidation process, and the site - directed modification of bis - phenothiazine on the porphyrin is realized through the synergistic catalysis of trifluoroacetic acid / 2,3 - dichloro - 5,6 - dicyanobenzoquinone. Although the content of the present invention has been introduced in detail through the above - mentioned preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art read the above content, various modifications and substitutions of the present invention will be obvious. Therefore, fluorescent parent nuclei with technical features similar to those described herein all fall within the protection scope of this patent. Therefore, donor - acceptor porphyrin fluorescent parent nuclei with technical features similar to those described herein all fall within the protection scope of this patent. meso Although the content of the present invention has been introduced in detail through the above - mentioned preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art read the above content, various modifications and substitutions of the present invention will be obvious. Therefore, fluorescent parent nuclei with technical features similar to those described herein all fall within the protection scope of this patent. Therefore, donor - acceptor porphyrin fluorescent parent nuclei with technical features similar to those described herein all fall within the protection scope of this patent.

Claims

1. A highly sensitive hypochlorous acid fluorescent probe based on donor-acceptor porphyrin molecular engineering and its synthesis method, characterized in that Its molecular structure is shown in formula (TM): Wherein: (i) Two phenothiazine groups are introduced at the meso-site of porphyrin to form an intramolecular donor-acceptor system; (ii) The sulfur atom in the phenothiazine group can be specifically oxidized by hypochlorous acid (HClO), resulting in the hindrance of photoexcited electron transfer, thereby generating a 667 nm red fluorescence signal.

2. The highly sensitive hypochlorous acid fluorescent probe based on donor-acceptor porphyrin molecular engineering and its synthesis method according to claim 1, characterized in that The synthesis method of the probe comprises the following steps: Step I. Synthesis of methyl 4-(bis(1H-pyrrol-2-yl)methyl)benzoate Under nitrogen protection and in the dark, 1 equivalent of methyl p-formylbenzoate and 25 equivalents of pyrrole monomer are successively added to a degassed two-necked flask. After three degassing-nitrogen replacement cycles, 0.1 equivalent of trifluoroacetic acid catalyst is injected through a syringe, sealed and wrapped with tin foil to avoid light, and stirred at the reflux temperature. After reacting for 1 hour, 1 equivalent of solid sodium hydroxide is added to neutralize the system, and stirring is continued for 15 minutes to terminate the reaction; the reaction solution is vacuum filtered in the dark, the filtrate is transferred to a round-bottom flask, and the excess pyrrole is removed by distillation under reduced pressure. The distillation residue is cooled to room temperature, added with anhydrous ethanol and ultrasonically dispersed, filtered in the dark and washed with anhydrous ethanol to obtain a white solid (methyl 4-(bis(1H-pyrrol-2-yl)methyl)benzoate); Step II. Synthesis of 10-methyl-10H-phenothiazine-3-carbaldehyde Under nitrogen protection, DMF (10 mL) is successively added to a degassed round-bottom flask, and 2.5 equivalents of NaH are slowly added under an ice-water bath. Subsequently, 1.1 equivalents of methyl iodide and 1 equivalent of phenothiazine are successively added. The reaction flask is transferred to room temperature and stirred for 2 h. After the reaction is complete, water is added to quench the reaction, and the mixture is extracted with dichloromethane. The combined organic phases are dried over anhydrous magnesium sulfate, concentrated under reduced pressure by rotary evaporation, and separated by silica gel column chromatography (petroleum ether / ethyl acetate gradient) to obtain a white solid (N-methylphenothiazine); Under nitrogen protection, an appropriate amount of dry and redistilled N,N-dimethylformamide (DMF) is slowly added to an equal volume of phosphorus oxychloride (POCl3), and stirred at 20-50 °C for 30-60 minutes to obtain a yellow solution. N-methylphenothiazine is dissolved in an appropriate amount of DMF and added dropwise to the yellow mixed solution. The mixture is continuously stirred at 60 °C for 12 hours under nitrogen protection; after the reaction is complete, the reaction solution is poured into an appropriate amount of ice water to precipitate a solid, filtered by suction, washed with cold water, and the solid is separated by silica gel column chromatography (petroleum ether / ethyl acetate = 9:1) to obtain a yellow solid (10-methyl-10H-phenothiazine-3-carbaldehyde); Step III. Synthesis of the target fluorescent probe TM Under a light - avoiding inert atmosphere, methyl 4 - (bis(1H - pyrrol - 2 - yl)methyl)benzoate and 10 - methyl - 10H - phenothiazine - 3 - carbaldehyde in equimolar ratio were dissolved in dichloromethane. After degassing, 1 - 3% equivalent of trifluoroacetic acid was injected. After stirring at room temperature for 1 hour, a methanol solution containing 3 equivalents of 2,3 - dichloro - 5,6 - dicyanobenzoquinone (DDQ) was injected into the reaction solution, and stirring was continued under light - avoidance for 6 hours. After the reaction was completed, purification was carried out by silica gel column chromatography with 200 - 300 mesh silica gel, using dichloromethane as the eluent. The main red band was collected under ultraviolet lamp monitoring, and the solution was concentrated by rotary evaporation to obtain a purplish - red solid. The solid was dissolved in methanol and ultrasonically dispersed, and then obtained the high - purity purplish - red probe product through vacuum filtration and methanol washing.

3. The preparation method according to claim 2, characterized in that: In step I, the molar ratio of methyl p - formylbenzoate, pyrrole monomer and trifluoroacetic acid is 1:25:0.1; in step III, the dosage of trifluoroacetic acid is 1 - 3% equivalent.

4. A highly sensitive hypochlorous acid fluorescent probe based on donor-acceptor porphyrin molecular engineering and its synthesis method according to claim 1, characterized in that, The unique donor - acceptor molecular structure design of the fluorescent molecular probe can detect the fluorescence intensity emitted at 667 nm under an excitation wavelength of 450 nm for hypochlorous acid fluorescence sensing analysis.

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