Bodipy and NBD dual fluorescent probe and synthesis method and application thereof

Abstract

This invention relates to the field of organic synthesis technology, and particularly to a dual-fluorescent probe for Bodipy and NBD, its synthesis method, and its application. The method includes: first, synthesizing NBD coumarin compound 6; then, using nitrobenzaldehyde f and dimethylpyrrole as starting materials, undergoing a four-step one-pot cascade reaction with catalytic amounts of trifluoroacetic acid, DDQ oxidation, and excess triethylamine and Lewis acid boron trifluoride diethyl ether to obtain the fluorescent compound Bodipy h; finally, subjecting the fluorescent compound Bodipy h to reduction and acylation to obtain Bodipy fluorescent probe 8; and finally, after lithium hydroxide demethylation of Bodipy fluorescent probe 8 to obtain a carboxylic acid, which is then amidated with the deBoc-protected NBD compound 6 by trifluoroacetic acid to obtain the target product, dual-fluorescent probe 9. This invention anticipates that the fluorescence of coumarin and naphthalimide, through this dual quenching process, can efficiently achieve the quenching strategy, and can be applied to the simultaneous detection of H2S and hNQO1 in vivo or in vitro.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and in particular to a Bodipy and NBD dual fluorescent probe, its synthesis method, and its application. Background Technology

[0002] Hydrogen sulfide (H2S) is the third endogenous gaseous signaling molecule, after carbon monoxide and nitric oxide, to exert physiological effects in living organisms. This gas plays a crucial role in the physiological and pathological regulation of the cardiovascular and nervous systems. Therefore, the selective recognition and highly sensitive detection of H2S in organisms is of great biomedical significance. Among biodetection techniques, fluorescent probe methods possess unique advantages such as high selectivity, high sensitivity, minimal damage to biological samples, and the ability to achieve real-time in-situ detection. Therefore, the application of fluorescent probe methods to detect changes in intracellular H2S concentration has become a research hotspot in recent years. Developing detection methods capable of in-situ detection of bioactive species is of great importance for understanding their functions and mechanisms of action in biological systems. Compared with conventional analytical methods, small molecule fluorescent probes offer advantages such as real-time detection, sensitivity, and non-invasiveness. Therefore, imaging techniques based on small molecule fluorescent probes have become an effective means of visually detecting various bioactive species in biological systems.

[0003] Bodipy exhibits a narrow spectral peak width, which enhances detection sensitivity and reduces sample volume in optical and biological analyses. The measured fluorescence spectrum shows more pronounced changes in fluorescence absorption and emission, leading to more accurate sample concentration. The absence of negative charge within the Bodipy molecule prevents electrostatic interactions between ions and the uncharged molecule, effectively avoiding interference and ensuring a more stable and accurate measurement process. Bodipy fluorescent dyes exhibit excellent photostability; once excited to the excited state, they do not decompose or undergo other changes during analysis, resulting in more accurate and reliable fluorescence spectra. The fluorescence quantum yield of Bodipy molecules is very high, typically reaching 0.6. Other modified Bodipy dyes even approach 1.0. In contrast, the fluorescence quantum yield of common dyes decreases in aqueous solutions, and some are even quenched. While the fluorescence quantum yield of Bodipy molecules decreases somewhat in aqueous solution, it still maintains a relatively high level. As the parent structure of Bodipy reveals, its substituents generally do not include acid- or base-sensitive reactive groups such as amino or carboxyl groups. Therefore, the influence of external pH levels is significantly reduced, and Bodipy dyes do not easily deteriorate.

[0004] NBD fluorescent probes are a class of fluorescent dyes with 7-nitrobenzo-2-oxo-1,3-diazole (NBD) as their core backbone. Their most significant characteristic is their high sensitivity to the environment (especially solvent polarity), coupled with their small molecular weight and minimal impact on the function of the labeled target, making them widely used in bioimaging and molecular detection. The outstanding performance of NBD probes stems primarily from the following characteristics: ① Environmental sensitivity: The fluorescence intensity and emission wavelength of NBD significantly change with variations in the surrounding environment (such as solvent polarity and membrane microenvironment). This characteristic makes them an ideal tool for studying membrane protein interactions, membrane fluidity, and the dynamic behavior of molecules within membranes. ② Small molecular weight, minimal interference: Compared to other green fluorescent dyes (such as GFP and FITC), NBD molecules are much smaller. This means that when attached to target molecules (such as lipids and small molecule drugs), it has less impact on the properties, conformation, and function of the target molecule itself, resulting in more reliable experimental results. ③ High designability: The molecular structure of NBD is easily chemically modified. By introducing different reactive groups (such as carboxyl groups, maleimide, etc.) onto the NBD backbone, “turn-on” probes that can specifically recognize different targets (such as ions, reactive oxygen species, small molecules) can be designed. The fluorescence is very weak before the target is detected, and the fluorescence is significantly enhanced after binding.

[0005] Current medical evidence shows that when cells are subjected to acute oxidative stress induced by exogenous H2O2, endogenous H2S and hNQO1 can be spontaneously produced intracellularly. According to current knowledge, hNQO1 is generated via the Keap1 (Kelch-like ECH-associated protein 1) / Nrf2 (nucleoerythrocyte cytokine 2-associated factor 2) / ARE (antioxidant response element) pathway. Nrf2 protein levels can rapidly increase the response to ROS, triggering hNQO1 expression to inhibit free radical formation. This leads to a proposed mechanism for the synergistic antioxidant effect of H2S and hNQO1 under oxidative stress. Therefore, this application proposes a synergistic antioxidant effect study investigating the role of H2S and hNQO1 in treating oxidative stress in living cells, suggesting that the role may be regulated by Nrf2. Nrf2 can trigger hNQO1 to directly and indirectly increase endogenous H2S levels by controlling glutathione, etc. Figure 12 As shown.

[0006] To simultaneously detect H2S and hNQO1, this application incorporates two chemically selective triggering groups, one responding to H2S and the other to hNQO1, into a single fluorophore. This dual-activity probe is superior to traditional single-analyte detection probes because it offers several advantages, including: (1) avoiding uneven distribution of different probes within the cell; (2) providing an enhanced on-response due to the double quenching effect; and (3) establishing a simple method to study the cooperative relationships between analytes. The design of the dual biomarker-triggered fluorescent probe is illustrated. This probe should only be activated through a synergistic chemical reaction with H2S and an enzymatic reaction with hNQO1, as shown below. Figure 13 As shown.

[0007] Significant progress has been made in the research of H2S fluorescent molecular probes in recent years, with many probe molecules already capable of detecting H2S concentrations in cells and organisms. However, novel fluorescent molecular probes suitable for quantitative detection of H2S in living cells and in vivo still require further development. Furthermore, because H2S fluorescent molecular probes are easily interfered with by other endogenous biological thiols, the development of fluorescent molecular probes with high selectivity for H2S is also a key research focus for researchers.

[0008] Most existing bioactive species-responsive fluorescent probes have shortcomings, such as short excitation and emission wavelengths, shallow imaging depth, poor photostability, and high cytotoxicity. Furthermore, the low steady-state concentrations and short lifetimes of bioactive species in biological systems make the construction of fluorescent probes with excellent responsiveness for in-situ detection of bioactive species in vivo a significant challenge. Organic fluorescent probes have attracted widespread attention due to their advantages, including simple synthesis, high structural controllability, high sensitivity, and rapid real-time in-situ detection. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing technologies by proposing a Bodipy and NBD dual-fluorescent probe, its synthesis method, and its application. The dual-fluorescence response system is evaluated using phthalimide fluorescence. The fluorescence of the phthalimide moiety can be quenched by the fluorescence-induced electron transfer (PET) effect when the fluorescent lamp is turned off, while the fluorescence of the NBD moiety can be quenched by the fluorescence resonance energy transfer (FRET) effect. This invention anticipates that the fluorescence of coumarin and phthalimide, through this dual quenching, can efficiently achieve a quenching strategy, applicable to the simultaneous detection of H2S and hNQO1 in vivo or in vitro.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A dual fluorescent probe combining Bodipy and NBD has the following chemical structure:

[0012] .

[0014] A method for synthesizing a Bodipy and NBD dual fluorescent probe, comprising the following steps:

[0015] First, piperazine phenol reacts with phosphorus oxychloride to add an aldehyde group to obtain compound 2, namely piperazine benzaldehyde. Then, benzaldehyde 2 is condensed with dimethyl malonate to obtain coumarin compound 3. The methyl ester is demethylated with sodium hydroxide alkaline solution to obtain carboxylic acid 4. Finally, it reacts with NBD piperazine 5 under the action of condensing agent EDCI to generate NBD coumarin compound 6.

[0016] The synthetic route for the NBD coumarin compound 6 is shown in the following formula: .

[0017] Preferably, the synthesis method further includes the following steps:

[0018] Starting from nitrobenzaldehyde f and dimethylpyrrole as raw materials, the fluorescent compound Bodipy h was obtained through a four-step one-pot cascade reaction under the action of catalytic amounts of trifluoroacetic acid (TFA), DDQ oxidation, and excess base triethylamine and excess Lewis acid boron trifluoride diethyl ether.

[0019] Specifically, the process of obtaining Bodipy fluorescent probe 8 by reducing and esterifying the fluorescent compound Bodipy h with an acylate involves the following steps: the nitro group h of the parent ring structure of the fluorescent compound Bodipy is reduced with hydrogen under palladium-carbon catalysis to obtain the amino compound i of Bodipy; then, benzoquinone acyl chloride and the amino compound i of Bodipy are amidated to obtain Bodipy fluorescent probe 8; finally, Bodipy fluorescent probe 8 is demethylated with lithium hydroxide to obtain carboxylic acid, which is then amidated with NBD compound 6 deprotected by trifluoroacetic acid to obtain the target product dual fluorescent probe 9.

[0020] The synthesis route of the Bodipy and NBD dual fluorescent probe synthesis method is shown in the following formula: .

[0021] The present invention also provides an application of the Bodipy and NBD dual fluorescent probe obtained by the above synthesis method for the simultaneous detection of H2S and hNQO1 in vivo or in vitro.

[0022] The detection principle is shown in the following formula:

[0023] This invention utilizes benzoquinone to block the amino functional groups of the Bodipy and NBD dual-fluorescent probe, which exhibit fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) mechanisms, thereby shutting down the parent fluorescence. When this fluorescent probe is used to detect thiol-containing compounds such as H₂S, a Michael addition reaction occurs between the carbonyl double bond and the thiol group at the probe's terminal chain, opening the blocked amino functional groups and generating chromophores. This achieves fluorescent recognition of thiol-containing compounds with high detection sensitivity, as shown in the figure below.

[0024]

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The novel structure of the Bodipy and NBD dual-fluorescent probe obtained by the reaction of this invention involves a conjugated addition reaction between the benzoquinone group and the thiol group in the probe to generate a chromophore. Based on the dual-effect principle of fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET), this invention designed and synthesized a fluorescent probe 9 containing both Bodipy and coumarin in 11 chemical steps. The probe was designed to test the coupling of hydrogen sulfide (H2S) with quinone oxidoreductase 1 (NAD(P)H: Quinone Oxidoreductase, NQO1), especially bioactive components containing thiol groups (such as glutathione GSH), and the compound structure is relatively stable.

[0027] 2. The synthesis method provided by this invention has a high yield of Bodipy and NBD dual fluorescent probes, and the overall yield of the 11-step reaction is high.

[0028] 3. The unique structure of the fluorescent probe Bodipy of the present invention, especially the Bodipy carboxylic acid obtained by deesterifying the methyl ester with lithium hydroxide, provides a basis for subsequent protein coupling.

[0029] 4. The Bodipy fluorescent probe of this invention has significant fluorescence color development effect at a wavelength of 485nm, and has good selectivity. Its fluorescence signal does not change much when interacting with other common amino acids. It has high anti-interference ability, short reaction time, large Stokes shift, and low detection limit. Attached Figure Description

[0030] Figure 1This is a synthetic route diagram for NBD coumarin compound 6 of the present invention;

[0031] Figure 2 This is a synthesis route diagram of the Bodipy and NBD dual fluorescent probe 9 of this invention;

[0032] Figure 3 This is the proton NMR spectrum of NBD coumarin compound 6 in Example 1 of the present invention;

[0033] Figure 4 This is the carbon spectrum of NBD coumarin compound 6 in Example 1 of the present invention;

[0034] Figure 5 The hydrogen spectrum of compound i in Example 2 of this invention;

[0035] Figure 6 This is the carbon spectrum of compound i of Bodipy in Example 2 of the present invention;

[0036] Figure 7 The hydrogen spectrum of compound 8 of Bodipy in Example 3 of this invention;

[0037] Figure 8 This is the carbon spectrum of compound 8 of Bodipy in Example 3 of the present invention;

[0038] Figure 9 The hydrogen spectrum of the Bodipy and NBD dual fluorescent probe 9 in Example 4 of this invention;

[0039] Figure 10 The carbon spectrum of the Bodipy and NBD dual fluorescent probe 9 in Example 4 of this invention;

[0040] Figure 11 The images show fluorescence spectra of the Bodipy and NBD dual-fluorescent probe 9 at different concentrations and times with H2S donors; where A represents 10 μM probe and 1 mM Na2S (H2S donor). The absorption peaks of the probe (0 min) are at 420 and 490 nm, corresponding to the fluorescent group and the NBD amine structure, respectively; the reaction with H2S is rapid, and the reaction is close to saturation at 20 min. After the reaction, the absorbance at 420 nm increases significantly, and the absorption peak at 490 nm redshifts significantly to 530 nm, proving the formation of NBD-SH.

[0041] B: Probe 1 μM, Na₂S 100 μM, excitation wavelength 400 nm, emission wavelength approximately 485 nm. The background fluorescence intensity of the probe is very low. After reacting with 100 eq H₂S for 30 minutes, it reaches equilibrium, and the fluorescence intensity increases by approximately 42 times, demonstrating the strong quenching efficiency (FRET effect) of the NBD amino group on the fluorescent group coumarin.

[0042] Figure 12 This is a possible hypothetical mechanism blueprint for the synergistic antioxidant effect of H2S and hNQO1 under oxidative stress in this invention. (It is inferred that the synergistic antioxidant effect of H2S and hNQO1 in treating oxidative stress in living cells may be regulated by Nrf2. Nrf2 can trigger the expression of hNQO1 (of a certain gene or protein) to directly exert its function and indirectly increase the level of endogenous H2S by controlling glutathione (GSH).)

[0043] Figure 13 This is a schematic diagram of the design of the dual biomarker-triggered fluorescent probe of the present invention. The probe should only be activated through a synergistic chemical reaction with H2S and an enzymatic reaction with hNQO1. Detailed Implementation

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the scope of protection of the present invention. The embodiments described in this invention are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0045] Example 1: Synthesis of NBD compound 6

[0046] Synthesis of NBD fragment 6 of coumarin -- First, piperazine benzaldehyde is obtained by reacting piperazine phenol with the aldehyde group of phosphorus oxychloride. Then, benzaldehyde 2 is condensed with dimethyl malonate to obtain coumarin compound 3. The methyl group is then demethylated with alkaline sodium hydroxide solution to obtain carboxylic acid 4. Finally, NBD piperazine 5 is reacted with the condensing agent EDCI to generate NBD coumarin compound 6. Figure 4 For details on the synthesis of this compound, please refer to the literature: Changyu Zhang, Qiang-Zhe Zhang, Kun Zhang, et al., Dual-biomarker-triggered fluorescence probes for differentiating cancer cells and revealing synergistic antioxidant effects under oxidative stress; Chem. Sci., 2019, 10, 1945–1952.

[0047] Example 2: Synthesis of Bodipy compound i

[0048] The Bodipy compound (i) is referenced in Liu Wei; Lu Yang; Shen Haoliang; Xu Jian; et al., "A Bodipy Fluorescent Probe and Its Synthesis Method", Patent No.: ZL 2022 1 0272240.1

[0049] Example 3: Synthesis of Bodipy compound 8

[0050] Benzoquinone 7 (55 mg, 0.22 mmol) was dissolved in 10 mL of anhydrous dichloromethane in a 25 mL flask. Under anhydrous and oxygen-free nitrogen protection, an excess of 0.5 mL of thionyl chloride was added dropwise. The mixture was stirred electromagnetically for 10 min, and then the dichloromethane and excess thionyl chloride were removed by rotary evaporation. Under anhydrous and oxygen-free nitrogen protection, 15 mL of anhydrous dichloromethane solution was added. The reduced Bodipy product i (80 mg, 0.2 mmol) was added to triethylamine (505 mg, 1.0 mmol), and the mixture was stirred at room temperature. The reaction was monitored by TLC. After 4 hours, 10 mL of water was added to quench the reaction. The solvent dichloromethane was removed by rotary evaporation, and 10 mL of water was added. The mixture was extracted three times with 30 mL of ethyl acetate each time. The ethyl acetate organic layer was collected, dried over anhydrous sodium sulfate for more than one hour, and then purified by column chromatography after rotary evaporation to give 97.0 mg of a red solid compound, Bodipy compound 8, in 77% yield.

[0051] Example 4: Synthesis of Bodipy and NBD dual fluorescent probe 9

[0052] Bodipy compound 8 (63.1 mg, 0.1 mmol) was dissolved in a 25 mL flask in a mixture of 5 mL tetrahydrofuran, 5 mL methanol, and 5 mL water. Lithium hydroxide (12.0 mg, 0.5 mmol) was added, and the mixture was stirred electromagnetically for 2 hours. All solvent was removed using a rotary evaporator. The mixture was then rapidly washed with methanol through a 1 cm silica gel column, and all solvent was removed again using a rotary evaporator. The mixture was dried under vacuum using an oil pump (this step removed the methyl ester to obtain the carboxylic acid compound). Simultaneously, NBD compound 6 (60.6 mg, 0.1 mmol) was... Add 2 mL of trifluoroacetic acid and 8 mL of dichloromethane solution, stir electromagnetically for 2 hours, remove all solvent using a rotary evaporator, and dry under vacuum using an oil pump (this step removes the Boc protecting group to obtain the amino compound); under anhydrous and oxygen-free nitrogen protection, add the above-treated Bodipy compound 8 and treated NBD compound 6, add N,N-diisopropylethylamine (64.7 mg, 0.5 mmol) and EDCI (38.3 mg, 0.2 mmol), add 10 mL of DMF, stir at room temperature, monitor the reaction by TLC, and quench the reaction with 10 mL of water after 6 hours. Post-treatment: remove the solvent DMF by vacuum distillation, add 20 mL of water, extract three times with 30 mL of ethyl acetate each time, collect the ethyl acetate organic layer, dry with anhydrous sodium sulfate for more than one hour, remove the solvent ethyl acetate by rotary evaporation, and purify by column chromatography to obtain 58.0 mg of red solid compound, the final product being Bodipy and NBD dual fluorescent compound 9, with a yield of 53%.

[0053] Materials: 9 μM Bodipy and NBD dual fluorescent probes, 100 μM Na2S

[0054] Methods: A 1 μmol / L Bodipy and NBD dual-fluorescent probe 9-dimethyl sulfoxide standard solution was prepared; 0.1 mmol / L Na₂S-dimethyl sulfoxide standard solution was added. Results are shown in [Figure number missing]. Figure 11 , Figure 11 The graph shows the fluorescence emission intensity changes of the Bodipy and NBD dual fluorescent probe 9 at different times; from Figure 11 As can be seen, after about 25 minutes, the reaction between Bodipy and NBD dual fluorescent probe 9 and H2S is basically complete, and the fluorescence intensity remains basically unchanged. This indicates that the fluorescent probe can respond to H2S rapidly.

[0055] in, Figure 11A: Probe 10 μM, Na₂S 1 mM (H₂S donor). The absorption peaks of the probe (0 min) are at 420 and 490 nm, corresponding to the fluorescent group and the NBD amine structure, respectively; the reaction with H₂S is rapid, and the reaction is close to saturation at 20 min. After the reaction, the absorbance at 420 nm increases significantly, and the absorption peak at 490 nm red-shifts significantly to 530 nm, proving the formation of NBD-SH.

[0056] B: Probe 1 μM, Na₂S 100 μM, excitation wavelength 400 nm, emission wavelength approximately 485 nm. The background fluorescence intensity of the probe is very low. After reacting with 100 eq H₂S for 30 minutes, it reaches equilibrium, and the fluorescence intensity increases by approximately 42 times, demonstrating the strong quenching efficiency (FRET effect) of the NBD amino group on the fluorescent group coumarin.

[0057] In summary, this invention evaluates a dual-fluorescence response system using phthalimide fluorescence. The fluorescence of the naphthalimide moiety can be quenched through a fluorescence-induced electron transfer (PET) effect when the fluorescent lamp is turned off, while the fluorescence of the NBD moiety can be quenched through a fluorescence resonance energy transfer (FRET) effect. This invention anticipates that the fluorescence of coumarin and naphthalimide, through this dual quenching, can efficiently implement a quenching strategy, applicable to the simultaneous detection of H2S and hNQO1 in vivo or in vitro.

[0058] The descriptions and practices disclosed in this invention are readily apparent and understandable to those skilled in the art, and various modifications and refinements can be made without departing from the principles of this invention. Therefore, any modifications or improvements made without departing from the spirit of this invention should also be considered within the scope of protection of this invention.

Claims

1. A Bodipy and NBD dual fluorescent probe, characterized in that, The chemical structure of the fluorescent probe is shown below: 。 2. The method for synthesizing a Bodipy and NBD dual fluorescent probe according to claim 1, characterized in that, The synthesis method includes the following steps: First, piperazine phenol reacts with phosphorus oxychloride to add an aldehyde group to obtain compound 2, namely piperazine benzaldehyde. Then, benzaldehyde 2 is condensed with dimethyl malonate to obtain coumarin compound 3. The methyl ester is demethylated with sodium hydroxide alkaline solution to obtain carboxylic acid 4. Finally, it reacts with NBD piperazine 5 under the action of condensing agent EDCI to generate NBD coumarin compound 6. The synthetic route for the NBD coumarin compound 6 is shown in the following formula: .

3. The method for synthesizing a Bodipy and NBD dual fluorescent probe according to claim 2, characterized in that, The synthesis method also includes the following steps: Starting from nitrobenzaldehyde f and dimethylpyrrole as raw materials, the fluorescent compound Bodipy h was obtained through a four-step one-pot cascade reaction under the catalytic oxidation of trifluoroacetic acid TFA and DDQ and the action of excess triethylamine and excess Lewis acid boron trifluoride diethyl ether. Specifically, the process of obtaining Bodipy fluorescent probe 8 by reducing and esterifying the fluorescent compound Bodipy h with an acylate involves the following steps: the nitro group h of the parent ring structure of the fluorescent compound Bodipy is reduced with hydrogen under palladium-carbon catalysis to obtain the amino compound i of Bodipy; then, benzoquinone acyl chloride and the amino compound i of Bodipy are amidated to obtain Bodipy fluorescent probe 8; finally, Bodipy fluorescent probe 8 is demethylated with lithium hydroxide to obtain carboxylic acid, which is then amidated with NBD compound 6 deprotected by trifluoroacetic acid to obtain the target product dual fluorescent probe 9. The synthesis route of the Bodipy and NBD dual fluorescent probe synthesis method is shown in the following formula: .

4. An application of a dual fluorescent probe of Bodipy and NBD obtained by the synthesis method described in any one of claims 2-3 for the simultaneous detection of H2S and hNQO1 in vivo or in vitro.

Images