Synthesis method of NBD fluorescent probe and method for detecting and imaging cysteine in living cells by using probe

Abstract

According to the synthesis method of the NBD fluorescent probe disclosed by the invention, the fluorescent probe is synthesized based on nitrobenzoxadiazole, and selective detection of cysteine is realized by connecting electron-deficient 4-hydroxyl-N, N, N-trimethyl phenyl ammonium to a nitrobenzoxadiazole fluorophore. The NBD fluorescent probe can be used for selectively detecting cysteine in a pure water environment in a detection and imaging method of cysteine in living cells, has a high signal-to-noise ratio and a low detection limit, and has a good distinguishing capability on other biological mercaptans. In addition, due to low cytotoxicity and excellent biocompatibility, endogenous and exogenous cysteine in living cells can be imaged in real time. The multifunctional probe provides a powerful tool for dynamic monitoring of cysteine in a complex biological system, and has a wide application prospect in the fields of disease diagnosis and precision medical treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a method for synthesizing an NBD fluorescent probe and a method for using the probe to detect and image cysteine in living cells. Background Art

[0002] Cysteine (Cys), a thiol-containing amino acid, participates in vital processes such as protein synthesis, metabolism, and detoxification. Cysteine plays a crucial role in many physiological processes, including metabolic regulation and cellular redox homeostasis. The normal concentration of cysteine in the human body ranges from 30 to 200 μM. Abnormal fluctuations and imbalances in cysteine levels are associated with a variety of diseases, including growth retardation, Alzheimer's disease, Parkinson's disease, immune dysfunction, cancer, neurodegenerative diseases, and cardiovascular disease. Therefore, real-time monitoring of cysteine level fluctuations in complex biological systems is not only applicable for early diagnosis and monitoring of diseases, but also facilitates in-depth research into biochemical processes inside and outside cells, revealing their dynamic changes in health and disease, thereby developing personalized treatment plans and improving therapeutic efficacy.

[0003] Currently, commonly used methods for measuring cysteine levels include high-performance liquid chromatography, electrochemical sensing, mass spectrometry, colorimetry, and spectroscopy. These methods often have limitations in terms of equipment cost, operational complexity, real-time monitoring capabilities, and detection sensitivity. Fluorescent sensors offer high specificity, high sensitivity, portability, ease of use, and real-time detection capabilities. However, the number of fluorescent sensors that specifically respond to cysteine is limited, they cannot distinguish between Cys, HCY, and GSH, and most operate in the presence of mixed aqueous / organic solvents. Therefore, there is an urgent need to develop fluorescent sensors with good water solubility, high sensitivity, and strong anti-interference capabilities. Therefore, improvements to this technology are necessary. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a method for synthesizing an NBD fluorescent probe to improve sensitivity and anti-interference ability.

[0005] In order to solve the above technical problems, the present invention is solved by the following technical solutions:

[0006] An NBD fluorescent probe, the compound synthesis formula of the NBD fluorescent probe is:

[0007]

[0008] The present invention also discloses a method for synthesizing an NBD-A fluorescent probe, comprising the following steps:

[0009] S1. Under nitrogen atmosphere, add 4-trimethylaminophenol and triethylamine to a reaction flask containing DMF;

[0010] S2. After stirring at 0°C for 30 minutes, a DMF solution of NBD-Cl was added dropwise to the mixture;

[0011] S3, the reaction mixture was continued to be stirred at room temperature overnight;

[0012] S4. The reaction mixture was then slowly added to acetone, followed by filtration and washing with acetone multiple times to obtain a yellow solid precipitate product NBD-A.

[0013] The present invention also discloses a method for synthesizing an NBD-B fluorescent probe, comprising the following steps:

[0014] S1. Under nitrogen atmosphere, add 4-dimethylaminophenol and triethylamine to a reaction flask containing DMF;

[0015] S2. After stirring at 0°C for 30 minutes, a DMF solution of NBD-Cl was added dropwise to the mixture;

[0016] S3, the reaction mixture was then stirred at room temperature overnight;

[0017] S4. To terminate the reaction, the mixture was added with water and extracted three times with ethyl acetate. The organic phase was washed three times with a saturated aqueous sodium bicarbonate solution, once with pure water, and once with a saturated sodium chloride solution, and finally dried over anhydrous sodium sulfate for 30 minutes.

[0018] S5. After removing the solvent under reduced pressure, the crude product was purified by silica gel column chromatography to obtain black solid NBD-B.

[0019] The present invention also discloses a method for synthesizing an NBD-PFP fluorescent probe, comprising the following steps:

[0020] S1. Under nitrogen atmosphere, add 4-dimethylaminophenol and triethylamine to a reaction flask containing DMF;

[0021] S2. After stirring at 0°C for 30 minutes, a DMF solution of NBD-Cl was added dropwise to the mixture;

[0022] S3, the reaction mixture was then stirred at room temperature overnight;

[0023] S4. To terminate the reaction, the mixture was added with water and extracted three times with ethyl acetate. The organic phase was washed three times with a saturated aqueous sodium bicarbonate solution, once with pure water, and once with a saturated sodium chloride solution, and finally dried over anhydrous sodium sulfate for 30 minutes.

[0024] S5. After removing the solvent under reduced pressure, the crude product was subjected to silica gel column chromatography to synthesize dark yellow solid NBD-PFP.

[0025] The present invention also discloses a method for detecting cysteine in living cells using an NBD fluorescent probe, which is characterized by comprising the following steps:

[0026] S1. The reactivity and fluorescence response of NBD fluorescent probe in water were investigated. In the presence of cysteine, the UV-visible absorption spectrum and fluorescence emission spectrum of NBD fluorescent probe were recorded.

[0027] S2. After adding cysteine, the UV-visible absorption spectrum of the NBD fluorescent probe red-shifted from 375 nm to 480 nm, and the color changed from colorless to brown, indicating the formation of amino-substituted NBD;

[0028] S3. In the fluorescence spectrum, the NBD fluorescent probe exhibited weak fluorescence due to insufficient electron donation ability and free rotation of the aromatic ring at the R site. After reacting with cysteine, a significant fluorescence enhancement was observed, accompanied by a red shift in the UV-visible absorption peak, with an emission wavelength of 550 nm.

[0029] S4. In the presence of biothiols, the fluorescence intensity of the NBD fluorescent probe was monitored over time. The NBD fluorescent probe showed high selectivity for cysteine, and the fluorescence signal reached saturation within 30 minutes.

[0030] S5, the fluorescence intensity of the NBD fluorescent probe at 550 nm remained stable within 60 min, and NBD-cysteine formed a stable adduct;

[0031] S6. Conduct fluorescence titration experiments to understand the interaction between NBD-A and Cys to evaluate the sensitivity and response dynamics of the probe;

[0032] S7. Analysis of the reaction mechanism by ultra-performance liquid chromatography (UPLC);

[0033] S8. Compare the reaction kinetics with the reaction of homocysteine (HCY) under the same conditions.

[0034] In the above technical solution, the experimental process of S6 includes:

[0035] S61. With the increase of cysteine concentration, the fluorescence intensity at 550 nm increased, indicating a strong fluorescence turn-on effect;

[0036] S62. The effect of the NBD fluorescent probe on the Cys fluorescence response in the reaction environment was studied by detecting changes in fluorescence intensity at different pH values. The fluorescent probe maintained optimal fluorescence activation within the pH range of 6-8, which covers the slightly acidic environment of diseased tissue and physiological pH values.

[0037] S63, the specificity of NBD fluorescent probe for Cys, including metal ions and amino acids, the fluorescence intensity of NBD fluorescent probe at 550 nm in PBS buffer in the presence of different metal ions, anions and amino acids;

[0038] The S64 and NBD fluorescent probes exhibit selective fluorescence responses to cysteine, while the fluorescence changes are negligible in the presence of other biological thiols or potential interfering substances.

[0039] In the above technical solution, the reaction process of the NBD fluorescent probe and Cys in S7 includes:

[0040] S71, first forming an S-linked NBD-Cys intermediate, in which the sulfhydryl group (-SH) of Cys attacks the electrophilic center of the probe through a nucleophilic substitution reaction;

[0041] S72, this intermediate undergoes an intramolecular rearrangement, specifically a Smiles rearrangement, to generate an N-linked NBD-Cys product.

[0042] In the above technical solution, S8 includes:

[0043] S81 and the NBD fluorescent probe formed an S-linked NBD-HCY intermediate, and only a small amount was converted into the corresponding N-linked amide product after 1 h of incubation;

[0044] S82, in the presence of cysteine, the conversion efficiency of internal thioester to amide is higher than that of HCY;

[0045] The S83 and NBD fluorescent probes can promote Smiles rearrangement in the presence of cysteine, but are insensitive to structurally similar biological thiols.

[0046] The present invention also discloses a method for imaging cysteine in living cells using an NBD fluorescent probe, which is characterized by comprising the following steps:

[0047] S1. When cells were pre-treated with N-methylmaleimide before incubation with the NBD fluorescent probe to inactivate intracellular biothiols and thiol-containing proteins, no fluorescence was detected;

[0048] S2. After cells were pretreated with N-methylmaleimide, they were incubated with Cys, HCY, or GSH, and finally treated with the NBD fluorescent probe. Only the Cys-treated cells showed strong fluorescence, while no fluorescence was observed in the HCY- or GSH-treated cells.

[0049] The S3 and NBD fluorescent probes can selectively monitor endogenous and exogenous cysteine in living cells.

[0050] Beneficial effects: Compared with the prior art, the present invention has the following beneficial effects:

[0051] The synthesis method of the NBD fluorescent probe of the present invention is a fluorescent probe synthesized based on nitrobenzoxadiazole, by connecting electron-deficient 4-hydroxy-N, N, N-trimethylphenylammonium to a nitrobenzoxadiazole fluorophore, to achieve selective detection of cysteine. The probe is used for cysteine detection and imaging methods in living cells and can selectively detect cysteine in a pure water environment, with a high signal-to-noise ratio (1000 times) and a low detection limit (0.01ppm), and has good distinguishing ability to other biological thiols (such as HCY and GSH). In addition, its low cytotoxicity and superior biocompatibility enable it to image endogenous and exogenous cysteine in living HeLa cells in real time. This multifunctional probe provides a powerful tool for the dynamic monitoring of cysteine in complex biological systems, making it also have a wide range of applications in disease diagnosis and precision medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those skilled in the art, other relevant drawings can be obtained based on these drawings without creative work.

[0053] Figure 1a Schematic diagram of the synthesis route of the NBD-A fluorescent probe of the present invention;

[0054] Figure 1b Schematic diagram of NBD-A aqueous solution with different concentrations of the present invention;

[0055] Figure 2a is the chemical structural formula of the NBD derivative probe of the present invention;

[0056] Figure 2b : is the normalized UV absorption spectrum of NBD-A (10 μM) of the present invention in Cys (50 μM) aqueous solution;

[0057] Figure 2c is the fluorescence emission spectrum of the present invention;

[0058] Figure 2d Fluorescence images of NBD-A (10 μM) and Cys (50 μM) at different reaction times of the present invention;

[0059] Figure 2eSchematic diagram of the fluorescence intensity of NBD-A (10 μM) and various biothiols (cysteine, HCY, GSH and H2S, 50 μM) at 550 nm under different reaction times;

[0060] Figure 2f Schematic diagram showing the fluorescence intensity comparison of the NBD derivative probe (10 μM) of the present invention and different biothiols (50 μM) after incubation at 25° C. for 30 minutes;

[0061] Figure 3a The fluorescence intensity of Cys (50 μM) and NBD-A (10 μM) at 550 nm at different pH values of the present invention;

[0062] Figure 3b The fluorescence intensity of NBD-A (10 μM) of the present invention changes with the Cys concentration in PBS buffer (10 mM, pH 7.4);

[0063] Figure 3c is the fluorescence intensity of NBD-A (10 μM) of the present invention in PBS buffer (10 mM, pH 7.4) at 550 nm in the presence of different metal ions, anions and amino acids (50 μM);

[0064] Figure 4a is the sensing mechanism of the NBD-A probe of the present invention, wherein Smiles rearrangement activates fluorescence;

[0065] Figure 4b The kinetic line graph of the reaction between NBD-A probe (20 μM) and Cys (100 μM) in water was analyzed by UPLC for the present invention;

[0066] Figure 4c The kinetic line graph of the reaction between NBD-A probe (20 μM) and HCY (100 μM) in water was analyzed by UPLC in the present invention;

[0067] FIG5 a is a confocal fluorescence image of the present invention showing the fluorescence detection of endogenous cysteine in HeLa cells after treatment with NBD-A probe;

[0068] FIG5 b is a confocal fluorescence image of the present invention showing fluorescence detection of HeLa cells pretreated with NEM (1 mM) for 30 min and then incubated with NBD-A probe for 30 min;

[0069] FIG5 c is a confocal fluorescence image of the present invention showing the detection of exogenous biosulfur in HeLa cells that were pretreated with NEM (1 mM) for 30 min, then incubated with 200 μM cysteine, and finally treated with NBD-A probe for 30 min;

[0070] FIG5 d is a confocal fluorescence image of the present invention showing the detection of exogenous biosulfur in HeLa cells that were pretreated with NEM (1 mM) for 30 min, then incubated with HCY, and finally treated with NBD-A probe for 30 min;

[0071] FIG5e is a confocal fluorescence image of the present invention showing the detection of exogenous biosulfur in HeLa cells that were pretreated with NEM (1 mM) for 30 min, then incubated with GSH, and finally treated with NBD-A probe for 30 min. DETAILED DESCRIPTION

[0072] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0073] Please refer to the accompanying drawings of the specification for an NBD fluorescent probe. The compound synthesis formula of the NBD fluorescent probe is as follows. In this embodiment, it includes NBD-A or NBD-B or NBD-PFP.

[0074]

[0075] The synthesis method of NBD-A comprises the following steps:

[0076] (1) Under nitrogen atmosphere, 4-trimethylaminophenol (0.1 g, 0.36 mmol) and triethylamine (50 μL, 0.36 mmol) were added to a reaction flask containing DMF;

[0077] (2) After the mixture in step (1) was stirred at 0°C for 30 minutes, a DMF solution of NBD-Cl (90 mg, 0.45 mmol) was added dropwise to the mixture;

[0078] (3) continuing to stir the reaction mixture in step (2) at room temperature overnight;

[0079] (4) The reaction mixture was then slowly added to acetone, followed by filtration and multiple washings with acetone to obtain the yellow solid precipitate product NBD-A (0.16 g, 81%). H NMR (400 MHz, DMSO-D6) δ 8.69, 8.67, 8.17, 8.14, 7.68, 7.66, 6.86, 6.84, 3.64. C NMR (101 MHz, DMSO-D6) δ 154.43, 152.37, 145.96, 145.45, 135.70, 131.72, 124.01, 122.32, 116.39, 112.04, 57.26.

[0080] The synthesis method of the NBD-B fluorescent probe includes the following steps:

[0081] (1) Under nitrogen, add 4-dimethylaminophenol (68.5 mg, 0.5 mmol) and triethylamine (70 μL, 0.5 mmol) to a reaction flask containing DMF;

[0082] (2) After the mixture in step (1) was stirred at 0°C for 30 minutes, a DMF solution of NBD-Cl (0.1 g, 0.5 mmol) was added dropwise to the mixture;

[0083] (3) the reaction mixture in step (2) was then stirred at room temperature overnight;

[0084] (4) To terminate the reaction, the mixture was added with water and extracted three times with ethyl acetate. The organic phase was washed three times with a saturated sodium bicarbonate aqueous solution, once with pure water, and once with a saturated sodium chloride solution, and finally dried over anhydrous sodium sulfate for 30 minutes.

[0085] (5) After removing the solvent under reduced pressure, the crude product was purified by silica gel column chromatography (PE / EA=4 / 1) to give NBD-B (0.13 g, 87%) as a black solid. H NMR (400 MHz, CDCl3) δ 8.41, 8.39, 7.25, 7.10, 7.08, 6.78, 6.76, 6.52, 6.50, 3.00, 3.00. C NMR (101 MHz, CDCl3) δ 155.86, 149.43, 145.31, 144.27, 143.15, 133.94, 130.10, 121.46, 113.49, 107.16, 40.87.

[0086] The synthesis method of the NBD-PFP fluorescent probe includes the following steps:

[0087] (1) Under nitrogen, add 4-dimethylaminophenol (68.5 mg, 0.5 mmol) and triethylamine (70 μL, 0.5 mmol) to a reaction flask containing DMF;

[0088] (2) After the mixture in step (1) was stirred at 0°C for 30 minutes, a DMF solution of NBD-Cl (0.1 g, 0.5 mmol) was added dropwise to the mixture;

[0089] (3) the reaction mixture in step (2) was then stirred at room temperature overnight;

[0090] (4) To terminate the reaction, the mixture was added with water and extracted three times with ethyl acetate. The organic phase was washed three times with a saturated sodium bicarbonate aqueous solution, once with pure water, and once with a saturated sodium chloride solution, and finally dried over anhydrous sodium sulfate for 30 minutes.

[0091] (5) After removing the solvent under reduced pressure, the crude product was purified by silica gel column chromatography (PE / EA = 4 / 1) to obtain NBD-PFP (0.144 g, 82%) as a dark yellow solid. H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 8.2 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H).

[0092] In addition, the following performance tests were conducted on the NBD fluorescent probe in this example. Figure 1a-Figure 3c shown.

[0093] (1) UV-visible absorption spectroscopy and fluorescence spectroscopy analysis

[0094] A 1 mM stock solution of probe NBD-A was prepared in deionized water, while stock solutions of probes NBD-B and NBD-C were prepared in DMSO. Stock solutions of cysteine (Cys), homocysteine (HCY), and other analytes (10 mM) were prepared in deionized water. The mixed solutions containing 10 μM probes were incubated at room temperature and analyzed at an excitation wavelength of 487 nm.

[0095] (2) Cell culture and imaging experiments

[0096] HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO₂ and 95% air. Images were taken using a Zeiss LSM900 laser confocal microscope using an NBD-A probe with an excitation wavelength of 487 nm and fluorescence collection in the range of 500–575 nm.

[0097] (3) Cytotoxicity test

[0098] The biotoxicity of the probe was evaluated using the CCK-8 kit. 3 HeLa cells were seeded in 96-well plates and incubated for 24 hours in DMEM medium with final NBD-A concentrations of 2, 5, 10, 20, and 50 μM. CCK-8 solution (1 mg / mL) was added to each well and incubated for another 2 hours. After thorough mixing, the absorbance was measured at 450 nm using a microplate reader. Each experiment was repeated three times, and cell viability was normalized to untreated cells (100%).

[0099] NBD-A probe detection capabilities

[0100] To evaluate the detection ability of NBD-A for Cys in water, we first investigated its reactivity and fluorescence response in water. In the presence of cysteine, the UV-visible absorption and fluorescence emission spectra of NBD-A were recorded. Figure 2b As shown in the figure, after adding cysteine, the UV-visible absorption spectrum of NBD-A red-shifted from 375nm to 480nm, and the color changed from colorless to brown, indicating the formation of amino-substituted NBD. In the fluorescence spectrum, NBD-A exhibited weak fluorescence due to insufficient electron donor ability and free rotation of the aromatic ring at the R site. After reacting with cysteine, a significant fluorescence enhancement (1000 times) was observed, accompanied by a red shift of the UV-visible absorption peak, with a maximum emission wavelength of 550nm ( Figure 2c-2d In the presence of biothiols, the fluorescence intensity of NBD-A was monitored over time. NBD-A showed high selectivity for cysteine, and the fluorescence signal reached saturation within 30 minutes (see Figure 2e In contrast, NBD-B and NBD-PFP also showed some selectivity for cysteine, but the fluorescence intensity changes were smaller (up to 15 times), indicating that the smile rearrangement was affected by both the electron density of the leaving group (R) and the water solubility of the probe (see Figure 2f Importantly, the fluorescence intensity of NBD-A at 550 nm remained stable for 60 minutes, demonstrating the stability of the NBD-cysteine adduct. In summary, NBD-A not only exhibits high selectivity for cysteine but also forms a highly stable fluorescent product, highlighting its potential in cell fluorescence imaging.

[0101] To gain a deeper understanding of the interaction between NBD-A and Cys, fluorescence titration experiments were performed to evaluate the sensitivity and response kinetics of the probe. As the concentration of cysteine increased, the fluorescence intensity at 550 nm increased significantly, indicating a strong fluorescence turn-on effect (see Figure 3aNBD-A has high sensitivity and excellent signal-to-noise ratio, with a detection limit of 0.01ppm. Subsequently, the effect of the reaction environment on the fluorescence response of NBD-A to Cys was studied by detecting the changes in fluorescence intensity at different pH values (see Figure 3b The results showed that the probe maintained its optimal fluorescence activation state within the pH range of 6-8, which covers the slightly acidic environment of diseased tissue (pH 6-7) and physiological pH (pH 7.4). Since the selectivity of fluorescent probes in complex biological environments is crucial for practical applications, we further investigated the specificity of NBD-A for Cys in the presence of various competing species, including metal ions and amino acids. Figure 3c As shown, the fluorescence intensity of NBD-A (10 μM) in PBS buffer (10 mM, pH 7.4) at 550 nm in the presence of different metal ions, anions and amino acids (50 μM), including (1) NBD-A, (2) KI, (3) KBr, (4) KF, (5) KNO3, (6) K2CO3, (7) KSO4, (8) LiCl, (9) Mn(SO4)2, (10) CaCl2, (11) ,(12)PbCl2,(13)MgSO4,(14)NaCl,(15)Fe(NO3)3,(16)Ala,(17)Asp,(18)His,(19)Val,(20)Leu,(21)Arg,(22)Gly,(23)Trp,(24)Lys,(25)Glu,(26)Pro,(27)Thr,(28)Cys,(29)HCY,(30)GSH. NBD-A exhibits a selective fluorescence response to cysteine, while the fluorescence change is almost negligible in the presence of other biological thiols or potential interfering substances.

[0102] Elucidation of the sensing mechanism

[0103] After confirming the specific sensing ability of NBD-A for Cys, we further elucidated the reaction mechanism by ultra-performance liquid chromatography (UPLC) analysis (see Figure 4b The results showed that NBD-A reacts with Cys to first form an S-linked NBD-Cys intermediate, in which the sulfhydryl group (-SH) of Cys attacks the electrophilic center of the probe via a nucleophilic substitution reaction. Subsequently, the intermediate undergoes an intramolecular rearrangement, specifically a Smiles rearrangement, to generate an N-linked NBD-Cys product (please refer to Figure 4a This structural transition is accompanied by a significant increase in fluorescence intensity at 550 nm, providing strong evidence that the reaction mechanism is responsible for the observed fluorescence turn-on response.

[0104] To further elucidate the selectivity of NBD-A, we compared its reaction kinetics with that of homocysteine (HCY) under the same conditions. UPLC analysis showed that NBD-A primarily formed the S-linked NBD-HCY intermediate, with only a small amount converted to the corresponding N-linked amide product even after 1 hour of incubation (see also Figure 4c ). This significant difference indicates that in the presence of cysteine, the conversion efficiency of internal thioesters to amides is significantly higher than that of HCY. The slower and incomplete conversion of HCY may be attributed to its additional methylene (-CH2-) unit, which may introduce steric hindrance and reduce the required nucleophilicity, thereby affecting the efficient rearrangement. These findings provide compelling mechanistic insights into the strong selectivity of NBD-A for cysteine. NBD-A can quickly and efficiently promote Smiles rearrangement in the presence of cysteine, while being almost insensitive to structurally similar biological thiols (such as HCY and GSH), highlighting its potential as a highly selective and sensitive probe for cysteine detection in complex biological environments.

[0105] Live cell imaging of NBD-A

[0106] To evaluate its biocompatibility, the cytotoxicity of NBD-A at various concentrations was determined in HeLa cells. After 24 hours of incubation, cell viability remained above 90%, even at concentrations as high as 50 μM, indicating that NBD-A has low cytotoxicity and excellent biocompatibility, making it suitable for biological applications.

[0107] Selective imaging of cysteine in HeLa cells was then performed. Initially, cells not incubated with the NBD-A probe exhibited almost no fluorescence. After 30 minutes of incubation with the NBD-A probe, strong fluorescence was observed in the green channel (see Figure 5a). However, when cells were pretreated with N-methylmaleimide (NEM) to inactivate intracellular biothiols and thiol-containing proteins before incubation with the NBD-A probe, no fluorescence was detected (see Figure 5b). To further evaluate the probe's ability to discriminate and image exogenous biothiols, cells were pretreated with NEM, then incubated with Cys, HCY, or GSH, and finally treated with the NBD-A probe. As shown in Figures 5c-5e, only cells treated with Cys exhibited strong fluorescence, while no fluorescence was observed in cells treated with HCY or GSH. These results confirm that the NBD-A probe can selectively monitor endogenous and exogenous cysteine in living cells.

[0108] In summary, the synthesis method of the NBD fluorescent probe of the present invention is a fluorescent probe synthesized based on nitrobenzoxadiazole, by connecting electron-deficient 4-hydroxy-N, N, N-trimethylphenylammonium to a nitrobenzoxadiazole fluorophore, achieving selective detection of cysteine. The probe is used for cysteine detection and imaging methods in living cells and can selectively detect cysteine in a pure water environment, with a high signal-to-noise ratio (1000 times) and a low detection limit (0.01ppm), and has good distinguishing ability to other biological thiols (such as HCY and GSH). In addition, its low cytotoxicity and superior biocompatibility enable it to image endogenous and exogenous cysteine in living HeLa cells in real time. This multifunctional probe provides a powerful tool for the dynamic monitoring of cysteine in complex biological systems, making it also have a wide range of applications in disease diagnosis and precision medicine.

[0109] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the concept and scope of the present invention. Any modifications and improvements made to the technical solution of the present invention by a person of ordinary skill in the art without departing from the design concept of the present invention shall fall within the scope of protection of the present invention. The technical content for which protection is sought in the present invention is fully set forth in the claims.

Claims

1. A NBD fluorescent probe, characterized in that The compound synthesis formula of the NBD fluorescent probe is:

2. A method for synthesizing an NBD-A fluorescent probe, characterized in that: The following steps are involved: S1. Under nitrogen atmosphere, add 4-trimethylaminophenol and triethylamine to a reaction flask containing DMF; S2. After stirring at 0°C for 30 minutes, a DMF solution of NBD-Cl was added dropwise to the mixture; S3, the reaction mixture was continued to be stirred at room temperature overnight; S4. The reaction mixture was then slowly added to acetone, followed by filtration and washing with acetone multiple times to obtain a yellow solid precipitate product NBD-A.

3. A method for synthesizing an NBD-B fluorescent probe, characterized in that: The following steps are involved: S1. Under nitrogen atmosphere, add 4-dimethylaminophenol and triethylamine to a reaction flask containing DMF; S2. After stirring at 0°C for 30 minutes, a DMF solution of NBD-Cl was added dropwise to the mixture; S3, the reaction mixture was then stirred at room temperature overnight; S4. To terminate the reaction, the mixture was added with water and extracted three times with ethyl acetate. The organic phase was washed three times with a saturated aqueous sodium bicarbonate solution, once with pure water, and once with a saturated sodium chloride solution, and finally dried over anhydrous sodium sulfate for 30 minutes. S5. After removing the solvent under reduced pressure, the crude product was purified by silica gel column chromatography to obtain black solid NBD-B.

4. A method for synthesizing a NBD-PFP fluorescent probe, characterized in that: The following steps are involved: S1. Under nitrogen atmosphere, add 4-dimethylaminophenol and triethylamine to a reaction flask containing DMF; S2. After stirring at 0°C for 30 minutes, a DMF solution of NBD-Cl was added dropwise to the mixture; S3, the reaction mixture was then stirred at room temperature overnight; S4. To terminate the reaction, the mixture was added with water and extracted three times with ethyl acetate. The organic phase was washed three times with a saturated aqueous sodium bicarbonate solution, once with pure water, and once with a saturated sodium chloride solution, and finally dried over anhydrous sodium sulfate for 30 minutes. S5. After removing the solvent under reduced pressure, the crude product was subjected to silica gel column chromatography to synthesize dark yellow solid NBD-PFP.

5. A method for detecting cysteine in living cells using an NBD fluorescent probe, characterized in that: The following steps are involved: S1. The reactivity and fluorescence response of NBD fluorescent probe in water were investigated. In the presence of cysteine, the UV-visible absorption spectrum and fluorescence emission spectrum of NBD fluorescent probe were recorded. S2. After adding cysteine, the UV-visible absorption spectrum of the NBD fluorescent probe red-shifted from 375 nm to 480 nm, and the color changed from colorless to brown, indicating the formation of amino-substituted NBD; S3. In the fluorescence spectrum, the NBD fluorescent probe exhibited weak fluorescence due to insufficient electron donation ability and free rotation of the aromatic ring at the R site. After reacting with cysteine, a significant fluorescence enhancement was observed, accompanied by a red shift in the UV-visible absorption peak, with an emission wavelength of 550 nm. S4. In the presence of biothiols, the fluorescence intensity of the NBD fluorescent probe was monitored over time. The NBD fluorescent probe showed high selectivity for cysteine, and the fluorescence signal reached saturation within 30 minutes. S5, the fluorescence intensity of the NBD fluorescent probe at 550 nm remained stable within 60 min, and NBD-cysteine formed a stable adduct; S6. Conduct fluorescence titration experiments to understand the interaction between NBD-A and Cys to evaluate the sensitivity and response dynamics of the probe; S7. Analysis of the reaction mechanism by ultra-performance liquid chromatography (UPLC); S8. Compare the reaction kinetics with the reaction of homocysteine (HCY) under the same conditions.

6. The detection method according to claim 5, wherein The experimental process of S6 includes: S61. With the increase of cysteine concentration, the fluorescence intensity at 550 nm increased, indicating a strong fluorescence turn-on effect; S62. The effect of the NBD fluorescent probe on the Cys fluorescence response in the reaction environment was studied by detecting changes in fluorescence intensity at different pH values. The fluorescent probe maintained optimal fluorescence activation within the pH range of 6-8, which covers the slightly acidic environment of diseased tissue and physiological pH values. S63, the specificity of NBD fluorescent probe for Cys, including metal ions and amino acids, the fluorescence intensity of NBD fluorescent probe at 550 nm in PBS buffer in the presence of different metal ions, anions and amino acids; The S64 and NBD fluorescent probes exhibited selective fluorescence responses to cysteine, while the fluorescence changes were negligible in the presence of other biological thiols or potential interferents.

7. The detection method according to claim 5, wherein The reaction process between the NBD fluorescent probe and Cys in S7 includes: S71, first forming an S-linked NBD-Cys intermediate, in which the sulfhydryl group (-SH) of Cys attacks the electrophilic center of the probe through a nucleophilic substitution reaction; S72, this intermediate undergoes an intramolecular rearrangement, specifically a Smiles rearrangement, to generate an N-linked NBD-Cys product.

8. The detection method according to claim 5, wherein The S8 includes: S81 and the NBD fluorescent probe formed an S-linked NBD-HCY intermediate, and only a small amount was converted into the corresponding N-linked amide product after 1 h of incubation; S82, in the presence of cysteine, the conversion efficiency of internal thioester to amide is higher than that of HCY; The S83 and NBD fluorescent probes can promote Smiles rearrangement in the presence of cysteine, but are insensitive to structurally similar biological thiols.

9. A method for imaging cysteine in living cells using an NBD fluorescent probe, characterized in that: The following steps are involved: S1. When cells were pre-treated with N-methylmaleimide before incubation with the NBD fluorescent probe to inactivate intracellular biothiols and thiol-containing proteins, no fluorescence was detected; S2. After cells were pretreated with N-methylmaleimide, they were incubated with Cys, HCY, or GSH, and finally treated with the NBD fluorescent probe. Only the Cys-treated cells showed strong fluorescence, while no fluorescence was observed in the HCY- or GSH-treated cells. The S3 and NBD fluorescent probes can selectively monitor endogenous and exogenous cysteine in living cells.

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