Ultrafast fluorescent probe group for detecting cell stress pulse and application of ultrafast fluorescent probe group

Abstract

The invention relates to a cell stress pulse detection ultrafast fluorescent probe group and application thereof, belongs to the field of organic fluorescent probes, and provides specific micromolecular fluorescent probes C1 and T1 for detecting Cu < 2 + > and tyrosinase of CSP, the probe C1 is designed based on a specific recognition group of Cu < 2 + >, and the probe T1 is designed based on an active cavity structure of tyrosinase and a specific recognition group of the tyrosinase. All the compounds have ultrafast response speed, excellent light stability, good pH stability and anti-interference ability to other ions and amino acids in cells. After the probe C1 reacts with Cu < 2 + >, a lactam spiro ring is opened to emit strong near-infrared fluorescence, similarly, after the probe T1 reacts with tyrosinase, a spirolactone ring is opened, and a near-infrared fluorescence signal is remarkably enhanced. The probe C1 and the probe T1 can specifically and rapidly detect the Cu < 2 + > content and the tyrosinase activity in vitro, and CSP detection is achieved by monitoring the changes of Cu < 2 + > and tyrosinase in cells under the stress condition in real time.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of organic fluorescent probes, in particular to a cell stress pulse detection superfast fluorescent probe set and application thereof. BACKGROUND

[0002] Cell stress pulse (CSP) is a dynamic feedback regulation phenomenon when cells respond to damage beyond the homeostatic range, which is manifested as a pulsatile change in physiological phenotypes (such as copper ion concentration, tyrosinase activity). This process can be divided into three stages: the positive feedback stage from the initial stimulation time (t s ) to the time when the phenotype reaches the peak (t p ), the phenotype signal is amplified to the peak (P max ); then into the negative feedback stage from t p to the end of the stimulation time (t e ), the phenotype expression is finely regulated to terminate the stress response. At the cellular level, CSP is related to key physiological processes such as mitochondrial autophagy and cell proliferation. For example, during the sperm capacitation process, when external stimulation drives the phenotype change to the optimal activity level (t p ), the success rate of fertilization is significantly enhanced.

[0003] Accurate detection and understanding of CSP has great application potential in the biomedical field. However, there is still a lack of tools and methods for accurately detecting CSP. Small molecule fluorescent probe detection technology has the advantages of high sensitivity and good biocompatibility, and is widely used in the detection of biomarkers. The development of a superfast response fluorescent probe set can achieve rapid capture and definition of marker changes, thereby accurately monitoring the CSP process. Copper ions (Cu² + ) and tyrosinase are core regulatory modules of the melanin synthesis pathway: Cu² + As the third most abundant transition metal, it is a signal mediator of kinase activity and mitochondrial autophagy, and can enhance the ability of tyrosinase to catalyze the oxidation of catechol by stabilizing its binuclear copper active center (CuA / CuB), driving melanin synthesis to cope with oxidative stress. The sensitive biomarker set coupled by the two is directly related to melanin lesions.

[0004] Therefore, how to design and synthesize specific superfast fluorescent probe sets with Cu 2+ concentration and tyrosinase activity as marker phenotypes to realize real-time monitoring of Cu² + concentration and tyrosinase activity in living cells is crucial for analyzing CSP mechanisms. The superfast fluorescent probe set can rapidly capture changes in markers in a short time, track and define the peak of the phenotype at t p , and reveal the spatiotemporal characteristics of stress adaptation, providing precise molecular tools for studying physiological and pathological processes such as mitochondrial autophagy and oxidative damage defense. Summary of the Invention

[0005] The purpose of this invention is to solve the aforementioned problems in the prior art and provide an ultrafast fluorescent probe set for detecting cell stress pulses and its applications. The probes are respectively reacted with Cu... 2+ The fluorescence properties of the products reacting with tyrosinase are significantly different, making the signal easily detectable. This probe reaction can be used to detect Cu in various biological samples. 2+ The content of [specific substance] and the activity of tyrosinase were detected, and the CSP process was defined.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An ultrafast fluorescent probe set for detecting cellular stress pulses, comprising probe C1 of a compound as shown in formula (I) and probe T1 of a compound as shown in formula (II):

[0008] Formula (I)

[0009] Formula (II).

[0010] The probe C1 specifically recognizes Cu² + Upon response, the spironolactone ring opens and emits near-infrared fluorescence. The probe T1 specifically recognizes tyrosinase and, upon response, the spironolactone ring opens and emits near-infrared fluorescence.

[0011] The method for preparing the ultrafast fluorescent probe set for detecting cell stress pulses includes the following steps:

[0012] The steps for preparing probe C1 are as follows: 2'-(N,N-dimethylamino)-5-hydroxy-5,5-dimethyl-5H,10'H-spiro[dibenzo[b,e]silane-10,1'(3'H)-isobenzofuran]-3'-one was reacted with methanol and hydrazine hydrate under nitrogen protection, and then purified by reflux, vacuum distillation, extraction, drying, and column chromatography.

[0013] The steps for preparing probe T1 are as follows: 3-[(tert-butyldimethylsilyl)oxy]benzyl[7-(N,N-dimethylamino)-5,5-dimethyl-3'-oxo-5H,10'H-spiro[5,6]dibenzo[b,e]silane-10,1'(3'H)-isobenzofuran]-9-yl carbonate was reacted with tetrabutylammonium fluoride in tetrahydrofuran solution under ice-water bath and nitrogen protection. The mixture was then purified by heating and stirring, column chromatography, and vacuum concentration.

[0014] The aforementioned ultrafast fluorescent probe set for detecting cell stress pulses in living cells was used to detect Cu²⁺. + Applications in content measurement. Specifically, Cu²⁺ can be achieved in living cells within 1 minute.+ Content detection.

[0015] This invention uses a probe as a Cu 2+ The specific recognition molecule allows for the quantitative detection of the fluorescence signal of the product per unit time after the reaction to determine the Cu content in different biological systems. 2+ The content of [something] was determined, and the cell's pulse stress state was assessed.

[0016] The aforementioned ultrafast fluorescent probe set for detecting cell stress pulses is applied to the detection of tyrosinase activity in live cells. Specifically, it can detect tyrosinase activity in live cells within 15 minutes.

[0017] This invention uses a probe as a specific substrate for tyrosinase. After the reaction occurs, the activity of tyrosinase in different biological systems is determined by quantitatively detecting the fluorescence signal of the product per unit time, and at the same time, the cellular pulse stress state is determined.

[0018] The application of the aforementioned ultrafast fluorescent probe set for detecting cell stress pulses involves quantitatively detecting the probe and Cu²⁺. + The intensity and rate of change of fluorescence signals of the products after tyrosinase reaction are used to determine the state of cellular stress pulses.

[0019] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0020] 1. High specificity (unaffected by amino acids, reactive oxygen species, and various anions and cations): C1 and T1 can react with Cu respectively. 2+ It reacts with tyrosinase with high specificity to generate fluorescent dye;

[0021] 2. Inexpensive and readily available: C1 and T1 and their oxidation products can all be obtained through chemical synthesis, and the synthesis process is simple and easy to carry out;

[0022] 3. High sensitivity (in vitro detection concentration as low as 2 μM): C1 and T1 can react with Cu respectively. 2+ It reacts with tyrosinase with high specificity to generate the corresponding near-infrared fluorescent dye Dye1, which has excellent fluorescence emission spectral characteristics (620–750 nm). The probe molecule shows no detectable fluorescence signal, while the reaction product exhibits excellent fluorescence properties, allowing for good differentiation and detection. Quantitative determination can also be performed by plotting a standard curve. The product possesses high brightness and near-infrared fluorescence properties, reducing interference from biological background fluorescence. It is also suitable for super-resolution fluorescence imaging under structured light illumination to detect changes in target substances in living cells. Attached Figure Description

[0023] Figure 1 C1 and T1 are the effects of Cu on the cellular stress pulse, respectively. 2+Flowchart for content and tyrosinase activity detection;

[0024] Figure 2 C1 to Cu 2+ The detection spectrum (Ex = 580 nm);

[0025] Figure 3 C1 to Cu 2+ Fluorescence spectrum with increased concentration;

[0026] Figure 4 The results show the anti-interference capability test results for C1.

[0027] Figure 5 The detection spectrum of T1 against tyrosinase (Ex = 580 nm).

[0028] Figure 6 The fluorescence spectrum of T1 with increased tyrosinase protein concentration;

[0029] Figure 7 The results of the anti-interference capability test for T1;

[0030] Figure 8 CCK8 assay for cytotoxicity of C1;

[0031] Figure 9 Results of a live-cell imaging experiment in which C1 and B16 cells were co-incubated for 1 minute;

[0032] Figure 10 The results of the CCK8 assay for T1 cytotoxicity;

[0033] Figure 11 Results of a live-cell imaging experiment in which T1 and B16 cells were co-incubated for 15 minutes;

[0034] Figure 12 The results of confocal fluorescence microscopy imaging experiments were obtained after C1 and T1 cells were co-incubated with B16 cells for 0-2 hours.

[0035] Figure 13 Cu 2+ Results of quantitative analysis of fluorescence intensity from confocal imaging with tyrosinase. Detailed Implementation

[0036] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0037] The Cu designed and synthesized in this invention for detecting CSP 2+ The specific small molecule fluorescent probes C1 and T1 for tyrosinase have the following structural formulas: Figure 1 As shown above. Probe C1 is based on Cu.2+ Designed with specific recognition groups, probe T1 is based on the active cavity structure of tyrosinase and its specific recognition groups. Both exhibit ultrafast response speed, excellent photostability, good pH stability, and resistance to interference from other intracellular ions and amino acids. Probe C1 and Cu 2+ Following the reaction, the spironolactone ring opens, emitting strong near-infrared fluorescence. Similarly, after probe T1 reacts with tyrosinase, the spironolactone ring opens, significantly enhancing the near-infrared fluorescence signal. Probes C1 and T1 can specifically and rapidly detect Cu in vitro. 2+ Cu content and tyrosinase activity in cells under stress were monitored in real time. 2+ CSP detection is achieved through changes in tyrosinase, the mechanism of which is as follows: Figure 1 As shown below.

[0038] Example 1

[0039] Chemical synthesis methods of C1 and T1

[0040] The reaction formula is shown below:

[0041]

[0042] Step 1, 3-[(tert-butyldimethylsilyl)oxy]benzaldehyde (WH2)

[0043] 3-hydroxybenzaldehyde (32 g, 0.26 mmol) and imidazole (173 g, 2.6 mmol) dissolved in dichloromethane (5 mL) were added to a round-bottom flask. Then, di-tert-butyl dicarbonate (5.28 g, 27.83 mmol) was added, followed by tert-butyldimethylchlorosilane (394 mg, 2.6 mmol). The reaction mixture was stirred at 25 °C for 3 hours under nitrogen protection. The reaction was terminated with saturated sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous Na₂SO₄, concentrated under vacuum, and used directly in the next reaction step.

[0044] Step 2, [3-[(tert-butyldimethylsilyl)oxy]phenyl]methanol (WH3)

[0045] WH2 (1.77 g, 7.5 mmol) was dissolved in methanol (23 mL). NaBH4 (318 mg, 9.0 mmol) was added to the solution under ice-water bath conditions. The reaction was heated to 25 °C and stirred continuously for 1 hour. The reaction was slowly terminated at 0 °C with ultrapure water (10 mL), extracted with dichloromethane, and the organic phase was collected and dried over anhydrous Na2SO4. The organic phase was purified by column chromatography to give WH3 (1.52 g, 85%).

[0046] Step 3, 3-Bromo-N,N-diallylaniline (D2)

[0047] In a round-bottom flask, 13.76 g (80 mmol) of 3-bromoaniline, 33.87 g (280 mmol) of 3-bromopropene, and 22 g (160 mmol) of K₂CO₃ were added sequentially, followed by 80 mL of acetonitrile. The mixture was heated to 80 °C and refluxed for 14 hours. After cooling, the reaction was terminated by adding pure water. The organic phase was extracted with ethyl acetate, dried over anhydrous Na₂SO₄, and purified by rapid column chromatography to give a yellow oily compound D₂ (14 g, 90%).

[0048] Step 4, 4-(N,N-diallylamino)-2-bromobenzaldehyde (D3)

[0049] N,N-dimethylformamide (17.5 mL) and dichloromethane (37 mL) were added to a flask, and phosphorus oxychloride (25.5 g, 165 mmol) was slowly added dropwise under stirring in an ice-water bath. After the addition was complete, stirring was continued for 15 minutes. D2 was dissolved in dichloromethane (15 mL) and added dropwise to the flask. The reaction was allowed to proceed at room temperature for 18 hours. The reaction was terminated by adding sodium hydroxide solution (1 N) in an ice-water bath. The organic phase was extracted with dichloromethane and dried over anhydrous Na2SO4, and purified by rapid column chromatography to give a yellow oily compound D3 (13.9 g, 90%).

[0050] Step 5, [2-bromo-4-(N,N-diallylamino)phenyl]methanol (D4)

[0051] D3 was added to a flask, dissolved in methanol (30 mL) and dichloromethane (30 mL), and sodium borohydride (150 mmol) dissolved in dichloromethane (20 mL) was added dropwise to the flask. The reaction was allowed to proceed at room temperature for 24 hours. The reaction was terminated by adding pure water (80 mL). The organic phase was extracted with dichloromethane and dried over anhydrous Na2SO4, then purified by rapid column chromatography to give a yellow oily compound D4 (11 g, 80%).

[0052] Step 6, 4-[4-(N-methylamino)-2-bromobenzyl]-N,N-diallyl-3-bromoaniline (D5)

[0053] D4 (2 g, 7.08 mmol) and 3-bromo-1-N,N-dimethylaniline (1.42 g, 7.08 mmol) were added to a flask and dissolved in dry dichloromethane (30 mL). Boron trifluoride diethyl ether (1 mL) was then added under nitrogen protection. The reaction was allowed to proceed for 24 hours at room temperature. The reaction was terminated by adding saturated sodium bicarbonate solution, and the mixture was extracted with dichloromethane, dried over anhydrous Na₂SO₄, and purified by rapid column chromatography to give D5 (350 mg, 34%).

[0054] Step 7, 5,5-Dimethyl-N 3 N 3 -diallyl-N 7 N 7 -Dimethyl-5,10-dihydrodibenzo[b,e]silane-3,7-diamine (D6)

[0055] Add D5 (1 g, 2.76 mmol) to a two-necked flask under nitrogen protection. Add dry tetrahydrofuran (8 mL) using a syringe. Stir at -78°C for 20 min, then slowly add sec-butyllithium (5.5 mL) and continue stirring for 30 min. Next, add dichlorodimethylsilane (0.5 mL). Continue stirring at room temperature for 2 h. Terminate the reaction with aqueous hydrochloric acid (2 N). Neutralize the reaction solution with saturated sodium bicarbonate solution. Extract the mixture with dichloromethane, dry with anhydrous Na2SO4, concentrate under vacuum to collect D6, and proceed directly to the next step.

[0056] Step 8, 3-[N,N-diallylamino]-7-[N,N-dimethylamino]-5,5-dimethyl-5,10-dihydrodibenzo[b,e]silane-10-one (D7)

[0057] D6 and potassium permanganate (1.15 g, 7 mmol) were added to a flask, along with acetone (10 mL). The reaction was carried out overnight in an ice-water bath under nitrogen protection. The mixture was filtered through diatomaceous earth, the solid was washed with ethyl acetate, and the filtrate was collected, dried over anhydrous Na2SO4, and purified by rapid column chromatography to give compound D7 (0.21 g, 20%).

[0058] Step 9, 3-amino-7-[N,N-dimethylamino]-5,5-dimethyl-5,10-dihydrodibenzo[b,e]silane-10-one (D8)

[0059] D7 (1 g, 2.7 mmol) and 1,3-dimethylbarbituric acid (0.86 g, 5.4 mmol) were added to a flask and dissolved in dry dichloromethane (35 mL). The reaction was carried out overnight at room temperature under nitrogen protection. The reaction was terminated by adding saturated sodium carbonate aqueous solution (30 mL). The mixture was extracted with dichloromethane, dried over anhydrous Na2SO4, and purified by rapid column chromatography to give compound D8 (0.58 g, 72%).

[0060] Step 10, 3-(dimethylamino)-7-hydroxy-5,5-dimethyl-5,10-dihydrodibenzo[b,e]silane-10-one (D9)

[0061] D8 (258 mg, 0.87 mmol) was dissolved in methanol (10 mL) and sulfuric acid solution (6 N, 10 mL). The mixture was stirred in an ice-water bath, and sodium nitrite (600 mg, 8.7 mmol) dissolved in pure water (4 mL) was added dropwise. The reaction mixture was stirred in an ice-water bath for 1 hour under nitrogen protection, followed by the addition of sulfuric acid solution (1 N, 100 mL) and continued stirring for 10 minutes. After the reaction reached ambient temperature, the reaction mixture was extracted with dichloromethane. The organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous Na₂SO₄, and purified by rapid column chromatography to give D9 (81 mg, 31%).

[0062] Step 11, 3-[(tert-butyldimethylsilyl)oxy]-7-[N,N-dimethylamino]-5,5-dimethyl-5,10-dihydrodibenzo[b,e]silane-10-one (D15)

[0063] D9 (77 mg, 0.26 mmol), imidazole (176 mg, 2.6 mmol), and tert-butyldimethylchlorosilane (390 mg, 2.6 mmol) were added to a flask, and dissolved in 25 mL of dry dichloromethane. The reaction was carried out at room temperature for 3 hours under nitrogen protection, and the reaction was terminated by adding saturated sodium bicarbonate aqueous solution. The mixture was extracted with dichloromethane, dried over anhydrous Na2SO4, and purified by rapid column chromatography to give D15 (53 mg, 50%), a yellow oily compound.

[0064] Step 12, 3-[(tert-butyldimethylsilyl)oxy]-7-(dimethylamino)-5,5-dimethyl-3'H-spiro[5,10-dihydrodibenzo[b,e]silane-10,1'-isobenzofuran]-3'-one (D16)

[0065] 309 mg (1.5 mmol) of tert-butyl 2-bromobenzoate was added to a two-necked flask and dissolved in dry tetrahydrofuran (2 mL). The mixture was stirred at -15 °C for 10 min under nitrogen protection, and then iPrMgCl-LiCl (1.3 M dissolved in tetrahydrofuran, 924 μL, 1.2 mmol) was added. The temperature was gradually increased to -5 °C. In another flask, D15 (400 mg, 1 mmol) was added, dissolved in dry tetrahydrofuran (2 mL), and the mixture was protected under nitrogen. The D15 solution was added dropwise to the two-necked flask using a syringe. After stirring for 10 min, the reaction was heated to room temperature and stirred for 30 min. The reaction was terminated by adding saturated ammonium chloride solution. The mixture was extracted with ethyl acetate, dried over anhydrous Na2SO4, and purified by rapid column chromatography to obtain the oily compound D16, which was then directly proceeded to the next step.

[0066] Step 13, 2'-(N,N-dimethylamino)-5-hydroxy-5,5-dimethyl-5H,10'H-spiro[dibenzo[b,e]silane-10,1'(3'H)-isobenzofuran]-3'-one (Dye1)

[0067] D16 (0.015 mmol, 13 mg) was added to a flask and dissolved in tetrahydrofuran (0.5 mL). Tetrabutylammonium fluoride (1.0 M dissolved in tetrahydrofuran, 0.045 mmol, 45 μL) was then added with stirring at room temperature. After 1 hour, the solvent was removed by rotary evaporation, and the product was purified by rapid column chromatography to obtain Dye1 (6.5 mg, 67%). 1 H NMR (400 MHz, 298 K, CDCl3)δ 7.98 (d, J = 7.6 Hz, 1H), 7.66 (m, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.30 (d,J = 7.7 Hz, 1H), 7.14 (d, J = 2.8 Hz, 2H), 6.95 (d, J = 2.9 Hz, 2H), 6.81 (d,J = 8.9 Hz, 2H), 6.71 (d, J = 8.7 Hz, 2H), 6.63 (d, J = 2.8 Hz, 1H), 6.55 (m,2H), 2.96 (s, 9H), 0.59 (s, 3H), 0.51 (s, 3H). 13 C NMR (101 MHz, 298 K, CDCl3)δ 171.57, 155.53, 154.38, 149.48, 138.34, 136.78, 136.18, 133.64, 130.58,129.50, 128.67, 128.30, 126.61, 125.94, 125.09, 120.02, 117.25, 116.72,113.50, 92.11, 78.71, 77.14, 76.82, 40.38, 0.23, -1.52.

[0068] Step 14, 3-(dimethylamino)-5,5-dimethyl-7-[(4-nitrophenoxy)carbonyloxy]-5,10-dihydrospiro[dibenzo[b,e]silane-10,1'-isobenzofuran]-3'-one (D18)

[0069] Compound Dye1 (20 mg, 0.05 mmol) was added to a flask and dissolved in dry dichloromethane (2 mL). Pyridine (59 mg, 0.75 mmol) and p-nitrophenyl chloroformate (20 mg, 0.1 mmol) were added under stirring in an ice-water bath. The reaction mixture was brought to room temperature and stirred for 24 hours. The reaction mixture was then diluted with dichloromethane (10 mL), washed successively with saturated sodium bicarbonate and saturated sodium chloride aqueous solutions, and dried over anhydrous Na₂SO₄. Purification by rapid column chromatography yielded a white solid, D18 (11.3 mg, 40%). 1 H NMR (400 MHz, 298 K, CDCl3) delta 8.31 (d, J = 9.2 Hz,1H), 8.07 (s, 1H), 8.00 (d, J = 6.7 Hz, 1H), 7.71 (s, 2H), 7.59 (d, J = 7.0Hz, 2H), 7.48 (d, J = 9.2 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.05 (d, J = 8.8Hz, 1H), 6.99 (s, 1H), 6.87 (d, J = 6.9 Hz, 1H), 6.59 (d, J = 6.1 Hz, 1H), 2.98 (s, 6H), 0.66 (d, J = 17.7 Hz, 6H).

[0070] Step 15, 3-[(tert-butyldimethylsilyl)oxy]benzyl[7-(N,N-dimethylamino)-5,5-dimethyl-3'-oxo-5H,10'H-spiro[5,6]dibenzo[b,e]silane-10,1'(3'H)-isobenzofuran]-9-yl carbonate (D19)

[0071] A solution of triethylamine (0.3 mmol) in dry dichloromethane (3 mL) was slowly added dropwise at 0 °C to a round-bottom flask containing D18 (80 mg, 0.16 mmol) and WH3 (0.3 mmol, 72 mg) in dry dichloromethane (5 mL). The reaction was carried out at room temperature for 12 h. The product was dried over anhydrous Na2SO4, concentrated under vacuum, and used directly in the next reaction step.

[0072] Step 16, 2'-amino-3-(dimethylamino)-5-hydroxy-5,5-dimethyl-5,10-dihydrospiro[dibenzo[b,e]silane-10,1'-isoindoline]-3'-one (C1)

[0073] Dye1 (500 mg, 1.25 mmol) was placed in a dry 250 mL round-bottom flask. Methanol (20 mL) was added under nitrogen protection, followed by dropwise addition of hydrazine hydrate (80%, 5.6 mL). The solution immediately turned pink. The reaction system was stirred at room temperature for 10 min, then refluxed. The reaction was complete when the system turned pale yellow. Methanol was removed by vacuum distillation to obtain the crude compound, which was dissolved in dichloromethane. The organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous Na₂SO₄, concentrated under vacuum, and separated by column chromatography to obtain C1 (26 mg, 50%). 1 H NMR (400 MHz, 298K, MeOD) delta 8.10 (s,1H), 7.59 - 7.45 (m, 2H), 7.07 (d, J = 29.7 Hz, 3H), 6.73 - 6.61 (m, 2H), 6.50 (s, 2H), 2.86 (s, 6H), 0.51 (d, J = 21.9 Hz, 6H). 13 C NMR (101 MHz, 298K, MeOD) delta 168.49, 157.15, 150.51, 147.12, 141.10, 140.44, 139.57, 137.29, 132.79, 131.68, 131.17, 130.94, 130.58, 128.64, 121.34, 118.80, 115.71, 113.56, 68.28, 40.77, 1.34, -2.31.

[0074] Step 17, [9-({[(3-hydroxyphenyl)methoxy]carbonyl}oxy)-7-(N,N-dimethylamino)-5,5-dimethyl-5H,10'H-spiro[5.6]dibenzo[b,e]silane-10,1'(3'H)-isobenzofuran]-3'-one (T1)

[0075] D19 (60 mg, 0.06 mmol) was dissolved in dry dichloromethane (5 mL) and placed in an ice-water bath under nitrogen protection. Tetrabutylammonium fluoride (24 mg, 0.09 mmol) was dissolved in dry tetrahydrofuran (3 mL) and slowly added dropwise to a round-bottom flask containing D19, stirring for 10 minutes. The mixture was then heated to room temperature and stirred for 2 hours. The reaction mixture was purified by column chromatography and concentrated under vacuum to give T1 (22 mg, 67%). 1 H NMR (400 MHz, 298K, DMSO- d 6 ) delta 7.96 (t, J =10.5 Hz, 1H), 7.77 (td, J = 7.6, 1.0 Hz, 1H), 7.69 - 7.62 (m, 1H), 7.56 (t, J = 3.7 Hz, 1H), 7.38 - 7.31 (m, 1H), 7.22 (ddd, J = 21.9, 10.9, 4.9 Hz, 1H),7.15 - 7.05 (m, 2H), 7.01 - 6.94 (m, 1H), 6.86 (dd, J = 8.4, 4.8 Hz, 2H), 6.82 - 6.74 (m, 2H), 6.69 - 6.61 (m, 1H), 5.15 (d, J = 31.3 Hz, 2H), 3.03 –2.89 (m, 6H), 0.64 (d, J = 10.6 Hz, 3H), 0.60 - 0.55 (m, 3H). 13 C NMR (101MHz, 298K, MeOD) delta 170.96, 157.44, 154.08, 153.51, 150.78, 149.81, 142.33, 138.66, 136.54, 135.62, 134.33, 130.48, 129.35, 129.22, 127.87, 127.72, 125.85, 125.73, 125.33, 124.41, 122.02, 118.94, 116.43, 115.20, 114.66, 113.49, 91.10, 69.98, 39.01, -1.24, -2.95.

[0076] Example 2

[0077] C1 to Cu 2+ responsiveness test

[0078] Prepare Cu 2+ The reaction system (200 μL) included PBS buffer at pH 7.4 and Cu. 2+ (50 μM), C1 (5 μM), after shaking and pre-incubation at 37℃ for 3 minutes, fluorescence scanning detection (Ex = 580 nm) was performed. C1 and Cu 2+ The response produces enhanced fluorescence, see Figure 2 .

[0079] Example 3

[0080] C1 to Cu 2+ Concentration gradient response

[0081] (1) Prepare Cu 2+ The reaction system (200 μL) included PBS buffer at pH 7.4 and Cu. 2+ (0~50 μM), C1 (5 μM), pre-incubated at 37°C with shaking for 3 minutes.

[0082] (2) Fluorescence scanning was performed 3 minutes after the reaction (Ex = 580 nm). The fluorescence intensity increased with Cu 2+ The concentration increases with increasing concentration, see Figure 3 .

[0083] Example 4

[0084] C1 to Cu 2+ Selective testing

[0085] Prepare reaction systems (200 μL) for different interfering factors, including PBS buffer (pH = 7.4), C1 (5 μM), and incubate with shaking at 37°C for 1 hour. After 1 hour of reaction, detect the fluorescence intensity at 630 nm (Ex = 580 nm). The fluorescence intensity of C1 and Cu... 2+ The response produces enhanced fluorescence, while the reaction system of C1 with other interfering factors shows almost no increase in fluorescence. See Figure 4 .

[0086] Example 5

[0087] T1 response test to tyrosinase

[0088] Prepare a tyrosinase reaction system (200 μL) including PBS buffer (pH = 7.4), tyrosinase (50 U / mL), and T1 (2 μM). Incubate at 37°C with shaking for 1 hour. After 1 hour of reaction, perform fluorescence scanning (Ex = 580 nm). T1 reacts with tyrosinase, producing enhanced fluorescence. See [link to relevant documentation]. Figure 5 .

[0089] Example 6

[0090] T1 response to tyrosinase concentration gradient test

[0091] (1) Prepare a tyrosinase reaction system (200 μL), including PBS buffer at pH = 7.4, tyrosinase (0-100 μM), T1 (2 μM), and pre-incubate at 37°C with shaking for 1 hour.

[0092] (2) Fluorescence scanning was performed 1 hour after the reaction (Ex = 580 nm). The fluorescence intensity increased with the increase of tyrosinase concentration, see [reference needed]. Figure 6 .

[0093] Example 7

[0094] Selectivity test of T1 for tyrosinase

[0095] Prepare reaction systems (200 μL) for different interfering factors, including PBS buffer (pH = 7.4) and T1 (2 μM), and incubate with shaking at 37°C for 1 hour. After 1 hour of reaction, measure the fluorescence intensity at 630 nm (Ex = 580 nm). T1 reacts with tyrosinase, producing enhanced fluorescence. In the reaction systems of T1 with other interfering factors, fluorescence shows almost no increase. See [link to relevant documentation]. Figure 7 .

[0096] Example 8

[0097] C1 cytotoxicity test

[0098] B16 cells were seeded into 96-well plates in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100.0 mg / L streptomycin, and 100 IU / mL penicillin. When the cell density reached approximately 60%, the medium was aspirated from the 96-well plates, and different concentrations of C1 (0–30 μM) were dissolved in DMEM and added to the 96-well plates. After incubation for 24 hours, cell viability was tested using the CCK8 cell viability assay kit. Figure 8 .

[0099] Example 9

[0100] C1 affects Cu in living cells2+ Content detection

[0101] B16 cells were seeded into confocal culture dishes in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100.0 mg / L streptomycin, and 100 IU / mL penicillin. After complete cell adhesion, the medium was aspirated, and the cells were incubated with mitochondrial-targeting and lysosome-targeting commercial dyes according to the instructions for 20 minutes. Then, medium containing C1 (5 μM) was added to the culture dishes and incubated for 3 minutes. Imaging was performed using confocal microscopy (C1 excitation light 561 nm, commercial dye excitation light 488 nm). The imaging results showed the presence of Cu in both lysosomes and mitochondria. 2 + ,See Figure 9 .

[0102] Example 10

[0103] T1 cytotoxicity test

[0104] B16 cells were seeded into 96-well plates in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100.0 mg / L streptomycin, and 100 IU / mL penicillin. When the cell density reached approximately 60%, the medium was aspirated from the 96-well plates, and different concentrations of T1 (0–30 μM) were dissolved in DMEM and added to the 96-well plates. After incubation for 24 hours, cell viability was tested using the CCK8 cell viability assay kit. Figure 10 .

[0105] Example 11

[0106] T1 assay for tyrosinase activity in living cells

[0107] B16 cells were seeded into confocal culture dishes in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100.0 mg / L streptomycin, and 100 IU / mL penicillin. After complete cell adhesion, the medium was aspirated, and the cells were incubated with mitochondrial-targeting and lysosome-targeting commercial dyes according to the instructions for 20 minutes. Then, medium containing T1 (2 μM) was added to the culture dishes and incubated for 15 minutes. Imaging was performed using a confocal microscope (T1 excitation light 561 nm, commercial dye excitation light 488 nm). The imaging results showed the presence of tyrosinase in both lysosomes and mitochondria. Figure 11 .

[0108] Example 12

[0109] C1 and T1 were used to monitor intracellular Cu in B16 cells during CSP using fluorescence imaging. 2+ Changes in tyrosinase

[0110] B16 cells were seeded into confocal culture dishes in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100.0 mg / L streptomycin, and 100 IU / mL penicillin. After complete cell adhesion, the medium was aspirated, and C1 (5 μM) and T1 (2 μM) were added to culture dishes containing CCCP (40 μM) and incubated for 2 hours. Imaging was performed using confocal microscopy and structured illumination fluorescence microscopy (Ex = 561 nm). The imaging results showed that C1 and T1 could inhibit Cu during the CSP process. 2+ Real-time imaging was performed to capture changes in the content of [specific substance] and the activity of tyrosinase. (See attached image.) Figure 12 .

[0111] Example 13

[0112] Cu 2+ Quantitative analysis using fluorescence imaging of tyrosinase confirmed the phenotypic peak time (t) in CSP. p )

[0113] For Cu 2+ The fluorescence intensity was quantified, and the rate of increase in fluorescence in the tyrosinase imaging map was calculated. This allowed for real-time tracking of changes in the catalytic rate of tyrosinase during CSP. The two methods corroborated each other, successfully confirming the presence of t in CSP. p ,See Figure 13 .

Claims

1. An ultrafast fluorescent probe set for detecting cellular stress pulses, characterized in that, Probe C1 having a compound as shown in formula (I) and probe T1 having a compound as shown in formula (II): Formula (I) Formula (II).

2. The ultrafast fluorescent probe set for detecting cell stress pulses as described in claim 1, characterized in that: The probe C1 specifically recognizes Cu² + Upon response, the spironolactone ring opens and emits near-infrared fluorescence. The probe T1 specifically recognizes tyrosinase and, upon response, the spironolactone ring opens and emits near-infrared fluorescence.

3. The method for preparing the ultrafast fluorescent probe set for detecting cell stress pulses according to claims 1-2, characterized in that, Includes the following steps: The steps for preparing probe C1 are as follows: 2'-(N,N-dimethylamino)-5-hydroxy-5,5-dimethyl-5H,10'H-spiro[dibenzo[b,e]silane-10,1'(3'H)-isobenzofuran]-3'-one was reacted with methanol and hydrazine hydrate under nitrogen protection, and then purified by reflux, vacuum distillation, extraction, drying, and column chromatography. The steps for preparing probe T1 are as follows: 3-[(tert-butyldimethylsilyl)oxy]benzyl[7-(N,N-dimethylamino)-5,5-dimethyl-3'-oxo-5H,10'H-spiro[5,6]dibenzo[b,e]silane-10,1'(3'H)-isobenzofuran]-9-yl carbonate was reacted with tetrabutylammonium fluoride in tetrahydrofuran solution under ice-water bath and nitrogen protection. The mixture was then purified by heating and stirring, column chromatography, and vacuum concentration.

4. The ultrafast fluorescent probe set for detecting cell stress pulses according to any one of claims 1 to 2 in detecting Cu²⁺ in living cells. + Application of content.

5. The application as described in claim 4, characterized in that: Cu²⁺ in living cells within 1 minute + Content detection.

6. The application as described in claim 4, characterized in that: Using the probe as Cu 2+ The specific recognition molecule allows for the quantitative detection of the fluorescence signal of the product per unit time after the reaction to determine the Cu content in different biological systems. 2+ The content of [something] was determined, and the cell's pulse stress state was assessed.

7. The application of the ultrafast fluorescent probe set for detecting cell stress pulses according to any one of claims 1 to 2 in detecting tyrosinase activity in living cells.

8. The application as described in claim 7, characterized in that: The activity of tyrosinase in live cells can be detected within 15 minutes.

9. The application as described in claim 7, characterized in that: Using the probe as a specific substrate for tyrosinase, the activity of tyrosinase in different biological systems is determined by quantitatively detecting the fluorescence signal of the product per unit time after the reaction occurs, and the cellular pulse stress state is also assessed.

10. The application of the ultrafast fluorescent probe set for detecting cell stress pulses according to any one of claims 1 to 2, characterized in that: Quantitative detection probe with Cu² + The intensity and rate of change of fluorescence signals of the products after tyrosinase reaction are used to determine the state of cellular stress pulses.

Images