Beta-galactosidase fluorescent probe, preparation method and application thereof

Abstract

The application belongs to the technical field of biological analysis detection, and specifically discloses a kind of beta-galactosidase fluorescent probe and a preparation method and application thereof.The molecular structural formula of the fluorescent probe provided by the application includes one of TCFM-Gal1, TCFM-Gal2, TCFM-Gal3 or TCFM-Gal4.By introducing an eliminable fluorine-substituted methyl (difluoromethyl, monofluoromethyl) at the ortho position of the enzymatic substrate of the fluorescent probe, the generation of an electrophilic quinone methyl intermediate under enzymatic hydrolysis can be realized;and further covalently combined with the adjacent nucleophilic protein, the specific opening and in-situ retention of the fluorescent signal in the senescent cells are realized.The fluorescent probe also has excellent beta-galactosidase detection sensitivity and specificity, and the detection fluorescence has a good linear relationship with the enzyme concentration, and the detection range is wide.

Description

Technical Field

[0001] This invention relates to the field of bioanalytical detection technology, and in particular to a class of β-galactosidase fluorescent probes, their preparation methods, and applications. Background Technology

[0002] Cellular senescence is a stress response mechanism triggered by intrinsic or extrinsic damage, ultimately leading to cells entering a stable state of cell cycle arrest. Studies have shown that cellular senescence participates in regulating various age-related diseases, such as cancer, fibrosis, and cardiovascular diseases. Furthermore, senescence-related growth arrest in cancer cells has been considered an effective therapeutic strategy; however, research has found that long-term treatment-induced cellular senescence can alter the tumor microenvironment through senescence-related secretory phenotypes, thereby promoting tumor invasion, metastasis, and recurrence. The dual nature of tumor senescence significantly complicates its role in cancer biology. Therefore, exploring the specific roles and mechanisms of cellular senescence in aging and related diseases is of great significance, and achieving real-time, specific detection and visualization of cellular senescence will be a key technical challenge.

[0003] Cellular senescence is characterized by phenotypic heterogeneity, and the abnormal accumulation of β-galactosidase in the lysosomes of senescent cells is currently the most widely used biomarker. Traditional detection methods (such as X-Gal staining) can effectively identify senescent cells, but they suffer from problems such as the need for tissue fixation, low sensitivity, and diffusion artifacts, making real-time monitoring impossible. Fluorescent detection methods can specifically image senescent cells in a non-invasive manner; for example, β-galactosidase (β-Gal)-activated fluorescent probes (C... 12 FDG, DDAOG). Existing β-Gal fluorescent probes mainly rely on the specificity of enzyme catalytic activity—hydrolyzing the β-D-galactopyranoside bond on the fluorophore to release the electron-donating group of the fluorophore, "turning on" the fluorescence signal, and achieving specific detection of senescent cells. However, in long-term tracking and flow cytometry analysis of senescent cells, this "fluorescent signal substance" produced by enzymatic hydrolysis is prone to extravasation and intercellular diffusion, leading to false negative / false positive results and reduced imaging signal-to-noise ratio, severely limiting the resolution and sensitivity of cell senescence detection.

[0004] Therefore, there is an urgent need to develop a fluorescent probe that can detect cellular senescence in situ and achieve long-term in situ retention of fluorescent signals for the study of mechanisms of aging-related diseases and screening of anti-aging drugs. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention provides an enzyme-controlled labeled β-galactosidase fluorescent probe. The fluorescent probe of this invention has excellent β-galactosidase detection sensitivity and specificity, and exhibits a good linear relationship between detection fluorescence and enzyme concentration, a wide detection range, and can also achieve in-situ detection of β-galactosidase in cells, and achieve long-term in-situ retention of the fluorescence signal.

[0006] The present invention also provides a method for preparing the fluorescent probe.

[0007] This invention also provides applications of the fluorescent probe.

[0008] In a first aspect, the present invention provides a β-galactosidase fluorescent probe, wherein the molecular structure of the fluorescent probe comprises one of the following: TCFM-Gal1, TCFM-Gal2, TCFM-Gal3, or TCFM-Gal4: ; ; ; .

[0009] According to a specific embodiment of the present invention, the fluorescent probe provided by the present invention utilizes a unique enzyme-controlled labeling strategy: by introducing an eliminable fluorinated substituted methyl group (difluoromethyl, monofluoromethyl) at the ortho position of the enzyme-catalyzed substrate of the fluorescent probe, the generation of an electrophilic quinone methyl (QM) intermediate under enzymatic hydrolysis can be achieved; and further, it can covalently bind with a nearby nucleophilic reagent (such as a thiol group on a protein) to achieve specific activation and in-situ retention of the fluorescent signal in senescent cells.

[0010] A second aspect of the present invention provides a method for preparing a β-galactosidase fluorescent probe as described in the first aspect of the present invention, characterized in that the synthesis of the fluorescent probe comprises the following route:

[0011] Route 1: ;

[0012] Starting with p-hydroxybenzaldehyde and 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile, intermediate m1 was generated by Knoevenagel condensation reaction. m1 was then converted to intermediate m2 by Duff reaction, and then the key intermediate m3 was obtained by nucleophilic substitution reaction. m3 underwent fluorination reaction to obtain compound TCFM-Gal1, and further obtained probe TCFM-Gal2 by alcoholysis reaction.

[0013] Route 2: ;

[0014] Using intermediate m3 as a raw material, intermediate m4 is generated by reduction reaction with sodium borohydride, and then fluorinated to obtain compound TCFM-Gal3. Further, probe TCFM-Gal4 is obtained through alcoholysis reaction.

[0015] According to some embodiments of the present invention, the preparation steps of route one include:

[0016] S1. Dissolve p-hydroxybenzaldehyde in a mixture with 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile, add base 1, and stir under nitrogen protection and reflux to react; cool, adjust the pH of the system to 5-8, let stand at low temperature, filter, wash and dry to obtain intermediate m1;

[0017] S2. Mix and dissolve intermediate m1 with hexamethylenetetramine, and stir the reaction under nitrogen protection; cool, pour the reaction solution into water, adjust the pH of the system to 7-8, extract, concentrate and separate and purify to obtain intermediate m2;

[0018] S3. Mix and dissolve intermediate m2 with base 2, add 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl-1-bromide, and stir the reaction under nitrogen protection; concentrate and separate and purify to obtain intermediate m3;

[0019] S4. Dissolve intermediate m3, cool it down and slowly add diethylaminosulfur trifluoride solution dropwise, and stir the reaction under nitrogen protection; quench the reaction with ice water, extract, concentrate and separate and purify to obtain compound TCFM-Gal1.

[0020] S5. Dissolve compound TCFM-Gal1, cool it down, and slowly add alkali 3 solution dropwise. Stir the reaction under nitrogen protection. Quench the reaction with ice water, extract, concentrate, and separate and purify to obtain compound TCFM-Gal2.

[0021] According to some embodiments of the present invention, the preparation steps of route two include:

[0022] S10. Dissolve intermediate m3, cool it down, add sodium borohydride in multiple portions, and stir the reaction under nitrogen protection; quench the reaction with dilute hydrochloric acid, extract, concentrate and separate and purify to obtain intermediate m4.

[0023] S20. Dissolve intermediate m4, cool it down and slowly add diethylaminosulfur trifluoride solution, stir the reaction under nitrogen protection; quench the reaction with ice water, extract, concentrate and separate and purify to obtain compound TCFM-Gal3.

[0024] S30. Dissolve compound TCFM-Gal3, cool it down and slowly add alkali 3 solution dropwise, and stir the reaction under nitrogen protection; quench the reaction with ice water, extract, concentrate and separate and purify to obtain compound TCFM-Gal4.

[0025] According to some embodiments of the present invention, in step S1, the molar ratio of p-hydroxybenzaldehyde to 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile is 1:(0.5~6), the solvent for mixing and dissolving includes at least one of dichloromethane, acetonitrile, anhydrous ethanol, N,N-dimethylformamide or dimethyl sulfoxide, and the base 1 includes at least one of pyridine, piperidine or diethylamine.

[0026] According to some embodiments of the present invention, in step S2, the molar ratio of the intermediate m1 to hexamethylenetetramine is 1:(1~6), and the solvent for mixing and dissolving includes trifluoroacetic acid or acetic acid.

[0027] According to some embodiments of the present invention, in step S3, the base 2 includes at least one of sodium carbonate, potassium carbonate, or cesium carbonate; the molar ratio of intermediate m2, base 2, and 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl-1-bromide is 1:(1~6):(1~3), and the solvent for mixing and dissolving includes at least one of dichloromethane, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, or acetone.

[0028] According to some embodiments of the present invention, in step S4, the solvent in which the intermediate m3 is dissolved includes dichloromethane or tetrahydrofuran, and the molar ratio of intermediate m3 to diethylaminosulfur trifluoride is 1:(1~10).

[0029] According to some embodiments of the present invention, in step S5, the solvent in which the compound TCFM-Gal1 is dissolved includes at least one of dichloromethane, tetrahydrofuran, acetone, ethanol or methanol, and the base 3 includes at least one of sodium ethoxide, sodium methoxide or sodium hydride, and the molar ratio of the compound TCFM-Gal1 to the base 3 is 1:(1~6).

[0030] According to some embodiments of the present invention, in step S10, the solvent in which the intermediate m3 is dissolved includes at least one of methanol, ethanol, dichloromethane or tetrahydrofuran; the molar ratio of intermediate m3 to sodium borohydride is 1:(1~5).

[0031] According to some embodiments of the present invention, in step S20, the solvent in which the intermediate m4 is dissolved includes dichloromethane and / or tetrahydrofuran, and the molar ratio of intermediate m4 to diethylaminosulfur trifluoride is 1:(1~10).

[0032] According to some embodiments of the present invention, in step S30, the solvent in which the compound TCFM-Gal3 is dissolved includes at least one of dichloromethane, tetrahydrofuran, acetone, ethanol or methanol, and the base 3 includes at least one of sodium ethoxide, sodium methoxide or sodium hydride, and the molar ratio of the compound TCFM-Gal3 to the base 3 is 1:(1~6).

[0033] A third aspect of the present invention provides the use of the β-galactosidase fluorescent probe as described in the first aspect of the present invention in the detection of β-galactosidase, the use comprising using the fluorescent probe to detect β-galactosidase, or to use it to prepare a reagent for detecting β-galactosidase.

[0034] A fourth aspect of the present invention provides the application of the β-galactosidase fluorescent probe as described in the first aspect of the present invention in biolabeling, the application including using the fluorescent probe for β-galactosidase-catalyzed protein labeling or cell labeling, or for preparing reagents for β-galactosidase-catalyzed protein labeling or cell labeling.

[0035] A fifth aspect of the present invention provides the application of the β-galactosidase fluorescent probe as described in the first aspect of the present invention in the detection of senescent cells, the application including using the fluorescent probe for the detection, fluorescence imaging or fluorescence labeling of senescent cells, or for the preparation of reagents for fluorescence imaging or fluorescence labeling of senescent cells.

[0036] A sixth aspect of the invention provides the use of the β-galactosidase fluorescent probe as described in the first aspect of the invention, the use of the fluorescent probe in the preparation of diagnostic tool molecules or reagents for detecting aging.

[0037] The beneficial effects of this invention are:

[0038] (1) The fluorescent probe for detecting aging-related β-galactosidase provided by this invention has inexpensive and readily available raw materials, a simple synthesis process, and high yield, making it suitable for large-scale production and widespread application. (2) This fluorescent probe responds rapidly to β-galactosidase, has a wide detection range, high sensitivity, good photostability, and high resistance to interference from common coexisting substances in the sample. (3) Dependent on the fluoromethyl group modified at the ortho position of the enzyme substrate, this fluorescent probe can generate a highly reactive quinone methylate (QM) intermediate in situ after enzymatic hydrolysis, which then covalently binds to the adjacent nucleophilic protein to form a stable "fluorescent-protein" complex. This enables the specific activation and long-term in situ retention of the fluorescent signal in aging cells, effectively solving the signal diffusion problem of traditional diffusible fluorescent probes. (4) This probe can not only achieve specific detection of aging cells, but also achieve long-term labeling of aging cells through covalent anchoring, facilitating long-term tracking of aging cells and flow cytometry cell sorting. (5) This enzyme-controlled fluorescent probe, which relies on the widespread expression of β-galactosidase in senescent cells, can sensitively detect and label senescent cells induced by different stimuli.

[0039] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0041] Figure 1 The absorption and emission spectra of the fluorescent probe in Example 5 of this invention are shown.

[0042] Figure 2 This is a kinetic diagram of the enzyme response time of the fluorescent probe in Example 6 of the present invention;

[0043] Figure 3 This is a linear response graph of the fluorescent probe in Example 7 of the present invention;

[0044] Figure 4 This is a selectivity and anti-interference response diagram of the fluorescent probe in Example 8 of the present invention;

[0045] Figure 5 This is a diagram showing the in vitro enzyme-controlled covalent labeling of the fluorescent probe in Example 9 of the present invention;

[0046] Figure 6 This is a diagram illustrating the enzyme-controlled nucleophilic fluorescent covalent labeling mechanism of the fluorescent probe in Example 9 of the present invention;

[0047] Figure 7 This is an in vitro enzyme-controlled protein covalent labeling diagram of the fluorescent probe in Example 10 of the present invention;

[0048] Figure 8 This is a cell senescence imaging image of the fluorescent probe in Example 11 of the present invention;

[0049] Figure 9 This is a diagram of the covalently labeled cellular senescence markers of the fluorescent probe in Example 12 of the present invention. Detailed Implementation

[0050] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0051] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0052] Example 1

[0053] This embodiment provides a method for synthesizing a β-galactosidase fluorescent probe—compound TCFM-Gal1. The synthetic route is as follows: ;

[0054] The specific steps are as follows:

[0055] 1) Synthesis of intermediate m1: 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile (1.20 g, 6 mmol) and p-hydroxybenzaldehyde (0.61 g, 5 mmol) were dissolved in 25 mL of anhydrous ethanol, and 100 μL of piperidine was added. The mixture was stirred and refluxed at 90 °C for 3 h. After the reaction was completed, the mixture was cooled to room temperature, and 100 μL of acetic acid was added to neutralize the piperidine in the system. A large amount of red solid precipitated immediately. The precipitate was collected by suction filtration and washed thoroughly with ice-cold ethanol to obtain 1.13 g of orange-red crystalline product, which is intermediate m1, with a yield of 75%.

[0056] 2) Synthesis of intermediate m2: Intermediate m1 (303 mg, 1 mmol) and hexamethylenetetramine (210 mg, 1.5 mmol) were dissolved in 10 mL of trifluoroacetic acid and stirred at 90 °C for 4 h under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, the reaction was quenched with ice water, and the pH of the system was adjusted to 7.5. The reaction mixture was extracted with water and dichloromethane, and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography, which was intermediate m2, with a yield of 28.4%.

[0057] 3) Synthesis of intermediate m3: Intermediate m2 (331 mg, 1 mmol) and potassium carbonate (146.6 mg, 4.5 mmol) were dissolved in 10 mL of acetonitrile and stirred at room temperature for 10 min; then 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl-1-bromide (616 mg, 1.5 mmol) was added and stirred at room temperature overnight; after the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 351 mg of yellow powder, which was intermediate m3, with a yield of 53%.

[0058] 4) Synthesis of compound TCFM-Gal1: Intermediate m3 (300 mg, 0.45 mmol) was dissolved in 10 mL of ultra-dry dichloromethane and stirred in an ice bath for 10 min; diethylaminosulfur trifluoride (725 mg, 4.5 mmol) was dissolved in 2 mL of dichloromethane solution and slowly added dropwise to the system in an ice bath; the reaction system was stirred at room temperature under nitrogen protection for 8 h, and the reaction progress was monitored by TLC; after the reaction was complete, the reaction was quenched with ice water, and the reaction solution was extracted with water and dichloromethane. The combined organic layers were dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 168 mg of yellow solid product, which was compound TCFM-Gal1, with a yield of 54.6%.

[0059] The nuclear magnetic resonance and high-resolution mass spectrometry data of the product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 8.17 (d, J= 8.8 Hz, 2H) , 7.95 (d, J = 16.4 Hz, 1H) , 7.32 (d, J = 8.5 Hz, 1H) , 7.24(d, J = 16.5 Hz, 1H) , 6.92 (t, J = 54.9 Hz, 1H) , 5.69 (d, J = 7.5 Hz, 1H) ,5.41 (d, J = 3.2 Hz, 1H) , 5.34 – 5.29 (m, 2H) , 4.55 (t, J = 6.5 Hz, 1H) ,4.18 – 4.13 (m, 2H) , 2.17 (s, 3H) , 2.06 (s, 3H) , 2.04 (s, 3H) , 1.97 (s,3H) , 1.82 (s, 6H). HRMS: theoretical for C 33 H 31 N3O 11 F2Na [M+Na]+ , 706.1824;found, 706.1816.

[0060] Example 2

[0061] This embodiment provides a method for synthesizing a β-galactosidase fluorescent probe—compound TCFM-Gal2. The synthetic route is as follows: ;

[0062] The specific steps are as follows:

[0063] Compound TCFM-Gal1 (100 mg, 0.15 mmol) was dissolved in 10 mL of methanol and stirred in an ice bath for 10 min. Sodium methoxide (11 mg, 0.2 mmol) was dissolved in 10 mL of methanol and slowly added dropwise to the system in an ice bath. The mixture was stirred overnight at room temperature under nitrogen protection, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction was quenched with ice water, and the reaction solution was extracted with water and dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to give 49 mg of yellow solid, which was the product TCFM-Gal2, with a yield of 65.5%.

[0064] The nuclear magnetic resonance and high-resolution mass spectrometry data of the product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 8.15 (s,1H) , 8.12 (d, J = 9.0 Hz, 1H) , 7.95 (d, J = 16.4 Hz, 1H) , 7.46 – 7.25 (m,2H) , 7.23 (d, J = 16.6 Hz, 1H) , 5.38 (d, J = 4.9 Hz, 1H) , 4.99 (d, J = 7.7Hz, 1H) , 4.96 (d, J = 5.1 Hz, 1H) , 4.71 (t, J = 5.3 Hz, 1H) , 4.62 (d, J =4.0 Hz, 1H) , 3.73 (s, 1H) , 3.70 – 3.67 (m, 1H) , 3.67 – 3.63 (m, 1H) , 3.58 – 3.50 (m, 2H) , 3.48 – 3.44 (m, 1H) , 1.81 (s, 6H) . HRMS: theoretical forC 25 H 23 N3O7F2Na [M+Na] +, 538.1402; found, 538.1401.

[0065] Example 3

[0066] This embodiment provides a method for synthesizing a β-galactosidase fluorescent probe—compound TCFM-Gal3. The synthetic route is as follows: ;

[0067] The specific steps are as follows:

[0068] 1) Synthesis of intermediate m4: Intermediate m3 (300 mg, 0.45 mmol) prepared in Example 1 was dissolved in 10 mL of tetrahydrofuran and stirred in an ice bath for 10 min; sodium borohydride (85 mg, 2.24 mmol) was added to the reaction system in 5 portions, and the reaction system was stirred in an ice bath for 2 h under nitrogen protection and the reaction progress was monitored by TLC; after the reaction was completed, 1.0 mmol / L dilute hydrochloric acid was added to quench the reaction, and the reaction solution was extracted with water and dichloromethane. The combined organic layers were dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 597 mg of yellow solid product, which was intermediate m4, with a yield of 90%.

[0069] 2) Synthesis of compound TCFM-Gal3: The synthesis of compound TCFM-Gal3 is based on Example 1, except that intermediate m3 in step 4) of Example 1 is replaced with an equimolar amount of intermediate m4, and the remaining reaction conditions and methods are the same as in Example 1.

[0070] The nuclear magnetic resonance and high-resolution mass spectrometry data of the product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.98 (d, J= 8.8 Hz, 2H) , 7.95 (d, J = 16.4 Hz, 1H) , 7.30 (d, J = 8.5 Hz, 1H) , 7.24(d, J = 16.5 Hz, 1H) , 6.92 (t, J = 54.9 Hz, 1H), 5.66 (d, J = 7.5 Hz, 1H), 5.41 (m, 2H), 5.34 – 5.29 (m, 2H), 4.55 (t, J = 6.5 Hz, 1H), 4.18 – 4.13(m, 2H), 2.17 (s, 3H) , 2.06 (s, 3H) , 2.04 (s, 3H) , 1.97 (s, 3H) , 1.81(s, 6H). HRMS: theoretical for C 33H 40 N3O7F [M+H] + , 609.2850; found, 609.2856.

[0071] Example 4

[0072] This embodiment provides a method for synthesizing a β-galactosidase fluorescent probe—compound TCFM-Gal4. The synthetic route is as follows: ;

[0073] The specific steps are as follows:

[0074] Referring to Example 2, compound TCFM-Gal1 was replaced with an equimolar amount of compound TCFM-Gal3, and the remaining reaction conditions and methods were the same as in Example 2, to obtain product TCFM-Gal4.

[0075] The nuclear magnetic resonance and high-resolution mass spectrometry data of the product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.94 (d, J= 16.3 Hz, 1H) , 7.91 (d, J = 8.6 Hz, 2H) , 7.15(d, J = 8.6 Hz, 2H) , 7.11(d, J = 16.3 Hz, 1H) , 5.38 (m, 2H) , 4.99 (d, J = 7.7 Hz, 1H) , 3.74 (d, J =2.9 Hz, 2H) , 3.65 (dd, J = 11.0, 4.6 Hz, 2H) , 3.60 –3.54 (m, 2H) , 3.50(dd, J = 10.7, 6.5 Hz, 2H), 3.44 (dd, J = 9.5, 3.2 Hz, 2H), 1.81 (s, 6H). HRMS theoretical for C 25 H 24 FN3NaO7 + [M+Na] + : 520.1490; found: [M+Na] + : 520.1493.

[0076] Example 5

[0077] This embodiment explores the spectral response performance of a fluorescent probe to β-galactosidase.

[0078] The UV-Vis absorption and fluorescence emission spectra of TCFM-Gal2 prepared in Example 2, TCFM-Gal4 prepared in Example 4, and the control probe without fluoromethyl substitution were compared and analyzed.

[0079] The control probe is: This is the product obtained by omitting steps 2) and 4) in the technical solution of Example 1.

[0080] Four test systems were prepared, containing 10 μM of different probes, 10 μM of different probes and 0.5 mg / mL BSA, 10 μM of different probes and 5 U / mL β-Gal, and 10 μM of different probes, 0.5 mg / mL BSA, and 5 U / mL β-Gal, respectively. After mixing each system, they were placed in a water bath at 37℃ for 30 min, and then their UV-Vis absorption and fluorescence emission spectra were measured.

[0081] like Figure 1 As shown, the introduction of β-Gal into the response system triggers a significant redshift in the absorption spectrum of TCFM-Gal2 from 415 nm to 555 nm, and the presence of BSA has negligible influence on these absorption spectra (e.g., Figure 1 As shown in Figure (a). Under 550 nm excitation conditions, without BSA, β-Gal activation of TCFM-Gal2 only showed a 193-fold fluorescence enhancement; while the introduction of BSA triggered a fluorescence surge of up to 594 times (as shown in Figure (a)). Figure 1 As shown in Figure (c). TCFM-Gal4 showed fluorescence enhancements of 200-fold and 360-fold, respectively (as shown in Figure (c)). Figure 1 (as shown in Figure (d)). Conversely, the fluorescence response of the control probe without difluoromethyl substitution to β-Gal was largely unaffected by BSA (e.g., as shown in Figure (d)). Figure 1 (As shown in Figure (b)). This illustrates that the ortho-fluoromethyl substitution structural modification endows the probe with additional response to the protein, significantly restricts intramolecular motion, and improves the fluorescence quantum yield of the fluorophore. Furthermore, the difluoromethyl-substituted fluorescent probe TCFM-Gal2 exhibits a stronger enzyme-controlled labeling response and can be considered a more preferred technical solution of this invention.

[0082] Example 6

[0083] This embodiment explores the enzyme response kinetics of fluorescent probes.

[0084] Four assay systems were prepared, each containing 10 μM probe TCFM-Gal2 and 0.5 mg / mL BSA, with β-Gal concentrations of 0 U / mL, 1.0 U / mL, 2.0 U / mL, and 5.0 U / mL, respectively. A control probe without difluoromethyl substitution was also included for comparison. The fluorescence intensity of the probe at 611 nm was measured over time using a fluorescence spectrophotometer.

[0085] like Figure 2 As shown, the fluorescence change of TCFM-Gal2 was negligible within 20 min. In contrast, upon the addition of BSA and 5 U / mL β-Gal, the fluorescence enhancement effect of TCFM-Gal2 plateaued within 10 min, while the control probe reached its maximum signal intensity within 5 min. This established a 10-min enzyme incubation time for subsequent signal characterization. Despite the longer response kinetics, TCFM-Gal2 consistently exhibited a higher signal intensity than the control probe after 1 min, indicating that the nucleophilic addition reaction between enzyme-activated TCFM-Gal2 and the protein significantly enhanced the fluorescence signal intensity and improved the signal-to-noise ratio during detection, consistent with previous observations. Furthermore, the response time of TCFM-Gal2 was enzyme concentration-dependent, shortening with increasing β-Gal concentration.

[0086] Example 7

[0087] This embodiment examines the linear response and detection limit of the fluorescent probe.

[0088] The concentration sensitivity of TCFM-Gal2 to β-Gal was evaluated by titration. Assay systems containing 10 μM TCFM-Gal2 probe and different concentrations of β-Gal (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 U / mL) were prepared, incubated at 37℃ for 30 min, and fluorescence emission spectra were recorded.

[0089] like Figure 3 As shown, TCFM-Gal2 exhibits only weak fluorescence at 611 nm, while the fluorescence intensity significantly increases with increasing β-Gal concentration. Linear fitting of the fluorescence emission intensity at 611 nm and β-Gal concentration in the fluorescence emission spectrum reveals a good linear relationship between fluorescence intensity and β-Gal concentration within the range of 0-1.0 U / mL. The regression equation is fitted as y = 193.8x + 2.54 (R²). 2 = 0.9980), the detection limit of the fluorescent probe TCFM-Gal2 for β-Gal was calculated to be 5.7 × 10⁻⁶. -3The U / mL indicates that TCFM-Gal2 has high sensitivity for detecting β-Gal activity.

[0090] Example 8

[0091] This embodiment examines the selectivity and anti-interference properties of the fluorescent probe.

[0092] Prepare a 10 μM TCFM-Gal2 probe assay system by sequentially adding interfering component solutions, including blank PBS, ions (sodium, potassium, calcium, magnesium, sulfate, sulfite), amino acids (cysteine, homocysteine, glutathione), other active substances (hydrogen peroxide, hypochlorous acid, human serum albumin, heparin, glucose), and enzymes (alkaline phosphatase, gamma-glutamyl transferase, glucose oxidase, and nitroreductase), all at a concentration of 100 μM. Incubate at 37°C for 30 min. Record the fluorescence intensity of TCFM-Gal2 at 611 nm for different substances in each assay system using a fluorescence spectrophotometer.

[0093] Prepare a test system containing 10 μM TCFM-Gal2 probe and 5 U / mL β-Gal, repeatedly adding the above interfering component solution, and incubate in a water bath at 37℃ for 30 min. Record the fluorescence intensity of TCFM-Gal2 in response to β-Gal at 611 nm in each test system under different interfering substances using a fluorescence spectrophotometer.

[0094] like Figure 4 As shown, only the addition of β-Gal significantly enhanced the fluorescence intensity of TCFM-Gal2 at 611 nm, while the other analytes had no significant effect on the fluorescence signal. Furthermore, compared to the addition of β-Gal alone, the presence of these interfering substances had minimal impact on the fluorescence intensity. This indicates that the probe TCFM-Gal2 exhibits good selectivity and anti-interference properties in response to β-Gal.

[0095] Example 9

[0096] This embodiment investigates the in vitro enzymatic labeling of nucleophiles with fluorescent probes.

[0097] Assay systems were prepared with 20 μM TCFM-Gal2 probe, 5 U / mL β-Gal and 20 μM TCFM-Gal2 probes responding to 3 and 10 min respectively, and 1 U / mL β-Gal, 20 μM mercaptoethanol and 20 μM TCFM-Gal2 probes responding to 30 min respectively. Mercaptoethanol was used to simulate the nucleophiles in the systems, and the systems were analyzed using high-performance liquid chromatography (HPLC).

[0098] like Figure 5As shown, a retention peak at 10.5 min was observed after β-Gal activation of the probe in the system. This retention peak increased with increasing response time, but disappeared at 10.5 min after the addition of mercaptoethanol, and a new retention peak appeared at 9.48 min. This demonstrates the enzymatic labeling process of the fluoromethyl-substituted probe from a molecular mechanism perspective. Figure 6 As shown: Since the control probe does not have an ortho-fluoromethyl substitution, the fluorophore can only release a fluorescent signal after β-Gal hydrolysis of the galactosidic bond; while the TCFM-Gal2 or TCFM-Gal4 with an ortho-fluoromethyl substitution can undergo an intramolecular rearrangement reaction after β-Gal hydrolysis of the galactosidic bond, generating an electrophilic intermediate methylquinone (QM), which further covalently binds to nucleophilic substances (such as mercaptoethanol) in the medium to generate a probe-nucleophilic complex, thereby achieving enzyme-controlled labeling of the probe.

[0099] Example 10

[0100] This embodiment investigates the in vitro enzymatic labeling of proteins by fluorescent probes.

[0101] Six test systems were prepared: ① containing 10 U / mL β-Gal and 20 μM control probe; ② containing 10 U / mL β-Gal, 1 mg / mL BSA and 20 μM control probe; ③ containing 10 U / mL β-Gal and 20 μM TCFM-Gal2 probe; ④ containing 10 U / mL β-Gal, 1 mg / mL BSA and 20 μM TCFM-Gal2 probe; ⑤ containing 1 mg / mL BSA and 20 μM TCFM-Gal2 probe; and ⑥ containing 10 U / mL β-Gal and 1 mg / mL BSA. After incubating the systems at 37°C for 20 min, proteins were extracted, dissolved in PBS, mixed with loading buffer, and boiled for 10 min to prepare samples. 10 μL of each sample was added to a 12% polyacrylamide gel, proteins were separated by electrophoresis, and fluorescence images were captured using an XRS gel imaging system. The gel was then stained with Coomassie Brilliant Blue, and a white light image of the gel was captured.

[0102] like Figure 7 As shown, a significantly strong fluorescent band was observed at the expected molecular weight of BSA only when TCFM-Gal2, β-Gal, and BSA were present simultaneously. Furthermore, no fluorescent signal was detected at the BSA position in the control probe. This indicates that the fluorescence of TCFM-Gal2 with ortho-fluoromethyl substitution, after activation by β-Gal, undergoes structural rearrangement to form the QM intermediate, which further nucleophilically couples with BSA, achieving fluorescent labeling of the protein. This demonstrates the crucial role of fluoromethyl structural modification in fluorescent labeling.

[0103] Example 11

[0104] This embodiment examines the imaging of cell senescence induced by different drugs using fluorescent probes.

[0105] A549 cells were seeded in covered glass-bottomed culture dishes and cultured overnight. The culture medium was discarded, and the cells were gently washed three times with pre-cooled PBS. The experimental groups were treated with 2 mL of culture medium containing 0.5 μM doxorubicin, 0.5 μM mitomycin C, or 100 μM hydrogen peroxide, respectively, while the control group was treated with pure culture medium. Three days after treatment, the cells were incubated for 30 min in the dark with culture medium containing 1 μM nuclear dye (Hoechst 33342) and 10 μM probe TCFM-Gal2 or a control probe without difluoromethyl substitution. Images of the probe and cell nuclei co-stained were captured under a laser confocal microscope.

[0106] like Figure 8 As shown, obvious red dotted fluorescence signals were observed in senescent cells, while the fluorescence signal intensity was significantly reduced in normal cells. This indicates that both probes, due to their β-galactose structure, can achieve specific detection of β-Gal in senescent cells. However, compared with the control probe without ortho-fluoromethyl substitution, TCFM-Gal2 produced a stronger signal intensity and exhibited a denser dotted distribution pattern. This suggests that more precise subcellular targeting and improved imaging fidelity were achieved through nucleophilic covalent anchoring, significantly enhancing the imaging resolution of the probe in senescent cells.

[0107] Example 12

[0108] This embodiment investigates the covalent labeling of cell senescence by fluorescent probes.

[0109] A549 cells were seeded in 6-well plates and cultured overnight. The culture medium was discarded, and the cells were treated with medium containing 0.5 μM doxorubicin for three days. The control group was treated with normal medium. Cells were then incubated for 30 min with medium containing 10 μM of the compound or a control probe without difluoromethyl substitution. Cells were gently washed three times with PBS, lysed on ice for 30 min with RIPA lysis buffer, mixed with loading buffer, and boiled for 10 min to prepare samples. The samples were then added to a 12% polyacrylamide gel, and proteins were separated by electrophoresis. Fluorescence images were captured using an XRS gel imaging system. The gels were then stained with Coomassie brilliant blue, and white light images were captured.

[0110] like Figure 9As shown, bright red fluorescent bands were observed only in senescent cells incubated with TCFM-Gal2, while no obvious fluorescent signal was observed in normal cells. This indicates that the enzyme-controlled labeling probe can be activated by senescence-associated β-galactosidase in senescent cells and label intracellular proteins. Conversely, the control probe without enzyme-controlled labeling ability did not show fluorescent bands in either cell type, indicating that although the control probe without difluoromethyl substitution can be activated by senescent cells, it cannot anchor to intracellular nucleophilic proteins. These results demonstrate that enzyme-controlled labeling probes can be activated by highly expressed senescence-associated β-galactosidase in senescent cells, forming an active intermediate and then covalently linking to adjacent nucleophilic proteins, achieving specific labeling of senescent cells and long-term imaging.

[0111] In summary, this invention utilizes a simple synthetic process to prepare an enzyme-controlled fluorescent probe for detecting aging-related β-galactosidases in high yield. This fluorescent probe exhibits rapid response, high sensitivity, and strong specificity to β-galactosidases, with a clear enzyme concentration-dependent response time and a good linear relationship between fluorescence intensity and enzyme concentration. Upon hydrolysis by β-galactosidase, the probe generates a highly reactive quinone methylation (QM) intermediate in situ, which then covalently binds to a nearby nucleophilic protein, forming a stable fluorescent-protein complex. This complex enables specific activation and long-term in situ retention of the fluorescent signal in senescent cells. This probe not only allows for specific detection of senescent cells but also enables long-term labeling of senescent cells through covalent anchoring, facilitating long-term tracking and flow cytometry cell sorting of senescent cells.

[0112] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A β-galactosidase fluorescent probe, characterized in that, The molecular structure of the fluorescent probe includes one of the following: TCFM-Gal1, TCFM-Gal2, TCFM-Gal3, or TCFM-Gal4: ； ； ； 。 2. The method for preparing the β-galactosidase fluorescent probe as described in claim 1, characterized in that, The synthesis of the fluorescent probe includes the following route: Route 1: ； Starting with p-hydroxybenzaldehyde and 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile, intermediate m1 was generated by Knoevenagel condensation reaction. m1 was then converted to intermediate m2 by Duff reaction, and then the key intermediate m3 was obtained by nucleophilic substitution reaction. m3 underwent fluorination reaction to obtain compound TCFM-Gal1, and further obtained probe TCFM-Gal2 by alcoholysis reaction. Route 2: ； Using intermediate m3 as a raw material, intermediate m4 is generated by reduction reaction with sodium borohydride, and then fluorinated to obtain compound TCFM-Gal3. Further, probe TCFM-Gal4 is obtained through alcoholysis reaction.

3. The preparation method according to claim 2, characterized in that, The preparation steps of Route 1 include: S1. Dissolve p-hydroxybenzaldehyde in a mixture with 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile, add base 1, and stir under nitrogen protection and reflux to react; cool, adjust the pH of the system to 5-8, let stand at low temperature, filter, wash and dry to obtain intermediate m1; S2. Mix and dissolve intermediate m1 with hexamethylenetetramine, and stir the reaction under nitrogen protection; cool, pour the reaction solution into water, adjust the pH of the system to 7-8, extract, concentrate and separate and purify to obtain intermediate m2; S3. Mix and dissolve intermediate m2 with base 2, add 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl-1-bromide, stir the reaction under nitrogen protection; cool, concentrate and separate and purify to obtain intermediate m3; S4. Dissolve intermediate m3, cool it down and slowly add diethylaminosulfur trifluoride solution dropwise, and stir the reaction under nitrogen protection; quench the reaction with ice water, extract, concentrate and separate and purify to obtain compound TCFM-Gal1. S5. Dissolve compound TCFM-Gal1, cool it down and slowly add alkali 3 solution dropwise, and stir the reaction under nitrogen protection; quench the reaction with ice water, extract, concentrate and separate and purify to obtain compound TCFM-Gal2.

4. The preparation method according to claim 2, characterized in that, The preparation steps of Route 2 include: S10. Dissolve intermediate m3, cool it down, add sodium borohydride in multiple portions, and stir the reaction under nitrogen protection; quench the reaction with dilute hydrochloric acid, extract, concentrate and separate and purify to obtain intermediate m4. S20. Dissolve intermediate m4, cool it down and slowly add diethylaminosulfur trifluoride solution, stir the reaction under nitrogen protection; quench the reaction with ice water, extract, concentrate and separate and purify to obtain compound TCFM-Gal3. S30. Dissolve compound TCFM-Gal3, cool it down and slowly add alkali 3 solution dropwise, and stir the reaction under nitrogen protection; quench the reaction with ice water, extract, concentrate and separate and purify to obtain compound TCFM-Gal4.

5. The preparation method according to claim 3, characterized in that, In step S1, the base 1 includes at least one of pyridine, piperidine, or diethylamine; the solvent in which the p-hydroxybenzaldehyde is dissolved in 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile includes at least one of dichloromethane, acetonitrile, anhydrous ethanol, N,N-dimethylformamide, or dimethyl sulfoxide. In step S2, the molar ratio of intermediate m1 to hexamethylenetetramine is 1:(1~6), and the solvent for mixing and dissolving includes trifluoroacetic acid and / or acetic acid; In step S3, the base 2 includes at least one of sodium carbonate, potassium carbonate, or cesium carbonate; the molar ratio of intermediate m2, base 2, and 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl-1-bromide is 1:(1~6):(1~3), and the solvent for mixing and dissolving includes at least one of dichloromethane, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, or acetone; In step S4, the solvent in which the intermediate m3 is dissolved includes dichloromethane and / or tetrahydrofuran, and the molar ratio of intermediate m3 to diethylaminosulfur trifluoride is 1:(1~10). In step S5, the solvent in which the compound TCFM-Gal1 is dissolved includes at least one of dichloromethane, tetrahydrofuran, acetone, ethanol or methanol, and the base 3 includes at least one of sodium ethoxide, sodium methoxide or sodium hydride. The molar ratio of the compound TCFM-Gal1 to the base 3 is 1:(1~6).

6. The preparation method according to claim 4, characterized in that, In step S10, the solvent in which the intermediate m3 is dissolved includes at least one of methanol, ethanol, dichloromethane or tetrahydrofuran; the molar ratio of intermediate m3 to sodium borohydride is 1:(1~5). In step S20, the solvent in which the intermediate m4 is dissolved includes dichloromethane and / or tetrahydrofuran, and the molar ratio of intermediate m4 to diethylaminosulfur trifluoride is 1:(1~10). In step S30, the solvent in which the compound TCFM-Gal3 is dissolved includes at least one of dichloromethane, tetrahydrofuran, acetone, ethanol or methanol, and the base 3 includes at least one of sodium ethoxide, sodium methoxide or sodium hydride. The molar ratio of the compound TCFM-Gal3 to the base 3 is 1:(1~6).

7. The application of the β-galactosidase fluorescent probe as described in claim 1 in the detection of β-galactosidase, characterized in that, The applications include using the fluorescent probe to detect β-galactosidase, or to prepare reagents for detecting β-galactosidase.

8. The application of the β-galactosidase fluorescent probe as described in claim 1 in biolabeling, characterized in that, The applications include using the fluorescent probe for β-galactosidase-catalyzed protein or cell labeling, or for preparing reagents for β-galactosidase-catalyzed protein or cell labeling.

9. The application of the β-galactosidase fluorescent probe as described in claim 1 in the detection of senescent cells, characterized in that, The applications include using the fluorescent probe for the detection, fluorescence imaging, or fluorescence labeling of senescent cells, or for the preparation of reagents for fluorescence imaging or fluorescence labeling of senescent cells.

10. The application of the β-galactosidase fluorescent probe as described in claim 1, characterized in that, Used to prepare diagnostic tool molecules or reagents for detecting aging.

Images