A class of β-galactosidase fluorescent probes, their preparation methods and applications

Abstract

This invention discloses a class of fluorescent probes for detecting β-galactosidase, their preparation method, and their applications. The structure of these probes is shown in general formula A. The β-galactosidase fluorescent probes provided by this invention have multiple applications, including in vitro, cellular, tissue / organ, and animal-level sensing and detection of β-galactosidase. After recognizing β-galactosidase, the fluorescence emission range extends from green to near-infrared light (445–662 nm). Some of the β-galactosidase fluorescent probes provided by this invention exhibit species selectivity, correctly recognizing human β-galactosidase without interference from bacterial species-specific β-galactosidases. This discovery provides a rapid, sensitive, multicolor, and species-specific β-galactosidase fluorescent probe, with broad application prospects in the field of biomolecular detection.

Description

Technical Field

[0001] This invention relates to a class of β-galactosidase fluorescent probes, their preparation methods, and their applications. More specifically, it relates to their biological use in the detection of β-galactosidase, particularly as a diagnostic tool molecule for aging. Background Technology

[0002] β-galactosidase (β-gal) is a glycosidic hydrolase that hydrolyzes galactose into galactose and glucose, and can also hydrolyze many other biochemical molecules with β-galactosidic bonds. β-galactosidase is a ubiquitous hydrolase found in various organisms, including bacteria, fungi, and mammals, and has wide-ranging biological applications. It is an important biomarker, and abnormalities in its activity are closely related to certain pathological processes and environmental factors. Detecting the bacterial β-galactosidase content in a sample can indicate the bacterial density in the environment. In the pathogenesis of certain cancers, such as primary ovarian cancer, tumor cells express increased levels of β-galactosidase compared to normal cells; therefore, β-galactosidase can serve as a diagnostic marker for ovarian cancer. With aging, the expression of β-galactosidase in senescent cells continuously increases, termed aging-associated β-galactosidase (SA-β-gal). SA-β-galactosidase is an important biomarker for monitoring and studying the aging process. Therefore, developing detection methods for β-galactosidase is of great significance. Enzyme activity detection methods include fluorescence methods, colorimetric methods, enzyme-linked immunosorbent assays (ELISA), and electrochemical methods. Among these, fluorescent probe detection methods have advantages such as high selectivity, high sensitivity, real-time detection, ease of operation, non-destructive testing, and good biocompatibility, and have become an important means of biological enzyme detection. The basic design principle of β-galactosidase fluorescent detection probes is to link a fluorophore to a β-D-galactose residue via a glycosidic bond. After the probe molecule binds to β-galactosidase, the β-galactosidic bond is cleaved under the enzyme's catalytic hydrolysis, exposing the fluorophore molecule and producing a fluorescence change. The fluorescence change is used to qualitatively and quantitatively detect β-galactosidase.

[0003] Aging is the gradual decline and deterioration of bodily functions after an organism reaches maturity. It is a complex physiological and pathological process influenced by multiple mechanisms, primarily manifested as damage to the functions of various tissues and organs, a decline in metabolic and stress response capabilities, and the development of various age-related diseases, including diabetes, tumors, cardiovascular diseases, and neurodegenerative diseases. Aging and age-related diseases seriously threaten human health and quality of life, and increase the burden on social healthcare. Therefore, accurate detection of aging is of great significance for aging-related research. Aging-related β-galactosidase is currently recognized as the most classic aging-related biomarker. However, currently reported β-galactosidase probes cannot distinguish between β-galactosidases from different species. It should be noted that β-galactosidases from different species have certain differences, and indiscriminate identification may lead to false positive results. Bacteria are ubiquitous prokaryotes in the natural environment, and bacterial infection is a common pathological condition. This presents an opportunity for bacterial β-galactosidase secretion to interfere with the detection results of human samples. Therefore, developing species-selective fluorescent probes for β-galactosidase to eliminate interference from bacterial β-galactosidase is of great research significance.

[0004] Currently, there are no publicly available reports in the literature on species-selective β-galactosidase fluorescent probes. Summary of the Invention

[0005] The purpose of this invention is to provide a class of β-galactosidase fluorescent probes, their preparation method, and their applications in the detection of aging-related β-galactosidases.

[0006] In a first aspect, the present invention provides a fluorescent probe having the structure shown in Formula A:

[0007]

[0008] In the formula, X is S, O, or NH;

[0009] Y is Or it may not exist;

[0010] R a R b R c R d Each can be independently H, C1-C4 alkyl, -CHO, -COOH, -CN, -NO2, -COOC1-C4 alkyl, -CH=CH (N-methylpyridinium salt).

[0011] In another preferred embodiment, R a R b Rc Each is independently H or C1-C4 alkyl, R d It can be -CHO, -COOH, -CN, -NO2, -COOC1-C4 alkyl, or -CH=CH (N-methylpyridinium salt).

[0012] In another preferred embodiment, R a R c R d Each is independently H or C1-C4 alkyl; R b It can be -CHO, -COOH, -CN, -NO2, -COOC1-C4 alkyl, or -CH=CH (N-methylpyridinium salt).

[0013] In another preferred embodiment, the fluorescent probe has the structure shown in Formula I or II:

[0014]

[0015] Where X is S, O or NH;

[0016] R can be -CHO, -COOH, -CN, -NO2, -COOC1-C4 alkyl, or -CH=CH (N-methylpyridinium salt).

[0017] In another preferred example, R is selected from the following group:

[0018]

[0019] In another preferred embodiment, the fluorescent probe is selected from the group consisting of:

[0020]

[0021] A second aspect of the present invention provides a method for preparing the fluorescent probe described in the first aspect, the method comprising the following steps:

[0022]

[0023] Route 1: Starting with 2-aminobenzylthiol and 5-methylsalicylaldehyde, the intermediate m1 is generated by reacting under concentrated hydrochloric acid and hydrogen peroxide conditions. m1 undergoes the Duff reaction to generate intermediate KSLOH01, which then undergoes a nucleophilic substitution reaction with intermediate m2 to obtain the key intermediate m3. m3 is deacetylated from the galactose residue under sodium methoxide conditions to obtain the probe KSL01.

[0024]

[0025] Route 2: Using KSLOH01 as raw material, intermediate KSLOH02 is obtained through Wittig reaction, and then undergoes nucleophilic substitution reaction with m2 to obtain intermediate m4. Under sodium methoxide conditions, the acetyl protecting group on the galactose residue of m4 is removed to obtain probe KSL02.

[0026]

[0027] Route 3: Using probe KSL01 as a raw material, probe KSL03 was prepared by Knoevenagel condensation reaction with 1,4-dimethylpyridinium iodide.

[0028]

[0029] Route 4: Intermediate m4 and malononitrile undergo a Knoevenagel condensation reaction to obtain the key intermediate m5. Under sodium methoxide conditions, m5 is deacetylated from the galactose residue to obtain the probe KSL04.

[0030]

[0031] Route 5: Starting with benzothiazole and 3-bromo-4-methoxybenzaldehyde, intermediate m6 is obtained by the Suzuki reaction. Then, the methyl protecting group is removed in aqueous hydrobromic acid to obtain intermediate KSLOH05. It undergoes a nucleophilic substitution reaction with 2,3,4,6-tetraacetoxy-α-D-pyranose bromide to obtain the key intermediate m7. Under sodium methoxide conditions, the acetyl protecting group on the galactose residue of m7 is removed to obtain probe KSL05.

[0032]

[0033] Route 6: Intermediate m7 is reacted with 2-(1-phenylethylidene)malononitrile via Knoevenagel condensation to obtain intermediate m8. Under sodium methoxide conditions, m8 is deacetyl-protecting group removed from galactose residues to obtain probe KSL06.

[0034]

[0035] Route 7: Using probe KSL05 as a raw material, probe KSL07 was prepared by Knoevenagel condensation reaction with 1,4-dimethylpyridinium iodide.

[0036]

[0037] Route 8: Using intermediate KSLOH05 as a raw material, intermediate KSLOH08 is obtained by Wittig reaction, and then undergoes nucleophilic substitution reaction with 2,3,4,6-tetraacetoxy-α-D-pyranose bromide to obtain key intermediate m10. Under sodium methoxide conditions, the acetyl protecting group on the galactose residue of m10 is removed to obtain probe KSL08.

[0038]

[0039] Route 9: Using intermediate KSLOH08 as a raw material, the aldehyde conjugated chain is extended by the Wittig reaction to obtain intermediate KSLOH09, which then undergoes a nucleophilic substitution reaction with 2,3,4,6-tetraacetoxy-α-D-pyranose bromide to obtain the key intermediate m11. Under sodium methoxide conditions, the acetyl protecting group on the galactose residue of m11 is removed to obtain probe KSL09.

[0040]

[0041] Route 10: Intermediate m7 and intermediate m12 (2-(2,6-dimethyl-4H-pyran-4-alkylene)malonitrile) undergo Knoevenagel condensation reaction to obtain intermediate m13. Under sodium methoxide conditions, m13 is deacetyl protecting group on galactose residue to obtain probe KSL10.

[0042]

[0043] Route 11: Intermediate m7 and intermediate m15 (2-(3-methylcyclohexyl-2-en-1-alkylene)malonitrile) undergo Knoevenagel condensation reaction to obtain intermediate m16. Under sodium methoxide conditions, m16 is deacetyl protecting group on galactose residue to obtain probe KSL11.

[0044]

[0045] Alternatively, route 12: intermediate m7 and intermediate m18 (2-(3,5,5-trimethylcyclohexyl-2-en-1-alkylene)malonitrile)propionate are condensed by Knoevenagel to obtain intermediate m19. Under sodium methoxide conditions, the acetyl protecting group on the galactose residue of m19 is removed to obtain probe KSL12.

[0046] The parameters of all the above-mentioned chemical reaction conditions are adjustable and replaceable to a certain extent. That is, the substitution of reaction solvents, acid-base substitutions, adjustment of reaction temperature, and adjustment of reaction time under the general chemical reaction guidelines are not enough to escape the protection scope of the claims of this patent.

[0047] In another preferred embodiment, the reaction solvents used in each reaction step are conventional chemical solvents such as methanol, ethanol, acetonitrile, tetrahydrofuran, dichloromethane, ethyl acetate, n-butanol, diethyl ether, and toluene.

[0048] In another preferred embodiment, the reaction temperature for each reaction step is -20 to 150°C, and the reaction time is 0.5 to 48 hours.

[0049] A third aspect of the invention provides the use of the fluorescent probe described in the first aspect for detecting β-galactosidase; or for preparing reagents for detecting β-galactosidase.

[0050] In another preferred embodiment, the fluorescent probe is used to detect β-galactosidase activity in vitro, in cells, and in tissues / organs. In another preferred embodiment, the fluorescent probe is used to detect its application in β-galactosidase in tumor cells. In another preferred embodiment, the tumor cells are ovarian cancer cells.

[0051] In another preferred embodiment, the fluorescent probe is used to detect β-galactosidases of different species, wherein the different species are selected from bacteria, fungi, mammals, and β-galactosidases; or to prepare a reagent for species-specific detection of β-galactosidases.

[0052] In another preferred embodiment, the different species of β-galactosidases include E. coli β-galactosidase, Aspergillus oryz β-galactosidase, mouse β-galactosidase, and human β-galactosidase.

[0053] In another preferred embodiment, the species-specific detection of β-galactosidase is used, meaning it can normally identify and detect human β-galactosidase but cannot identify and detect Escherichia coli β-galactosidase.

[0054] In another preferred embodiment, the species-specific fluorescent probes include KSL08–KSL12.

[0055] In another preferred embodiment, the fluorescent probe is used to prepare diagnostic tool molecules for detecting aging.

[0056] In another preferred embodiment, the aging includes bodily aging, tissue and organ aging, and cellular aging.

[0057] In another preferred embodiment, the fluorescent probe is used for fluorescence imaging or as a reagent for preparing fluorescence imaging.

[0058] In another preferred embodiment, the probe can be used to detect the content of β-galactosidase.

[0059] The present invention has the following significant advantages: (1) This series of fluorescent probes based on HBT-estimated structures has a large Stokes shift. (2) This series of fluorescent probes has good selectivity and detection sensitivity for β-galactosidase. (3) This series of fluorescent probes has good biocompatibility, no obvious cytotoxicity, and can be applied to live cell detection. (4) This series of fluorescent probes has a broad emission spectrum, from green light to near-infrared light, which can meet the needs of different imaging conditions. (5) This series of probes can be used to accurately detect senescent cells and determine the degree of senescence. (6) Some of the probes in this series are species-specific and can specifically recognize human β-galactosidase, but are not affected by bacterial species β-galactosidase, thus eliminating false positives caused by bacterial infection.

[0060] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0061] Figure 1 The UV-Vis absorption spectra of fluorescent probes KSL01-KSL12 after adding A. oryzae β-gal.

[0062] Figure 2 The fluorescence spectrum of A. oryzae β-gal after adding it to the KSL01-KSL12 fluorescent probe solution.

[0063] Figure 3 The fluorescence spectrum of E. coli β-gal after adding it to the KSL01-KSL12 fluorescent probe solution.

[0064] Figure 4 The changes in fluorescence spectra of fluorescent probes KSL01-KSL12 with increasing concentration of A. oryzae β-gal and the linear relationship between fluorescence intensity and enzyme concentration were investigated.

[0065] Figure 5 The time-kinetic changes in fluorescence intensity after adding A. oryzae β-gal to the KSL01-KSL12 fluorescent probe solution.

[0066] Figure 6 The results show the selectivity of fluorescent probes KSL04 and KSL11.

[0067] Figure 7 The survival rate of MRC5 cells for the fluorescent probes KSL01-KSL12.

[0068] Figure 8 The survival rate of SKOV3 cells for the fluorescent probes KSL01-KSL12.

[0069] Figure 9 The survival rate of HepG2 cells for the fluorescent probes KSL01-KSL12.

[0070] Figure 10 Cellular imaging of senescent MRC5 and SKOV3 cells using fluorescent probes KSL01-KSL12.

[0071] Figure 11 The image shows the results of detecting senescence in MRC5 cells at different passages using the fluorescent probe KSL04.

[0072] Figure 12 The image shows the results of detecting the degree of aging of kidney tissue sections from mice of different ages using fluorescent probes KSL04 and KSL11. Detailed Implementation

[0073] The inventors of this application, through extensive and in-depth research, constructed a series of β-galactosidase fluorescent probes based on the HBT backbone structure, and provided their preparation methods and applications in aging detection. Based on this, the present invention was completed.

[0074] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions (such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0076] Example 1

[0077] Preparation of probe KSL01 and hydrolysis product KSLOH01

[0078]

[0079] (1) Synthesis of compound m1:

[0080] 2-Aminophenylthiol (1.25 g, 10 mmol) and 5-methylsalicylic acid (1.36 g, 10 mmol) were dissolved in anhydrous ethanol. Concentrated HCl (2.5 mL, 30 mmol) was slowly added dropwise while stirring at 0 °C. After stirring for 10 min, a 30% H₂O₂ solution (6.8 mL, 60 mmol) was slowly added dropwise. The mixture was then stirred at room temperature for 1 h, resulting in the precipitation of yellow crystals. The solid was collected by filtration, washed with a small amount of anhydrous ethanol, and dried to give 1.31 g of compound m1, with a yield of 54%. 1 H NMR (400MHz, DMSO-d6) δ11.38(br,1H),8.14(d,J=7.6Hz,1H),8.06(d,J=8.0Hz,1H),7.98(d,J=1.4Hz,1H ),7.58–7.51(m,1H),7.49–7.42(m,1H),7.23(dd,J=8.3,1.8Hz,1H),6.99(d,J=8.3Hz,1H),2.33(s,3H). 13 C NMR(101MHz,DMSO-d6)δ165.74,154.67,151.89,134.73,133.71,128.87,12 8.73,126.91,125.49,122.50,122.45,118.38,117.34,20.50.ESI-HRMS:m / z calc.forC 14 H 12 NOS[M+H] + :242.0640,found:242.0641.

[0081] (2) Synthesis of compound KSLOH01

[0082] Compound m1 (1.11 g, 4.6 mmol) and hexamethylenetetramine (733 mg, 5.52 mmol) were dissolved in 10 mL of trifluoroacetic acid. The mixture was stirred at 70 °C for 10 h. The reaction solution was then cooled to room temperature, and the pH was adjusted to alkaline with 3 M NaOH solution, resulting in the precipitation of a yellow solid. The solid was collected by filtration, washed with a small amount of anhydrous ethanol, and dried to obtain the crude product. Column chromatography was used to separate the crude product into 780 mg of the yellow solid KSLOH01, with a yield of 63%. 1 H NMR(600MHz,DMSO-d6)δ12.75(s,1H),10.33(s,1H),8.23–8.19(m,2H),8.12(d,J=8 .1Hz,1H),7.72(d,J=1.6Hz,1H),7.62–7.58(m,1H),7.54–7.50(m,1H),2.39(s,3H). 13C NMR(151MHz,DMSO-d6)δ192.29,165.66,157.51,151.46,135.74,134.02,133.56 ,129.76,127.42,126.31,123.70,122.77,122.74,119.57,20.20.ESI-HRMS:m / z calc.for C 15 H 12 NO2S[M+H] + :270.0589,found:270.0590.

[0083] (3) Synthesis of compound m3

[0084] Compound m2 (234 mg, 0.454 mmol) and compound KSLOH01 (122 mg, 0.454 mmol) were dissolved in 2 mL of tetrahydrofuran, and K2CO3 (75 mg, 0.545 mmol) was added. After stirring overnight at room temperature, the mixture was neutralized with saturated NH4Cl solution and then extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous Na2SO4, and then evaporated to dryness. Column chromatography was used to separate the solid m3 into 264 mg of white solid, with a yield of 84%. 1 H NMR (400MHz, CDCl3) δ10.20(s,1H),8.49(d,J=1.8Hz,1H),8.17(d,J=8.1Hz,1H),7.95(d,J=7.8Hz,1H), 7.78(d,J=1.9Hz,1H),7.56(dd,J=11.3,4.1Hz,1H),7.46(t,J=7.2Hz,1H),7.34(d,J=8.6Hz,2H),7.00( d,J=8.6Hz,2H),5.54–5.45(m,2H),5.13(dd,J=10.5,3.4Hz,1H),5.06(d,J=7.9Hz,1H),4.99(s,2H),4. 28–4.13(m,2H),4.08(t,J=6.7Hz,1H),2.49(s,3H),2.19(s,3H),2.10(s,3H),2.06(s,3H),2.03(s,3H). 13C NMR (151MHz, CDCl3) δ189.10,170.35,170.25,170.13,169.42,161.95,15 7.27,157.19,152.36,136.65,136.05,135.24,131.72,130.21,130.16,1 30.13,128.15,126.43,125.50,123.25,121.57,117.06,99.53,79.06,71 .07,70.81,68.60,66.85,61.33,20.77,20.73,20.67,20.59.EI-HRMS:m / z calc.for C 36 H 35 NO 12 S[M] + :705.1880,found:705.1882.

[0085] (4) Synthesis of compound KSL01

[0086] Compound m3 (130 mg, 0.184 mmol) was placed in a flask and dissolved in anhydrous methanol. The mixture was stirred briefly at -20°C. Simultaneously, sodium methoxide (70 mg, 1.29 mmol) was dissolved in anhydrous methanol and slowly added dropwise to the flask while stirring. TLC monitoring was maintained. After the substrate reaction was complete, Amberlite IR-120plus (H) was added. + Adjust the pH to neutral. Filter to remove Amberlite IR-120plus (H) + After collecting the filtrate, the liquid was evaporated to dryness and separated by column chromatography to obtain 36 mg of white solid KSL01, with a yield of 45%. 1H NMR (600MHz, DMSO-d6) δ10.14(s,1H),8.48(d,J=1.8Hz,1H),8.18(d,J=7.9Hz,1H),8.12(d,J=8.1Hz,1 H),7.77(d,J=1.8Hz,1H),7.59(t,J=7.6Hz,1H),7.49(t,J=7.5Hz,1H),7.41(d,J=8.5Hz,2H),7.04(d,J =8.5Hz,2H),5.19(d,J=5.1Hz,1H),5.06(s,2H),4.90–4.82(m,2H),4.66(t,J=5.2Hz,1H),4.52(d,J=4 .5Hz,1H),3.72(t,J=3.6Hz,1H),3.62–3.54(m,3H),3.53–3.47(m,1H),3.44–3.40(m,1H),2.47(s,3H). 13 C NMR(151MHz,DMSO-d6)δ189.45,161.67,158.24,157.01,152.19,136.04,136.01,135.30,132.29,130.88,130.49,129.07 ,128.02,127.07,126.08,123.33,122.70,116.60,101.32,79.20,75.97,73.76,70.74,68.57,60.80,20.67.ESI-HRMS:m / z calc.C 28 H 27 NNaO8S[M+Na] + :560.1355,found 560.1354.

[0087] Example 2

[0088] Preparation of probe KSL02 and its hydrolysis product KSLOH02

[0089]

[0090] (1) Synthesis of compound KSLOHO2

[0091] (Formylmethylene)triphenylphosphine (304 mg, 1 mmol) and compound KSLOH01 (269 mg, 1 mmol) were placed in a flask, and 10 mL of anhydrous tetrahydrofuran was added. The mixture was refluxed overnight under nitrogen protection. After the reaction was complete, the mixture was cooled, the solvent was removed by rotary evaporation, and the solution was separated by column chromatography to obtain 236 mg of yellow solid KSLOH02, with a yield of 80%. 1H NMR (400MHz, CDCl3) δ13.24(br,1H),9.74(d,J=7.8Hz,1H),8.00(d,J=8.1Hz,1H),7.98–7. 89(m,2H),7.59–7.50(m,2H),7.48–7.40(m,2H),6.88(dd,J=16.1,7.8Hz,1H),2.38(s,3H). 13 C NMR (101MHz, CDCl3) δ194.51,168.77,155.11,151.52,147.41,132.55,132.07,131.53 ,129.30,128.70,126.95,125.92,122.90,122.27,121.59,117.28,20.50.EI-HRMS:m / z calc.for C 17 H 13 NO2S[M] + :295.0667,found:295.0664.

[0092] (2) Synthesis of compound m4

[0093] Compound m2 (142 mg, 0.249 mmol), compound KSLOHO2 (73 mg, 0.249 mmol), and K2CO3 (41 mg, 0.299 mmol) were added to a flask, along with 2 mL of DMF solvent. The mixture was reacted overnight at room temperature. After the reaction was complete, the mixture was neutralized with saturated NH4Cl solution, then extracted three times with ethyl acetate. The combined organic phases were dried over anhydrous Na2SO4, evaporated to dryness, and separated by column chromatography to give 169 mg of white solid m4, with a yield of 88%.

[0094] 1H NMR (400MHz, CDCl3) δ9.54(d,J=7.7Hz,1H),8.29(s,1H),8.13(d,J=8.1Hz,1H),7.95(d,J=7.8Hz,1H),7.64 (d,J=16.1Hz,1H),7.58–7.50(m,2H),7.44(t,J=7.5Hz,1H),7.33(d,J=8.5Hz,2H),7.00(d,J=8.5Hz,2H),6 .65(dd,J=16.1,7.7Hz,1H),5.50(dd,J=10.4,8.0Hz,2H),5.14(dd,J=10.4,3.4Hz,1H),5.04(d,J=7.9Hz,1 H),4.91–4.82(m,2H),4.29–4.13(m,3H),2.47(s,3H),2.19(s,3H),2.11(s,3H),2.06(s,3H),2.03(s,3H). 13 C NMR (151MHz, CDCl3) δ193.45,170.37,170.26,170.14,169.48,162.75,157.2 4,153.74,151.82,146.56,135.82,135.28,133.64,130.54,130.49,130.22,1 29.51,129.00,127.61,126.56,125.60,123.08,121.57,117.14,99.48,77.87 ,71.03,70.82,68.57,66.84,61.30,20.91,20.76,20.69,20.60.EI-HRMS:m / z calc.for C 38 H 37 NO 12 S[M] + :731.2036,found:731.2039.

[0095] (3) Synthesis of compound KSL02

[0096] Compound m4 (100 mg, 0.137 mmol) was placed in a flask and dissolved in anhydrous methanol. The mixture was stirred briefly at -20°C. Simultaneously, sodium methoxide (52 mg, 0.96 mmol) was dissolved in anhydrous methanol and slowly added dropwise to the flask while stirring. TLC monitoring was maintained. After the substrate reaction was complete, Amberlite IR-120plus (H) was added. + Adjust the pH to neutral. Filter to remove Amberlite IR-120plus (H) +After collecting the filtrate, the liquid was evaporated to dryness and separated by column chromatography to obtain 40 mg of white solid KSL02, with a yield of 52%. 1 H NMR (400MHz, DMSO-d6) δ9.68(d,J=7.7Hz,1H),8.28(d,J=1.6Hz,1H),8.18(d,J=7.8Hz,1H),8.11(d,J=8.1Hz ,1H),7.91(d,J=1.7Hz,1H),7.86(d,J=16.0Hz,1H),7.61–7.55(m,1H),7.51–7.48(m,1H),7.46(d,J=8.5Hz,2 H),7.06(d,J=8.6Hz,2H),6.92(dd,J=16.0,7.7Hz,1H),5.19(d,J=5.1Hz,1H),4.92–4.83(m,4H),4.66(t,J=5 .4Hz,1H),4.52(d,J=4.6Hz,1H),3.74–3.70(m,1H),3.62–3.54(m,3H),3.53–3.47(m,1H),3.47–3.39(m,1H). 13 C NMR(151MHz,DMSO-d6)δ195.07,162.26,158.18,153.87,152.25,146.74,136.02,135.26,132.88,131.66,130.78,130.36,129.37 ,129.24,127.56,127.01,125.98,123.26,122.67,116.65,101.34,78.10,75.95,73.75,70.73,68.56,60.78,20.78.ESI-HRMS:m / z calc.for C 30 H 29 NNaO8S[M+Na] + :586.1512,found 586.1513.

[0097] Example 3

[0098] Preparation of probe KSL03 and its hydrolysis product KSLOH03

[0099]

[0100] (1) Synthesis of compound KSL03

[0101] Compound KSL01 (120 mg, 0.223 mmol) and 1,4-dimethylpyridinium iodide (53 mg, 0.223 mmol) were added to a flask, followed by the addition of anhydrous ethanol. Piperidine (20 μL, 0.223 mmol) was added dropwise while stirring. The mixture was heated to reflux temperature and stirred overnight. A yellow solid precipitated from the reaction solution. The solid was collected by filtration, washed with a small amount of anhydrous ethanol, and dried to obtain 122.2 mg of yellow solid KSL03, with a yield of 73%. 1 H NMR (400MHz, DMSO-d6) δ8.83(d,J=6.7Hz,2H),8.27(d,J=1.6Hz,1H),8.19(d,J=7.8Hz,1H),8.12(d,J=8.1Hz,1H),7.98(d, J=6.7Hz,2H),7.84(d,J=1.7Hz,1H),7.77(d,J=16.4Hz,1H),7.62–7.56(m,1H),7.53–7.47(m,1H),7.43(d,J=16.4Hz,1H),7 .36(d,J=8.6Hz,2H),6.96(d,J=8.6Hz,2H),5.21(d,J=5.0Hz,1H),4.93(d,J=4.3Hz,3H),4.76(d,J=7.7Hz,1H),4.69(t,J= 5.3Hz,1H),4.59(d,J=4.5Hz,1H),3.74–3.70(m,1H),3.62–3.53(m,3H),3.53–3.45(m,1H),3.45–3.38(m,1H),2.47(s,3H). 13 CNMR(101MHz,DMSO-d6)δ162.41,158.01,154.03,152.38,152.24,145.64,136.04,135.10,134.76,131.97,130.92,130.87,130.70,129.55,12 7.51,127.03,125.99,125.24,124.08,123.27,122.66,116.54,101.06 ,78.09,75.99,73.75,70.65,68.60,60.89,47.53,20.92.ESI-HRMS:m / z calc.for C 35 H 35 N2O7S[MI] + :627.2159,found 627.2166.

[0102]

[0103] (2) Synthesis of compound KSLOHO3

[0104] Compound KSLOH01 (84 mg, 0.312 mmol) and 1,4-dimethylpyridinium iodide (73 mg, 0.312 mmol) were added to a flask, followed by the addition of anhydrous ethanol. Piperidine (29 μL, 0.312 mmol) was added dropwise while stirring. The mixture was heated to reflux temperature and stirred overnight. A yellow solid precipitated from the reaction solution. The solid was collected by filtration, washed with a small amount of anhydrous ethanol, and dried to obtain 106.1 mg of yellow solid KSLOH03, with a yield of 70%. 1 H NMR (400MHz, DMSO-d6) δ13.11(s,1H),8.86(d,J=6.7Hz,2H),8.28–8.22(m,3H),8.14(t,J=12.2Hz,2H),7.84 (s,1H),7.79(s,1H),7.69(d,J=16.4Hz,1H),7.66–7.60(m,1H),7.58–7.52(m,1H),4.27(s,3H),2.41(s,3H). 13 C NMR(151MHz,DMSO-d6)δ168.72,154.67,153.15,151.32,145.59,135.40,133.07,132.94,131.21,129.48,127.65,126.54,124.9 1,124.13,123.98,122.93,122.44,117.74,47.39,40.52,40.40,40.26,40.12,39.98,39.84,39.70,39.56,20.44.ESI-HRMS:m / z calc.forC 22 H 19 N2OS[MI] + :359.1213,found359.1219.

[0105] Example 4

[0106] Preparation of probe KSL04 and its hydrolysis product KSL0H04

[0107]

[0108] (1) Synthesis of compound m5

[0109] Compound m4 (200 mg, 0.273 mmol) was added to a dry two-necked flask, dissolved in toluene. Under nitrogen protection, malononitrile (18 μL, 0.287 mmol) and ammonium acetate (3 mg, 0.041 mmol) were added while stirring. Finally, acetic acid (5 μL, 0.082 mmol) was slowly added dropwise. The mixture was heated to reflux temperature and reacted for 12 h. After the reaction was complete, the mixture was cooled, the solvent was evaporated, and column chromatography was used to separate 150 mg of yellow solid m5, with a yield of 70%. 1 H NMR (600MHz, DMSO-d6) δ8.39(d,J=11.0Hz,1H),8.28(d,J=1.6Hz,1H),8.13(d,J=7.8Hz,1H),8.10(d,J=8.1Hz,1H),7 .99(d,J=1.9Hz,1H),7.74(d,J=15.2Hz,1H),7.60–7.55(m,1H),7.51–7.45(m,3H),7.34(dd,J=15.2,11.4Hz,1H),7.0 2–6.98(m,2H),5.48(d,J=7.9Hz,1H),5.36(d,J=3.6Hz,1H),5.31(dd,J=10.4,3.5Hz,1H),5.26–5.21(m,1H),4.92–4. 83(m,2H),4.44(t,J=6.6Hz,1H),4.15–4.07(m,2H),2.46(s,3H),2.16(s,3H),2.05(s,3H),2.01(s,3H),1.96(s,3H). 13 C NMR(151MHz,DMSO-d6)δ170.46,170.32,170.05,169.69,162.86,162.11,157.06,154.26,1 52.25,144.83,135.97,135.94,135.58,133.49,131.81,131.10,131.03,130.49,129.51,12 7.70,127.04,126.03,124.64,123.28,122.59,116.69,114.63,112.61,98.08,81.98,78.18 ,70.84,70.61,68.83,67.66,61.74,54.41,20.95,20.94,20.87,20.83,20.69.EI-HRMS:m / z calc.for C 41 H 37 N3O 11 S[M] +:779.2149,found 779.2152.

[0110] (2) Synthesis of compound KSL04

[0111] Compound m5 (123 mg, 0.158 mmol) was placed in a flask and dissolved in anhydrous methanol. The mixture was stirred briefly at -20°C. Simultaneously, sodium methoxide (60 mg, 1.104 mmol) was dissolved in anhydrous methanol and slowly added dropwise to the flask while stirring. TLC monitoring was maintained. After the substrate reaction was complete, Amberlite IR-120plus (H) was added. + Adjust the pH to neutral. Filter to remove Amberlite IR-120plus (H) + After collecting the filtrate, the solution was evaporated to dryness and separated by column chromatography to obtain 30.5 mg of yellow solid KSL04, with a yield of 31%. 1 H NMR (600MHz, DMSO-d6) δ8.43(d,J=11.3Hz,1H),8.28(s,1H),8.15(d,J=7.9Hz,1H),8.10(d,J=8.1Hz,1H) ,7.99(s,1H),7.76(d,J=15.2Hz,1H),7.59–7.55(m,1H),7.49–7.45(m,1H),7.42(d,J=8.6Hz,2H),7.34(d d,J=15.2,11.4Hz,1H),7.03(d,J=8.6Hz,2H),5.19(d,J=4.4Hz,1H),4.89–4.82(m,4H),4.65(s,1H),4.5 1(s,1H),3.72(s,1H),3.58(d,J=2.7Hz,4H),3.52–3.48(m,1H),3.45–3.41(m,1H),2.45(d,J=8.3Hz,4H). 13 C NMR(151MHz,DMSO-d6)δ162.88,162.14,158.23,154.28,152.25,144.73,136.00,135.51,133.47,131.64,131.04,129.56,129.15,127.71,12 7.02,126.01,124.52,123.27,122.64,116.51,114.69,112.64,101.44 ,82.08,78.49,76.00,73.74,70.73,68.55,60.79,20.71.ESI-HRMS:m / z calc.for C 33 H 29 N3NaO7S[M+Na]+ :634.1624,found 634.1623.

[0112]

[0113] (3) Synthesis of compound KSLOH04

[0114] Compound KSLOHO2 (50 mg, 0.169 mmol) and malononitrile (12 μL, 0.186 mmol) were placed in a flask and dissolved in anhydrous ethanol. Under nitrogen protection, piperidine (17 μL, 0.169 mmol) was added dropwise while stirring. The reaction was carried out at room temperature for 10 min, and a yellow solid precipitated. The solid was collected by filtration and washed with a small amount of anhydrous ethanol to obtain 40 mg of KSLOHO4, with a yield of 69%. 1 H NMR (600MHz, CDCl3) δ8.04(d,J=8.1Hz,1H),7.94(d,J=7.6Hz,1H),7.72(d,J=15.3Hz,1H) ,7.66(d,J=11.7Hz,1H),7.60(d,J=1.2Hz,1H),7.58–7.43(m,4H),2.41(s,J=9.9Hz,3H). 13 C NMR (151MHz, CDCl3) δ168.52,161.21,156.08,151.44,145.49,132.53,132.39,129.06,127.08 ,126.08,123.36,122.92,122.35,121.63,117.52,113.85,112.06,81.87,20.46.EI-HRMS:m / z calc.for C 20 H 13 N3OS[M] + :343.0779,found343.0781.

[0115] Example 5

[0116] Preparation of probe KSL05 and its hydrolysis product KSLOH05

[0117]

[0118] (1) Synthesis of compound m6

[0119] 3-Bromo-4-methoxybenzaldehyde (6.820 g, 30 mmol), palladium acetate (674 mg, 3 mmol), triphenylphosphine (3.934 g, 15 mmol), copper acetate monohydrate (1.198 g, 6 mmol), K₂CO₃ (8.292 g, 60 mmol), and benzothiazole (10 mL, 90 mmol) were added to a 250 mL flask. Toluene was added as a solvent, and the mixture was refluxed under nitrogen protection for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and the insoluble solids were removed by filtration. The product was then evaporated to dryness and separated by column chromatography to obtain 16 g of product m6, with a yield of 99%. 1 HNMR (400MHz, CDCl3) δ10.06(s,1H),9.05(d,J=2.1Hz,1H),8.13(d,J=8.2Hz,1H),8.04(dd,J=8.6,2.1H z,1H),7.95(d,J=7.9Hz,1H),7.55–7.50(m,1H),7.44–7.38(m,1H),7.20(d,J=8.6Hz,1H),4.16(s,3H). 13 C NMR (151MHz, CDCl3) δ190.72,161.59,161.33,151.78,135.95,133.34,131.71 ,130.16,126.30,125.14,122.99,122.61,121.31,112.09,56.22.EI-HRMS:m / z calc.for C 15 H 11 NO2S[M]+:269.0510,found 269.0513.

[0120] (2) Synthesis of compound KSLOH05

[0121] Compound m6 (6.3 g, 23.39 mmol) was added to a flask, followed by 60 mL of 40% HBr aqueous solution. The mixture was refluxed for 2 days and monitored by TLC. After the substrate reaction was complete, the mixture was cooled to room temperature, neutralized with 2N NaOH, and extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous Na2SO4, and then evaporated to dryness. Column chromatography was used to separate the solid KSLOHO5, yielding 3.11 g of white solid, with a yield of 52%. 1H NMR (400MHz, CDCl3) δ13.35(s,1H),9.95(s,1H),8.25(d,J=1.9Hz,1H),8.03(d,J=8.1Hz,1H),7.96(d, J=8.0Hz,1H),7.92(dd,J=8.6,1.9Hz,1H),7.59–7.52(m,1H),7.50–7.44(m,1H),7.23(d,J=8.6Hz,1H). 13 C NMR (151MHz, CDCl3) δ189.93,168.26,163.11,151.36,133.95,132.59,130 .70,128.85,127.07,126.17,122.37,121.74,118.72,117.13.EI-HRMS:m / z calc.for C 14 H9NO2S[M] + :255.0354,found 255.0356.

[0122] (3) Synthesis of compound m7

[0123] Prepare a dry flask, add compound KSLOHO5 (2 g, 7.83 mmol), cesium carbonate (12 g, 36.83 mmol), and a small amount of anhydrous Na2SO4, then add dichloromethane as a solvent. After stirring briefly at room temperature, add 2,3,4,6-tetraacetoxy-alpha-D-pyranose bromide (4.9 g, 11.88 mmol). Under nitrogen protection, react overnight at room temperature. After the substrate has reacted completely, filter to remove the insoluble solid, evaporate to dryness, and separate by column chromatography to obtain 3.8 g of m7 as a white solid, yield 82%. 1 H NMR (400MHz, CDCl3) δ10.10(s,1H),9.13(d,J=1.7Hz,1H),8.18(d,J=8.1Hz,1H),8.04(dd ,J=8.7,1.9Hz,1H),3.4Hz,1H),4.28–4.17(m,3H),2.24(s,3H),2.10(s,3H),2.04(s,7.9 7(d,J=7.9Hz,1H),7.56(t,J=7.7Hz,1H),7.45(t,J=7.6Hz,1H),7.36(d,J=8.7Hz,1H),5. 78(dd,J=10.1,8.0Hz,1H),5.52(d,J=3.4Hz,1H),5.46(d,J=8.0Hz,1H),5.18(dd,J=10.2 3H),1.88(s,3H). 13C NMR(101MHz,DMSO-d6)δ192.18,170.46,170.34,170.01,169.53,160.94,157.88,151.94,135.89,133.10,131.86,131.76,127.1 6,126.19,123.41,122.85,122.04,116.23,97.30,71.54,71.12,68.71,67.70,61.77,21.00,20.98,20.93,20.81.ESI-HRMS:m / z calc.for C 28 H 28 NO 11 S[M+H] + :586.1383,found:586.1384.

[0124] (4) Synthesis of compound KSL05

[0125] Compound m7 (215 mg, 0.37 mmol) was placed in a flask and dissolved in anhydrous methanol. The mixture was stirred briefly at -20°C. Simultaneously, sodium methoxide (139 mg, 2.57 mmol) was dissolved in anhydrous methanol and slowly added dropwise to the flask while stirring. TLC monitoring was maintained. After the substrate reaction was complete, Amberlite IR-120plus (H) was added. + Adjust the pH to neutral. Filter to remove AmberliteIR-120plus (H) + After collecting the filtrate, the liquid was evaporated to dryness and separated by column chromatography to obtain 68 mg of white solid KSL05, with a yield of 44%. 1 HNMR(600MHz,DMSO-d6)δ10.07(s,1H),9.00(d,J=2.0Hz,1H),8.14(t,J=7.2Hz, 2H),8.07(dd,J=8.7,2.0Hz,1H),7.58(dd,J=8.1,5.8Hz,2H),7.49(t,J=7.5Hz, 1H),5.37(d,J=7.7Hz,1H),5.29(d,J=5.7Hz,1H),5.04(d,J=5.7Hz,1H),4.75–4 .68(m,2H),3.99(dd,J=14.8,7.9Hz,1H),3.81–3.75(m,2H),3.63–3.50(m,3H). 13C NMR(151MHz,DMSO-d6)δ192.20,161.77,159.33,151.92,136.37,133.06,131.46,130.89,126.89, 125.77,123.15,122.61,122.32,116.04,101.27,76.41,73.97,70.56,68.49,60.75.ESI-HRMS:m / z calc.forC 20 H 20 NO7S[M+H] + :418.0960,found:418.0953.

[0126] Example 6

[0127] Preparation of probe KSL06 and its hydrolysis product KSLOH06

[0128]

[0129] (1) Synthesis of compound m8

[0130] Compound m7 (600 mg, 1.02 mmol) and 2-(1-phenylethylidene)malononitrile (344 mg, 2.04 mmol) were added to a flask, and anhydrous ethanol was added as a solvent. Piperidine (94 μL, 1.18 mmol) was added dropwise while stirring. The mixture was heated to reflux and reacted overnight. After the substrate had reacted completely, the mixture was evaporated to dryness and separated by column chromatography to obtain 294 mg of pale yellow solid m8, with a yield of 39%. 1 H NMR (400MHz, CDCl3) δ8.69(s,1H),8.18(d,J=8.0Hz,1H),7.96(d,J=7.9Hz,1H),7.80(d,J=9.0 Hz,1H),7.68–7.51(m,5H),7.48–7.43(m,1H),7.40(d,J=6.6Hz,2H),7.29(d,J=9.0Hz,1H),7.0 1(d,J=15.5Hz,1H),5.80–5.71(m,1H),5.52(d,J=3.2Hz,1H),5.43(d,J=8.0Hz,1H),5.18(dd, J=10.2,3.3Hz,1H),4.23(t,J=8.8Hz,3H),2.23(s,3H),2.11(s,3H),2.04(s,3H),1.86(s,3H). 13C NMR (151MHz, CDCl3) δ171.12,170.31,170.16,170.08,169.25,161.07,156.01,151 .88,147.75,136.11,132.99,132.07,131.26,130.07,129.72,129.16,128.85,126 .40,125.39,124.49,124.05,123.07,121.39,115.70,113.33,112.84,98.78,82.5 1,71.72,71.21,68.49,66.77,61.40,20.74,20.72,20.56.ESI-HRMS:m / zcalc.for C 39 H 34 N3O 10 S[M+H] + :736.1965,found:736.1960.

[0131] (2) Synthesis of compound KSL06

[0132] Compound m8 (100 mg, 0.14 mmol) was placed in a flask and dissolved in anhydrous methanol. The mixture was stirred briefly at -20°C. Simultaneously, sodium methoxide (51 mg, 0.95 mmol) was dissolved in anhydrous methanol and slowly added dropwise to the flask while stirring. TLC monitoring was maintained. After the substrate reaction was complete, Amberlite IR-120plus (H) was added. + Adjust the pH to neutral. Filter to remove AmberliteIR-120plus (H) + After collecting the filtrate, the liquid was evaporated to dryness and separated by column chromatography to obtain 36 mg of yellow solid KSL06, with a yield of 45%. 1 HNMR (600MHz, DMSO-d6) δ8.64(s,1H),8.12(d,J=7.9Hz,1H),8.07(d,J=8.1Hz,1H),7.98(d,J= 8.7Hz,1H),7.69–7.62(m,4H),7.57–7.52(m,4H),7.47(dd,J=7.8,5.2Hz,2H),7.06(d,J=15.5 Hz,1H),5.31(d,J=7.6Hz,1H),5.25(d,J=5.6Hz,1H),5.02(d,J=5.8Hz,1H),4.71(t,J=5.4Hz, 1H), 4.67 (d, J=4.2Hz, 1H), 3.96 (dd, J=14.6, 8.0Hz, 1H), 3.78–3.72 (m, 2H), 3.60–3.49 (m, 4H).13 C NMR(151MHz,DMSO-d6)δ171.58,161.91,157.45,151.85,148.52,136.35,133.54,132.50,131.63,130.58,129.58,129.43,128.61,126 .84,125.70,124.00,123.05,122.69,122.27,116.38,114.51,113.75,101.20,81.40,76.35,73.95,70.57,68.49,60.76.ESI-HRMS:m / z calc.for C 31 H 25 N3NaO6S[M+Na] + :590.1362,found:590.1365.

[0133]

[0134] (3) Synthesis of compound m9

[0135] Compound m6 (1.16 g, 4.31 mmol) and 2-(1-phenylethylidene)malononitrile (1.3 g, 7.72 mmol) were added to a flask, and anhydrous ethanol was added as a solvent. Piperidine (473 μL, 5.17 mmol) was added dropwise while stirring. The mixture was heated to reflux and reacted overnight. After the substrate had reacted completely, the mixture was evaporated to dryness and separated by column chromatography to obtain 145 mg of pale yellow solid m9, with a yield of 8%. 1 H NMR (400MHz, CDCl3) δ8.70(s,1H),8.18(d,J=8.2Hz,1H),7.94(d,J=7.9Hz,1H),7.84(d,J=8.6Hz,1H ),7.65–7.51(m,5H),7.47–7.37(m,3H),7.16(d,J=8.8Hz,1H),7.01(d,J=15.5Hz,1H),4.16(s,3H). 13 C NMR (101MHz, CDCl3) δ171.41,161.73,159.50,148.42,135.71,133.13,132.01,131.17,130.79,129.11,128.87 ,127.75,126.47,125.27,123.59,122.70,122.42,121.33,113.56,113.04,112.51,81.63,56.22.ESI-HRMS:m / z calc.forC 26 H 18N3OS[M+H] + :420.1171,found:420.1170.

[0136] (4) Synthesis of compound KSLOH06

[0137] Prepare a dry two-necked flask, add m9 (100 mg, 0.24 mmol), and add anhydrous dichloromethane as a solvent. Under nitrogen protection, slowly add C2H6BBr3S (1 M in CH2Cl2, 715 μL, 0.72 mmol) at 0 °C. Stir for 1 h, then bring the mixture to room temperature and continue stirring for 23 h. Monitor by TLC. After the substrate reaction is complete, pour the reaction solution into 50 mL of ice water, then extract three times with dichloromethane. Combine the organic phases, dry with anhydrous Na2SO4, evaporate to dryness, and separate by column chromatography to obtain 25 mg of pale yellow solid KSLOHO6, with a yield of 26%.

[0138] 1 H NMR (400MHz, DMSO-d6) δ12.21(s,1H),9.11(s,1H),8.33(d,J=8.7Hz,1H),8.25(s,1H),8.17(t,J=7.0Hz,2H), 7.80(d,J=3.5Hz,2H),7.65–7.60(m,3H),7.57(t,J=7.6Hz,1H),7.48(t,J=7.5Hz,1H),7.24(d,J=8.8Hz,1H). 13 C NMR(151MHz,DMSO-d6)δ164.05,159.24,159.08,156.55,151.87,144.85,135.90,135.34,132.18,130.85,129.36 ,128.61,127.54,126.97,125.66,122.88,122.51,119.97,119.46,118.07,117.10,109.33,65.53.ESI-HRMS:m / z calc.for C 25 H 16 N3OS[M+H] + :406.1014,found:406.1016.

[0139] Example 7

[0140] Preparation of probe KSL07 and its hydrolysis products

[0141]

[0142] (1) Synthesis of probe KSL07

[0143] Compound KSL05 (120 mg, 0.29 mmol) and 1,4-dimethylpyridinium iodide (68 mg, 0.29 mmol) were added to a flask, and anhydrous ethanol was added as a solvent. Piperidine (26 μL, 0.29 mmol) was added dropwise while stirring. The mixture was heated to reflux and reacted overnight. A pale yellow solid precipitated out. The solid was collected by filtration and washed with a small amount of anhydrous ethanol. A total of 50 mg of pale yellow solid KSL07 was obtained, with a yield of 27%. 1 H NMR (400MHz, DMSO-d6) δ8.85(d,J=6.5Hz,2H),8.80(d,J=1.9Hz,1H),8.25(d,J=6.6Hz,2H),8.15(t ,J=11.2Hz,2H),8.09(d,J=8.1Hz,1H),7.96(dd,J=8.9,1.8Hz,1H),7.58(t,J=7.3Hz,1H),7.50(dd, J=17.6,8.7Hz,3H),5.33(d,J=7.7Hz,1H),5.29(d,J=5.6Hz,1H),5.06(d,J=5.8Hz,1H),4.73(dd,J =10.8,5.0Hz,2H),4.25(s,3H),3.98(dd,J=14.3,8.3Hz,1H),3.82–3.74(m,2H),3.62–3.50(m,3H). 13 C NMR(101MHz,DMSO-d6)δ162.23,156.54,153.02,151.89,145.38,140.19,136.37,131.58,129.53,129.48,126.84,125 .62,123.81,122.93,122.75,122.50,122.29,116.18,101.18,76.36,74.01,70.62,68.55,60.85,47.31.ESI-HRMS:m / z calc.for C 27 H 27 N2O6S[MI] + :507.1584,found:507.1591.

[0144]

[0145] (2) Synthesis of compound KSLOAc07

[0146] Compound KSLOHO5 (255 mg, 1 mmol), 1,4-dimethylpyridinium iodide (235 mg, 1 mmol), and NaOAc (246 mg, 3 mmol) were added to a flask, followed by 15 mL of acetic anhydride. The mixture was stirred at 90 °C for 36 h. After the substrate reaction was complete, 10 mL of diethyl ether was added to the reaction solution, resulting in the precipitation of a brown solid. The solid was collected by filtration and washed with a small amount of water, yielding a total of 145 mg of KSLOAcO7 solid, with a yield of 28%. 1 H NMR (600MHz, DMSO-d6) δ8.92(d,J=6.6Hz,2H),8.67(s,1H),8.29(d,J=6.6Hz,2H),8.24–8.18(m,2H),8.14(d,J=8 .1Hz,1H),8.05(dd,J=8.5,1.9Hz,1H),7.68–7.60(m,2H),7.55(dd,J=14.6,7.7Hz,2H),4.29(s,3H),2.53(s,3H). 13 C NMR(151MHz,DMSO-d6)δ169.45,161.64,152.66,152.54,149.56,145.72,139.30,135.40,134.20,130.8 8,129.91,127.39,126.51,126.42,125.74,124.99,124.23,123.48,122.82,47.52,22.04.ESI-HRMS:m / z calc.for C 23 H 19 N2O2S[MI] + :387.1162,found:387.1166.

[0147] (3) Synthesis of compound KSLOH07

[0148] Compound KSLOAc07 (43 mg, 0.08 mmol) was placed in a flask and dissolved in anhydrous methanol. The mixture was stirred at room temperature. Simultaneously, NH4OAc (51 mg, 0.66 mmol) was dissolved in anhydrous methanol and slowly added dropwise to the flask. The mixture was stirred for 3 hours and monitored by TLC. After the substrate reaction was complete, Amberlite IR-120plus (H) was added. + Adjust the pH to neutral. Filter to remove Amberlite IR-120plus (H) + After collecting the filtrate, the solution was evaporated to dryness, and column chromatography was used to separate it into 39 mg of yellow solid KSLOHO7, with a yield of 99%. 1H NMR (600MHz, DMSO-d6) δ12.26(s,1H),8.86(d,J=6.7Hz,2H),8.64(d,J=2.1Hz,1H),8.23(d,J=6.8Hz,2H),8.20–8.12(m,2H), 8.08(d,J=8.1Hz,1H),7.87(dd,J=8.6,2.1Hz,1H),7.60–7.55(m,1H),7.50–7.41(m,2H),7.30(t,J=6.3Hz,1H),4.25(s,3H). 13 C NMR(151MHz,DMSO-d6)δ163.80,158.64,153.34,151.87,145.42,140.87,135.47,131.65,129.89 ,127.42,126.97,125.58,123.61,122.68,122.56,121.61,119.90,118.36,47.22.ESI-HRMS:m / z calc.for C 21 H 17 N2OS[MI - ] + :345.1056,found:345.1063.

[0149] Example 8

[0150] Preparation of probe KSL08 and its hydrolysis product KSLOH08

[0151]

[0152] (1) Synthesis of compound KSLOH08

[0153] Compound KSLOH05 (1 g, 3.92 mmol) was dissolved in 25 mL of tetrahydrofuran. While stirring at 0 °C, (1,3-dioxane-2-methyl)triphenylphosphine bromide (3.363 g, 7.84 mmol), sodium hydride (60% oil dispersion) (784 mg, 19.6 mmol), and 18-crown ether (100 mg, 25 mg / mmol) were added. Stirring continued for 10 min, then the temperature was raised to 50–60 °C, and the reaction was allowed to proceed overnight. The next day, a small amount of water was added to quench the reaction mixture, followed by the addition of 1 M HCl solution and stirring. TLC monitoring was performed. After the reaction was complete, the pH was adjusted to 7 with ammonia. The mixture was extracted three times with ethyl acetate. The combined organic phases were dried over anhydrous Na₂SO₄, evaporated to dryness, and separated by column chromatography to obtain 782 mg of KSLOH08 as a pale yellow solid, with a yield of 71%. 1H NMR (400MHz, CDCl3) δ13.01(s,1H),9.70(d,J=7.7Hz,1H),8.01(d,J=8.1Hz,1H),7.94(d,J=8.0Hz,1H),7.85(d,J=2.1Hz,1 H),7.61(dd,J=8.7,2.1Hz,1H),7.57–7.51(m,1H),7.49–7.41(m,3H),7.16(d,J=8.7Hz,1H),6.67(dd,J=15.9,7.7Hz,1H). 13 C NMR (101MHz, CDCl3) δ193.43,168.23,160.57,151.55,151.52,132.49,132.25,129. 23,127.04,127.03,126.08,125.80,122.36,121.69,119.00,117.22.ESI-HRMS:m / z calc.for C 16 H 12 NO2S[M+H] + :282.0589,found:282.0591.

[0154] (2) Synthesis of compound m10

[0155] Prepare a dry flask, add compound KSLOHO8 (400 mg, 1.422 mmol), cesium carbonate (2.316 g, 7.108 mmol), and a small amount of anhydrous Na2SO4, then add dichloromethane as a solvent. After stirring briefly at room temperature, add 2,3,4,6-tetraacetoxy-alpha-D-pyranose bromide (880 mg, 1.939 mmol). Under nitrogen protection, react overnight at room temperature. After the substrate has reacted completely, filter to remove the insoluble solid, evaporate to dryness, and separate by column chromatography to obtain 700 mg of m10 as a yellow solid, with a yield of 81%. 1H NMR (600MHz, CDCl3) δ9.74(d,J=7.6Hz,1H),8.80(d,J=2.2Hz,1H),8.15(d,J=8.1Hz,1H),7.96(d,J=7.7Hz,1 H),7.69(dd,J=8.7,2.3Hz,1H),7.58–7.53(m,2H),7.46–7.42(m,1H),7.29(d,J=8.7Hz,1H),6.80(dd,J=15. 9,7.6Hz,1H),5.76(dd,J=10.2,8.0Hz,1H),5.52(d,J=3.2Hz,1H),5.43–5.39(m,1H),5.17(dd,J=10.2,3.5H z,1H),4.27(dd,J=13.2,9.2Hz,1H),4.22–4.18(m,2H),2.24(s,3H),2.10(s,3H),2.04(s,3H),1.85(s,3H). 13 C NMR (151MHz, CDCl3) δ193.42,170.30,170.19,170.11,169.28,161.38,155.79,151.59,150.92,135.98,130.97,130.89,129.53,128.77 ,126.49,125.47,123.79,123.05,121.40,115.51,98.88,71.62,71.25,68.51,66.75,61.33,20.76,20.73,20.69,20.57.ESI-HRMS:m / z calc.for C 30 H 30 NO 11 S[M+H] + :612.1540,found:612.1539.

[0156] (3) Synthesis of compound KSL08

[0157] Compound m10 (300 mg, 0.491 mmol) was placed in a flask, dissolved in anhydrous methanol, stirred briefly at 0°C, and then slowly added dropwise in sodium methoxide solution (30% in MeOH, 546 μL, 2.946 mmol). Stirring continued, and TLC monitoring was performed. After the substrate reaction was complete, Amberlite IR-120plus (H) was added. + Adjust the pH to neutral. Filter to remove Amberlite IR-120plus (H) +After collecting the filtrate, the liquid was evaporated to dryness and separated by column chromatography to obtain 130 mg of yellow solid KSL08, with a yield of 60%. 1 H NMR(400MHz,DMSO-d6)δ9.69(d,J=7.8Hz,1H),8.72(d,J=2.2Hz,1H),8.12(dd,J=10.6,8.0Hz,2H) ,8.02(dd,J=8.9,2.2Hz,1H),7.91(d,J=15.8Hz,1H),7.60–7.53(m,1H),7.51–7.44(m,2H),6.88(d d,J=15.9,7.8Hz,1H),5.32(d,J=7.7Hz,1H),5.25(d,J=5.7Hz,1H),5.03(d,J=5.9Hz,1H),4.72(t ,J=5.5Hz,1H),4.68(d,J=4.5Hz,1H),4.00–3.91(m,1H),3.76(t,J=5.7Hz,2H),3.62–3.47(m,3H). 13 C NMR(101MHz,DMSO-d6)δ194.72,162.11,157.13,152.95,151.94,136.39,131.88,130.67,128.49,128.30, 126.80,125.64,123.06,122.53,122.28,116.23,101.23,76.36,73.99,70.58,68.54,60.82.ESI-HRMS:m / z calc.forC 22 H 22 NO7S[M+H] + :444.1117,found:444.1118.

[0158] Example 9

[0159] Preparation of probe KSL09 and its hydrolysis product KSLOH09

[0160]

[0161] (1) Synthesis of compound KSLOH09

[0162] Compound KSLOH08 (2 g, 7.11 mmol) was dissolved in 50 mL of tetrahydrofuran. While stirring at 0 °C, (1,3-dioxane-2-methyl)triphenylphosphine bromide (6.104 g, 14.228 mmol), sodium hydride (60% oil dispersion) (1.422 g, 35.55 mmol), and 18-crown ether (178 mg, 25 mg / mmol) were added. Stirring continued for 10 min, then the temperature was raised to 50–60 °C, and the reaction was allowed to proceed overnight. The next day, a small amount of water was added to quench the reaction mixture, followed by the addition of 1 M HCl solution and stirring. TLC monitoring was performed. After the reaction was complete, the pH was adjusted to 7 with ammonia. The mixture was extracted three times with ethyl acetate. The combined organic phases were dried over anhydrous Na₂SO₄, evaporated to dryness, and separated by column chromatography to obtain 2 g of KSLOH09 as a yellow solid, with a yield of 92%. 1 H NMR (400MHz, CDCl3) δ9.63(d,J=7.9Hz,1H),8.02(d,J=8.1Hz,1H),7.94(d,J=7.7Hz,1H),7.79(d,J=2.0Hz,1H),7.58(dd,J=8.7,2. 1Hz,1H),7.56–7.52(m,1H),7.49–7.42(m,1H),7.32–7.24(m,1H),7.13(d,J=8.7Hz,1H),6.97(m,2H),6.29(dd,J=15.1,7.9Hz,1H). 13 C NMR (101MHz, CDCl3) δ193.61,168.53,159.31,152.19,151.63,141.19,132.52,131.35,131. 07,127.98,127.46,126.98,125.95,124.69,122.33,121.64,118.77,117.07.ESI-HRMS:m / z calc.forC 18 H 14 NO2S[M+H] + :308.0745,found:308.0746.

[0163] (2) Synthesis of compound m11

[0164] Prepare a dry flask, add compound KSLOH09 (500 mg, 1.361 mmol), cesium carbonate (2.65 g, 8.133 mmol), and a small amount of anhydrous Na2SO4, then add dichloromethane as a solvent. After stirring briefly at room temperature, add 2,3,4,6-tetraacetoxy-alpha-D-pyranose bromide (1 g, 2.424 mmol). Under nitrogen protection, react overnight at room temperature. After the substrate has reacted completely, filter to remove the insoluble solid, evaporate to dryness, and separate by column chromatography to obtain 830 mg of m11 as a yellow solid, with a yield of 96%. 1 H NMR (400MHz, CDCl3) δ9.69(d,J=7.9Hz,1H),8.74(s,1H),8.18(d,J=8.1Hz,1H),8.01(d,J=7.9Hz, 1H),7.76–7.55(m,3H),7.52–7.45(m,1H),7.38–7.26(m,1H),7.16–7.12(m,2H),6.37(dd,J=15.1, 7.9Hz,1H),5.83–5.75(m,1H),5.56(d,J=2.8Hz,1H),5.43(d,J=8.0Hz,1H),5.21(dd,J=10.2,3.2 Hz,1H),4.36–4.28(m,1H),4.27–4.21(m,2H),2.28(s,3H),2.15(s,3H),2.08(s,4H),1.88(s,3H). 13 C NMR (101MHz, CDCl3) δ193.49,170.28,170.18,170.09,169.26,161.71,154.74, 151.95,151.65,140.58,136.14,132.13,132.04,131.80,131.14,130.30,129. 17,128.55,128.43,126.49,126.29,125.25,123.87,123.03,121.35,115.43,9 9.01,71.52,71.27,68.58,66.80,61.34,20.69,20.54.ESI-HRMS:m / zcalc.for C 32 H 32 NO 11 S[M+H] + :638.1696,found:638.1697.

[0165] (3) Synthesis of compound KSL09

[0166] Compound m11 (300 mg, 0.470 mmol) was placed in a flask and dissolved in anhydrous methanol. The mixture was stirred briefly at 0°C. Simultaneously, sodium methoxide (177.7 mg, 3.29 mmol) was dissolved in anhydrous methanol and slowly added dropwise to the flask while stirring. TLC monitoring was maintained. After the substrate reaction was complete, Amberlite IR-120plus (H) was added. + Adjust the pH to neutral. Filter to remove Amberlite IR-120plus (H) + After collecting the filtrate, the solution was evaporated to dryness and separated by column chromatography to obtain 196.2 mg of white solid KSL09, with a yield of 89%. 1 H NMR (400MHz, DMSO-d6) δ9.61(d,J=8.1Hz,1H),8.11(t,J=8.6Hz,2H),7.85(dd,J=8.8,2 .2Hz,1H),7.58–7.52(m,2H),7.51–7.40(m,3H),7.34–7.29(m,2H),6.35(dd,J=15.0,8 .1Hz,1H),5.28(d,J=7.7Hz,1H),5.23(d,J=5.7Hz,1H),5.02(d,J=5.9Hz,1H),4.72(t, J=5.5Hz,1H),4.67(d,J=4.4Hz,1H),3.95(m,1H),3.78–3.70(m,2H),3.62–3.46(m,3H). 13 C NMR(101MHz,DMSO-d6)δ194.50,162.39,156.02,153.33,151.96,141.62,136.40,131.67,131.28,130.11,128.56, 126.73,126.59,125.54,122.98,122.52,122.22,116.08,101.31,76.34,74.05,70.66,68.54,60.83.ESI-HRMS:m / z calc.for C 24 H 24 NO7S[M+H] + :470.1273,found:470.1274.

[0167] Example 10

[0168] Synthesis of probe KSL10

[0169]

[0170] (1) Synthesis of compound m13

[0171] Compound m7 (600 mg, 1.02 mmol) and compound m12 (440 mg, 2.56 mmol) were added to a flask, and anhydrous ethanol was added as a solvent. Piperidine (94 μL, 1.18 mmol) was added dropwise while stirring. The mixture was heated to reflux and reacted overnight. After the substrates had reacted completely, the mixture was evaporated to dryness and separated by column chromatography to obtain 153 mg of pale yellow solid m13, with a yield of 23%. 1 H NMR (600MHz, CDCl3) δ8.72(d,J=2.1Hz,1H),8.12(d,J=8.1Hz,1H),7.96(d,J=7.9Hz,1H),7.66–7.62(m,1H),7.56 –7.50(m,2H),7.43(t,J=7.6Hz,1H),7.31–7.27(m,1H),6.78(d,J=16.0Hz,1H),6.72(d,J=1.9Hz,1H),6.54(d,J=0 .8Hz,1H),5.76(dd,J=10.1,8.0Hz,1H),5.53(d,J=3.4Hz,1H),5.43(d,J=8.0Hz,1H),5.19(dd,J=10.2,3.4Hz,1H) ,4.28(dd,J=8.6,4.6Hz,1H),4.25–4.19(m,2H),2.42(s,3H),2.24(s,3H),2.11(s,3H),2.04(s,3H),1.86(s,3H). 13 CNMR(151MHz, CDCl3)δ170.32,170.20,170.13,169.29,162.04,161.41,158.82, 156.26,155.22,151.95,136.42,136.17,130.13,129.94,129.88,126.40,125.3 5,124.00,123.08,121.41,118.24,115.58,114.97,107.50,106.53,98.92,71.6 0,71.28,68.55,66.77,61.37,59.71,20.77,20.73,20.58,20.01.ESI-HRMS:m / z calc.for C 38 H 34 N3O 11 S[M+H] + :740.1914,found:740.1908.

[0172] (2) Synthesis of compound KSL10

[0173] Compound m13 (228 mg, 0.35 mmol) was placed in a flask and dissolved in anhydrous methanol. The mixture was stirred briefly at -20°C. Simultaneously, sodium methoxide (132 mg, 2.44 mmol) was dissolved in anhydrous methanol and slowly added dropwise to the flask while stirring. TLC monitoring was maintained. After the substrate reaction was complete, Amberlite IR-120plus (H) was added. + Adjust the pH to neutral. Filter to remove Amberlite IR-120plus (H) + After collecting the filtrate, the liquid was evaporated to dryness, and column chromatography was used to separate it into 70 mg of KSL10, which was a dark yellow solid with a yield of 35%. 1 H NMR (600MHz, DMSO-d6) δ8.74(s,1H),8.13(d,J=7.8Hz,1H),8.09(d,J=8.0Hz,1H),7.90(d,J =8.0Hz,1H),7.70(d,J=16.1Hz,1H),7.57(t,J=7.4Hz,1H),7.47(t,J=9.0Hz,2H),7.41(d,J =16.1Hz,1H),7.00(s,1H),6.70(s,1H),5.31(d,J=7.6Hz,1H),5.26(br,1H),5.06(br,1H), 4.79–4.68(m,2H),3.97(t,J=8.0Hz,1H),3.82–3.74(m,2H),3.62–3.50(m,3H),2.49(s,3H). 13 C NMR(151MHz,DMSO-d6)δ164.67,162.30,160.39,157.29,156.45,151.96,137.11,136.42,131.71,129.26,129.08,126.78,125.58,122 .95,122.60,122.27,118.74,116.14,115.96,107.44,106.32,101.27,76.34,74.03,70.63,68.52,60.81,56.15,19.87.ESI-HRMS:m / z calc.for C 30 H 26 N3O7S[M+H] + :572.1491,found:572.1490.

[0174]

[0175] (3) Synthesis of compound m14

[0176] Compound m6 (225 mg, 0.84 mmol) and compound m12 (288 mg, 1.67 mmol) were added to a flask, and anhydrous ethanol was added as a solvent. Piperidine (114 μL, 1.24 mmol) was added dropwise while stirring. The mixture was heated to reflux and reacted overnight. After the substrates had reacted completely, the mixture was evaporated to dryness and separated by column chromatography to obtain 186 mg of m14 as a deep yellow solid, with a yield of 54%. 1 H NMR (600MHz, CDCl3) δ8.75(s,1H),8.13(d,J=8.1Hz,1H),7.95(d,J=7.9Hz,1H),7.66(d,J=7.4Hz,1H),7.56–7.49(m,2H),7.42(t ,J=7.5Hz,1H),7.14(d,J=8.5Hz,1H),6.74(d,J=16.0Hz,1H),6.68(d,J=1.7Hz,1H),6.55–6.51(m,1H),4.14(s,3H),2.40(s,3H). 13 C NMR (101MHz, CDCl3) δ162.02,159.18,158.60,156.36,136.91,130.96,129.58,127.90,126.49,125.25,12 2.65,122.32,121.38,117.28,115.13,115.09,112.34,107.06,106.42,77.24,56.13,19.99.ESI-HRMS:m / z calc.for C 25 H 18 N3O2S[M+H] + :424.1120,found:424.1121.

[0177] (4) Synthesis of compound KSLOH10

[0178] Prepare a dry two-necked flask, add M14 (186 mg, 0.44 mmol), and add anhydrous dichloromethane as a solvent. Under nitrogen protection, slowly add BBr3 (1 M in CH2Cl2, 2.1 mL, 2.1 mmol) at 0 °C. Stir for 1 h, then bring the mixture to room temperature and continue stirring overnight. Monitor by TLC. After the substrate reaction is complete, pour the reaction mixture into 50 mL of ice water, then extract three times with dichloromethane. Combine the organic phases, dry with anhydrous Na2SO4, evaporate to dryness, and separate by column chromatography to obtain 38 mg of pale yellow solid KSLOH10, with a yield of 21%. 1H NMR (600MHz, DMSO-d6) δ12.09(s,1H),8.56(s,1H),8.17(d,J=7.9Hz,1H),8.08(d,J=8.1Hz,1H),7.80(d,J=7.1Hz,1H),7.66(d,J=16.1Hz ,1H),7.57(t,J=7.5Hz,1H),7.47(t,J=7.4Hz,1H),7.32(d,J=16.1Hz,1H),7.17(d,J=8.5Hz,1H),6.95(s,1H),6.69(s,1H),2.48(s,3H). 13 C NMR(151MHz,DMSO-d6)δ164.60,164.28,160.63,158.34,157.29,151.85,137.54,135.23,131.93,129.37,127.28,12 7.02,125.67,122.69,122.54,119.69,118.23,117.58,116.03,107.04,106.26,55.88,19.86.ESI-HRMS:m / zcalc.for C 24 H 16 N3O2S[M+H] + :410.0963,found:410.0964.

[0179] Example 11

[0180] Preparation of probe KSL11 and its hydrolysis products

[0181]

[0182] (1) Synthesis of compound m16

[0183] Compound m7 (500 mg, 0.85 mmol) and compound m15 (202 mg, 1.27 mmol) were added to a flask, and anhydrous ethanol was added as a solvent. Piperidine (78 μL, 0.85 mmol) was added dropwise while stirring. The mixture was heated to reflux and reacted overnight. After the substrates had reacted completely, the mixture was evaporated to dryness and separated by column chromatography to obtain 145 mg of m16 as a deep yellow solid, with a yield of 23%. 1H NMR (400MHz, CDCl3) δ8.69(d,J=2.1Hz,1H),8.15(d,J=8.1Hz,1H),7.96(d,J=7.8Hz,1H),7.63(dd,J=8.7,2.1Hz,1H),7. 57–7.51(m,1H),7.45–7.40(m,1H),7.27(d,8.8Hz,1H),7.15(d,J=16.1Hz,1H),7.03(d,J=16.2Hz,1H),6.89(s,1H),5.76 (dd,J=10.2,8.0Hz,1H),5.52(d,J=3.3Hz,1H),5.40(d,J=8.0Hz,1H),5.17(dd,J=10.2,3.4Hz,1H),4.31–4.17(m,4H),2. 84–2.79(m,2H),2.66(t,J=5.9Hz,2H),2.24(s,3H),2.11(s,3H),2.04(s,3H),1.98(dd,J=12.2,6.0Hz,2H),1.85(s,3H). 13 C NMR (101MHz, CDCl3) δ170.35,170.23,170.13,169.69,169.32,161.68,155.38,154.79,15 1.80,136.07,135.46,131.13,129.89,129.67,129.10,126.38,125.32,124.84,123.73,12 3.01,121.38,115.52,113.42,112.74,98.92,78.07,77.36,77.25,77.05,76.73,71.57,7 1.29,68.56,66.81,61.42,29.48,25.10,21.23,20.77,20.75,20.74,20.58.ESI-HRMS:m / z calc.for C 38 H 36 N3O 10 S[M+H] + :726.2121,found:726.2123.

[0184] (2) Synthesis of compound KSL11

[0185] Compound m16 (237 mg, 0.33 mmol) was placed in a flask and dissolved in anhydrous methanol. The mixture was stirred briefly at -20°C. Simultaneously, sodium methoxide (124 mg, 2.30 mmol) was dissolved in anhydrous methanol and slowly added dropwise to the flask while stirring. TLC monitoring was maintained. After the substrate reaction was complete, Amberlite IR-120plus (H) was added. + Adjust the pH to neutral. Filter to remove Amberlite IR-120plus (H) + After collecting the filtrate, the liquid was evaporated to dryness, and column chromatography was used to separate it into a total of 81 mg of KSL11, which was a dark yellow solid with a yield of 44%. 1 H NMR(600MHz, DMSO-d6)δ8.69(d,J=1.6Hz,1H),8.11(dd,J=13.7,8.0Hz,2H),7.97–7.91(m,1H),7.57(t,J= 7.6Hz,1H),7.47(dd,J=9.3,5.4Hz,1H),7.45–7.38(m,3H),6.92(s,1H),5.30(d,J=7.7Hz,1H),5.23(d,J= 5.7Hz,1H),5.01(d,J=5.8Hz,1H),4.73(t,J=5.4Hz,1H),4.67(d,J=4.3Hz,1H),3.96(dd,J=14.5,8.3Hz,1 H),3.79–3.73(m,2H),3.62–3.49(m,3H),2.78(t,J=6.2Hz,2H),2.71(t,J=5.6Hz,2H),1.93–1.85(m,2H). 13 C NMR(151MHz,DMSO-d6)δ171.71,162.43,158.49,156.09,151.97,137.38,136.40,131.26,130.43,129.31,129.10,126.75,125.54,124.0 8,122.98,122.47,122.24,116.04,114.48,113.66,101.23,76.33,75.62,74.02,70.63,68.57,60.85,29.63,24.98,21.32.ESI-HRMS:m / z calc.for C 30 H 26 N3O6S - [MH] - :556.1548,found:556.1544.

[0186]

[0187] (3) Synthesis of compound m17

[0188] Compound m6 (705 mg, 2.62 mmol) and compound m15 (620 mg, 3.92 mmol) were added to a flask, and anhydrous ethanol was added as a solvent. Piperidine (360 μL, 3.93 mmol) was added dropwise while stirring. The mixture was heated to reflux and reacted overnight. After the substrates had reacted completely, the mixture was evaporated to dryness and separated by column chromatography to obtain 95 mg of m17 as a deep yellow solid, with a yield of 9%. 1 H NMR (400MHz, CDCl3) δ8.76(s,1H),8.18(d,J=8.2Hz,1H),7.95(d,J=8.0Hz,1H),7.67(dd,J=8.7,2.0Hz,1H),7.54(t,J=7.6Hz,1H),7.42(t,J=7.5Hz ,1H),7.14(dd,J=16.8,12.4Hz,2H),7.01(d,J=16.1Hz,1H),6.86(s,1H), 4.13(s,3H),2.82–2.77(m,2H),2.65(t,J=6.0Hz,2H),2.02–1.93(m,2H). 13 C NMR (101MHz, CDCl3) δ169.78,158.20,155.79,136.12,130.47,129.46,129.06,128.18,126.3 6,125.10,124.36,122.73,121.33,112.31,56.08,29.71,25.15,21.27,14.25.ESI-HRMS:m / z calc.for C 25 H 20 N3OS[M+H] + :410.1327,found:410.1325.

[0189] (4) Synthesis of compound KSLOH11

[0190] Prepare a dry two-necked flask, add M17 (95 mg, 0.23 mmol), and add anhydrous dichloromethane as a solvent. Under nitrogen protection, slowly add BBr3 (1 M in CH2Cl2, 1.15 mL, 1.15 mmol) at 0 °C. Stir for 1 h, then bring to room temperature and continue stirring overnight. Monitor by TLC. After the substrate reaction is complete, pour the reaction solution into 50 mL of ice water, then extract three times with dichloromethane. Combine the organic phases, dry with anhydrous Na2SO4, evaporate to dryness, and separate by column chromatography to obtain 78 mg of pale yellow solid KSLOH11, with a yield of 85%.1 H NMR (600MHz, DMSO-d6) δ12.02(s,1H),8.49(d,J=2.1Hz,1H),8.17(d,J=7.8Hz,1 H),8.09(d,J=8.1Hz,1H),7.85(dd,J=8.6,2.1Hz,1H),7.59–7.54(m,1H),7.49– 7.45(m,1H),7.42(d,J=16.1Hz,1H),7.32(d,J=16.1Hz,1H),7.14(d,J=8.6Hz,1 H),6.88(s,1H),2.77(t,J=6.4Hz,2H),2.69(t,J=5.9Hz,2H),1.91–1.84(m,2H). 13 C NMR(151MHz,DMSO-d6)δ171.69,164.56,158.73,157.97,151.86,137.85,135.12,131.63,129.47,128.47,128.06,1 27.00,125.65,123.63,122.70,122.53,119.47,118.17,114.55,113.74,75.17,29.62,24.99,21.34.ESI-HRMS:m / z calc.for C 24 H 18 N3OS[M+H] + :396.1171,found:396.1172.

[0191] Example 12

[0192] Preparation of probe KSL12 and its hydrolysis product KSLOH12

[0193]

[0194] (1) Synthesis of compound m19

[0195] Compound m7 (530 mg, 0.91 mmol) and compound m18 (185 mg, 1 mmol) were added to a flask, and anhydrous ethanol was added as a solvent. Piperidine (72 μL, 0.78 mmol) was added dropwise while stirring. The mixture was heated to reflux and reacted overnight. After the substrates had reacted completely, the mixture was evaporated to dryness and separated by column chromatography to obtain 232 mg of m19 as a yellow solid, with a yield of 34%. 1H NMR (600MHz, DMSO-d6) δ8.66(d,J=2.1Hz,1H),8.11(d,J=8.1Hz,1H),8.05(d,J=8.0Hz,1H),8.01(dd,J=8.9,2.0Hz ,1H),7.62–7.56(m,1H),7.50(dd,J=11.5,4.4Hz,1H),7.47(s,2H),7.42(d,J=8.9Hz,1H),6.96(s,1H),5.95(d,J= 8.0Hz,1H),5.50(dd,J=10.1,8.1Hz,1H),5.42(d,J=3.4Hz,1H),5.32(dd,J=10.2,3.6Hz,1H),4.59(t,J=6.4Hz,1H ),4.14(d,J=6.3Hz,2H),2.62(d,J=21.3Hz,4H),2.18(s,3H),2.05(s,3H),1.96(s,3H),1.90(s,3H),1.04(s,6H). 13 C NMR(151MHz,DMSO-d6)δ170.48,170.35,170.02,169.52,161.63,156.44,154.68, 152.05,136.92,135.94,131.56,131.31,129.89,129.71,127.04,126.00,123.28 ,122.79,122.00,116.27,114.37,113.58,97.43,76.82,71.41,71.17,68.75,67. 77,61.88,32.16,31.43,27.95,22.54,21.05,21.00,20.94,20.82.ESI-HRMS:m / z calc.for C 40 H 40 N3O 10 S[M+H] + :754.2434,found:754.2435.

[0196] (2) Synthesis of compound KSL12

[0197] Compound m19 (220 mg, 0.29 mmol) was placed in a flask and dissolved in anhydrous methanol. The mixture was stirred briefly at -20°C. Simultaneously, sodium methoxide (110 mg, 2.04 mmol) was dissolved in anhydrous methanol and slowly added dropwise to the flask while stirring. TLC monitoring was maintained. After the substrate reaction was complete, Amberlite IR-120plus (H) was added. +Adjust the pH to neutral. Filter to remove Amberlite IR-120plus (H) + After collecting the filtrate, the liquid was evaporated to dryness, and column chromatography was used to separate it into 38 mg of KSL12, which was a red solid with a yield of 22%. 1 H NMR (600MHz, DMSO-d6) δ8.70(d,J=2.2Hz,1H),8.13(d,J=7.9Hz,1H),8.09(d,J=8.1Hz,1H),7.95( dd,J=8.9,2.2Hz,1H),7.59–7.55(m,1H),7.50–7.41(m,4H),6.96(s,1H),5.29(d,J=7.7Hz,1H),5. 23(d,J=5.5Hz,1H),5.01(d,J=4.6Hz,1H),4.73(s,1H),4.67(d,J=4.3Hz,1H),3.96(dd,J=13.1,9 .0Hz,1H),3.76(dd,J=12.3,5.4Hz,2H),3.61–3.49(m,3H),2.64(s,2H),2.61(s,2H),1.04(s,6H). 13 C NMR(151MHz,DMSO-d6)δ170.92,162.43,156.67,156.11,151.98,137.42,136.40,131.25,130.42,129.34,129.24,126.75,125.55,122.98,1 22.48,122.25,116.06,114.44,113.62,101.27,76.47,76.33,74.03, 70.64,68.55,60.84,42.82,38.69,32.16,27.96,27.94.ESI-HRMS:m / z calc.forC 32 H 31 N3NaO6S[M+Na] + :608.1831,found:608.1833.

[0198]

[0199] (3) Synthesis of compound m20

[0200] Compound m6 (610 mg, 2.26 mmol) and compound m18 (540 mg, 3.42 mmol) were added to a flask, and anhydrous ethanol was added as a solvent. Piperidine (210 μL, 2.29 mmol) was added dropwise while stirring. The mixture was heated to reflux and reacted overnight. After the substrates had reacted completely, the mixture was evaporated to dryness and separated by column chromatography to obtain 750 mg of m20 as a red solid, with a yield of 80%. 1 H NMR (400MHz, CDCl3) δ8.73(s,1H),8.14(d,J=8.0Hz,1H),7.95(d,J=8.0Hz,1H),7.66(dd,J=8.7,1.8Hz,1H),7.53(t,J=7.6Hz,1H),7. 41(t,J=7.4Hz,1H),7.14(t,J=13.4Hz,2H),7.03(d,J=16.1Hz,1H),6.87(s,1H),4.12(s,3H),2.61(s,2H),2.48(s,2H),1.09(s,6H). 13 CNMR(101MHz, CDCl3)δ169.32,162.28,158.18,154.01,136.09,135.95,130.37,129.45,129.03,128.32,126.30,125. 05,123.28,122.80,122.49,121.32,113.63,112.87,112.29,78.33,56.06,43.00,39.22,32.05,28.06.ESI-HRMS:m / z calc.for C 27 H 24 N3OS[M+H] + :438.1640,found:438.1641.

[0201] (4) Synthesis of compound KSLOH12

[0202] Prepare a dry two-necked flask, add m20 (300 mg, 0.69 mmol), and add anhydrous dichloromethane as a solvent. Under nitrogen protection, slowly add BBr3 (1 M in CH2Cl2, 3.43 mL, 3.43 mmol) at 0 °C. Stir for 1 h, then bring the mixture to room temperature and continue stirring overnight. Monitor by TLC. After the substrate reaction is complete, pour the reaction mixture into 50 mL of ice water, then extract three times with dichloromethane. Combine the organic phases, dry with anhydrous Na2SO4, evaporate to dryness, and separate by column chromatography to obtain 121 mg of KSLOH12 as a red solid, with a yield of 29%. 1H NMR (600MHz, DMSO-d6) δ12.05(s,1H),8.49(d,J=2.1Hz,1H),8.17(d,J=7.9Hz,1H),8.08(d,J=8.1Hz,1H),7.85(dd,J=8.6,2.1Hz,1H),7.59–7.54 (m,1H),7.49–7.46(m,1H),7.45–7.39(m,1H),7.35(d,J=16.1Hz,1H),7. 14(d,J=8.6Hz,1H),6.90(s,1H),2.62(s,2H),2.58(s,2H),1.03(s,7H). 13 C NMR(151MHz,DMSO-d6)δ170.87,164.64,157.99,156.90,151.85,137.86,135.08,131.61,129.54,128.47,128.22, 127.03,125.68,122.68,122.54,119.42,118.19,114.51,113.69,76.01,42.80,38.69,32.15,27.94.ESI-HRMS:m / z calc.for C 26 H 22 N3OS[M+H] + :424.1484,found:424.1485.

[0203] Example 13

[0204] The changes in UV-Vis absorption and fluorescence spectra of the fluorescent probe before and after the addition of Aspergillus oryzae β-galactosidase and Escherichia coli β-galactosidase were detected.

[0205] The fluorescent probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mmol / L stock solution. 40 μL of the stock solution was added to a 5 mL centrifuge tube and diluted to 3 mL with PBS buffer (10 mmol / L; pH 4.5 for A. oryzae β-gal assay and pH 7.4 for E. coli β-gal assay). Then, 1 mL of 40 U / mL β-galactosidase standard solution was added, and the mixture was incubated at 37 °C for 10 min to create the experimental group. For the blank group, 40 μL of the probe stock solution was added to a 5 mL centrifuge tube and diluted directly to 4 mL with PBS without adding β-galactosidase. The mixture was incubated at 37 °C for 10 min. The UV absorption and fluorescence spectra of the experimental and blank group samples were measured.

[0206] like Figure 1 As shown, the fluorescent probe KSL01-KSL12 solution exhibited significant UV-Vis absorption spectrum changes after the addition of A. oryzae β-gal. The trend of these changes was similar to that of the UV-Vis absorption spectrum of the fluorescent probe hydrolysis product KSLOH01-KSLOH12, indicating that A. oryzae β-gal cleaved the glycosidic bond of the probe substrate, releasing the fluorophore.

[0207] like Figure 2 As shown, the fluorescent probe KSL01-KSL12 solution exhibited a significant change in fluorescence spectrum after the addition of *A. oryzae* β-gal, showing a significant red shift. This shift trended towards the fluorescence spectrum of the probe hydrolysis product KSLOH01-KSLOH12, indicating that *A. oryzae* β-gal cleaved the glycosidic bond of the probe substrate, releasing the fluorophore. The maximum emission wavelengths of the probe KSL01-KSL12 solution after co-incubation with *A. oryzae* β-gal are shown in Table 1.

[0208] Table 1. Maximum emission wavelengths after co-incubation of probe KSL01-KSL12 solution with A. oryzae β-gal

[0209]

[0210] like Figure 3 As shown, after the addition of E. coli β-gal, the fluorescence spectra of fluorescent probes KSL01-KSL06 in the KSL01-KSL12 solution changed significantly, exhibiting a significant red shift. This shift trended towards the fluorescence spectra of the hydrolysis products KSLOH01-KSLOH06, indicating that E. coli β-gal can cleave the glycosidic bonds in probes KSL01-KSL06, releasing the fluorophore. The fluorescence spectrum of probe KSL07 also changed after the addition of E. coli β-gal, showing fluorescence at the maximum emission wavelength of both the probe itself and the fluorophore (KSLOH07), indicating that probe KSL07 was not completely hydrolyzed by E. coli β-gal. The fluorescence spectrum of the probe KSL08-KSL12 solution remained essentially unchanged after the addition of E. coli β-gal, and was completely different from that of the fluorescent probe hydrolysis product KSLOH08-KSLOH12, indicating that E. coli β-gal cannot cleave the glycosidic bonds in the probe KSL08-KSL12. In summary... Figure 2 The experimental results show that the probes KSL08-KSL12 are species-specific and can recognize eukaryotic A. oryzae β-gal, but not bacterial E. coli β-gal.

[0211] Example 14

[0212] Changes in fluorescence spectra and linear relationships of fluorescent probes with increasing concentration of A. oryzae β-gal.

[0213] Fluorescent probes KSL01-KSL12 were dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mmol / L stock solution. 40 μL of this stock solution was added to a 5 mL centrifuge tube and diluted with PBS buffer (10 mmol / L, pH 4.5). Then, different concentrations of *A. oryzae* β-gal standard solution (0-1 U / mL) were added, bringing the total sample volume to 4 mL. The samples were incubated at 37 °C for the same duration at different enzyme concentrations using the same probe, and then their fluorescence spectra were measured.

[0214] like Figure 4 As shown, significant changes in fluorescence spectra occurred after co-incubation of fluorescent probes KSL01-KSL12 solutions with standard solutions of different concentrations of *A. oryzae* β-gal. With increasing enzyme concentration, the fluorescence at the maximum emission wavelength of the probe-released fluorophores gradually increased. Specifically, probes KSL04, KSL06, KSL07, KSL10, KSL11, and KSL12 exhibited a gradual redshift in emission wavelength. Furthermore, within a certain concentration range of *A. oryzae* β-gal, the fluorescence intensity at the maximum emission wavelength of the probe showed a good linear relationship with the enzyme concentration. Specifically, probe KSL11 showed a good linear ratio between the fluorescence intensity at 662 nm and the fluorescence intensity at 570 nm.

[0215] The lowest detection range of probes KSL01-KSL12 for A. oryzae β-gal concentration can be calculated using the formula LOD = 3σ / k, where σ is the standard deviation of 10 blank control groups and k is the slope of the linear equation.

[0216] The detection limits of the 12 probe molecules were calculated according to the formula, as shown in Table 2.

[0217] Table 2. Detection limits of probes KSL01-KSL12 for A. oryzae β-gal

[0218]

[0219] Example 15

[0220] The relationship between fluorescence spectrum and time after adding fluorescent probe A. oryzae β-gal.

[0221] The fluorescence intensity of fluorescent probes KSL01-KSL12 (10 μM) and A. oryzae β-gal (10 U / mL) in PBS buffer solution at pH 4.5 was investigated over time at a temperature of 37 °C. The time-kinetic curves of the probe molecules are shown below. Figure 5 As shown, when A. oryzae β-gal was added, the fluorescence spectrum showed significant fluorescence enhancement and / or a red shift in the maximum emission wavelength, reaching its maximum value within 40 s–20 min.

[0222] Example 16

[0223] Selectivity experiment of fluorescent probes for β-gal.

[0224] The selectivity of fluorescent probes for *A. oryzae* β-gal, *E. coli* β-gal, and other biological enzymes and small molecules was investigated to verify whether the probe molecules would be interfered with by other biological and chemical molecules. Representative probe molecules KSL04 (10 μM) and KSL11 (10 μM) were incubated for 20 min with biological enzymes such as *A. oryzae* β-gal, *E. coli* β-gal, estradiol, pepsin, trypsin, and cellulase (all 10 U / mL), and small molecules such as LZM, DTT, GSH, L-Cys, Hcy, and H2O2 (10 μM). The results are as follows: Figure 6 As shown, probe KSL04 selectively responds to *A. oryzae* β-gal and *E. coli* β-gal, exhibiting a significant enhancement of fluorescence at its maximum emission wavelength, unaffected by interference from other analytes. Probe KSL11 selectively responds to *A. oryzae* β-gal, exhibiting a significant enhancement of fluorescence at its maximum emission wavelength, unaffected by interference from *E. coli* β-gal and other analytes.

[0225] Example 17

[0226] Cytotoxicity assays using fluorescent probes.

[0227] The cytotoxicity of fluorescent probes against three cell lines was assessed using the CCK-8 assay to evaluate the biocompatibility of the probe molecules. Fluorescent probes KSL01-KSL12 were incubated with MRC5 cells at 37°C for 48 hours at concentrations ranging from 5-100 μM. Figure 7 As shown, probes KSL01, KSL02, and KSL04 caused significant cell death at high concentrations of 50 μM and 100 μM, while the remaining probe molecules did not show significant cytotoxicity. However, no cell death was observed at the fluorescent probe concentration (10 μM). Fluorescent probes KSL01-KSL12, when incubated with SKOV3 cells at 37°C for 48 h at concentrations of 5-100 μM, showed... Figure 8As shown, none of the probe molecules caused cell death at the experimental concentration (10 μM). However, probes KSL01, KSL02, KSL04, and KSL12 showed significant cell death at high concentrations of 50 μM and 100 μM. The remaining probe molecules did not show significant cytotoxicity. Fluorescent probes KSL01-KSL12, when incubated with HepG2 cells at 37°C for 48 h at concentrations of 5-100 μM, showed... Figure 9 As shown, none of the probe molecules exhibited cell death at the experimental concentration (10 μM). However, probes KSL01, KSL02, and KSL04 showed significant cell death at a high concentration of 100 μM, while the remaining probe molecules did not show significant cytotoxicity. In summary, the HBT-based β-gal fluorescent probe molecules provided in this study exhibit low cytotoxicity and good biocompatibility.

[0228] Example 18

[0229] Application of fluorescent probes in live-cell fluorescence imaging.

[0230] Senescent MRC5 cells (human embryonic lung fibroblasts) and SKOV3 cells (human ovarian cancer cells) were incubated with 10 μM fluorescent probes KSL01-KSL12 at 37°C for 30 min, respectively. Imaging effects were observed using fluorescence confocal microscopy. The results are as follows: Figure 10 As shown in Table 3, the excitation and emission wavelength ranges of the fluorescent probes KSL01-KSL12 are also shown. Both MRC5 and SKOV3 are cells that highly express β-galactosidase. MRC5 is a normal human cell that overexpresses SA-β-gal with cellular senescence, while SKOV3 is a tumor cell that highly expresses β-gal. Figure 10 It can be seen that probes KSL01-KSL12 produced significant fluorescence after incubation with both types of cells, with fluorescence emission ranging from green to near-infrared light.

[0231] Table 3. Imaging conditions for confocal microscopy of fluorescent probes KSL01-KSL12

[0232]

[0233] Example 19

[0234] Application of fluorescent probes in detecting senescent cells and the degree of cell senescence.

[0235] The changes in fluorescence intensity of normal cells with aging were detected using fluorescent probes. The representative fluorescent probe KSL04 was incubated with continuously cultured MRC5 cells from passage 22 to passage 29, representing the natural process of normal cells from youth to aging. 10 μM KSL04 was incubated with MRC5 cells of different passage numbers at 37°C for 20 min, and the imaging effects were observed using a fluorescence confocal microscope. Figure 11 As shown in Figure a, with increasing cell culture passages, cells gradually age, and the fluorescence intensity gradually increases. Figure 11 The quantitative results of cell fluorescence intensity in section b clearly show the trend of fluorescence intensity gradually increasing with the number of cell culture passages. This indicates that β-gal expression gradually increases with cell senescence, and the fluorescent probe molecule provided by this invention can accurately detect cell senescence qualitatively and quantitatively. Incubation of 29th passage (P29) MRC5 cells with the anti-aging drug rapamycin (Rapa, 10 nM, 25 nM) 3 days in advance yielded the following results: Figure 11 As shown, compared with the untreated P29 blank group, the fluorescence intensity of the P29+Rapa group decreased significantly. The anti-aging efficacy of rapamycin was demonstrated by the fluorescence quantitative method, indicating that the fluorescent probe molecule provided by the present invention can be used as a tool molecule to detect anti-aging efficacy and establish an anti-aging efficacy evaluation method based on fluorescence visualization.

[0236] Example 20

[0237] Application of fluorescent probes in detecting aging of tissues and organs.

[0238] Frozen sections of tissues and organs from mice of different ages were prepared, incubated with fluorescent probes, and their fluorescence intensity was measured to investigate the application of probe molecules in detecting tissue and organ aging. Kidneys from 1-month-old, 13-month-old, and 23-month-old C57BL / 6J mice were collected and frozen sections were prepared. These sections were then incubated with representative probe molecules KSL04 (10 μM) and KSL11 (10 μM) at 37°C for 1 h, and the imaging effects were observed using a fluorescence confocal microscope. Figure 12 As shown in Figure a, after co-incubation with probe molecules KSL04 and KSL11, the fluorescence intensity of kidney slices gradually increased with age, i.e., fluorescence intensity 23 months > 13 months > 1 month. Figure 12 The bar chart of the quantitative fluorescence intensity results of the tissue sections described in section b provides a clear visual conclusion. The results demonstrate that the β-gal content in kidney tissue gradually increases with age, and the fluorescent probe provided by this invention can detect aging at the tissue and organ level.

[0239] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A fluorescent probe, characterized in that, The fluorescent probe is selected from the following group:

2. The method for preparing the fluorescent probe as described in claim 1, characterized in that, The preparation method includes the following route: Route 1: Starting with 2-aminobenzylthiol and 5-methylsalicylaldehyde, the intermediate m1 is generated by reacting under concentrated hydrochloric acid and hydrogen peroxide conditions. m1 undergoes the Duff reaction to generate intermediate KSLOH01, which then undergoes a nucleophilic substitution reaction with intermediate m2 to obtain the key intermediate m3. m3 is deacetylated from the galactose residue under sodium methoxide conditions to obtain the probe KSL01. Route 2: Using KSLOH01 as a raw material, intermediate KSLOH02 is obtained through Wittig reaction, and then undergoes nucleophilic substitution reaction with m2 to obtain intermediate m4. Under methanol and sodium methoxide conditions, the acetyl protecting group on the galactose residue of m4 is removed to obtain probe KSL02. Route 3: Using probe KSL01 as a raw material, probe KSL03 was prepared by Knoevenagel condensation reaction with 1,4-dimethylpyridinium iodide. Route 4: Intermediate m4 and malononitrile undergo a Knoevenagel condensation reaction to obtain the key intermediate m5. Under methanol and sodium methoxide conditions, the acetyl protecting group on the galactose residue of m5 is removed to obtain the probe KSL04. Route 5: Starting with benzothiazole and 3-bromo-4-methoxybenzaldehyde, intermediate m6 is obtained by the Suzuki reaction. Then, the methyl protecting group is removed in aqueous hydrobromic acid to obtain intermediate KSLOH05. It undergoes a nucleophilic substitution reaction with 2,3,4,6-tetraacetoxy-α-D-pyranose bromide to obtain the key intermediate m7. Under methanol and sodium methoxide conditions, the acetyl protecting group on the galactose residue of m7 is removed to obtain probe KSL05. Route 6: Intermediate m7 is reacted with 2-(1-phenylethylidene)malononitrile via Knoevenagel condensation to obtain intermediate m8. Under methanol and sodium methoxide conditions, m8 is deacetyl protecting group on galactose residue to obtain probe KSL06. Route 7: Using probe KSL05 as a raw material, probe KSL07 was prepared by Knoevenagel condensation reaction with 1,4-dimethylpyridinium iodide. Route 8: Using intermediate KSLOH05 as a raw material, intermediate KSLOH08 is obtained through Wittig reaction, and then undergoes nucleophilic substitution reaction with 2,3,4,6-tetraacetoxy-α-D-pyranose bromide to obtain key intermediate m10. Under methanol and sodium methoxide conditions, the acetyl protecting group on the galactose residue of m10 is removed to obtain probe KSL08. Route 9: Using intermediate KSLOH08 as a starting material, the aldehyde conjugated chain is extended by the Wittig reaction to obtain intermediate KSLOH09, which then undergoes a nucleophilic substitution reaction with 2,3,4,6-tetraacetoxy-α-D-pyranose bromide to obtain the key intermediate m11. Under methanol and sodium methoxide conditions, the acetyl protecting group on the galactose residue of m11 is removed to obtain probe KSL09. Route 10: Intermediate m7 and intermediate m12 undergo a Knoevenagel condensation reaction to obtain intermediate m13. Under methanol and sodium methoxide conditions, m13 is deacetyl-protecting group removed from galactose residues to obtain probe KSL10. Route 11: Intermediate m7 and intermediate m15 undergo a Knoevenagel condensation reaction to obtain intermediate m16. Under methanol and sodium methoxide conditions, the acetyl protecting group on the galactose residue of m16 is removed to obtain probe KSL11. Alternatively, route 12: intermediate m7 and intermediate m18 undergo a Knoevenagel condensation reaction to obtain intermediate m19. Under methanol and sodium methoxide conditions, the acetyl protecting group on the galactose residue of m19 is removed to obtain probe KSL12.

3. The use of the fluorescent probe as described in claim 1, characterized in that, This reagent is used to prepare reagents for detecting Aspergillus oryzae β-galactosidase or for detecting human β-galactosidase.

4. The use as described in claim 3, characterized in that, The reagent for detecting human β-galactosidase is used to detect β-galactosidase activity in in vitro cells or tissues / organs.

5. The use as described in claim 4, characterized in that, The cells in question are tumor cells.

6. The use as described in claim 5, characterized in that, The tumor cells were ovarian cancer cells.

7. The use of the fluorescent probe as described in claim 1, characterized in that, The reagent is used to prepare a test for detecting Escherichia coli β-galactosidase E. coli β-gal, wherein the fluorescent probe is selected from: KSL01, KSL02, KSL03, KSL04, KSL05, KSL06, KSL07.

8. The use of the fluorescent probe as described in claim 1, characterized in that, Reagents for preparing species-specific detection of β-galactosidase The species-specific β-galactosidase detection method can normally identify and detect human β-galactosidase, but cannot identify and detect Escherichia coli β-galactosidase. The fluorescent probes are selected from: KSL08, KSL09, KSL10, KSL11, and KSL12.

9. The use of the fluorescent probe as described in claim 1, characterized in that, Used to prepare diagnostic tool molecules for detecting aging.

10. The use as described in claim 9, characterized in that, The aging referred to here is the aging of the body, the aging of tissues and organs, or the aging of cells.

11. The use of the fluorescent probe as described in claim 1, characterized in that, It is used as a reagent for preparing fluorescence imaging for the detection of Aspergillus oryzae β-galactosidase or human β-galactosidase.

Images