CN113801179A - A kind of β-galactosidase fluorescent probe, preparation method and application thereof - Google Patents
A kind of β-galactosidase fluorescent probe, preparation method and application thereof Download PDFInfo
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- CN113801179A CN113801179A CN202010532586.1A CN202010532586A CN113801179A CN 113801179 A CN113801179 A CN 113801179A CN 202010532586 A CN202010532586 A CN 202010532586A CN 113801179 A CN113801179 A CN 113801179A
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- probe
- galactosidase
- beta
- reaction
- fluorescent probe
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Abstract
The invention discloses a fluorescent probe for detecting beta-galactosidase, a preparation method and application thereof, and the structure of the fluorescent probe is shown as a general formula A. The beta-galactosidase fluorescent probe provided by the invention has multiple purposes, can be used for sensing and detecting beta-galactosidase in vitro, cells, tissues and organs and animal level, and can identify the fluorescent emission range of the beta-galactosidase from green light to near infrared light (445-662 nm). The invention provides a partial beta-galactosidase fluorescent probeHas species selectivity, can be the common sense other human source beta-galactosyl glycinase, and is not interfered by the beta-galactosidase of the bacterial species. The discovery provides a rapid, sensitive and multicolor beta-galactosidase fluorescent probe with species specificity, and the probe has a wide application prospect in the field of biomolecule detection.
Description
Technical Field
The invention relates to a beta-galactosidase fluorescent probe, a preparation method and application thereof, in particular to biological application in the field of beta-galactosidase detection, and especially application in the aspect of serving as an aging diagnosis tool molecule.
Background
Beta-galactosidase (beta-gal) is a glycoside hydrolase that is capable of hydrolyzing galactose to galactose and glucose, as well as other various biochemical molecules having beta-galactoside linkages. Beta-galactosidase is a hydrolase that is ubiquitous in many organisms, such as bacteria, fungi, and mammals, and has a wide range of biological applications. Beta-galactosidase is an important biomarker, and the abnormality of the activity thereof is closely related to certain pathological processes and environmental factors. By detecting the bacterial beta-galactosidase content in the sample, the bacterial density in the environment can be detected. In some cancer cases, such as primary ovarian cancer, the expression level of beta-galactosidase in tumor cells is increased compared with that in normal cells, so that the beta-galactosidase can be used as a diagnostic marker of ovarian cancer. As the organism ages, the expression level of beta-galactosidase in aging cells increases, called senescence-associated beta-galactosidase (SA-beta-gal). Senescence-associated beta-galactosidase is an important biomarker for monitoring the senescence process and for studying senescence. Therefore, the development of a detection method of beta-galactosidase is of great significance. The enzyme activity detection method comprises a fluorescence method, a colorimetric method, an enzyme-linked immunosorbent assay, an electrochemical method and the like, wherein the fluorescence probe detection method has the advantages of high selectivity, high sensitivity, real-time detection, easy operation, nondestructive detection, good biological sample compatibility and the like, and becomes an important means for biological enzyme detection. The basic design principle of the beta-galactosidase fluorescence detection probe is that a fluorophore and a beta-D-galactose residue are connected through glycosidic bonds, after a probe molecule is combined with beta-galactosidase, the beta-galactose bonds are cut off under the action of enzyme catalytic hydrolysis, the fluorophore molecule is exposed, and therefore fluorescence change is generated, and the beta-galactosidase is qualitatively and quantitatively detected through the fluorescence change.
Aging is the process of gradual decline and decline of body functions after the development of organisms reaches maturity. Aging is a complex physiological and pathological process, is affected by various mechanisms, and is mainly characterized in that the functions of various tissues and organs of an organism begin to be damaged, the metabolism and the stress response capability decline along with the continuous increase of the age, and various aging-related diseases are accompanied, such as diabetes, tumors, cardiovascular diseases, neurodegenerative diseases and the like. Aging and aging-related diseases seriously threaten the human life health and quality of life and aggravate the social medical burden. Therefore, accurate detection of aging is of great significance for aging-related studies. Senescence-associated beta-galactosidase is currently recognized as the most classical senescence-associated biomarker. However, it has been reported that none of the β -galactosidase probes can detect β -galactosidase of different species differentially. It is required that beta-galactosidase from different species has certain difference, and no difference in identification may bring false positive result. The bacteria are ubiquitous prokaryotes in the natural environment, bacterial infection is a common pathological state, and interference caused by beta-galactosidase secreted by the bacteria on the detection result of human body samples exists. Therefore, the development of a beta-galactosidase fluorescent probe with species selectivity and the elimination of interference brought by bacterial beta-galactosidase have important research significance.
At present, no literature discloses a beta-galactosidase fluorescent probe with species selectivity.
Disclosure of Invention
The invention aims to provide a beta-galactosidase fluorescent probe, a preparation method thereof, and application of the beta-galactosidase fluorescent probe in detection of aging-related beta-galactosidase and the like.
In a first aspect of the present invention, a fluorescent probe is provided, which has a structure as shown in formula a:
wherein X is S, O or NH;
y is
Or is absent;
Ra、Rb、Rc、Rdeach independently is H, C1-C4Alkyl, -CHO, -COOH, -CN, -NO2、-COOC1-C4Alkyl, -CH ═ CH (N-methylpyridinium),
In another preferred embodiment, Ra、Rb、RcEach independently is H or C1-C4Alkyl radical, Rdis-CHO, -COOH, -CN, -NO2、-COOC1-C4Alkyl, -CH ═ CH (N-methylpyridinium),
In another preferred embodiment, Ra、Rc、RdEach independently is H or C1-C4An alkyl group; rbis-CHO, -COOH, -CN, -NO2、-COOC1-C4Alkyl, -CH ═ CH (N-methylpyridinium),
In another preferred embodiment, the fluorescent probe has a structure represented by formula I or II below:
wherein X is S, O or NH;
r is-CHO, -COOH, -CN, -NO2、-COOC1-C4Alkyl, -CH ═ CH (N-methylpyridinium),
In another preferred embodiment, R is selected from the group consisting of:
in another preferred embodiment, the fluorescent probe is selected from the group consisting of:
in a second aspect of the present invention, there is provided a method for preparing the fluorescent probe of the first aspect, the method comprising the following steps:
route one: 2-amino benzenethiol and 5-methyl salicylaldehyde are used as starting materials, and react under the conditions of concentrated hydrochloric acid and hydrogen peroxide to generate an intermediate m1, m1 generates an intermediate KSLOH01 through Duff reaction, and then the intermediate m2 generates a nucleophilic substitution reaction to obtain a key intermediate m3, and m3 removes acetyl protecting groups on galactose residues under the condition of sodium methoxide to obtain a probe KSL 01;
and a second route: KSLOH01 is used as a raw material, an intermediate KSLOH02 is obtained through Wittig reaction, then nucleophilic substitution reaction is carried out on the intermediate KSLOH02 and m2 to obtain an intermediate m4, and the acetyl protecting group on galactose residues is removed from m4 under the condition of sodium methoxide to obtain a probe KSL 02;
and a third route: the probe KSL01 is used as a raw material and is subjected to Knoevenagel condensation reaction with 1, 4-dimethyl pyridine iodide to prepare a probe KSL 03;
and a fourth route: performing Knoevenagel condensation reaction on the intermediate m4 and malononitrile to obtain a key intermediate m5, and removing acetyl protecting groups on galactose residues from m5 under the condition of sodium methoxide to obtain a probe KSL 04;
and a fifth route: benzothiazole and 3-bromo-4-methoxybenzaldehyde are used as starting materials, a intermediate m6 is obtained through a Suzuki reaction, then methyl protecting groups are removed from a hydrobromic acid aqueous solution to obtain an intermediate KSLOH05, the intermediate KSLOH05 and 2,3,4, 6-tetraacetoxy-alpha-D-pyranose bromide undergo a nucleophilic substitution reaction to obtain a key intermediate m7, and acetyl protecting groups on galactose residues are removed from the m7 under the condition of sodium methoxide to obtain a probe KSL 05;
route six: carrying out Knoevenagel condensation reaction on the intermediate m7 and 2- (1-phenylethylene) malononitrile to obtain an intermediate m8, and removing acetyl protecting groups on galactose residues from m8 under the condition of sodium methoxide to obtain a probe KSL 06;
a seventh route: the probe KSL05 is used as a raw material and is subjected to Knoevenagel condensation reaction with 1, 4-dimethyl pyridine iodide to prepare a probe KSL 07;
and a route eight: taking an intermediate KSLOH05 as a raw material, obtaining an intermediate KSLOH08 through a Wittig reaction, then carrying out nucleophilic substitution reaction with 2,3,4, 6-tetraacetoxy-alpha-D-pyranose bromide to obtain a key intermediate m10, and removing acetyl protecting groups on galactose residues by m10 under the condition of sodium methoxide to obtain a probe KSL 08;
the route is nine: taking an intermediate KSLOH08 as a raw material, prolonging an aldehyde conjugated chain through a Wittig reaction to obtain an intermediate KSLOH09, carrying out nucleophilic substitution reaction with 2,3,4, 6-tetraacetoxy-alpha-D-pyranose bromide to obtain a key intermediate m11, and removing acetyl protecting groups on galactose residues from m11 under the condition of sodium methoxide to obtain a probe KSL 09;
a route ten: carrying out Knoevenagel condensation reaction on the intermediate m7 and the intermediate m12(2- (2, 6-dimethyl-4H-pyran-4-alkylidene) malononitrile) to obtain an intermediate m13, and removing acetyl protecting groups on galactose residues from m13 under the condition of sodium methoxide to obtain a probe KSL 10;
the route eleven: carrying out Knoevengel condensation reaction on the intermediate m7 and the intermediate m15(2- (3-methylcyclohex-2-en-1-alkylene) malononitrile) to obtain an intermediate m16, and removing acetyl protecting groups on galactose residues by using m16 under the condition of sodium methoxide to obtain a probe KSL 11;
or route twelve: and (3) performing Knoevenagel condensation reaction on the intermediate m7 and the intermediate m18(2- (3,5, 5-trimethylcyclohex-2-en-1-alkylene) malononitrile) propane to obtain an intermediate m19, and removing acetyl protecting groups on galactose residues by using m19 under the condition of sodium methoxide to obtain the probe KSL 12.
All the parameters of the chemical reaction conditions have a certain degree of adjustability and replaceability, that is, the reaction solvent replacement, the acid-base replacement, the reaction temperature adjustment, the reaction time adjustment and the like under the general chemical reaction guiding principle are not enough to be out of the protection scope of the patent claims.
In another preferred example, the reaction solvent used in each reaction step is selected from conventional chemical solvents such as methanol, ethanol, acetonitrile, tetrahydrofuran, dichloromethane, ethyl acetate, n-butanol, diethyl ether, toluene, etc.
In another preferred example, the reaction temperature adopted in each reaction step is-20 to 150 ℃, and the reaction time is 0.5 to 48 hours.
In a third aspect of the invention, there is provided the use of a fluorescent probe according to the first aspect for detecting β -galactosidase; or used for preparing a reagent for detecting beta-galactosidase.
In another preferred embodiment, the fluorescent probe is used for detecting beta-galactosidase activity of cells and tissues and organs in vitro. In another preferred embodiment, the fluorescent probe is used for detecting tumor cell beta-galactosidase. In another preferred embodiment, the tumor cell is an ovarian cancer cell.
In another preferred embodiment, the fluorescent probe is used for detecting beta-galactosidase of different species, wherein the different species is selected from the group consisting of bacterial genus, fungal genus and mammalian genus beta-galactosidase; or used for preparing a reagent for species-specific detection of beta-galactosidase.
In another preferred embodiment, the beta-galactosidase of different species includes e.coli beta-galactosidase (e.coli beta-gal), aspergillus oryzae beta-galactosidase (a.oryz beta-gal), mouse beta-galactosidase (mouse beta-gal), and human beta-galactosidase (human beta-gal).
In another preferred embodiment, the species-specific detection of beta-galactosidase is that human beta-galactosidase can be detected normally and Escherichia coli beta-galactosidase cannot be detected.
In another preferred embodiment, the species-specific fluorescent probe comprises KSL08-KSL 12.
In another preferred embodiment, the fluorescent probe is used for preparing a diagnostic tool molecule for detecting aging.
In another preferred embodiment, the aging includes body aging, tissue organ aging, and cell aging.
In another preferred embodiment, the fluorescent probe is used for fluorescence imaging or as a reagent for preparing fluorescence imaging.
In another preferred embodiment, the probe can be applied to the detection of the content of beta-galactosidase.
The invention has the following obvious advantages: (1) the series of fluorescent probes based on the HBT estimation structure has larger Stokes shift. (2) The series of fluorescent probes have good selectivity and detection sensitivity for beta-galactosidase. (3) The series of fluorescent probes have good biocompatibility and no obvious cytotoxicity, and can be applied to living cell detection. (4) The series of fluorescent probes have a wide luminescence spectrum from green light to near infrared light, and can meet the requirements of different imaging conditions. (5) The series of probes can be used for accurately detecting the aging cells and judging the aging degree. (6) The series of probe parts have species specificity, can specifically identify human beta-galactosidase, but are not interfered by bacterial beta-galactosidase, and can eliminate false positive caused by bacterial infection.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the UV-VIS absorption spectrum of a fluorescent probe KSL01-KSL12 after addition of A.oryzae beta-gal to the solution.
FIG. 2 shows the fluorescence spectra of the fluorescent probe KSL01-KSL12 after adding A.oryzae beta. -gal to the solution.
FIG. 3 shows the fluorescence spectra of fluorescent probes KSL01-KSL12 after E.coli beta-gal was added to the solution.
FIG. 4 shows the change of fluorescence spectrum of the fluorescent probe KSL01-KSL12 with the increase of A.oryzae beta. -gal concentration and the linear relationship between the fluorescence intensity and the enzyme concentration.
FIG. 5 is a graph showing the time-kinetic change in fluorescence intensity of the fluorescent probe KSL01-KSL12 after the addition of A.oryzae β -gal to a solution of the fluorescent probe KSL01-KSL 12.
FIG. 6 shows the results of selective experiments with fluorescent probes KSL04 and KSL 11.
FIG. 7 shows the survival rate of MRC5 cells by the fluorescent probe KSL01-KSL 12.
FIG. 8 shows the survival rate of SKOV3 cells by fluorescent probe KSL01-KSL 12.
FIG. 9 shows the survival rate of fluorescent probe KSL01-KSL12 against HepG2 cells.
FIG. 10 is a photograph showing the cytographic imaging of the fluorescent probes KSL01-KSL12 on senescent MRC5 cells and SKOV3 cells.
FIG. 11 is a graph showing the results of detecting different generations of MRC5 cell senescence with the fluorescent probe KSL 04.
FIG. 12 is a graph showing the results of fluorescent probes KSL04 and KSL11 in detecting the degree of aging of kidney tissue sections of mice of different ages.
Detailed Description
The inventor of the application extensively and deeply researches, constructs a series of beta-galactosidase fluorescent probes based on HBT framework structure, and provides a preparation method and application thereof in aging detection. On the basis of this, the present invention has been completed.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for which specific conditions are not indicated in the following examples are generally carried out according to conventional conditions (e.g.as described in Sambrook et al, molecular cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Example 1
Preparation of Probe KSL01 and hydrolysate KSLOH01
(1) Synthesis of compound m 1:
dissolving 2-aminothiophenol (1.25g, 10mmol) and 5-methylsalicylic acid (1.36g, 10mmol) in anhydrous ethanol, slowly adding concentrated HCl (2.5mL,30mmol) dropwise at 0 deg.C under stirring, stirring for 10min, and slowly adding 30% H dropwise2O2The solution (6.8mL,60mmol) was stirred at room temperature for 1h, whereupon yellow crystals precipitated. Suction filtration was carried out, and the solid was collected, washed with a small amount of anhydrous ethanol, and dried to obtain 1.31g of compound m1 in 54% yield.1H 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).13C NMR(101MHz,DMSO-d6)δ165.74,154.67,151.89,134.73,133.71,128.87,128.73,126.91,125.49,122.50,122.45,118.38,117.34,20.50.ESI-HRMS:m/z calc.for C14H12NOS[M+H]+:242.0640,found:242.0641.
(2) Synthesis of Compound KSLOH01
Compound M1(1.11g, 4.6mmol) and hexamethylenetetramine (733mg, 5.52mmol) were dissolved in 10mL of trifluoroacetic acid, and after stirring at 70 ℃ for 10 hours, the reaction mixture was cooled to room temperature, and 3M NaOH solution was added to adjust the reaction mixture to be alkaline, whereby a yellow solid precipitated. Suction filtration, collection of the solid, washing with a small amount of absolute ethanol, drying to give the crude product, column chromatography separation to give 780mg of a yellow solid KSLOH01, 63% yield.1H 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 C15H12NO2S[M+H]+:270.0589,found:270.0590.
(3) Synthesis of Compound m3
Compound m2(234mg, 0.454mmol), compound KSLOH01(122mg, 0.454mmol) were dissolved in 2mL tetrahydrofuran and K was added2CO3(75mg, 0.545mmol) and after stirring overnight at room temperature saturated NH was added4Neutralizing with Cl solution, extracting with ethyl acetate three times, combining organic phases, and adding anhydrous Na2SO4After drying, spin-drying and column chromatography, 264mg of m3 was obtained as a white solid in 84% yield.1H 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)δ189.10,170.35,170.25,170.13,169.42,161.95,157.27,157.19,152.36,136.65,136.05,135.24,131.72,130.21,130.16,130.13,128.15,126.43,125.50,123.25,121.57,117.06,99.53,79.06,71.07,70.81,68.60,66.85,61.33,20.77,20.73,20.67,20.59.EI-HRMS:m/z calc.for C36H35NO12S[M]+:705.1880,found:705.1882.
(4) Synthesis of Compound KSL01
Compound m3(130mg, 0.184mmol) was placed in a flask, and anhydrous methanol was added to dissolve it, and stirred at-20 ℃ for a while. At the same time, sodium methoxide (70mg, 1.29mmol) is dissolved in anhydrous methanol, slowly added dropwise into the flask, stirring is continued, TLC monitoring is carried out, after the substrate reaction is finished, Amberlite IR-120plus (H) is added+) The pH was adjusted to neutral. Filtration removed Amberlite IR-120plus (H)+) The filtrate was collected, spin-dried and isolated by column chromatography to give 36mg of KSL01 as a white solid 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,1H),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).13C 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.C28H27NNaO8S[M+Na]+:560.1355,found 560.1354.
Example 2
Preparation of Probe KSL02 and its hydrolyzate KSLOH02
(1) Synthesis of Compound KSLOH02
Will (formyl methylene oxide)Methyl) triphenylphosphine (304mg, 1mmol) and the compound KSLOH01(269mg, 1mmol) were placed in a flask, 10mL anhydrous tetrahydrofuran was added and refluxed overnight under nitrogen. After the reaction was completed, it was cooled, the solvent was removed by rotary drying, and column chromatography was performed to obtain 236mg of KSLOH02 as a yellow solid in 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).13C 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 C17H13NO2S[M]+:295.0667,found:295.0664.
(2) Synthesis of Compound m4
The compounds m2(142mg, 0.249mmol), KSLOH02(73mg, 0.249mmol), K2CO3(41mg, 0.299mmol) was charged into the flask, 2mL of DMF solvent was added, and the reaction was allowed to proceed overnight at room temperature. After the reaction is finished, saturated NH is used4Neutralizing with Cl solution, extracting with ethyl acetate three times, combining organic phases, and adding anhydrous Na2SO4After drying, spin drying and column chromatography gave 169mg of m4 as a white solid with a yield of 88%.
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,1H),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).13C NMR(151MHz,CDCl3)δ193.45,170.37,170.26,170.14,169.48,162.75,157.24,153.74,151.82,146.56,135.82,135.28,133.64,130.54,130.49,130.22,129.51,129.00,127.61,126.56,125.60,123.08,121.57,117.14,99.48,77.87,71.03,70.82,68.57,66.84,61.30,20.91,20.76,20.69,20.60.EI-HRMS:m/z calc.for C38H37NO12S[M]+:731.2036,found:731.2039.
(3) Synthesis of Compound KSL02
Compound m4(100mg, 0.137mmol) was placed in a flask and dissolved by adding anhydrous methanol, and stirred at-20 ℃ for a while. At the same time, sodium methoxide (52mg, 0.96mmol) was dissolved in anhydrous methanol, slowly added dropwise to the flask, stirring was continued, TLC was monitored, and after the substrate had reacted, Amberlite IR-120plus (H) was added+) The pH was adjusted to neutral. Filtration removed Amberlite IR-120plus (H)+) The filtrate was collected, spin-dried and isolated by column chromatography to yield 40mg of KSL02 as a white solid with a yield of 52%.1H 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,2H),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).13C 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 C30H29NNaO8S[M+Na]+:586.1512,found 586.1513.
Example 3
Preparation of Probe KSL03 and its hydrolyzate KSLOH03
(1) Synthesis of Compound KSL03
Compound KSL01(120mg, 0.223mmol), 1, 4-dimethylpyridinium iodide (53mg, 0.223mmol) was added to the flask, absolute ethanol was added, piperidine (20. mu.L, 0.223mmol) was added dropwise with stirring, the temperature was raised to reflux temperature, and stirring was carried out overnight. Separating out yellow solid in the reaction solution, collecting the solid by suction filtration, washing with a small amount of absolute ethyl alcohol,after drying, 122.2mg of KSL03 were obtained as a yellow solid in 73% yield.1H 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).13C NMR(101MHz,DMSO-d6)δ162.41,158.01,154.03,152.38,152.24,145.64,136.04,135.10,134.76,131.97,130.92,130.87,130.70,129.55,127.51,127.03,125.99,125.24,124.08,123.27,122.66,116.54,101.06,78.09,75.99,73.75,70.65,68.60,60.89,47.53,20.92.ESI-HRMS:m/z calc.for C35H35N2O7S[M-I]+:627.2159,found 627.2166.
(2) Synthesis of Compound KSLOH03
Compound KSLOH01(84mg, 0.312mmol), 1, 4-dimethylpyridinium iodide (73mg, 0.312mmol) was added to the flask, absolute ethanol was added, piperidine (29. mu.L, 0.312mmol) was added dropwise with stirring, the temperature was raised to reflux temperature, and stirring was carried out overnight. Yellow solid is separated out from the reaction liquid, the solid is collected by suction filtration and washed by a small amount of absolute ethyl alcohol, and 106.1mg of yellow solid KSLOH03 is obtained after drying, wherein the yield is 70%.1H 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).13C NMR(151MHz,DMSO-d6)δ168.72,154.67,153.15,151.32,145.59,135.40,133.07,132.94,131.21,129.48,127.65,126.54,124.91,124.13,123.98,122.93,122.44,117.74,47.39,40.52,40.40,40.26,40.12,39.98,39.84,39.70,39.56,20.44.ESI-HRMS:m/z calc.for C22H19N2OS[M-I]+:359.1213,found359.1219.
Example 4
Preparation of Probe KSL04 and its hydrolyzate KSL0H04
(1) Synthesis of Compound m5
Compound m4(200mg, 0.273mmol) was added to a dry two-neck flask, toluene was added to dissolve it, malononitrile (18. mu.L, 0.287mmol), ammonium acetate (3mg, 0.041mmol) were added with stirring under nitrogen, and finally acetic acid (5. mu.L, 0.082mmol) was slowly added dropwise. The temperature is increased to the reflux temperature for reaction for 12 h. After the reaction was completed, cooling, spin-drying the solvent, and column chromatography separation gave 150mg of m5 as a yellow solid with a yield of 70%.1H 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),7.02–6.98(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).13C NMR(151MHz,DMSO-d6)δ170.46,170.32,170.05,169.69,162.86,162.11,157.06,154.26,152.25,144.83,135.97,135.94,135.58,133.49,131.81,131.10,131.03,130.49,129.51,127.70,127.04,126.03,124.64,123.28,122.59,116.69,114.63,112.61,98.08,81.98,78.18,70.84,70.61,68.83,67.66,61.74,54.41,20.95,20.94,20.87,20.83,20.69.EI-HRMS:m/z calc.for C41H37N3O11S[M]+:779.2149,found 779.2152.
(2) Synthesis of Compound KSL04
Compound m5(123mg, 0.158mmol) was placed in a flask and dissolved by adding anhydrous methanol, and stirred at-20 ℃ for a while. While sodium methoxide (60mg, 1.104mmol) was dissolved in anhydrous methanol and slowly added dropwise to the flask, stirring was continued, TLMonitoring with C, adding Amberlite IR-120plus (H) after the substrate reaction is finished+) The pH was adjusted to neutral. Filtration removed Amberlite IR-120plus (H)+) The filtrate was collected, spin-dried and isolated by column chromatography to give KSL04 as a yellow solid, 30.5mg, 31% yield.1H 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(dd,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),4.51(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).13C NMR(151MHz,DMSO-d6)δ162.88,162.14,158.23,154.28,152.25,144.73,136.00,135.51,133.47,131.64,131.04,129.56,129.15,127.71,127.02,126.01,124.52,123.27,122.64,116.51,114.69,112.64,101.44,82.08,78.49,76.00,73.74,70.73,68.55,60.79,20.71.ESI-HRMS:m/z calc.for C33H29N3NaO7S[M+Na]+:634.1624,found 634.1623.
(3) Synthesis of Compound KSLOH04
The compound KSLOH02(50mg, 0.169mmo) and malononitrile (12. mu.L, 0.186mmol) were put into a flask, and anhydrous ethanol was added thereto to dissolve the compound, piperidine (17. mu.L, 0.169mmol) was added dropwise with stirring under nitrogen protection, and the reaction was carried out at room temperature for 10min, whereby a yellow solid precipitated, and the solid was collected by suction filtration and washed with a small amount of anhydrous ethanol to obtain a total of 40mg of KSLOH04 with a yield of 69%.1H 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).13C 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 C20H13N3OS[M]+:343.0779,found343.0781.
Example 5
Preparation of Probe KSL05 and its hydrolyzate KSLOH05
(1) Synthesis of Compound m6
3-bromo-4-methoxybenzaldehyde (6.820g, 30mmol), palladium acetate (674mg, 3mmol), triphenylphosphine (3.934g, 15mmol), copper acetate monohydrate (1.198g, 6mmol), K2CO3(8.292g, 60mmol), and benzothiazole (10mL, 90mmol) were added to a 250mL flask, toluene was added as a solvent, and the reaction was refluxed under nitrogen for 12 h. After the reaction is finished, the reaction product is cooled to room temperature, insoluble solids are removed by filtration, and after the product is dried by spinning, column chromatography separation is carried out to obtain 16g of the product m6 with the yield of 99%.1H NMR(400MHz,CDCl3)δ10.06(s,1H),9.05(d,J＝2.1Hz,1H),8.13(d,J＝8.2Hz,1H),8.04(dd,J＝8.6,2.1Hz,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).13C 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 C15H11NO2S[M]+:269.0510,found 269.0513.
(2) Synthesis of Compound KSLOH05
Compound m6(6.3g, 23.39mmol) was added to the flask, 40% aqueous HBr (60mL) was added, the reaction was refluxed for 2 days, monitored by TLC, after completion of the substrate reaction, cooled to room temperature, neutralized to neutrality with 2N NaOH, extracted three times with ethyl acetate, the organic phases were combined, and dried over anhydrous Na2SO4After drying, spin drying and column chromatography gave 3.11g total of KSLOH05 as a white solid in 52% yield.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).13C 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 C14H9NO2S[M]+:255.0354,found 255.0356.
(3) Synthesis of Compound m7
A dry flask was prepared and the compound KSLOH05(2g, 7.83mmol), cesium carbonate (12g, 36.83mmol) and a small amount of anhydrous Na were added2SO4Then, dichloromethane was added as a solvent, and after stirring at room temperature for a while, 2,3,4, 6-tetraacetoxy-alpha-D-pyranosylbromide (4.9g, 11.88mmol) was added, and the reaction was allowed to proceed overnight at room temperature under nitrogen. After the substrate reaction was completed, insoluble solids were removed by filtration, spin-dried, and separated by column chromatography to obtain 3.8g in total of m7 as a white solid with a yield of 82%.1H 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,7.97(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)δ192.18,170.46,170.34,170.01,169.53,160.94,157.88,151.94,135.89,133.10,131.86,131.76,127.16,126.19,123.41,122.85,122.04,116.23,97.30,71.54,71.12,68.71,67.70,61.77,21.00,20.98,20.93,20.81.ESI-HRMS:m/z calc.for C28H28NO11S[M+H]+:586.1383,found:586.1384.
(4) Synthesis of Compound KSL05
Compound m7(215mg, 0.37mmol) was placed in a flask, and anhydrous methanol was added to dissolve it, and stirred at-20 ℃ for a while. At the same time, sodium methoxide (139mg, 2.57mmol) was dissolved in anhydrous methanol, slowly added dropwise to the flask, stirring was continued, TLC was monitored, and after the substrate had reacted, Amberlite IR-120plus (H) was added+) The pH was adjusted to neutral. Filtration removed Amberlite IR-120plus (H)+) The filtrate was collected, spin-dried and isolated by column chromatography to yield 68mg of KSL05 as a white solid with a yield of 44%.1H NMR(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.for C20H20NO7S[M+H]+:418.0960,found:418.0953.
Example 6
Preparation of Probe KSL06 and its hydrolyzate KSLOH06
(1) Synthesis of Compound m8
Compound m7(600mg, 1.02mmol), 2- (1-phenylethylidene) malononitrile (344mg, 2.04mmol) were added to a flask, absolute ethanol was added as a solvent, piperidine (94 μ L, 1.18mmol) was added dropwise with stirring, the temperature was raised to reflux, the reaction was allowed to stand overnight, after the substrate had reacted completely, spin-dried, and column chromatography was performed to obtain m8 as a pale yellow solid and 294mg in total, with a yield of 39%.1H 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＝9.0Hz,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),7.01(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)δ171.12,170.31,170.16,170.08,169.25,161.07,156.01,151.88,147.75,136.11,132.99,132.07,131.26,130.07,129.72,129.16,128.85,126.40,125.39,124.49,124.05,123.07,121.39,115.70,113.33,112.84,98.78,82.51,71.72,71.21,68.49,66.77,61.40,20.74,20.72,20.56.ESI-HRMS:m/z calc.for C39H34N3O10S[M+H]+:736.1965,found:736.1960.
(2) Synthesis of Compound KSL06
Compound m8(100mg, 0.14mmol) was placed in a flask and dissolved by adding anhydrous methanol, and stirred at-20 ℃ for a while. At the same time, sodium methoxide (51mg, 0.95mmol) was dissolved in anhydrous methanol, slowly added dropwise to the flask, stirring was continued, TLC was monitored, and after the substrate had reacted, Amberlite IR-120plus (H) was added+) The pH was adjusted to neutral. Filtration removed Amberlite IR-120plus (H)+) The filtrate was collected, spin-dried and isolated by column chromatography to give 36mg of KSL06 as a yellow solid in 45% yield.1H NMR(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＝15.5Hz,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).13C 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 C31H25N3NaO6S[M+Na]+:590.1362,found:590.1365.
(3) Synthesis of Compound m9
Compound m6(1.16g, 4.31mmol), 2- (1-phenylethylidene) malononitrile (1.3g, 7.72mmol) were added to a flask, absolute ethanol was added as a solvent, piperidine (473 μ L, 5.17mmol) was added dropwise with stirring, the temperature was raised to reflux, the reaction was allowed to stand overnight, after the substrate had reacted completely, spin-dried, and column chromatography was performed to obtain m9 as a pale yellow solid in total 145mg with a yield of 8%.1H 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).13C 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.for C26H18N3OS[M+H]+:420.1171,found:420.1170.
(4) Synthesis of Compound KSLOH06
A dry two-neck flask was prepared, m9(100mg,0.24mmol) was added, anhydrous dichloromethane was added as solvent, C was slowly added at 0 ℃ under nitrogen protection2H6BBr3S(1M in CH2Cl2715 μ L, 0.72mmol), stirring for 1h, then switching to room temperature and stirring for a further 23 h. TLC monitoring, after the substrate reaction is completed, the reaction solution is poured into 50mL of ice water, then dichloromethane is used for extraction for three times, organic phases are combined, and anhydrous Na is used2SO4After drying, spin-drying and column chromatography gave 25mg total of KSLOH06 as a pale yellow solid in 26% yield.
1H 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).13C 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 C25H16N3OS[M+H]+:406.1014,found:406.1016.
Example 7
Preparation of Probe KSL07 and its hydrolyzate
(1) Synthesis of Probe KSL07
Compound KSL05(120mg, 0.29mmol), 1, 4-dimethylpyridinium iodide (68mg, 0.29mmol) was addedIn a flask, absolute ethanol was added as a solvent, piperidine (26. mu.L, 0.29mmol) was added dropwise with stirring, the mixture was heated to reflux, reacted overnight, a pale yellow solid precipitated, and the solid was collected by suction filtration and washed with a small amount of absolute ethanol to obtain KSL07 as a pale yellow solid in a total amount of 50mg with a yield of 27%.1H 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).13C 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 C27H27N2O6S[M-I]+:507.1584,found:507.1591.
(2) Synthesis of the Compound KSLOAc07
The compound KSLOH05(255mg, 1mmol), 1, 4-dimethylpyridinium iodide (235mg, 1mmol) and NaOAc (246mg,3mmol) were added to the flask, 15mL of acetic anhydride were added and the mixture was stirred at 90 ℃ for 36 h. After the substrate reaction, 10mL of diethyl ether was added to the reaction solution to precipitate a brown solid, which was collected by suction filtration and washed with a small amount of water to obtain 145mg of KSLOAc07 as a solid with a yield of 28%.1H 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).13C NMR(151MHz,DMSO-d6)δ169.45,161.64,152.66,152.54,149.56,145.72,139.30,135.40,134.20,130.88,129.91,127.39,126.51,126.42,125.74,124.99,124.23,123.48,122.82,47.52,22.04.ESI-HRMS:m/z calc.for C23H19N2O2S[M-I]+:387.1162,found:387.1166.
(3) Synthesis of Compound KSLOH07
The compound KSLOAc07(43mg, 0.08mmol) was placed in a flask, and anhydrous methanol was added thereto to dissolve it, and stirred at room temperature while NH was added4OAc (51mg,0.66mmol) was dissolved in anhydrous methanol and slowly added dropwise to the flask, stirring was continued for 3H, monitored by TLC, and after substrate reaction was complete Amberlite IR-120plus (H)+) The pH was adjusted to neutral. Filtration removed Amberlite IR-120plus (H)+) The filtrate was collected, spin-dried and isolated by column chromatography to give 39mg of KSLOH07 as a yellow solid in 99% yield.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).13C 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 C21H17N2OS[M-I-]+:345.1056,found:345.1063.
Example 8
Preparation of Probe KSL08 and its hydrolyzate KSLOH08
(1) Synthesis of Compound KSLOH08
Compound KSLOH05(1g, 3.92mmol) was dissolved in 25mL of tetrahydrofuran, and (1, 3-dioxane-2-methyl) triphenylphosphonium bromide (3.363g, 7.84mmol), sodium hydride (60% oil dispersion) (784mg, 19.6mmol) and 18-crown ether (100mg, 25mg/mmol) were added under stirring at 0 ℃ and stirring was continued for 10min, after which the temperature was raised to 50 to 60 ℃ and the reaction was allowed to proceed overnight. The next day, a small amount of water was added to the reaction solution to quench, and then 1M HCl solution was added to the reaction solution to stir. TLC monitoring, after the reaction is finished, adjusting with ammonia waterThe pH was adjusted to 7, the mixture was extracted three times with ethyl acetate, the organic phases were combined and freed from anhydrous Na2SO4After drying, spin drying and column chromatography gave a total of 782mg 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,1H),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).13C 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 C16H12NO2S[M+H]+:282.0589,found:282.0591.
(2) Synthesis of Compound m10
A dry flask was prepared and the compound KSLOH08(400mg, 1.422mmol), cesium carbonate (2.316g, 7.108mmol) and a small amount of anhydrous Na were added2SO4Then, dichloromethane was added as a solvent, and after stirring at room temperature for a while, 2,3,4, 6-tetraacetoxy-alpha-D-pyranosyl bromide (880mg, 1.939mmol) was added, and the reaction was allowed to proceed overnight at room temperature under nitrogen. After the substrate reaction was complete, insoluble solids were removed by filtration, spin-dried, and separated by column chromatography to yield m10 as a yellow solid in 81% yield.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,1H),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＝10.2,3.5Hz,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).13C 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 C30H30NO11S[M+H]+:612.1540,found:612.1539.
(3) Synthesis of Compound KSL08
Compound m10(300mg, 0.491mmol) was placed in a flask, and anhydrous methanol was added to dissolve it, stirring was carried out at 0 ℃ for a while, sodium methoxide solution (30% in MeOH, 546. mu.L, 2.946mmol) was slowly added dropwise, stirring was continued, TLC monitoring was carried out, and after completion of the substrate reaction, Amberlite IR-120plus (H)+) The pH was adjusted to neutral. Filtration removed Amberlite IR-120plus (H)+) The filtrate was collected, spin-dried and isolated by column chromatography to yield 130mg of KSL08 as a yellow solid in 60% yield.1H 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(dd,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).13C 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.for C22H22NO7S[M+H]+:444.1117,found:444.1118.
Example 9
Preparation of Probe KSL09 and its hydrolyzate KSLOH09
(1) Synthesis of Compound KSLOH09
The compound KSLOH08(2g, 7.11mmol) was dissolved in 50mL of tetrahydrofuran, and (1, 3-dioxane-2-methyl) triphenylphosphonium bromide (6.104g,14.228mmol), sodium hydride (60% oil dispersion) (1.422g, 35.55mmol) and 18-crown ether (178mg,25mg/mmol) were added under stirring at 0 ℃ and after stirring for 10min, the temperature was raised to 50 to 60 ℃ and the reaction was continued overnight. The next day, a small amount of water was added to the reaction solution to quench, and then 1M HCl solution was added to the reaction solution to stir. TLC monitoring, after the reaction is finished, the pH is adjusted by ammonia waterTo 7, the mixture was extracted three times with ethyl acetate, the organic phases were combined and freed from anhydrous Na2SO4After drying, spin drying and column chromatography gave a total of 2g of KSLOH09 as a yellow solid in 92% yield.1H 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).13C 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.for C18H14NO2S[M+H]+:308.0745,found:308.0746.
(2) Synthesis of Compound m11
A dry flask was prepared and the compound KSLOH09(500mg, 1.361mmol), cesium carbonate (2.65g, 8.133mmol) and a small amount of anhydrous Na were added2SO4Then, dichloromethane was added as a solvent, and after stirring at room temperature for a while, 2,3,4, 6-tetraacetoxy-alpha-D-pyranosyl bromide (1g, 2.424mmol) was added, and the reaction was allowed to proceed overnight at room temperature under nitrogen. After the substrate reaction was complete, insoluble solids were removed by filtration, spin-dried, and separated by column chromatography to obtain a total of 830mg of m11 as a yellow solid in 96% yield.1H 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＝10.2,3.2Hz,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).13C NMR(101MHz,CDCl3)δ193.49,170.28,170.18,170.09,169.26,161.71,154.74,151.95,151.65,140.58,136.14,132.13,132.04,131.80,131.14,130.30,129.17,128.55,128.43,126.49,126.29,125.25,123.87,123.03,121.35,115.43,99.01,71.52,71.27,68.58,66.80,61.34,20.69,20.54.ESI-HRMS:m/z calc.for C32H32NO11S[M+H]+:638.1696,found:638.1697.
(3) Synthesis of Compound KSL09
Compound m11(300mg, 0.470mmol) was placed in a flask, and anhydrous methanol was added thereto for dissolution, followed by stirring at 0 ℃ for a while. At the same time, sodium methoxide (177.7mg, 3.29mmol) was dissolved in anhydrous methanol, slowly added dropwise to the flask, stirring was continued, monitored by TLC, and after the substrate had reacted, Amberlite IR-120plus (H) was added+) The pH was adjusted to neutral. Filtration removed Amberlite IR-120plus (H)+) The filtrate was collected, spin-dried and isolated by column chromatography to give 196.2mg of KSL09 as a white solid with a yield of 89%.1H 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).13C 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 C24H24NO7S[M+H]+:470.1273,found:470.1274.
Example 10
Synthesis of Probe KSL10
(1) Synthesis of Compound m13
Adding a compound m7(600mg, 1.02mmol) and a compound m12(440mg, 2.56mmol) into a flask, adding absolute ethyl alcohol as a solvent, dropwise adding piperidine (94 mu L, 1.18mmol) while stirring, heating to reflux, reacting overnight, after the substrate is completely reacted, performing spin-drying, and performing column chromatography to obtain a light yellow solid m13 of 153mg in total and the yield of 23%.1H 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).13C NMR(151MHz,CDCl3)δ170.32,170.20,170.13,169.29,162.04,161.41,158.82,156.26,155.22,151.95,136.42,136.17,130.13,129.94,129.88,126.40,125.35,124.00,123.08,121.41,118.24,115.58,114.97,107.50,106.53,98.92,71.60,71.28,68.55,66.77,61.37,59.71,20.77,20.73,20.58,20.01.ESI-HRMS:m/z calc.for C38H34N3O11S[M+H]+:740.1914,found:740.1908.
(2) Synthesis of Compound KSL10
Compound m13(228mg, 0.35mmol) was placed in a flask, and anhydrous methanol was added to dissolve it, and stirred at-20 ℃ for a while. At the same time, sodium methoxide (132mg, 2.44mmol) was dissolved in anhydrous methanol, slowly added dropwise to the flask, stirring was continued, TLC was monitored, and after the substrate had reacted, Amberlite IR-120plus (H) was added+) The pH was adjusted to neutral. Filtration removed Amberlite IR-120plus (H)+) The filtrate was collected, spin-dried and column chromatographed to give a total of 70mg of KSL10 as a dark yellow solid with a yield of 35%.1H 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).13C 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 C30H26N3O7S[M+H]+:572.1491,found:572.1490.
(3) Synthesis of Compound m14
Compound m6(225mg, 0.84mmol), compound m12(288mg, 1.67mmol) were added to the flask, absolute ethanol was added as a solvent, piperidine (114 μ L, 1.24mmol) was added dropwise with stirring, the temperature was raised to reflux, the reaction was overnight, after the substrate reaction was complete, spin-dried, and column chromatography was performed to obtain m14 as a dark yellow solid in a total amount of 186mg, with a yield of 54%.1H 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).13C NMR(101MHz,CDCl3)δ162.02,159.18,158.60,156.36,136.91,130.96,129.58,127.90,126.49,125.25,122.65,122.32,121.38,117.28,115.13,115.09,112.34,107.06,106.42,77.24,56.13,19.99.ESI-HRMS:m/z calc.for C25H18N3O2S[M+H]+:424.1120,found:424.1121.
(4) Synthesis of Compound KSLOH10
A dry two-neck flask was prepared, m14(186mg, 0.44mmol) was added, anhydrous dichloromethane was added as solvent, BBr was slowly added at 0 ℃ under nitrogen protection3(1M in CH2Cl22.1mL, 2.1mmol), stirred for 1h, then turned to room temperature and stirred further overnight. TLC monitoring, after the substrate reaction is completed, the reaction solution is poured into 50mL of ice water, then dichloromethane is used for extraction for three times, organic phases are combined, and anhydrous Na is used2SO4After drying, spin-drying and column chromatography gave a pale yellow solid, KSLOH10, in total 38mg, in 21% yield.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).13C NMR(151MHz,DMSO-d6)δ164.60,164.28,160.63,158.34,157.29,151.85,137.54,135.23,131.93,129.37,127.28,127.02,125.67,122.69,122.54,119.69,118.23,117.58,116.03,107.04,106.26,55.88,19.86.ESI-HRMS:m/z calc.for C24H16N3O2S[M+H]+:410.0963,found:410.0964.
Example 11
Preparation of Probe KSL11 and its hydrolyzate
(1) Synthesis of Compound m16
Compound m7(500mg, 0.85mmol), compound m15(202mg,1.27mmol) were added to a flask, absolute ethanol was added as a solvent, piperidine (78 μ L,0.85mmol) was added dropwise with stirring, the temperature was raised to reflux, the reaction was overnight, after the substrate reaction was complete, spin-dried, and column chromatography was performed to obtain m16 as a dark yellow solid in 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).13C NMR(101MHz,CDCl3)δ170.35,170.23,170.13,169.69,169.32,161.68,155.38,154.79,151.80,136.07,135.46,131.13,129.89,129.67,129.10,126.38,125.32,124.84,123.73,123.01,121.38,115.52,113.42,112.74,98.92,78.07,77.36,77.25,77.05,76.73,71.57,71.29,68.56,66.81,61.42,29.48,25.10,21.23,20.77,20.75,20.74,20.58.ESI-HRMS:m/z calc.for C38H36N3O10S[M+H]+:726.2121,found:726.2123.
(2) Synthesis of Compound KSL11
The compound m16(237mg, 0.33mmol) was placed in a flask, and anhydrous methanol was added thereto for dissolutionStirring at 20 ℃ for a while. At the same time, sodium methoxide (124mg, 2.30mmol) was dissolved in anhydrous methanol, slowly added dropwise to the flask, stirring was continued, TLC was monitored, and after the substrate had reacted, Amberlite IR-120plus (H) was added+) The pH was adjusted to neutral. Filtration removed Amberlite IR-120plus (H)+) The filtrate was collected, spin dried and column chromatographed to yield a total of 81mg of KSL11 as a dark yellow solid in 44% yield.1H 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,1H),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).13C NMR(151MHz,DMSO-d6)δ171.71,162.43,158.49,156.09,151.97,137.38,136.40,131.26,130.43,129.31,129.10,126.75,125.54,124.08,122.98,122.47,122.24,116.04,114.48,113.66,101.23,76.33,75.62,74.02,70.63,68.57,60.85,29.63,24.98,21.32.ESI-HRMS:m/z calc.for C30H26N3O6S-[M-H]-:556.1548,found:556.1544.
(3) Synthesis of Compound m17
Compound m6(705mg, 2.62mmol), compound m15(620mg, 3.92mmol) were added to a flask, absolute ethanol was added as a solvent, piperidine (360 μ L, 3.93mmol) was added dropwise with stirring, the temperature was raised to reflux, the reaction was overnight, after the substrate reaction was complete, spin-dried, and column chromatography was performed to obtain m17 of 95mg in total as a dark yellow solid with a yield of 9%.1H 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).13C NMR(101MHz,CDCl3)δ169.78,158.20,155.79,136.12,130.47,129.46,129.06,128.18,126.36,125.10,124.36,122.73,121.33,112.31,56.08,29.71,25.15,21.27,14.25.ESI-HRMS:m/z calc.for C25H20N3OS[M+H]+:410.1327,found:410.1325.
(4) Synthesis of Compound KSLOH11
A dry two-neck flask was prepared, m17(95mg, 0.23mmol) was added, anhydrous dichloromethane was added as solvent, BBr was slowly added at 0 ℃ under nitrogen protection3(1M in CH2Cl21.15mL, 1.15mmol), stirred for 1h, then turned to room temperature and stirred further overnight. TLC monitoring, after the substrate reaction is completed, the reaction solution is poured into 50mL of ice water, then dichloromethane is used for extraction for three times, organic phases are combined, and anhydrous Na is used2SO4After drying, spin-drying and column chromatography gave a pale yellow solid, KSLOH11 mg in 85% yield.1H NMR(600MHz,DMSO-d6)δ12.02(s,1H),8.49(d,J＝2.1Hz,1H),8.17(d,J＝7.8Hz,1H),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,1H),6.88(s,1H),2.77(t,J＝6.4Hz,2H),2.69(t,J＝5.9Hz,2H),1.91–1.84(m,2H).13C NMR(151MHz,DMSO-d6)δ171.69,164.56,158.73,157.97,151.86,137.85,135.12,131.63,129.47,128.47,128.06,127.00,125.65,123.63,122.70,122.53,119.47,118.17,114.55,113.74,75.17,29.62,24.99,21.34.ESI-HRMS:m/z calc.for C24H18N3OS[M+H]+:396.1171,found:396.1172.
Example 12
Preparation of Probe KSL12 and its hydrolyzate KSLOH12
(1) Synthesis of Compound m19
Compound m7(530mg,0.91mmol), compound m18(185mg,1mmol) was added to the flask, and anhydrous ethanol was added asSolvent, piperidine (72 μ L,0.78mmol) was added dropwise with stirring, the temperature was raised to reflux, the reaction was carried out overnight, after the substrate had reacted completely, spin-drying and column chromatography separation gave 232mg of m19 in total 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).13C 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 C40H40N3O10S[M+H]+:754.2434,found:754.2435.
(2) Synthesis of Compound KSL12
Compound m19(220mg,0.29mmol) was placed in a flask and dissolved by adding anhydrous methanol, and stirred at-20 ℃ for a while. At the same time, sodium methoxide (110mg, 2.04mmol) is dissolved in absolute methanol, slowly added dropwise into the flask, stirring is continued, TLC monitoring is carried out, after the substrate reaction is finished, Amberlite IR-120plus (H) is added+) The pH was adjusted to neutral. Filtration removed Amberlite IR-120plus (H)+) The filtrate was collected, spin-dried and column chromatographed to give a total of 38mg of KSL12 as a red solid, 22% yield.1H 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).13C NMR(151MHz,DMSO-d6)δ170.92,162.43,156.67,156.11,151.98,137.42,136.40,131.25,130.42,129.34,129.24,126.75,125.55,122.98,122.48,122.25,116.06,114.44,113.62,101.27,76.47,76.33,74.03,70.64,68.55,60.84,42.82,38.69,32.16,27.96,27.94.ESI-HRMS:m/z calc.for C32H31N3NaO6S[M+Na]+:608.1831,found:608.1833.
(3) Synthesis of Compound m20
Compound m6(610mg, 2.26mmol), compound m18(540mg, 3.42mmol) were added to a flask, absolute ethanol was added as a solvent, piperidine (210 μ L, 2.29mmol) was added dropwise with stirring, the temperature was raised to reflux, the reaction was overnight, after completion of the substrate reaction, spin-drying, column chromatography separation gave a total of 750mg of m20 as a red solid with a yield of 80%.1H 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).13C NMR(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 C27H24N3OS[M+H]+:438.1640,found:438.1641.
(4) Synthesis of Compound KSLOH12
A dry two-neck flask was prepared, m20(300mg, 0.69mmol) was added, anhydrous dichloromethane was added as solvent, BBr was slowly added at 0 ℃ under nitrogen protection3(1M in CH2Cl23.43mL, 3.43mmol), stirred for 1h, then turned to room temperature and stirred further overnight. TLC monitoring, after the substrate reaction is completed, the reaction solution is poured into 50mL of ice water, then dichloromethane is used for extraction for three times, organic phases are combined, and anhydrous Na is used2SO4Drying, spin drying, and separating by column chromatography to obtain KSLOH12 total 121mg as a red solid, 29% yield.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).13C 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 C26H22N3OS[M+H]+:424.1484,found:424.1485.
Example 13
Detecting the change of the ultraviolet-visible absorption spectrum and the fluorescence spectrum of the fluorescent probe before and after adding aspergillus oryzae beta-galactosidase (A. oryzae beta-gal) and escherichia coli beta-galactosidase (E. coli beta-gal).
The fluorescent probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1mmol/L stock solution. From the stock solution, 40 μ L was added to a 5mL centrifuge tube, diluted to 3mL with a PBS buffer solution (10mmol/L, a. oryzae β -gal assay using PBS buffer at pH 4.5, e. coli β -gal assay using PBS buffer at pH 7.4), and 1mL of a β -galactosidase standard solution with a concentration of 40U/mL was added, and incubated at 37 ℃ for 10min to prepare an experimental group. Blank group take 40u L above probe stock solution to 5mL centrifuge tube, directly use PBS dilution to 4mL, do not add beta-galactosidase, 37 degrees C were incubated for 10 min. And measuring the ultraviolet absorption and fluorescence spectrum properties of the experimental group sample and the blank group sample.
As shown in FIG. 1, the fluorescent probe KSL01-KSL12 solution underwent a significant change in the UV-visible absorption spectrum after the addition of A.oryzae β -gal, with a trend toward the UV-visible absorption spectrum of the fluorescent probe hydrolysate KSLOH01-KSLOH12, indicating that A.oryzae β -gal cleaves the glycosidic bond of the probe substrate to release the fluorophore.
As shown in FIG. 2, the fluorescent probe KSL01-KSL12 solution underwent a significant change in fluorescence spectrum after the addition of A.oryzae beta-gal, and underwent a significant red shift, with the trend of the change tending towards the fluorescence spectrum of the fluorescent probe hydrolysate KSLOH01-KSLOH12, indicating that A.oryzae beta-gal cleaves the glycosidic bond of the probe substrate to release the fluorophore. The maximum emission wavelengths after co-incubation of the probe KSL01-KSL12 solution with A.oryzae beta. -gal are shown in Table 1.
TABLE 1 maximum emission wavelength after co-incubation of probes KSL01-KSL12 solution with A.oryzae beta. -gal
As shown in FIG. 3, after the fluorescent probe KSL01-KSL12 solution is added with E.coli beta-gal, the fluorescent spectrum of the probe KSL01-KSL06 changes significantly, and the change trend of the fluorescent spectrum changes towards the fluorescent spectrum of the fluorescent probe hydrolysate KSLOH01-KSLOH06, which shows that the E.coli beta-gal can cut off the glycosidic bond in the probe KSL01-KSL06, and release the fluorophore. The fluorescence spectrum of the probe KSL07 solution also changed after the E.coli beta-gal was added, and the fluorescence occurred at the maximum emission wavelength of the probe itself and the fluorophore (KSLOH07), indicating that the probe KSL07 was not completely hydrolyzed by the E.coli beta-gal. The fluorescence spectrum of the probe KSL08-KSL12 solution is not changed basically after E.coli beta-gal is added, and is completely different from the fluorescence spectrum of the fluorescent probe hydrolysate KSLOH08-KSLOH12, which indicates that the E.coli beta-gal can not cut off the glycosidic bond in the probe KSL08-KSL 12. The results of the experiments in FIG. 2 show that the probes KSL08-KSL12 have species specificity and can recognize A.oryzae beta-gal of eukaryotic origin, but not E.coli beta-gal of bacterial origin.
Example 14
Change of fluorescence spectrum of fluorescent probe along with increase of A.oryzae beta-gal concentration and linear relation.
The fluorescent probe KSL01-KSL12 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1mmol/L stock solution. mu.L of the stock solution was added to a 5mL centrifuge tube, diluted with PBS buffer (10mmol/L, pH 4.5), and added with various concentrations of A.oryzae β -gal standard solutions (final concentrations varied from 0 to 1U/mL) in a total sample volume of 4 mL. The samples were incubated at 37 ℃ for the same incubation time at different enzyme concentrations with the same probe, and then their fluorescence spectra were measured.
As shown in fig. 4, the fluorescent probes KSL01-KSL12 solution underwent significant changes in fluorescence spectra after co-incubation with different concentrations of a. oryzae β -gal standard solution. The fluorescence at the maximum emission wavelength of the fluorophore released by the probe is gradually increased along with the increase of the enzyme concentration, wherein the emission wavelengths of the probes KSL04, KSL06, KSL07, KSL10, KSL11 and KSL12 have a gradual red shift phenomenon, and in addition, the fluorescence intensity at the maximum emission wavelength of the probe and the enzyme concentration show a good linear relation in a certain A.oryzae beta-gal concentration range, and the fluorescence intensity of the probe KSL11 at 662nm and 570nm have a good ratio linear relation.
The lowest detectable range of probes KSL01-KSL12 for a. oryzae β -gal concentration can be calculated from the formula limit of detection (LOD) of 3 σ/k, where σ is the standard deviation of the 10 blanks and k is the slope of the linear equation.
The detection limit for 12 probe molecules was calculated according to the formula, as shown in table 2.
TABLE 2 detection limits of probes KSL01-KSL12 on A.oryzae beta. -gal
Example 15
Fluorescence spectra as a function of time after addition of a.oryzae β -gal to the fluorescent probe.
The fluorescence intensity of the fluorescent probes KSL01-KSL12 (10. mu.M) and A.oryzae. beta. -gal (10U/mL) in PBS buffer at pH 4.5 was examined as a function of time at a test temperature of 37 ℃. The time kinetic profile of the probe molecule is shown in fig. 5, when a. oryzae β -gal is added, the fluorescence spectrum undergoes significant fluorescence enhancement and/or red shift of the maximum emission wavelength and reaches a maximum within 40s-20 min.
Example 16
Fluorescence probe selectivity assay for β -gal.
The selectivity of fluorescent probe to A.oryzae beta-gal, E.coli beta-gal, other biological enzyme and small molecule is examined to verify that the probe molecule isWhether it will be interfered by other biological and chemical molecules. Representative probe molecules KSL04 (10. mu.M) and KSL11 (10. mu.M), and biological enzymes A.oryzae.beta. -gal, E.coli.beta. -gal, Esterase, Pepsin, Trypsin, Cellulase, etc. (all 10U/mL), and LZM, DTT, GSH, L-Cys, Hcy, H2O2The results of 20min incubation with small molecules (10. mu.M) are shown in FIG. 6, which shows that the probe KSL04 selectively responds to A.oryzae. beta. -gal and E.coli. beta. -gal, and fluorescence is significantly enhanced at the maximum emission wavelength without interference from other analytes. Probe KSL11 selectively responds to a. oryzae β -gal with significant enhancement of fluorescence at the maximum emission wavelength without interference from e.coli β -gal and other analytes.
Example 17
Cytotoxicity assay of fluorescent probes.
The CCK-8 method is adopted to detect the cytotoxicity of the fluorescent probe to 3 cell lines, and the biocompatibility of the probe molecule is inspected. The fluorescent probes KSL01-KSL12 were incubated with MRC5 cells at 37 ℃ for 48h at concentrations of 5-100. mu.M, as shown in FIG. 7, in which probes KSL01, KSL02, KSL04 exhibited significant cell death at high concentrations of 50. mu.M and 100. mu.M, and the remaining probe molecules exhibited no significant cytotoxicity. However, no cell death occurred at the concentration tested for the fluorescent probe (10. mu.M). The fluorescent probes KSL01-KSL12 were incubated with SKOV3 cells at 37 ℃ for 48h at a concentration of 5-100. mu.M, as shown in FIG. 8, and all probe molecules showed no cell death at the experimental concentration (10. mu.M), wherein the probes KSL01, KSL02, KSL04 and KSL12 showed significant cell death at high concentrations of 50. mu.M and 100. mu.M, and no significant cytotoxicity was observed for the remaining probe molecules. The fluorescent probes KSL01-KSL12 were incubated with HepG2 cells at 37 ℃ for 48h at a concentration of 5-100. mu.M, as shown in FIG. 9, all probe molecules showed no cell death at the experimental concentration (10. mu.M), wherein the probes KSL01, KSL02 and KSL04 showed significant cell death at a high concentration of 100. mu.M, and the rest probe molecules showed no significant cytotoxicity. Comprehensive analysis shows that the beta-gal fluorescent probe molecule based on HBT framework has low cytotoxicity and good biocompatibility.
Example 18
The application of the fluorescent probe in living cell fluorescence imaging.
The fluorescent probes KSL01-KSL12 were incubated with senescent MRC5 cells (human embryonic lung fibroblasts) and SKOV3 cells (human ovarian cancer cells), respectively, at a concentration of 10. mu.M for 30min at 37 ℃ and the imaging effect was observed by a fluorescence confocal microscope, and the results are shown in FIG. 10. The excitation and emission wavelength ranges for the fluorescent probes KSL01-KSL12 are shown in Table 3. MRC5 and SKOV3 are both highly beta-galactosidase expressing cells, where MRC5 is a normal cell of human origin, SA-beta-gal is overexpressed as the cell ages, and SKOV3 is a tumor cell highly expressing beta-gal. As can be seen from FIG. 10, the probes KSL01-KSL12 showed significant fluorescence generation after incubation with both cells, and emitted fluorescence from green to near-infrared light.
TABLE 3 photographic conditions of fluorescent probes KSL01-KSL12 confocal microscope
Example 19
The fluorescent probe is used for detecting senescent cells and the senescence degree of the cells.
The fluorescent probe is used to detect the change of the fluorescence intensity of normal cells along with aging. A representative fluorescent probe KSL04 was incubated with MRC5 cells in continuous culture, MRC5 cells were continuously cultured from 22 passages to 29 passages, representing the natural process of normal cells from young to senescent. KSL04 was incubated with different generations of MRC5 cells at a concentration of 10. mu.M for 20min at 37 ℃ and the imaging effect was observed with a fluorescence confocal microscope. As shown in FIG. 11 a, the cells gradually senesced and the fluorescence intensity gradually increased as the number of generations of cell culture increased. The change trend of the fluorescence intensity gradually increasing with the generation number of the cell culture can be visually seen from the result of the quantification of the fluorescence intensity of the cells in b of FIG. 11. The invention shows that the expression level of beta-gal gradually rises along with the cell aging, and the fluorescent probe molecule provided by the invention can accurately, qualitatively and quantitatively detect the cell aging. The anti-aging drug rapamycin (Rapa,10nM, 25nM) is used for incubation with 29 generation (P29) MRC5 cells 3 days ahead of time, the result is shown in figure 11, compared with a P29 blank group without drug administration, the fluorescence intensity of a P29+ Rapa group is obviously reduced, the anti-aging drug effect of rapamycin is proved by a fluorescence quantification method, and the fluorescent probe molecule provided by the invention can be used as a tool molecule for detecting the anti-aging drug effect and is established as an anti-aging drug effect evaluation method based on fluorescence visualization.
Example 20
The application of the fluorescent probe in detecting the aging of tissues and organs.
Preparing frozen sections from the tissues and organs of mice of different ages, incubating the frozen sections with a fluorescent probe, detecting the fluorescence intensity, and inspecting the application of the probe molecules in detecting the aging of the tissues and organs. C57BL/6J mice, 1 month old, 13 months old, and 23 months old, were subjected to kidney harvest to prepare cryosections, which were then incubated with representative probe molecules KSL04 (10. mu.M) and KSL11 (10. mu.M) at 37 ℃ for 1h, and the imaging effect was observed using a confocal fluorescence microscope. As shown in fig. 12 a, after co-incubation with probe molecules KSL04 and KSL11, the fluorescence intensity of the kidney sections gradually increased with age, i.e. fluorescence intensity 23 months >13 months >1 month old. This can be visually concluded from the histogram of slice fluorescence intensity quantification depicted in b of FIG. 12. The result proves that the fluorescent probe provided by the invention can detect the aging of tissues and organs at the level as the content of beta-gal in kidney tissues is gradually increased with the age.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
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