CN114478473A - Synthesis and application of leucine aminopeptidase chemiluminescence detection reagent - Google Patents
Synthesis and application of leucine aminopeptidase chemiluminescence detection reagent Download PDFInfo
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Abstract
The invention belongs to the technical field of small molecule chemiluminescence probes, and particularly relates to a chemiluminescence probe for detecting Leucine Aminopeptidase (LAP), and a synthesis method and application thereof. The invention comprises an amino acid substrate L-Leu which can be specifically hydrolyzed by LAP, a 1, 2-dioxetane chemiluminescence group and a self-cleavable connecting group. The invention also discloses a synthetic method and application of the chemiluminescent probe. The chemiluminescent probe can be used for detecting LAP in vivo and in vitro, including the detection and imaging of LAP in cells, animal living bodies and tissues and liver cancer tissue samples. The chemiluminescence probe obtained by the invention has the characteristics of high sensitivity and signal-to-noise ratio, and is an important technical breakthrough. The invention belongs to the technical field of small molecule chemiluminescence probes, and particularly relates to a chemiluminescence probe for detecting Leucine Aminopeptidase (LAP), and a synthesis method and application thereof.
Description
Technical Field
The invention belongs to the field of biological probes, and relates to a chemiluminescent probe and a synthetic method and application thereof.
Background
In 1987, the group Schaap reports chemically and enzymatically activated 1, 2-dioxygen hybridized butane chemiluminescent probes, which do not need an excitation light source and have the advantages of high-efficiency luminescence, high sensitivity, high signal-to-noise ratio, high chemiluminescent quantum yield and the like. In 2017, for the transformation of the system, particularly, electron-withdrawing groups such as methyl acrylate and the like are introduced into the ortho-position of a phenol oxygen group, the chemiluminescence efficiency of the system can be obviously enhanced in aqueous solution and organisms, so that the system is widely applied to the fields of chemistry and biology and has important application prospect.
Leucine aminopeptidase (LAP; EC 3.4.11.1) is an important proteolytic enzyme of the M1 and M17 peptidase families, and specifically catalyzes the hydrolysis of the N-terminal leucine residue of proteins or peptides. And LAP is associated with various physiological and pathological processes such as tumor cell proliferation, invasion and angiogenesis. Over-expressed LAP can be detected in malignant cells (e.g., HEPG2 hepatoma cells). Thus, LAP can be tumor-chased as a cancer-associated biomarker. The in-situ dynamic identification and monitoring of intracellular LAP activity in a living system can provide important clues for the diagnosis of tumors and the research of pathophysiology.
To date, while a variety of assays have been developed to monitor LAP activity, efficient tracking of LAP activity in vitro and in vivo remains a significant challenge. Commercial detection methods have certain drawbacks and are not suitable for real-time tracking of LAP in live samples. The fluorescent probe is a very effective detection method, has the advantages of super sensitivity, high space-time resolution, simple and convenient operation, in-situ response and the like, and is very helpful for specifically monitoring the activity of LAP. Although the existing fluorescent probe can perform imaging detection on LAP activity at the living body level, the existing fluorescent probe has the defects of low tissue penetrability, easy photobleaching, easy generation of fluorescent background interference under external light excitation and the like.
In view of the advantages of chemiluminescence patterns in optical analysis and the great development of 1, 2-dioxetane chemiluminescence systems, the development of a 1, 2-dioxetane chemiluminescence probe for detecting LAP is an important technical breakthrough, and the application range of the probe is further expanded.
Disclosure of Invention
In order to overcome the defects of a fluorescent probe, the invention provides the chemiluminescent probe which has good sensitivity and high accuracy and is suitable for detecting Leucine Aminopeptidase (LAP) in vitro and in vivo, and the preparation method and the application thereof. The chemiluminescent probe for detecting LAP provided by the invention is a 1, 2-dioxetane chemiluminescent probe, and has the following structure:
the invention also provides a preparation method of the chemiluminescent probe, which comprises the following specific steps:
(1) mixing the compounds 6 and K2CO3And dissolving KI in anhydrous N, N-Dimethylformamide (DMF), stirring at room temperature under the protection of nitrogen, adding a compound 7 after 10min, after complete reaction, adding ethyl acetate and saturated saline water into a reaction system for extraction, collecting an organic phase, drying by anhydrous sodium sulfate, concentrating under reduced pressure to remove a solvent, and purifying the obtained solid by column chromatography to obtain a compound 8;
(2) the crude compound 8 was dissolved in dry dichloromethane (CH)2Cl2) In (1), ZnBr is added2Carrying out Boc removal reaction, after the reaction is finished, adding ethyl acetate and saturated saline solution into a reaction system for extraction, collecting an organic phase, drying the organic phase by using anhydrous sodium sulfate, carrying out reduced pressure concentration to remove a solvent, and purifying the obtained solid by column chromatography to obtain a compound 9;
(3) dissolving the compound 9 in Dichloromethane (DCM), adding methylene blue, introducing oxygen into a reaction system in a bubbling mode under an ice bath condition, irradiating the reaction system by using a white light LED lamp, after the reaction is completed, adding ethyl acetate and water into the reaction system for extraction, collecting an organic phase, drying the organic phase by using anhydrous sodium sulfate, decompressing, carrying out rotary evaporation and concentration, and purifying the obtained solid by column chromatography to obtain the compound of the probe 1.
The chemiluminescent probe of the present invention can be used for the measurement of LAP at different concentrations in vitro, with the preferred concentration of the probe being 10 μ Μ.
The chemiluminescent probe of the present invention can be used for detecting LAP in living cells and distinguishing tumor cells from normal cells. The preferred concentration of the probe is 10 μm, and the cells include human liver cancer cell HepG2 and human normal liver cell L02.
The chemiluminescent probe of the present invention may be used in the detection of LAP activity in tissue. The preferred concentration of the probe is 10 μm, and the tissue includes various organ tissues derived from different species of organism.
The chemiluminescent probe of the present invention may be used for the purpose of LAP imaging in vivo in animals. The animal is normal
Nude mice, tumor-bearing nude mice.
The chemiluminescence probe can be used for detecting and imaging LAP in a human liver cancer sample. Excellent quality of probe
Selecting the liver cancer sample with the concentration of 10 μ Μ, wherein the liver cancer sample comprises liver tissues of different liver cancer patients.
The invention obtains the chemiluminescence probe by improving the adamantane-dioxetane, realizes the detection of LAP, is an important technical breakthrough, further expands the application range of the LAP, and has important application value.
Brief description of the drawings
FIG. 1. chemical structure of LAP activated chemiluminescent probe 1 and its possible chemical transformation process after its response to LAP.
FIG. 2 Synthesis of chemiluminescent probe 1.
FIG. 3 (A) UV-VISIBLE absorbance spectrum (dotted line) and fluorescence spectrum (solid line) of Probe 1(10 μm) after incubation for 6 hours (blue) with LAP (red) and LAP (100U/L) in enzyme assay buffer (PBS containing 10% DMSO, pH 7.4), and UV-VISIBLE absorbance spectrum (black dotted line) and fluorescence spectrum (black solid line) of Compound 5. (B) HPLC analysis of Probe 1(10 μm) before addition of LAP (black) and after incubation at 37 ℃ for 1h after addition of LAP (100U/L) (red). (C) Chemiluminescence spectra and chemiluminescence photographs (inset) of probe 1(10 μm) before addition of LAP (lower curve) and after incubation for 10min at 37 ℃ with addition of LAP (100U/L) (upper curve). (D) Probe 1(10 μm) chemiluminescence kinetics curves were incubated for 100min at 37 ℃ before addition of LAP (lower curve) and after addition of LAP (100U/L) (upper curve).
FIG. 4 (A) chemiluminescence images of Probe 1(10 μ M) incubated with different LAP concentrations (0,10,20,40, and 80U/L) for 10min at 37 ℃. (B) The average chemiluminescence intensity versus LAP concentration for each group in panel a is a linear fit curve. Values are expressed as mean ± standard deviation (n ═ 3). (C) Chemiluminescence spectra of probe 1(10 μ M) after incubation with different classes of interfering substances in digestion buffer for 10min at 37 ℃. (D) Quantification of the 550nm mean chemiluminescence intensity in panel C. Values are expressed as mean ± standard deviation (n ═ 3). (E) Chemiluminescence spectra of probe 1 (10. mu.M) and LAP (100U/L) incubated at 25 ℃ and 37 ℃ for 10 minutes, respectively, in digestion buffer. (F) Quantification of the 550nm mean chemiluminescence intensity in panel E. Values are expressed as mean ± standard deviation (n ═ 3).
FIG. 5 (A) cell survival of HepG2 cells after 24 h incubation with varying concentrations of probe 1(0,5,10, 20. mu.M). (B) Cell survival of LO2 cells after 24 h incubation with different concentrations of probe 1(0,5,10,20 μ M). (C) Indicated viable cells (about 4X 10 per well)4One) real-time measurement of chemiluminescence intensity after incubation with probe 1(10 μ M).
FIG. 6 confocal fluorescence imaging of 1 hour incubation with Probe 1(10 μ M) with LO2 and HepG2 cells, respectively, or HepG2 cells pre-treated with Ube (40 μ M) for 1 hour. Fluorescence was obtained at 500-650nm with excitation at 405 nm.
FIG. 7 fluorescent image of 20min staining after 1h incubation with HepG2 cells with Probe 1 (10. mu.M) or fluorescent product 5 (10. mu.M), respectively, followed by Lyso Tracker Green (10. mu.g/mL).
FIG. 8.(A) chemiluminescence images of HepG2 and LO2 cells with different densities, respectively, after incubation with 10. mu.M probe for 30min at 37 ℃. (B) Quantification of the intensity of the chemiluminescence was averaged in panel a. Values are expressed as mean ± standard deviation (n ═ 3). (C) HepG2 tumor mice were imaged by real-time chemiluminescence of the whole body at 10 minutes in mice intratumorally injected with PBS (100. mu.L), intratumorally injected with Probe 1 (100. mu.M, 100. mu.L), or previously injected with Ube (200. mu.M, 100. mu.L) followed by Probe 1 (100. mu.M, 100. mu.L). (D) LAP levels in different tissues of BALB/c nude mice were determined using probe 1 (red) and Leu-AMC (black). Values are expressed as mean ± standard deviation (n ═ 3).
FIG. 9 chemiluminescence detection and imaging of LAP in human tissue samples. (A) Chemiluminescence images of LAP in normal and liver cancer tissues. Probe 1 was incubated with 10% homogenate supernatant (diluted 1:10, 1:20, 1:40, and 1:80 fold with saline) at 37 ℃ for 10 minutes. (B) LAP activity in human tissue samples was measured using Probe 1 and Leu-AMC. The 10% homogenate supernatant (diluted 1:10 with saline) was incubated with probe 1(10 min incubation) or Leu-AMC (30 min incubation) at 37 ℃. Chemiluminescence and fluorescence intensity were obtained with a microplate reader. Values are expressed as mean ± SD (n ═ 3, p < 0.005).
Detailed Description
The present invention is illustrated in detail by the following examples.
Reagent and apparatus
All reagents and solvents were analytical grade available from commercial sources, used as is or purified by standard techniques. Thin Layer Chromatography (TLC) was used to monitor the reaction. Nuclear magnetic spectrum (1HNMR and13c NMR) from a 400MHz (Hertz) spectrometer (Bruker Co., Ltd., Swiss) in CDCl3Chemical shift δ in ppm, singlet, doublet, triplet, quartet, dd (doublets) peak, multiplet expressed as s, d, t, q, dd, m, respectively; coupling constants (J) are in Hz; the number of hydrogens was determined by integration of the map and marked nH. High Performance Liquid Chromatography (HPLC) analysis was analyzed by an instrument (Orbitrap fusion Tribridge, Thermo scienfic); UV-visible absorption spectra were measured with an instrument (U-3010, Hitachi, Japan); fluorescence spectra were measured using an instrument (Hitachi FluoroMax-4/PLUS); the cell fluorescence pictures were collected by laser confocal fluorescence microscope (LSM 880); chemiluminescence spectra were collected using an instrument (Spectramax M5 (MDC)); chemiluminescence images used Xenogen
The Spectrum imaging System (Caliper Life Science, USA). Leucine aminopeptidase, cathepsin B and Ubenimex were purchased from Sigma-Aldrich, Boc-L-leucine and other reagents from Macklin.
General operation method for detecting LAP by probe
The probes were dissolved in DMSO to prepare 20mM stock solutions and stored at-20 ℃ until use. When used, the buffer solution (containing 10% DMSO) is digested with LAP&10mM PBS, pH 7.4) to the desired concentration. Typically, probes (20. mu.M) are dissolved in 100. mu.L of LAP cleavage buffer, 100. mu.L of LAP cleavage buffer containing different LAP concentrations are added, the probe final working concentration is 10. mu.M, and incubated at 37 ℃ for the indicated time. The reaction of the probe with LAP was characterized by high performance liquid chromatography, ultraviolet-visible absorption spectroscopy, fluorescence spectroscopy, and chemiluminescence spectroscopy, respectively. Wherein the collection range of the ultraviolet-visible absorption spectrum is 200-1000 nm; the excitation wavelength of the fluorescence spectrum is 400nm, and the emission wavelength range is 450nm-700 nm. The chemiluminescence spectrum is collected by an enzyme-labeling instrument, and the collection range is 450-700 nm. Using xenogens
The Spectrum imaging system detects chemiluminescent images of cells and mice.
Design, Synthesis and characterization of probes (see FIG. 1)
Fig. 1 shows the structural design of the probe. The probe mainly comprises an amino acid substrate L-Leu which can be specifically hydrolyzed by LAP, a 1, 2-dioxetane chemiluminescence group (3) with strong emission and a self-cleavable connecting group p-aminobenzyl alcohol. The enzyme digestion substrate L-Leu is widely used for a recognition group of the LAP fluorescent probe, and the 1, 2-dioxetane chemiluminescence group (3) can emit yellow green fluorescence under physiological conditions, has strong chemiluminescence efficiency, and can be used for living body imaging. In addition, the self-breaking connecting group on the aminobenzyl alcohol helps to reduce the influence of the steric hindrance of the probe and helps the L-Leu part to extend into the deep and narrow active site of LAP enzyme. When the L-Leu substrate is hydrolyzed by LAP, the probe is converted to intermediate 2, which in turn triggers a 1, 6-elimination reaction of the aminobenzyl alcohol linker to generate the caged dioxetane phenol chemiluminescent group (3). Under physiological conditions with p Η ═ 7.4, chemiluminescent group (3) will deprotonate allowing cleavage of the peroxygen bond to generate fluorescent product 5, with a process of chemoexcitation accompanied by light emission with a maximum emission wavelength of 550 nm. Thus, LAP enzyme can selectively activate and enhance the chemiluminescence of probe 1, allowing the detection of LAP activity in cells and in vivo.
Example 1 Synthesis of adamantane-dioxetane-based chemiluminescent Probe 1 for detection of LAP (see FIG. 2 for synthetic route).
The synthesis of the compound 8 comprises the following specific steps:
compound 6(50mg,0.13mmol), KI (216mg,1.3mmol) and K2CO3(35mg,0.26mmol) was dissolved in DMF (1mL) and stirred for 10 min. Under nitrogen protection, compound 7(52mg,0.13mmol) was added to the above reaction solution. The reaction mixture was stirred at room temperature for 6 hours and the progress of the reaction was monitored by TLC (PE: EA ═ 5: 1). After completion of the reaction, the reaction mixture was extracted with ethyl acetate (50mL) and saturated brine (20mL), the organic layer was collected and concentrated under reduced pressure, and the mixture was purified by silica gel column chromatography (PE: EA ═ 6:1) to obtain a solid product 8(76mg, yield: 83%).1H NMR(400MHz,CDCl3)δ9.01(s,1H),7.93(s,1H),7.54(d,J＝8.1Hz,2H),7.42(d,J＝8.0Hz,1H),7.37(s,2H),7.06(d,J＝8.0Hz,1H),6.45(d,J＝16.2Hz,1H),5.39(s,1H),4.93(s,2H),4.35(d,J＝30.3Hz,1H),3.79(s,3H),3.29(d,J＝13.3Hz,4H),2.06(s,1H),1.93(d,J＝15.2Hz,5H),1.86–1.61(m,10H),1.45(s,9H),0.97(t,J＝6.8Hz,6H).13C NMR(101MHz,CDCl3)δ171.39,167.00,156.35,153.64,139.34,138.80,138.30,138.02,132.16,131.48,129.49,127.61,125.03,119.82,119.60,80.33,75.70,57.09,51.69,40.98,39.11,38.95,38.52,36.98,32.84,29.61,28.27,24.73,22.94,21.79.
The synthesis of the compound 9 comprises the following specific steps:
compound 8(110mg 0.156mmol) was dissolved in dichloromethane (10mL) and stirred for 10min, then ZnBr was added2(146mg, 0.65mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 6 hours and the progress of the reaction was monitored by TLC (PE: EA ═ 5: 1). After completion of the reaction, ethyl acetate (50mL) and saturated brine (20mL) were added for extraction, and the organic layer was collected, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM: MeOH ═ 50:1) to obtain product 9(85mg, yield: 90%) as a solid.1H NMR(400MHz,CDCl3)δ9.60(s,1H),7.90(d,J＝16.2Hz,1H),7.61(d,J＝8.3Hz,2H),7.41(dd,J＝8.1,4.1Hz,3H),7.06(d,J＝8.0Hz,1H),6.42(d,J＝16.2Hz,1H),4.97(d,J＝4.5Hz,2H),3.79(s,3H),3.52(s,1H),3.31(s,3H),3.27(s,1H),2.06(s,1H),1.93(d,J＝14.6Hz,5H),1.87–1.76(m,8H),1.73(s,2H),1.03–0.88(m,6H).13C NMR(101MHz,CDCl3)δ173.71,153.56,138.82,132.19,131.24,129.73,129.65,127.64,125.00,119.75,119.13,75.77,57.12,53.87,51.70,43.79,39.10,38.95,38.53,36.98,32.85,29.62,28.13,24.90,23.34,21.29.
(III) the synthesis of the chemiluminescent probe 1 comprises the following specific steps:
compound 9(91mg, 0.15mmol) and a catalytic amount of methylene blue (2mg) were dissolved in dichloromethane (20mL) and stirred in an ice bath for 10 min. After the reaction was completed by introducing oxygen gas into the reaction system by bubbling and irradiating with a white LED (150W) lamp for 0.5 hour, ethyl acetate and water were added to the reaction system to conduct extraction, the organic phase was collected and dried over anhydrous sodium sulfate, the solvent was removed by concentration under reduced pressure, and the obtained solid was purified by column chromatography (DCM: MeOH ═ 50:1) to obtain probe 1(67mg, yield: 70%). MS (ESI +), m/z calculated for C35H44O7N2Cl:639.2831；found:639.2832.1H NMR(400MHz,CDCl3)δ9.61(s,1H),7.96–7.81(m,2H),7.63(d,J＝8.2Hz,2H),7.56(d,J＝8.3Hz,1H),7.40(d,J＝8.2Hz,2H),6.47(d,J＝16.2Hz,1H),4.91(s,2H),3.81(s,3H),3.52(d,J＝10.4Hz,1H),3.22(s,3H),3.02(s,1H),2.01(s,1H),1.76(d,J＝24.0Hz,9H),1.63(d,J＝15.6Hz,3H),1.48(d,J＝10.4Hz,2H),1.33(s,2H),1.03–0.93(m,6H).13C NMR(101MHz,CDCl3)δ173.72,154.06,138.27,131.59,129.82,128.79,125.19,120.97,119.16,96.27,75.97,53.88,51.79,49.63,43.81,36.51,33.81,33.51,32.53,32.15,31.44,26.10,25.74,24.93,23.36,21.28.
Example 2 chemiluminescence response assay of probes for LAP (see FIG. 3)
The response of the probe (10. mu.M) to LAP was first investigated make internal disorder or usurp in LAP cleavage buffer (10% DMSO in PBS, pH 7.4). As shown in FIG. 3A, the probe itself has weak UV absorption and fluorescence emission, and after 6h incubation with LAP, a strong UV absorption band appears at 400nm, accompanied by a significant increase in fluorescence at 550 nm. Meanwhile, HPLC analysis of a reaction solution of the probe (10 μ M) and LAP (100U/L) showed that the probe (retention time TR ═ 5.13min) was mostly converted into compound 5 (retention time TR ═ 4.30min, fig. 3B) after the reaction with LAP. Next, the change in the reaction of the probe before and after addition of LAP was investigated. As shown in FIG. 3C, the chemiluminescence of the probe is in a quenched state, and after LAP (100U/L) is added for reaction for 10min, the chemiluminescence intensity is significantly enhanced, and the maximum emission wavelength is 550 nm. The chemiluminescence intensity at the maximum emission wavelength is significantly enhanced, so that the reaction solution exhibits a strong yellow-green light emission in the absence of excitation light (fig. 3C inset). Next, a time-dependent change in chemiluminescence between the probe (10. mu.M) and LAP (100U/L) before and after the reaction was examined make internal disorder or usurp. FIG. 3D shows the kinetics of a typical reaction of probe 1 with LAP, with the chemiluminescence intensity of the probe reaching a maximum around 20min and then beginning to decay gradually. Whereas in the absence of LAP, little chemiluminescence was produced by the probe throughout the measurement. The above results indicate that LAP can efficiently activate the probe, resulting in a significant increase in chemiluminescence intensity.
Example 3 detection sensitivity of probes for LAP (see FIG. 4 A.B)
Adding a probe 1(20 mu M,100 mu L) solution into a 96-well plate, then adding 100 mu L PBS enzyme digestion buffer solution containing different LAP concentrations, wherein the final working concentration of the probe is 10 mu M, the final concentration of the LAP is 0,10,20,40 and 80U/L respectively, and each concentration group has three multiple wells. Incubating the 96-well plate at 37 ℃ for 10min, and passing through Xenogen
The Spectrum imaging system performs chemiluminescence imaging. The chemiluminescence imaging adopts a full-acceptance optical filter mode for acquisition, and the acquisition time is 0.75 second. The chemiluminescence intensity was measured by ROI measurement with IVIS Lumia XR III system software and fitted linearly to LAP concentration. After fitting, the slope k of each fitting straight line is obtained, and the detection limit LOD is 3 δ/k. Wherein δ isThe standard deviation of the intensity values of the 11 blank probe well solutions was measured.
Example 4 specificity of probes for LAP detection (see FIG. 4 C.D)
Add probe 1 (20. mu.M, 100. mu.L) solution to a 96-well plate, then add 100. mu.L PBS digestion buffer solution containing different kinds of enzymes, with a final working concentration of probe of 10. mu.M, incubate with various potential interfering substances: (1) control ( only probe 1, 10. mu.M); (2) hcy (100. mu.M); (3) L-Cys (100. mu.M); (4) glu (100. mu.M); (5) KCl (100. mu.M); (6) na (Na)2S(100μM)；(7)Al2(SO4)3(100μM)；(8)CuSO4(100μM)；(9)ZnCl2(100μM)；(10)FeSO4(100μM)；(11)MgSO4(100μM)；(12)CaCl2(100. mu.M); (13) beta-galactositase (100U/L); (14) cathepsin B (100U/L); (15) alkaline phosphatase (100U/L); (16) pyroglutamyl aminopeptidase I (100U/L); (17) LAP (100U/L); and (18) LAP (100U/L) and its inhibitor Ube (40. mu.M), each group having three duplicate wells. After incubating the 96-well plate at 37 ℃ for 10 minutes, the chemiluminescence intensity of 360-700nm was immediately collected by a microplate reader.
After incubating the probes with various potential interfering substances for 10 minutes at 37 ℃ respectively, the chemiluminescence changes of the comparative probes were observed. As shown in fig. 4C and D, only the addition of LAP can activate the probe and cause a significant increase in the chemiluminescence intensity, while in the presence of the LAP inhibitor Ube (40 μ M), the activation of the probe by LAP is significantly inhibited. The addition of other enzymes did not cause significant increase in the chemiluminescence intensity of the probe, indicating that the probe has strong specificity for LAP.
EXAMPLE 5 determination of the hydrolysis Effect of LAP at different temperatures (see FIG. 4 E.F)
Temperature is an important factor affecting the hydrolytic capacity of the enzyme. Thus, the enzymatic hydrolysis of LAP (100U/L) at different temperatures on probe 1 (10. mu.M) was investigated. Probe 1 (10. mu.M) was incubated with LAP (100U/L) for 10 minutes in white 96-well plates at 25 ℃ and 37 ℃ respectively. The chemiluminescence spectra in each well were recorded in chemiluminescence mode on a microplate reader with an integration time of 1 s. As shown in FIGS. 4E and F, probe 1 has greater enzymatic activity and greater catalytic hydrolysis capacity at higher temperatures over the temperature range of the experiment.
Example 6 cytotoxicity assay (see FIG. 5 A.B)
HepG2 and LO2 cells (-5000 cells/well) were seeded in 96-well plates, respectively. After incubation 24, the medium was removed and 100 μ L of DMEM complete medium containing varying concentrations of probe 1(0,5,10,20,40 μ M) was added again. After an additional 24 hours of incubation, 50 μ L of MTT (1 in PBS) reagent was added to each well and incubation was continued for 4 hours at 37 ℃. Subsequently, the culture containing the MTT reagent was removed, 150. mu.L of DMSO was added to each well to dissolve the formed crystalline formazan solid, and the absorbance (OD) at 490nm was detected by a microplate reader. Absorbance (OD) of blank wells without any treatmentcontrol) The percentage of viability activity of the cells per well can be determined from OD/OD, set as control wellscontrolX 100% was calculated and each experiment was repeated three times. As shown in fig. 5a.b, after adding probe 1 at different concentrations (0,5,10,20,40 μ M) and incubating for 24 hours, probe 1 showed no strong biotoxicity for both cell lines, indicating that the probe did not affect the viability of the cells and could be used to detect LAP activity in living cells.
Example 7 chemiluminescent detection of LAP Activity in Living cells (see FIG. 5C)
To verify the ability of probe 1 to detect LAP activity in cells, HepG2 cells (. about.4X 10)4One/well) were inoculated in 96-well plates, each setAnd (4) three multiple holes. After 24 hours incubation, the medium was removed and 200. mu.L of LAP cleavage buffer containing 10. mu.M probe was added to each well. To inhibit LAP activity in cells, HepG2 cells were incubated with their inhibitor Ube (40 μ M) for 1 hour, after which the Ube-containing medium was removed and 200 μ L of LAP cleavage buffer containing 10 μ M probe was added to each well. Immediately after the probe is added, the chemiluminescence intensity of each hole at 37 ℃ is collected in real time by using a microplate reader, the time interval is 1min, and the duration is about 1 h.
Since probe 1 has very low background signal and high chemiluminescence activation times after reaction with LAP, the probe can detect LAP activity on cells in real time without washing after addition. As shown in fig. 5C, LAP-over-expressed HepG2 cells all showed a gradual increase in chemiluminescence intensity over time after incubation with probe 1(10 μ M), with the chemiluminescence intensity reaching a maximum at about 30 minutes and then continuing to emit for more than 60 minutes. In contrast, the chemiluminescence intensity of these cells was greatly reduced after the addition of the inhibitor Ube (40 μ M) to inhibit the LAP activity in the cells beforehand, indicating that the probe can be used for the determination of LAP in living cells.
Example 8 fluorescence imaging of LAP Activity in Living cells (see FIG. 6)
HepG2 or LO2 cells (5X 10)4One) were seeded on glass-bottom cell culture dishes and kept for overnight growth. To inhibit LAP activity, HepG2 cells were pretreated with Ube (40 μ M) for 1 hour. Probe 1 (10. mu.M) in FBS-free DMEM medium was then added to the petri dish and incubated at 37 ℃ for 1 hour. After washing 3 times with cold PBS (1X), 500-650nm cellular fluorescence images were obtained at 405nm excitation using confocal laser scanning microscope LSM 880. As shown in FIG. 6, the fluorescence intensity of HepG2 cells was significantly enhanced compared to LO2 and Ube-pretreated HepG2 cells. Therefore, the change in fluorescence intensity was caused by intracellular LAP, indicating that expression of LAP was higher in HepG2 cells than in LO2 cells.
Example 9 subcellular localization (see FIG. 7)
HepG2 cells were first incubated with probe 1 or product 5 (10. mu.M) for 1 hour, washed 3 times with cold PBS, then incubated with the lysosomal dye Lyso Tracker Green (10. mu.g/mL) for 20 minutes, washed 3 times with PBS (1X, pH 7.4), and fluorescence images of the cells were taken on an LSM880 confocal laser scanning microscope. Fluorescence of probe 1 or product 5 was obtained in the range of 500nm to 550nm with an excitation wavelength of 405 nm. The fluorescence of the lysosome dye LysoTracker Green was collected in the range of 550nm to 600nm with an excitation wavelength of 488 nm. As shown in fig. 7, in HepG2 cells, the fluorescence signal of probe 1 activation overlapped with the lysosomal dye signal. This phenomenon is similar to the fluorescence profile of fluorescent product 5 incubated directly with HepG2 cells. This is probably because the LAP-activated fluorescent product 5 has a naked hydroxyl group, and weakly acidic interactions with lysosomes, resulting in a large accumulation of the hydrolysate 5 in the lysosomes.
Example 10 differentiation of probes between tumor cells and Normal cells (see FIG. 8 A.B)
For in vitro detection of tumor cell numbers, different numbers of HepG2 or LO2 cells (0, 2.5X 10, respectively) were first introduced3,5×103,1×104,2×104,4×104And Ube (40. mu.M) pretreatment 4X 104) Seeded in 96-well plates and incubated overnight to allow adherent growth of the cells. The medium was then removed from each well and 200. mu.L of LAP digestion buffer containing 10. mu.M probe was added to each well. After incubating the cell solution at 37 ℃ for 30min, collecting chemiluminescence images of the cell solution by an IVIS LuminaXR III system for 1 min. Chemiluminescence intensity by Xenogen
The ROI measurements were taken by the Spectrum imaging system software and plotted against cell number. As shown in FIG. 8A.B, the chemiluminescence intensity in each well gradually increased with the increase in cell number, and the chemiluminescence image of HepG-2 hepatoma cells was much brighter than that of LO2 normal cells, indicating that the probe has high detection sensitivity for HepG-2 cells and can be used to distinguish tumor cells from normal cells.
Example 11 chemiluminescence assay for LAP Activity at the level of live animals (see FIG. 8 C.D)
Immunodeficient 6-8 week-old female BALB/C nude mice were injected subcutaneously into the forelimb at 1X 107HepG2 liver cancer cell, establishment of xenograft HepG2 tumor. When the average tumor volume reaches about 150mm3When the mice were randomly divided into two groups (n-3). Probe 1 (100. mu.M, 100. mu.LPBS) was injected in situ. To inhibit LAP activity in tumors, the LAP inhibitor Ube (200 μ M,100 μ L) was injected directly into the tumor 1 hour before the in situ injection of the probe. Xenogen was used 10 minutes after in situ probe injection
The Spectrum imaging system (full-open filter mode) collects the chemiluminescence images of the whole body of the mouse, and the collection time is 60 seconds. As shown in fig. 8C, after 10 minutes of tail in situ injection of the probe, a strong chemiluminescence signal appeared at the tumor site of the mouse, whereas the previous intratumoral injection of LAP inhibitor significantly suppressed the chemiluminescence intensity within the tumor. The results show that the probe 1 can be activated by the LAP endogenously expressed by the tumor after being injected into a mouse body in situ, and is used for real-time and non-invasive chemiluminescence imaging detection of LAP positive tumors in animal living bodies.
EXAMPLE 12 Probe 1 measurement of LAP in tissue (see FIG. 8D)
After the tumor-bearing nude mice die, urine, blood, heart, liver, spleen, lung, kidney, brain and tumor tissues of the nude mice are respectively taken to prepare 10% physiological saline tissue homogenate, after centrifugation for 10 minutes at 10000 rpm, 10 mu L of supernatant is taken, diluted to 100 mu L by physiological saline, 100 mu L of chemiluminescent probe with the concentration of 20 mu M is added, and Xenogen is added
The Spectrum imaging system takes pictures and carries out quantitative analysis by the self-contained software of the instrument. The commercially available fluorescent probe Leu-AMC fluoresces at 450nm after cleavage by LAP. As shown in FIG. 8D, the results are consistent with the trend of Leu-AMC measurements, and higher chemiluminescence enhancement was obtained in serum, liver and tumor tissues compared to other tissues, indicating higher LAP levels. The results show that probe 1 can be used to detect LAP activity in tissues,has excellent chemiluminescence property, accuracy and sensitivity for LAP analysis.
EXAMPLE 13 chemiluminescent detection of LAP Activity in human tissue samples (see FIG. 9)
Human tissue samples were provided by the national hospital of Guangdong province. Preparing 10% physiological saline tissue homogenate from normal tissue and liver cancer tissue, centrifuging at 10000 rpm for 20min, collecting supernatant 10 μ L, diluting with physiological saline 10,20,40, 80 times to 100 μ L, adding 100 μ L chemiluminescence probe 1 with concentration of 20 μ M, incubating the solution at 37 deg.C for 10min, and immediately using Xenogen
The Spectrum imaging system was photographed and quantitatively analyzed by the instrument's own software. As shown in FIG. 9A, a very strong chemiluminescent signal was observed after incubation of probe 1 with the supernatant of 10% liver cancer homogenate, whereas a weak chemiluminescent signal was detected in normal tissue.
Respectively taking 10 μ L of tissue homogenate supernatant, diluting with normal saline 10 times to 100 μ L, adding 100 μ L of chemiluminescent probe 1 with concentration of 20 μ M, incubating the above solution at 37 deg.C for 10min, and immediately using Xenogen
The Spectrum imaging system was photographed and quantitatively analyzed by the instrument's own software. The probe 1 is used for detecting the LAP activity in a human tissue sample (normal and liver cancer tissues), and the result is consistent with the measurement trend of Leu-AMC, which shows that the probe 1 is a reliable detection method, can be used for distinguishing the normal tissue from the liver cancer tissue and detecting the LAP activity (figure 9B), and can be used for detecting and imaging the LAP in the human liver cancer sample.
Claims (10)
1. A chemiluminescent probe for detecting leucine aminopeptidase is characterized by being a 1, 2-dioxetane chemiluminescent probe and having the following structure:
2. the chemiluminescent probe of claim 1 wherein the adamantane-1, 2-dioxetane chemiluminescent group is substituted with a methyl acrylate group.
3. The chemiluminescent probe of claim 1 wherein the adamantane-1, 2-dioxetane chemiluminescent is 3- (2' -spiroadamantane) -4-methoxy-4- (2 "-chloro-3" -hydroxy-4 "-methyl acrylate) -phenyl-1, 2-dioxetane.
4. The chemiluminescent probe of claim 1 wherein the self-cleavable linking group is para-aminobenzyl alcohol.
5. The method for synthesizing a chemiluminescent probe of claim 1 is achieved by the following scheme Sl,
the compound 6 and the compound 7 directly react to obtain a compound 8, and then the compound 8 passes through ZnBr2Removing the Boc protection to obtain 9, and oxidizing the compound 9 by singlet oxygen to obtain a probe compound 1.
6. The method for synthesizing a chemiluminescent probe of claim 1 comprising the steps of:
(1) mixing the compounds 6 and K2CO3And dissolving KI in anhydrous N, N-dimethylformamide, stirring at room temperature under the protection of nitrogen, adding compound 7 after 10min, adding ethyl acetate and saturated saline solution into the reaction system for extraction after complete reaction, collecting organic phase, drying with anhydrous sodium sulfate, concentrating under reduced pressure to remove solvent, and purifying the obtained solid by column chromatography to obtain the final productCompound 8;
(2) dissolving the crude compound 8 in dry dichloromethane, adding ZnBr2Carrying out Boc removal reaction, after the reaction is finished, adding ethyl acetate and saturated saline solution into a reaction system for extraction, collecting an organic phase, drying the organic phase by using anhydrous sodium sulfate, carrying out reduced pressure concentration to remove a solvent, and purifying the obtained solid by column chromatography to obtain a compound 9;
(3) dissolving the compound 9 in dichloromethane, adding methylene blue, introducing oxygen into a reaction system in a bubbling mode under an ice bath condition, irradiating by using a white light LED lamp, after the reaction is completed, adding ethyl acetate and water into the reaction system for extraction, collecting an organic phase, drying by using anhydrous sodium sulfate, concentrating under reduced pressure to remove a solvent, and purifying the obtained solid by column chromatography to obtain the compound of the probe 1.
7. Use of the chemiluminescent probe of claim 1 for detecting LAP in living cells, differentiating between tumor cells and normal cells.
8. Use of the chemiluminescent probe of claim 1 for detecting LAP activity in a tissue.
9. Use of the chemiluminescent probe of claim 1 for in vivo LAP imaging in an animal.
10. Use of the chemiluminescent probe of claim 1 for LAP detection and imaging in human liver cancer samples.
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