A Fluorescent Probe for Detecting Carboxylesterase by Two-Photon, Preparation Method and Application

Abstract

The present invention discloses a fluorescence probe for two - photon detection CE, its preparation, screening and application, belonging to the technical field of synthesis and detection. The structural formula of the fluorescence probe is as follows: #imgabs0# wherein, R1 is H, R2 is H; or, R1 is H, R2 is F; or, R1 is F, R2 is H. The fluorescence probe is synthesized in one step by reacting 7 - amino - 4 - methylcoumarin with differently substituted chloroacetyl chloride and triethylamine to obtain the fluorescence probe P. By comparing the fluorescence response multiples, response times and stabilities of the three fluorescence probes, the fluorescence probe P2 with R1 being H and R2 being F is selected for subsequent testing and application. The fluorescence probe P2 can react with CE to break the amide bond, and the ICT effect disappears, resulting in enhanced fluorescence. Therefore, the fluorescence probe P2 can be used to detect CE, and the probe has a simple structure, and has the advantages of high selectivity, high sensitivity and rapid response, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthesis and detection, and particularly relates to a two-photon detection fluorescent probe for carboxylesterase, a preparation method and an application thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to increase the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Carboxylesterase (CE) is an important member of the serine hydrolase superfamily, which can catalyze the cleavage of a large number of structurally diverse ester- or amide-containing substrates into corresponding alcohols and carboxylic acids. Therefore, it plays a key role in biological metabolism and detoxification. CE can metabolize many ester drugs (such as oseltamivir, clopidogrel, irinotecan and capecitabine) and environmental toxins (such as pyrethroids). In addition, CE can also metabolize endogenous esters, including cholesterol esters, triglycerides and other endogenous lipids, thus playing an important physiological function in lipid homeostasis. Therefore, CE dysfunction is closely related to atherosclerosis, cholesterol-induced liver injury, type 2 diabetes and non-alcoholic fatty liver disease.

[0004] In view of the important role of CE in biological metabolism, establishing a fluorescent probe that can rapidly, highly sensitively and highly selectively visualize CE at the in vivo level is of great significance for predicting drug pharmacokinetics, rationally designing prodrugs activated by CE, and further studying the pathogenesis of lipid metabolism diseases. Currently, the existing fluorescent probes for detecting CE have disadvantages such as poor selectivity, long response time and inability to perform in vivo monitoring. Summary of the Invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a two-photon detection fluorescent probe for carboxylesterase, a preparation method and an application thereof. The fluorescent probe P provided by the present invention can react with CE and present a change in fluorescence signal. The probe has a simple structure and has advantages such as high selectivity, high sensitivity and fast response time.

[0006] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, a two-photon detection fluorescent probe P for carboxylesterase is provided. The structural formula of the fluorescent probe is as follows:

[0008]

[0009] Wherein, R 1 is H, R 2 is H, and the obtained fluorescent probe is named P1;

[0010] Or, R1 is H, R 2 is F, and the resulting fluorescent probe is named P2;

[0011] or, R 1 is F, R 2 is F, and the resulting fluorescent probe is named P3.

[0012] In some embodiments of the present invention, R 1 is H, R 2 is F. The present invention uses 7-amino-4-methylcoumarin as the fluorescent parent and different fluorine-substituted chloroacetyl groups as the recognition sites of CE to design and synthesize three fluorescent probes. After detection, the fluorescent probe P2 with R 1 being H and R 2 being F has a high fluorescence response multiple, rapid response, and good stability of the probe itself. The fluorescent probe P2 can react with CE and show changes in fluorescence signals, and has the advantages of high selectivity, high sensitivity, and fast response time.

[0013] The second aspect of the present invention provides a preparation method of the above-mentioned fluorescent probe for two-photon detection of carboxylesterase, and the preparation route is as follows:

[0014]

[0015] It includes the following steps:

[0016] Dissolve 7-amino-4-methylcoumarin and fluorine- and / or chlorine-substituted acetyl chloride in an organic solvent, add triethylamine, and react at 0 °C for 3.5 - 4.5 h. After the reaction is completed, the fluorescent probe for two-photon detection of carboxylesterase is obtained.

[0017] When the fluorine- and / or chlorine-substituted acetyl chloride is chloroacetyl chloride, the fluorescent probe P1 is obtained.

[0018] When the fluorine- and / or chlorine-substituted acetyl chloride is chloro(fluoro)acetyl chloride, the fluorescent probe P2 is obtained. The structural formula of the chloro(fluoro)acetyl chloride is as follows:

[0019]

[0020] When the fluorine- and / or chlorine-substituted acetyl chloride is chlorodifluoroacetyl chloride, the fluorescent probe P3 is obtained. The structural formula of the chlorodifluoroacetyl chloride is as follows:

[0021]

[0022] In some embodiments of the present invention, the molar ratio of 7-amino-4-methylcoumarin, fluorine- and / or chlorine-substituted acetyl chloride, and triethylamine is 1:1.8 - 2.2:7.

[0023] Preferably, the molar ratio of 7-amino-4-methylcoumarin, fluoro- and / or chloro-substituted acetyl chloride, and triethylamine is 1:1.9-2.1:7.

[0024] In some embodiments of the present invention, the organic solvent includes tetrahydrofuran, and in the organic solvent, the concentration of 7-amino-4-methylcoumarin is 0.08-0.12 mmol / mL.

[0025] Preferably, the concentration of 7-amino-4-methylcoumarin is 0.09-0.11 mmol / mL.

[0026] In some embodiments of the present invention, after the reaction is completed, the reaction mixture is rotary evaporated and then separated and purified to obtain a fluorescent probe for two-photon detection of carboxylesterase.

[0027] Preferably, the product is separated and purified by silica gel column chromatography, and the separation and purification reagents are petroleum ether and ethyl acetate; more preferably, the volume ratio of petroleum ether to ethyl acetate is 8-12:1.

[0028] In the third aspect of the present invention, there is provided the use of the above-mentioned fluorescent probe for two-photon detection of carboxylesterase in the detection of carboxylesterase.

[0029] In the fourth aspect of the present invention, there is provided a method for detecting carboxylesterase, which uses the above-mentioned fluorescent probe for two-photon detection of carboxylesterase, and includes the following steps:

[0030] Adding the fluorescent probe to the sample to be tested, incubating at 35-40 °C for 25-35 min, and performing cell imaging under a two-photon confocal microscope; the excitation wavelength is 720 nm, and the blue channel wavelength collection range is 400 to 490 nm;

[0031] The method is not for the purpose of diagnosing diseases.

[0032] In some embodiments of the present invention, the incubation is carried out at 37 °C for 30 min.

[0033] In some embodiments of the present invention, the concentration of the fluorescent probe in the sample to be tested is 8-12 μM.

[0034] The beneficial effects of the present invention are as follows:

[0035] In this invention, 7-amino-4-methylcoumarin is used as the fluorescent parent, and chloroacetyl groups substituted with different fluorines are used as the recognition sites for CE. Three fluorescent probes are designed and synthesized. Finally, the fluorescent probe P2 with a high fluorescence response multiple, rapid response, and good stability of the probe itself is selected for subsequent applications. The fluorescent probe P2 can react with CE to break the amide bond, and the ICT effect disappears, resulting in enhanced fluorescence. The structure of the fluorescent probe P2 is simple, the fluorescence at 445 nm is enhanced by 41 times, and the fluorescence intensity at 445 nm reaches the reaction plateau in about 30 min. It can detect CE in a physiological environment, can react with CE and show changes in fluorescence signals. When applied to detect CE, it has the advantages of high selectivity, high sensitivity, and fast response time. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0037] Figure 1 It is the absorption spectrum diagram when the three small-molecule fluorescent probes P of the invention react with CE. The abscissa is the wavelength (nm), and the ordinate is the absorbance.

[0038] Figure 2 It is the fluorescence response spectrum diagram when the three small-molecule probes P of the invention react with CE. The abscissa is the wavelength (nm), and the ordinate is the fluorescence intensity.

[0039] Figure 3 It is the kinetic experiment of the response of the three small-molecule fluorescent probes P of the invention to CE. The abscissa is time, and the ordinate is the fluorescence intensity.

[0040] Figure 4 It is the fluorescence response spectrum diagram of the small-molecule fluorescent probe P2 of the invention to different concentrations of CE. The excitation wavelength of the probe is 350 nm. The abscissa is the wavelength (nm), and the ordinate is the fluorescence intensity.

[0041] Figure 5 It is the response of the small-molecule fluorescent probe P2 of the invention to CE in different pH environments. The excitation wavelength of the probe is 350 nm. The abscissa is the pH value, and the ordinate is the fluorescence intensity at the wavelength of 445 nm.

[0042] Figure 6 It is the selectivity experiment of the small-molecule fluorescent probe P2 of the invention to CE. The excitation wavelength of the probe is 350 nm. The abscissa is the probe blank, the interferent, and the analyte to be measured, and the ordinate is the fluorescence intensity at the wavelength of 445 nm.

[0043] Figure 7It is the selectivity experiment of the small molecule fluorescent probe P2 of the present invention for CE. The excitation wavelength of the probe is 350 nm. The abscissa represents the probe blank, interferents and analytes to be measured, and the ordinate represents the fluorescence intensity at a wavelength of 445 nm.

[0044] Figure 8 It is the selectivity experiment of the small molecule fluorescent probe P2 of the present invention for CE. The excitation wavelength of the probe is 350 nm. The abscissa represents the probe blank, interferents and analytes to be measured, and the ordinate represents the fluorescence intensity at a wavelength of 445 nm.

[0045] Figure 9 It is the mass spectrometry characterization of the reaction mechanism between the small molecule fluorescent probe P2 of the present invention and CE;

[0046] Figure 10 It is the high performance liquid chromatography characterization of the reaction mechanism between the small molecule fluorescent probe P2 of the present invention and CE. The abscissa represents the retention time;

[0047] Figure 11 It is the schematic diagram of the reaction mechanism between the small molecule fluorescent probe P2 of the present invention and CE;

[0048] Figure 12 It is the two-photon confocal imaging diagram of the small molecule fluorescent probe P2 of the present invention for detecting exogenous CE in human normal hepatocytes;

[0049] Figure 13 It is the two-photon confocal imaging diagram of the small molecule fluorescent probe P2 of the present invention for detecting endogenous CE in human normal hepatocytes and human hepatoma cells. Detailed implementation manners

[0050] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0051] The chloroacetyl chloride, chloro(fluoro)acetyl chloride, and chlorodifluoroacetyl chloride used in the following examples can be commercially available products or synthesized by oneself.

[0052] For example: Chloro(fluoro)acetyl chloride can be prepared by the method disclosed in the patent US04122115 Preparation of chloro-and bromo-fluoroacetyl chloride. Chloro(fluoro)acetyl chloride is generated by reacting ethyl chloro(fluoro)acetate with chlorosulfonic acid at 120 °C to 200 °C. The synthesis route is as follows:

[0053]

[0054] Or, chloro(fluoro)acetyl chloride is generated by reacting ethyl chloro(fluoro)acetate, chlorosulfonic acid and phthaloyl chloride at 120 °C to 200 °C. The synthesis route is as follows:

[0055]

[0056] For example, chlorodifluoroacetyl chloride can be prepared by the method described in Zard S S, Z.A Practical Source of Chlorodifluoromethyl Radicals. Convergent Routes to gem-Difluoroalkenes and -dienes and (2,2-Difluoroethyl)-indoles, -azaindoles, and -naphthols[J]. Organic letters, 2014, 16(11). Treating chlorodifluoroacetic acid with thionyl chloride produces chlorodifluoroacetyl chloride, and the synthetic route is as follows:

[0057]

[0058] Example 1

[0059] 1) Synthesis of fluorescent probe P1:

[0060] Dissolve 7-amino-4-methylcoumarin (0.5 mmol, 88 mg) and chloroacetyl chloride (1.0 mmol, 112 mg) in 5 mL of tetrahydrofuran, then add triethylamine (3.5 mmol, 285 μL) to the reaction solution, and react at 0 °C for 4 h. After the reaction is completed, a white solution is obtained. After rotary evaporation of the reaction mixture, the product is separated and purified by silica gel column chromatography. The eluent is petroleum ether:ethyl acetate = 10:1, and white solid P1, 52.9 mg, with a conversion rate of 42.3% is obtained.

[0061] 2) Synthesis of fluorescent probe P2:

[0062] Dissolve 7-amino-4-methylcoumarin (0.5 mmol, 88 mg) and chloro(fluoro)acetyl chloride (1.0 mmol, 131 mg) in 5 mL of tetrahydrofuran, then add triethylamine (3.5 mmol, 285 μL) to the reaction solution, and react at 0 °C for 4 h. After the reaction is completed, a white solution is obtained. After rotary evaporation of the reaction mixture, the product is separated and purified by silica gel column chromatography. The eluent is petroleum ether:ethyl acetate = 10:1, and white solid P2, 42.0 mg, with a conversion rate of 31.3% is obtained.

[0063] 3) Synthesis of fluorescent probe P3:

[0064] Dissolve 7-amino-4-methylcoumarin (0.5 mmol, 88 mg) and chlorodifluoroacetyl chloride (1.0 mmol, 113 mg) in 5 mL of tetrahydrofuran. Then add triethylamine (3.5 mmol, 285 μL) to the reaction solution and react at 0 °C for 4 h. After the reaction is completed, a white solution is obtained. After rotary evaporation of the reaction mixture, the product is separated and purified by silica gel column chromatography. The eluent is petroleum ether:ethyl acetate = 10:1, and a white solid P3, 60.0 mg, is obtained with a conversion rate of 42.0%.

[0065] Mass spectrometry and NMR characterization of the fluorescent probe P1 obtained in Example 1:

[0066] HRMS(ESI): calculated for C 12 H 11 ClNO 3 + (M + H) + 252.0419, found 252.0463.

[0067] 1 H NMR(400 MHz, DMSO-d 6 ): δ10.71(s, 1H), 7.78 - 7.71(m, 2H), 7.49(dd, J = 8.7, 2.1 Hz, 1H), 6.29(d, J = 1.3 Hz, 1H), 4.32(s, 2H), 2.40(d, J = 1.2 Hz, 3H).

[0068] 13 C NMR(101 MHz, DMSO-d 6 ): δ165.33, 159.89, 153.58, 153.00, 141.69, 126.07, 115.48, 115.30, 112.58, 105.88, 43.55, 17.94.

[0069] Mass spectrometry and NMR characterization of the fluorescent probe P2 obtained in Example 1:

[0070] HRMS(ESI): calculated for C 12 H 9 ClFNNaO 3 + (M + Na) + 292.0147, found 292.0188.

[0071] 1 H NMR(400 MHz, DMSO-d 6): δ 11.05 (s, 1H), 7.82 - 7.73 (m, 2H), 7.61 (dd, J = 8.7, 2.1 Hz, 1H), 6.93 (d, J = 48.9 Hz, 1H), 6.33 (d, J = 1.3 Hz, 1H), 2.42 (d, J = 1.3 Hz, 3H).

[0072] 13 C NMR (101 MHz, DMSO - d 6 ): δ 162.60, 162.35, 159.78, 153.42, 152.93, 140.61, 126.17, 116.18, 115.92, 113.05, 106.74, 94.69, 92.19, 17.95.

[0073] Mass spectrometry and NMR characterization of the fluorescent probe P3 obtained in Example 1:

[0074] HRMS (ESI): calculated for C 12 H 9 ClF 2 NO 3 + (M + H) + 288.0233, found 288.0286.

[0075] 1 H NMR (400 MHz, DMSO - d 6 ): δ 11.49 (s, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.75 (d, J = 2.1 Hz, 1H), 7.69 (dd, J = 8.7, 2.1 Hz, 1H), 6.36 (q, J = 1.3 Hz, 1H), 2.42 (d, J = 1.3 Hz, 3H).

[0076] 13 C NMR (101 MHz, DMSO - d 6 ) δ 159.67, 153.24, 152.84, 139.67, 126.18, 116.86, 116.64, 113.52, 107.76, 17.95.

[0077] The fluorescent probes P1, P2, and P3 used in Example 2, Example 3, and Example 4 were all prepared in Example 1.

[0078] Example 2

[0079] Response experiments of three fluorescent probes P to CE:

[0080] First, the absorption changes of fluorescent probes P1, P2, and P3 were studied.

[0081] In PBS buffer solution (0.1 M, pH = 7.4), the absorption spectral changes of the fluorescent probe (10 μM) in response to CE (1.0 U / mL) were measured. The experimental results are as Figure 1 shown: After adding CE (1.0 U / mL) to the fluorescent probe P1 (10 μM), the absorption peak at 280 nm increased; after adding CE (1.0 U / mL) to the fluorescent probe P2 (10 μM), the absorption peak at 330 nm redshifted to 350 nm; after adding CE (1.0 U / mL) to the fluorescent probe P3 (10 μM), the absorption peak at 330 nm redshifted to 350 nm, and the absorption peak at 350 nm increased.

[0082] After that, the fluorescence responses of fluorescent probes P1, P2, and P3 to CE were studied.

[0083] In PBS buffer solution (0.1 M, pH = 7.4), the fluorescence spectral changes of the fluorescent probe (10 μM) before and after adding CE (1.0 U / mL) were measured. The excitation wavelength of the fluorescent probe P1 was 330 nm, and the excitation wavelengths of the fluorescent probes P2 and P3 were 350 nm. The experimental results are as Figure 2 shown: A new fluorescence peak appeared at 445 nm for the fluorescent probe P1, and the fluorescence intensity increased by 3 times; the fluorescence of the fluorescent probe P2 at 445 nm increased by 41 times; the fluorescence of the fluorescent probe P3 at 445 nm increased by 5 times.

[0084] In addition, the reaction kinetics of probes P1, P2, and P3 with CE were studied.

[0085] In PBS buffer solution (0.1 M, pH = 7.4), the changes in fluorescence intensity at 445 nm of the fluorescent probe (10 μM) before and after adding CE (1.0 U / mL) were measured. The excitation wavelength of the fluorescent probe P1 was 330 nm, and the excitation wavelengths of the fluorescent probes P2 and P3 were 350 nm. The experimental results are as Figure 3As shown: It can be seen that within 600 s, the fluorescence intensity of the fluorescent probe P1 (Figure a) and the fluorescent probe P2 (Figure b) themselves at 445 nm did not change significantly, indicating that the fluorescent probes P1 and P2 themselves have good stability; within 600 s, the fluorescence intensity of the fluorescent probe P3 (Figure c) itself at 445 nm increased slightly, indicating that the stability of the fluorescent probe P3 itself is poor. When CE (1.0 U / mL) was added at 600 s, the fluorescence intensity of the fluorescent probe P1 at 445 nm increased slowly and did not reach the reaction plateau even after about 2 h. The fluorescence intensity of the fluorescent probe P2 at 445 nm reached the reaction plateau at about 30 min, and the fluorescence intensity of the fluorescent probe P3 at 445 nm reached the reaction plateau at about 10 min.

[0086] Therefore, the fluorescent probe P2 with the highest fluorescence enhancement multiple, good stability and fast reaction time was selected for subsequent tests and applications.

[0087] Example 3

[0088] Response experiment of the fluorescent probe P2 to CE:

[0089] First, the fluorescence response of the fluorescent probe P2 to different concentrations of CE was studied.

[0090] In a PBS buffer solution (0.1 M, pH = 7.4), the fluorescence spectral changes of the fluorescent probe P2 (10 μM) in response to a series of concentrations of CE (0 - 1.0 U / mL) were measured, and the excitation wavelength was 350 nm. The experimental results are as Figure 4 shown: As the concentration of CE added to the fluorescent probe P2 (10 μM) increased, the fluorescence at 445 nm gradually increased, and it was enhanced by 41 times compared to the fluorescent probe P2 itself.

[0091] After that, the response of the fluorescent probe P2 to CE in different pH environments was studied.

[0092] In different pH buffer solutions, the fluorescence intensity changes at 445 nm of the fluorescent probe P2 (10 μM) in response to CE (1.0 U / mL) were measured. The excitation wavelength was 350 nm. The ordinate was the fluorescence intensity at 445 nm, and the abscissa was the pH value. The experimental results are as Figure 5 shown: The fluorescent probe P2 itself has good stability between pH = 4.0 - 8.0, and the response of the fluorescent probe P2 (10 μM) to CE (1.0 U / mL) has good stability between pH = 7.0 - 8.0. Therefore, it can be proved that the fluorescent probe P2 can detect CE in a physiological environment.

[0093] In addition, the selectivity of the fluorescent probe P2 to CE was studied.

[0094] The fluorescence intensity changes at 445 nm were measured for the fluorescent probe P2 (10 μM) in response to CE (1.0 U / mL) and the selected interferents in different pH buffer solutions. The ·OH, HClO, H 2 O 2 、NO, ONOO - 、S 2- 、HS - 、HSO 3 - 、Vc, Na + 、K + 、Ca 2+ 、Mg 2+ 、Zn 2+ 、Cu 2+ 、Fe 2+ 、Fe 3+ 、Co 2+ 、Al 3+ 、Cys, Hcy, GSH, BSA, HSA, lipase, lysozyme, trypsin, proteinase K, acetylcholinesterase were used as interferents. Among them, Figure 6 the corresponding concentration of Vc was 1 mM, and the concentrations of the remaining interferents were all 200 μM; the excitation wavelength was 350 nm, and the ordinate was the fluorescence intensity at 445 nm. Figure 7 the corresponding concentrations of Cys, Hcy, and GSH were 1 mM, and the concentrations of the remaining interferents were all 500 μM; the excitation wavelength was 350 nm, and the ordinate was the fluorescence intensity at 445 nm. Figure 8 the corresponding concentrations of BSA and HSA were 5 mg / mL, the concentration of lipase was 10 U / mL, the concentration of lysozyme was 0.1 mg / mL, the concentration of trypsin was 0.2 mg / mL, the concentration of proteinase K was 1 U / mL, and the concentration of acetylcholinesterase was 0.5 mg / mL; the excitation wavelength was 350 nm, and the ordinate was the fluorescence intensity at 445 nm. The selectivity of the fluorescent probe P2 for them was measured. The experimental results are as Figure 6 、 7 、8 shown, indicating that the fluorescent probe P2 can selectively recognize CE.

[0095] Finally, the reaction mechanism between the fluorescent probe P2 and CE was studied.

[0096] Through the combined characterization of high-resolution mass spectrometry spectra and high-performance liquid chromatography spectra, the experimental results are as Figure 9 、 10 、11 shown, proving that the fluorescent probe P2 generates the compound Coumarin-120 after enzymatic hydrolysis by CE.

[0097] Among them, Figure 9Mass spectrometry characterization of the reaction mechanism between the fluorescent probe P2 prepared according to the present invention and CE. Experimental conditions: Add 1 mL of PBS buffer (0.1 M, pH = 7.4) to a 2 mL EP tube, then add 20 μL of the fluorescent probe P2 (1 mM) and 200 μL of CE (10 U / mL), and make up the volume to 2 mL with secondary water. React at 37 °C for 30 min. Take 20 μL of this sample and dilute it to 2 mL with chromatographic methanol, and then perform mass spectrometry detection.

[0098] Figure 10 High performance liquid chromatography characterization of the reaction mechanism between the fluorescent probe P2 prepared according to the present invention and CE. Experimental conditions: The concentration of the fluorescent probe P2 is 100 μM( Figure 10 a), the concentration of the fluorescent probe P2 in the mixture of the fluorescent probe P2 and CE is 100 μM, and the concentration of CE is 10 U / mL( Figure 10 b), and the concentration of the compound Coumarin-120 is 100 μM( Figure 10 c).

[0099] Figure 11 Schematic diagram of the reaction mechanism between the fluorescent probe P2 prepared according to the present invention and CE.

[0100] Example 4 Fluorescent imaging experiment of the fluorescent probe P2 in cells

[0101] 1. Detection of exogenous CE

[0102] To study the fluorescent imaging ability of the fluorescent probe P2 for exogenous CE in cells, normal hepatocytes were selected as the experimental research object. The normal hepatocytes were first washed with PBS and then incubated with the fluorescent probe P2 (10 μM) at 37 °C for 30 min as the experimental control group. Other groups of normal hepatocytes were treated with different substances, and then the fluorescent probe P2 (10 μM) was added and incubated at 37 °C for 30 min. Finally, cell imaging was performed under a two-photon confocal microscope.

[0103] The experimental results are as Figure 12 shown. Figure 12 a, Figure 12 b and Figure 12 c are normal hepatocytes incubated with the fluorescent probe P2 (10 μM) for 30 min. Figure 12 d, Figure 12 e and Figure 12 f are normal hepatocytes incubated with CE (1.0 U / mL) for 1 h and then incubated with the fluorescent probe P2 (10 μM) for 30 min. Figure 12 g, Figure 12 h and Figure 12Group i: Normal hepatocytes were incubated with CE (1.0 U / mL) for 1 h, then with the CE inhibitor bis(4-nitrophenyl) phosphate (BNPP) (500 μM) for 1 h, and finally with the fluorescent probe P2 (10 μM) for 30 min. Figure 12 Group j: Quantitative fluorescence intensity map of the blue channel for three groups of experiments. The excitation wavelength was 720 nm, and the wavelength collection range of the blue channel was 400 to 490 nm. Scale bar: 10 μm. Among them, normal hepatocytes in the blue channel were blue, those in the bright-field channel were colorless, and normal hepatocytes in the overlay channel were blue.

[0104] Figure 12 In it, under two-photon excitation at 720 nm, when normal hepatocytes were incubated with the fluorescent probe P2 (10 μM) for 30 min, there was almost no fluorescence in the blue channel; when normal hepatocytes were incubated with CE (1.0 U / mL) for 1 h, the cells were washed three times with physiological PBS buffer solution and then incubated with the fluorescent probe P2 (10 μM) for 30 min. It could be seen that after adding CE, the fluorescence in the blue channel was significantly enhanced, indicating that the fluorescent probe P2 could detect exogenous CE at the cellular level; when normal hepatocytes were incubated with CE (1.0 U / mL) for 1 h, the cells were washed three times with physiological PBS buffer solution, then incubated with the CE inhibitor bis(4-nitrophenyl) phosphate (BNPP) (500 μM) for 1 h, the cells were washed three times with physiological PBS buffer solution, and finally incubated with the fluorescent probe P2 (10 μM) for 30 min. It could be seen that after adding CE and then the CE inhibitor BNPP, there was almost no fluorescence in the blue channel, further indicating that the fluorescent probe P2 could detect exogenous CE at the cellular level.

[0105] 2. Detection of endogenous CE

[0106] To study the fluorescence imaging ability of the fluorescent probe P2 for endogenous CE in cells, normal hepatocytes and hepatoma cells were selected as experimental research objects. Normal hepatocytes and hepatoma cells were first washed with PBS and then added with the fluorescent probe P2 (10 μM) and incubated at 37 °C for 30 min. Finally, cell imaging was performed under a two-photon confocal microscope.

[0107] The experimental results are as Figure 13 shown. Figure 13 a. Figure 13 b: Normal hepatocytes were incubated with the fluorescent probe P2 (10 μM) for 30 min. Figure 13 c. Figure 13 d: Hepatoma cells were incubated with the fluorescent probe P2 (10 μM) for 30 min. Figure 13e is a quantification graph of the fluorescence intensity of the blue channel in two groups of experiments. The excitation wavelength is 720 nm, and the wavelength collection range of the blue channel is 400 to 490 nm. Scale bar: 10 μm. Among them, the cells in the blue channel are blue, the bright-field channel is colorless, and the cells in the overlay channel are blue.

[0108] Figure 13 Among them, also under 720 nm two-photon excitation, normal liver cells were incubated with the fluorescent probe P2 (10 μM) for 30 min, and there was almost no fluorescence in the blue channel; while liver cancer cells were incubated with the fluorescent probe P2 (10 μM) for 30 min, and it could be clearly observed that the fluorescence in the blue channel was significantly enhanced. It shows that the fluorescent probe P2 can detect intracellular endogenous CE.

[0109] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A fluorescence probe for two-photon detection of carboxylesterase, characterized in that, the structural formula of the fluorescence probe is as follows: Among them, R 1 is H, R 2 is F.

2. A preparation method of the fluorescence probe for two-photon detection of carboxylesterase according to claim 1, characterized in that, it comprises the following steps: Dissolve 7-amino-4-methylcoumarin and chloro(fluoro)acetyl chloride in an organic solvent, add triethylamine, and react at 0 °C for 3.5 - 4.5 h. After the reaction is completed, the fluorescence probe for two-photon detection of carboxylesterase is obtained.

3. A preparation method of the fluorescence probe for two-photon detection of carboxylesterase according to claim 2, characterized in that, the molar ratio of 7-amino-4-methylcoumarin, chloro(fluoro)acetyl chloride, and triethylamine is 1:1.8 - 2.2:

7.

4. A preparation method of the fluorescence probe for two-photon detection of carboxylesterase according to claim 3, characterized in that, the molar ratio of 7-amino-4-methylcoumarin, chloro(fluoro)acetyl chloride, and triethylamine is 1:1.9 - 2.1:

7.

5. A preparation method of the fluorescence probe for two-photon detection of carboxylesterase according to claim 2, characterized in that, the organic solvent includes tetrahydrofuran, and in the organic solvent, the concentration of 7-amino-4-methylcoumarin is 0.08 - 0.12 mmol / mL.

6. A preparation method of the fluorescence probe for two-photon detection of carboxylesterase according to claim 5, characterized in that, the concentration of 7-amino-4-methylcoumarin is 0.09 - 0.11 mmol / mL.

7. A preparation method of the fluorescence probe for two-photon detection of carboxylesterase according to claim 2, characterized in that, after the reaction is completed, spin-dry the reaction mixture, separate and purify to obtain the fluorescence probe for two-photon detection of carboxylesterase.

8. A preparation method of the fluorescence probe for two-photon detection of carboxylesterase according to claim 7, characterized in that, separate and purify the product by silica gel column chromatography, and the separation and purification reagents are petroleum ether and ethyl acetate.

9. A preparation method of the fluorescence probe for two-photon detection of carboxylesterase according to claim 8, characterized in that, the volume ratio of petroleum ether to ethyl acetate is 8 - 12:

1.

10. Application of the fluorescence probe for two-photon detection of carboxylesterase according to claim 1 in the preparation of a reagent for detecting carboxylesterase.

11. The application according to claim 10, characterized in that, the carboxylesterase includes exogenous carboxylesterase and endogenous carboxylesterase.

12. A method for detecting carboxylesterase, characterized in that, using the fluorescence probe for two-photon detection of carboxylesterase according to claim 1, and it comprises the following steps: Add the fluorescence probe to the sample to be tested, incubate at 35 - 40 °C for 25 - 35 min, and perform cell imaging under a two-photon confocal microscope; the excitation wavelength is 720 nm, and the wavelength collection range of the blue channel is 400 to 490 nm; This method is not for the purpose of disease diagnosis.

13. A method for detecting carboxylesterase according to claim 12, characterized in that, incubate at 37 °C for 30 min.

14. A method for detecting carboxylesterase according to claim 12, characterized in that the concentration of the fluorescent probe in the sample to be tested is 8-12 μM.

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