CN114478578A - Specific bioluminescent probe substrate for detecting carboxylesterase 1 and preparation method and application thereof - Google Patents
Specific bioluminescent probe substrate for detecting carboxylesterase 1 and preparation method and application thereof Download PDFInfo
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- CN114478578A CN114478578A CN202111597847.9A CN202111597847A CN114478578A CN 114478578 A CN114478578 A CN 114478578A CN 202111597847 A CN202111597847 A CN 202111597847A CN 114478578 A CN114478578 A CN 114478578A
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- carboxylesterase
- probe substrate
- ces1
- fluorescent probe
- detection
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- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 1
- 229960004768 irinotecan Drugs 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229940116369 pancreatic lipase Drugs 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 125000000951 phenoxy group Chemical group [H]C1=C([H])C([H])=C(O*)C([H])=C1[H] 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- POGQSBRIGCQNEG-UHFFFAOYSA-N rufinamide Chemical compound N1=NC(C(=O)N)=CN1CC1=C(F)C=CC=C1F POGQSBRIGCQNEG-UHFFFAOYSA-N 0.000 description 1
- 229960003014 rufinamide Drugs 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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Abstract
The invention belongs to the technical field of biological medicines, and discloses a specific fluorescent probe substrate for determining carboxylesterase 1 activity, and a preparation method and application thereof. The probe substrate can be used for quantitative detection of the activity of the CES1 enzyme in cells or tissue samples of different species sources, can also be used for rapid screening of a CES1 regulator, and can be used as a probe substrate of an experimental animal in vivo and whole CES1 to evaluate individual and species differences of a metabolic enzyme CES 1. The invention also discloses a preparation method of the probe substrate. The CES1 fluorescent probe substrate detection CES1 single-enzyme in-vitro activity has high specificity, high sensitivity, high detection convenience and high flux detection, has different detection modes compared with a fluorescent probe, is basically not interfered by a biological matrix, and has higher selectivity and accuracy.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a specific bioluminescent probe substrate for detecting carboxylesterase 1, and a preparation method and application of the probe.
Background
Carboxylesterase 1(Carboxylesterase 1, CES1, EC 3.1.1.1) belongs to the serine hydrolase family and is widely found in mammals. In humans, CES1 is distributed mainly to the liver and exhibits significant tissue specificity. CES1 is localized to the endoplasmic reticulum in subcellular organelles, CES1 is fixed to the endoplasmic reticulum by a C-terminal connexin, and its active center is located at the N-terminus of the protein. CES1 is a key enzyme for catalyzing and metabolizing ester substances and is biased to hydrolyze ester, amide and thioester compounds with large acyl groups connected with small alcohol group structures. Most of the substrate drugs of carboxylesterases are esters (such as rufinamide, irinotecan and capestatin), and thioesters, amides and carbamates are potential substrates of carboxylesterases. Most cardiovascular drugs, statins and phenoxy acids, are substrates for carboxylesterases. More than 80% of clopidogrel is metabolically inactivated by CES1, the activity of CES1 directly influences the concentration of active products of clopidogrel in vivo, and influences the effectiveness and safety of clopidogrel administration. In endogenous metabolism, CES1 is a key enzyme in cholesterol ester and fatty ester metabolism. These are associated with disorders of lipid metabolism. The CES1 is found to be closely related to the occurrence and development of various liver lipid metabolism diseases, and CES1 knockout mice cause the increase of liver generation and the increase of liver low-density lipoprotein excretion, which cause the increase of hyperlipidemia and fat deposition in peripheral tissues. The detection of the activity of the CES1 can provide guidance for researching the metabolism process of the drug in vivo.
Due to the characteristics of liver-specific distribution and high distribution of CES1, it is assumed that when liver cells are damaged and destroyed, intracellular CES1 may be released to the extracellular space or even the blood circulation system like transaminase markers, and it is an important way to perform non-invasive examination by analyzing liver-derived molecules in the system. The CES1 is secreted to cytoplasm or extracellular and still maintains hydrolytic activity, and due to the characteristic, the activity of the CES1 can be doubly characterized by residual activity in liver tissues and the activity of the CES1 in blood or extracellular body fluid, and the characterization method of the activity of the CES1 can characterize the percentage of residue and hepatocyte damage, which indicates that the CES1 possibly becomes an evaluation index of liver injury.
At present, the main detection methods of CES1 include immunoblotting, proteomics, ultraviolet spectroscopy, capillary electrophoresis and fluorescence detection. Immunoblotting is usually based on antigen-antibody specific binding, and this method usually has good specificity, but the antibody needs to be stored at low temperature, and is easy to inactivate after multiple uses, and is expensive. Proteomics methods require a specific instrument LC-MS, which quantifies by detecting specific peptide fragments after proteolysis. This method is expensive in equipment, complicated in operation steps, and like the immunological method, can quantify only the absolute amount of enzyme or semiquantify it. It has been reported previously that protein expression of CEs is not always positively correlated with their activity. The ultraviolet spectrum law expresses the activity of enzyme based on the change of absorbance after hydrolysis, and the specificity of the substrates is poor, so that the ultraviolet spectrum law cannot be used for detecting the activity of CES1 in a complex system. Still other substrates are detected by liquid phase detection. Compared with other technologies, the fluorescence detection method has the advantages of high sensitivity, small wound and real-time imaging, and has great advantages in analyzing CES1 in cells and tissues. The development of the CES1 probe substrate with high sensitivity and strong specificity can not only better explore the important role played by the CES1 in related diseases, but also provide powerful technical support for screening CES1 target drugs and quantitatively measuring the activity of CES1 in a biological system.
The fluorescent probes currently used for measuring CES1 mainly comprise 2- (benzothiazole-2-yl) -6-methoxyphenyl benzoate (BMBT) and D-fluorescein methyl ester (DME) bioluminescent probes, wherein the probe substrate of the BMBT ratio type can realize the quantitative detection of CES1 in a biological sample. However, this probe substrate is not selective, and is hydrolyzed by proteins having an ester bond hydrolysis function such as albumin in addition to CES 1. And the maximum emission wavelength of the product is 488nm, and the detection is easily interfered by a biological matrix. Separation by means of high performance liquid chromatography coupled with a fluorescence detector is required to realize the CES1 functional analysis of complex samples. While the bioluminescent probe DME has great advantages in the quantification of CES1 in a variety of complex biological systems, such as cell and tissue preparations, the probe has certain drawbacks in selectivity. At equal protein concentrations, the selectivity of DME to CES1 and butyrylcholinesterase is about 30 times different, which means that the quantitative result of activity of DME on CES1 is not reliable in samples (such as plasma/serum) with more abundant butyrylcholinesterase. In addition, the hydroxyl group of DME is easy to metabolize by glucuronyl transferase (UGTs), an important drug metabolizing enzyme in human body, and the existence of UGTs influences the detection result in living cells, living tissues or living body researches.
The above various drawbacks are the further exploration of the limitations of CES1 function with DME. Therefore, the development of a high-selectivity CES1 probe reaction and a matched high-throughput detection method thereof have important practical value.
Disclosure of Invention
The invention aims to provide a specific bioluminescent probe substrate for measuring carboxylesterase 1(CES1) activity and application thereof, wherein the probe has a different detection mode compared with a fluorescent probe, is basically interfered by a biological matrix to be 0, and has higher selectivity and sensitivity compared with the conventional bioluminescent probe. The distribution and the function of CES1 in various biological systems can be quantitatively evaluated by utilizing the probe reaction.
The invention is realized by the following technical scheme:
a specific fluorescent probe substrate for detecting carboxylesterase 1(CES1), which can be specifically catalyzed by CES1 to carry out ester bond hydrolysis reaction and generate corresponding (S) -2- (6,7-dihydro- [4,5-f ] indole-thiazolyl) -4, 5-dihydrothiazole-4-formic acid derivative (DDC for short), and the structural general formula of the probe is as follows:
wherein R1 is selected from H, alkyl, - (C)1-C8Alkylene) -carboxyl, - (C)1-C8Alkylene) -ester radicals, - (C)1-C8Alkylene) -amino, - (C)1-C8Alkylene) -cyano radicals, - (C)1-C8Alkylene) -nitro, - (C)1-C3Alkylene) -O- (C)1-C3Alkyl), carbocyclyl, - (C)1-C3Alkylene) -carbocyclyl, aryl, - (C)1-C3Alkylene) -aryl, heteroaryl, - (C)1-C3Alkylene) -heteroaryl, heterocyclyl or- (C)1-C3Alkylene) -heterocyclyl. R2Is selected from C1-C10An alkyl group.
Preferably, R is H or C1-C6 alkyl, more preferably H, methyl, ethyl, propyl or isopropyl.
The structure of the product after hydrolysis is as follows:
in a preferred embodiment of the present invention, when R1 ═ H, R2 ═ CH3When the probe substrate is:
(S) -2- (6,7-dihydro- [4, 5-f)]Indole-thiazolyl) -4, 5-dihydrothiazole-4-carboxylic acid methyl ester (methyl (S) -2- (6,7-dihydro-5H-thiazolo [4, 5-f)]indol-2-yl) -4,5-dihydrothiazole-4-carboxylate, abbreviated as DDM); or R1 ═ CH3，R2＝CH3When (S) -2- (7-methyl-6, 7-dihydro- [4, 5-f)]Indole-thiazolyl) -4, 5-dihydrothiazole-4-carboxylic acid methyl ester (DDM-2).
The invention also provides a preparation method of the specific bioluminescent probe substrate for measuring the activity of carboxylesterase 1(CES1), which comprises the following steps:
1) carrying out cyano substitution reaction on the compound 2 to obtain a compound 3;
2) the compound 3 is subjected to cyano hydrolysis with D-cysteine and alkali to generate corresponding acid (compound 4);
3) a specific fluorescent probe substrate (compound 5) for detecting carboxylesterase 1 is generated through esterification reaction.
In the step 1), adding a catalyst into the compound 2 and cyanide, reacting at the temperature of 120-140 ℃ until the reaction is complete, and extracting and washing to obtain a compound 3; the molar ratio of the compound 2 to cyanide ions in cyanide salt is 1: 1-3, preferably 1: 2; the catalyst is an iodide salt, preferably NaI. The solvent used was DMSO.
In the step 2), the molar ratio of the compound 3 to the D-cysteine is 1: 1-3: 1-2, preferably 1: 2: 1.5; (ii) occurs; the alkali is carbonate or bicarbonate; the solvent used is an alcohol, preferably methanol.
In the step 3), the compound 4 and methanol are subjected to esterification reaction, preferably EDCI and DMAP are used as catalysts; the solvent used was dichloromethane.
The specific fluorescent probe substrate can be used for detecting carboxylesterase 1 and qualitatively or quantitatively detecting the activity of the carboxylesterase.
A method for detecting carboxylesterase 1 comprises the steps of mixing the specific fluorescent probe substrate with a sample to be detected, carrying out enzymatic reaction, and detecting fluorescence intensity to qualitatively or quantitatively detect the activity of CES 1. The activity of CES1 in different biological systems can be quantitatively determined by quantitatively detecting the elimination rate of the substrate or the generation rate of its carboxyl product per unit time.
The determination method comprises the following steps:
adding a carboxylesterase 1 specific fluorescent probe substrate into a phosphate buffer system, wherein the reaction temperature is 20-60 ℃, the pH of the incubation system is 5.5-10.5, adding luciferase to start a bioluminescence reaction, and the reaction time is 10-20 min; the fluorescence intensity was measured, and the amount of decrease in the substrate or the amount of the carboxyl product produced from the specific fluorescent probe was used as an index for evaluating the carboxylesterase 1 activity.
Or adding a carboxylesterase 1 specific fluorescent probe substrate into a phosphate buffer system, reacting at 20-60 ℃, and reacting for 10-20 min, wherein the pH of the incubation system is 5.5-10.5; detecting the specific fluorescent probe substrate or carboxyl product at 256-365nm by using a liquid phase, and taking the reduction amount of the specific fluorescent probe substrate or the generation amount of the carboxyl product as an evaluation index of the activity of the carboxylesterase 1.
The specific fluorescent probe substrate directly reacts with luciferase, a carboxyl product of the specific fluorescent probe substrate can be metabolized and oxidized by the luciferase, chemical energy is converted into light energy, a bioluminescent signal is detected by using an enzyme-labeling instrument, and full-wavelength scanning is carried out; the probe substrate and the carboxyl product after ester hydrolysis can also be directly detected by liquid phase, such as HPLC or LCMS, and has signal value at 256-365 nm.
The method can measure the decrease of the probe substrate or the amount of the carboxyl product generated in a unit time as an evaluation index of the carboxylesterase 1 activity.
Preferably, the reaction temperature is 34-42 ℃, and more preferably 37 ℃; the pH of the incubation system is 5.5 to 7.0, and more preferably 6.5.
The sample to be detected is a biological sample and is any one of recombinant single enzyme containing CES1, human or animal tissue preparation liquid, various mammalian tissue cells and preparations thereof.
The probe substrate can also be used for quantitative detection of the activity of the CES1 enzyme in body fluid, cell or tissue samples from different species. Or can be used for CES1 detection of experimental animals to evaluate individual and species differences of metabolic enzyme CES 1.
The probe has high substrate specificity and sensitivity, and can be used for screening and evaluating CES1 inhibitor; especially for the rapid screening of CES1 inhibitor and the quantitative evaluation of the inhibition ability.
The specific fluorescent probe substrate of the invention can also be used for fluorescence bioimaging, in particular for fluorescence bioimaging to locate carboxylesterase 1CES1 in biological tissues.
The bioluminescent fluorescent probe substrate of CES1 and the hydrolysis product of the carboxyl ester bond do not have spontaneous bioluminescence property, do not interfere the detection, and can realize the quick and sensitive detection of the product by adopting a bioluminescent detector; and (3) carrying out secondary reaction on the carboxyl ester bond cleavage product and luciferase, and then detecting by full-wavelength scanning. Moreover, the detection process of the CES1 activity is not easily interfered by a biological system matrix and impurities, and the method can be used for quantitative determination of the CES1 enzyme activity in various recombinant CES1, human and animal tissue preparation solutions and various tissue cells; meanwhile, the probe substrate can be used as a probe substrate of the animal integral CES1 to evaluate the individual and species difference of the metabolic enzyme CES 1. The chemiluminescence detection method after the secondary reaction of the probe carboxyl product and luciferase can also be used for rapid screening of CES1 inhibitor and quantitative evaluation of inhibition capability.
As a high-specificity CES1 single enzyme fluorescent probe substrate, the compound can be used for detecting the activity of CES1, and is particularly suitable for measuring the activity of CES1 produced by bacteria, insect cells, mammalian cells and yeast clone expression systems, and calibrating the activity of CES1 in preparations such as microsomes of various mammalian tissues and organs sources, S9 and the like.
The invention has the beneficial effects that the specific fluorescent probe substrate has the following characteristics:
(1) high specificity: can be metabolized into a metabolite, namely a carboxyl ester bond cleavage product, by CES1 with high specificity, thereby greatly improving the result reliability in biochemical index detection.
(2) The sensitivity is high: the quantitative determination of CES1 was performed by establishing a ratiometric standard curve with a lower detection limit of 5 ng/mL.
(3) The preparation method is simple and easy to obtain: can be obtained by a chemical synthesis method, and the synthesis process is simple and feasible.
(4) Can realize high-flux detection, and has wide application: the kit can be used for measuring on various common fluorescent enzyme labeling instruments and biochemical instruments in a laboratory, and can be used for batch detection by using 96 or 386 microporous plates; in addition to measuring enzyme activity, fluorescence bioimaging can be achieved to localize CES1 in biological tissues, and to screen or assess inhibitors or inducers of CES 1.
Drawings
FIG. 1 is a diagram of DDM1H-NMR spectrum;
FIG. 2 shows a DDM13A C-NMR spectrum;
FIG. 3 is a drawing of DDM21H-NMR spectrum;
FIG. 4 is a drawing of DDM213A C-NMR spectrum;
FIG. 5 shows the selectivity of DDM;
FIG. 6 is a linear reaction time curve for CES1 catalyzed DDM hydrolysis;
fig. 7 is a quantitative standard curve for CES 1;
FIG. 8 is a graph of the enzymatic kinetics of CES1 catalyzed hydrolysis of DDM;
figure 9 is a curve for quantitative assessment of CES1 activity in human tissue microsomes;
figure 10 is CES1 inhibitor screen:
FIG. 10(a) is a graph showing the result of the inhibition of a probe substrate (DDM) by betulinic acid,
FIG. 10(b) is a graph showing the results of the inhibition of the probe substrate (DDM) by betulinic acid,
FIG. 10(c) is a graph showing the result of the inhibition of a probe substrate (DDM) by liquidambaric acid,
FIG. 10(d) is a graph showing the results of the inhibitory effect of oleanolic acid on probe substrates (DDM);
figure 11 is CES1 inhibitor screen:
FIG. 11(a) is a graph showing the results of the inhibition of the probe substrate (DDM2) by betulinic acid,
FIG. 11(b) is a graph showing the results of the inhibition of the probe substrate (DDM2) by betulinic acid,
FIG. 11(c) is a graph showing the results of the inhibition of a probe substrate (DDM2) by liquidambaric acid,
FIG. 11(d) is a graph showing the results of the inhibitory effect of oleanolic acid on the probe substrate (DDM 2);
figure 12 residual activity measurement of liver CES1 following ANIT-induced cholestatic liver injury in rats;
figure 13 serum CES1 activity measurements after ANIT induced cholestatic liver injury in rats;
FIG. 14 use of DDM for transfection of LUCs+And different numbers of cells CES 1;
FIG. 15 comparison of DME and DDM for transfection of LUC+Mice were imaged whole-body CES1 with DME probe on the left and DDM probe on the right.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
The equipment adopted by the invention and the types thereof are as follows: the fluorescence emission/excitation spectrum is detected by a SynergyH1 full-function micropore plate detector;1the H-NMR spectrum was obtained by detection with a nuclear magnetic resonance spectrometer (Avance II 400 MHz).
The structural general formula of the bioluminescent probe is as follows:
example 1
Synthesis of DDM
The synthesis route of DDM is as follows:
1) synthesis of Compound (2):
100mg (0.57mmol,1eq) of compound (1) was put into a 100mL reaction flask, dissolved by adding 10mL DMSO, and then 55.49mg NaCN (1.14mmol,2eq) was added, stirred in a 130 ℃ oil bath for reaction for 3-4 hours, detected by TLC, and the developing solvent was petroleum ether: ethyl acetate 5:1, NaHCO after reaction3Adjusting pH of the reaction solution to 7-8, adding ethyl acetate and water, extracting the water phase with ethyl acetate for 2 times (30mL × 2), washing the organic phase with water for 1 time (30mL), washing with saturated salt water for 1 time (30mL), and adding anhydrous Na2SO4Drying (30min), concentrating by rotary evaporation, passing through silica gel column, and eluting with petroleum ether: ethyl acetate ═ 5:1, the yellow solid compound is the compound (2), and the yield is 30-40%.
2) Synthesis of Compound (3):
55.82mg (0.28mmol,1eq) of compound (2) was put into a 100mL reaction flask, dissolved by adding 10mL of methanol, added with 67.85mg of D-cysteine (0.56mmol,2eq), added with 3mL of water, and 44.52mg of Na2CO3(0.42mmol,1.5eq), stirring at normal temperature for reaction for 45 minutes to 1 hour, detecting by TLC, and taking petroleum ether as a developing agent: ethyl acetate 5:1, after the reaction was complete, the pH was adjusted to 8-9, and the reaction mixture was stirred with petroleum ether: ethyl acetate ═ 5:1 with water to remove impurities, adjusting the pH of the aqueous phase to 5-6, extracting the product with ethyl acetate (30mL, 20mL, 10mL), and then with anhydrous Na2SO4Drying (30min), evaporating to dryness to obtain compound (3) with yield of 50-60%.
3) Synthesis of Compound (4): 40mg (0.13mmol, 1eq) of the compound (3) was put into a 100mL reaction flask, and 10mL of CH was added2Cl2Dissolving, adding 10mL (1.3mmol,10eq) of methanol, adding 4.79mg of EDCI (0.03mmol, 0.2eq), adding 31.76mg of DMAP (0.26mmol, 2eq), detecting by TLC with petroleum ether and ethyl acetate of 10:1, extracting with ethyl acetate and water after reaction, extracting the aqueous phase with ethyl acetate for 2-3 times, and saturatingWashed with brine 1 time (30mL) and anhydrous Na2SO4Drying (30min), concentrating by rotary evaporation, passing through silica gel column, and eluting with petroleum ether: ethyl acetate 10:1, obtaining a reddish-brown compound (4) with a yield of 30-40%, of DDM1The H-NMR spectrum is shown in figure 1,13the C-NMR spectrum is shown in figure 2.
Synthesis of DDM2
The synthetic route of DDM2 is as follows:
synthesis of Compound 2-b: dissolving 120mg (0.57mmol,1eq) of compound 1-b in 10ml of dichloromethane, adding 62.95 μ L (1.71mmol,3eq) of formaldehyde, then adding 211.94mg (1.71mmol,3eq) of sodium borohydride, reacting at room temperature for about 45min, detecting by TLC, developing solvent petroleum ether: ethyl acetate 10:1, when the reaction was completed, dichloromethane (30 mL. times.2) and water (20 mL. times.2) were added and extracted twice, and the mixture was washed once with saturated brine (20 mL. times.1) and anhydrous Na2SO4Drying (30min), loading on silica gel column with 200 meshes, and eluting with petroleum ether: ethyl acetate 10:1, the product is a yellow-green oil, yield 85-95%.
Synthesis of Compound 3-b: 63mg (0.28mmol,1eq) of compound 2-b was dissolved in 10ml DMSO, 27.48mg (0.56mmol,2eq) NaCN was added, a catalytic amount of NaI was added, the reaction was carried out in an oil bath at 130 ℃ for about 4 hours, and TLC was carried out using petroleum ether as a developing solvent: ethyl acetate ═ 5:1, the reaction is terminated when the reaction product does not increase any more, with NaHCO3Adjusting pH of the reaction solution to 7-8, adding ethyl acetate and water, extracting the water phase with ethyl acetate for 2 times (30mL × 2), washing the organic phase with water for 1 time (30mL), washing with saturated salt water for 1 time (30mL), and adding anhydrous Na2SO4Drying (30min), concentrating by rotary evaporation, passing through silica gel column, and eluting with petroleum ether: ethyl acetate ═ 5:1, the yellow solid compound is the compound 3-b, and the yield is 30-40%.
Synthesis of Compound 4-b: 24mg (0.11mmol, 1eq) of the compound (c) was dissolved in 10ml of dichloromethane, followed by the addition of 27.02mg (0.22mmol,2eq) of D-Cysteine, 17.65mg (0.1665mmol, 1.5eq)Na2CO3And 10mL of water, stirring at normal temperature for reaction for about 1 hour, detecting by TLC, and taking petroleum ether as a developing agent: ethyl acetate ═ 5: after the reaction was completed, the pH was adjusted to 8 to 9, and the reaction mixture was stirred with petroleum ether: ethyl acetate ═ 5:1 with water to remove impurities, adjusting the pH of the aqueous phase to 5-6, extracting the product with ethyl acetate (30mL, 20mL, 10mL), and then with anhydrous Na2SO4Drying (30min), evaporating to dryness to obtain compound 4-b with yield of 35-45%.
Synthesis of Compound 5-b 14mg (0.044mmol,1eq) of Compound 4-b was dissolved in 5mL of dichloromethane, 4mL (0.44mmol,10eq) of methanol was added, 16.82mg of EDCI (0.088mmol,2eq) was added, 11mg of DMAP (0.09mmol, 2eq) was added and the reaction was carried out at room temperature for 2 hours, TLC detection was carried out using petroleum ether as a developing agent, ethyl acetate 10:1, after completion of the reaction, extraction was carried out using ethyl acetate and water, the aqueous phase was extracted 2-3 times with ethyl acetate, washing with saturated brine 1 time (30mL) and anhydrous Na2SO4Drying (30min), concentrating by rotary evaporation, passing through silica gel column, eluting with petroleum ether and ethyl acetate (10: 1) to obtain reddish brown compound 5-b (DMM-2) with yield of 40-50%, and DDM21The H-NMR spectrum is shown in figure 3,13the C-NMR spectrum is shown in figure 4.
Example 2 in vitro determination of Single enzyme Selectivity of human recombinant CES1
(1) The following zymogens were used in the single enzyme screen: catechol-O-methyltransferase (COMT), Human Serum Albumin (HSA), α -chymotrypsin, lysozyme (Lysaozyme), human carboxyesterase 1(CES1A), human carboxyesterase 2(CES2A), acetylcholinesterase (Ache), butyrylcholinesterase (Bche), Carbonic Anhydrase (CA), Human Pancreatic Lipase (HPL), thrombin, α -glycosidase, α -Amylase (α -Amylase), Porcine Pancreatic Lipase (PPL), pepsin, pancreatin. The reaction system used the same amount of enzyme, and mainly comprised 5. mu.L of enzyme (final concentration 10. mu.g/mL), 2. mu.L of DDM probe substrate (final concentration 10. mu.M), and 93. mu.L of PBS buffer (pH 6.5). The reaction process is that 5 mu L of different single enzymes are respectively added into 93 mu L of PBS buffer solution, incubated for 3 minutes at 37 ℃, then 2 mu L of DDM probe substrate is added to start the reaction, and the reaction lasts 20 minutes.
(2) Mixing 50 μ L of reaction solution with 50 μ L of Luciferase (LDR), placing into microplate reader to detect chemiluminescence signal, scanning at full wavelength, detecting once in 1min, detecting for 30min, and comparing with the highest value. Each experiment was set up with 3 sets of parallel experiments, and the mean was taken for calculation. The probe is only hydrolyzed specifically by the single enzyme of the recombinant human CES1, and other single enzymes have almost no hydrolysis reaction. The selectivity of DDM is shown in FIG. 5, and it can be seen that the substrate has very high activity selectivity for human carboxylesterase 1.
EXAMPLE 3CES1 time Standard Curve determination
The reaction system mainly comprised 2.5. mu.L of CES1 monoose (final concentration 0.5. mu.g/mL), 2. mu.L of DDM probe substrate (final concentration 1.5. mu.M), and 45.5. mu.L of PBS buffer (0.1M, pH 6.5). The reaction process is as follows: firstly, 2.5 mu L of CES1 single enzyme is added into 45.5 mu L of PBS buffer solution to be mixed with 50 mu L of Luciferase (LDR), the mixture is incubated for 3min at 37 ℃, then 2 mu L of DDM probe substrate is added to start reaction, the mixture is put into an enzyme labeling instrument to detect a luminescent signal, the whole wavelength scanning is carried out, the detection is carried out once every 1min, the detection is continued for 40min, the highest value of parallel reaction is taken as comparison, the generation amount-time curve of the metabolite is measured, and the linear reaction time range is determined. Each experiment was set up in 3 replicates and the linear reaction time curve for the CES1 catalyzed hydrolysis of DDM is shown in figure 6.
Example 4 lower limit of detection of CES1 in vitro assay
The reaction system mainly contained 5. mu.L of the enzyme (final concentrations were 0.005, 0.010, 0.020, 0.050, 0.100, 0.200, 0.500, 1.000, 2.000, 4.000, 8.000, 16.000, and 20.000. mu.g/mL, respectively), 2. mu.L of the DDM probe substrate (final concentration: 10. mu.M), and 93. mu.L of PBS buffer (0.1M, pH 6.5). The reaction process comprises the steps of firstly adding 5 mu L of enzyme into 93 mu L of PBS buffer solution, incubating for 3min at 37 ℃, then adding 2 mu L of DDM probe substrate to initiate reaction, reacting for 20min, mixing 50 mu L of reaction solution with 50 mu L of Luciferase (LDR), putting the reaction solution into an enzyme labeling instrument to detect a luminescent signal, detecting once in 1min, continuously detecting for 30min, taking the highest value as comparison, determining the metabolite generation amount-time curve, and determining the concentration range of the linear reaction enzyme. Each experiment was set up in 3 replicates. The average value of each group was compared with that of the control group without CES1, and the result showed statistical significance at 0.005. mu.g/mL (R)2＝0.9982，P＜
0.0001), and a quantitative standard curve of CES1 is shown in figure 7, so that the detection lower limit of the in-vitro CES1 of the probe is determined to be 5 ng/mL.
Example 5CES1 enzyme kinetic testing
The assay was performed on a microplate reader using 96-well plates with substrate 1-400. mu.M, CES1 monoose 0.02mg/mL, pH 6.5 in 100mM PBS buffer, total volume 100. mu.L, incubation at 37 ℃ for 3min, analysis by microplate reader for 30min, and detection every 1 min. Detection conditions are as follows: full wavelength scanning. Substituting the obtained fluorescence intensity into a standard curve to obtain V of Human Liver Microsome (HLM) to DDMmaxAnd KmThe enzymatic kinetics of CES 1-catalyzed hydrolysis of DDM is shown in FIG. 8.
Example 6 quantitative determination of CES1 Activity in human liver microsomes
The assay is carried out on a microplate reader by using a 96-well plate, wherein the reaction system mainly comprises 2 μ L (4 μ M, final concentration) of a DDM probe substrate, 5 μ L (final concentration is 0.5, 1, 2.5, 5, 10, 20, 50, 100, 200 and 400 μ g/mL) of a CES1 single enzyme, and 93 μ L of PBS buffer (0.1M, pH 6.5), and the reaction process comprises the following steps: firstly, adding 5 mu L of enzyme into 93 mu L of PBS buffer solution, incubating for 3min at 37 ℃, then adding 2 mu L of DDM probe substrate to initiate reaction, reacting for 20min, taking 50 mu L of reaction solution to mix with 50 mu L of Luciferase (LDR), immediately putting into an enzyme labeling instrument to detect a luminescent signal, detecting once in 1min, continuously detecting for 30min, taking the highest value as comparison, determining a metabolite generation amount-time curve, determining an enzyme activity linear reaction range, and obtaining an activity quantitative evaluation curve of CES1 in human tissue microsomes as shown in figure 9.
Example 7CES1 Primary Screen for inhibitor
4 natural compounds are selected to test the inhibition effect of two CES1 mediated probe hydrolyzation, the final concentration of the inhibitor is set to be 1 mu M, 10 mu M and 100 mu M, the final concentration of two probe substrates is 2 mu M, the final concentration of the enzyme is 1 mu g/mL, the experiment is carried out on an enzyme-labeling instrument by using a 96-pore plate for determination, firstly, the enzyme, each concentration inhibitor and buffer solution are pre-incubated for 3min at 37 ℃, then the probe substrates are added for initiating reaction, 50 mu L of reaction solution is taken after 10min, 50 mu L of luciferase detection reagent is added, then the reaction solution is immediately placed into the enzyme-labeling instrument for detection for 30min, and the detection is carried out once every 1 min. Detection conditions are as follows: full wavelength scanning. Each set of experiments was set up in 3 sets of data in parallel. CES1 inhibitor screening is shown in FIGS. 10 and 11, wherein FIG. 10(a) is a graph showing the result of the inhibition of betulinic acid on the probe substrate (DDM), FIG. 10(b) is a graph showing the result of the inhibition of betulinic acid on the probe substrate (DDM), FIG. 10(c) is a graph showing the result of the inhibition of liquidambaric acid on the probe substrate (DDM), and FIG. 10(d) is a graph showing the result of the inhibition of oleanolic acid on the probe substrate (DDM);
FIG. 11(a) is a graph showing the results of the inhibition of the probe substrate (DDM2) by betulinic acid, FIG. 11(b) is a graph showing the results of the inhibition of the probe substrate (DDM2) by betulinic acid, FIG. 11(c) is a graph showing the results of the inhibition of the probe substrate (DDM2) by liquidaric acid, and FIG. 11(d) is a graph showing the results of the inhibition of the probe substrate (DDM2) by oleanolic acid.
Example 8 detection of residual enzyme Activity of animal tissue Carboxylic esterase 1(CES1)
Preparation of homogenate of S9 preparation of tissue S9: weighing the tissues, shearing the tissues, adding PBS (5 times volume) into the tissues, preparing tissue homogenate in a low-temperature homogenate medium device, centrifuging the tissue homogenate for 20min at 4 ℃ by 9000g, taking supernatant to be the tissue S9, and quickly freezing the tissue at-80 ℃ for subsequent detection.
Detection of carboxylesterase 1(CES1) serum frozen at-80 ℃ and prepared liver tissue S9 are taken to detect the activity level of CES1, and a specific probe substrate NLMe independently developed by the laboratory and used for CES1 detection is used.
Preparing: PBS phosphate buffer solution with pH of 6.5, CES1 probe substrate NLMe (final concentration 2. mu.M), reaction temperature of 37 ℃, and the system is formed as follows:
1) the CES1 total reaction concentration system (volume 100. mu.L) included: 2 μ L of substrate (NLMe), 5 μ L of tissue S9, 93 μ L of PBS phosphate buffer;
2) adding luciferase to start luminescence reaction for 10 min;
3) and (4) placing the mixture into a microplate reader for full-wavelength detection, and measuring the hydrolysis generation amount of the ester bond in unit time as an evaluation index of CES1 activity.
The whole study was performed in triplicate, and the measurement of residual activity of liver CES1 after ANIT-induced cholestatic liver injury in rats is shown in fig. 12, and the measurement of activity of serum CES1 after ANIT-induced cholestatic liver injury in rats is shown in fig. 13.
Example 9 DDM for transfection of LUCs+And different number of cells CES1
3 different cells are selected to test the CES1 imaging effect of the DDM on a cell level, wherein a HepG2 cell is a human liver cancer cell, an HCT116 cell is a human colon cancer cell, a U87 cell is a human brain astrocytoma cell, and the three cells are transfected with a Luciferase reporter gene. According to the principle of probe luminescence, when CES1 exists in cells, DDM is hydrolyzed into carboxyl products by CES1, and then undergoes oxidation reaction with luciferase (Luciferin) to release fluorescence.
All three cells used 5X 105、2.5×105、1×105For three different quantities, we can observe quantity-dependent imaging brightness, and as a contrast we have done a column of 5 × 105Control experiment of number cells plus the CES1 positive inhibitor BNPP, DDM as shown in FIG. 14, was used to transfect LUCs+The results of experiments of imaging of CES1 show that human liver cancer cells have the most CES1, next to colon cancer cells, almost no CES1 exists in brain astrocytoma cells, and positive inhibitors inhibit CES1 at the cell level.
Example 10 DDM for transfection of LUCs+Whole body CES1 imaging in mice
Mice transfected with the Luciferase reporter gene systemically are selected, and the DDM and the previous generation probe DME are injected into the mice simultaneously for imaging and comparison. The left mouse was injected intraperitoneally with 0.1mL, 5mM DME probe, the right mouse was injected intraperitoneally with 0.1mL, 5mM DDM probe, and after injection, both mice were imaged simultaneously, as shown in fig. 15 for DME and DDM contrast for whole-body CES1 imaging of transfected LUC + mice, the DME probe on the left and DDM probe on the right.
Discussion:
according to the results of the above embodiments, it can be seen that, in the application of the bioluminescent fluorescent probe reaction of CES1 provided by the present invention, neither the probe substrate nor the hydrolysis product of the carboxyl ester bond has spontaneous bioluminescence property, and neither interference on detection can be caused, and a bioluminescence detector can be used to realize rapid and sensitive detection of the product. In addition, the kit is not easily interfered by a biological system matrix and impurities in a CES1 activity detection process, can be used for quantitative determination of CES1 enzyme activity in various recombinant CES1, human and animal tissue preparation solutions and various tissue cells, and can also be used for rapid screening of a CES1 inhibitor and quantitative evaluation of inhibition capacity; meanwhile, the probe substrate can be used as a probe substrate of the animal integral CES1 to evaluate the individual and species difference of the metabolic enzyme CES 1.
Furthermore, as a high-specificity CES1 single enzyme fluorescent probe substrate, the compound can be used for detecting the activity of CES1, and is particularly suitable for measuring the activity of CES1 produced by bacteria, insect cells, mammalian cells and yeast clone expression systems, and calibrating the activity of CES1 in preparations such as microsomes derived from various mammalian tissues and organs, S9 and the like.
Whatever the application, the CES1 fluorescent probe substrate has high specificity in detecting the single-enzyme in-vitro activity of CES1, and in the embodiment, DDM can be metabolized into a metabolite by CES1 with high specificity; the kit has easy high-flux detection, can be measured on various common fluorescent enzyme labeling instruments and biochemical instruments in a laboratory in the embodiment, and can carry out batch detection by utilizing a 96 or 386 micropore plate; and the detection result also shows that the kit has high sensitivity, the CES1 can be quantitatively determined by establishing a ratio type standard curve, and the lower limit of detection can reach 5 ng/mL. Therefore, overall, the single-enzyme in vitro activity of the CES1 for detecting the CES1 fluorescent probe substrate has high specificity, high sensitivity, high detection convenience and high flux detection, has different detection modes compared with the fluorescent probe, is basically not interfered by a biological matrix, and has higher selectivity and accuracy.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Claims (9)
1. A specific fluorescent probe substrate for detecting carboxylesterase 1 is characterized by having the following structural general formula:
wherein R is1Selected from H, C1-C10Alkyl, - (C1-C8 alkylene) -carboxy, - (C1-C8 alkylene) -ester, - (C1-C8 alkylene) -amino, - (C1-C8 alkylene) -cyano, - (C1-C8 alkylene) -nitro, - (C1-C3 alkylene) -O- (C1-C3 alkyl), carbocyclyl, - (C1-C3 alkylene) -carbocyclyl, aryl, - (C1-C3 alkylene) -aryl, heteroaryl, - (C1-C3 alkylene) -heteroaryl, heterocyclyl or- (C1-C3 alkylene) -heterocyclyl; r2Is selected from C1-C10An alkyl group.
2.A method for preparing a substrate for a fluorescent probe specific for measuring carboxylesterase 1 according to claim 1, comprising the steps of:
1) carrying out cyano substitution reaction on the compound 2 to obtain a compound 3;
2) the compound 3 undergoes cyano hydrolysis to generate corresponding acid;
3) and carrying out esterification reaction to generate a specific fluorescent probe substrate for detecting carboxylesterase 1.
3. Use of a substrate for a fluorescent probe specific for the detection of carboxylesterase 1 as claimed in claim 1 for the identification of carboxylesterase 1 or for the detection of carboxylesterase 1 activity.
4. A method for measuring carboxylesterase 1 activity, comprising reacting the specific fluorogenic probe substrate of claim 1 with a sample to be measured, measuring the specific fluorogenic probe substrate or a hydrolyzed carboxyl product thereof, and qualitatively or quantitatively measuring carboxylesterase 1.
5. The method of claim 4, wherein the steps comprise:
adding a carboxylesterase 1 specific fluorescent probe substrate into a phosphate buffer system, wherein the reaction temperature is 20-60 ℃, the pH of the incubation system is 5.5-10.5, adding luciferase to start a bioluminescence reaction, and the reaction time is 10-20 min; detecting fluorescence intensity, and taking the reduction amount of the specific fluorescent probe substrate or the generation amount of the carboxyl product as an evaluation index of the activity of the carboxylesterase 1;
or adding a carboxylesterase 1 specific fluorescent probe substrate into a phosphate buffer system, reacting at 20-60 ℃, and reacting for 10-20 min, wherein the pH of the incubation system is 5.5-10.5; detecting the specific fluorescent probe substrate or carboxyl product at 256-365nm by using a liquid phase, and taking the reduction amount of the specific fluorescent probe substrate or the generation amount of the carboxyl product as an evaluation index of the activity of the carboxylesterase 1.
6. The method according to claim 5, wherein the reaction temperature is 34-42 ℃, and the pH of the incubation system is 5.5-7.0.
7. The method of claim 5, wherein the specific fluorescent probe substrate or carboxyl product is detected by HPLC or LCMS at 256-nm.
8. Use of a fluorescent probe substrate specific for the detection of carboxylesterase 1 as claimed in claim 1 for screening or evaluating inhibitors or inducers of carboxylesterase 1.
9. Use of a fluorescent probe substrate specific for the detection of carboxylesterase 1 as claimed in claim 1 for fluorescence bioimaging.
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