Summary of the invention
The object of the present invention is to provide a kind of human carboxylatase CES2(Carboxylesterase2, CES2) specificity fluorescent probe substrate and application, the fluorescence emission wavelengths of this substrate prototype and hydrolysate has notable difference, and product more easily detects.Utilize this probe reaction can carry out quantitative evaluation to the distribution of CES2 in multiple living things system and function.
The invention provides the high specific fluorescent probe substrate of a kind of human carboxylatase CES2, the ester bond of this substrate can be corresponding hydrolysate by CES2 specific for hydrolysis and show the emission spectrum different from substrate;
This substrate is for raw material with 3-hydroxyflavone compounds, corresponding 3-hydroxy ester analog derivative is obtained by esterification, its general structure as the formula (1), wherein, R be phenyl, p-methylphenyl, to ethylphenyl, to propyl group phenyl, to the one in hexyl propyl group, p-nitrophenyl, rubigan, 1-how base, 2-furyl, 2-thienyl, (4-phenyl) phenyl, 4-ethoxyl phenenyl substituting group.
As when R is 4-ethylphenyl, this substrate is 3-hydroxyflavone ester derivative.
Present invention also offers the application of the high specific fluorescent probe substrate of described human carboxylatase CES2, this substrate is as the specific substrate of CES2, its hydrolysis reaction activity can be utilized, measure the activity of CES2 in different enzyme source by the growing amount of hydrolysate in the detection by quantitative unit time; Concrete measuring method is:
---using 3-hydroxyflavone ester derivative as Specific probe in system; Concentration of substrate selects 1/10 ~ 10K
m; Concentration of substrate preferred K during single point assay
m.
---in the conventional damping fluids such as PBS or Tris-HCl, temperature of reaction is between 20 DEG C to 60 DEG C, and preferably 37 DEG C is the peak optimization reaction time; Incubation system pH is between 5.5 ~ 10.5, and preferred pH7.4 is peak optimization reaction pH value;
---the reaction times is 5 ~ 120 minutes, the termination reaction when guaranteeing that the corresponding hydrolysate of above substrate reaches quantitative limit and substrate conversion efficiency is no more than 20%;
---in the analytical unit time, hydrolysate growing amount is as the evaluation index of Procaine esterase CES2 activity.
The application of the high specific fluorescent probe substrate of human carboxylatase 2 provided by the invention, described enzyme source is the recombinant expressed mono-enzyme of CES2, human or animal tissues prepares liquid or various biological sample.
The application of the high specific fluorescent probe substrate of human carboxylatase CES2 provided by the invention, the production rate of described substrate elimination factor or hydrolysate should between 0.1% ~ 20%.
The application of the high specific fluorescent probe substrate of human carboxylatase CES2 provided by the invention, probe substrate and hydrolysate thereof all have fluorescence properties, and the rapid sensitive that fluorimetric detector can be adopted to realize product and substrate detects; Fluoroscopic examination condition is: excitation wavelength 308nm, carries out the detection of fluorescence emission spectrum at 380 ~ 560nm.In addition, this Specific probe and corresponding CES2 Activity determination process can not be subject to the interference of living things system matrix and impurity, can be used for the quantitative assay that in various living things system, CES2 enzyme is lived.
The reaction of this specific probe can be used for restructuring Procaine esterase, people and animal tissues and prepares the quantitative assay that in liquid and various, CES2 enzyme is lived, and also can be used for the inhibitor of rapid screening CES2 enzyme and the quantitative evaluation of rejection ability.
Adopt the mono-enzyme of restructuring Procaine esterase CES2, liver microsomes incubation system is investigated, pass through correlation analysis, specificity Inhibition test, single enzymes metabolism of recombinating reacts, and many-sided evidence such as enzymatic reaction kinetics, prove that 3-hydroxyflavone ester derivative can be specific through Procaine esterase CES2 metabolism (as shown in Figure 5-Figure 8), generate corresponding hydrolysate.
As the fluorescent probe substrate of the Procaine esterase CES2 of high specific, this compound can be used for detecting the activity of Procaine esterase CES2, be especially suitable for the enzyme activity determination to the Procaine esterase CES2 recombinase that bacterium, insect cell, mammalian cell and yeast clonal expression system are produced, and in the prepared product such as tissue microsomal, S-9 of multiple mammalian tissues organ origin, the activity of CES2 is demarcated.
Select the Specific probe of Procaine esterase CES2 of the present invention to detect Procaine esterase CES2 enzyme external activity and there is following outstanding advantage:
(1) high specific: 3-hydroxyflavone ester derivative can be metabolized to a meta-bolites with high specificity by Procaine esterase CES2, i.e. the hydrolysate of 3 ester linkage breakings.
(2) cheap and easy to get: 3-hydroxyflavone ester derivative and hydrolysate thereof all can obtain through chemosynthesis, and synthesis technique is simple.
(3) highly sensitive: the compound with 3-hydroxyl mother nucleus structure all has good fluorescence emission spectral property (380 ~ 560nm), this substrate and hydrolysis meta-bolites thereof have different fluorescence emission spectrum signatures, differentiation can be carried out preferably detect, quantitative assay can be carried out through ratio method by drawing standard curve simultaneously.
Embodiment
The following examples will be further described the present invention, but not thereby limiting the invention.
The chemosynthesis of embodiment 13-flavonol 4-ethylbenzoyl ester
(1) contain in the 3-hydroxyflavone of 0.5mmol and the tetrahydrofuran solution of 0.625mmol triethylamine to 10mL, slowly drip the p-ethylbenzoyl chloride (being dissolved in the tetrahydrofuran (THF) of 5mL) of 0.6mmol, control temperature is at 0 DEG C;
(2), after ice bath stirs 1h, solution is to room temperature, and stirring is spent the night;
(3) reaction solution is through removal of solvent under reduced pressure, and residual solid adopts silica gel chromatography to carry out purifying, adopts ethyl acetate-hexane (1:3v/v) to carry out wash-out, obtains 155mg white solid powder shape sterling.δ (
1h-NMR, 400MHz, CDCl
3): 8.29 (1H, dd), 8.15-8.09 (2H, m), 7.98-7.90 (2H, m), 7.73 (1H, ddd), 7.59 (1H, dd), 7.50-7.42 (4H, m), 7.32 (2H, d), 2.74 (2H, q), 1.35-1.22 (3H, m) .(synthetic route chart is shown in Fig. 2) (hydrogen spectrogram is shown in Fig. 3).
The chemosynthesis of embodiment 23-flavonol 4-chlorobenzoyl ester
(1) contain in the 3-hydroxyflavone of 0.5mmol and the tetrahydrofuran solution of 0.625mmol triethylamine to 10mL, slowly drip the parachlorobenzoyl chloride (being dissolved in the tetrahydrofuran (THF) of 5mL) of 0.6mmol, control temperature is at 0 DEG C;
(2), after ice bath stirs 1h, solution is to room temperature, and stirring is spent the night;
(3) reaction solution is through removal of solvent under reduced pressure, and residual solid adopts silica gel chromatography to carry out purifying, adopts ethyl acetate-hexane (1:3v/v) to carry out wash-out, obtains 152mg white solid powder shape sterling.δ (
1h-NMR, 400MHz, CDCl
3): 8.29 (1H, dd), 8.13 (2H, m), 7.92-.89 (2H, m), 7.77-7.72 (1H, ddd), 7.60 (1H, dd), 7.49-7.46 (6H, m) .(synthetic route chart is shown in Fig. 2) (hydrogen spectrogram is shown in Fig. 4).
The chemosynthesis of embodiment 33-flavonol 4-toluyl ester
(1) contain in the 3-hydroxyflavone of 0.5mmol and the tetrahydrofuran solution of 0.625mmol triethylamine to 10mL, slowly drip 0.6mmol to methyl benzoyl chloride (being dissolved in the tetrahydrofuran (THF) of 5mL), control temperature is at 0 DEG C;
(2), after ice bath stirs 1h, solution is to room temperature, and stirring is spent the night;
(3) reaction solution is through removal of solvent under reduced pressure, and residual solid adopts silica gel chromatography to carry out purifying, adopts ethyl acetate-hexane (1:3v/v) to carry out wash-out, obtains 151mg white solid powder shape sterling.δ (
1h-NMR, 400MHz, CDCl
3): 8.30 (1H, dd), 8.10-8.12 (2H, m), 7.96-7.94 (2H, m), 7.75 (1H, ddd), 7.61 (1H, dd), 7.50-7.44 (4H, m), 7.31 (2H, d), 2.46 (2H, s) .(synthetic route chart is shown in Fig. 2) (hydrogen spectrogram is shown in Fig. 5).
The chemosynthesis of embodiment 43-flavonol 4-propyl ester
(1) contain in the 3-hydroxyflavone of 0.5mmol and the tetrahydrofuran solution of 0.625mmol triethylamine to 10mL, slowly drip 0.6mmol to propyl chlorine (being dissolved in the tetrahydrofuran (THF) of 5mL), control temperature is at 0 DEG C;
(2), after ice bath stirs 1h, solution is to room temperature, and stirring is spent the night;
(3) reaction solution is through removal of solvent under reduced pressure, and residual solid adopts silica gel chromatography to carry out purifying, adopts ethyl acetate-hexane (1:3v/v) to carry out wash-out, obtains 158mg white solid powder shape sterling.δ (
1h-NMR, 400MHz, CDCl
3): 8.29 (1H, dd), 8.12-8.10 (2H, m), 7.95-7.93 (2H, m), 7.72 (1H, ddd), 7.59 (1H, dd), 7.48-7.43 (4H, m), 7.30 (2H, d), 1.70-1.68 (2H, m), 0.98-0.95 (3H, t) .(synthetic route chart is shown in Fig. 2) (hydrogen spectrogram is shown in Fig. 6).
The chemosynthesis of embodiment 53-flavonol 4-oil of mirbane carbamoyl ester
(1) contain in the 3-hydroxyflavone of 0.5mmol and the tetrahydrofuran solution of 0.625mmol triethylamine to 10mL, slowly drip the paranitrobenzoyl chloride (being dissolved in the tetrahydrofuran (THF) of 5mL) of 0.6mmol, control temperature is at 0 DEG C;
(2), after ice bath stirs 1h, solution is to room temperature, and stirring is spent the night;
(3) reaction solution is through removal of solvent under reduced pressure, and residual solid adopts silica gel chromatography to carry out purifying, adopts ethyl acetate-hexane (1:3v/v) to carry out wash-out, obtains 165mg white solid powder shape sterling.δ (
1h-NMR, 400MHz, CDCl
3): 8.39-9.33 (4H, m), 8.29 (1H, dd), 7.89-7.79 (2H, m), 7.77-7.75 (1H, ddd), 7.62 (1H, dd), 7.53-7.46 (4H, m) .(synthetic route chart is shown in Fig. 2) (hydrogen spectrogram is shown in Fig. 7).
Selectivity in the single enzyme of people that embodiment 6 is recombinant expressed
(1) 99 μ l metabolic reaction systems are prepared in advance, comprise the PBS damping fluid (10mM) of pH7.4, recombinant expressed people CES2(5 μ g/ml)/serum albumin (500 μ g/ml)/blood plasma (1%)/butyrylcholine esterase (25U/L)/phosphoric acid buffer, shakes and incubates 10 minutes in advance under 37 DEG C of conditions;
(2) in reaction system, add the 3-hydroxyflavone-to ethylbenzoyl ester/to chlorobenzoyl ester/to toluyl ester/to propyl ester/p-nitrophenyl carbamoyl ester initial action that 1 μ l final concentration is 25 μMs;
After (3) 40 minutes, add 100 μ l ice acetonitriles, after concuss, termination reaction;
(4) fluoroscopic examination (Ex=308nm, Em=392,528nm) is carried out; Calculate fluorescence intensity in each system (see Fig. 8-Figure 12) and (for 3-hydroxyflavone 4-ethylbenzoyl ester by the metabolic pathway of CES2 catalytic hydrolysis, see Figure 13; The ester metabolic pathway of all the other synthesis is consistent with 3-hydroxyflavone 4-ethylbenzoyl ester metabolic pathway);
Embodiment 7 is recombinated the protein concentration of CES2 catalyzed linear reaction in single enzyme
(1) prepare 99 μ l CES2 metabolic reaction systems in advance, comprise the PBS damping fluid (10mM) of pH7.4, recombinant human CES2 (0-100ug/ml), shake under 37 DEG C of conditions and incubate 10 minutes in advance;
(2) in reaction system, add the 3-hydroxyflavone-to ethylbenzoyl ester initial action that 1 μ l final concentration is 25 μMs;
After (3) 40 minutes, add 100 μ l ice acetonitriles, after concuss, termination reaction;
(4) fluoroscopic examination (Ex=308nm, Em=392,528nm) is carried out; Calculate the linear protein concentration (see Figure 14) of recombinant human CES2 enzyme.
Embodiment 8 is recombinated the linear incubation time in single enzyme CES2
(1) prepare 99 μ l CES2 metabolic reaction systems in advance, comprise the PBS damping fluid (10mM) of pH7.4, recombinant human CES2 (8ug/ml), shake under 37 DEG C of conditions and incubate 10 minutes in advance;
(2) in reaction system, add the 3-hydroxyflavone-to ethylbenzoyl ester initial action that 1 μ l final concentration is 25 μMs;
(3) first order fluorescence Scanning Detction (Ex=308nm, Em=392,528nm) was carried out every 0.5 minute; Calculate the linear response time (see Figure 15) of recombinant human CES2 enzyme.
The enzyme that embodiment 9 Quantitative in vitro measures CES2 in the single enzyme of restructuring is lived
(1) prepare 99 μ l CES2 metabolic reaction systems in advance, comprise the PBS damping fluid (10mM) of pH7.4, recombinant human CES2 (2ug/ml), shake under 37 DEG C of conditions and incubate 10 minutes in advance;
(2) in reaction system, add the 3-hydroxyflavone-to ethylbenzoyl ester initial action that 1 μ l final concentration is 25 μMs;
After (3) 40 minutes, add 100 μ l ice acetonitriles, after concuss, termination reaction;
(4) fluoroscopic examination (Ex=308nm, Em=392,528nm) is carried out; The maximum catalytic rate calculating recombinant human CES2 enzyme is 407 ± 31nmol/min/mg.(see Figure 16)
The enzyme that embodiment 10 Quantitative in vitro measures CES2 in people's intestines microsome is lived
(1) prepare 99 μ l people small intestine microsomal metabolism reaction systems in advance, comprise the Tris-HCl damping fluid (50mM) of pH7.4, people's small intestine microsome (20ug/ml), shake under 37 DEG C of conditions and incubate 10 minutes in advance;
(2) in reaction system, add the 3-hydroxyflavone-to ethylbenzoyl ester initial action that 1 μ l final concentration is 25 μMs;
After (3) 40 minutes, add 100 μ l ice acetonitriles, after concuss, termination reaction;
(4) fluoroscopic examination (Ex=308nm, Em=392,528nm) is carried out; Calculating in people small intestine the maximum catalytic rate of this probe compound is 342 ± 23nmol/min/mg.(see Figure 16)
The activity of CES2 in embodiment 11 quantitative assay human pulmonary epithelial cells
(1) human pulmonary epithelial cells system is incubated on cover glass, and the substratum of employing is DMEM substratum (containing 10% calf serum) and 100ug/ml's is dual anti-, and culture environment is in 5% CO2gas incubator of 37 DEG C.
(2) before using, attached cell adopts does not rinse 3 times containing the DMEM substratum of serum, adds 3-hydroxyflavone that final concentration is 10uM-to ethylbenzoyl ester, incubates 40 minutes in 37 DEG C of temperature.
(3), after, PBS wash buffer 3 times are adopted.Observation of cell under laser confocal microscope, carry out the distribution of CES2 in showed cell by fluorescence distribution position and intensity and relative content how many.(see Figure 17)
The inhibitor of the external rapid screening CES2 of embodiment 12
(1) the recombinant expressed single enzyme of CES2 (5 μ g/mL) 196 μ l, shake and incubate 10 minutes in advance under 37 DEG C of conditions;
(2) in reaction system, add 2 μ l Chinese medicine 95% ethanol extracts, continue to hatch 10 minutes;
(3) final concentration adding 2 μ l is the 3-hydroxyflavone of 10 μMs-to ethylbenzoyl ester initial action;
After (3) 40 minutes, add 200 μ l ice acetonitriles, after concuss, termination reaction;
(4) fluoroscopic examination (Ex=308nm, Em=392,528nm) is carried out; Calculate fluorescence intensity, calculate the inhibition strength of CES2 according to the fluorescence intensity ratio of fluorescence intensity ratio and DMSO group under the fluorescence intensity under Chinese medicine extract group 528nm and 392nm.(see Figure 18).