CN1594284A - Enzyme-linked clenbuterol and method for producing same - Google Patents

Abstract

The invention relates to an enzyme-labeled clenbuterol and a preparation method thereof. An enzyme-labeled clenbuterol of the present invention has a structure (as shown in the figure): wherein, the structure of E is: wherein, the preparation method of the present invention comprises: 1. Clenbuterol derivative hemiglutarate Clenbuterol 2. Synthesis of long-arm clenbuterol derivative of clenbuterol, 3. Cross-linking of long-arm clenbuterol with horseradish peroxidase. The enzyme-labeled clenbuterol prepared by the present invention can quickly and accurately detect the β ₂ -stimulant through its affinity with the β ₂ -adrenergic receptor, and the method is simple to operate, low in cost and convenient to use .

Description

Enzyme-labeled clenbuterol and its preparation method

technical field

The invention relates to an enzyme-labeled clenbuterol and a preparation method thereof, in particular to a horseradish peroxidase-labeled long-arm clenbuterol and a preparation method thereof.

Background technique

Among the β ₂ -adrenergic stimulants (abbreviated as β ₂ -stimulants), there is a class of synthetic hormone drugs, such as Clenbuterol (Clenbuterol), Salbutamol (Salbutamol), etc., which are clinically used to treat asthma and bronchospasm, etc. related diseases. However, if the dose is increased by 5-10 times and used for a long time, it also has the side effect of strengthening muscles and reducing fat. This feature makes them often used as growth promoters and added to feed to increase the lean meat rate of livestock (commonly known as clenbuterol). The abuse of these drugs poses a health hazard to consumers.

At present, there are many methods for detecting _β2 -adrenergic stimulants, and the more commonly used methods mainly fall into two categories: one is instrumental analysis, including HPLC, GC/MS, etc., and the second is immune analysis, including RIA, ELISA, etc. , these methods are still unsatisfactory, each has its own shortcomings. Immunoassay is an antigen-antibody combination method based on immunological principles, and has some inherent weaknesses. An antibody can only detect one or several drugs with a common antigenic determinant, and cannot detect all β ₂ -agonists; instruments The analysis is accurate and sensitive, but the operation is cumbersome and cannot be used for routine screening.

Contents of the invention

One of the objectives of the present invention is to provide a horseradish peroxidase-labeled long-armed clenbuterol, which can be used to detect β ₂ -adrenergic receptors through a receptor and ligand detection system. The purpose of detecting β ₂ -agonist is achieved.

The second object of the present invention is to provide the above-mentioned preparation method of enzyme-labeled clenbuterol.

The technical scheme of the present invention is: first synthesize the clenbuterol derivative clenbuterol hemiglutarate; then synthesize the long-arm clenbuterol derivative of clenbuterol, and finally combine the long-arm clenbuterol with horseradish Oxidase cross-linking. Its specific reaction mechanism is as follows:

1. Synthesis of clenbuterol derivative clenbuterol hemiglutarate:

(the same below)

2. Synthesis of Clenbuterol Derivative Long Arm Clenbuterol

3. Cross-linking of long-arm clenbuterol with horseradish peroxidase

To achieve the above object, the present invention adopts the following technical solutions:

An enzyme-labeled clenbuterol, characterized in that the structure of the compound is:

E-NH-HRP

Among them, the structure of E is:

in,

A method for preparing the enzyme-labeled clenbuterol of claim 1, characterized in that the method comprises the steps of:

a. Preparation of clenbuterol hemiglutaric acid A: In the pyridine of clenbuterol, under constant stirring, slowly add the pyridine solution of glutaric anhydride dropwise, wherein the molar ratio of clenbuterol to glutaric anhydride The ratio is 1:1-1.5, and the reaction time is 24 hours. After the reaction is completed, distill under reduced pressure to remove the solvent pyridine; dissolve the residue in ethanol, add 20 times the volume of n-hexane of the above solution and reconstitute at -20°C. Crystallization, so repeated several times, until thin-layer chromatography analysis shows that clenbuterol is not contained in the sample, the developing solvent used is: ethyl acetate and methanol, the volume ratio is 6:4, and the Rf of clenbuterol is 0.82 , the Rf of A is 0.32; A is finally obtained as a white powder, and the yield is about 80%;

b. Preparation of ethylenediamine succinic acid B: prepare ethylenediamine succinic acid B by using a conventional reaction method of amine and acid anhydride;

c. Preparation of long-arm clenbuterol D: use dimethylformamide as solvent, add equimolar amounts of A, N-hydroxysuccinimide and dicyclohexylcarbodiimide, stir for 24 hours, until Thin layer chromatography analysis shows that A has been converted into its active ester form C; then add 1.1-1.2 times of B and triethylamine to the above reaction product, continue the reaction for 24 hours, distill under reduced pressure, and remove the solvent dimethylformamide , then extract the reaction mixture with tetrahydrofuran, retain the tetrahydrofuran layer, distill the extract under reduced pressure, remove the solvent tetrahydrofuran, and obtain the long-arm clenbuterol D; finally dissolve D with dimethylformamide, and use spectrophotometry to determine The concentration of clenbuterol, the developer that adopts is ethyl acetate and methyl alcohol, and its volume ratio is 6: 4, wherein the Rf of reactant A is 0.32, and the Rf of C is 0.84, and the Rf of D is 0.52;

d. Preparation of enzyme-labeled clenbuterol: put the dimethylformamide solution of D in an eppendorf tube, add equimolar amounts of triethylamine and isobutyl chloroformate under ice cooling, and shake at 0°C for 5 -10 minutes, get the mixed acid anhydride E of D, set aside, then add horseradish peroxidase in the eppendorf tube, then add the phosphate buffer solution with a pH value of 7.4, and then press the moles of horseradish peroxidase and E Slowly add the dimethylformamide solution of E dropwise at a ratio of 1:10-20, shake and react at 0°C for 15-30 minutes, separate the reaction mixture with Sephadex G-50, and monitor it with a nucleic acid and protein detector. The yellow fraction was collected, which was the prepared horseradish peroxidase-labeled clenbuterol.

Compared with the prior art, the present invention has the following obvious features and significant advantages: the affinity of the enzyme-labeled clenbuterol prepared by the present invention and the β ₂ -adrenergic receptor can be detected by using the receptor and ligand detection system , can quickly and accurately detect the beta ₂ -stimulant, and the method is simple in operation, low in cost and convenient in use.

Detailed ways

Embodiment one: a preferred embodiment of the present invention is:

1) Preparation of clenbuterol hemiglutarate (A)

Dissolve 100mg of clenbuterol in 2ml of pyridine, slowly add 1.84ml of 20mg/ml glutaric anhydride pyridine solution dropwise under continuous stirring, stir and react for 24 hours, after the reaction is completed, distill under reduced pressure with a water pump to remove the solvent pyridine , add 200 μl of ethanol to dissolve the reactant, then add 4ml of n-hexane to crystallize at -20°C. This crystallization was carried out several times until thin-layer chromatography analysis showed that clenbuterol was no longer contained in the sample, and a white powdery substance was finally obtained with a yield of about 80%. The Rf of clenbuterol was 0.82, and the Rf of clenbuterol hemiglutarate (A) was 0.32 (developing solvent: ethyl acetate:methanol=6:4).

2) Preparation of ethylenediaminosuccinic acid (B)

Dissolve 1.68ml of ethylenediamine in 25ml of water, adjust the pH to 6.0 with 5M sulfuric acid, and slowly add 80ml of 0.5g/ml succinic anhydride in dioxane solution dropwise under vigorous stirring in an ice bath (0°C), and continuously Adjust the pH to 6.0 with 5M NaOH. The reaction solution was reacted at 0°C for 2 hours, and after reacting at 4°C for 16 hours, filtered to remove inorganic salts, the filtrate was concentrated under reduced pressure to about 12ml, adjusted to pH 3.0 with 5M H ₂ SO ₄ , added 60ml of methanol, and filtered to remove inorganic salts , The filtrate precipitated crystals at -20°C, and the crude product was obtained after filtration and drying. After the crude product was dissolved in 3ml of water, 15ml of methanol was added for recrystallization at -20°C to obtain white crystals. The Rf of ethylenediamine succinic acid (B) is 0.56, (developer: isopropanol: glacial acetic acid: water = 3:1:1, developer: 0.25% ninhydrin ethanol solution).

3) Preparation of long arm clenbuterol (D)

Dissolve 15mg of clenbuterol hemiglutarate (A) in 2ml of dimethylformamide, add 4.1mg of N-hydroxysuccinimide and 7.3mg of dicyclohexylcarbodiimide (DCC) and stir at room temperature After at least 24 hours of reaction, TLC analysis showed that clenbuterol hemiglutarate (A) had been converted to its active ester form (C). Add 8mg of B and 14.75ml of triethylamine to the above reaction product, continue the reaction at room temperature for at least 24 hours, distill under reduced pressure with a water pump, remove the solvent dimethylformamide, and then dissolve the final reaction product (D) with tetrahydrofuran , Excess unreacted ethylenediaminosuccinic acid (B) is removed by being more polar and insoluble in tetrahydrofuran. The solution of the reaction product was distilled under reduced pressure with a water pump, and the solvent tetrahydrofuran was removed to obtain the long-armed clenbuterol (D). The long arm clenbuterol (D) was dissolved in dimethylformamide, and the concentration of clenbuterol was determined by spectrophotometry. The Rf of reactant clenbuterol hemiglutarate (A) is 0.32, the Rf of its active ester form (C) is 0.84, and the Rf of long-arm clenbuterol (D) is 0.52 (developing agent: ethyl acetate ester:methanol=6:4).

4) Preparation of enzyme-labeled clenbuterol

Take 16.8 μl of 18.9 mg/ml long-arm clenbuterol (D) in dimethylformamide solution in an eppendorf tube, add 1 μl of 10% triethylamine in dimethylformamide solution and 1.1 μl of 10 % dimethylformamide solution of isobutyl chloroformate, shaken and reacted at 0° C. for 5 to 10 minutes to obtain the mixed anhydride (E) of long-armed clenbuterol (D). The Rf of the long-arm clenbuterol (D) is 0.52, and the Rf of the mixed anhydride (E) is 0.82 (developing solvent: ethyl acetate:methanol=6:4).

Take 1 mg of horseradish peroxidase (HRP) in an eppendorf tube, add 100 μl of 0.01M phosphate buffer (7.4) to dissolve it, place it in an ice bath, and slowly add the above-mentioned 10- to 20-fold molar amount of mixed anhydride (E) dimethylformamide solution, 0 ° C shaking reaction for 15 to 30 minutes, the reaction mixture was separated with Sephadex G-50, and monitored with a nucleic acid protein detector, and the yellow fraction was collected, which was the prepared horseradish peroxidation Biozyme-labeled clenbuterol.

Identification of enzyme-labeled clenbuterol

i) Optical identification

Use 0.01M phosphate buffer (7.4) to configure the solutions of clenbuterol and horseradish peroxidase with concentrations of 0.1mg/ml and 0.2mg/ml respectively, and use a Shimadzu spectrophotometer to test the two solutions respectively Scan between 180nm-700nm, the results show that they have the largest characteristic absorption peaks at 295nm and 403nm respectively, and then measure the 0.1mg/ml clenbuterol solution and 0.2mg/ml horseradish peroxidase solution at 295nm and the absorbance at 403nm are as follows: concentration A _295nm A _403nm Clenbutero 0.1mg/ml 0.796 0 Horseradish peroxidase 0.2mg/ml 0.164 0.492 Enzyme-labeled clenbuterol m mol/l 0.179 0.576

Clenbuterol: KAam＝0.796/(0.1/313)＝2491/mol/l 295nm

KAbm＝0/mol/l 403nm

Horseradish peroxidase: KBam＝0.114/(0.2/40000)＝22725/mol/l 295nm

      KBbm＝0.492/(0.2/40000)＝98400/mol/l 403nm

Wherein, KAam, KAbm respectively represent the molar absorptivity of clenbuterol at 295nm and 403nm, KBam, KBbm respectively represent the molar absorptivity of horseradish peroxidase at 295nm and 403nm, and n represents the gram The number of renbuterol, m represents the molar concentration of the enzyme standard ligand (no need to know the specific concentration). List the following equation, the molar concentration of the cross-linked clenbuterol × its molar absorptivity + the molar concentration of the labeled enzyme × its molar absorptivity = the absorptivity of the enzyme-labeled clenbuterol (two wavelengths list two equation)

That is, one molecule of horseradish peroxidase is labeled with 1.53 molecules of clenbuterol.

ii) Immunological detection

Principle: Use the clenbuterol antibody immobilized on the microtiter plate and the affinity of clenbuterol in the enzyme-labeled clenbuterol to identify the success of clenbuterol and horseradish peroxidase, that is, in the package Add enzyme-labeled clenbuterol diluted with diluent buffer to the microtiter plate with clenbuterol antibody, and wash the microtiter plate with washing buffer several times after incubation reaction, if clenbuterol and horseradish peroxide If the enzyme is cross-linked successfully, the horseradish peroxidase will not be washed off on the microtiter plate due to the labeling of clenbuterol, and the enzyme substrate TMB will be added to develop color; if clenbuterol and horseradish are cross-linked If the oxidase cross-linking fails, the horseradish peroxidase will not be able to bind to the microtiter plate and be washed away. Adding the enzyme substrate TMB will not develop color. The specific method is as follows:

① Prepare diluent/washing buffer

② Dilute enzyme-labeled clenbuterol (0.114mg/ml) and horseradish peroxidase (0.114mg/ml) with diluent buffer (1:200)

③Take six ELISA plates coated with clenbuterol antibody, divide them into two groups, add 120 μl of 1:200 dilution of enzyme-labeled clenbuterol and horseradish peroxidase, and incubate at 37°C for one hour

④ Pour off the enzyme solution and blot dry with filter paper

⑤Wash the plate 4 times with washing buffer, 10 minutes each time

⑥Add 125μl substrate TMB to each well and react for 20 minutes

⑦Add 50μl 5M sulfuric acid to stop the reaction

⑧Use a microplate reader to read as follows: number 1 number 2 number 3 Enzyme-labeled clenbuterol 0.807 0.823 0.812 Horseradish peroxidase 0.033 0.027 0.031

In the above experiments, the experimental group that added enzyme-labeled clenbuterol had a color reaction, while the blank control group that added horseradish peroxidase had no color reaction, which indicated that the interaction between horseradish peroxidase and clenbuterol Union is a success.

The sequence of HRP is:

qltptfydns cpnvsnivrd tivnelrsdp riaasilrlh fhdcfvngcd asilldntts

frtekdafgn ansargfpvi drmkaavesa cprtvscadl ltiaaqqsvt lagpswrvp

lgrrdslqaf ldlananlpa pfftlpqlkd sfrnvglnrs sdlvalsggh tfgknqcrfi

mdrlynfsnt glpdptlntt ylqtlrglcp lngnlsalvd fdlrtptifd nkyyvnleeq

kgliqsdqel fsspnatdti plvrsfanst qtffnafvea mdrmgnitpl tgtqgqirln

crvvnsns

Claims (2)

1. an enzyme-labeled clenbuterol, characterized in that the structure of the compound is: E——NH——HRP Among them, the structure of E is:

in,

2. A method for preparing the enzyme-labeled clenbuterol of claim 1, characterized in that the method comprises the steps of: a. Preparation of clenbuterol hemiglutaric acid A: In the pyridine of clenbuterol, under constant stirring, slowly add the pyridine solution of glutaric anhydride dropwise, wherein the molar ratio of clenbuterol to glutaric anhydride The ratio is 1:1-1.5, and the reaction time is 24 hours. After the reaction is completed, distill under reduced pressure to remove the solvent pyridine; dissolve the residue in ethanol, add 20 times the volume of n-hexane of the above solution and reconstitute at -20°C. Crystallization, so repeated several times, until thin-layer chromatography analysis shows that clenbuterol is not contained in the sample, the developing solvent used is: ethyl acetate and methanol, the volume ratio is 6:4, and the Rf of clenbuterol is 0.82 , the Rf of A is 0.32; the final white powder is obtained; b. Preparation of ethylenediamine succinic acid B: prepare ethylenediamine succinic acid B by using a conventional reaction method of amine and acid anhydride; c. Preparation of long-arm clenbuterol D: use dimethylformamide as solvent, add equimolar amounts of A, N-hydroxysuccinimide and dicyclohexylcarbodiimide, stir for 24 hours, until Thin layer chromatography analysis shows that A has been converted into its active ester form C; then add 1.1-1.2 times of B and triethylamine to the above reaction product, continue the reaction for 24 hours, distill under reduced pressure, and remove the solvent dimethylformamide , then extract the reaction mixture with tetrahydrofuran, retain the tetrahydrofuran layer, distill the extract under reduced pressure, remove the solvent tetrahydrofuran, and obtain the long-arm clenbuterol D; finally dissolve D with dimethylformamide, and use spectrophotometry to determine The concentration of clenbuterol, the developer that adopts is ethyl acetate and methyl alcohol, and its volume ratio is 6: 4, wherein the Rf of reactant A is 0.32, and the Rf of C is 0.84, and the Rf of D is 0.52; d. Preparation of enzyme-labeled clenbuterol: put the dimethylformamide solution of D in an eppendorf tube, add equimolar amounts of triethylamine and isobutyl chloroformate under ice cooling, and shake at 0°C for 5 -10 minutes, get the mixed acid anhydride E of D, set aside, then add horseradish peroxidase in the eppendorf tube, then add the phosphate buffer solution with a pH value of 7.4, and then press the moles of horseradish peroxidase and E Slowly add the dimethylformamide solution of the mixed acid anhydride E in a ratio of 1:10-20, shake and react at 0°C for 15-30 minutes, separate the reaction mixture with Sephadex G-50, and use a nucleic acid and protein detector Monitor and collect the yellow fraction, which is the prepared horseradish peroxidase-labeled clenbuterol.