CN106928079A - Trefanocide haptens and its preparation method and application - Google Patents
Trefanocide haptens and its preparation method and application Download PDFInfo
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- CN106928079A CN106928079A CN201710177395.6A CN201710177395A CN106928079A CN 106928079 A CN106928079 A CN 106928079A CN 201710177395 A CN201710177395 A CN 201710177395A CN 106928079 A CN106928079 A CN 106928079A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/18—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to carbon atoms of six-membered aromatic rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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Abstract
The invention discloses trefanocide haptens and its preparation method and application, it has following structural formula：Wherein, R is (CH2)n, n=1 4 or CH=CH or
Description
Technical field
The present invention relates to immunological technique field, and in particular to trefanocide haptens and its preparation method and application.
Background technology
Trefanocide (trifluralin) is dinitroaniline (dinitroaniline) before a kind of important selective bud
Herbicide, is widely used in the crops such as soybean, barley, vegetables and fruit, prevents and kill off annual gramineous weed and part broad leaved weed.
Though trefanocide belongs to low-toxin farm chemicals, long-term exposure experiment shows that liver and kidney can be caused to damage, the meeting under high dose exposure
Cause the body weight and height of fetus relatively low and increase risk of miscarriage.Because of widely using for trefanocide agricultural chemicals, make it in soil and water
Residual generally existing in the middle of body, particularly can in vivo be enriched with, in aquatic products (fish, shrimp, crab) so as to be eaten to aquatic products
Safety belt carrys out huge hidden danger.
In order to avoid the potential hazard of Basalin, many countries are used for being limited.European Union 2015
Formulate trefanocide new limit standard, water fruits and vegetables, cereal and in animal-derived food trefanocide MRL
(MRLs) it is 0.01mg/kg.Japan is 0.001mg/kg to trefanocide MRL in import aquatic products.
The method of the usually used Instrumental Analysis of the detection of trefanocide, such as ultraviolet spectra, high-efficient liquid phase color in current agricultural product
Spectrum and LC-MS, gas-chromatography, Capillary Electrophoresis etc..The characteristics of analysis method above has accurate, sensitive, but due to instrument
Device is expensive, operating cost is high-leveled and difficult with all-round popularization.It was verified that primary dcreening operation is carried out to a large amount of samples with method for quick, then
It is to improve detection efficiency, the effective means of reduces cost that instrument confirmation is carried out to wherein positive, is tallied with the national condition.Existing
In having a Fast Detection Technique, the immunoassay technology based on Ag-Ab specific recognition due to it is sensitive, quick, accurate,
It is with low cost and the advantages such as Site Detection can be realized, it is considered to be the ultramicron detection skill of 21 century most competitive and challenge
Art.
The external only several reports of document on trefanocide immune detection.Bruce Riggle were attempted in 1991 first
Construct the enzyme-linked immune analytic method for trefanocide, 0.1~1.0ppm of the range of linearity (ng/mL), while can be with test section
Divide the metabolin of trefanocide.Sz é k á cs et al have carried out again attempting to and establishing for 2000 to trefanocide immune detection
ELISA method, the linear scope of detection increases (0.1~100ng/mL), the cross reaction with other structures analog
Rate≤5%, and have detected the trefanocide content in surface water and fruit juice.Sz é k á cs seminars established and are based on again in 2003
Spectral detection (the optical waveguide lightmode spectroscopic of the light wave mediation of immunosensor
Detection, OWLS) method, although the method substantially increases detection sensitivity (2 × 10-7~3 × 10-5ng/mL), but
It is this kind of method because instrument condition problem is difficult in actually detected middle application.At present, the country has no relevant trefanocide immune detection
Relevant report.Country's detection in the market is more the absence of the product of trefanocide residues of pesticides immunity detection reagent.
Currently the majority medicine, toxin, environmental contaminants molecular mass are smaller, belong to haptens, most of haptens
Need that with after protein molecule immunogenicity could be produced.The conventional method for preparing hapten antibody at present is that selection has toxicity
The original shape medicine of meaning is learned as determinand, determinand molecular characterization or similar structures feature are retained simultaneously by designing synthesis
Haptens with active group, by haptens and macro-molecular protein carrier conjugation, prepares artificial immunogen, while through dynamic
Thing is immunized the specific antibody prepared for haptens.And the offer of haptens is the most key step in immunochemistry analysis
Suddenly, rational hapten design and preparation is beneficial to the raising of the quality of antibody.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, there is provided a series of to can be universally used in the partly anti-of trefanocide detection
Original, can make body produce potency high after the coupled carrier protein of haptens, can recognize the special of trefanocide agricultural chemicals apokoinou construction
Property antibody.
It is a further object to provide the preparation method and application of the haptens.
The technical problems to be solved by the invention are achieved by the following technical programs：
A kind of trefanocide haptens, it is characterised in that with following structural formula：
Wherein, R is-(CH2)n-, n=1-4 or-CH=CH- or
Trefanocide haptens of the present invention specifically has the structural formula shown in following 1c, 2c, 3c, 4c, 5c and 6c：
It is below the description of the preparation process of hapten molecule of the invention.
1. the reaction equation involved by hapten synthesis of the present invention is expressed as follows：
1c：R=- (CH2)-1
2c：R=- (CH2)-2N=1
3c：R=- (CH2)-3N=2
4c：R=- (CH2)-4N=3
5c：R=- (CH=CH)-
6c：
2. six kinds of trefanocide hapten synthesis steps involved in the present invention are as follows：
(1) when trefanocide haptens structure is 1c, 2c, 3c or 4c, its synthesis step includes：In the presence of a catalyst, by substrate
Reaction is alkylated with alkylating reagent, secondary amine intermediate is obtained after purification, the secondary amine intermediate reaction that is hydrolyzed obtains the
Two intermediates, the second intermediate 3,5- dinitro-p-trifluorotoluenes hybrid reaction chloro- with 4- again, it is purified after obtain target haptens
Molecule.
Further, ethyl acrylate is dissolved in chloroform, adds n-propylamine, wherein, ethyl acrylate and n-propylamine rub
You are than being 1: 1.5,40 DEG C of oil baths, stirring reaction, secondary amine intermediate of the acquisition containing ester group；Secondary amine intermediate containing ester group is dissolved
In tetrahydrofuran, sodium hydrate aqueous solution is added, be stirred at room temperature and be hydrolyzed, obtain the second intermediate；In the middle of above-mentioned second
Chloro- 3, the 5- dinitro-p-trifluorotoluenes of 4- are added in body, are stirred at room temperature, reaction carries out soda acid and falls to extract after terminating, merge organic phase,
With anhydrous sodium sulfate drying, solvent evaporated is rotated, obtain hapten molecule (1c).
Further, 2-Pyrrolidone is dissolved in DMF, sequentially adds 60% sodium hydride solution and n-Propyl Bromide, wherein,
The mol ratio of 2-Pyrrolidone, sodium hydride and n-Propyl Bromide is：1: 1.2: 1.3, under nitrogen protection, 70 DEG C of oil baths, stirring is anti-
Should；After reaction terminates, extractive reaction mixed liquor merges organic phase, and anhydrous sodium sulfate drying rotates solvent evaporated, obtains and contain ester
The secondary amine intermediate of base；To 4N sodium hydrate aqueous solutions are added in the secondary amine intermediate containing ester group, it is stirred overnight in 100 DEG C of oil baths
It is hydrolyzed, obtains the second intermediate；Chloro- 3, the 5- dinitro-p-trifluorotoluenes of 4- are dissolved in tetrahydrofuran, above-mentioned second is added
The mol ratio of intermediate, chloro- 3, the 5- dinitro-p-trifluorotoluenes of 4- and the second intermediate is 1: 1.5, and reaction is stirred at room temperature；React
It is diluted with water after complete, plus watery hydrochloric acid is acidified and is extracted with ethyl acetate, organic phase saturated common salt water washing, anhydrous sodium sulfate is done
It is dry, solvent evaporated is rotated, obtain hapten molecule (2c).
Further, valerolactam is dissolved in DMF, sequentially adds 60% sodium hydride solution and n-Propyl Bromide, wherein, penta
The mol ratio of lactams, sodium hydride and n-Propyl Bromide is：1: 1.2: 1.2, under nitrogen protection, 70 DEG C of oil baths, stirring reaction；Instead
After should terminating, it is diluted with water, and adds acid for adjusting pH to 2-3, sediment occur, is extracted with ethyl acetate, then use saturated aqueous common salt
Washing, anhydrous sodium sulfate drying rotates solvent evaporated, obtains the secondary amine intermediate containing ester group；To the secondary amine intermediate containing ester group
It is middle to add 37% hydrochloric acid and water, 100 DEG C of oil baths, condensing reflux to be hydrolyzed, obtain the second intermediate；By the chloro- 3,5- dinitros of 4-
Base benzotrifluoride is dissolved in tetrahydrofuran, adds above-mentioned second intermediate, triethylamine and water, chloro- 3, the 5- dinitros fluoroforms of 4-
The mol ratio of benzene and the second intermediate is 1:1.2, reaction is stirred at room temperature；It is diluted with water after reaction completely, plus watery hydrochloric acid acidifying is simultaneously
It is extracted with ethyl acetate, organic phase saturated common salt water washing, anhydrous sodium sulfate drying, rotates solvent evaporated, obtains haptens
Molecule (3c).
Further, caprolactam is dissolved in DMF, sequentially adds 60% sodium hydride solution and n-Propyl Bromide, wherein, penta
The mol ratio of lactams, sodium hydride and n-Propyl Bromide is：1:1.2:1.2, under nitrogen protection, 70 DEG C of oil baths, stirring reaction；Instead
It is diluted with water after answering completely, plus watery hydrochloric acid is acidified and is extracted with ethyl acetate, organic phase saturated common salt water washing, anhydrous slufuric acid
Sodium is dried, and rotates solvent evaporated, obtains the secondary amine intermediate containing ester group；To adding 37% hydrochloric acid in the secondary amine intermediate containing ester group
And water, 100 DEG C of oil baths, condensing reflux is hydrolyzed, and obtains the second intermediate；By the chloro- 3,5- dinitro-p-trifluorotoluenes dissolvings of 4-
In tetrahydrofuran, above-mentioned second intermediate, triethylamine and water, chloro- 3, the 5- dinitro-p-trifluorotoluenes of 4- and the second intermediate are added
Mol ratio be 1:3, reaction is stirred at room temperature；It is diluted with water after reaction completely, plus watery hydrochloric acid is acidified and is extracted with ethyl acetate, and has
Machine mutually uses saturated common salt water washing, anhydrous sodium sulfate drying to rotate solvent evaporated, obtain hapten molecule (4c).
The present inventor attempts finding by many-sided research, when trefanocide haptens structure is 1c, by appropriate
Its preparation process condition of variable and parameter, secondary amine intermediate of the generation containing ester group, protect carboxyl not reacted with other groups,
This step substantially increases the purity and yield of trefanocide haptens.When trefanocide haptens structure is 2c, 3c or 4c, inventor
Selected in numerous substrates and alkylating reagent, by its preparation process condition of appropriate variable and parameter, greatly improved
The purity and yield of trefanocide haptens.
(2) when trefanocide haptens structure is 5c or 6c, its synthesis step includes：Substrate is with n-propylamine by being acylated
Reaction, secondary amine intermediate of the generation containing ester group, 3,5- dinitro-p-trifluorotoluenes chloro- with 4- are anti-again for the secondary amine intermediate containing ester group
Should, it is purified after obtain the second intermediate, the second intermediate is hydrolyzed reaction again.
Further, 4- crotonic ethyl bromides are dissolved in ethyl acetate, reactor are placed in ice-methyl alcohol and are cooled to -15
DEG C, n-propylamine is slowly added to syringe, 4- crotonic ethyl bromides are 1 with the mol ratio of n-propylamine:3, stirring at normal temperature reaction.Instead
After should terminating, ethyl acetate extractive reaction mixed liquor, then washed with saturated nacl aqueous solution, organic phase is dry with anhydrous sodium sulfate
It is dry, solvent evaporated is rotated, obtain the secondary amine intermediate containing ester group；Secondary amine intermediate containing ester group is dissolved in tetrahydrofuran, plus
Enter chloro- 3, the 5- dinitro-p-trifluorotoluenes of 4- and triethylamine, be stirred at room temperature reaction, chloro- 3, the 5- dinitro-p-trifluorotoluenes of 4- with contain ester
The mol ratio of the secondary amine intermediate of base is 1:1.5；It is diluted with water after reaction completely, plus watery hydrochloric acid is acidified and is extracted with ethyl acetate
Take, organic phase saturated common salt water washing, anhydrous sodium sulfate drying, rotate solvent evaporated, residue through column chromatographic isolation and purification,
Rotation solvent evaporated obtains hapten molecule (5c).
Further, 4- bromomethyl-benzoic acid methyl esters are dissolved in ethyl acetate, reactor are placed in ice-methyl alcohol and are lowered the temperature
To -15 DEG C, n-propylamine is slowly added to syringe, 4- bromomethyl-benzoic acid methyl esters are 1 with the mol ratio of n-propylamine:2.5.Reaction
After end, reaction mixture is extracted with ethyl acetate, then is washed with saturated nacl aqueous solution, organic phase merges, anhydrous sodium sulfate
Dry, rotate solvent evaporated, obtain the secondary amine intermediate containing ester group；Secondary amine intermediate containing ester group is dissolved in tetrahydrofuran,
Add chloro- 3, the 5- dinitro-p-trifluorotoluenes of 4- and triethylamine, be stirred at room temperature reaction, chloro- 3, the 5- dinitro-p-trifluorotoluenes of 4- with contain
The mol ratio of the secondary amine intermediate of ester group is 1:1.5；It is diluted with water after reaction completely, plus watery hydrochloric acid is acidified and is extracted with ethyl acetate
Take, organic phase saturated common salt water washing, anhydrous sodium sulfate drying, rotate solvent evaporated, residue through column chromatographic isolation and purification,
Rotation solvent evaporated obtains hapten molecule (6c).
The present invention also provides a kind of trefanocide artificial immunogen and coating antigen, and it is by above-mentioned trefanocide haptens and carrier
Albumen coupling is obtained.
The present invention also provides a kind of trefanocide antibody, and the trefanocide antibody is prepared using above-mentioned trefanocide artificial immunogen
Obtain, the trefanocide antibody is polyclonal antibody, monoclonal antibody or genetic engineering antibody.
The present invention has the advantages that：
(1) six kinds of haptens of the invention are derivative connection arm, active carboxyl, with carrier protein in amino sites
Body can be made to produce potency high after coupling, the specific antibody of trefanocide agricultural chemicals apokoinou construction can be recognized.
(2) hapten compound of the invention, not only simple synthetic method, purity are higher, and it is suitable to can apply to synthesis
In the antigen system of animal immune, the blank of domestic trefanocide immunological detection method technical field is compensate for, be that trefanocide is exempted from
The further development of epidemic disease detection method is laid a good foundation.
(3) present invention is improved to the preparation method of trefanocide haptens, and synthesis step is simple, required experiment condition
Gently, preparation method is simple of the present invention is feasible, cost is relatively low, and the purity and yield of haptens are higher.
Specific embodiment
With reference to embodiment, the present invention will be described in detail, and embodiment is only the preferred embodiment of the present invention,
It is not limitation of the invention.
Embodiment 1：The synthetic method of trefanocide hapten compound 1c is as follows：
(1) ethyl acrylate (3600mg, 36mmol) is dissolved in the chloroform of 30mmol, adds n-propylamine (3191.94mg, 54mmol)
40 DEG C of stirring 12h of oil bath, obtain the secondary amine intermediate 1a containing ester group.
(2) 1a (239mg, 1.5mmol) is dissolved in 6ml tetrahydrofurans, adds the NaOH aqueous solution (120mg, 3mmol),
It is stirred at room temperature, is hydrolyzed, 1b is obtained after hydrolysis.
(3) directly toward adding the chloro- benzotrifluorides of 3,5- dinitros -4- (270mg, 1mmol), stirring at normal temperature in 1b.With than
Example is n-hexane:Ethyl acetate=2:1 chromatographic solution carries out a plate tracking, soda acid is carried out after the completion of question response and falls to extract, acid extraction
Organic phase merges, and uses anhydrous sodium sulfate drying.40 DEG C are spin-dried for, and are n-hexane with chromatographic solution:Ethyl acetate:Acetic acid=2:1:
Silica gel plate is separated on 0.015, obtains object 1c, is weighed, and yield is 75.6%.ESI-MS analysis(negative):m/
z 366.2[M+H]—；1H NMR(600MHz,CDCl3)δ8.03(s,2H)；3.29 (t, J=6.3Hz, 2H)；2.89–2.84(m,
2H)；2.57–2.51(m,2H)；1.60–1.50(m,2H)；0.81 (t, J=7.4Hz, 3H).
Embodiment 2：The synthetic method of trefanocide hapten compound 2c is as follows：
(1) 2-Pyrrolidone (1000mg, 11mmol) is dissolved in 10mlDMF, adds 13.2mmol NaH (60%), n-Propyl Bromide
(1758.9mg, 14.3mmol), under nitrogen protection, 70 DEG C of oil baths are stirred overnight.70ml ethyl acetate is added in reaction solution,
50ml saturated ammonium chloride solutions are extracted, and organic phase merges, anhydrous sodium sulfate drying.40 DEG C are spin-dried for, and obtain secondary containing ester group
Amine intermediate 2a.
(2) 2a (846mg, 6mmol) the addition 13ml 4N NaOH aqueous solution is stirred overnight into water-filling in 100 DEG C of oil baths
Solution, obtains 2b.
(3) the chloro- benzotrifluorides of 3,5- dinitros -4- (270mg, 1mmol) is dissolved in 6ml tetrahydrofurans, adds 2b
(190.5mg, 1.5mmol), is stirred at room temperature.It is diluted with 30ml water, plus 1M hydrochloric acid adjusts pH to 2-3, sediment to occur.Use second
Acetoacetic ester is extracted twice, and each ethyl acetate consumption is 30ml, and precipitation dissolving, 10ml saturated aqueous common salts are washed, organic phase
Merge, use anhydrous sodium sulfate drying.40 DEG C are spin-dried for, and are n-hexane with chromatographic solution:Ethyl acetate=1:Silica gel plate is divided on 1
From, object 2c is obtained, to weigh, yield is 82.4%.ESI-MS analysis(negative):m/z380.1[M+H]—；1H
NMR(600MHz,CDCl3)δ8.00(s,2H)；3.07–2.98(m,2H)；2.94–2.86(m,2H)；2.34 (t, J=7.2Hz,
2H)；1.89–1.79(m,2H)；1.55 (dd, J=14.9,7.5Hz, 2H)；0.81 (t, J=7.4Hz, 3H).
Embodiment 3：The synthetic method of trefanocide hapten compound 3c is as follows：
(1) valerolactam (2180mg, 20mmol) is dissolved in 10mlDMF, adds 24mmol NaH (60%), n-Propyl Bromide
(2950mg, 24mmol), 70 DEG C of oil bath stirrings, reacts 4-5 hours under nitrogen protection.It is diluted with 50ml water, plus 1M salt
Acid adjusts pH to 2-3, sediment to occur.It is extracted with ethyl acetate twice, each ethyl acetate consumption is 50ml, precipitation dissolves,
15ml saturated aqueous common salts are washed, and organic phase merges, and uses anhydrous sodium sulfate drying.40 DEG C are spin-dried for, and obtain the secondary amine containing ester group
3a, weighs, and yield is 55%.
(2) hydrochloric acid of 2.5ml 37% and 2.5ml water, 100 DEG C of oil bath condensing refluxes is added to enter in 3a (846mg, 6mmol)
Water-filling solution 4-5 hours, obtains hydrolysate 3b.
(3) the chloro- benzotrifluorides of 3,5- dinitros -4- (270mg, 1mmol) is dissolved in 6ml tetrahydrofurans, adds 3b
(235mg, 1.2mmol), triethylamine (404mg, 4mmol) and 1ml water, stirring at normal temperature is overnight.It is diluted with 30ml water, plus
1M hydrochloric acid adjusts pH to 2-3, sediment to occur.It is extracted with ethyl acetate twice, each ethyl acetate consumption is 30ml, is precipitated molten
Solution, is washed with 10ml saturated aqueous common salts, and organic phase merges, and uses anhydrous sodium sulfate drying.40 DEG C are spin-dried for, with chromatographic solution for just
Hexane：Ethyl acetate：Acetic acid=4：1：Silica gel plate is separated on 0.02, obtains object 3c, and weigh 72mg, and yield is
65.3%.ESI-MS analysis(negative):m/z 394.3[M+H]—；1H NMR(600MHz,CDCl3)δ8.06(s,
2H)；3.04 (t, J=6.9Hz, 2H)；3.01–2.94(m,2H)；2.36 (t, J=6.8Hz, 2H)；1.74–1.50(m,6H)；
0.88 (t, J=7.4Hz, 3H).
Embodiment 4：The synthetic method of trefanocide hapten compound 4c is as follows：
(1) caprolactam (2260mg, 20mmol) is dissolved in 10mlDMF, adds 24mmol NaH (60%), n-Propyl Bromide
(2950mg, 24mmol), 70 DEG C of oil bath stirrings, reacts 2h under nitrogen protection.It is diluted with 50ml water, plus 1M hydrochloric acid adjusts pH
To 2-3, sediment occurs.It is extracted with ethyl acetate twice, each ethyl acetate consumption is 50ml, precipitation dissolving is full with 15ml
Washed with saline solution, organic phase merges, and uses anhydrous sodium sulfate drying.40 DEG C are spin-dried for, and obtain the secondary amine intermediate containing ester group
4a, weighs, and yield is 55%.
(2) 4a (930mg, 6mmol) the additions hydrochloric acid of 2.5ml 37% and 2.5ml water, 100 DEG C of oil bath condensing refluxes are carried out
Hydrolyse over night, obtains hydrolysate 4b.
The chloro- benzotrifluorides of (3) 3,5- dinitro -4- (450mg, 1.67mmol) is dissolved in 15ml tetrahydrofurans, adds 4b
(1047.5mg, 5mmol), triethylamine (1686.7mg, 16.7mmol) and 2ml water, stirring at normal temperature is overnight.Carried out with 90ml water
Dilution, plus 1M hydrochloric acid adjusts pH to 2-3, sediment to occur.It is extracted with ethyl acetate twice, each ethyl acetate consumption is 90m, is sunk
Form sediment and dissolve, washed with 30ml saturated aqueous common salts, organic phase merges, and uses anhydrous sodium sulfate drying.40 DEG C are spin-dried for, with just oneself
Alkane：Ethyl acetate=20：1、10：1、5：1、4：1 chromatography liquid proportional carries out column chromatography for separation, and 40 DEG C are spin-dried for, and obtains object
4c, weigh 73.2mg, and yield is 58%.ESI-MS analysis(negative)m/z 408.3[M+H]—；1H NMR
(600MHz,CDCl3)δ8.06(s,2H)；3.1 (t, J=7.2Hz, 2H)；2.98-2.93(m,2H)；2.34 (t, J=7.2Hz,
2H)；1.64-1.57(m,6H)；1.37-1.29(m,2H)；0.88 (t, J=7.8Hz, 3H).
Embodiment 5：The synthetic method of trefanocide hapten compound 5c is as follows：
(1) 4- crotonic ethyl bromides (965mg, 5mmol) are dissolved in 15ml ethyl acetate, and it is (cold in advance that reactor is placed in into ice-methyl alcohol
Freeze) in be cooled to -15 DEG C, be slowly added to n-propylamine (885mg, 15mmol), stirring at normal temperature 4-5 hours with syringe.Use 80ml
Ethyl acetate is extracted, 30ml washings, the washing of 10ml saturated nacl aqueous solutions, organic phase anhydrous sodium sulfate drying.Less than 30 DEG C
It is spin-dried for, obtains the secondary amine intermediate 5a containing ester group, is weighed as 874mg.
(2) 5a (256.5mg, 1.5mmol) is dissolved in 5ml tetrahydrofurans, adds 3, the 5- dinitros chloro- benzotrifluorides of -4-
(270mg, 1mmol), triethylamine (101mg, 1mmol), is stirred overnight at room temperature.It is n-hexane with chromatographic solution：Ethyl acetate=10：
1 is tracked, and adds 70ml water to be diluted after reaction completely, plus 1M hydrochloric acid adjusts pH to 2-3, sediment to occur.Use acetic acid second
Ester is extracted twice, and each ethyl acetate consumption is 70ml, and precipitation dissolving is washed with 20ml saturated aqueous common salts, organic to be harmonious
And, use anhydrous sodium sulfate drying.40 DEG C are spin-dried for, and use n-hexane:Ethyl acetate=40:1、20:1、10:1 chromatography liquid proportional enters
Row column chromatography for separation, 40 DEG C are spin-dried for, and obtain intermediate 5b, and weigh 276.6mg, and yield is 68.29%.
(3) 5b (352mg, 0.87mmol) adds 10ml tetrahydrofurans, the hydrochloric acid of 13ml 37% stirred in 50 DEG C of oil bath pans
At night, be n-hexane with chromatographic solution：Ethyl acetate：Acetic acid=2:1:0.01 is tracked, and adds 120ml water to carry out after reaction completely
Dilution, plus 1M hydrochloric acid adjusts pH to 2-3, sediment to occur.Extracted with 120ml*2 ethyl acetate, precipitation dissolving is eaten with 40ml saturations
Salt solution is washed, and organic phase merges, and uses anhydrous sodium sulfate drying.40 DEG C are spin-dried for, and are petroleum ether with chromatographic solution:Ethyl acetate:
Acetic acid=10:1:0.05、10:2:0.05、10:4:0.05 order carries out column chromatography for separation, is spin-dried for, and weigh 122.9mg, yield
It is 76.9%.ESI-MS analysis(negative)m/z 378.2[M+H]—；1H NMR(600MHz,CDCl3)δ8.10(s,
2H)；7.12–6.72(m,1H)；6.03 (d, J=15.7Hz, 1H)；3.02–2.90(m,2H)；2.17(s,2H)；1.66–1.48
(m,2H)；0.86 (dd, J=15.9,8.5Hz, 3H).
Embodiment 6：The synthetic method of trefanocide hapten compound 6c is as follows：
(1) 4- bromomethyl-benzoic acid methyl esters (916.28mg, 4mmol) are dissolved in 12ml ethyl acetate, by reactor as ice-methyl alcohol
In be cooled to -15 DEG C, with syringe be slowly added to n-propylamine (591.1mg, 10mmol) react 4-5 hours.With 90ml acetic acid second
Ester is extracted, and 30ml washings, 10ml saturated nacl aqueous solutions washing, organic phase merges, anhydrous sodium sulfate drying.Less than 30 DEG C of rotations
It is dry, the secondary amine intermediate 6a containing ester group is obtained, weigh 725mg, and yield is 87.5%.
(2) 6a (310.77mg, 1.5mmol) is dissolved in 6ml tetrahydrofurans, adds 3, the 5- dinitros chloro- benzotrifluorides of -4-
(270mg, 1mmol), triethylamine (101mg, 1mmol), is stirred overnight at room temperature.Reaction solution adds 80ml water to be diluted, plus 1M
Hydrochloric acid adjusts pH to 2-3, sediment to occur.It is extracted with ethyl acetate twice, each ethyl acetate consumption is 80ml, precipitation dissolves,
Washed with 30ml saturated aqueous common salts, organic phase merges, and uses anhydrous sodium sulfate drying.40 DEG C are spin-dried for, with chromatographic solution for just oneself
Alkane：Ethyl acetate=40:1、20:1、10:1 order carries out column chromatography for separation, is spin-dried for obtaining 6b, is weighed as 338.9mg, yield
It is 76.9%.
(3) 6b (150mg, 0.34mmol) is dissolved in 7.5ml tetrahydrofurans, 6.8ml 1mol/L sodium hydroxide solutions, room temperature
It is stirred overnight, is n-hexane with chromatographic solution：Ethyl acetate：Acetic acid=2:1:0.01 is tracked, and reaction is completed, and carries out soda acid
, acid extraction organic phase merges, and 40 DEG C are spin-dried for, and obtains crude product 118.8mg, carries out thin-layer chromatography separation, obtains object 6c, claims
Weight 105.8mg, yield is 86%.ESI-MS analysis(negative)m/z 428.4[M+H]—；1H NMR(600MHz
CDCl3)δ8.08(s,2H)；8.06 (d, J=8.4Hz, 2H)；4.28(s,2H)；2.93–2.88(m,2H)；0.87 (d, J=
7.1Hz,2H)；0.82 (d, J=7.2Hz, 3H).
Embodiment described above only expresses embodiments of the present invention, and its description is more specific and detailed, but can not
Therefore the limitation to the scope of the claims of the present invention is interpreted as, as long as the skill obtained in the form of equivalent or equivalent transformation
Art scheme, all should fall within the scope and spirit of the invention.
Claims (9)
1. a kind of trefanocide haptens, it is characterised in that with following structural formula：
Wherein, R is-(CH2)n-, n=1-4 or-CH=CH- or
2. trefanocide haptens as claimed in claim 1, it is characterised in that molecular structure is specially：
3. trefanocide haptens as claimed in claim 1, it is characterised in that molecular structure is specially：
4. the synthetic method of trefanocide haptens as claimed in claim 2, it is characterised in that comprise the following steps：In catalysis
In the presence of agent, substrate and alkylating reagent are alkylated reaction, secondary amine intermediate is obtained after purification, secondary amine intermediate is carried out
Hydrolysis obtains the second intermediate, the second intermediate 3,5- dinitro-p-trifluorotoluenes hybrid reaction chloro- with 4- again, it is purified after
Obtain target hapten molecule.
5. the synthetic method of trefanocide haptens as claimed in claim 4, it is characterised in that when haptens structure is 1c, bottom
Thing is ethyl acrylate, and alkylating reagent is n-propylamine；When haptens structure is 2c, 3c, 4c, substrate is respectively 2- pyrrolidines
Ketone, valerolactam, caprolactam, alkylating reagent are n-Propyl Bromide.
6. the synthetic method of trefanocide haptens as claimed in claim 3, it is characterised in that comprise the following steps：Substrate with
N-propylamine by acylation reaction, secondary amine intermediate of the generation containing ester group, 3, the 5- chloro- with 4- bis- again of the secondary amine intermediate containing ester group
Nitro-trifluoromethyl toluene reacts, it is purified after obtain the second intermediate, the second intermediate is hydrolyzed reaction again.
7. the synthetic method of trefanocide haptens as claimed in claim 6, it is characterised in that when haptens structure is 5c, bottom
Thing is 4- crotonic ethyl bromides；When haptens structure is 6c, substrate is 4- bromomethyl-benzoic acid methyl esters.
8. a kind of trefanocide artificial antigen, it is characterised in that：By trefanocide haptens described in claim any one of 1-3 or by weighing
Profit requires that the trefanocide haptens that any methods describeds of 4-7 are directly obtained is obtained with carrier protein couplet.
9. a kind of trefanocide antibody, it is characterised in that：The trefanocide antibody is prepared using the artificial antigen described in claim 8
Obtain, the trefanocide antibody is polyclonal antibody, monoclonal antibody or genetic engineering antibody.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061170A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | It is a kind of detect pendimethalin enzyme linked immunological kit and its application |
| CN109061147A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of test strips and its preparation method and application detecting pendimethalin |
| CN109575123A (en) * | 2018-11-08 | 2019-04-05 | 中国农业大学 | The preparation method and application of a kind of Fluorakil 100 haptens and monoclonal antibody |
| CN109593044A (en) * | 2018-12-06 | 2019-04-09 | 盐城工学院 | A kind of alkyl fatty acid amide and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4525199A (en) * | 1981-05-04 | 1985-06-25 | Nelson Research & Development Co. | Method of improved pest control |
| US5189043A (en) * | 1989-10-27 | 1993-02-23 | Takeda Chemical Industries, Ltd. | Isoquinolone derivatives, their production and use |
| WO2001017947A1 (en) * | 1999-09-06 | 2001-03-15 | Takeda Chemical Industries, Ltd. | Process for the preparation of 2,3-dihydroazepine compounds |
| JP2003166991A (en) * | 2001-11-30 | 2003-06-13 | Otsuka Chemical Holdings Co Ltd | Trifluralin compound, immunological reagin, hybridoma, and method of measuring trifluralin |
-
2017
- 2017-03-21 CN CN201710177395.6A patent/CN106928079B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4525199A (en) * | 1981-05-04 | 1985-06-25 | Nelson Research & Development Co. | Method of improved pest control |
| US5189043A (en) * | 1989-10-27 | 1993-02-23 | Takeda Chemical Industries, Ltd. | Isoquinolone derivatives, their production and use |
| WO2001017947A1 (en) * | 1999-09-06 | 2001-03-15 | Takeda Chemical Industries, Ltd. | Process for the preparation of 2,3-dihydroazepine compounds |
| EP1211239A1 (en) * | 1999-09-06 | 2002-06-05 | Takeda Chemical Industries, Ltd. | Process for the preparation of 2,3-dihydroazepine compounds |
| JP2003166991A (en) * | 2001-11-30 | 2003-06-13 | Otsuka Chemical Holdings Co Ltd | Trifluralin compound, immunological reagin, hybridoma, and method of measuring trifluralin |
Non-Patent Citations (1)
| Title |
|---|
| HEGEDUS, G.ETAL: "Development of an enzyme-linked immunosorbent assay (ELISA) for the herbicide trifluralin", 《 ANALYTICA CHIMICA ACTA》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061170A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | It is a kind of detect pendimethalin enzyme linked immunological kit and its application |
| CN109061147A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of test strips and its preparation method and application detecting pendimethalin |
| CN109061170B (en) * | 2018-09-21 | 2021-04-06 | 中国烟草总公司郑州烟草研究院 | Enzyme linked immunosorbent assay kit for detecting pendimethalin and application thereof |
| CN109575123A (en) * | 2018-11-08 | 2019-04-05 | 中国农业大学 | The preparation method and application of a kind of Fluorakil 100 haptens and monoclonal antibody |
| CN109593044A (en) * | 2018-12-06 | 2019-04-09 | 盐城工学院 | A kind of alkyl fatty acid amide and preparation method thereof |
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