CN107325174A - A kind of synthetic method of robenidine hydrochloride artificial antigen - Google Patents
A kind of synthetic method of robenidine hydrochloride artificial antigen Download PDFInfo
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- 239000000427 antigen Substances 0.000 title claims abstract description 36
- 102000036639 antigens Human genes 0.000 title claims abstract description 36
- 108091007433 antigens Proteins 0.000 title claims abstract description 36
- LTWIBTYLSRDGHP-HCURTGQUSA-N 1,2-bis[(e)-(4-chlorophenyl)methylideneamino]guanidine;hydrochloride Chemical compound [Cl-].C=1C=C(Cl)C=CC=1\C=N\N=C(/N)N\[NH+]=C\C1=CC=C(Cl)C=C1 LTWIBTYLSRDGHP-HCURTGQUSA-N 0.000 title claims abstract description 22
- 229960005445 robenidine hydrochloride Drugs 0.000 title claims abstract description 22
- 238000010189 synthetic method Methods 0.000 title claims abstract description 8
- 230000008878 coupling Effects 0.000 claims abstract description 15
- 238000010168 coupling process Methods 0.000 claims abstract description 15
- 238000005859 coupling reaction Methods 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 13
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 7
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 7
- HAZRIBSLCUYMQP-UHFFFAOYSA-N 1,2-diaminoguanidine;hydron;chloride Chemical compound Cl.NN\C(N)=N/N HAZRIBSLCUYMQP-UHFFFAOYSA-N 0.000 claims abstract description 4
- 150000003839 salts Chemical class 0.000 claims abstract description 4
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 3
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 3
- 239000000376 reactant Substances 0.000 claims abstract description 3
- 239000007788 liquid Substances 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 239000013067 intermediate product Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 7
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229960001866 silicon dioxide Drugs 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000000502 dialysis Methods 0.000 claims description 4
- -1 4- benzenebutanoic acids methyl esters Chemical class 0.000 claims description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical class OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 239000012065 filter cake Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- BXGTVNLGPMZLAZ-UHFFFAOYSA-N n'-ethylmethanediimine;hydrochloride Chemical compound Cl.CCN=C=N BXGTVNLGPMZLAZ-UHFFFAOYSA-N 0.000 claims description 3
- AVPYQKSLYISFPO-UHFFFAOYSA-N 4-chlorobenzaldehyde Chemical compound ClC1=CC=C(C=O)C=C1 AVPYQKSLYISFPO-UHFFFAOYSA-N 0.000 claims description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 claims description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims 1
- 102000002322 Egg Proteins Human genes 0.000 claims 1
- 108010000912 Egg Proteins Proteins 0.000 claims 1
- 235000014103 egg white Nutrition 0.000 claims 1
- 210000000969 egg white Anatomy 0.000 claims 1
- 238000001291 vacuum drying Methods 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 4
- 238000003018 immunoassay Methods 0.000 abstract description 3
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- 238000012797 qualification Methods 0.000 description 4
- 241000287828 Gallus gallus Species 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 108010042653 IgA receptor Proteins 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 2
- 229940125904 compound 1 Drugs 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- MOOFYEJFXBSZGE-QJUDHZBZSA-N 1,2-bis[(z)-(4-chlorophenyl)methylideneamino]guanidine Chemical compound C=1C=C(Cl)C=CC=1\C=N/N=C(/N)N\N=C/C1=CC=C(Cl)C=C1 MOOFYEJFXBSZGE-QJUDHZBZSA-N 0.000 description 1
- JGGFDEJXWLAQKR-UHFFFAOYSA-N 1,2-diaminoguanidine Chemical compound NNC(N)=NN JGGFDEJXWLAQKR-UHFFFAOYSA-N 0.000 description 1
- FPYUJUBAXZAQNL-UHFFFAOYSA-N 2-chlorobenzaldehyde Chemical compound ClC1=CC=CC=C1C=O FPYUJUBAXZAQNL-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 241000224483 Coccidia Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- NMRHMBPYSDZGGI-UHFFFAOYSA-N NC(=N)N.NNC(NN)=O Chemical class NC(=N)N.NNC(NN)=O NMRHMBPYSDZGGI-UHFFFAOYSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 230000001165 anti-coccidial effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- VKYKSIONXSXAKP-UHFFFAOYSA-N hexamethylenetetramine Chemical compound C1N(C2)CN3CN1CN2C3 VKYKSIONXSXAKP-UHFFFAOYSA-N 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229960004591 robenidine Drugs 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000002893 slag Substances 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
- C07C281/16—Compounds containing any of the groups, e.g. aminoguanidine
- C07C281/18—Compounds containing any of the groups, e.g. aminoguanidine the other nitrogen atom being further doubly-bound to a carbon atom, e.g. guanylhydrazones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/09—Preparation of carboxylic acids or their salts, halides or anhydrides from carboxylic acid esters or lactones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
- C07C67/333—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton
- C07C67/343—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Engineering & Computer Science (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
A kind of synthetic method of robenidine hydrochloride artificial antigen, belongs to technical field of biochemical industry.The present invention is with robenidine hydrochloride and 1, and 3 diaminoguanidine hydrochloric acid reactant salts obtain the product with carboxyl for haptens, and with carbodlimide method by haptens and carrier protein couplet, synthesis obtains robenidine hydrochloride artificial antigen, and the coupling ratio of conjugate is determined with ultraviolet method.The present invention successfully synthesizes robenidine hydrochloride artificial antigen, and synthesis step is succinct, effectively, is fully available in immunoassay, and required artificial antigen is provided for the research of later people.
Description
Technical field
The present invention relates to a kind of synthetic method of robenidine hydrochloride artificial antigen, belong to technical field of biochemical industry.
Background technology
Robenidine hydrochloride（Robenidine hydrochloride）It is a kind of new and effective anticoccidial drug.The medicine is in previous generation
Record and list and be widely used as feed addictive in the U.S. beginning of the seventies, be mainly used in preventing and treating the coccidia sense of chicken
Dye.It is mainly used in preventing and treating the parasitic infection of chicken, rabbit and aquatic livestock in China.Long-term use can produce serious anti-medicine
Property, MRL of No. 235 bulletin regulation robenidine hydrochloride of the Ministry of Agriculture in chicken fat, skin, Edible tissues is respectively
200、200、100 μg/kg.In recent years, people are to the various chronic diseases caused by extended low level intake residue of veterinary drug and induction
The problems such as pathogen produces drug resistance pays special attention to.To ensure that fish quality and consumer are healthy, promote aquatic products
The health of aquaculture, sustainable development, it is necessary to be monitored to the medicament residue in aquatic products.Therefore, set up quick effective
Detection robenidine hydrochloride content method is significant and market value.
ELISA（ELISA）It is a kind of extremely efficient, sensitive, quick detection method, but obtains high-affinity
Monoclonal antibody with high specific is the premise of immunology detection, and the synthesis of artificial antigen is a wherein important step.
The content of the invention
It is an object of the invention to provide a kind of synthetic method of robenidine hydrochloride artificial antigen, prepared product is used for salt
Sour Robenidine immunoassay method research, required artificial antigen is provided for the research of people from now on.
Technical scheme, a kind of synthetic method of robenidine hydrochloride artificial antigen, its by robenidine hydrochloride and 1,
3- diaminoguanidine hydrochloric acid reactant salts obtain the product with carboxyl, i.e. haptens Rob, with carbodlimide method by haptens Rob with
Carrier protein couplet, that is, obtain robenidine hydrochloride artificial antigen；Step is：
（1）Haptens Rob synthesis：
Synthetic route is as follows：
By 4- benzenebutanoic acid methyl esters（Compound 1）(1.50 g, 8.42 mmol) are dissolved in 20 mL trifluoroacetic acids, add six sub-
Tetramine (1.29 g, 9.26 mmol), reaction solution is stirred overnight under the conditions of 80 DEG C, and room temperature is cooled to afterwards, decompression
Concentration, is purified with silicagel column and obtains yellow oily intermediate product 2；
Intermediate product 2 (700 mg, 3.39 mmol) is dissolved in 10 mL hydrochloric acid（6 mol/L）In, reaction solution is 60
It is stirred overnight under the conditions of DEG C, by reacting liquid filtering, filter cake is rinsed with water, is dried in vacuo, obtains white solid intermediate product 3；
Intermediate product 3 (400 mg, 2.08 mmol) is dissolved in the pure methanol solutions of 10 mL, 1,3- diaminoguanidine is added
Hydrochloride (261 mg, 2.08 mmol), reaction solution stirs 1 h under the conditions of 60 DEG C, and reaction solution is cooled to after room temperature, plus
Enter 4-chloro-benzaldehyde (292 mg, 2.08 mmol), 1 h is stirred under the conditions of 60 DEG C, mixture is concentrated under reduced pressure, residual
Slag obtains white solid target compound 4 through silicagel column purifying, is prepared into haptens Rob.
（2）The preparation of comlete antigen：Step（1）Haptens Rob and the BSA coupling of preparation obtain conjugate comlete antigen
Rob-BSA。
Step（2）The preparation method of the comlete antigen Rob-BSA is as follows：
A, weigh step（1）The mg of Rob 3.5 mg, 1- ethyl-carbodiimide hydrochloride 5.2 of preparation, n-hydroxysuccinimide
3.2 mg, are dissolved with the anhydrous DMFs of 300 μ L, and 4~5h of reaction (being referred to as A liquid) is stirred at room temperature；Weigh cow's serum
The mg of protein B SA 10（Rob is 30 with BSA mol ratios:1）, add isometric borate buffer solution（Referred to as B liquid）；In room temperature bar
A liquid, is added in B liquid, room temperature reaction is stayed overnight by part dropwise, produces conjugate Rob-BSA mixed liquors；
B, dialysis：10cm bag filter is taken, 5min is boiled in boiling water, then with 60 DEG C of deionized water rinsing 3min, is stored in 4
It is standby in DEG C deionized water；Conjugate Rob-BSA mixed liquors are put into bag filter to dialyse 3 days in 0.01mol/L PBS, often
Change liquid it three times, produce comlete antigen Rob-BSA.
The identification of robenidine hydrochloride artificial antigen
（1）Using nuclear magnetic resonance and LC-MS technical appraisement haptens.
（2）Artificial antigen identifies that it is coupled result using ultraviolet method, utilizes the concentration of conjugate small molecular and albumen, meter
Calculate its coupling ratio.
Coupling ratio is determined：The ratio for two kinds of molecules being coupled in estimation conjugate（Coupling ratio）Method, although survey
Determine method species a lot, but be in accordance with detecting two kinds of molecule contents being coupled in conjugate（Or relative amount）Principle build
Erect what is come.Ultraviolet method is to determine coupling ratio according to the little molecular concentration and the ratio of protein concentration in the artificial antigen synthesized.
0.5 mg/mL protein solution and comlete antigen solution is prepared with PBS, baseline is set up with PBS, wavelength 200 ~
Scanned between 500 nm, obtain light absorption value of the protein solution with comlete antigen solution at the characteristic ultraviolet absorption peak of haptens and distinguish
For A1, A2；The haptens that 5 ~ 50 μ g/mL are prepared with methanol is X μ g/mL, baseline is set up with methanol, in wavelength 200 ~ 500
Scanned between nm, obtain the light absorption value A at haptens characteristic ultraviolet absorption peak3, the relative molecular mass of haptens is M1, albumen
Relative molecular mass is M2, then coupling ratio=[(A2-A1) (X/M1)/A3]/(0.5/M2)
Beneficial effects of the present invention：The present invention successfully synthesizes robenidine hydrochloride artificial antigen, and synthesis step is succinct, effectively, complete
It can be used for entirely in immunoassay, convenient approach provided for the research of later people.
Brief description of the drawings
Fig. 1 haptens Rob NMR qualification figures.
Fig. 2 haptens Rob LC-MS qualification figures.
The ultraviolet qualification figure of immunogene of Fig. 3 Rob-BSA artificial antigens.
Embodiment
The haptens Rob of embodiment 1 synthesis
Synthetic route is as follows：
By 4- benzenebutanoic acid methyl esters（Compound 1）(1.50 g, 8.42 mmol) are dissolved in 20 mL trifluoroacetic acids, add six methylenes
Urotropine (1.29 g, 9.26 mmol), reaction solution is stirred overnight under the conditions of 80 DEG C, and room temperature is cooled to afterwards, is depressurized dense
Contracting, is purified with silicagel column and obtains yellow oily intermediate product 2；
Intermediate product 2 (700 mg, 3.39 mmol) is dissolved in 10 mL hydrochloric acid（6 mol/L）, reaction solution is in 60 DEG C of bars
It is stirred overnight under part, by reacting liquid filtering, filter cake is rinsed with water, is dried in vacuo, obtains white solid intermediate product 3；
Intermediate product 3 (400 mg, 2.08 mmol) is dissolved in the pure methanol solutions of 10 mL, 1,3- diaminourea guanidinesalts are added
Hydrochlorate (261 mg, 2.08 mmol), reaction solution stirs 1 h under the conditions of 60 DEG C, and reaction solution is cooled to after room temperature, addition pair
Chlorobenzaldehyde (292 mg, 2.08 mmol), stirs 1 h, mixture is concentrated under reduced pressure, and residue is through silicon under the conditions of 60 DEG C
The purifying of glue post obtains white solid target compound 4, is prepared into haptens Rob.
The preparation of the comlete antigen of embodiment 2
Comlete antigen Rob-BSA preparation methods are as follows：
A, weigh the mg of Rob3.5 mg, 1- ethyl-carbodiimide hydrochloride 5.2, N- hydroxysuccinimidyl acyls are weighed made from embodiment 1
The mg of imines 3.2, is dissolved with the anhydrous DMFs of 300 μ L and (is referred to as A liquid), anti-4~5h is stirred at room temperature.Weigh ox blood
The mg of albumin BSA 10（Rob is 30 with BSA mol ratios:1）, add isometric borate buffer solution（Referred to as B liquid）, in room temperature
A liquid, is added in B liquid, room temperature reaction is stayed overnight by condition dropwise, produces conjugate Rob-BSA mixed liquors；
B, dialysis：10cm bag filter is taken, 5min is boiled in boiling water, then with 60 DEG C of deionized water rinsing 3min, is stored in 4
It is standby in DEG C deionized water；Conjugate Rob-BSA mixed liquors are put into bag filter to dialyse 3 days in 0.01mol/L PBS, often
Change liquid it three times, produce comlete antigen Rob-BSA.The ultraviolet qualification figure of Rob-BSA immunogenes is as shown in Figure 3.
The identification of the robenidine hydrochloride artificial antigen of embodiment 3
（1）Using nuclear magnetic resonance and LC-MS technical appraisement haptens.
（2）Artificial antigen identifies that it is coupled result using ultraviolet method, utilizes the concentration of conjugate small molecular and albumen, meter
Calculate its coupling ratio.
Coupling ratio is determined：The ratio for two kinds of molecules being coupled in estimation conjugate（Coupling ratio）Method, although survey
Determine method species a lot, but be in accordance with detecting two kinds of molecule contents being coupled in conjugate（Or relative amount）Principle build
Erect what is come.Ultraviolet method is to determine coupling ratio according to the little molecular concentration and the ratio of protein concentration in the artificial antigen synthesized.
Conjugate determination of protein concentration：The quality of albumen divided by the volume of conjugate after dialysis are that can obtain idol before reacting
The content of albumen in connection.
Conjugate Rob-BSA characteristic absorption peak is at 328 nm, and light absorption value A2=0.929581, BSA herein is here
Light absorption value A1=0.021483, Rob characteristic absorption peak A3=1.612249, Rob relative molecular mass M1=385.5, BSA
Relative molecular mass M2=67000, X be 10 μ g/mL, then according to coupling ratio formula：
Haptens Rob and protein B SA coupling ratio are in coupling ratio=[(A2-A1) (X/M1)/A3]/(0.5/M2), Rob-BSA
119.932:1。
Claims (2)
1. a kind of synthetic method of robenidine hydrochloride artificial antigen, it is characterised in that it is by robenidine hydrochloride and 1,3- diaminoguanidine
Hydrochloric acid reactant salt obtains the product with carboxyl, i.e. haptens Rob, with carbodlimide method that haptens Rob and carrier protein is even
Connection, that is, obtain robenidine hydrochloride artificial antigen；Step is：
（1）Haptens Rob synthesis：
Synthetic route is as follows：
2222
The g of 4- benzenebutanoic acids methyl esters 1.50 is dissolved in 20 mL trifluoroacetic acids, the g of hexa 1.29 is added, reaction solution exists
It is stirred overnight under the conditions of 80 DEG C, room temperature is cooled to afterwards, be concentrated under reduced pressure, is purified with silicagel column and obtain yellow oily intermediate product
2；
700 mg intermediate products 2 are dissolved in the mol/L hydrochloric acid of 10 mL 6, reaction solution is stirred overnight under the conditions of 60 DEG C,
By reacting liquid filtering, filter cake is rinsed with water, vacuum drying, obtains white solid intermediate product 3；
400 mg intermediate products 3 are dissolved in the pure methanol solutions of 10 mL, the mg of 1,3- diaminoguanidine hydrochloride 261 are added, instead
Answer liquid to stir 1 h under the conditions of 60 DEG C, reaction solution is cooled to after room temperature, the mg of 4-chloro-benzaldehyde 292 is added, in 60 DEG C of conditions
1 h of lower stirring, mixture is concentrated under reduced pressure, and residue obtains white solid target compound 4 through silicagel column purifying, is prepared into
Haptens Rob
（2）The preparation of comlete antigen：Step（1）Haptens Rob and the BSA coupling of preparation obtain conjugate comlete antigen Rob-
BSA。
2. the synthetic method of robenidine hydrochloride artificial antigen as claimed in claim 1, it is characterised in that：Step（2）It is described complete
Antigen Rob-BSA preparation method is as follows：
A, weigh step（1）The mg of Rob 3.5 mg, 1- ethyl-carbodiimide hydrochloride 5.2 of preparation, n-hydroxysuccinimide
3.2 mg, are dissolved with the anhydrous DMFs of 300 μ L, 4~5h of reaction, referred to as A liquid are stirred at room temperature；Weigh cow's serum egg
White BSA 10 mg, Rob and BSA mol ratio is 30:1, add isometric borate buffer solution, referred to as B liquid；In room temperature condition, by
A liquid is added in B liquid by drop, and room temperature reaction is stayed overnight, and produces conjugate Rob-BSA mixed liquors；
B, dialysis：10cm bag filter is taken, 5min is boiled in boiling water, then with 60 DEG C of deionized water rinsing 3min, is stored in 4
It is standby in DEG C deionized water；Conjugate Rob-BSA mixed liquors are put into bag filter to dialyse 3 days in 0.01mol/L PBS, often
Change liquid it three times, produce comlete antigen Rob-BSA.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108794428A (en) * | 2018-06-19 | 2018-11-13 | 广东工业大学 | A kind of Trimetazidine haptens, artificial antigen and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3980705A (en) * | 1975-05-19 | 1976-09-14 | American Cyanamid Company | Purification of robenidine hydrochloride |
| US20020123649A1 (en) * | 1999-01-06 | 2002-09-05 | Paul Hanselmann | Process for the preparation of robenidine and salts thereof |
| CN105294511A (en) * | 2015-11-13 | 2016-02-03 | 浙江汇能生物股份有限公司 | Preparation method of robenidine hydrochloride |
| CN106905428A (en) * | 2017-04-25 | 2017-06-30 | 江南大学 | A kind of synthetic method of florfenicol amine artificial antigen |
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- 2017-07-12 CN CN201710566363.5A patent/CN107325174A/en active Pending
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