CN112876554B - Fluoxetine antigen and preparation method thereof - Google Patents
Fluoxetine antigen and preparation method thereof Download PDFInfo
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- CN112876554B CN112876554B CN202110117646.8A CN202110117646A CN112876554B CN 112876554 B CN112876554 B CN 112876554B CN 202110117646 A CN202110117646 A CN 202110117646A CN 112876554 B CN112876554 B CN 112876554B
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- 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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- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4717—Plasma globulins, lactoglobulin
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- 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
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention relates to the technical field of synthesis of whole antigens, and discloses a fluoxetine antigen and a preparation method thereof. The preparation method of the fluoxetine antigen comprises the following steps: (1) protecting a secondary amine group in fluoxetine by using 2-trimethylsilylethoxycarbonyl chloride to obtain N-trimethylsilylethoxycarbonyl-fluoxetine; (2) carrying out Friedel-crafts acylation reaction on N-trimethylsilyl ethoxycarbonyl-fluoxetine and succinic anhydride to obtain fluoxetine hapten; (3) coupling the fluoxetine hapten with carrier protein, and removing a protecting group to obtain the fluoxetine antigen. The fluoxetine antigen of the invention completely reserves the active group secondary amino group in the molecular structure of the fluoxetine, so the antigen has higher specificity and sensitivity.
Description
Technical Field
The invention relates to the technical field of synthesis of whole antigens, and particularly relates to a fluoxetine antigen and a preparation method thereof.
Background
Fluoxetine (Fluoxetine), also known as fluorophenoxypropylamine, is known by the scientific name "N-methyl-3-phenyl-3 (4-trifluoromethylphenoxy) propylamine" and has the following structural formula:
fluoxetine is a second-generation antidepressant drug widely used in clinic at present, is a selective 5-hydroxytryptamine (5-HT) reuptake inhibitor, can selectively inhibit 5-HT transporters, blocks the reuptake of 5-HT by presynaptic membranes, prolongs and increases the effect of 5-HT, and thus generates antidepressant effect. It has obvious antidepressant and antianxiety effects on various depression disorder patients, and also has the clinical effects of treating migraine, obesity, premature ejaculation, eating disorder, panic disorder, childhood autism and other diseases. In addition, in recent years, it has been found that fluoxetine has been used as an anti-obesity agent because of significant weight loss in depression patients receiving fluoxetine. The research control test result proves that the fluoxetine is 40-80 mg per day, the body weight is reduced by 2-6 kg after 8 weeks, and the effect is obviously better than that of the common placebo. In the research on the dose-effect of over 600 cases of obesity, the weight reduction range caused by 0, 10, 20, 40 and 60mg of fluoxetine per day is mainly determined by the drug dose, and illegal vendors can freely increase the dose of the component in weight-reducing drugs and various weight-reducing health-care foods under the drive of interest due to obvious weight-reducing effect, so that the detection research on the fluoxetine in the weight-reducing drugs and the weight-reducing health-care foods can effectively control the illegal abuse of the drugs, and is very necessary for maintaining the health safety of people.
The common detection and analysis method for fluoxetine mainly comprises the following steps: gas Chromatography (GC), gas-mass spectrometry (GC-MS), High Performance Liquid Chromatography (HPLC), liquid-mass spectrometry (LC-MS), High Performance Capillary Electrophoresis (HPCE), and Immunoassay (IA). Instrumental analysis methods have extremely high sensitivity and precision, but require expensive instrumentation, equipment and specially trained technicians, and are not suitable for screening and on-site real-time testing of batch samples. The immunoassay developed in recent years has the advantages of simple and convenient operation, high efficiency, sensitivity, suitability for large-scale detection and the like, and is widely applied to the detection of various drugs.
To establish an immune detection method of fluoxetine, a fluoxetine holoantigen with antigenic activity must be obtained, but the existing preparation method of the fluoxetine holoantigen generally has the problems of low specificity and low sensitivity. For example, chinese patent publication No. CN108264551A discloses a method for preparing a fluoxetine artificial antigen, in which a carboxyl-containing coupling arm is first led out from a secondary amine group in a fluoxetine molecule, and then reacted with an amino group in a carrier protein by an active ester method to obtain a fluoxetine whole antigen. Although the preparation process is simple, the active group secondary amine group in fluoxetine cannot be reserved, and the obtained holoantigen has low specificity and sensitivity.
Disclosure of Invention
In order to solve the technical problems, the invention provides a fluoxetine antigen and a preparation method thereof. The fluoxetine antigen of the invention completely reserves the active group secondary amino group in the fluoxetine molecular structure, so that the fluoxetine antigen has higher specificity and sensitivity.
The specific technical scheme of the invention is as follows:
a fluoxetine antigen of the structural formula:
wherein R is carrier protein.
Preferably, the carrier protein is one of Bovine Serum Albumin (BSA), hemocyanin (KLH), chicken Ovalbumin (OVA), and Bovine Gamma Globulin (BGG).
A preparation method of the fluoxetine antigen comprises the following steps:
(1) protecting the secondary amine group in fluoxetine with 2-trimethylsilylethyloxycarbonylchloride (Teoc-Cl) to obtain N-trimethylsilylethyloxycarbonyl-fluoxetine;
(2) carrying out Friedel-crafts acylation reaction on N-trimethylsilyl ethoxycarbonyl-fluoxetine and succinic anhydride to obtain fluoxetine hapten;
(3) coupling the fluoxetine hapten with carrier protein, and removing a protecting group to obtain the fluoxetine antigen.
The mechanism for preparing the fluoxetine antigen is as follows: in the step (2), carboxyl can be grafted on a benzene ring of the N-trimethylsilyl ethoxycarbonyl-fluoxetine through Friedel-crafts acylation reaction, and the carboxyl is utilized to react with amino in carrier protein in the step (3) to realize the coupling of the fluoxetine and the carrier protein, so as to obtain the fluoxetine antigen with immunogenicity.
Because the secondary amine group of the active group in the fluoxetine can react with succinic anhydride in the step (2) and can react with the carboxyl group in the fluoxetine hapten and carrier protein in the step (3) to influence the performance of the finally obtained fluoxetine antigen, the secondary amine group in the fluoxetine is protected by Teoc-Cl before the carboxyl group is grafted, and the protecting group Teoc is removed after the secondary amine group is coupled with the carrier protein, so that the active group in the molecular structure of the fluoxetine can be completely reserved, and the obtained fluoxetine antigen has stronger specificity and higher sensitivity.
In addition, the preparation method of the invention can ensure that the reaction conditions in the whole preparation process are mild, the yield is high, and reagents with strong toxicity and corrosivity are not needed.
Preferably, in the step (1), the fluoxetine is obtained by free extraction of fluoxetine hydrochloride, and the specific process is as follows: mixing fluoxetine hydrochloride and water, mashing, adjusting the pH value to 8.0-9.0, adding ethyl acetate for extraction, washing an organic layer with water, drying the organic layer with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain the fluoxetine.
Preferably, the specific process of step (1) is as follows: dissolving fluoxetine in an organic solvent, adding an acid-binding agent, cooling to 0-5 ℃, adding 2-trimethylsilyl ethoxycarbonyl chloride, reacting at 20-30 ℃ for 10-24h, and separating a product to obtain the N-trimethylsilyl ethoxycarbonyl-fluoxetine.
Further, in the step (1), the organic solvent is dichloromethane.
Further, in the step (1), the acid-binding agent is imidazole, and the mass ratio of the imidazole to the fluoxetine is 1: 1.5-1.7.
Preferably, in the step (1), the mass ratio of the fluoxetine to the 2-trimethylsilylethyloxycarbonylchloride is 1: 1.5-2.0.
Further, in the step (1), the specific process for separating the product is as follows: adding saturated sodium bicarbonate solution, stirring, extracting with dichloromethane, washing the organic layer with water, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and performing column chromatography.
Preferably, the specific process of step (2) is as follows: dissolving N-trimethylsilyl ethoxycarbonyl-fluoxetine in a reaction solvent, adding succinic anhydride, cooling to 0-5 ℃ in a protective atmosphere, adding a Friedel-crafts acylation reaction catalyst, reacting at 0-5 ℃ for 1-1.5h, heating to 20-30 ℃ for 9.5-10.5h, stopping the reaction, and separating a product to obtain the fluoxetine hapten.
Further, in the step (2), the reaction solvent is dichloromethane.
Further, in the step (2), the Friedel-crafts acylation reaction catalyst is anhydrous aluminum trichloride, and the mass ratio of the anhydrous aluminum trichloride to the N-trimethylsilyl ethoxycarbonyl-fluoxetine is 1: 1.5-8.0; the anhydrous aluminum trichloride is added in batches.
Further, in the step (2), the mass ratio of the N-trimethylsilylethoxycarbonyl-fluoxetine to the succinic anhydride is 1: 0.9-1.2.
Further, in the step (2), the specific process for separating the product is as follows: cooling to 0-5 deg.C, adjusting pH to 3.6-3.8, washing the organic layer with water, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and performing column chromatography.
Preferably, in the step (3), the specific process for coupling the fluoxetine hapten and the carrier protein is as follows: dissolving fluoxetine hapten in a coupling solvent, adding Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), reacting for 14.5-15.5h at 20-30 ℃, centrifuging, taking clear liquid, adding the clear liquid into a PBS (phosphate buffer solution) solution of carrier protein, and reacting for 14.5-15.5h at 4-10 ℃.
Further, in the step (3), the carrier protein is bovine serum albumin, and the mass ratio of the fluoxetine hapten to the N-hydroxysuccinimide to the dicyclohexylcarbodiimide to the bovine serum albumin is 1:0.21-0.26:0.38-0.40: 1.0-1.5.
The amount of NHS and DCC relative to the reactants will affect product yield and titer: if the relative dosage of NHS and DCC is too large, the mutual coupling between carrier proteins is easily caused, and the precipitation is too much, so that the product titer and the product yield are reduced; if the relative dosage of NHS and DCC is too small, the carboxyl in the hapten is easy to be incompletely activated, and the product titer is reduced.
The amount of carrier protein relative to fluoxetine hapten also affects product yield and potency: if the relative amount of the carrier protein is too small, the product yield is too low; if the relative dosage of the carrier protein is too large, excessive fluoxetine molecules are wrapped by the carrier protein, the combination of the antigen and the antibody is influenced, and the product titer is too low.
According to the invention, the mass ratio of the fluoxetine hapten to the N-hydroxysuccinimide to the dicyclohexylcarbodiimide to the carrier protein BSA is controlled within the range of 1:0.21-0.26:0.38-0.40:1.0-1.5, so that higher yield can be obtained, and the prepared fluoxetine antigen has higher titer, thus higher sensitivity is obtained.
Further, in the step (3), the coupling solvent is N, N-dimethylformamide.
Preferably, in step (3), the specific procedure for removing the protecting group is as follows: after coupling fluoxetine hapten and carrier protein, tetrabutylammonium fluoride (TBAF) is added into the reaction solution and reacts for 11.5-12.5h at the temperature of 10-20 ℃.
Further, in the step (3), the mass ratio of the fluoxetine hapten to the tetrabutylammonium fluoride is 1: 2.3-3.6.
Compared with the prior art, the invention has the following advantages:
(1) the fluoxetine antigen completely reserves the active group secondary amino group in the molecular structure of the fluoxetine, thereby having stronger specificity and higher sensitivity;
(2) the reaction conditions in the whole preparation process of the fluoxetine antigen are mild, and a reagent with strong toxicity and corrosivity is not used.
Detailed Description
The present invention will be further described with reference to the following examples.
General examples
A preparation method of fluoxetine antigen comprises the following steps:
(1) extraction of fluoxetine (I):
mixing fluoxetine hydrochloride and water, mashing, adjusting the pH value to 8.0-9.0, adding ethyl acetate for extraction, washing an organic layer with water, drying the organic layer with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain fluoxetine (I).
(2) Synthesis of N-trimethylsilylethoxycarbonyl-fluoxetine (II):
dissolving fluoxetine (I) in dichloromethane, adding an acid-binding agent imidazole, wherein the mass ratio of imidazole to fluoxetine is 1:1.5-1.7, cooling to 0-5 ℃, adding 2-trimethylsilyl ethoxycarbonyl chloride, wherein the mass ratio of fluoxetine to 2-trimethylsilyl ethoxycarbonyl chloride is 1:1.5-2.0, and reacting for 10-24h at 20-30 ℃; and after the reaction is stopped, adding a saturated sodium bicarbonate solution, fully stirring, adding dichloromethane for extraction, washing an organic layer with water, drying by anhydrous sodium sulfate, concentrating under reduced pressure, and then carrying out column chromatography to obtain the N-trimethylsilyl ethoxycarbonyl-fluoxetine (II).
(3) Synthesis of fluoxetine hapten (III):
dissolving N-trimethylsilylethoxycarbonyl-fluoxetine (II) in dichloromethane, adding succinic anhydride, wherein the mass ratio of the N-trimethylsilylethoxycarbonyl-fluoxetine to the succinic anhydride is 1:0.9-1.2, cooling to 0-5 ℃ in a protective atmosphere, adding a Friedel-crafts acylation catalyst of anhydrous aluminum trichloride in batches, wherein the mass ratio of the anhydrous aluminum trichloride to the N-trimethylsilylethoxycarbonyl-fluoxetine is 1:1.5-8.0, reacting for 1-1.5h at 0-5 ℃, and then heating to 20-30 ℃ for reacting for 9.5-10.5 h; and (3) after the reaction is stopped, cooling to 0-5 ℃, adjusting the pH to 3.6-3.8, washing the organic layer with water, drying the organic layer with anhydrous sodium sulfate, concentrating the organic layer under reduced pressure, and performing column chromatography to obtain the fluoxetine hapten (III).
(4) Synthesis of fluoxetine antigen (IV):
dissolving fluoxetine hapten (III) in N, N-Dimethylformamide (DMF), adding Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), wherein the mass ratio of the fluoxetine hapten to the NHS to the DCC is 1:0.21-0.26:0.38-0.40, reacting for 14.5-15.5 hours at 20-30 ℃, centrifuging, taking clear liquid, adding the clear liquid into a PBS (phosphate buffer solution) solution of carrier protein, wherein the carrier protein is one of bovine serum albumin, hemocyanin, egg albumin and bovine gamma globulin (when the carrier protein is the bovine serum albumin, the mass ratio of the fluoxetine hapten to the bovine serum albumin is 1:1.0-1.5), and reacting for 14.5-15.5 hours at 4-10 ℃. Then adding tetrabutylammonium fluoride (TBAF) into the reaction solution, wherein the mass ratio of the fluoxetine hapten to the TBAF is 1:2.3-3.6, and reacting for 11.5-12.5h at the temperature of 10-20 ℃; placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; and (4) centrifuging the dialyzed reaction solution, and taking a supernatant to obtain the fluoxetine antigen (IV).
Example 1
A preparation method of fluoxetine antigen comprises the following steps:
(1) and (3) extracting fluoxetine:
36 tablets of fluoxetine hydrochloride (20 mg per tablet) were mixed with 150mL of water, triturated, adjusted to pH 8.2 with 1mol/L sodium hydroxide solution, extracted twice with 150mL of ethyl acetate, and the separated organic layer was washed once with 50mL of water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 630mg of fluoxetine (used in example 1 and comparative examples 1-2 for the preparation of fluoxetine antigen).
(2) Synthesis of N-trimethylsilylethoxycarbonyl-fluoxetine:
dissolving 465mg of fluoxetine in 20mL of dichloromethane, adding 300mg of imidazole, cooling to 0 ℃, adding 300mg of 2-trimethylsilylethyloxycarbonylchloride, and reacting for 15 hours at 20 ℃; after the reaction was terminated, 200mL of a saturated sodium bicarbonate solution was added, the mixture was stirred at room temperature for 10min, 1000mL of dichloromethane was added to conduct extraction, the separated organic layer was washed twice with 200mL of water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography to obtain 455mg of N-trimethylsilylethoxycarbonyl-fluoxetine.
(3) Synthesis of fluoxetine hapten:
dissolving 455mg of N-trimethylsilylethoxycarbonyl-fluoxetine in 100mL of dichloromethane, adding 450mg of succinic anhydride, cooling to 0 ℃ under the protection of nitrogen, adding 60mg of anhydrous aluminum trichloride in 3 batches in 10min, reacting for 1h at 0 ℃, and heating to 20 ℃ for 10 h; after the reaction was stopped, the temperature was reduced to 0 ℃ and the pH was adjusted to 3.8 with 1mol/L hydrochloric acid, and the organic layer was washed twice with 100mL of water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography to obtain 210mg of a fluoxetine hapten (used in example 1 and comparative examples 3 to 6 for the preparation of fluoxetine antigens).
(4) Synthesis of fluoxetine antigen:
42mg of fluoxetine hapten is dissolved in 0.8 mg of DMF, 10mg of NHS and 16mg of DCC are added, the mixture is reacted for 15 hours at room temperature, after centrifugation, a clear solution is taken, added to 4mL of BSA in PBS (wherein, the concentration of BSA is 12mg/mL), and the mixture is reacted for 15 hours at 4 ℃. Then adding 100mg of TBAF into the reaction solution, and reacting for 12h at 10 ℃; placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; and centrifuging the dialyzed reaction solution, and taking a supernatant to obtain 4.5mL6.5mg/mL fluoxetine antigen.
Example 2
A preparation method of fluoxetine antigen comprises the following steps:
(1) and (3) extracting fluoxetine:
6 tablets of fluoxetine hydrochloride (each tablet has a content of 20mg) are mixed with 30mL of water, after mashing, the pH is adjusted to 8.5 by 1mol/L sodium hydroxide solution, then the mixture is extracted twice by 40mL of ethyl acetate, and the separated organic layer is washed once by 10mL of water, dried by anhydrous sodium sulfate and concentrated under reduced pressure to obtain 112mg of fluoxetine.
(2) Synthesis of N-trimethylsilylethoxycarbonyl-fluoxetine:
dissolving 110mg of fluoxetine in 25mL of dichloromethane, adding 65mg of imidazole, cooling to 0 ℃, adding 62mg of 2-trimethylsilylethyloxycarbonylchloride, and reacting at 25 ℃ for 16 hours; after the reaction was terminated, 40mL of a saturated sodium bicarbonate solution was added, the mixture was stirred at room temperature for 10min, 220mL of dichloromethane was added for extraction, the separated organic layer was washed twice with 40mL of water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography to obtain 95mg of N-trimethylsilylethoxycarbonyl-fluoxetine.
(3) Synthesis of fluoxetine hapten:
dissolving 95mg of N-trimethylsilylethoxycarbonyl-fluoxetine in 100mL of dichloromethane, adding 96mg of succinic anhydride, cooling to 0 ℃ under the protection of nitrogen, adding 60mg of anhydrous aluminum trichloride in 3 batches in 10min, reacting for 1h at 0 ℃, and heating to 25 ℃ for 10 h; after the reaction was stopped, the temperature was reduced to 0 ℃ and the pH was adjusted to 3.5 with 1mol/L hydrochloric acid, and the organic layer was washed twice with 20mL of water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography to obtain 44mg of a fluoxetine hapten (used in example 1 and comparative examples 2 to 5 for the preparation of fluoxetine antigens).
(4) Synthesis of fluoxetine antigen:
44mg of fluoxetine hapten was dissolved in 1mL of DMF, 11mg of NHS and 17mg of DCC were added to the solution, and the mixture was reacted at room temperature for 15 hours, centrifuged to obtain a clear solution, which was added to 5mL of BSA in PBS (where BSA concentration was 12mg/mL) and reacted at 10 ℃ for 15 hours. Then adding 110mg of TBAF into the reaction solution, and reacting for 12h at 20 ℃; placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; the dialyzed reaction solution was centrifuged, and the supernatant was collected to obtain 4.5mL of 6.8mg/mL of fluoxetine antigen.
Example 3
A preparation method of fluoxetine antigen comprises the following steps:
(1) and (3) extracting fluoxetine:
6 tablets of fluoxetine hydrochloride (each tablet has a content of 20mg) are mixed with 32mL of water, after mashing, the pH is adjusted to 8.6 by 1mol/L sodium hydroxide solution, then the mixture is extracted twice by 40mL of ethyl acetate, and the separated organic layer is washed once by 10mL of water, dried by anhydrous sodium sulfate and concentrated under reduced pressure to obtain 105mg of fluoxetine.
(2) Synthesis of N-trimethylsilylethoxycarbonyl-fluoxetine:
dissolving 104mg of fluoxetine in 25mL of dichloromethane, adding 63mg of imidazole, cooling to 0 ℃, adding 62mg of 2-trimethylsilylethyloxycarbonylchloride, and reacting at 30 ℃ for 18 hours; after the reaction was terminated, 50mL of a saturated sodium bicarbonate solution was added, the mixture was stirred at room temperature for 10 minutes, 200mL of dichloromethane was added for extraction, the separated organic layer was washed twice with 40mL of water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography to obtain 40mg of N-trimethylsilylethoxycarbonyl-fluoxetine.
(3) Synthesis of fluoxetine hapten:
dissolving 40mg of N-trimethylsilylethoxycarbonyl-fluoxetine in 100mL of dichloromethane, adding 92mg of succinic anhydride, cooling to 0 ℃ under the protection of nitrogen, adding 60mg of anhydrous aluminum trichloride in 3 batches within 10min, reacting for 1h at 0 ℃, and then heating to 30 ℃ for 10 h; after the reaction was stopped, the temperature was reduced to 0 ℃ and the pH was adjusted to 3.6 with 1mol/L hydrochloric acid, and the organic layer was washed twice with 20mL of water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography to obtain 43mg of a fluoxetine hapten (used in example 1 and comparative examples 2 to 5 for the preparation of fluoxetine antigens).
(4) Synthesis of fluoxetine antigen:
43mg of fluoxetine hapten was dissolved in 1mL of DMF, 11mg of NHS and 17mg of DCC were added to the solution, and the mixture was reacted at room temperature for 15 hours, centrifuged to obtain a clear solution, which was added to 5mL of BSA in PBS (where BSA concentration was 12mg/mL) and reacted at 10 ℃ for 15 hours. Then adding 110mg of TBAF into the reaction solution, and reacting for 12h at 20 ℃; placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and changing the PBS solution every 24 hours; the dialyzed reaction solution was centrifuged, and the supernatant was collected to obtain 4.6mL of 6.7mg/mL of fluoxetine antigen.
Comparative example 1
A preparation method of a fluoxetine antigen comprises the following steps:
42mg of fluoxetine (prepared in example 1) was dissolved in 0.8mL of DMF, 10mg of NHS and 16mg of DCC were added and reacted at room temperature for 15 hours, and after centrifugation, the supernatant was added to a 4mLBSA PBS solution (wherein the concentration of BSA was 12mg/mL) and reacted at 4 ℃ for 15 hours. Then adding 100mg of TBAF into the reaction solution, and reacting for 12h at 10 ℃; placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; and centrifuging the dialyzed reaction solution, and taking supernatant to obtain 4.5mL of 4.2mg/mL fluoxetine antigen.
Comparative example 2
A preparation method of fluoxetine antigen comprises the following steps:
(1) synthesis of fluoxetine hapten:
and (2) taking 110mg of fluoxetine (prepared in example 1), sequentially adding 5mL of anhydrous methanol and 34mg of methyl acrylate, refluxing and stirring for reaction for 12h, after the reaction is finished, decompressing and drying the solvent, and purifying the product by using a silica gel column chromatography to extract the fluoxetine hapten intermediate. Weighing 43mg of fluoxetine hapten intermediate, adding 2mL of 10 wt% NaOH solution, carrying out reflux reaction for 2h, adjusting the pH of the solution to 5.0 by using 6mol/L HCl, extracting with ethyl acetate for 3 times, combining extracts, and carrying out reduced pressure evaporation to dryness to obtain 34mg of fluoxetine hapten.
(2) Synthesis of fluoxetine antigen:
30mg of fluoxetine hapten was dissolved in 0.8mL of DMF, 10mg of NHS and 16mg of DCC were added, and the mixture was reacted at room temperature for 15 hours, and after centrifugation, the supernatant was added to 4mL of BSA in PBS (where BSA was 12mg/mL) and reacted at 4 ℃ for 15 hours. Then placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; and centrifuging the dialyzed reaction solution, and taking a supernatant to obtain 4.5mL6.5mg/mL fluoxetine antigen.
Comparative example 3
A preparation method of fluoxetine antigen comprises the following steps:
42mg of fluoxetine hapten (prepared in example 1) was dissolved in 0.8mL of DMF and added with 10mg of NHS and 15mg of DCC to react at room temperature for 15h, and after centrifugation, the supernatant was added to 4mL of BSA in PBS (where BSA was 12mg/mL) and reacted at 4 ℃ for 15 h. Then adding 100mg of TBAF into the reaction solution, and reacting for 12h at 10 ℃; placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; the dialyzed reaction solution was centrifuged, and the supernatant was collected to obtain 4.6mL of 7.0mg/mL fluoxetine antigen.
Comparative example 4
A preparation method of fluoxetine antigen comprises the following steps:
42mg of fluoxetine hapten (prepared in example 1) was dissolved in 0.8mL of DMF, 10mg of NHS and 18mg of DCC were added and reacted at room temperature for 15 hours, and after centrifugation, the supernatant was added to 4mL of PBS solution (where BSA concentration was 12mg/mL) and reacted at 4 ℃ for 15 hours. Then adding 100mg of TBAF into the reaction solution, and reacting for 12h at 10 ℃; placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; the dialyzed reaction solution was centrifuged, and the supernatant was collected to obtain 4.3mL of 4.0mg/mL fluoxetine antigen.
Comparative example 5
A preparation method of fluoxetine antigen comprises the following steps:
42mg of fluoxetine hapten (prepared in example 1) was dissolved in 0.8mL of DMF, 10mg of NHS and 16mg of DCC were added and reacted at room temperature for 15 hours, and after centrifugation, the supernatant was added to 3mL of BSA in PBS (where BSA was 12mg/mL) and reacted at 4 ℃ for 15 hours. Then adding 100mg of TBAF into the reaction solution, and reacting for 12h at 10 ℃; placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; the dialyzed reaction solution was centrifuged, and the supernatant was collected to obtain 3.2mL of 6.0mg/mL fluoxetine antigen.
Comparative example 6
A preparation method of a fluoxetine antigen comprises the following steps:
42mg of fluoxetine hapten (prepared in example 1) was dissolved in 0.8mL of DMF, 10mg of NHS and 16mg of DCC were added and reacted at room temperature for 15 hours, and after centrifugation, the supernatant was added to 6mL of BSA in PBS (where BSA was 12mg/mL) and reacted at 4 ℃ for 15 hours. Then adding 100mg of TBAF into the reaction solution, and reacting for 12h at 10 ℃; placing the reaction solution in a PBS solution with the pH value of 7.4 for dialysis for 72 hours, and replacing the PBS solution every 24 hours; the dialyzed reaction solution was centrifuged, and the supernatant was collected to obtain 6.6mL of 8.0mg/mL fluoxetine antigen.
The activity of the fluoxetine antigens prepared in the examples 1-3 and the comparative examples 1-6 is detected by using a colloidal gold immunochromatography method, and the detection conditions are as follows: and (3) spraying the fluoxetine antigen IV on an NC membrane by a spraying amount of 1.0 mu g/cm through a membrane spraying machine, marking colloidal gold by a fluoxetine antibody, combining glass fiber paper and absorbent paper to assemble a test strip, and detecting the test strip. The results of the measurements are shown in tables 1 and 2.
TABLE 1
TABLE 2
From table 1, it can be seen that: in the embodiment 1-3, the preparation parameters of the hapten are adjusted within the parameter range, the negative detection T-line color intensity of the obtained antigen reaches G8.5, and the result proves that the synthesized fluoxetine antigen has higher activity and can be identified by the antibody and efficiently combined; positive detection proves that the fluoxetine PBS solution has competitive inhibition on the prepared fluoxetine antigen and has higher sensitivity and gradient.
Comparative example 1 differs from example 1 in that comparative example 1 is not modified but directly coupled to a carrier protein, whereas example 1 is modified by the process of the invention and then coupled to a carrier protein. From table 2, it can be seen that: the negative detection T-line color intensity and the positive detection sensitivity of the antigen obtained in comparative example 1 are lower than those of example 1. The invention proves that the fluoxetine can not be directly coupled with the protein, the fluoxetine can be coupled with the carrier protein after being modified by the method, and the prepared fluoxetine antigen has stronger specificity and higher sensitivity.
Comparative example 2 fluoxetine antigen was prepared by the method of the prior art (patent CN108264551A), i.e. a coupling arm containing a carboxyl group was first led out at the position of the secondary amine group in the fluoxetine molecule, and then reacted with an amino group in the carrier protein by an active ester method. From table 2, it can be seen that: the negative detection T-line color intensity and the positive detection sensitivity of the antigen obtained in comparative example 2 were lower than those of example 1. The method provided by the invention has the advantages that the fluoxetine antigen obtained by the method has better specificity and sensitivity compared with the prior art. The reason is that: the existing fluoxetine antigen preparation method cannot keep the active group secondary amine group in the fluoxetine; in the invention, before grafting carboxyl, Teoc-Cl is adopted to protect a secondary amine group in fluoxetine, and after coupling with carrier protein, the Teoc protecting group is removed, so that an active group in a fluoxetine molecular structure can be completely reserved, and the obtained fluoxetine antigen has stronger specificity and higher sensitivity.
Comparative example 3 differs from example 1 in that in step (4), the mass ratio of fluoxetine hapten, NHS, DCC, carrier protein BSA in example 1 is 1:0.238:0.381:1.143, and in comparative example 3 is 1:0.238:0.357: 1.143. As can be seen from tables 1 and 2: the negative detection T line color intensity of the antigen obtained in the comparative example 3 is only G7.0, which is obviously lower than that of the antigen obtained in the example 1, and the positive detection sensitivity is also reduced. The reason is that: when the dosage of NHS and DCC is too small, the carboxyl in hapten is incompletely activated, and the product titer is reduced.
Comparative example 4 differs from example 1 in that in step (4), the mass ratio of fluoxetine hapten, NHS, DCC, carrier protein BSA in example 1 was 1:0.238:0.381:1.143, and in comparative example 4 was 1:0.238:0.429: 1.143. As can be seen from tables 1 and 2: the negative detection T line color intensity of the antigen obtained in the comparative example 4 is only G7.5, which is obviously lower than that of the antigen obtained in the example 1, and the positive detection sensitivity is also reduced. In addition, the yield of fluoxetine antigen in comparative example 4 was also reduced compared to example 1. The reason is that: the excessive use of NHS and DCC easily causes the mutual coupling between carrier proteins, resulting in excessive precipitation and reducing the product titer and the product yield.
Comparative example 5 differs from example 1 in that in step (4), the mass ratio of fluoxetine hapten to carrier protein BSA in example 1 was 1:1.143, and in comparative example 5 was 1: 0.857. As can be seen from tables 1 and 2: the negative detection T line color intensity of the antigen obtained in the comparative example 5 can reach G9.0, and the positive detection result is the same as that of the antigen obtained in the example 1. However, the yield of fluoxetine antigen in comparative example 5 was lower compared to example 1. The reason is that: relatively less carrier protein can expose more fluoxetine molecules and be more easily combined with antibodies, so that the titer is improved, but the product yield is too low.
Comparative example 6 differs from example 1 in that in step (4), the mass ratio of fluoxetine hapten to carrier protein BSA in example 1 is 1:1.143, and in comparative example 6 is 1: 1.714. As can be seen from tables 1 and 2: the negative detection T line color intensity of the antigen obtained in the comparative example 6 is only G7.0, which is obviously lower than that of the antigen obtained in the example 1, and the positive detection sensitivity is also reduced. The reason is that: the carrier protein is excessively used, so that excessive fluoxetine molecules are wrapped by the carrier protein, the combination of an antigen and an antibody is influenced, and the product titer is excessively low.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (7)
1. A fluoxetine antigen characterized by the structural formula:
wherein R is carrier protein, and the carrier protein is one of bovine serum albumin, hemocyanin, egg albumin and bovine gamma globulin.
2. A preparation method of fluoxetine antigen is characterized by comprising the following steps:
(1) protecting a secondary amine group in fluoxetine by using 2-trimethylsilylethoxycarbonyl chloride to obtain N-trimethylsilylethoxycarbonyl-fluoxetine;
(2) carrying out Friedel-crafts acylation reaction on N-trimethylsilyl ethoxycarbonyl-fluoxetine and succinic anhydride to obtain fluoxetine hapten;
(3) coupling the fluoxetine hapten with carrier protein, and then removing a protecting group to obtain a fluoxetine antigen;
in the step (3), the specific process of coupling the fluoxetine hapten and the carrier protein is as follows:
dissolving fluoxetine hapten in a coupling solvent, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide, reacting for 14.5-15.5h at 20-30 ℃, centrifuging, taking clear liquid, adding the clear liquid into a PBS (phosphate buffer solution) solution of carrier protein, and reacting for 14.5-15.5h at 4-10 ℃;
the carrier protein is bovine serum albumin, and the mass ratio of the fluoxetine hapten to the N-hydroxysuccinimide to the dicyclohexylcarbodiimide to the bovine serum albumin is 1:0.21-0.26:0.38-0.40: 1.0-1.5;
wherein the fluoxetine antigen has a structural formula
And R is bovine serum albumin.
3. The preparation method of claim 2, wherein in the step (1), the fluoxetine is obtained by free extraction of fluoxetine hydrochloride by the following specific process: mixing fluoxetine hydrochloride and water, mashing, adjusting the pH value to 8.0-9.0, adding ethyl acetate for extraction, washing an organic layer with water, drying the organic layer with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain the fluoxetine.
4. The method according to claim 2, wherein the specific process of step (1) is as follows: dissolving fluoxetine in an organic solvent, adding an acid-binding agent, cooling to 0-5 ℃, adding 2-trimethylsilyl ethoxycarbonyl chloride, reacting at 20-30 ℃ for 10-24h, and separating a product to obtain the N-trimethylsilyl ethoxycarbonyl-fluoxetine.
5. The method according to claim 4, wherein in step (1), the mass ratio of fluoxetine to 2-trimethylsilylethyloxycarbonyl chloride is 1: 1.5-2.0.
6. The method according to claim 2, wherein the specific process of step (2) is as follows: dissolving N-trimethylsilyl ethoxycarbonyl-fluoxetine in a reaction solvent, adding succinic anhydride, cooling to 0-5 ℃ in a protective atmosphere, adding a Friedel-crafts acylation reaction catalyst, reacting at 0-5 ℃ for 1-1.5h, heating to 20-30 ℃ for 9.5-10.5h, stopping the reaction, and separating a product to obtain the fluoxetine hapten.
7. The process according to claim 2, wherein in the step (3), the protecting group is removed by the following procedure: after coupling fluoxetine hapten and carrier protein, tetrabutylammonium fluoride is added into the reaction liquid and reacts for 11.5-12.5h at the temperature of 10-20 ℃.
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