CN113214132A - Preparation method of hapten iodoacetyl thyroxine active coupling reagent - Google Patents
Preparation method of hapten iodoacetyl thyroxine active coupling reagent Download PDFInfo
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- CN113214132A CN113214132A CN202110530221.XA CN202110530221A CN113214132A CN 113214132 A CN113214132 A CN 113214132A CN 202110530221 A CN202110530221 A CN 202110530221A CN 113214132 A CN113214132 A CN 113214132A
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- hapten
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- iodothyroxine
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 38
- 230000008878 coupling Effects 0.000 title claims abstract description 35
- 238000010168 coupling process Methods 0.000 title claims abstract description 35
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 35
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 title claims abstract description 19
- 229940034208 thyroxine Drugs 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- -1 iodoacetyl thyroxine Chemical compound 0.000 title abstract description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 42
- ZBGMJXAWZLQCPJ-LBPRGKRZSA-N (2s)-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]-2-(iodoamino)propanoic acid Chemical compound IC1=CC(C[C@@H](C(=O)O)NI)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 ZBGMJXAWZLQCPJ-LBPRGKRZSA-N 0.000 claims abstract description 32
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 30
- 229940125904 compound 1 Drugs 0.000 claims abstract description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims abstract description 11
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 11
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 9
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 claims abstract description 8
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims abstract description 8
- 150000002148 esters Chemical class 0.000 claims abstract description 6
- 238000000605 extraction Methods 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims description 46
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 39
- 239000000243 solution Substances 0.000 claims description 27
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- 239000007864 aqueous solution Substances 0.000 claims description 24
- 230000035484 reaction time Effects 0.000 claims description 22
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 20
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 15
- 239000007810 chemical reaction solvent Substances 0.000 claims description 15
- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 11
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 239000002274 desiccant Substances 0.000 claims description 9
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 8
- 239000007832 Na2SO4 Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 7
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical group Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- LEKJTGQWLAUGQA-UHFFFAOYSA-N acetyl iodide Chemical compound CC(I)=O LEKJTGQWLAUGQA-UHFFFAOYSA-N 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 5
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 5
- 230000002378 acidificating effect Effects 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 3
- 239000003513 alkali Substances 0.000 claims description 3
- 239000012670 alkaline solution Substances 0.000 claims description 3
- 239000001257 hydrogen Substances 0.000 claims description 3
- 229910052739 hydrogen Inorganic materials 0.000 claims description 3
- 229910052740 iodine Inorganic materials 0.000 claims description 3
- 239000011630 iodine Substances 0.000 claims description 3
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 2
- 238000004440 column chromatography Methods 0.000 claims 1
- 238000010898 silica gel chromatography Methods 0.000 claims 1
- 239000002994 raw material Substances 0.000 abstract description 13
- 239000000427 antigen Substances 0.000 abstract description 8
- 102000036639 antigens Human genes 0.000 abstract description 8
- 108091007433 antigens Proteins 0.000 abstract description 8
- 230000015572 biosynthetic process Effects 0.000 abstract description 7
- 238000000746 purification Methods 0.000 abstract description 5
- WVMNBFKLJMCFEB-AWEZNQCLSA-N CC(N([C@@H](CC(C=C1I)=CC(I)=C1OC(C=C1I)=CC(I)=C1O)C(O)=O)I)=O Chemical compound CC(N([C@@H](CC(C=C1I)=CC(I)=C1OC(C=C1I)=CC(I)=C1O)C(O)=O)I)=O WVMNBFKLJMCFEB-AWEZNQCLSA-N 0.000 abstract description 4
- 150000003384 small molecules Chemical class 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 12
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N dichloromethane Natural products ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 4
- 229920006395 saturated elastomer Polymers 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 235000011152 sodium sulphate Nutrition 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 3
- 108010078791 Carrier Proteins Proteins 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 230000008570 general process Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 231100000481 chemical toxicant Toxicity 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 150000007529 inorganic bases Chemical class 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- ZHSOTLOTTDYIIK-ZDUSSCGKSA-N (2S)-2-amino-3-[4-(4-hydroxyphenoxy)-3,5-diiodophenyl]propanoic acid Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C=C1 ZHSOTLOTTDYIIK-ZDUSSCGKSA-N 0.000 description 1
- XUIIKFGFIJCVMT-LBPRGKRZSA-N L-thyroxine Chemical compound IC1=CC(C[C@H]([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-LBPRGKRZSA-N 0.000 description 1
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- DEZRYPDIMOWBDS-UHFFFAOYSA-N dcm dichloromethane Chemical compound ClCCl.ClCCl DEZRYPDIMOWBDS-UHFFFAOYSA-N 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 229950008325 levothyroxine Drugs 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 235000015320 potassium carbonate Nutrition 0.000 description 1
- 235000011181 potassium carbonates Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 229940035722 triiodothyronine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a preparation method of a hapten acetyl iodothyroxine active coupling reagent, which comprises the steps of preparing acetic acid active ester; reacting the prepared active acetate with iodothyroxine to synthesize an intermediate compound 1; and (3) reacting the intermediate compound 1 with N-hydroxysuccinimide and a condensing agent to synthesize the hapten iodoacetyl thyroxine active coupling reagent. The invention has the advantages of low price and easy obtainment of raw materials, few synthesis steps, simple operation, convenient extraction and purification and high yield, provides a brand-new solution for synthesizing the hapten iodoacetyl thyroxine active coupling reagent, and is beneficial to solving the problems of synthesizing key raw materials of artificial antigens by using small-molecule haptens in the preparation of monoclonal antibodies and developing key raw materials in clinical detection kit for detecting the thyroxine activity.
Description
Technical Field
The invention relates to the technical field of chemical synthesis, in particular to a preparation method of a hapten iodoacetyl thyroxine active coupling reagent.
Background
The current methods for synthesizing hapten iodoacetyl methide (T4 and anti-T4, T3 and anti-T3, and T2 and anti-T2) active coupling reagents are rare, mainly because of the following technical difficulties: acetic anhydride is available for reaction in solvent DMF in T4 acetylation, but iodothyroxine (T4 and trans T4, T3 and trans T3, and T2 and trans T2) has poor solubility in organic solvents or water, resulting in long reaction time, low yield, many side reactions, and difficult purification. And the acetic anhydride used in the process is a chemical product which is easily prepared and disinfected and is regulated by the state, and the raw material is not easy to obtain.
Iodothyroxine is a hapten with a molecular mass of less than 1000u, which is only reactive and non-immunogenic. Iodothyroidin has amino, carboxyl and phenolic hydroxyl groups. Hapten which retains original molecular structure characteristics and is provided with active groups is designed and synthesized, the hapten is coupled with a macromolecular mass protein carrier through a covalent bond to prepare artificial immunogen, and a specific antibody aiming at the hapten is prepared through an animal immunization procedure.
Iodothyroxine is a small molecule hapten, and is lack of more than two sites which can be used for an immunoassay sandwich method, so that the iodothyroxine cannot be measured by a double-antibody sandwich method and can only be detected by adopting a competition method mode, while in the development of the existing mainstream chemiluminescence immunoassay kit, an active derivative reagent with a structure similar to that of the iodothyroxine hapten needs to be used to be coupled with carrier protein to form an antigen marker, and the antigen marker and free antigen in a sample to be detected carry out competition reaction.
Therefore, the method for synthesizing the hapten iodothyroxine active coupling reagent by using the general process with low cost, easy operation and cheap and easily available raw materials is very important.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provides a preparation method of a hapten acetyl iodothyroxine (comprising T4 and reverse T4, T3 and reverse T3, and T2 and reverse T2) active coupling reagent.
In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:
the invention discloses a preparation method of a hapten iodoacetyl thyroxine active coupling reagent, which comprises the following steps:
s1, preparing acetic acid active ester;
s2, reacting the prepared active acetate with iodothyroxine to synthesize an intermediate compound 1;
s3, reacting the intermediate compound 1 with N-hydroxysuccinimide and a condensing agent to synthesize the hapten iodomethacin active coupling reagent 2;
the synthesis reaction formula is as follows:
as a further improvement of the embodiment of the present invention, the general formula of the iodothyroxine is represented by formula a and formula B, wherein formula B is trans of formula a;
wherein R is1And R2The radical is hydrogen or iodine; formula a includes T4, T3, or T2; formula B includes reverse T4, reverse T3, and reverse T2;
the specific chemical formula is shown as follows:
as a further improvement of the embodiment of the present invention, the specific steps of preparing the active acetate in step S1 are adding acetic acid and N-hydroxysuccinimide into a first reaction solvent, slowly adding a condensing agent below a first preset reaction temperature, and reacting at a second preset reaction temperature for a first preset reaction time or longer; the condensing agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.
As a further improvement of the embodiment of the present invention, the specific steps of synthesizing intermediate compound 1 in step S2 include:
adding iodothyroxine to the first reaction solvent and further mixing and dissolving the iodothyroxine in an aqueous alkaline solution to obtain an iodothyroxine solution;
and (2) under the protection of nitrogen, dropwise adding the active acetate solution prepared in the step (S1) into the iodothyroxine solution, reacting at room temperature for more than a second preset reaction time, adjusting the pH to be acidic by using dilute acid after the reaction is finished, extracting for 3 times by using an extracting agent, washing for 2 times by using saturated NaCl aqueous solution, drying the extract by using a drying agent, and removing the extracting agent in a rotary evaporator to obtain an intermediate compound 1.
As a further improvement of the embodiment of the present invention, in the step S3, under the protection of nitrogen, adding intermediate compound 1 and hou into a second reaction solvent, adding a condensing agent below the first preset reaction temperature, reacting at a third preset reaction temperature for more than a third preset reaction time, after the reaction is completed, diluting with water, adding a preset amount of an extraction agent, extracting for 3 times, adding a saturated aqueous NaCl solution, washing for 2 times, drying the extract with a drying agent, removing the extraction agent in a rotary evaporator, purifying with a column, and obtaining a white-like solid powder, i.e., the hapten iodoacetyl methine active coupling reagent, with an eluent EA: DCM: PE: 1:1: 1.
As a further improvement of the embodiment of the present invention, in the step S1, the first reaction solvent is DMF or DMSO; the first preset reaction temperature and the second preset reaction temperature are 10-50 ℃; the first preset reaction time is 0.5-24 h.
As a further improvement of the embodiment of the present invention, the second preset reaction time is 0.5h to 24 h; the second reaction solvent in the step S3 is DMF or DMSO; the third preset reaction time is 0.5h-24 h;
the third preset reaction temperature is 10-50 ℃; the condensing agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.
As a further improvement of the embodiment of the present invention, the first preset reaction temperature in the step S1 is 20 ℃, and the second preset reaction temperature is 25 ℃; the second preset reaction time in the step S2 is 1 h.
As a further improvement of the embodiment of the present invention, the extractant in the steps S2 and S3 is EA or DCM; the drying agent in the steps S2 and S3 is anhydrous Na2SO4Or anhydrous MgSO4；
As a further improvement of the embodiment of the present invention, in the step S2, the diluted acid is a diluted hydrochloric acid aqueous solution or a diluted sulfuric acid aqueous solution, a diluted citric acid aqueous solution;
as a further improvement of an embodiment of the present invention, the alkali in the basic aqueous solution is K2CO3、Na2CO3、NaHCO3At least one of them or a mixture of any of them.
As a further improvement of the embodiment of the invention, the product after the step S3 is purified by passing through a 200-300 mesh silica gel column to obtain the hapten iodomethacin active coupling reagent.
The invention has the beneficial effects that:
the invention provides a brand-new process for preparing hapten acetyl iodothyroxine (T4 and inverse T4, T3 and inverse T3, T2 and inverse T2) active coupling reagent, which takes cheap acetic acid and iodothyroxine (T4 and inverse T4, T3 and inverse T3, T2 and inverse T2) as starting raw materials, replaces the raw materials of easily prepared toxic chemicals regulated by China, synthesizes different target products by a general process method for dissolving the iodothyroxine in a mixed solvent DMF and water by using inorganic base, does not need to purify an intermediate compound 1, has simple steps and convenient treatment, omits the operation of purifying the intermediate compound 1, reduces the cost, shortens the production period, is simple to operate, and also improves the yield; the prepared active coupling reagent of the acetyl iodide thyroxine can easily react with amino in carrier protein to obtain artificial antigen, and the invention firstly provides a solution for synthesizing the active coupling reagent of the hapten acetyl iodide thyroxine, and solves the problems that the small molecule hapten synthesizes the key raw material of the artificial antigen in the preparation of monoclonal antibody and the key raw material in the development of clinical detection thyroxine kits.
It should be noted that the abbreviations of the reagents used in the present invention are as follows:
t4: tetraiodothyronine
T3: triiodothyronine
T2: diiodothyronine
DMF: n, N-dimethylformamide
EA ethyl acetate
DCM dichloromethane
HOSU: n-hydroxysuccinimide
EDC. HCl: 1-Ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride
K2CO3: potassium carbonate
Na2CO3: sodium carbonate
NaHCO3: sodium bicarbonate
HCl: hydrogen chloride
Na2SO4: sodium sulfate
MgSO4: magnesium sulfate.
Detailed Description
The present invention will be described in detail with reference to examples.
The invention discloses a preparation method of a hapten iodoacetyl thyroxine active coupling reagent, which comprises the following steps:
s1, preparing acetic acid active ester;
s2, reacting the prepared active acetate with iodothyroxine to synthesize an intermediate compound 1;
s3, reacting the intermediate compound 1 with N-hydroxysuccinimide and a condensing agent to synthesize a hapten iodoacetyl thyroxine active coupling reagent 2;
the synthesis reaction formula is as follows:
the general formula of the iodothyroxine is shown as formula A and formula B, wherein the formula B is trans of the formula A;
wherein R is1And R2The radical is hydrogen or iodine; formula A includes T4、T3Or T2(ii) a Formula B includes a reverse T4Reverse T3And reverse T2；
The specific chemical formula is shown as follows:
specifically, the step S1 of preparing the active acetate includes the steps of adding acetic acid and N-hydroxysuccinimide into a first reaction solvent, slowly adding a condensing agent below a first preset reaction temperature, and reacting at a second preset reaction temperature for a first preset reaction time or longer; the condensing agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.
Wherein, the first reaction solvent in the step S1 is DMF or DMSO; the first preset reaction temperature and the second preset reaction temperature are 10-50 ℃; the first preset reaction time is 0.5-24 h.
Further, the specific steps for synthesizing the intermediate compound 1 in step S2 include:
adding iodothyroxine to the first reaction solvent and further mixing and dissolving the iodothyroxine in an aqueous alkaline solution to obtain an iodothyroxine solution;
and (2) under the protection of nitrogen, dropwise adding the active acetate solution prepared in the step (S1) into the iodothyroxine solution, reacting at room temperature for more than a second preset reaction time, adjusting the pH to be acidic by using dilute acid after the reaction is finished, extracting for 3 times by using an extracting agent, washing for 2 times by using saturated NaCl aqueous solution, drying the extract by using a drying agent, and removing the extracting agent in a rotary evaporator to obtain an intermediate compound 1.
Specifically, the specific step of step S3 is to add intermediate compound 1 and hou to a second reaction solvent under the protection of nitrogen, add a condensing agent below the first preset reaction temperature, react for more than a third preset reaction time at a third preset reaction temperature, dilute the mixture with water after the reaction is completed, add a preset amount of an extracting agent to extract the mixture for 3 times, add a saturated aqueous solution of NaCl to wash the mixture for 2 times, dry the extract with a drying agent, remove the extracting agent in a rotary evaporator, purify the mixture through a column, and obtain an eluent with EA: DCM: PE: 1:1 to obtain a white-like solid powder, namely the hapten iodoacetyl thyroxine active coupling reagent.
Wherein the second preset reaction time is 0.5h-24h, preferably more than 1 h; the second reaction solvent in the step S3 is DMF or DMSO; the third preset reaction time is 0.5h-24 h;
preferably, the third preset reaction temperature is 10-50 ℃; the condensing agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.
Preferably, the first preset reaction temperature in step S1 is 20 ℃, and the second preset reaction temperature is 25 ℃; in step S2, the second preset reaction time is 1 h.
As a further improvement of the embodiment of the present invention, the steps S2 and S3 are performed in the same mannerTaking EA or DCM as a reagent; the drying agent in the steps S2 and S3 is anhydrous Na2SO4Or anhydrous MgSO4；
In step S2, the dilute acid is a dilute hydrochloric acid aqueous solution or a dilute sulfuric acid aqueous solution, or a dilute citric acid aqueous solution;
and the alkali in the alkaline aqueous solution is K2CO3、Na2CO3、NaHCO3At least one of them or a mixture of any of them.
In the embodiment of the invention, the product after the step S3 is purified by passing through a 200-300 mesh silica gel column to obtain the hapten iodoacetyl thyroxine active coupling reagent.
Example 1
Specific experimental conditions for the synthesis of intermediate compound 1 in S1 and S2 include: acetic acid (0.7g, 3eq) and HOSU (1.3g, 3eq) were added to DMF (10ml) under nitrogen protection, EDC & HCl (2.2g, 3eq) was added slowly at below 20 ℃ and the reaction was carried out for 1 hour or more at 25 ℃. T4(3g, 1eq) was then added to DMF (15ml) and K was added2CO3(1.6g, 3eq) was dissolved in water (20ml) and then dissolved T4 was mixed. Dropwise adding the reacted active acetate solution into a T4 solution, reacting at room temperature for 1h, adjusting the pH to acidity with a dilute HCl aqueous solution after the reaction is finished, extracting with proper amount of EA for 3 times, washing with a saturated NaCl aqueous solution for 2 times, and using anhydrous Na for the EA solution2SO4Drying, removing EA in a rotary evaporator without purification, and directly using the EA in the next reaction;
s3, synthesis of product 2: adding intermediate compound 1(1.5g, 1eq) and HOSU (0.26g, 1.2eq) into DMF (20ml) under nitrogen protection, adding EDC & HCl (0.43g,1.2eq) at 20 deg.C or below, reacting at 25 deg.C for 1 hr or more, adding appropriate amount of water after reaction, extracting with appropriate amount of EA for 3 times, washing with appropriate amount of saturated aqueous NaCl solution for 2 times, drying the EA solution with anhydrous Na2SO4, removing EA in rotary evaporator, purifying with column, eluting with EA: DCM: PE: 1:1:1, to obtain 1.57g of white-like solid powderThe total yield of the two steps is 83 percent, the HPLC purity is 98.08 percent,1H NMR(DMSO-d6)9.29(s,1H),8.65(d，1H)， 7.93(s,2H)，7.06(s，2H),5.01(m，1H)，3.23(dd，1H)，2.96 (m，1H)，2.83(s，4H)，1.82(s，3H)。
example 2
S1 and S2, synthesis of intermediate compound 1: acetic acid (0.82g, 3eq) and HOSU (1.6g, 3eq) were added to DMF (10ml) under nitrogen protection, EDC & HCl (2.65g, 3eq) was added slowly at below 20 ℃ and the reaction was carried out for 1 hour or more at 25 ℃. T3(3g, 1eq) was added to DMF (15ml), K2CO3(1.9g, 3eq) was added to water (20ml) and dissolved, followed by mixing and dissolving T3. Dripping the reacted active acetate solution into T3 solution, reacting at room temperature for more than 1h, adjusting pH to acidity with dilute HCl aqueous solution, extracting with appropriate amount of EA for 3 times, washing with saturated NaCl aqueous solution for 2 times, and adding anhydrous Na into the EA solution2SO4Drying, removing EA in a rotary evaporator without purification, and directly using the EA in the next reaction;
s3, synthesis of product 2: adding compound 1(1.5g, 1eq) and HOSU (0.3g, 1.2eq) into DMF (20ml) under nitrogen protection, adding EDC & HCl (0.5g,1.2eq) below 20 ℃, reacting at 25 ℃ for more than 1h, adding a proper amount of water after the reaction is finished, extracting with a proper amount of EA for 3 times, washing with a proper amount of saturated NaCl aqueous solution for 2 times, and using anhydrous Na for the EA solution2SO4Drying, removing EA in rotary evaporator, purifying with column, eluting with EA: DCM: PE: 1:1:1 to obtain 1.55g white solid powder with two-step total yield of 81% and HPLC purity of 98.23%,1H NMR(DMSO-d6)9.99(s，1H)，8.62 (d,1H),7.91(s，2H)，6.96(d，1H),6.82(d，1H)，6.60(dd， 1H)，4.99(m，1H)，3.21(dd，1H)，2.95(m，1H)，2.83(s，4H)， 1.82(s，3H)。
example 3
Specific experimental conditions for the synthesis of S1, S2 intermediate compound 1: acetic acid (1g, 3eq) and HOSU (2g, 3eq) were added to DMF (10ml) under nitrogen protection, EDC & HCl (3.3g, 3eq) was added slowly at below 20 ℃ and the reaction was carried out for more than 1h at 25 ℃. T2(3g, 1eq) was then added to DMF (15ml) and K was added2CO3(2.4g, 3eq) was dissolved in water (20ml) and then dissolved T2 was mixed. Dropwise adding the reacted acetic acid active ester solution into a T2 solution, reacting at room temperature for more than 1h, adjusting the pH to be acidic by using a dilute HCl aqueous solution after the reaction is finished, extracting with proper amount of EA for 3 times, washing with a saturated NaCl aqueous solution for 2 times, drying the EA solution with anhydrous Na2SO4, removing the EA in a rotary evaporator without purification, and directly using the EA solution for the next reaction;
s3, synthesis of product 2: adding compound 1(1.5g, 1eq) and HOSU (0.37g, 1.2eq) into DMF (20ml) under the protection of nitrogen, adding EDC & HCl (0.62g,1.2eq) at 20 ℃ below, reacting at 25 ℃ for more than 1h, adding a proper amount of water after the reaction is finished, extracting with a proper amount of EA for 3 times, washing with a proper amount of saturated aqueous NaCl solution for 2 times, drying the EA solution with anhydrous Na2SO4, removing EA in a rotary evaporator, purifying by passing through a column, eluting with EA, DCM, PE (proportion) 1:1: 1:1 to obtain 1.68g of white solid powder, the total yield of the two steps is 84%, and the HPLC purity is 98.36%,1H NMR(DMSO-d6)9.10(s，1H)，8.62 (d,1H),7.90(s，2H)，6.67(d，2H),6.52(d，2H)，4.97(m，1H)， 3.21(dd，1H)，2.96(m，1H)，2.83(s，4H)，1.83(s，3H)。
the invention has the beneficial effects that:
the invention provides a brand-new process for preparing hapten acetyl iodothyroxine (T4 and inverse T4, T3 and inverse T3, T2 and inverse T2) active coupling reagent, which takes cheap acetic acid and iodothyroxine (T4 and inverse T4, T3 and inverse T3, T2 and inverse T2) as starting raw materials, replaces the raw materials of easily prepared toxic chemicals regulated by China, synthesizes different target products by a general process method for dissolving the iodothyroxine in a mixed solvent DMF and water by using inorganic base, does not need to purify an intermediate compound 1, has simple steps and convenient treatment, omits the operation of purifying the intermediate compound 1, reduces the cost, shortens the production period, is simple to operate, and also improves the yield; the prepared active coupling reagent of the acetyl iodide thyroxine can easily react with amino in carrier protein to obtain artificial antigen, and the invention firstly provides a solution for synthesizing the active coupling reagent of the hapten acetyl iodide thyroxine, and solves the problems that the small molecule hapten synthesizes the key raw material of the artificial antigen in the preparation of monoclonal antibody and the key raw material in the development of clinical detection thyroxine kits.
Claims (10)
1. A preparation method of a hapten acetyl iodide thyroxine active coupling reagent is characterized by comprising the following steps:
s1, preparing acetic acid active ester;
s2, reacting the prepared active acetate with iodothyroxine to synthesize an intermediate compound 1;
s3, reacting the intermediate compound 1 with N-hydroxysuccinimide and a condensing agent to synthesize the hapten iodomethacin active coupling reagent 2;
the synthesis reaction formula is as follows:
2. the method for preparing the hapten iodothyroxine active coupling reagent according to claim 1, wherein the iodothyroxine has a general formula shown as formula a and formula B, wherein the formula B is trans of formula a;
wherein R is1And R2The radical is hydrogen or iodine; formula a includes T4, T3, or T2; formula B includes reverse T4, reverse T3, and reverse T2;
the specific chemical formula is shown as follows:
3. the method for preparing a hapten iodomethacin active coupling reagent as claimed in claim 1, wherein the specific steps of preparing the acetic acid active ester in the step S1 are adding acetic acid and N-hydroxysuccinimide into a first reaction solvent, slowly adding a condensing agent below a first preset reaction temperature, and reacting at a second preset reaction temperature for a first preset reaction time or longer; the condensing agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.
4. The method for preparing a hapten iodomethacin active coupling reagent according to claim 3, wherein the specific steps of synthesizing intermediate compound 1 in step S2 comprise:
adding iodothyroxine to the first reaction solvent and further mixing and dissolving the iodothyroxine in an aqueous alkaline solution to obtain an iodothyroxine solution;
and (2) under the protection of nitrogen, dropwise adding the active acetate solution prepared in the step (S1) into the iodothyroxine solution, reacting at room temperature for more than a second preset reaction time, adjusting the pH to be acidic by using dilute acid after the reaction is finished, extracting for 3 times by using an extracting agent, washing for 2 times by using saturated NaCl aqueous solution, drying the extract by using a drying agent, and removing the extracting agent in a rotary evaporator to obtain an intermediate compound 1.
5. The method for preparing a hapten iodothyroxine active coupling reagent according to claim 4, wherein the step S3 comprises adding intermediate compound 1 and HOSU into a second reaction solvent under the protection of nitrogen, adding a condensing agent below a first preset reaction temperature, reacting at a third preset reaction temperature for more than a third preset reaction time, diluting with water after the reaction is completed, adding a preset amount of an extraction agent, extracting for 3 times, adding saturated NaCl aqueous solution, washing for 2 times, drying the extract with a drying agent, removing the extraction agent in a rotary evaporator, purifying by column chromatography, and obtaining a white-like solid powder as the hapten iodothyroxine active coupling reagent, wherein the extraction agent is EA: DCM: PE: 1: 1.
6. The method for preparing a hapten-iodomethacin-active coupling reagent according to claim 3, wherein the first reaction solvent in step S1 is DMF or DMSO; the first preset reaction temperature and the second preset reaction temperature are 10-50 ℃; the first preset reaction time is 0.5-24 h.
7. The method for preparing a hapten iodomethacin active coupling reagent according to claim 5, wherein said second predetermined reaction time is 0.5h-24 h; the second reaction solvent in the step S3 is DMF or DMSO; the third preset reaction time is 0.5h-24 h;
the third preset reaction temperature is 10-50 ℃; the condensing agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.
8. The method for preparing a hapten iodomethacin-active coupling reagent of claim 6, wherein said first predetermined reaction temperature of step S1 is 20 ℃ and said second predetermined reaction temperature is 25 ℃; the second preset reaction time in the step S2 is 1 h.
9. The method for preparing the hapten iodomethacin active coupling reagent according to claim 5, wherein the extracting agent in steps S2 and S3 is EA or DCM; the drying agent in the steps S2 and S3 is anhydrous Na2SO4Or anhydrous MgSO4；
In the step S2, dilute acid is dilute hydrochloric acid aqueous solution or dilute sulfuric acid aqueous solution or dilute citric acid aqueous solution;
the alkali in the alkaline aqueous solution is K2CO3、Na2CO3、NaHCO3At least one of them or a mixture of any of them.
10. The method as claimed in claim 5, wherein the product of step S3 is purified by 200-300 mesh silica gel column chromatography to obtain the hapten iodomethacin active coupling reagent.
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