CN104262250A - Triiodothyroxin hapten luminous marker and synthetic method thereof - Google Patents
Triiodothyroxin hapten luminous marker and synthetic method thereof Download PDFInfo
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- CN104262250A CN104262250A CN201410441542.2A CN201410441542A CN104262250A CN 104262250 A CN104262250 A CN 104262250A CN 201410441542 A CN201410441542 A CN 201410441542A CN 104262250 A CN104262250 A CN 104262250A
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- iodo
- thyroxine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
- C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
Abstract
The invention discloses a triiodothyroxin hapten luminous marker and a synthetic method thereof. The synthetic method of the triiodothyroxin hapten luminous marker comprises the following steps: A) preparing a trifluoroacetamide crude product; B) preparing trifluoroacetamide; C) further preparing trifluoroacetamide; D) preparing triiodothyroxin methyl ester; E) further preparing triiodothyroxin methyl ester; F) further preparing triiodothyroxin methyl ester; and G) preparing a target product, namely acridine-marked triiodothyroxin methyl ester. The invention discloses a novel compound for detecting triiodothyroxin hapten by virtue of a chemical luminescence method as well as a synthetic route and a synthetic method of the compound. The marker of the small molecules is easy to purify, can be industrially produced and improves the detection sensitivity. The defects that the protein macromolecular compounds used by the conventional detection reagent have high price, difference easily produced in a production process, and the like are overcome.
Description
Technical field
The invention belongs to organic synthesis field, especially relate to the synthetic method of three iodo thyroxine haptens luminous markers.
Background technology
Chemiluminescence immune assay is the on-radiation immuno analytical method that development is very fast in world wide, be continue enzyme immune technology, put immune technology, immunofluorence technic after the supersensitive Micrometry of one that grows up.It has highly sensitive, linearity range is wide, easy and simple to handle fast, marker good stability, the advantage such as pollution-free, result is stable, be that current immune quantitative analyzes optimal method.In chemiluminescence immune assay, conventional luminophore comprises luminol,3-aminophthalic acid cyclic hydrazide, different luminol,3-aminophthalic acid cyclic hydrazide, acridinium ester, 1,2-dioxetane compounds (1,2-dioxetane compound).Wherein different luminol,3-aminophthalic acid cyclic hydrazide and acridinium ester directly mark as labelled molecule, belong to flash type chemiluminescence reaction.Luminol,3-aminophthalic acid cyclic hydrazide and 1,2-dioxetane compounds rely on enzyme to mark as labelled molecule, belong to enzymatic wide variety of glow-type chemiluminescence reaction.1,2-dioxetane compounds is up-to-date overdelicate alkaline phosphatase substrate, in suitable damping fluid, along with the catalytic hydrolysis effect of alkaline phosphatase, 1, the decomposition of 2-dioxetane compounds sends the very high optical signal of intensity, and sustainable more than 20 hours of signal is desirable chemiluminescent substance.It is marker that external manufacturer develops with alkaline phosphatase, and 1,2-dioxetane compounds is the test kit of substrate, and what match with it has DPC, Beckman, BioMerieux, Olympus Full-automatic chemiluminescence system.Chemoluminescence is the optical radiation that chemical substance produces in specified chemical reaction.In chemical reaction, excite singlet molecule to be formed by the decomposition of high energy intermediate, get back to ground state after molecule is excited, some of energy discharges with light emitting species.Therefore, any one chemiluminescence reaction comprises two processes: excite and luminescence process.Some excited state molecule energy also can be jumped by string between being and be that interior string jumps and disappears.
Chemoluminescence is as external widely used immuno analytical method in recent years, and it has highly sensitive, detects linear wide ranges and the feature such as pollution-free.Luminol,3-aminophthalic acid cyclic hydrazide is mainly contained, different luminol,3-aminophthalic acid cyclic hydrazide, the systems such as acridine and epidioxy ethane derivative (as AMPPD, CSPD etc.) in current chemiluminescence immune assay.Wherein in acridine system, the multiplex competition law of the thyroxinic detection of three iodos realizes, but in conventional kit, competition antigen used mostly is protein macromole, and this type of material is expensive, and production process easily produces difference, affects downstream and uses.
Many employing protein three iodo thyroxine markers in existing mensuration three iodo thryomimetic agent box, this type of marker not easily purifying, produces differences between batches large, expensive, directly enhances unit testing cost.
Summary of the invention
The present invention proposes a kind of novelly to detect the thyroxinic compound of three iodos and synthetic route and method for chemoluminescence method, and this type of micromolecular marker is easy to purifying, can suitability for industrialized production, and improves detection sensitivity.Solve the protein macromolecular cpd that detection reagent is in the past used, expensive, easily produce the shortcomings such as difference in production process.
The present invention synthesizes and a kind ofly novelly detects the thyroxinic compound of three iodos for chemoluminescence method, has following chemical structural formula:
The invention also discloses a kind of method of three iodo thyroxine haptens luminous markers, comprise step:
A) be dissolved in acetonitrile by two nonyl triamine (compound 1) and add Trifluoroacetic Acid Ethyl Ester, stirred overnight at room temperature, terminate with tlc monitoring reaction, concentrating under reduced pressure, drains, and obtains trifluoroacetamide (compound 2) crude product;
B) trifluoroacetamide (compound 2) crude product acetonitrile is dissolved, first add triethylamine, then add Succinic anhydried and add, stirred overnight at room temperature, with column chromatography purification after concentrating under reduced pressure, obtain trifluoroacetamide (compound 3);
C) trifluoroacetamide (compound 3) is dissolved with acetonitrile, first add N-Hydroxysuccinimide, then add EDC.HCl, stirred overnight at room temperature, terminate rear column chromatography purification with tlc monitoring reaction, obtain trifluoroacetamide (compound 4);
D) compound T3 anhydrous methanol is dissolved; Add SOCl
2reflux stirs spends the night, and concentrating under reduced pressure, drains, and obtains three iodo thyroxine methyl esters (compound 5);
E) trifluoroacetamide (compound 4) and three iodo thyroxine methyl esters (compound 5) are dissolved in DMF, stirred overnight at room temperature, terminate rear column chromatography purification with tlc monitoring reaction, obtain three iodo thyroxine methyl esters (compound 6);
F) three iodo thyroxine methyl esters (compound 6) are dissolved in methyl alcohol to add water again, add the reaction of salt of wormwood stirring at room temperature, liquid chromatography monitoring reaction, stirring at room temperature reaction is spent the night, through preparative chromatography purifying, obtain three iodo thyroxine methyl esters (compound 7);
G) three iodo thyroxine methyl esters (compound 7) are dissolved in dry DMF, add DIPEA, acridinium ester (compound 8) is added reaction solution, stirring at room temperature is reacted, after liquid chromatography monitoring reaction terminates, obtain acridine through preparative chromatography purifying and mark three iodo thyroxine methyl esters (compound 9).
Further: steps A) described pair of nonyl triamine compound 1 is replaced by two (hexamethylene) triamine or dipropyl triamine.
Further: step B) described Succinic anhydried is replaced by Pyroglutaric acid.
Further: step G) described acridinium ester (compound 8) is replaced, as NSP-DMAE-NHS, NSP-SA-NHS etc. by other acridinium ester compounds.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
It is of the present invention that novel to detect the synthetic route of the thyroxinic compound of three iodos for chemoluminescence method as follows:
Concrete:
Prepared by compound 2
Two nonyl triamine 11.55g is dissolved in 100ml acetonitrile, adds Trifluoroacetic Acid Ethyl Ester 1.56g.
1. stirred overnight at room temperature.
2. terminate with tlc monitoring reaction.
3. concentrating under reduced pressure, drains, and obtains trifluoroacetyl amine compound 2 (Formula:C20H39F6N3O4S2, MW:563.66) crude product 2g.
Prepared by compound 3
1. trifluoroacetamide (compound 2) crude product acetonitrile 100ml is dissolved, first add triethylamine 0.875ml.
2.0.74g Succinic anhydried adds, stirred overnight at room temperature.
3. after concentrating under reduced pressure with column chromatography purification, obtain trifluoroacetamide (compound 3) (Formula:C24H43F6N3O7S2:, MW:663.73) 1.38g yield 40%.
Prepared by compound 4
1. trifluoroacetyl amine compound 30.25g acetonitrile is dissolved, first add N-Hydroxysuccinimide 43.6mg.
2. add EDC.HCl 72.3mg again, stirred overnight at room temperature.
3. terminate rear column chromatography purification with tlc monitoring reaction, obtain trifluoroacetamide (compound 4) (Formula:C28H46F6N4O9S2:, MW:760.81) 0.25g yield 87%.
Prepared by compound 5
1. T3 0.5g anhydrous methanol is dissolved.
2. add SOCl
2reflux stirs spends the night.
3. concentrating under reduced pressure, drains, and obtains three iodo thyroxine methyl compounds 5 (Formula:C16H14I3NO4:, MW:665.00) 0.48g yield 97.5%.
Prepared by compound 6
1. trifluoroacetamide (compound 4) (100mg) is dissolved in 3mlDMF with three iodo thyroxine methyl compound 589mg, stirred overnight at room temperature.
2. terminate rear column chromatography purification with tlc monitoring reaction, obtain target product three iodo thyroxine methyl compound 6 (Formula:C40H55F6I3N4O10S2:, MW:1310.72) 80mg yield 46%.
Prepared by compound 7
1. three iodo thyroxine methyl esters (compound 6) (180mg) are dissolved in 3.3ml methyl alcohol and add 6.6ml water again.
2. add the reaction of salt of wormwood 189mg stirring at room temperature.
3. liquid chromatography monitoring reaction, stirring at room temperature reaction is spent the night.
4., through preparative chromatography purifying, obtain three iodo thyroxine methyl compounds 7 (Formula:C38H57I3N4O6:, MW:1046.60) 134mg productive rate 77%.
Prepared by compound 9
1. three iodo thyroxine methyl compounds 7 (29mg) are dissolved in dry DMF.
2. add DIPEA 19.8ml.
3. acridinium compound 8 (32.9mg) is added reaction solution, stirring at room temperature is reacted.
4. liquid chromatography monitoring is after reaction terminates, and obtains target product acridine mark three iodo thyroxine methyl compounds (Formula:C88H99I3N6O20S2:, MW:2005.60) 9 15mg yields 27% through preparative chromatography purifying.
Claims (5)
1. three iodo thyroxine haptens luminous markers, have chemical structural formula as follows:
2. synthesize a method for three iodo thyroxine haptens luminous markers described in claim 1, comprise step:
A) amination of two nonyl three be dissolved in acetonitrile and add Trifluoroacetic Acid Ethyl Ester, stirred overnight at room temperature, terminate with tlc monitoring reaction, concentrating under reduced pressure, drains, and obtains trifluoroacetamide crude product;
B) trifluoroacetamide crude product acetonitrile is dissolved, first add triethylamine, then add Succinic anhydried, stirred overnight at room temperature, with column chromatography purification after concentrating under reduced pressure, obtain trifluoroacetamide;
C) trifluoroacetamide acetonitrile is dissolved, first add N-Hydroxysuccinimide, then add EDC.HCl, stirred overnight at room temperature, terminate rear column chromatography purification with tlc monitoring reaction, obtain trifluoroacetamide;
D) compound T3 anhydrous methanol is dissolved; Add SOCl
2reflux stirs spends the night, and concentrating under reduced pressure, drains, and obtains three iodo thyroxine methyl esters;
E) trifluoroacetamide and three iodo thyroxine methyl esters are dissolved in DMF, stirred overnight at room temperature, terminate rear column chromatography purification with tlc monitoring reaction, obtain target product three iodo thyroxine methyl esters;
F) three iodo thyroxine methyl esters are dissolved in methyl alcohol to add water again, add the reaction of salt of wormwood stirring at room temperature, liquid chromatography monitoring reaction, stirring at room temperature reaction is spent the night, and through preparative chromatography purifying, obtains three iodo thyroxine methyl esters;
G) be dissolved in dry DMF by three iodo thyroxine methyl esters, add DIPEA, acridinium ester is added reaction solution, stirring at room temperature is reacted, and after liquid chromatography monitoring reaction terminates, obtains target product acridine mark three iodo thyroxine methyl esters through preparative chromatography purifying.
3. the method for three iodo thyroxine haptens luminous markers as claimed in claim 2, is characterized in that: steps A) the described pair of nonyl triamine replaced by two (hexamethylene) triamine or dipropyl triamine.
4. the method for three iodo thyroxine haptens luminous markers as claimed in claim 2, is characterized in that: step B) described Succinic anhydried replaced by Pyroglutaric acid.
5. the method for three iodo thyroxine haptens luminous markers as claimed in claim 2, is characterized in that: step G) described acridinium ester replaced by NSP-DMAE-NHS or NSP-SA-NHS acridinium ester compound.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109239347A (en) * | 2018-10-24 | 2019-01-18 | 苏州长光华医生物医学工程有限公司 | A kind of HBeAb chemiluminescence immune detection reagent kit and its preparation method and application |
| CN109575123A (en) * | 2018-11-08 | 2019-04-05 | 中国农业大学 | The preparation method and application of a kind of Fluorakil 100 haptens and monoclonal antibody |
| CN113214132A (en) * | 2021-05-14 | 2021-08-06 | 英科新创（苏州）生物科技有限公司 | Preparation method of hapten iodoacetyl thyroxine active coupling reagent |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109239347A (en) * | 2018-10-24 | 2019-01-18 | 苏州长光华医生物医学工程有限公司 | A kind of HBeAb chemiluminescence immune detection reagent kit and its preparation method and application |
| CN109575123A (en) * | 2018-11-08 | 2019-04-05 | 中国农业大学 | The preparation method and application of a kind of Fluorakil 100 haptens and monoclonal antibody |
| CN109575123B (en) * | 2018-11-08 | 2020-12-15 | 中国农业大学 | Preparation method and application of fluoroacetamide hapten and monoclonal antibody |
| CN113214132A (en) * | 2021-05-14 | 2021-08-06 | 英科新创（苏州）生物科技有限公司 | Preparation method of hapten iodoacetyl thyroxine active coupling reagent |
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Application publication date: 20150107 |