CN103319626A - Method for covalent linkage of hyaluronic acid and isothiocyano-containing organic molecule - Google Patents
Method for covalent linkage of hyaluronic acid and isothiocyano-containing organic molecule Download PDFInfo
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- CN103319626A CN103319626A CN2013102785581A CN201310278558A CN103319626A CN 103319626 A CN103319626 A CN 103319626A CN 2013102785581 A CN2013102785581 A CN 2013102785581A CN 201310278558 A CN201310278558 A CN 201310278558A CN 103319626 A CN103319626 A CN 103319626A
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- hyaluronic acid
- isothiocyano
- organic molecule
- adipic dihydrazide
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Abstract
The invention relates to a method for covalent linkage of hyaluronic acid and an isothiocyano-containing organic molecule. The method comprises the following steps of: (1), reacting hyaluronic acid with adipic dihy drazides to synthesize a hyaluronic acid-adipic dihy drazides derivate; (2) reacting the hyaluronic acid-adipic dihy drazides derivate with the isothiocyano-containing organic molecule to obtain a covalent linkage derivate of hyaluronic acid and the isothiocyano-containing organic molecule. The method is mild in reaction condition, simple in step and low in cost. A product prepared by the method can be used for detecting the content of hyaluronic acid in human serum.
Description
Technical field
The invention belongs to the organic chemical synthesis technical field, be particularly related to a kind of hyaluronic acid method covalently bound with the organic molecule that contains isothiocyano, be used for using the organic molecule mark hyaluronan molecule that contains isothiocyano, the product that the method obtains is as the marker that detects people's hepatic fibrosis test kit.
Background technology
The knot that hyaluronic acid extensively is present in humans and animals forms that maggot is knitted, its existence form has free and forms in conjunction with two kinds in albumen in the vitreum of eyeball, some intercellular substance, synovium of joint liquid, mucous epithelium, cornea, malignant tumor tissue and the bacteria wall.Many organs of humans and animals all are rich in hyaluronic acid, such as rooster comb, people's umbilical cord, ox, sheep vitreum, shark, pig and baby's skin, the cartilage of ox and whale and chicken embryo etc.
Though hyaluronic acid was just found before the sixties, because lacking enough, responsive special measuring method face has limited its clinical application research, along with continuing to bring out of various measuring methods, make hyaluronic acid contents measure the accurate degree that reaches, and reflect that clearly hyaluronic acid all obviously increases in rheumatoid arthritis, hepatopathy, some tumour and the scleroderma patient sample, especially hyaluronic acid is determined at and is better than all conventional liver function tests on the liver cirrhosis diagnosis, and serum of cirrhosis patients hyaluronic acid calibration ordinary person raises 10-100 doubly.The domestic people of having is reported in chronic hepatopathy, the liver cirrhosis group hyaluronic acid increases amplitude greater than III procollagen type Ⅲ and flaggy element (LamP1,) hyaluronic acid increases greater than the III procollagen type Ⅲ in the chronic active hepatitis group, in chronic persistent hepatitis then with the control group indifference.This shows, it is an extremely valuable diagnosing hepatism index that hyaluronic acid is measured.
Hyaluronic acid is a chain polymer, is comprised of multiply-connected the connecing of disaccharide unit weight of (1-β-4) D-glucuronic acid (1-β-3) N-ethanoyl-D-glucosamine.
The purpose of this invention is to provide a kind of organic molecule that will contain isothiocyano and be connected to method on the hyaluronan molecule, the hyaluronic acid that contains the organic molecule mark of isothiocyano can be used for measuring hyaluronic content in the human body.
Contain carboxyl on the hyaluronan molecule, can react with the hydrazide group of adipic dihydrazide, can also keep a hydrazide group after the reaction, this hydrazide group can with the organic molecule that contains isothiocyano on isothiocyano reaction, the organic molecule that is equivalent to contain isothiocyano is connected on the hyaluronan molecule.
The hyaluronic acid that contains the organic molecule mark of isothiocyano can be used for measuring hyaluronic content in the human body, cardinal principle is: its reaction system is first hyaluronic acid binding protein antibody to be fixed on the microwell plate, add again hyaluronic acid binding protein, the hyaluronic acid that contains the organic molecule mark of isothiocyano, the human serum sample, the hyaluronic acid and the hyaluronic acid in the human serum sample that contain the organic molecule mark of isothiocyano are competed in conjunction with hyaluronic acid binding protein, be attached to again on the hyaluronic acid binding protein antibody, mixture is fixed on the microwell plate, measures the fluorescence counting of the organic molecule that contains isothiocyano and can measure hyaluronic content in the human serum.
Summary of the invention
The object of the present invention is to provide a kind of hyaluronic acid method covalently bound with the organic molecule that contains isothiocyano, the product that the method makes is used for measuring the hyaluronic content of human serum.
The technical solution used in the present invention is:
The method that a kind of hyaluronic acid is covalently bound with the organic molecule that contains isothiocyano comprises the steps:
(1): hyaluronic acid and adipic dihydrazide reaction, synthetic hyaluronic acid-adipic dihydrazide derivative,
(2): hyaluronic acid-adipic dihydrazide derivative and the organic molecule that contains isothiocyano are reacted, obtain hyaluronic acid and the covalently bound derivative of organic molecule that contains isothiocyano.
Further, described step (1) is: get hyaluronic acid, be dissolved in the distilled water, the adipic dihydrazide that adds 10 times of hyaluronic acid quality, stir, regulate pH value 4.0-5.5 (HCl with 0.1M regulates), the EDC(1-(3-dimethylamino-propyl) of 5 times of hyaluronic acid quality of adding-3-ethyl-carbodiimide hydrochloride), regulate the pH value and make it to maintain the HCl adjusting that 4.0-5.5(uses 0.1M), react after 2 hours, regulate pH value to 7.0 (NaOH with 1M regulates), will react after product and dialyse with distilled water, the dialysis postlyophilization gets hyaluronic acid-adipic dihydrazide derivative.
Further, described step (2) is: get step (1) gained hyaluronic acid-adipic dihydrazide derivative, be dissolved in the distilled water, add the organic molecule that contains isothiocyano with hyaluronic acid-quality such as adipic dihydrazide derivative after the dissolving, then add the partly 0.01M pH=8.0-10.0 yellow soda ash of amount-sodium bicarbonate buffer liquid of distilled water volume, the lucifuge room temperature was placed 36 hours, after reaction finishes, use sepharose (Sepharose6B) filtration or use dialysis tubing to dialyse product after the reaction and remove the unreacted organic molecule that contains isothiocyano, collect end product (end product of acquisition is clear liquid).
Further, the described organic molecule that contains isothiocyano is N
1-(p-isothiocyano benzyl)-Diethylenetriamine-N
1, N
2, N
3, N
4– tetraacethyl europium sodium or fluorescein isothiocyanate.
The present invention also provides a kind of hyaluronic acid and contains the covalently bound derivative of organic molecule of isothiocyano, is prepared by each method of claim 1-4.
Beneficial effect of the present invention:
The inventive method reaction conditions is gentle, and step is simple, and is with low cost.The product that the inventive method makes is for detection of hyaluronic content in the human serum.
Description of drawings
Fig. 1: hyaluronic acid primary structure figure.
Fig. 2: hyaluronic acid and adipic dihydrazide reaction schematic diagram.
Fig. 3: hyaluronic acid-adipic dihydrazide derivative and the reactive organic molecule schematic diagram that contains isothiocyano.
Embodiment
The present invention will be further described below in conjunction with specific embodiment, but do not limit protection scope of the present invention.
Embodiment 1
A kind of use N
1-(p-isothiocyano benzyl)-Diethylenetriamine-N
1, N
2, N
3, N
4The method of – tetraacethyl europium sodium mark hyaluronan molecule comprises the steps:
(1) preparation of hyaluronic acid-adipic dihydrazide derivative: take by weighing 100 milligrams hyaluronic acid, be dissolved in 50 milliliters of distilled waters, add 1 gram adipic dihydrazide, stir, HCl with 0.1M regulates pH value 4.5, add 0.5 EDC(1-(3-dimethylamino-propyl) that restrains-3-ethyl-carbodiimide hydrochloride), HCl with 0.1M regulates the pH value, make it to maintain 4.5, react after 2 hours, with the NaOH adjusting pH value to 7.0 of 1M, will react after product and dialyse with distilled water, the dialysis postlyophilization gets hyaluronic acid-adipic dihydrazide derivative.
(2) hyaluronic acid and N
1-(p-isothiocyano benzyl)-Diethylenetriamine-N
1, N
2, N
3, N
4The preparation of the covalently bound derivative of – tetraacethyl europium sodium: take by weighing hyaluronic acid after 0.5 milligram of lyophilize-adipic dihydrazide derivative, add in the 250 microlitre distilled waters, add 0.5 milligram N after the dissolving
1-(p-isothiocyano benzyl)-Diethylenetriamine-N
1, N
2, N
3, N
4– tetraacethyl europium sodium adds the 0.01M of 125 microlitres again, the yellow soda ash of pH=9.0-sodium bicarbonate buffer liquid, and lucifuge room temperature reaction 36 hours uses sepharose (Sepharose6B) to filter product after the reaction, collects product.And get final product: hyaluronic acid and N
1-(p-isothiocyano benzyl)-Diethylenetriamine-N
1, N
2, N
3, N
4The covalently bound derivative of – tetraacethyl europium sodium, the product of acquisition is colourless transparent liquid.
Application Example 1
Utilize N
1-(p-isothiocyano benzyl)-Diethylenetriamine-N
1, N
2, N
3, N
4The hyaluronan molecule of – tetraacethyl europium sodium mark (is hyaluronic acid and the N that embodiment 1 makes
1-(p-isothiocyano benzyl)-Diethylenetriamine-N
1, N
2, N
3, N
4The covalently bound derivative of – tetraacethyl europium sodium) detect hyaluronic content in the human serum: step is as follows:
(1) coated hyaluronic acid binding protein antibody: get a certain amount of hyaluronic acid binding protein antibody, be dissolved in the phosphate buffered saline buffer of 0.01MpH=5.6, concentration 5 mcg/ml, solution is added in the transparent microwell plate, add 200 microlitres in every hole, room temperature was placed after 48 hours, outwell solution in the hole, use the phosphate buffered saline buffer of 0.01M pH=5.6 to wash plate 2 times, then 300 microlitres/hole add and contain 1% sucrose, the phosphate buffered saline buffer closed porosity plate of 2% bovine serum albumin, room temperature is placed after 2 hours and is outwelled confining liquid, drying.
(2) with hyaluronic acid and N
1-(p-isothiocyano benzyl)-Diethylenetriamine-N
1, N
2, N
3, N
4– tetraacethyl europium sodium (N
1100 times of the covalently bound derivative dilutions of-(p-isothiocyano benzyl)-DTTAEuNa), get 100 microlitres, with 50 microlitre human serums, 50 microlitre hyaluronic acid binding protein solution together add in the microwell plate for preparing by step (1), room temperature concussion reaction 2 hours, outwell mixed solution after the reaction, wash plate 6 times with damping fluid, add 200 microlitres and contain β-naphthoyltrifluoroacetone (enhancing liquid of β-NTA), room temperature concussion 5 minutes, duration of service, identifier was measured the fluorescence counting, can calculate hyaluronic content in the human serum according to typical curve.
Claims (5)
1. the method that hyaluronic acid is covalently bound with the organic molecule that contains isothiocyano is characterized in that: comprise the steps:
(1): hyaluronic acid and adipic dihydrazide reaction, synthetic hyaluronic acid-adipic dihydrazide derivative,
(2): hyaluronic acid-adipic dihydrazide derivative and the organic molecule that contains isothiocyano are reacted, obtain hyaluronic acid and the covalently bound derivative of organic molecule that contains isothiocyano.
2. method according to claim 1, it is characterized in that: described step (1) is: get hyaluronic acid, be dissolved in the distilled water, the adipic dihydrazide that adds 10 times of hyaluronic acid quality, stir, regulate pH value 4.0-5.5, the 1-(3-dimethylamino-propyl) of 5 times of hyaluronic acid quality of adding-3-ethyl-carbodiimide hydrochloride, regulate the pH value, make it to maintain 4.0-5.5, react after 2 hours, regulate pH value to 7.0, to react after product and dialyse with distilled water, the dialysis postlyophilization gets hyaluronic acid-adipic dihydrazide derivative.
3. method according to claim 1 and 2, it is characterized in that: described step (2) is: get the hyaluronic acid that step (1) obtains-adipic dihydrazide derivative, be dissolved in the distilled water, add the organic molecule that contains isothiocyano with hyaluronic acid-quality such as adipic dihydrazide derivative after the dissolving, then add the partly 0.01M pH=8.0-10.0 yellow soda ash of amount-sodium bicarbonate buffer liquid of distilled water volume, the lucifuge room temperature was placed 36 hours, after reaction finishes, using sepharose (Sepharose6B) filtration or use dialysis tubing to dialyse product removes the unreacted organic molecule that contains isothiocyano, collects end product.
4. each described method according to claim 1-3, it is characterized in that: the described organic molecule that contains isothiocyano is N
1-(p-isothiocyano benzyl)-Diethylenetriamine-N
1, N
2, N
3, N
4– tetraacethyl europium sodium or fluorescein isothiocyanate.
5. a hyaluronic acid and the covalently bound derivative of organic molecule that contains isothiocyano is characterized in that: prepared by each method of claim 1-4.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085817A (en) * | 2015-08-31 | 2015-11-25 | 华南理工大学 | Anionic polymer brush based on hyaluronic acid, preparation method of anionic polymer brush and application of anionic polymer brush |
| CN108210689A (en) * | 2017-11-27 | 2018-06-29 | 南京天纵易康生物科技股份有限公司 | A kind of canker sore film containing hyaluronic acid and preparation method thereof |
| WO2020000916A1 (en) * | 2018-06-28 | 2020-01-02 | 华南理工大学 | Hyaluronic acid-based zwitterionic polymer brush, preparation method thereof, and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS55161801A (en) * | 1979-06-06 | 1980-12-16 | Seikagaku Kogyo Co Ltd | Fluorescence-labeled acid mucopolysaccharide and preparation thereof |
| CN102095847A (en) * | 2009-12-10 | 2011-06-15 | 北京北方生物技术研究所 | Method for detecting content of hyaluronic acid in human serum |
-
2013
- 2013-07-04 CN CN2013102785581A patent/CN103319626A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS55161801A (en) * | 1979-06-06 | 1980-12-16 | Seikagaku Kogyo Co Ltd | Fluorescence-labeled acid mucopolysaccharide and preparation thereof |
| CN102095847A (en) * | 2009-12-10 | 2011-06-15 | 北京北方生物技术研究所 | Method for detecting content of hyaluronic acid in human serum |
Non-Patent Citations (4)
| Title |
|---|
| RAFAL FUDALA ET AL: "Lifetime-based Sensing of the Hyaluronidase using Flurescein Labeled Hyaluronic Acid", 《JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B: BIOLOGY》 * |
| 刘祥: ""透明质酸-喜树碱及叶酸-透明质酸-喜树碱复合体的合成及表征"", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 王浩丹等: "《生物医学标记示踪技术》", 31 December 1995, 人民卫生出版社 * |
| 蒋中华等: "《生物分子固定化技术及应用》", 31 July 1998, 化学工业出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085817A (en) * | 2015-08-31 | 2015-11-25 | 华南理工大学 | Anionic polymer brush based on hyaluronic acid, preparation method of anionic polymer brush and application of anionic polymer brush |
| CN105085817B (en) * | 2015-08-31 | 2017-06-20 | 华南理工大学 | A kind of anionic polymer brush based on hyaluronic acid and its preparation method and application |
| CN108210689A (en) * | 2017-11-27 | 2018-06-29 | 南京天纵易康生物科技股份有限公司 | A kind of canker sore film containing hyaluronic acid and preparation method thereof |
| WO2020000916A1 (en) * | 2018-06-28 | 2020-01-02 | 华南理工大学 | Hyaluronic acid-based zwitterionic polymer brush, preparation method thereof, and application thereof |
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Application publication date: 20130925 |