CN108218926A - Detect substrate of N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes and preparation method thereof and kit - Google Patents
Detect substrate of N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes and preparation method thereof and kit Download PDFInfo
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- CN108218926A CN108218926A CN201810068078.5A CN201810068078A CN108218926A CN 108218926 A CN108218926 A CN 108218926A CN 201810068078 A CN201810068078 A CN 201810068078A CN 108218926 A CN108218926 A CN 108218926A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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Abstract
The invention discloses a kind of for detecting the substrate of 2 deoxidation β D glucopyranoside enzymes of N acetylaminos, specially 2 acetylamino of thymolphthalein, 2 deoxidation β D glucopyranosides, preparation method includes the following steps：By thymolphthalein and 2 acetylaminos 3; 4; thymolphthalein N acetyl group 3 is made in the reaction of 6 three O acetyl, 2 deoxidation A D glucopyranoses acid chloride; 4; then 6 O triacetyl β D glucopyranosides remove acetyl group and obtain 2 acetylamino of thymolphthalein, 2 deoxidation β D glucopyranosides.Substrate of 2 acetylamino of thymolphthalein, the 2 deoxidation β D glucopyranosides of the present invention as detection 2 deoxidation β D glucopyranoside enzymes of N acetylaminos has the advantages that colour developing is sensitive and is not easy to miss inspection, and building-up process is simple, reduce cost.
Description
Technical field
The present invention relates to field of biological detection, and in particular to one kind is used to detect N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- pyrans
Substrate of glucuroide and preparation method thereof and for detecting N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes
Kit.
Background technology
N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes are a kind of acid hydrolases in lysosome, point
Son amount about 140000, be present in a organized way in, with prostate and renal tubule content highest.It is special when autologous tissue is damaged
When not being that nearly bent renal tubule comes to harm, the activity of N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes significantly increases in urine
Height, and it is more other urine increasing for enzyme occur earlier, therefore can by the N- acetylaminohydroxyphenylarsonic acid 2- deoxidations in vitro detection urine-
β-D- glucopyranosides enzymes provide foundation to the early diagnosis of tubular injury, state of illness monitoring.
However currently the majority uses 4- nitrobenzophenone -2- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides the bottom of as
Object, detects N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes, this substrate is primarily present problems with：1st, it develops the color ineffective
It is quick, easily weakly positive missing inspection；2nd, colour developing is yellow, and easy and urine color, which is obscured, to be not easy to distinguish.
Invention content
The object of the present invention is to provide a kind of substrates for detecting N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes
And preparation method thereof and kit, to solve drawbacks described above existing for background technology.
The present invention realizes by the following technical solutions：
A kind of substrate for detecting N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes, specially thymolphthalein -2-
Acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides, chemical structural formula are as follows：
The compound physical parameter is as follows：
1HNMR(DMSO-d6,400MHz):δ1.35(m,12H),1.83(s,3H),2.37(s,6H),3.22(m,1H),
3.29(m,1H),3.32(m,1H),3.35(m,1H),3.76(m,2H),4.66(m,1H),4.74(m,1H),5.03(1H,m),
5.11(m,1H),6.38(s,2H),6.85(m,2H),7.45(m,4H),7.91(s,2H)。
Molecular weight：633.
The invention also discloses the preparation sides of above-mentioned thymolphthalein -2- acetylaminohydroxyphenylarsonic acids 2- deoxidation-β-D- glucopyranosides
Method includes the following steps：By thymolphthalein and tri--O- acetyl -2- deoxidation-A-D- glucopyranoses of 2- acetylaminohydroxyphenylarsonic acids 3,4,6-
Thymolphthalein-N- acetyl group -3,4 is made in acid chloride reaction, then 6-O- triacetyl-β-D- glucopyranosides remove second
Acyl group obtains thymolphthalein -2- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides.
Preferably, the thymolphthalein-N- acetyl group -3,4,6-O- triacetyl-β-D- glucopyranosides are to pass through
Prepared by following method：By tri--O- acetyl of thymolphthalein, 4-butyl ammonium hydrogen sulfate (TBAHS) and 2- acetylaminohydroxyphenylarsonic acids 3,4,6--
2- deoxidation-A-D- glucopyranose acid chlorides are dissolved in dichloromethane, are then added in wet chemical and are fully reacted, separation has
Thymolphthalein-N- acetyl group -3,4,6-O- triacetyl-β-D- the glucopyranosides are obtained after the mutually concentrated purification of machine.
Wherein, 3,4,6- tri--O- acetyl -2- deoxidations-A-D- of thymolphthalein, 4-butyl ammonium hydrogen sulfate and 2- acetylaminohydroxyphenylarsonic acids
The molar ratio of glucopyranose acid chloride is 1：1：1.
Preferably, thymolphthalein-N- acetyl group -3,4,6-O- triacetyl-β-D- are removed using methanol/sodium methoxide system
Acetyl group in glucopyranoside.
Specifically, thymolphthalein-N- acetyl group -3,4,6-O- triacetyl-β-D- pyrans Portugal are removed with the following method
Acetyl group in polyglycoside：Thymolphthalein-N- acetyl group -3,4,6-O- triacetyl-β-D- glucopyranosides are dissolved in nothing
In water methanol and 5-10 DEG C is cooled to, room temperature is fully reacted after addition sodium methoxide, is then directly purified to obtain thymol with HPLC
Phthalein -2- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides.
Wherein mole of thymolphthalein-N- acetyl group -3,4,6-O- triacetyl-β-D- glucopyranosides and sodium methoxide
Than being 1：1.
The invention also discloses a kind of for detecting the reagent of N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes
Box is using thymolphthalein -2- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides as substrate.Concrete principle is as follows：Hundred
In phenolphthalein -2- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides and N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides
Enzyme reacts, and generates N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides and thymol, thymol is under alkaline condition
Become au bleu.When N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside azymias, do not occur more than react, solution is in alkali
Property under the conditions of be yellow.
Thymolphthalein -2- acetylaminohydroxyphenylarsonic acids 2- deoxidation-β-D- glucopyranosides of the present invention are as detection N- acetylaminohydroxyphenylarsonic acids
The substrate of 2- deoxidation-β-D- glucopyranoside enzymes, have the advantages that colour developing it is sensitive be not easy to miss inspection, and building-up process is simple,
Cost can be reduced.
Specific embodiment
The present invention is illustrated, but be not intended to limit the present invention below by specific embodiment.
The raw materials such as thymolphthalein, tri--O- acetyl -2- deoxidation-A-D- glucopyranose acid chlorides of 2- acetylaminohydroxyphenylarsonic acids 3,4,6-
Commercial product can be used.
First, the synthesis of thymolphthalein-N- acetyl group -3,4,6-O- triacetyl-β-D- glucopyranosides.It can use as follows
Reaction equation represents：
Specific preparation process is as follows：By thymolphthalein (4.3g, 10mmol), TBAHS (3.4g, 10mmol), 2- acetyl ammonia
Three-O- acetyl -2- deoxidation-Α-D- glucopyranoses acid chlorides (3.65g, 10mmol) of base -3,4,6- are dissolved in 30ml dichloromethane
And 10 degree are cooled to, it adds in 30ml 1N wet chemicals and is then stirred at room temperature 3 hours, liquid separation is simultaneously dried with anhydrous sodium sulfate.
Organic phase was concentrated to dryness column and obtains (the production of thymolphthalein-N- acetyl group -3,4,6-O- triacetyl-β-D- glucopyranosides
Measure 4.5g, yield 61.2%).
2nd, the synthesis of thymolphthalein -2- acetylaminohydroxyphenylarsonic acids 2- deoxidation-β-D- glucopyranosides.Following reaction formula table can be used
Show：
Specific preparation process is as follows：By thymolphthalein-N- acetyl group -3,4,6-O- triacetyl-β-D- glucopyranoses
Glycosides (1.5g, 2mmol) is dissolved in 10ml absolute methanols and is cooled to 10 degree, adds in sodium methoxide (88mg, 2mmol) and then room temperature is stirred
It mixes 3 hours, is then directly purified to obtain (the production of thymolphthalein -2- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides with HPLC
Measure 1.1g, yield 86.8%, purity 99.3%).
Its nuclear magnetic resonance measuring result is as follows：1HNMR(DMSO-d6,400MHz):δ1.35(m,12H),1.83(s,3H),
2.37(s,6H),3.22(m,1H),3.29(m,1H),3.32(m,1H),3.35(m,1H),3.76(m,2H),4.66(m,1H),
4.74(m,1H),5.03(1H,m),5.11(m,1H),6.38(s,2H),6.85(m,2H),7.45(m,4H),7.91(s,2H)。
MS Calcd.:633；MS Found:634(M+1)+。
Thymolphthalein -2- acetylaminohydroxyphenylarsonic acids 2- deoxidation-β-D- glucopyranosides of the present invention and N- acetylaminohydroxyphenylarsonic acid 2- deoxidations -
β-D- glucopyranoside enzymes react, and generate N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides and thymol, such as
Shown in lower reaction equation：
Thymol becomes au bleu under alkaline condition.When N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside azymias
When, do not occur more than react, solution under alkaline condition be yellow.Therefore thymolphthalein -2- acetylaminohydroxyphenylarsonic acids 2- can be used to take off
Oxygen-β-D- glucopyranosides are as substrate reagent preparation box, applied to N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoses
The detection of glycosides enzyme.
Basic principle, main feature and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (8)
1. detect the substrate of N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes, which is characterized in that be specially thymol
Phthalein -2- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides, chemical structural formula are as follows：
。
2. the preparation method of thymolphthalein -2- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides, which is characterized in that including such as
Lower step：Thymolphthalein is reacted with tri--O- acetyl -2- deoxidation-A-D- glucopyranose acid chlorides of 2- acetylaminohydroxyphenylarsonic acids 3,4,6-
Thymolphthalein-N- acetyl group -3,4 is made, then 6-O- triacetyl-β-D- glucopyranosides remove acetyl group and obtain hundred
In phenolphthalein -2- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides.
3. method as claimed in claim 2, which is characterized in that the thymolphthalein-N- acetyl group -3,4,6-O- triacetyls
Base-β-D- glucopyranosides are prepared via a method which：By thymolphthalein, 4-butyl ammonium hydrogen sulfate and 2- acetyl ammonia
Three-O- acetyl -2- deoxidation-A-D- glucopyranose acid chlorides of base -3,4,6- are dissolved in dichloromethane, then add in potash water
Solution fully reacts, and the thymolphthalein-N- acetyl group -3,4,6-O- triacetyls are obtained after detaching the concentrated purification of organic phase
Base-β-D- glucopyranosides.
4. method as claimed in claim 3, which is characterized in that thymolphthalein, 4-butyl ammonium hydrogen sulfate and 2- acetylaminohydroxyphenylarsonic acids 3,
The molar ratio of tri--O- acetyl -2- deoxidation-A-D- glucopyranose acid chlorides of 4,6- is 1：1：1.
5. method as claimed in claim 2, which is characterized in that thymolphthalein-N- acetyl is removed using methanol/sodium methoxide system
Acetyl group in base -3,4,6-O- triacetyl-β-D- glucopyranosides.
6. method as claimed in claim 5, which is characterized in that thymolphthalein-N- acetyl group -3,4 is removed with the following method,
Acetyl group in 6-O- triacetyl-β-D- glucopyranosides：By thymolphthalein-N- acetyl group -3,4,6-O- triacetyls -
β-D- glucopyranosides are dissolved in absolute methanol and are cooled to 5-10 DEG C, and room temperature is fully reacted after addition sodium methoxide, Ran Houzhi
It connects and is purified to obtain thymolphthalein -2- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides with HPLC.
7. method as claimed in claim 6, which is characterized in that thymolphthalein-N- acetyl group -3,4,6-O- triacetyls-β -
The molar ratio of D- glucopyranosides and sodium methoxide is 1：1.
8. a kind of kit for being used to detect N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes, which is characterized in that with
Thymolphthalein -2- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranosides are as substrate.
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| CN201810068078.5A CN108218926A (en) | 2018-01-24 | 2018-01-24 | Detect substrate of N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes and preparation method thereof and kit |
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| CN201810068078.5A CN108218926A (en) | 2018-01-24 | 2018-01-24 | Detect substrate of N- acetylaminohydroxyphenylarsonic acid 2- deoxidation-β-D- glucopyranoside enzymes and preparation method thereof and kit |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6419089A (en) * | 1987-07-15 | 1989-01-23 | Sapporo Breweries | Production of n-acetyl-beta-d-hexosamine derivative |
| US5126329A (en) * | 1988-12-29 | 1992-06-30 | Nitto Boseki Co., Ltd. | Glucosamine derivatives and compositions reagents and containing the same |
| CN101270104A (en) * | 2008-05-07 | 2008-09-24 | 大连理工大学 | Phenolphthalein type cyanate monomer, polymeric compounds and methods of formulating same |
| CN101738379A (en) * | 2009-12-31 | 2010-06-16 | 宁波美康生物科技有限公司 | Liquid reagent for determining N-acetyl-beta-D-glucosaminidase |
| CN103694292A (en) * | 2013-12-20 | 2014-04-02 | 宁波大学 | N-acetyl-3,4,6-triacetyl-beta-D-amino glucoside compound and hydrolyzates and preparation method of hydrolyzates |
-
2018
- 2018-01-24 CN CN201810068078.5A patent/CN108218926A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6419089A (en) * | 1987-07-15 | 1989-01-23 | Sapporo Breweries | Production of n-acetyl-beta-d-hexosamine derivative |
| US5126329A (en) * | 1988-12-29 | 1992-06-30 | Nitto Boseki Co., Ltd. | Glucosamine derivatives and compositions reagents and containing the same |
| CN101270104A (en) * | 2008-05-07 | 2008-09-24 | 大连理工大学 | Phenolphthalein type cyanate monomer, polymeric compounds and methods of formulating same |
| CN101738379A (en) * | 2009-12-31 | 2010-06-16 | 宁波美康生物科技有限公司 | Liquid reagent for determining N-acetyl-beta-D-glucosaminidase |
| CN103694292A (en) * | 2013-12-20 | 2014-04-02 | 宁波大学 | N-acetyl-3,4,6-triacetyl-beta-D-amino glucoside compound and hydrolyzates and preparation method of hydrolyzates |
Non-Patent Citations (1)
| Title |
|---|
| 张学铭，等: "《化学小辞典》", 31 August 1994, 科学技术文献出版社 * |
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Application publication date: 20180629 |