CN107759643A - A kind of glucosan derivative RG and preparation method thereof is with preparing the application on aspartic acid luciferase assay reagent - Google Patents
A kind of glucosan derivative RG and preparation method thereof is with preparing the application on aspartic acid luciferase assay reagent Download PDFInfo
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- CN107759643A CN107759643A CN201711225691.5A CN201711225691A CN107759643A CN 107759643 A CN107759643 A CN 107759643A CN 201711225691 A CN201711225691 A CN 201711225691A CN 107759643 A CN107759643 A CN 107759643A
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Abstract
A kind of glucosan derivative RG and preparation method thereof is with preparing the application on aspartic acid luciferase assay reagent, glycosyl is incorporated into fluorescence chromophoric group, specifically gluconic acid is incorporated on Rhodamine Derivatives, obtain water-soluble fabulous, the glycosyl rhodamine fluorescent color-developing agent selectively strong to aspartic acid, its synthetic method is simple, mild condition, product is easy to get, the aspartic acid detection that the compound is used for the present invention obtains good result, biomolecule is not coexisted by other, such as glycine, proline, valine, serine, the influence of the amino acid such as leucine, with high selectivity.Easy to use due to being tested in water, fluorescence intensity is high.
Description
Technical field
Field of molecular detection the present invention relates to identification combination and for optical detection aspartic acid, more particularly to one
Kind glucosan derivative RG and preparation method thereof is with preparing the application on aspartic acid luciferase assay reagent.
Background technology
Aspartic acid is a kind of important amino acid of needed by human body, has key effect to healthy metabolism, in energy
Key player is also responsible in terms of generation.The reasonable expression of human body aspartic acid dominates the conjunction of other biochemical substances and amino acid
Into（Galili, G.,2011. Plant Signal. Behav.6, 192–195）.Aspartic acid is in treatment confirmed fatigue
(Marquezi, M. L., Roschel, H. A., Costa, A. D., et al. International Journal
Of Sport Nutrition and Exercise Metabolism, 2003,13,65-75.) and strengthen immunity side
Also there are obvious effect (Li, P., Yin, Y. L., Li, D. F., et al. British Journal of in face
Nutrition, 2007, 98, 237–252.).It is very heavy to develop simple and efficient sensitive analysis method detection aspartic acid
Will.The method of detection aspartic acid mainly has high performance liquid chromatography, capillary electrophoresis, mass spectrography, fluorescence method etc. at present.Its
Middle fluorescence method high sensitivity simple to operate.But the compound water soluble of used identification aspartic acid is poor in these methods, selects
The low and corresponding slower development of selecting property.
The content of the invention
The defects of in order to overcome above method, especially with respect to it is water-soluble and selective the problem of, the invention provides one
Kind glucosan derivative RG and preparation method thereof is with preparing the application on aspartic acid luciferase assay reagent.
The technical solution that the present invention uses is：A kind of glucosan derivative RG, described glucosan derivative RG's
Structural formula is as follows：
。
A kind of glucosan derivative RG preparation method, comprises the following steps：
（1）Rhodamine Derivatives synthesize：479mg rhodamine is dissolved in the alcoholic solution of 20-40mL heat, by 0.33-0.6mL second
Diamine solution is added dropwise in above-mentioned solution dropwise, is stirred at reflux 10-15h and is disappeared to solution fluorescence, solution is cooled to room temperature, in ventilation
Cupboard evaporates into solvent almost completely without, obtaining product sieve with the mixed extractant solvent Rhodamine Derivatives of carbon trichloride and water three times
Red bright derivative；
（2）Glucosan derivative RG is synthesized：230-450mg gluconic acid is dissolved in 100mL ethanol and stirs 24h at room temperature extremely
All dissolvings, 0.1-0.2mL n-hydroxysuccinimide is added in the gluconic acid solution of dissolving, places 30-60min
Afterwards, the Rhodamine Derivatives of above-mentioned synthesis are added dropwise, room temperature is stirred for 24h after 50-60 oC heating water baths flow back 5h, ties
The solution of fruit, which is rotated, to be evaporated, and its product is refiltered with ethanol, obtains described glucosan derivative RG.
Described in the mixed solvent carbon trichloride and the volume ratio of water are 4-5:1.
A kind of applications of glucosan derivative RG on aspartic acid luciferase assay reagent is prepared.
Described aspartic acid luciferase assay reagent is prepared by following steps：Glucose described in claim 1 is spread out
Biological RG, water or alcohol solution are dissolved in, and are adjusted to pH value to 3.5, it is 1*10 to be made into glucosan derivative RG substance withdrawl syndromes- 5Mol/L aspartic acid luciferase assay reagent solution.
The beneficial effects of the invention are as follows：The invention provides a kind of glucosan derivative RG and preparation method thereof with preparing day
Application on winter propylhomoserin luciferase assay reagent, glycosyl is incorporated into fluorescence chromophoric group, specifically introduced gluconic acid
Onto Rhodamine Derivatives, water-soluble glycosyl rhodamine fluorescent color-developing agent fabulous, selectively strong to aspartic acid is obtained,
Its synthetic method is simple, mild condition, product are easy to get, and the aspartic acid detection that the compound is used for the present invention obtains good effect
Fruit, biomolecule, such as the influence of the amino acid such as glycine, proline, valine, serine, leucine is not coexisted by other,
With high selectivity.Easy to use due to being tested in water, fluorescence intensity is high.
Brief description of the drawings
Fig. 1 is that the compound of embodiment 1 responds in the pH3 aqueous solution to the fluorescence intensity of aspartic acid.
Fig. 2 be embodiment 1 compound in the pH3 aqueous solution in the presence of 5 times of interfering materials to the glimmering of aspartic acid
Photoresponse；Wherein 1 is blank, and 2 be histidine, and 3 be cysteine, and 4 be phenylalanine, and 5 be methionine, and 6 be arginine, and 7 are
Lysine, 8 be leucine, and 9 be serine, and 10 be histidine, and 11 be isoleucine, and 12 be valine, and 13 be threonine, and 14 are
Glutamine, 15 be glycine, and 16 be Glucosamine, and 17 be glucose, and wherein in figure in every group, bar-shaped mark is low for interference
The response of material, the high response to add after aspartic acid.
Embodiment
In order to illustrate more clearly of present invention, it is described as follows with specific embodiment, specific embodiment does not limit this hair
Bright context.
Embodiment 1（Compound R G synthesis）
（1）Compound R G synthesis
479mg rhodamine is dissolved in the methanol solution of 20mL heat, 0.33mL ethylenediamine solution is added dropwise to dropwise above-mentioned molten
In liquid, be stirred at reflux 15h and disappeared to solution fluorescence, solution is cooled to room temperature, in fume hood evaporate into solvent almost completely without.Use trichlorine
Change the mixed solvent of carbon and water（Volume ratio is 5:1）Extract Rhodamine Derivatives three times, obtain product Rhodamine Derivatives.Will
230mg gluconic acid is dissolved in 100mL ethanol stirs 24h to whole dissolvings at room temperature, and 0.2mL N- hydroxysuccinimidyls acyl is sub-
Amine is added in the gluconic acid solution of dissolving.After placing 30min, the Rhodamine Derivatives of above-mentioned synthesis are added dropwise, in 60
Room temperature is stirred for 24h after oC heating water baths backflow 5h, and solution as a result, which is rotated, to be evaporated, and its product is refiltered with ethanol, is obtained
Target compound RG.
（2）Compound R G synthesis
479mg rhodamine is dissolved in the methanol solution of 40mL heat, 0.6 mL ethylenediamine solution is added dropwise to dropwise above-mentioned molten
In liquid, be stirred at reflux 10h and disappeared to solution fluorescence, solution is cooled to room temperature, in fume hood evaporate into solvent almost completely without.Use trichlorine
Change the mixed solvent of carbon and water（Volume ratio is 4:1）Extract Rhodamine Derivatives three times, obtain product Rhodamine Derivatives.Will
450mg gluconic acid is dissolved in 100mL ethanol stirs 24h to whole dissolvings at room temperature, and 0.1mL N- hydroxysuccinimidyls acyl is sub-
Amine is added in the gluconic acid solution of dissolving.After placing 60min, the Rhodamine Derivatives of above-mentioned synthesis are added dropwise, in 50
Room temperature is stirred for 24h after oC heating water baths backflow 5h, and solution as a result, which is rotated, to be evaporated, and its product is refiltered with ethanol, is obtained
Target compound RG.
Embodiment 2（Fluorescence intensity curves）
The compound 100mg for weighing embodiment 1 is dissolved in the water, and is configured to 1*10-5M standard reserving solution, is adjusted to solution ph
For 3.5.100mg aspartic acid is weighed, is made into storing solution, and is adjusted to pH value as 3.5.Biomolecule is from histidine, bad ammonia
The materials such as acid, serine, proline, valine, leucine, the solution of all experiments are all for new configuration and solution ph
3.5, and immediately begin to test.The storing solution 2mL of embodiment 1 is measured, adds the aspartic acid of amount of calculation, obtains fluorescence intensity song
Line.
Detection aspartic acid experiment coexists in the interfering material of embodiment 3
Compound R G is made into 10 in fluorescence experiments-5M alcohol solution, solution ph is adjusted to as 3.5.Aspartic acid is made into 10-2M
Standard reserving solution, be adjusted to solution ph as 3.5.As biomolecule from histidine, lysine, serine, proline, figured silk fabrics
The materials such as propylhomoserin, leucine, solution ph is adjusted to as 3.5.The solution of all experiments is all new configuration, and test immediately.It is dry
Disturb in material experiment, first 10-55 times of interfering material is added in the M RG aqueous solution, its fluorescence is surveyed, adds aspartic acid,
Survey its change in fluorescence.Change in fluorescence is detected at 554 nm.
The method test aspartic acid of the present invention, is not coexisted biomolecule, such as glycine, proline, figured silk fabrics ammonia by other
The influence of the amino acid such as acid, serine, leucine, there is high selectivity.It is easy to use due to being tested in water, fluorescence intensity
It is high.In summary, the solution have the advantages that significantly, there is provided a kind of high selectivity high sensitivity testing aspartic acid
Method.
Mechanism of the present invention：Due to aspartic acid and the compound hydrogen bond action, the change that electronics is distributed in molecule is caused to be led
The notable rising of fluorescence intensity is caused, reaches the purpose of detection aspartic acid.And cysteine, phenylalanine, methionine, smart ammonia
The materials such as acid, lysine, leucine, serine can not function the change for producing fluorescence intensity.Show G pairs of the compound R
Aspartic acid has high selectivity.
Described above is only the preferred embodiment of the present invention, and protection scope of the present invention is not limited merely to above-mentioned implementation
Example, all technical schemes belonged under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that for the art
Those of ordinary skill for, some improvements and modifications without departing from the principles of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (5)
1. a kind of glucosan derivative RG, it is characterised in that described glucosan derivative RG structural formula is as follows：
。
2. the preparation method of the glucosan derivative RG described in a kind of claim 1, it is characterised in that comprise the following steps：
（1）Rhodamine Derivatives synthesize：479mg rhodamine is dissolved in the alcoholic solution of 20-40mL heat, by 0.33-0.6mL second
Diamine solution is added dropwise in above-mentioned solution dropwise, is stirred at reflux 10-15h and is disappeared to solution fluorescence, solution is cooled to room temperature, in ventilation
Cupboard evaporates into solvent almost completely without, obtaining product sieve with the mixed extractant solvent Rhodamine Derivatives of carbon trichloride and water three times
Red bright derivative；
（2）Glucosan derivative RG is synthesized：230-450mg gluconic acid is dissolved in 100mL ethanol and stirs 24h at room temperature extremely
All dissolvings, 0.1-0.2mL n-hydroxysuccinimide is added in the gluconic acid solution of dissolving, places 30-60min
Afterwards, the Rhodamine Derivatives of above-mentioned synthesis are added dropwise, room temperature is stirred for 24h after 50-60 oC heating water baths flow back 5h, ties
The solution of fruit, which is rotated, to be evaporated, and its product is refiltered with ethanol, obtains described glucosan derivative RG.
3. a kind of glucosan derivative RG according to claim 1 preparation method, it is characterised in that described mixing is molten
The volume ratio of carbon trichloride and water is 4-5 in agent:1.
A kind of 4. applications of the glucosan derivative RG on aspartic acid luciferase assay reagent is prepared described in claim 1.
5. applications of the glucosan derivative RG according to claim 4 on aspartic acid luciferase assay reagent is prepared, its
It is characterised by, described aspartic acid luciferase assay reagent is prepared by following steps：Glucose described in claim 1 is spread out
Biological RG, water or alcohol solution are dissolved in, and are adjusted to pH value to 3.5, it is 1*10 to be made into glucosan derivative RG substance withdrawl syndromes- 5Mol/L aspartic acid luciferase assay reagent solution.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112067591A (en) * | 2020-09-14 | 2020-12-11 | 江苏省农业科学院 | Nitrogen-doped graphene quantum dot fluorescent probe-based umami amino acid detection method and probe |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106543251A (en) * | 2016-10-08 | 2017-03-29 | 湖南科技大学 | Nitric oxide production water-soluble fluorescent probe and its application in a kind of detection hepatocyte |
| CN107941768A (en) * | 2017-11-29 | 2018-04-20 | 温州医科大学 | A kind of applications of glucosan derivative RG on histidine luciferase assay reagent is prepared |
-
2017
- 2017-11-29 CN CN201711225691.5A patent/CN107759643A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106543251A (en) * | 2016-10-08 | 2017-03-29 | 湖南科技大学 | Nitric oxide production water-soluble fluorescent probe and its application in a kind of detection hepatocyte |
| CN107941768A (en) * | 2017-11-29 | 2018-04-20 | 温州医科大学 | A kind of applications of glucosan derivative RG on histidine luciferase assay reagent is prepared |
Non-Patent Citations (1)
| Title |
|---|
| 郭炜 等: "近红外罗丹明荧光染料", 《山西大学学报（自然科学版）》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112067591A (en) * | 2020-09-14 | 2020-12-11 | 江苏省农业科学院 | Nitrogen-doped graphene quantum dot fluorescent probe-based umami amino acid detection method and probe |
| CN112067591B (en) * | 2020-09-14 | 2023-06-16 | 江苏省农业科学院 | Method for detecting delicious amino acid based on nitrogen-doped graphene quantum dot fluorescent probe and probe |
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Application publication date: 20180306 |