CN102675556A - Preparation method of fluorescent polymer simulating green fluorescent protein - Google Patents
Preparation method of fluorescent polymer simulating green fluorescent protein Download PDFInfo
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- CN102675556A CN102675556A CN2012101143925A CN201210114392A CN102675556A CN 102675556 A CN102675556 A CN 102675556A CN 2012101143925 A CN2012101143925 A CN 2012101143925A CN 201210114392 A CN201210114392 A CN 201210114392A CN 102675556 A CN102675556 A CN 102675556A
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Abstract
The invention discloses a preparation method of fluorescent polymer simulating a green fluorescent protein in the field of chemical technology. Inspired by the principle that the green fluorescent protein improves self-fluorescent performance through self-assembling, the preparation methodprovided by the invention introduces fluorescent chromophore containing hydroxy benzylidene imidazolinone into amphiphilic polymer to obtain a new type of amphiphilic fluorescent polymer. And fluorescence enhancement of the polymer is achieved by self-assembling of macromolecular. The new type of fluorescent polymer is simple in preparation and good in designability, obtains improved fluorescence enhancement by the self-assembling method, and is formed by connecting hydrophobic polymer, fluorescent chromophore and hydrophilic polymer. The ratio of hydrophilic polymer/hydrophobic polymer is between 0.2-0.8. The polymer is expected to be applied to the fields of biology, medicine and pharmacy.
Description
Technical field
The present invention relates to a kind of method of chemical technology field, specifically is a kind of preparation method who simulates the fluorescent polymer of green fluorescent protein.
Background technology
Fluorescent polymer has excellent optical property, thereby has extensively and important use at aspects such as medicine, biology, chemical industry.The ultimate principle that mimic biology is luminous is developed a kind of novel fluorescent polymer, has great importance.
Literature search through to prior art finds that the method for preparing fluorescent polymer at present mainly is: utilize the structural unit of fluorescent small molecule as polymkeric substance, prepare the conjugated type fluorescent polymer through linked reaction.For example; Yamamoto etc. are at " Polymer " (polymkeric substance; 2007; 48 volumes; 5449～5472 pages) on delivered " Synthesis of π-conjugated polymers bearing electronic and optical functionalities by organometallic polycondensations and their chemical properties " (pi-conjugated polymkeric substance and chemical property research thereof of utilizing organo-metallic condensation reaction preparation to have photoelectric properties), this article employing transition metal such as nickel, palladium etc. have prepared a series of fluorescent polymers with different structure and performance as catalyzer.
Yet the fluorescent polymer of this method preparation has weakened the optical property of fluorescence molecule itself.In addition, such conjugated polymers is difficult to degraded, and has very high cytotoxicity, and therefore can't be applied to living imaging can the bio-medical field.Ideal bio-imaging fluorescent material should be to make full use of fluorophor, and the better fluorescent material of obtained performance has excellent biocompatibility simultaneously.Learn to nature biotechnology is luminous, make full use of fluorophor, develop the preparation method of new biocompatibility fluorescent polymer, have important science and using value undoubtedly.
Green fluorescent protein is a kind ofly to utilize the protein technology and the synthetic novel protein can utilize this kind albumen to serve as a mark, and is significant in the biotechnology in modern times.Its chemical nature is a protein, is formed by the amino acid dehydrating condensation, is a kind of macromolecular compound.Because have approximate perfect cylindrical structure, green fluorescent protein shows unique with excellent photoluminescent property.When being in primary structure can rotate freely owing to produce the chromophore of fluorescence, the fluorescence of green fluorescent protein very a little less than.In case form cylindrical structure through self-assembly, rotating freely of chromophore is suppressed, green fluorescent protein has obtained excellent fluorescence.There is not the researchist to utilize this biological self-assembly to obtain excellent fluorescence property principle in the prior art, designs and develop one type of novel fluorescent polymer.
In sum, the preparation method of existing fluorescent polymer exists the fluorescent polymer make to be difficult to degrade, have very high cytotoxicity, can't to be applied to the technical problem in bio-medical fields such as living imaging thus.
Summary of the invention
The object of the present invention is to provide a kind of preparation method who simulates the fluorescent polymer of green fluorescent protein, to solve existing fluorescent polymer preparing method's circumscribed technical problem.
A kind of preparation method who simulates the fluorescent polymer of green fluorescent protein may further comprise the steps:
The first step: in green fluorescent protein chromophore or its analogue and p-methyl benzene sulfonic chloride activated polyglycol adding organic solvent, at high temperature stir, wash, precipitate, obtain the polyoxyethylene glycol that chromophore modifies;
Second step: the polyoxyethylene glycol that resulting chromophore is modified carries out polyreaction, obtains simulating the novel amphiphilic fluorescent polymer of green fluorescent protein.
Polyreaction may further comprise the steps:
Under triethylamine catalysis, with the polyoxyethylene glycol reaction of bromo isobutyl acylbromide and chromophore's modification, washing, ice ether sedimentation, preparation bromo chromophore polyoxyethylene glycol;
With bromo chromophore polyoxyethylene glycol is initiator, causes the ATRP of hydrophobic monomer, prepares amphipathic fluorescent polymer.
Polyreaction also can may further comprise the steps: the polyoxyethylene glycol that chromophore is modified is dissolved in the organic solvent, causes the ring-opening polymerization of hydrophobic monomer, prepares amphipathic fluorescent polymer.
At high temperature stir further to be included under the temperature that is higher than 80 ℃ and stirred 24 hours.
Green fluorescent protein chromophore in the said the first step or its analogue comprise the fluorescence chromophore that contains oxybenzene methylene radical imidazolone.
Organic solvent in the first step comprises one of them of following solvent: acetonitrile, DMSO 99.8MIN., N.
Hydrophobic monomer is following one of them or its combination: TEB 3K, Jia Jibingxisuanyizhi, methacrylic tert-butyl acrylate, methyl acrylate, other contains the compound of methacrylic ester or propenoate.
ATRP is: with bromo chromophore polyoxyethylene glycol was initiator, utilizes toluene as solvent, under cuprous bromide and dipyridyl effect, adds the compound that contains methacrylic ester or propenoate, in 80 ℃ of reactions 8 hours.
The polyoxyethylene glycol that chromophore is modified is dissolved in the organic solvent one of them for following solvent: toluene, methyl-sulphoxide or N.
Hydrophobic monomer in the ring-opening polymerization of initiation hydrophobic monomer is caprolactone or rac-Lactide.
Ring-opening polymerization in the ring-opening polymerization of initiation hydrophobic monomer is: stannous octoate or triethyl aluminum are initiator, the caprolactone under nitrogen or the argon shield or the polymerization of rac-Lactide.
Because have approximate perfect cylindrical structure, green fluorescent protein shows unique with excellent photoluminescent property.When being in primary structure can rotate freely owing to produce the chromophore of fluorescence, the fluorescence of green fluorescent protein very a little less than.In case form cylindrical structure through self-assembly, rotating freely of chromophore is suppressed, green fluorescent protein has obtained excellent fluorescence.The present invention utilizes this biological self-assembly to obtain excellent fluorescence property principle, has designed and developed one type of novel fluorescent polymer.Be about to the water-soluble excellent fluorescence that obtains of resulting novel amphiphilic fluorescent polymer.Therefore, the present invention is the structure of simulation green fluorescent protein and the principle that self-assembly strengthens fluorescence property, utilizes the chromophore similar with green fluorescent protein to make up amphipathic nature polyalcohol, and adopts self-assembling method to realize the enhancing of polymer fluorescent.Synthetic fluorescent polymer of the present invention has following characteristics: the one, have only a fluorescence chromophore on the polymer chain; The 2nd, polymkeric substance itself has amphipathic, can self-assembly; The 3rd, after the polymkeric substance assembling, its fluorescence property obviously strengthens.With respect to traditional fluorescent polymer, the polymkeric substance that present method obtains is design and preparation easily, and can be used for bio-imaging, has shown good prospects for application.
Description of drawings
Fig. 1 is the nucleus magnetic hydrogen spectrum of the fluorescent polymer of the present invention's preparation;
Fig. 2 strengthens figure for the fluorescent polymer of the present invention's preparation through self-assembly fluorescence: (a) fluorescent emission figure before the polymkeric substance assembling; (b) polymkeric substance assembling back fluorescent emission figure.
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.
Embodiment 1
The first step: 0.90-1.00 is restrained fluorescence chromophore analogue, 2.60-2.80 gram Anhydrous potassium carbonate and 2.00-2.20 gram Methyl benzenesulfonyl chlorating polyoxyethylene glycol, be dissolved in 100-200 milliliter acetonitrile, 110 ℃ were refluxed 24 hours.Be cooled to room temperature, remove acetonitrile, use the methylene dichloride lysate, washing, deposition obtains the polyoxyethylene glycol that fluorescence chromophore modifies.
Second step: the polyoxyethylene glycol that 600-700 milligram chromophore is modified is dissolved in the 15-20 milliliter methylene dichloride, is cooled to 0 ℃, slowly adds 0.30-0.40 milliliter triethylamine and 0.30-0.40 milliliter bromo isobutyl acylbromide respectively, reacts 24 hours.Mixture dilutes with methylene dichloride, and washing precipitates, and obtains the polyoxyethylene glycol of bromo chromophore.
The 3rd step: with 2.0-2.4 milliliter toluene dissolving 25-30 milligram cuprous bromide and 70-90 milligram dipyridyl, add the polyoxyethylene glycol and the 2.0-2.4 milliliter TEB 3K of 200-240 milligram bromo chromophore more respectively, in 80 ℃ of reactions 8 hours.Product dissolves with THF, and deposition obtains amphipathic fluorescent polymer.
The first step: 0.90-1.00 is restrained fluorescence chromophore analogue, 2.70-2.80 gram Anhydrous potassium carbonate and 2.00-2.10 gram Methyl benzenesulfonyl chlorating polyoxyethylene glycol, be dissolved in 100-200 milliliter acetonitrile, 110 ℃ were refluxed 24 hours.Be cooled to room temperature, remove acetonitrile, use the methylene dichloride lysate, washing, deposition obtains the polyoxyethylene glycol that fluorescence chromophore modifies.
Second step: with the polyoxyethylene glycol that 10-15 milliliter toluene dissolving 210-230 milligram chromophore modifies, add the stannous octoate of 20-30 milligram again, under the nitrogen protection,, obtain amphipathic fluorescent polymer in 80 ℃ of reactions 32 hours.
The resulting product of the present invention through one dimension nucleus magnetic hydrogen spectrum, one dimension nuclear-magnetism carbon spectrum, Fourier transform infrared spectroscopy, gel permeation chromatography, ultraviolet-visible absorption spectroscopy, fluorescence emission spectrum is tested proves its structure and character.
Embodiment result sees accompanying drawing.The one dimension nucleus magnetic hydrogen spectrum spectrogram of the fluorescent polymer of corresponding embodiment 1 preparation of Fig. 1.Wherein, 3.66-3.64 what ppm was corresponding is the peak of the methylene radical of polyoxyethylene glycol repeating unit; 3.63-3.56 the ppm representative is the peak of the methoxyl group of polymethylmethacrylate; 2.06-1.42 ppm is the peak of the methylene radical of polymethylmethacrylate, what 1.01-0.84 ppm was corresponding is the peak of the methyl of polymethylmethacrylate.This spectrogram has proved the successful preparation of fluorescent polymer.The assembling fluorescence of the fluorescent polymer of corresponding embodiment 1 preparation of Fig. 2 strengthens the result.In the drawings, (a) representative is the fluorescent emission spectrogram before the polymkeric substance assembling, and (b) representative is the fluorescent emission spectrogram after the polymkeric substance assembling, and both concentration is identical.From figure, can obviously observe fluorescence and strengthen phenomenon, the fluorescence intensity after the assembling is 24 times before the assembling.In a word, through the structure and the self-assembling method of simulation green fluorescent protein, we design and have synthesized a kind of novel fluorescent polymer, and utilize self-assembly obviously to improve its fluorescence property.
What the present invention innovated is one type of novel fluorescent polymer, is connected to form by hydrophobic polymer, fluorescence chromophore and hydrophilic polymer, and parent/hydrophobic polymer ratio is expected to be applied to fields such as biology, medical science, pharmacy between 0.2-0.8.
The preferred embodiment of the present invention disclosed herein just is used for helping to set forth the present invention.Preferred embodiment does not have all details of detailed descriptionthe, does not limit this invention yet and is merely described embodiment.Obviously, according to the content of this specification sheets, can do a lot of modifications and variation.These embodiment are chosen and specifically described to this specification sheets, is in order to explain principle of the present invention and practical application better, thereby makes one of ordinary skill in the art can utilize the present invention well.The present invention only receives the restriction of claims and four corner and equivalent.
Claims (11)
1. a preparation method who simulates the fluorescent polymer of green fluorescent protein is characterized in that, may further comprise the steps:
The first step: in green fluorescent protein chromophore or its analogue and p-methyl benzene sulfonic chloride activated polyglycol adding organic solvent, at high temperature stir, wash, precipitate, obtain the polyoxyethylene glycol that chromophore modifies;
Second step: the polyoxyethylene glycol that resulting chromophore is modified carries out polyreaction, obtains simulating the novel amphiphilic fluorescent polymer of green fluorescent protein.
2. the method for claim 1 is characterized in that, polyreaction may further comprise the steps:
Under triethylamine catalysis, with the polyoxyethylene glycol reaction of bromo isobutyl acylbromide and chromophore's modification, washing, deposition, preparation bromo chromophore polyoxyethylene glycol;
With bromo chromophore polyoxyethylene glycol is initiator, causes the ATRP of hydrophobic monomer, prepares amphipathic fluorescent polymer.
3. the method for claim 1 is characterized in that, polyreaction may further comprise the steps:
The polyoxyethylene glycol that chromophore is modified is dissolved in the organic solvent, causes the ring-opening polymerization of hydrophobic monomer, prepares amphipathic fluorescent polymer.
4. the method for claim 1 is characterized in that, at high temperature stirs further to be included under the temperature that is higher than 80 ℃ and stirs 24 hours.
5. the preparation method of the fluorescent polymer of simulation green fluorescent protein as claimed in claim 1 is characterized in that: the green fluorescent protein chromophore in the said the first step or its analogue comprise the fluorescence chromophore that contains oxybenzene methylene radical imidazolone.
6. the preparation method of the fluorescent polymer of simulation green fluorescent protein as claimed in claim 1 is characterized in that: the organic solvent in the first step comprises one of them of following solvent: acetonitrile, DMSO 99.8MIN., N.
7. the preparation method of the fluorescent polymer of simulation green fluorescent protein as claimed in claim 2 is characterized in that: hydrophobic monomer is following one of them or its combination: TEB 3K, Jia Jibingxisuanyizhi, methacrylic tert-butyl acrylate, methyl acrylate, other contains the compound of methacrylic ester or propenoate.
8. the preparation method of the fluorescent polymer of simulation green fluorescent protein as claimed in claim 2 is characterized in that: ATRP is:
With bromo chromophore polyoxyethylene glycol was initiator, utilizes toluene or N as solvent, under cuprous bromide and dipyridyl effect, adds the compound that contains methacrylic ester or propenoate, in 80 ℃ of reactions 8 hours.
9. the preparation method of the fluorescent polymer of simulation green fluorescent protein as claimed in claim 3 is characterized in that: the polyoxyethylene glycol that chromophore is modified is dissolved in the organic solvent one of them for following solvent: toluene, methyl-sulphoxide or N.
10. the preparation method of the fluorescent polymer of simulation green fluorescent protein as claimed in claim 3 is characterized in that: the hydrophobic monomer in the ring-opening polymerization of initiation hydrophobic monomer is caprolactone or rac-Lactide.
11. the preparation method of the fluorescent polymer of simulation green fluorescent protein as claimed in claim 1; It is characterized in that: the ring-opening polymerization in the ring-opening polymerization of initiation hydrophobic monomer is: stannous octoate or triethyl aluminum are initiator, the caprolactone under nitrogen or the argon shield or the polymerization of rac-Lactide.
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| CN101260219A (en) * | 2008-04-25 | 2008-09-10 | 华南理工大学 | Method for preparing triblock copolymer micelle system used for realizing reversible fluorescence regulation and control |
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Granted publication date: 20140115 Termination date: 20160418 |