CN101817908B - Preparation method of molecular imprinting polymer of PSD-95/nNOS uncoupler - Google Patents
Preparation method of molecular imprinting polymer of PSD-95/nNOS uncoupler Download PDFInfo
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- CN101817908B CN101817908B CN2010190260934A CN201019026093A CN101817908B CN 101817908 B CN101817908 B CN 101817908B CN 2010190260934 A CN2010190260934 A CN 2010190260934A CN 201019026093 A CN201019026093 A CN 201019026093A CN 101817908 B CN101817908 B CN 101817908B
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Abstract
The invention relates to a preparation method of a molecular imprinting polymer of a PSD-95/nNOS uncoupler. The preparation method comprises the following steps of: dissolving template molecules and functional monomers according to the mol ratio of 1:1-1:8 into an acetonitrile solution containing a cross-linking agent and an initiating agent and preparing into a mixed solution, wherein the concentration of the cross-linking agent in acetonitrile is 20-80mmol/L, and the concentration of the initiating agent in the acetonitrile is 0.1-1g/L; sealing the mixed solution, then performing polymerization reaction by adopting heating and stirring modes to generate a powdered polymer, wherein the polymerization reaction temperature is 50-70 DEG C, and the reaction time is 12-24h; adding the powdered polymer into a mixed solution containing methanol and triethylamine and carrying out ultrasonic treatment so as to remove the template molecules, centrifuging and then adding the powdered polymer to methanol and carrying out ultrasonic treatment so as to remove the triethylamine; putting the treated polymer in a vacuum drying chamber, and drying to balance weight to obtain the molecular imprinting polymer. The prepared molecular imprinting polymer can be used for identifying natural products with SD-95/nNOS uncoupling effect.
Description
One, technical field
The invention provides a kind of molecularly imprinted polymer that the PSD-95/nNOS uncoupling agents is had the specific recognition effect.Utilize this molecularly imprinted polymer, can discern compound with PSD-95/nNOS uncoupling.
Two, background technology
N-methyl D-aspartic acid (N-Methyl-D-aspartic acid after the cerebral ischemia, NMDA) acceptor excessive activation, (Neuronal Nitric Oxide Synthase, nNOS) the nitric oxide production pathologic release of synthetic is one of principal element of the neuronal damage behind the cerebral ischemia by the neuron pattern nitric oxide synthetase.(Postsynaptic Density Protein-95 PSD-95) has played the part of the key player to postsynaptic density albumen-95 in this path.Under the cerebral ischemia situation, the nmda receptor excessive activation forms the NMDA/PSD-95/nNOS mixture, and pathologic discharges NO, with superoxide anion (O
2 -) the stronger peroxynitrite anion (ONOO of reaction toxigenicity
-), cause neuronic damage.Because nmda receptor, PSD-95, nNOS have multiple important physical effect, suppress its function and may cause serious toxic side effect.The uncoupling of PSD-95/nNOS, the pathologic that can suppress NO under the prerequisite that does not influence nmda receptor, PSD-95, nNOS function discharges.Bibliographical information 4-N-(2-hydroxyl-3, the 5-dichloro benzyl) aminosallcylic acid (compound 1), 6-N-(2-hydroxyl-3,5-dichloro benzyl) amino benzotriazole (compound 2) has clear and definite PSD-95/nNOS uncoupling (Chinese patent 200910025698.1; British Journal ofPharmacology 2009,158,494-506).Baicalin can combine with PSD-95, and (Phytomedicine 2004,11:277-284) may to have the PSD-95/nNOS uncoupling.Compound 1, compound
2, the chemical structure of noroxylin:
Compound 1 compound 2 noroxylins
The activity rating of PSD-95/nNOS uncoupling agents can adopt the method for nucleus magnetic resonance, this method need obtain PSD-95 and nNOS respectively, by measuring the variation of the relevant spectrum of nucleus magnetic resonance N-H before and after its administration, judge the situation that combines of target compound and PSD-95; Perhaps adopt the co-immunoprecipitation method, investigate under the mouse MCAO condition, administration group and blank group nNOS and PSD95 bonding strength strengthen the difference of degree.Two kinds of methods all waste time and energy, and are difficult to fast and convenient molecularly imprinted polymer and have strong specificity and highly selective, can be used as the artificial antibody, the high-flux fast screening target compound.In the natural product, many effective constituents are often leaked sieve or are difficult to purifying because content is humble, utilize molecularly imprinted polymer directly to extract from crude extract by the compound that some content are very low.
Three, summary of the invention
Goal of the invention:, the invention provides a kind of compound molecule imprinted polymer of discerning PSD-95/nNOS uncoupling function and preparation method thereof that can be used in order to overcome the deficiencies in the prior art.This molecularly imprinted polymer can be discerned the compound with PSD-95/nNOS uncoupling, can be used for high-flux fast screening PSD-95/nNOS uncoupling target compound, also can discern the natural product with PSD-95/nNOS uncoupling.
Technical scheme: a kind of PSD-95/nNOS uncoupling agents molecularly imprinted polymer preparation method, its preparation process is: with template molecule and function monomer by 1: 1-1: 8 mol ratio is dissolved in the acetonitrile solution that contains linking agent, initiator, be prepared into mixed solution, the concentration of linking agent in acetonitrile is 20mmol/L-80mmol/L, the concentration 0.1g/L-1g/L of initiator in acetonitrile; After the mixed solution sealing, adopt the mode polyreaction of heated and stirred to generate polymer powder, polymeric reaction temperature is 50-70 ℃, the reaction times is 12-24h; Polymer powder is added in the mixing solutions of methyl alcohol and triethylamine and carry out supersound process, remove template molecule, polymer powder is added supersound process in the methyl alcohol after centrifugal, remove triethylamine; The polymkeric substance that above-mentioned processing is obtained places vacuum drying oven to be dried to weight, obtains molecularly imprinted polymer.
The mol ratio of described template molecule and function monomer is preferably 1: 4.
The concentration of described linking agent in acetonitrile is preferably 80mmol/L.
The concentration of described initiator in acetonitrile is preferably 0.8g/L.
Described template molecule is 6-N-(2-hydroxyl-3,5-dichloro benzyl) amino benzotriazole or 4-N-(2-hydroxyl-3,5-dichloro benzyl) aminosallcylic acid.
Described function monomer is a kind of or any several mixture in 4-vinylpyridine, 2-vinylpyridine or the acrylamide.
Described linking agent is a trimethylolpropane trimethacrylate.
Described initiator is a Diisopropyl azodicarboxylate.
Beneficial effect: beneficial effect of the present invention is, make molecularly imprinted polymer and not only template molecule is had specific adsorptive power, and there are specific absorption equally in other PSD-95/nNOS uncoupling agentss, can discern natural product with SD-95/nNOS uncoupling.
Four, description of drawings
Fig. 1 is the adsorption curve of PSD-95/nNOS uncoupling molecularly imprinted polymer to compound 1, compound 2, noroxylin, as seen from the figure, the PSD-95/nNOS uncoupling molecularly imprinted polymer of gained all has the good adsorption effect to compound 1, compound 2, noroxylin, for high-flux fast screening PSD-95/nNOS uncoupling target compound, find that the natural product with PSD-95/nNOS uncoupling has certain application prospect.
Five, embodiment
The following examples can make those skilled in the art comprehensively understand the present invention, but do not limit the present invention in any way.
Embodiment 1:
6-N-(2-hydroxyl-3 with 1mmol, the 5-dichloro benzyl) the function monomer acrylamide of amino benzotriazole and 6mmol is dissolved into 100mL and contains in the pure acetonitrile solution of initiator Diisopropyl azodicarboxylate of 8mmol trimethylolpropane trimethacrylate, 80mg, is prepared into mixed solution.With mixed solution sealing back ultrasonic degas 5min, logical nitrogen 10min removes oxygen molecule, then with after the mixed solution sealing, adopts the mode polyreaction of heated and stirred to generate polymer powder, and polymerization temperature is 55-70 ℃, and the reaction times is 16h.After polymerization is finished,, synthetic good polymkeric substance is carried out supersound process, remove template molecule, polymer powder is added supersound process in the methyl alcohol after centrifugal, remove triethylamine with the methyl alcohol of 100mL and the mixing solutions of triethylamine; Place vacuum drying oven to be dried to weight in polymkeric substance, obtain molecularly imprinted polymer.
Embodiment 2:
4-N-(2-hydroxyl-3 with 1mmol, the 5-dichloro benzyl) the function monomer acrylamide of aminosallcylic acid and 8mmol is dissolved into 100mL and contains in the pure acetonitrile solution of initiator Diisopropyl azodicarboxylate of 8mmol trimethylolpropane trimethacrylate, 80mg, is prepared into mixed solution.With mixed solution sealing back ultrasonic degas 5min, logical nitrogen 10min removes oxygen molecule, then with after the mixed solution sealing, adopts the mode polyreaction of heated and stirred to generate polymer powder, and polymerization temperature is 50-65 ℃, and the reaction times is 24h.After polymerization is finished,, synthetic good polymkeric substance is carried out supersound process, remove template molecule, polymer powder is added supersound process in the methyl alcohol after centrifugal, remove triethylamine with the methyl alcohol of 100mL and the mixing solutions of triethylamine.Place vacuum drying oven to be dried to weight in polymkeric substance, obtain PSD-95/nNOS uncoupling molecularly imprinted polymer.
Embodiment 3:
4-N-(2-hydroxyl-3 with 1mmol, the 5-dichloro benzyl) the function monomer 2-vinylpyridine of aminosallcylic acid and 4mmol is dissolved into 100mL and contains in the pure acetonitrile solution of initiator Diisopropyl azodicarboxylate of 8mmol trimethylolpropane trimethacrylate, 80mg, is prepared into mixed solution.With mixed solution sealing back ultrasonic degas 5min, logical nitrogen 10min removes oxygen molecule, then with after the mixed solution sealing, adopts the mode polyreaction of heated and stirred to generate polymer powder, and polymerization temperature is 55-65 ℃, and the reaction times is 24h.After polymerization is finished,, synthetic good polymkeric substance is carried out supersound process, remove template molecule, polymer powder is added supersound process in the methyl alcohol after centrifugal, remove triethylamine with the methyl alcohol of 100mL and the mixing solutions of triethylamine.Place vacuum drying oven to be dried to weight in polymkeric substance, obtain PSD-95/nNOS uncoupling molecularly imprinted polymer.
Embodiment 4:
4-N-(2-hydroxyl-3 with 1mmol, the 5-dichloro benzyl) the function monomer acrylamide of the function monomer 4-vinylpyridine of aminosallcylic acid and 1mmol, 4mmol is dissolved into 100mL and contains in the pure acetonitrile solution of initiator Diisopropyl azodicarboxylate of 5mmol trimethylolpropane trimethacrylate, 80mg, is prepared into mixed solution.With mixed solution sealing back ultrasonic degas 5min, logical nitrogen 10min removes oxygen molecule, then with after the mixed solution sealing, adopts the mode polyreaction of heated and stirred to generate polymer powder, and polymerization temperature is 55-65 ℃, and the reaction times is 24h.After polymerization is finished,, synthetic good polymkeric substance is carried out supersound process, remove template molecule, polymer powder is added supersound process in the methyl alcohol after centrifugal, remove triethylamine with the methyl alcohol of 100mL and the mixing solutions of triethylamine.Place vacuum drying oven to be dried to weight in polymkeric substance, obtain PSD-95/nNOS uncoupling molecularly imprinted polymer.
Embodiment 5:
4-N-(2-hydroxyl-3 with 1mmol, the 5-dichloro benzyl) the function monomer 4-vinylpyridine of aminosallcylic acid and 4mmol is dissolved into 100mL and contains in the pure acetonitrile solution of initiator Diisopropyl azodicarboxylate of 8mmol trimethylolpropane trimethacrylate, 80mg, is prepared into mixed solution.With mixed solution sealing back ultrasonic degas 5min, logical nitrogen 10min removes oxygen molecule, then with after the mixed solution sealing, adopts the mode polyreaction of heated and stirred to generate polymer powder, and polymerization temperature is 55-65 ℃, and the reaction times is 16h.After polymerization is finished,, synthetic good polymkeric substance is carried out supersound process, remove template molecule, polymer powder is added supersound process in the methyl alcohol after centrifugal, remove triethylamine with the methyl alcohol of 100mL and the mixing solutions of triethylamine.Place vacuum drying oven to be dried to weight in polymkeric substance, obtain PSD-95/nNOS uncoupling molecularly imprinted polymer.
Embodiment 6:
The function monomer 4-vinylpyridine of 4mmol is dissolved into 100mL contains in the pure acetonitrile solution of initiator Diisopropyl azodicarboxylate of 8mmol trimethylolpropane trimethacrylate, 80mg, be prepared into mixed solution.With mixed solution sealing back ultrasonic degas 5min, logical nitrogen 10min removes oxygen molecule, then with after the mixed solution sealing, adopts the mode polyreaction of heated and stirred to generate polymer powder, and polymerization temperature is 55-65 ℃, and the reaction times is 16h.After polymerization is finished,, synthetic good polymkeric substance is carried out supersound process, remove template molecule, polymer powder is added supersound process in the methyl alcohol after centrifugal, remove triethylamine with the methyl alcohol of 100mL and the mixing solutions of triethylamine.Place vacuum drying oven to be dried to weight in polymkeric substance, obtain the barren molecularly imprinted polymer.
Embodiment 7:
The polymkeric substance (NIP) of the molecularly imprinted polymer of 50mg embodiment 5 (MIP), 50mg embodiment 6, join the target molecule (compound 1 of different concns respectively, compound 2, noroxylin) 10mL acetonitrile solution, jolting is 24 hours under room temperature, (it is 284nm that compound 1, compound 2 detect wavelength to adopt high-efficient liquid phase technique, it is 276nm that noroxylin detects wavelength, moving phase is that volume ratio is 70: 30 a methanol aqueous solution, flow velocity 1mL/min), measuring its loading capacity, is X-coordinate with the strength of solution, loading capacity is an ordinate zou, does adsorption curve.
Loading capacity (μ mol/g)=(C
0-C
1) * 200
Wherein: C
0For adsorbing preceding strength of solution (mmol/L), C
1For adsorbing back strength of solution (mmol/L)
From Fig. 1 as seen, the PSD-95/nNOS uncoupling molecularly imprinted polymer of gained all has the good adsorption effect to compound 1, compound 2, noroxylin, and the natural product that has the PSD-95/nNOS uncoupling for exploitation has certain application prospect.
Claims (6)
1. PSD-95/nNOS uncoupling agents molecularly imprinted polymer preparation method is characterized by:
A. with template molecule and function monomer by 1: 1-1: 8 mol ratio is dissolved in the acetonitrile solution that contains linking agent, initiator, be prepared into mixed solution, the concentration of linking agent in acetonitrile is 20mmol/L-80mmol/L, the concentration 0.1g/L-1g/L of initiator in acetonitrile; Described template molecule is 6-N-(2-hydroxyl-3,5-dichloro benzyl) amino benzotriazole or 4-N-(2-hydroxyl-3,5-dichloro benzyl) aminosallcylic acid; Described function monomer is a kind of or any several mixture in 4-vinylpyridine, 2-vinylpyridine or the acrylamide;
B. with after the mixed solution sealing, adopt the mode polyreaction of heated and stirred to generate polymer powder, polymeric reaction temperature is 50-70 ℃, and the reaction times is 12-24h;
C. polymer powder is added in the mixing solutions of methyl alcohol and triethylamine and carry out supersound process, remove template molecule, polymer powder is added supersound process in the methyl alcohol after centrifugal, remove triethylamine;
D. the polymkeric substance that above-mentioned processing is obtained places vacuum drying oven to be dried to weight, obtains molecularly imprinted polymer.
2. according to the preparation method of the described PSD-95/nNOS uncoupling agents of claim 1 molecularly imprinted polymer, the mol ratio that it is characterized in that template molecule and function monomer is 1: 4.
3. according to the preparation method of the described PSD-95/nNOS uncoupling agents of claim 1 molecularly imprinted polymer, it is characterized in that the concentration of linking agent in acetonitrile is 80mmol/L.
4. according to the preparation method of the described PSD-95/nNOS uncoupling agents of claim 1 molecularly imprinted polymer, it is characterized in that the concentration of initiator in acetonitrile is 0.8g/L.
5. according to the preparation method of the described PSD-95/nNOS uncoupling agents of claim 1 molecularly imprinted polymer, it is characterized in that linking agent is a trimethylolpropane trimethacrylate.
6. according to the preparation method of the described PSD-95/nNOS uncoupling agents of claim 1 molecularly imprinted polymer, it is characterized in that initiator is a Diisopropyl azodicarboxylate.
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| CN102516107A (en) * | 2011-11-03 | 2012-06-27 | 南京医科大学 | N-benzylaniline derivatives and application thereof |
| CN103940932A (en) * | 2013-01-17 | 2014-07-23 | 中国农业科学院兰州畜牧与兽药研究所 | Non-biological method for screening effective antivirus components in traditional Chinese medicines |
| CN107344986B (en) * | 2017-06-21 | 2020-01-24 | 南京医科大学 | Magnetic artificial receptor and preparation method and application thereof |
| CN107353372B (en) * | 2017-06-21 | 2019-11-01 | 南京医科大学 | A kind of preparation method of the nNOS-PSD-95 uncoupler imprint surface polymer based on magnetic mesoporous molecular sieve |
| CN110498751B (en) * | 2019-08-15 | 2021-10-08 | 南京医科大学 | Imprinted template molecule and preparation method and application thereof |
| CN110627877A (en) * | 2019-09-25 | 2019-12-31 | 成都奥达生物科技有限公司 | PSD-95 inhibitor |
| CN111285923B (en) * | 2020-03-05 | 2023-02-03 | 成都奥达生物科技有限公司 | PSD-95 inhibitor |
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| CN1390862A (en) * | 2002-06-26 | 2003-01-15 | 天津大学 | Process for preparing microspheres of molecular blot polymer in water medium |
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