CN105399680A - Chiral polyoxometalate, preparation method and applications thereof - Google Patents
Chiral polyoxometalate, preparation method and applications thereof Download PDFInfo
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- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
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- C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
- C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
- C07C233/47—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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Abstract
The invention discloses a kind of chiral poly oxometallates and the preparation method and application thereof, belong to protein inhibitor technical field. It solves in the prior art, POMs lacks targeting, and recognition capability is poor under complicated biotic environment system, the technical issues of cannot achieve clinical application. Chiral poly oxometallate of the invention, shown in structural formula such as formula (I), in formula, R L-His, D-His, L-Asp, D-Asp, L-Leu, D-Leu, L-Phe or D-Phe take off the residue of an amino hydrogen. The chirality poly oxometallate can be used as the application of A beta inhibitor, can penetrate blood-brain barrier, realize under physiological environment, have high affinity and selectivity to A β, provide more abundant means for the treatment of AD.
Description
Technical field
The invention belongs to protein inhibitor technical field, be specifically related to a kind of chirality poly oxometallate and preparation method thereof and application.
Background technology
Alzheimer's disease (Alzheimer ' sdisease, be called for short AD) be the nervous system degenerative disease that the Progressive symmetric erythrokeratodermia of a kind of onset concealment develops.Clinical manifestation is that the acquired Premium Features of pallium are impaired, and namely dull-witted, mainly comprise hypomnesis in various degree, perceptibility, judgement, thinking ability, motor capacity etc. are impaired, and emotional responses obstacle and personality change.At present, AD has become in modern society the fourth-largest killer of the human health being only second to cardiovascular diseases, cancer and cerebral apoplexy.According to World Health Organization's statistics, the expense of whole world treatment beginning of this century AD patient accounts for 11.2% of more than 60 years old disease population total expenditure, far above above-mentioned three.By the end of 2013, have the AD patient of more than 40,000,000 in global range, wherein the AD patient of China was 10,000,000, and absolute quantity occupy first place, the world.According to EPDML prediction, the quantity to the year two thousand fifty patient can be increased to about 100,000,000.Along with the continuous improvement of people's living standard, comprised many countries of China or be about to enter aging society, the ratio of elderly population will rise day by day, objectively also increases the potential patient groups of AD.And be representative with AD, sickness rate that the nerve degenerative diseases that occurs with age growth is in the elderly remains high, most patients are the old man of over-65s, and course of disease length (is generally 10-20, even longer), without effective treatment means, all bring white elephant to society, family.
The major pathologic features of Alzheimer's disease is: the position such as temporal lobe and hippocampal cortex neuron loss, causes primarily of cholinergic function damage; Neurofibrillary tangles (neurofibrillarytangles, NFTs), condenses generation primarily of the Hyperphosphorylationof of microtubule-associated protein tau and glycoforms in cell; Senile plaque (senileplaques outside brain inner cell, SPs) deposit, typical senile plaque core assemble many diameters be about 8-10nm by amyloid-beta (β-amyloidpeptide, be called for short A β) fiber assembled, nutritious bad nervous process around, the astroglia cell of activation and microglia are around forming fine and close fibrous plaque.
The definite pathogenesis of AD is not also studied clear.The cause of disease for AD has multiple theory at present.Wherein, A β cascade hypothesis (amyloidcascadehypothesis) of Hardy and Higgins proposition in 1992 is of greatest concern, they think that the gathering of A β and Energy Deposition aggravate neurofibrillar entanglement, and cause necrocytosis, are the main reasons that AD is formed.Removed the patch of A beta peptide aggregation by immunization after, the nervous system lesion relevant to AD can be repaired, and this is that A β cascade hypothesis provides strong evidence.But in organic molecule of the prior art and peptide inhibitor, only have the Selective recognition that little a part of peptide inhibitor can realize A β, and this kind of inhibitor is often difficult to penetrate hemato encephalic barrier.This just makes above-mentioned inhibitor be difficult to obtain good curative effect when clinical treatment.
2011, Angew.Chem.Int.Ed. (German applied chemistry) magazine, disclose a series of poly oxometallate (also known as heteropolyacid, POMs), the poly oxometallate of this series is electronegative in physiological conditions, can produce an electrostatic interaction with positive electricity district His13-Lys16 (HHQK) of A β.Therefore, this poly oxometallate can regulate and control the gathering of AD paraprotein A β.But POMs is as mineral compound, lack targeting, under the coenocorrelation system of complexity, recognition capability is poor, and this just makes to still have certain distance between they and the clinical application of reality.
Summary of the invention
The object of the invention is to solve in prior art, POMs lacks targeting, and under the coenocorrelation system of complexity, recognition capability is poor, cannot realize the technical problem of clinical application, provides a kind of chirality poly oxometallate and preparation method thereof.
The technical scheme that the present invention solves the problems of the technologies described above employing is as follows.
Chirality poly oxometallate, structural formula is as follows:
In formula ,-R is the one in following structure:
The preparation method of chirality poly oxometallate, comprises the following steps:
(1) by amination Anderson type POM and Succinic anhydried by amount of substance 1:(2-10) be dissolved in anhydrous acetonitrile, stirring at room temperature reaction more than 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be carboxylated Anderson type POM;
(2) carboxylated Anderson type POM, N-hydroxy-succinamide (NHS) and dicyclohexylcarbodiimide (DCC) are dissolved in anhydrous N by amount of substance 1:1:1, in dinethylformamide (DMF), stirring at room temperature reaction more than 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be activated carboxylic Anderson type POM;
(3) by activated carboxylic Anderson type POM and chiral amino acid by amount of substance than 1:(1-20) be dissolved in dry DMF, stirring at room temperature reaction more than 48h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be chirality poly oxometallate;
Described chiral amino acid is L-His, D-His, L-Asp, D-Asp, L-Leu, D-Leu, L-Phe or D-Phe.
Preferably, in step (1), stirring velocity is more than 200r/min, and churning time is 24-48h.
Preferably, in step (2), stirring velocity is more than 200r/min, and churning time is 24-48h.
Preferably, in step (3), stirring velocity is more than 200r/min, and churning time is 48-72h.
Above-mentioned chirality poly oxometallate can as amyloid-beta inhibitor.
Compared with prior art, beneficial effect of the present invention:
1, chirality poly oxometallate of the present invention has POM structure and chiral amino acid structure, in physiological conditions, positive electricity district His13-Lys16 (HHQK) of electronegative POM structure and A β produces an electrostatic interaction, meanwhile, the little alkane chain of the upper modification of POM and Leu17 and Val18 two amino acid of A β produce water delivery and interact, the amino acid of the respective regions of chiral amino acid structure and A β produces chiral recognition effect, jointly playing a role of these three kinds of reactive forces makes to there is a very large binding constant between such chirality poly oxometallate and A β, thus realize the highly sensitive of chirality poly oxometallic acid salt pair A β of the present invention, strong specific recognition capability, detect through experiment, the binding ability of modifying the Anderson type POM of chiral amino acid is eager to excel 16-1090 doubly than not modified Anderson type POM, and A β sequence exists very strong amino acid Phe19 and Phe20 of two hydrophobicitys, if the chiral amino acid modified on POM is hydrophobic amino acid, can improve in conjunction with binding ability further,
2, the inhibition of chirality poly oxometallic acid salt pair A beta peptide aggregation of the present invention is good, is eager to excel 37-226 doubly than not modified Anderson type POM;
3, chirality poly oxometallate of the present invention, can eliminate the cytotoxicity that A is beta mediated;
4, chirality poly oxometallate of the present invention is as the application of A beta inhibitor, and can penetrate the inhibitor of hemato encephalic barrier, realize under physiological environment, to high affinity and the selectivity of A β, the treatment for AD provides the means of more horn of plenty.
Accompanying drawing explanation
Fig. 1 is the THT curve after the chirality POM of embodiment of the present invention 1-8 adds the hatching of A β substratum;
Fig. 2 is the chirality POM of the embodiment of the present invention 1-8 Cytotoxic impact beta mediated on A;
Fig. 3 is that the POM-D-Phe of different concns is to the effect of vigor of PC12 cell;
Fig. 4 is MnMo in chirality poly oxometallate of the present invention
6o
24floor map;
In figure, 1-6 all represents key.
Embodiment
Invention thought of the present invention: 2011, Germany's applied chemistry discloses a series of poly oxometallate (also known as heteropolyacid, POMs), the poly oxometallate of this series is electronegative in physiological conditions, can produce an electrostatic interaction with positive electricity district His13-Lys16 (HHQK) of A β.2009, PNAS reported little alkane chain and can produce water delivery with Leu17 and Val18 two amino acid of A β and interact.In A β sequence, ensuing two amino acid are Phe19 and Phe20, and this is the amino acid that two hydrophobicitys are very strong, if modify hydrophobic amino acid on POM, just may produce with them and interact, and then obtain better targeting.Meanwhile, because the natural amino acid in organism is the L-type amino acid of chirality, so for the natural protein of any one, all there is chirality microenvironment around it, this is equally applicable to A β.If modify chiral structure on POM, interact with the chirality microenvironment of A β, just likely obtain stronger binding ability.Consideration applied environment is physiological environment, so pay the utmost attention to chiral amino acid.But different amino acid has different character, be modified at after on POM, the binding ability for A β also can not be identical, so amino acid whose selection is very important.Even and if same amino acid, different chiralitys also has different binding abilities, a kind of compound of chirality can as medicine, but its enantiomorph is but invalid or even possibility poisonous (as world-shaking Japan " reaction stops " medical event), so the selection of amino acid chiral is also very important.Based on above imagination, we have designed and synthesized following POM derivative.
Chirality poly oxometallate of the present invention, structural formula is such as formula shown in (I):
In formula ,-R is the residue after L-His, D-His, L-Asp, D-Asp, L-Leu, D-Leu, L-Phe or D-Phe slough an amino hydrogen, and concrete R is the one in following structure:
Wherein, chemical formula is identical, two kinds of structures mapping structure each other of symmetrical configuration, namely different chiralitys is possessed, namely in said structure, from left to right, from top to bottom, represent successively L-His, D-His, L-Asp, D-Asp, L-Leu, D-Leu, L-Phe or D-Phe slough an amino hydrogen after residue.When-R is the residue after D-Phe sloughs an amino hydrogen, the effect of chirality poly oxometallate is carried out, and binding ability is eager to excel 10 than the Anderson type POM modified without chiral amino acid
3doubly, so preferred R is the residue after D-Phe sloughs an amino hydrogen.
It should be noted that, in chirality poly oxometallate of the present invention, due to MnMo
6o
24for cage structure, so cannot be embodied by structure, usually adopt formula (II) to its signal in prior art, specifically can see document Acollectionofrobustmethodologiesforthepreparationofasymm etrichybridMn – Andersonpolyoxometalatesformultifunctionalmaterials (Chem.Sci., 2013,4,3810 – 3817.).MnMo
6o
24structural plan schematic diagram as shown in Figure 4, as can be seen from Figure 4, MnMo
6o
246 MoO
4to be fenced up a Mn atom by coordinate bond, then 1,3,3 C of 5 number keys and the same side form C-O key respectively; 2,4, No. 6 form C-O key respectively with 3 C of opposite side.
The preparation method of chirality poly oxometallate of the present invention, comprises the following steps:
(1) by amination Anderson type POM and Succinic anhydried by amount of substance 1:(2-10) be dissolved in anhydrous acetonitrile, stirring at room temperature reaction more than 24h, general 200-400r/min rotating speed stirs 24-48h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be carboxylated Anderson type POM;
(2) carboxylated Anderson type POM, NHS and DCC are dissolved in dry DMF by amount of substance 1:1:1, stirring at room temperature reaction more than 24h, general 200-400r/min rotating speed stirs 24-48h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be activated carboxylic Anderson type POM;
(3) by activated carboxylic Anderson type POM and chiral amino acid by amount of substance than 1:(1-20) be dissolved in dry DMF, stirring at room temperature reaction more than 48h, general 200-400r/min rotating speed stirs 48-72h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be chirality poly oxometallate;
Wherein, chiral amino acid is L-His, D-His, L-Asp, D-Asp, L-Leu, D-Leu, L-Phe or D-Phe, preferred D-Phe.
It should be noted that, amination Anderson type POM is prior art, and structural formula is [N (C
4h
9)
4]
3[MnMo
6o
18{ (OCH
2)
3cNH
2}
2], can be prepared by existing method, see document Acollectionofrobustmethodologiesforthepreparationofasymm etrichybridMn – Andersonpolyoxometalatesformultifunctionalmaterials (Chem.Sci., 2013,4,3810 – 3817.).The invention provides the preparation method of a kind of amination Anderson type POM, but be not limited thereto, concrete steps are as follows:
(1) by Na
2moO
42H
2o is dissolved in intermediate water, adds excessive hydrochloric acid with vigorous stirring, then continues, at room temperature vigorous stirring 10min, to obtain solution A;
(2) Tetrabutyl amonium bromide is dissolved in intermediate water, obtains solution B;
(3) under vigorous stirring, solution B dropwise joined in solution A, heated and stirred reaction 1h, collected by suction precipitates, 3 times are washed with intermediate water, what obtain is precipitated and dissolved in the acetone of boiling, and heat filters insolubles, and filtrate is placed in-20 DEG C of precipitations, suction filtration, after 2 washed with diethylether, dry, obtain solid;
(4) by the solid that step (3) obtains, excessive MnAc
32H
2o and Tris, is dissolved in anhydrous acetonitrile jointly, reflux, and after being cooled to room temperature, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, and gained crystal is amination Anderson type POM.
Chirality poly oxometallate of the present invention can as A beta inhibitor, and chirality POM is combined in the HHQKLVFF region of A β.Utilize identical titration calorimetry (ITC) that the binding site of inhibitor on A β can be determined.
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1
Chirality poly oxometallate, structural formula is such as formula shown in (I), and in formula (I), R is
The preparation method of above-mentioned inhibitor, comprises the following steps:
(1) be dissolved in anhydrous acetonitrile by amination Anderson type POM and Succinic anhydried by amount of substance 1:2, stirring at room temperature reaction 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is carboxylated Anderson type POM;
(2) be dissolved in dry DMF by carboxylated Anderson type POM, NHS and DCC by amount of substance 1:1:1, stirring at room temperature reaction 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is activated carboxylic Anderson type POM;
(3) activated carboxylic Anderson type POM and L-His is dissolved in dry DMF by amount of substance than 1:1, stirring at room temperature reaction 48h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be chirality poly oxometallate, be designated as POM-L-His.
Embodiment 2
Chirality poly oxometallate, structural formula is such as formula shown in (I), and R is
The preparation method of above-mentioned inhibitor, comprises the following steps:
(1) be dissolved in anhydrous acetonitrile by amination Anderson type POM and Succinic anhydried by amount of substance 1:4, stirring at room temperature reaction 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is carboxylated Anderson type POM;
(2) be dissolved in dry DMF by carboxylated Anderson type POM, NHS and DCC by amount of substance 1:1:1, stirring at room temperature reaction 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is activated carboxylic Anderson type POM;
(3) activated carboxylic Anderson type POM and D-His is dissolved in dry DMF by amount of substance than 1:5, stirring at room temperature reaction 48h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be chirality poly oxometallate, be designated as POM-D-His.
Embodiment 3
Chirality poly oxometallate, structural formula is such as formula shown in (I), and R is
The preparation method of above-mentioned inhibitor, comprises the following steps:
(1) be dissolved in anhydrous acetonitrile by amination Anderson type POM and Succinic anhydried by amount of substance 1:5, stirring at room temperature reaction 36h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is carboxylated Anderson type POM;
(2) be dissolved in dry DMF by carboxylated Anderson type POM, NHS and DCC by amount of substance 1:1:1, stirring at room temperature reaction 36h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is activated carboxylic Anderson type POM;
(3) activated carboxylic Anderson type POM and D-Asp is dissolved in dry DMF by amount of substance than 1:5, stirring at room temperature reaction 48h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be chirality poly oxometallate, be designated as POM-D-Asp.
Embodiment 4
Chirality poly oxometallate, structural formula is such as formula shown in (I), and R is
The preparation method of above-mentioned inhibitor, comprises the following steps:
(1) be dissolved in anhydrous acetonitrile by amination Anderson type POM and Succinic anhydried by amount of substance 1:6, stirring at room temperature reaction 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is carboxylated Anderson type POM;
(2) be dissolved in dry DMF by carboxylated Anderson type POM, NHS and DCC by amount of substance 1:1:1, stirring at room temperature reaction 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is activated carboxylic Anderson type POM;
(3) activated carboxylic Anderson type POM and L-Asp is dissolved in dry DMF by amount of substance than 1:8, stirring at room temperature reaction 60h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be chirality poly oxometallate, be designated as POM-L-Asp.
Embodiment 5
Chirality poly oxometallate, structural formula is such as formula shown in (I), and R is
The preparation method of above-mentioned inhibitor, comprises the following steps:
(1) be dissolved in anhydrous acetonitrile by amination Anderson type POM and Succinic anhydried by amount of substance 1:6, stirring at room temperature reaction 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is carboxylated Anderson type POM;
(2) be dissolved in dry DMF by carboxylated Anderson type POM, NHS and DCC by amount of substance 1:1:1, stirring at room temperature reaction 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is activated carboxylic Anderson type POM;
(3) activated carboxylic Anderson type POM and L-Leu is dissolved in dry DMF by amount of substance than 1:20, stirring at room temperature reaction more than 72h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be chirality poly oxometallate, be designated as POM-L-Leu.
Embodiment 6
Chirality poly oxometallate, structural formula is such as formula shown in (I), and R is
The preparation method of above-mentioned inhibitor, comprises the following steps:
(1) be dissolved in anhydrous acetonitrile by amination Anderson type POM and Succinic anhydried by amount of substance 1:10, stirring at room temperature reaction 48h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is carboxylated Anderson type POM;
(2) be dissolved in dry DMF by carboxylated Anderson type POM, NHS and DCC by amount of substance 1:1:1, stirring at room temperature reaction 48h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is activated carboxylic Anderson type POM;
(3) activated carboxylic Anderson type POM and D-Leu is dissolved in dry DMF by amount of substance than 1:10, stirring at room temperature reaction 48h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be chirality poly oxometallate, be designated as POM-D-Leu.
Embodiment 7
Chirality poly oxometallate, structural formula is such as formula shown in (I), and R is
The preparation method of above-mentioned inhibitor, comprises the following steps:
(1) by amination Anderson type POM and Succinic anhydried by amount of substance 1:(2-10) be dissolved in anhydrous acetonitrile, stirring at room temperature reaction more than 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be carboxylated Anderson type POM;
(2) carboxylated Anderson type POM, NHS and DCC are dissolved in dry DMF by amount of substance 1:1:1, stirring at room temperature reaction more than 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be activated carboxylic Anderson type POM;
(3) by activated carboxylic Anderson type POM and L-Phe by amount of substance than 1:(1-20) be dissolved in dry DMF, stirring at room temperature reaction more than 48h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be chirality poly oxometallate, be designated as POM-L-Phe.
Embodiment 8
Chirality poly oxometallate, structural formula is such as formula shown in (I), and R is
The preparation method of above-mentioned inhibitor, comprises the following steps:
(1) be dissolved in anhydrous acetonitrile by amination Anderson type POM and Succinic anhydried by amount of substance 1:8, stirring at room temperature reaction 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is carboxylated Anderson type POM;
(2) be dissolved in dry DMF by carboxylated Anderson type POM, NHS and DCC by amount of substance 1:1:1, stirring at room temperature reaction 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtains crystal, is activated carboxylic Anderson type POM;
(3) activated carboxylic Anderson type POM and D-Phe is dissolved in dry DMF by amount of substance than 1:15, stirring at room temperature reaction 60h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be chirality poly oxometallate, be designated as POM-D-Phe.
Performance test is carried out to the chirality poly oxometallate that embodiment 1-8 obtains
1, the binding ability of the chirality poly oxometallate that obtains of embodiment 1-8 and A β is measured
The NanoITC instrument adopting TA company of the U.S. to produce is measured, and is the A β solution of 1.4ml20 μM below instrument in sample pool; POM derivative in over-injected pin synthesized by the Anderson type POM of 250ml448 μM or embodiment 1-8, carries out identical titration calorimetry (ITC).Result display, by modifying amino acid of different nature, can strengthen the binding ability of POM for A β really, even if simultaneously same amino acid, different chiralitys also has different binding abilities.Wherein the amino acid whose modification of water delivery is useful for the lifting of binding ability, and the amino acid whose modification of D type is useful for the lifting of binding ability too.So the POM (POM-D-Phe) that D-Phe modifies possesses the strongest binding ability.
2, thioflavin T hT tests
The kinetics of aggregation of A β can be detected by the change of ThT fluorescence.If A β assembles, so the fluorescence of ThT will rise; If the gathering of A β is suppressed, so would not there is obvious change in the fluorescence of ThT.ThT fluorescence exciting wavelength is at 444nm, and emission wavelength is at 482nm.
With 50mMHEPES (pH7.4, include 150mMNaCl) be solvent, configure 40 μMs of A β solution, a copy of it is hatched at 37 DEG C, all the other eight parts add amination Anderson type POM, POM-D-His, POM-L-His, POM-D-Asp, POM-L-Asp, POM-L-Leu, POM-D-Leu, POM-L-Phe, POM-D-Phe of 20 μMs respectively, hatch equally at 37 DEG C.Sample every 24h and add ThT, utilizing Fluorescence spectrophotometer to detect the ThT fluorescence of above-mentioned ten parts of solution.Testing conditions is, the A β final concentration in colorimetric pool is 1 μM, and the final concentration of probe molecule ThT is 10 μMs, cumulative volume 300ul.Result as shown in Figure 1.In Fig. 1, curve is followed successively by A β when hatching separately from top to bottom, add amination Anderson type POM after hatching, add POM-D-His after hatching, add POM-L-His after hatching, add POM-D-Asp after hatching, add POM-L-Asp after hatching, add POM-L-Leu hatching after, add POM-D-Leu hatching, add POM-L-Phe after hatching, add POM-D-Phe after the ThT fluorescence curve of hatching.As can be seen from Figure 1, A β is separately when hatching for 37 DEG C, and ThT fluorescence presents distinctive S type curve.After adding chiral POM, the formation of A beta peptide aggregation body is significantly inhibit.Can find out according to the speed that ThT fluorescence increases, what inhibition was best remains POM-D-Phe, compares with the POM of unmodified, and POM-D-Phe is to the 503nhibiting concentration (IC of A β
50) have dropped more than 200 times.
3, the Performance Detection of the bio-toxicity that chirality POM suppression A is beta mediated
PC12 cell is used to study the more a kind of neurocyte of A β toxicity, and we analyze chirality POM to the neurovirulent impact of A β with mtt assay.
Respectively by the A β of 2 μMs, 2 μMs of A β and 1 μM POM-D-Phe, 2 μMs of A β and 1 μM POM-D-Phe in vitro buffered soln 37 DEG C hatch 1h, then the three kinds of solution getting equivalent are distributed in the substratum of PC12 respectively cultivates 24h (blank sample is added in the substratum of PC12 by the damping fluid of equivalent), continue to cultivate 4h respectively to the MTT adding equivalent in substratum again, the purple crystals dimethyl sulfoxide (DMSO) (DMSO) obtained after removing substratum is dissolved, finally measure the absorption value under 490nm wavelength by microplate reader, result as shown in Figure 2.As can be seen from Figure 2, A beta peptide aggregation can cause cell viability reduction about 40%, chirality POM and A β to hatch the toxicity that significantly can reduce A β, and when especially hatching altogether with POM-D-Phe, cell viability can be promoted to more than 90% at most.
Get five parts of identical PC12 cells containing substratum, adding final concentration is respectively that the POM-D-Phe of 0 μM, 10 μMs, 20 μMs, 50 μMs, 100 μMs hatches 24h at 37 DEG C, the MTT adding equivalent again in substratum continues to cultivate 4h, the purple crystals dimethyl sulfoxide (DMSO) (DMSO) obtained after removing substratum is dissolved, measure the absorption value under 490nm wavelength by microplate reader, result as shown in Figure 3.As can be seen from Figure 3, the effect of vigor of chirality POM to PC12 cell itself is little.
Composition graphs 2 and Fig. 3 can illustrate, chirality POM not only can suppress A beta peptide aggregation in vitro, still effective in cell.
It should be noted that, the A β that test experiments of the present invention adopts is all through pre-treatment, and pretreatment process is first dissolved in hexafluoroisopropanol by A β powder with the concentration of 1mg/ml, then bottleneck is sealed, at 4 DEG C of vibration 2h, it is impelled to dissolve completely, then by frozen for storing solution in-20 DEG C of refrigerators.Before the use, by soft nitrogen gas stream, hexafluoroisopropanol is dried up, be then again dissolved in corresponding buffered soln, in 4 DEG C of refrigerators, leave standstill balance 6h.The PC12 cell that test experiments of the present invention uses is cultivated in RPMI1640 substratum, containing 5%CO in incubator
2. cultivate after 72h, the centrifugal 5min collecting cell of 1000rpm, by PBS buffer solution for cleaning twice.Cell is resuspended in pH7.4 buffered soln, with Petroff-Hausser cell counter, cell is counted.
Claims (6)
1. chirality poly oxometallate, is characterized in that, structural formula is as follows:
In formula ,-R is the one in following structure:
2. the preparation method of chirality poly oxometallate, is characterized in that, comprises the following steps:
(1) by amination Anderson type POM and Succinic anhydried by amount of substance 1:(2-10) be dissolved in anhydrous acetonitrile, stirring at room temperature reaction more than 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be carboxylated Anderson type POM;
(2) carboxylated Anderson type POM, N-hydroxy-succinamide and dicyclohexylcarbodiimide are dissolved in anhydrous N by amount of substance 1:1:1, in dinethylformamide, stirring at room temperature reaction more than 24h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be activated carboxylic Anderson type POM;
(3) by activated carboxylic Anderson type POM and chiral amino acid by amount of substance than 1:(1-20) be dissolved in anhydrous N, in dinethylformamide, stirring at room temperature reaction more than 48h, removing precipitation, filtrate is placed in ether atmosphere and carries out crystallization, obtain crystal, be chirality poly oxometallate;
Described chiral amino acid is L-His, D-His, L-Asp, D-Asp, L-Leu, D-Leu, L-Phe or D-Phe.
3. the preparation method of chirality poly oxometallate according to claim 2, is characterized in that, in step (1), stirring velocity is more than 200r/min, and churning time is 24-48h.
4. the preparation method of chirality poly oxometallate according to claim 2, is characterized in that, in step (2), stirring velocity is more than 200r/min, and churning time is 24-48h.
5. the preparation method of chirality poly oxometallate according to claim 2, is characterized in that, in step (3), stirring velocity is more than 200r/min, and churning time is 48-72h.
6. chirality poly oxometallate according to claim 1 can as amyloid-beta inhibitor.
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| CN111072722A (en) * | 2019-12-27 | 2020-04-28 | 湖北工业大学 | Anderson polyacid and application thereof as CVB3 virus inhibitor |
| CN111072723A (en) * | 2019-12-27 | 2020-04-28 | 湖北工业大学 | Organic derivative of Anderson polyacid modified by monoiodo benzoic acid through covalent bond and application of organic derivative in resisting ADV7 virus |
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| CN113600240A (en) * | 2021-07-16 | 2021-11-05 | 清华大学 | Amino acid or derivative modified polyoxometallate and preparation method and application thereof |
| CN115300527A (en) * | 2022-03-30 | 2022-11-08 | 河北医科大学 | Chiral heteropolyacid enantiomer selectively inhibits beta-amyloid peptide aggregation and application in preparation of medicine for treating Alzheimer's disease |
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| CN111072722A (en) * | 2019-12-27 | 2020-04-28 | 湖北工业大学 | Anderson polyacid and application thereof as CVB3 virus inhibitor |
| CN111072723A (en) * | 2019-12-27 | 2020-04-28 | 湖北工业大学 | Organic derivative of Anderson polyacid modified by monoiodo benzoic acid through covalent bond and application of organic derivative in resisting ADV7 virus |
| CN111138499A (en) * | 2019-12-27 | 2020-05-12 | 湖北工业大学 | Anderson polyacid and application thereof in resisting ADV7 virus |
| CN111138499B (en) * | 2019-12-27 | 2022-10-04 | 湖北工业大学 | Anderson polyacid and application thereof in resisting ADV7 virus |
| CN111072723B (en) * | 2019-12-27 | 2022-10-11 | 湖北工业大学 | Anderson polyacid organic derivative modified by monoiodo benzoic acid through covalent bond and application of Anderson polyacid organic derivative in resisting ADV7 virus |
| CN113600240A (en) * | 2021-07-16 | 2021-11-05 | 清华大学 | Amino acid or derivative modified polyoxometallate and preparation method and application thereof |
| CN115300527A (en) * | 2022-03-30 | 2022-11-08 | 河北医科大学 | Chiral heteropolyacid enantiomer selectively inhibits beta-amyloid peptide aggregation and application in preparation of medicine for treating Alzheimer's disease |
| CN115300527B (en) * | 2022-03-30 | 2023-09-26 | 河北医科大学 | Application of chiral heteropolyacid enantiomer-selective inhibition of beta-amyloid peptide aggregation in preparation of drug for treating Alzheimer's disease |
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