CN104829786A - Natural galactose and lysine side-hung cationic macromolecule, preparation method and application thereof - Google Patents
Natural galactose and lysine side-hung cationic macromolecule, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a natural galactose and lysine side-hung cationic macromolecule, its preparation method and an application of the cationic macromolecule used as a biological functional material in gene delivery. Through reversible addition-fragmentation chain transfer (RAFT), a cationic polymer is prepared. Conditions are mild; preparation is easy to operate; costs are low; and a polymer having a regular structure and narrow molecular weight distribution can be prepared. After deprotection, a galactose and lysine side-hung water-soluble methacrylate cationic polymer can be prepared. Side groups of the cationic polymer of the series are natural galactose and lysine blocks, and the cationic polymer has good biocompatibility. In addition, in comparison with a commercial carrier (bPEI-25k), the cationic polymer of the series has higher gene transfection efficiency in the presence of serum (10% FBS) when used as a gene delivery carrier. Thus, the polymer has a wide application prospect in fields of in vitro gene delivery and gene therapy and is a potential clinic gene transfection reagent.
Description
Technical field
The present invention relates to Biofunctional materials field.Be specifically related to the cation macromolecular of serial side extension semi-lactosi and Methionin, preparation method and the purposes as functional gene solid support material thereof.
Background technology
At present, the exploitation with the non-virus carrier of the advantages such as low cytotoxicity, reduced immunogenicity and high gene transportation performance causes the extensive concern of Biofunctional materials association area.Non-virus carrier conventional at present has polymine (PEI); polyamidoamine dendrimer (PAMAM); polymethyl acrylic acid N; N-dimethylaminoethyl (PDMAEMA) etc.; their feature is containing a large amount of cationic segment; energy and DNA or RNA with anionic backbone effectively combine, thus protection institute transporter gene is degraded to avoid the enzyme (as DNase etc.) in its body, improves gene delivery usefulness further.But above-mentioned polymkeric substance is all obtained by non-natural materials synthesis, and it mostly has poor biocompatibility, the shortcomings such as toxicity is high.Therefore utilize biocompatibility natural product Development of Novel Biofunctional materials, it is for significant in Materials science and clinical medicine.Some polymkeric substance be made up of natural amino acid, as polylysine, polyphenylalanine, polyhistidyl etc., with other non-natural polymer phase ratios, its efficiency gene transfection is in vitro high, there is good biocompatibility and hypotoxicity, in previous work of the present invention, develop a series of cationic polymers with side extension natural lysine, found that it can be applied as carrier that is efficient, low toxicity in vitro in gene transfection biomaterial.
But, up to now, it is found that the efficiency gene transfection of many solid support materials in vitro with high gene transfection performance in vivo in environment sharply declines, make it cannot meet the actual demand of clinical gene therapy.One of them major reason be cation carrier easily and in serum with the albumen generation non-specific interaction of negative charge, cause dissociating of carrier and cannot realize high efficiency gene transfection, these factors limit its practical application in clinical gene therapy.It is found that polyoxyethylene glycol (PEG) component introducing biocompatibility in cationic polymer carrier can increase the stability of cation carrier/gene composite in Biomedia in recent years, reduce the positive charge of nano grain surface, reduce and the absorption of protein ingredient in blood and aggregation, but, the coated meeting of PEG causes stronger electron screening and volume excluding effect, thus reduce endocytosis ability and the transfection efficiency of carrier/pDNA mixture, and there are some researches show that PEG may cause immune response, after injection easily remove by human recycle system.In order to address this problem, other the hydrophilic building block (or fragment) of biocompatibility of necessary designs is used as the supplementary material (or equivalent material) of PEG, at present, it is found that some amphoteric ion polymers and sugar-containing polymer have the performance similar with PEG, and the side effect that above-mentioned PEG causes can not be produced, very worth further investigation exploitation.Wherein, the saccharide compound adopted in sugar-containing polymer derives from natural animal-plant, and biocompatibility is high, has good wetting ability and excellent biological degradation and metabolism performance.Direct employing natural cationic polysaccharide (as chitosan, dextran and cyclodextrin etc.) builds the study hotspot that biocompatibility genophore is genophore field in recent years, but, natural polysaccharide have more complicated molecular structure, molecule regularity lower, be also difficult to realize fixed point, quantitative functional modification and modification, make the quality product between different batches be difficult to standardization control.And, because the structure of natural polysaccharide genophore can not obtain accuracy controlling, be thus difficult to the relation deeply illustrating its molecular structure and material property.Above-mentioned performance makes the research and development of the degree of depth of natural polysaccharide gene vector material and clinical application receive certain restriction.
In sum, the sugar-containing polymer that development preparation one class formation is regular, molecular weight is controlled, be very beneficial for the further optimization of glycosyl cationic polymer gene vector material and deep research and development, and necessary development certain operations is simple, the synthesis preparation method of mild condition prepares above-mentioned glycosyl cationic polymers.For these reasons, intend in the present invention with natural lysine and semi-lactosi as molecular building block constructing function monomer, the cationic polymers containing semi-lactosi and Methionin side base that one class formation is regular by reversible addion-fragmentation chain transfer radical polymerization (RAFT) design and synthesis, and above-mentioned polymkeric substance is further developed become that a class is new and effective, the gene delivery carrier material of low toxicity, particularly expect to strengthen the stability of such carrier in serum by the introducing of semi-lactosi hydrophilic radical, make it tentatively meet the requirement of gene transfection in vivo.This is also the target that the present invention endeavours.
Summary of the invention
An object of the present invention is to provide a kind of side to hang the water-soluble cationic function macromole of semi-lactosi and Methionin.
Two of object of the present invention is to provide a kind of side to hang the macromolecular preparation method of water-soluble cationic function of semi-lactosi and Methionin.
Three of object of the present invention is to provide a kind of side to hang the application of water-soluble cationic function macromole as gene function carrier of semi-lactosi and Methionin.
A kind of side provided by the present invention hangs the macromolecular chemical structure of cationic functional of semi-lactosi and Methionin as shown in general formula (I):
Wherein, A-represents negatively charged ion, comprises chlorion, bromide anion, iodide ion, trifluoromethanesulfonic acid radical ion or trifluoroacetic acid radical ion (TFA
-).M is the mean polymerisation degree containing Methionin building block monomer, and span is the preferential scope of 10 ~ 500, m is about 40.N is the mean polymerisation degree containing semi-lactosi building block monomer, and span is the preferential scope of 2 ~ 50, n is 3 ~ 20.
The preparation method that a kind of side provided by the present invention hangs the cation macromolecular of semi-lactosi and Methionin comprises the following steps:
The first step: by initiator Diisopropyl azodicarboxylate AIBN, Boc-Methionin methacrylate monomer
the methacrylic ester of fork ketone protection semi-lactosi
rAFT agent dithiobenzoic acid-(4-cyanopentanoic acid) ester is dissolved in organic solvent, after reacting 10-40h at a certain temperature, distillation removing organic solvent, be further purified the unreacted monomer of removing, be there is the methacrylic acid ester large molecule of Boc-Methionin side base and fork ketone protection galactose side simultaneously; The molecular weight and molecualr weight distribution of homopolymer is tested by nucleus magnetic resonance and gel permeation chromatography (GPC); The molecular weight (preferable range is between 8,000-30 ten thousand) between 2,000-40 ten thousand of Boc-Methionin methacrylate polymers, shown in the following formula II of its general formula:
Described Diisopropyl azodicarboxylate, Methionin methacrylate monomer, the fork ketone protection methacrylic ester of semi-lactosi and the mol ratio of dithiobenzoic acid-(4-cyanopentanoic acid) they are 1:10 ~ 5000:2 ~ 500:1 ~ 10; Described Boc is tertiary fourth oxygen formyl radical;
Second step: macromole gained being prepared by the first step is dissolved in organic solvent; add organic or inorganic acid and react 1-24h at a certain temperature to remove blocking group; obtain the cation macromolecular that a class side hangs semi-lactosi and Methionin, wherein, its structure is as shown in (I).The mol ratio of the polymkeric substance that the first step prepares and organic acid or mineral acid is 1:1.5 ~ 2.5.
Hang the preparation method of the cation macromolecular of semi-lactosi and Methionin according to a kind of side provided in the present invention, the organic or inorganic acid used in described synthesis step two comprises hydrochloric acid, Hydrogen bromide, hydroiodic acid HI, trifluoromethanesulfonic acid or trifluoroacetic acid.
The preparation method of the cation macromolecular of semi-lactosi and Methionin is hung according to a kind of side provided in the present invention, temperature of reaction in described synthesis step one, preferably but be not limited only to 10-95 DEG C, particularly preferably 40-80 DEG C, temperature of reaction in described synthesis step two, preferably but be not limited only to 10-50 DEG C, particularly preferably room temperature.
The preparation method of the cation macromolecular of semi-lactosi and Methionin is hung according to a kind of side provided in the present invention, in its step one, two, three, described organic solvent comprises methylene dichloride, ethyl acetate, 1,4-dioxane, tetrahydrofuran (THF), trichloromethane, dimethyl sulfoxide (DMSO), DMF, methyl alcohol, ethanol, acetonitrile, acetone, benzene or toluene.
The advantage of invention
Provide a kind of side in the present invention and hang cation macromolecular of semi-lactosi and Methionin and preparation method thereof, it adopts RAFT polymerization process, simple to operation, polymer architecture is regular, narrow molecular weight distribution, and do not need complicated last handling process to remove metal catalyst, therefore can not remain the metallic element that may cause toxic action to organism in product, be conducive to the application of product as biocompatible materials.This present invention is simple, easy handling, and material is easy to get, with low cost.With regard to the molecular structure of product, the main body of this amino acids acrylic ester polymer is the poly hydroxy ethyl acrylate being widely used in preparing Biofunctional materials, the side base be connected with main body is natural lysine and semi-lactosi building block, and therefore the cationic polymers of this series containing semi-lactosi and Methionin side base has good biocompatibility.Cell test result shows, this base polymer is easily degraded under cellular inclusion and lysosomal sour environment, discharge the gene of load, very high efficiency gene transfection has all been embodied in various kinds of cell strain, on the other hand, the introducing of hydrophilic semi-lactosi building block can form hydration layer at carrier surface, effectively reduces effect or the absorption of serum protein, this give the serum stable performance that such material is good, the high efficiency gene transfection under serum condition can be realized.This series polymer, compared with the business-like gene vector material bPEI-25K be widely used, has very low cytotoxicity, and in serum environment, have the efficiency gene transfection higher than bPEI-25k.These advantages can make it be applied in associated biomolecule medical field as excellent novel gene transport agent material.
Accompanying drawing explanation
Cation macromolecular/luciferase the DNA of Fig. 1, side extension semi-lactosi and Methionin, commercialization carrier bPEI-25K/luciferase DNA) in lung cancer cell line H1299, luciferase expression situation under 10%FBS exists, concrete testing method is see embodiment 6.
Fig. 2, in embodiment 3, side hangs the cation macromolecular/luciferase DNA of semi-lactosi and Methionin, commercialization carrier PEI-25K/luciferase DNA) in various kinds of cell strain as lung carcinoma cell (H1299), liver cancer cell (SK-HEP-1), breast cancer cell (MCF-7), cervical cancer cell (Hela), in Chinese hamster ovary cell (CHO), there iing blood serum medium (10%FBS) existence condition with commercialization " gold reference " transfection reagent PEI-25K sample in contrast, evaluate its luciferase expression on a cellular level, concrete testing method is see embodiment 7.
The cytotoxicity test result of cation macromolecular under series concentration of Fig. 3, side extension semi-lactosi and Methionin.Test cell is lung cancer cell line H1299, and concrete testing method is see embodiment 8.
Embodiment
Following embodiment will be described in detail the present invention, will contribute to the understanding of the present invention, but not be limitations on claims.
Embodiment 1
The first step: by Boc-Methionin methacrylate monomer
(4.58g, 10mmol), the methacrylic ester of fork ketone protection semi-lactosi
(0.28g; 0.8mmol); RAFT agent 4-cyano methyl valeric acid dithiobenzoic acid ester (5.2mg; 0.2mmol); Diisopropyl azodicarboxylate (2.5mg; 0.0155mmol) and the dioxane of 2ml drying join in dry Schlenk pipe, through three times freezing-vacuumize-melt logical nitrogen circulation after, react under 90 DEG C of nitrogen protections.After reaction terminates, Schlenk pipe is immersed in cancellation reaction in liquid nitrogen, reaction mixture concentrates, in normal hexane, precipitate twice, and the vacuum-drying side obtained containing protecting group hangs the polymkeric substance 3.2g of semi-lactosi and Methionin.
1H NMR(CDCl
3,δin ppm):5.68,5.17(br,NHCOO),5.51(s,Galactopyranose(Gal)-H at 1position),4.63(m,Gal-H at 3position),4.62-3.87(brm,OCH
2CH
2O,COCH(R)NH,Gal-H at 2,4,5position),3.10(m,NHCH
2COOR),2.06-1.49(brm,CH
2,CH
2-CCH
3),1.44(s,C(CH
3)
3),1.24-0.76(brm,C-CCH
3).
FTIR (in cm
-1): 3378,2976,2932,1715,1521,1456,1392,1366,1250,1169,1052,864,781.GPC (THF solvent, the reference of PS standard model): M
n=19.9KD, PDI=1.21.
Second step: be dissolved in a small amount of tetrahydrofuran (THF) by the multipolymer obtained by the first step, adds 1mL trifluoroacetic acid, after stirring at room temperature 24h, precipitates in cold diethyl ether, and filter, vacuum-drying obtains final product.Its structure is as follows:
1H NMR(DMSO-d
6,δin ppm):8.72,8.08(s,NH
3 +CF
3COO
-),4.62-3.83(brm,OCH
2CH
2O),3.17(s,Galactose hydroxyl groups),2.78(s,-NHCH
2COOR),2.07-1.21(brm,CH
2,CH
2-CCH
3),1.04-0.52(brm,C-CCH
3).
FTIR(in cm
-1):3378,2976,2932,1715,1521,1456,1392,1366,1250,1169,1052,864,781。
Embodiment 2
The first step: by Boc-Methionin methacrylate monomer
(4.58g, 10 mmol), the methacrylic ester of fork ketone protection semi-lactosi
(0.78g; 2.37mmol); RAFT agent 4-cyano methyl valeric acid dithiobenzoic acid ester (5.2mg; 0.02mmol); Diisopropyl azodicarboxylate (2.5mg; 0.0155mmol) and the dioxane of 2ml drying join in dry Schlenk pipe, through three times freezing-vacuumize-melt logical nitrogen circulation after, react under 90 DEG C of nitrogen protections.After reaction terminates, Schlenk pipe is immersed in cancellation reaction in liquid nitrogen, reaction mixture concentrates, in normal hexane, precipitate twice, and the vacuum-drying side obtained containing protecting group hangs the polymkeric substance 3.7g of semi-lactosi and Methionin.
1H NMR(CDCl
3,δin ppm):5.68,5.17(br,NHCOO),5.51(s,Galactopyranose(Gal)-H at 1position),4.63(m,Gal-H at 3position),4.62-3.87(brm,OCH
2CH
2O,COCH(R)NH,Gal-H at 2,4,5position),3.10(m,NHCH
2COOR),2.06-1.49(brm,CH
2,CH
2-CCH
3),1.44(s,C(CH
3)
3),1.24-0.76(brm,C-CCH
3).
FTIR (in cm
-1): 3378,2976,2932,1715,1521,1456,1392,1366,1250,1169,1052,864,781.GPC (THF solvent, the reference of PS standard model): M
n=21.4KD, PDI=1.26.
Second step: be dissolved in a small amount of tetrahydrofuran (THF) by the multipolymer obtained by the first step, adds 1mL trifluoromethanesulfonic acid, after stirring at room temperature 24h, precipitates in cold diethyl ether, and filter, vacuum-drying obtains final product.Its structure is as follows:
1H NMR(DMSO-d
6,δin ppm):8.72,8.08(s,NH
3 +CF
3COO
-),4.62-3.83(brm, OCH
2CH
2O),3.17(s,Galactose hydroxyl groups),2.78(s,-NHCH
2COOR),2.07-1.21(brm,CH
2,CH
2-CCH
3),1.04-0.52(brm,C-CCH
3).
FTIR(in cm
-1):3378,2976,2932,1715,1521,1456,1392,1366,1250,1169,1052,864,781。
Embodiment 3
The first step: by Boc-Methionin methacrylate monomer
(4.58g, 10mmol), the methacrylic ester of fork ketone protection semi-lactosi
(1.2g; 3.65mmol); RAFT agent 4-cyano methyl valeric acid dithiobenzoic acid ester (5.2mg; 0.02mmol); Diisopropyl azodicarboxylate (2.5mg; 0.0155mmol) and the dioxane of 2ml drying join in dry Schlenk pipe, through three times freezing-vacuumize-melt logical nitrogen circulation after, react under 90 DEG C of nitrogen protections.After reaction terminates, Schlenk pipe is immersed in cancellation reaction in liquid nitrogen, reaction mixture concentrates, in normal hexane, precipitate twice, and the vacuum-drying side obtained containing protecting group hangs the polymkeric substance 3.7g of semi-lactosi and Methionin.
1H NMR(CDCl
3,δin ppm):5.68,5.17(br,NHCOO),5.51(s,Galactopyranose(Gal)-H at 1position),4.63(m,Gal-H at 3position),4.62-3.87(brm,OCH
2CH
2O,COCH(R)NH,Gal-H at 2,4,5position),3.10(m,NHCH
2COOR),2.06-1.49(brm,CH
2,CH
2-CCH
3),1.44(s,C(CH
3)
3),1.24-0.76(brm,C-CCH
3).
FTIR (in cm
-1): 3378,2976,2932,1715,1521,1456,1392,1366,1250,1169,1052,864,781.GPC (THF solvent, the reference of PS standard model): M
n=23.4KD, PDI=1.32.
Second step: be dissolved in a small amount of tetrahydrofuran (THF) by the multipolymer obtained by the first step, adds 1mL trifluoromethanesulfonic acid, after stirring at room temperature 24h, precipitates in cold diethyl ether, and filter, vacuum-drying obtains final product.Its structure is as follows:
1H NMR(DMSO-d
6,δin ppm):8.72,8.08(s,NH
3 +CF
3COO
-),4.62-3.83(brm,OCH
2CH
2O),3.17(s,Galactose hydroxyl groups),2.78(s,-NHCH
2COOR),2.07-1.21(brm,CH
2,CH
2-CCH
3),1.04-0.52(brm,C-CCH
3).
FTIR(in cm
-1):3378,2976,2932,1715,1521,1456,1392,1366,1250,1169,1052,864,781。
Embodiment 4
The first step: by Boc-Methionin methacrylate monomer
(4.58g, 10mmol), the methacrylic ester of fork ketone protection semi-lactosi
(1.5g; 4.57mmol); RAFT agent 4-cyano methyl valeric acid dithiobenzoic acid ester (5.2mg; 0.02mmol); Diisopropyl azodicarboxylate (2.5mg; 0.0155mmol) and the dioxane of 2ml drying join in dry Schlenk pipe, through three times freezing-vacuumize-melt logical nitrogen circulation after, react under 90 DEG C of nitrogen protections.After reaction terminates, Schlenk pipe is immersed in cancellation reaction in liquid nitrogen, reaction mixture concentrates, in normal hexane, precipitate twice, and the vacuum-drying side obtained containing protecting group hangs the cationic polymers 3.5g of semi-lactosi and Methionin.
1H NMR(CDCl
3,δin ppm):5.68,5.17(br,NHCOO),5.51(s,Galactopyranose(Gal)-H at 1position),4.63(m,Gal-H at 3position),4.62-3.87(brm,OCH
2CH
2O,COCH(R)NH,Gal-H at 2,4,5position),3.10(m,NHCH
2COOR),2.06-1.49(brm,CH
2,CH
2-CCH
3),1.44(s,C(CH
3)
3),1.24-0.76(brm,C-CCH
3).
FTIR (in cm
-1): 3378,2976,2932,1715,1521,1456,1392,1366,1250,1169,1052,864,781.GPC (THF solvent, the reference of PS standard model): M
n=26.1KD, PDI=1.39.
Second step: be dissolved in a small amount of tetrahydrofuran (THF) by the multipolymer obtained by the first step, adds 1mL trifluoromethanesulfonic acid, after stirring at room temperature 24h, precipitates in cold diethyl ether, and filter, vacuum-drying obtains final product.Its structure is as follows:
1H NMR(DMSO-d
6,δin ppm):8.72,8.08(s,NH
3 +CF
3COO
-),4.62-3.83(brm,OCH
2CH
2O),3.17(s,Galactose hydroxyl groups),2.78(s,-NHCH
2COOR),2.07-1.21(brm,CH
2,CH
2-CCH
3),1.04-0.52(brm,C-CCH
3).
FTIR(in cm
-1):3378,2976,2932,1715,1521,1456,1392,1366,1250,1169,1052,864,781。
Embodiment 5
The side choosing preparation in embodiment 1 ~ 4 hangs the cationic polymers (GL-1, GL-2, GL-3, GL-4) of semi-lactosi and Methionin, respectively from luciferase reporter gene Luciferase pDNA with different N/P than compound, be then transfected in lung cancer cell line H1299.Specific as follows: by lung cancer cell line H1299 with 5 × 10
4the density in/hole is seeded in 24 well culture plates, cultivate after certain density, change the fresh substratum containing 10%FBS into, add the cationic polymers/Luciferase pDNA mixture of different N/P ratio, the fresh substratum containing 10%FBS is replaced by after 4 hours, continuation is cultivated 24 hours at 37 DEG C, uses Chemiluminescence Apparatus to measure transfection efficiency, and the cationic homopolymer PHML of employing side extension Methionin and commercialization carrier bPEI-25K in contrast.Its transfection results as shown in Figure 1.
Embodiment 6
The side choosing preparation in embodiment 3 hangs the cationic polymers GL-3 of semi-lactosi and Methionin, from luciferase reporter gene Luciferase pDNA with different N/P than compound, be then transfected into respectively in the clones such as lung carcinoma cell (H1299), liver cancer cell (SK-HEP-1), breast cancer cell (MCF-7), cervical cancer cell (Hela), Chinese hamster ovary cell (CHO).Specific as follows: each cell strain is seeded in 24 well culture plates with the density in 5 × 104/ holes, certain density is being cultivated containing under 10%FBS blood serum medium existent condition, then cationic polymers/Luciferase pDNA the mixture of different N/P ratio is directly added, continue to cultivate 24 hours at 37 DEG C, use Chemiluminescence Apparatus to measure transfection efficiency, and adopt commercialization carrier PEI25K in contrast.Can be found by result in accompanying drawing 2, in various kinds of cell system, the side extension semi-lactosi of preparation in embodiment 3 and the transfection efficiency of cationic polymers GL-3 under optimization charge ratio condition of Methionin have all exceeded " golden standard " bPEI-25K, illustrate that it can be applied as effective serum stable type gene delivery carrier.
Embodiment 7
CCK-8 test kit is adopted to assess, by lung carcinoma cell H1299 with every hole 5 × 10 toxicity containing the cationic polymers (GL-1, GL-2, GL-3, GL-4) of semi-lactosi and Methionin side base of preparation in embodiment 1 ~ 4
3the density of individual cell is inoculated in 96 well culture plates, cultivates after certain density, add the cationic polymers of different concns in 37 DEG C of incubators, continues cultivation 24 hours, add 10 μ L CCK-8 solution at 37 DEG C, places 3 hours for 37 DEG C.Then test by microplate reader.Adopt side to hang the cationic homopolymer PHML of Methionin, and commercialization carrier PEI25K in contrast.Result in accompanying drawing 3 shows, the cytotoxicity of the side extension semi-lactosi of preparation in embodiment 1 ~ 4 and the cationic polymers (GL-1, GL-2, GL-3, GL-4) of Methionin is all very low, its cell survival rate is mostly more than 60%, and the cell survival rate of PHML and b-PEI25K in contrast only has 20%-30%.
Claims (9)
1. a class contains the cationic polymers of semi-lactosi and Methionin side base, and it has following general structure:
Wherein, A-represents negatively charged ion: chlorion, bromide anion, iodide ion, trifluoromethanesulfonic acid radical ion or trifluoroacetic acid radical ion; N is the mean polymerisation degree containing semi-lactosi building block monomer, is 2-50; M is the mean polymerisation degree containing Methionin building block monomer, is 10 ~ 500.
2. a class as claimed in claim 1 contains the cationic polymers of semi-lactosi and Methionin side base, it is characterized in that described n is 2 ~ 50; Described m is 10 ~ 500.
3. a class as claimed in claim 1 contains the preparation method of the cationic polymers of semi-lactosi and Methionin side base, it is characterized in that adopting following steps to obtain:
The first step: by Boc-Methionin methacrylate monomer
the methacrylic ester of fork ketone protection semi-lactosi
initiator Diisopropyl azodicarboxylate, chain transfer agents dithiobenzoic acid-(4-cyanopentanoic acid) ester is dissolved in organic solvent, react 10-40h at 10-95 DEG C after, distillation removing organic solvent, purifying removes monomer, is had the methacrylate polymers of Boc-lysine side-chain and fork ketone protection galactose side simultaneously; The molecular weight and molecualr weight distribution of homopolymer is tested by nucleus magnetic resonance and gel permeation chromatography; The molecular weight of Boc-Methionin methacrylate polymers between 2,000-40 ten thousand, shown in the following formula II of its general formula:
Wherein, Boc represents tertiary fourth oxygen formyl radical; Described Diisopropyl azodicarboxylate, Methionin methacrylate monomer, the fork ketone protection methacrylic ester of semi-lactosi and the mol ratio of dithiobenzoic acid-(4-cyanopentanoic acid) they are 1:10 ~ 5000:2 ~ 500:1 ~ 10;
Second step: the polymkeric substance that the first step prepares adds organic acid or mineral acid reacts 1-5h to remove blocking group at 10-50 DEG C, obtain the cationic polymers containing semi-lactosi and Methionin side base, its structure is as shown in claim 1; Wherein, the mol ratio of the polymkeric substance for preparing of the first step and organic acid or mineral acid is 1:1.5 ~ 2.5.
4. preparation method as claimed in claim 2, is characterized in that the molecular weight of described Boc-Methionin methacrylate polymers is between 8,000-30 ten thousand.
5. preparation method as claimed in claim 2, it is characterized in that described organic solvent is methylene dichloride, ethyl acetate, 1,4-dioxane, tetrahydrofuran (THF), trichloromethane, dimethyl sulfoxide (DMSO), DMF, methyl alcohol, ethanol, acetonitrile, acetone, benzene or toluene.
6. preparation method as claimed in claim 2, is characterized in that the organic or inorganic acid used in described step 2 is hydrochloric acid, Hydrogen bromide, hydroiodic acid HI, trifluoromethanesulfonic acid or trifluoroacetic acid.
7. preparation method as claimed in claim 2, it is characterized in that the temperature of reaction in described synthesis step one is 40-80 DEG C, the temperature of reaction in described synthesis step two is room temperature.
8. a class as claimed in claim 1 cationic polymers that contains semi-lactosi and Methionin side base is for as efficient, low toxicity, the environment of resistance to serum Biofunctional materials.
9. a class as claimed in claim 7 contains the application of the cationic polymers of semi-lactosi and Methionin side base, it is characterized in that described application refers to the application in gene and medicament transport carrier field.
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| CN109796549A (en) * | 2016-11-14 | 2019-05-24 | 天津大学 | The preparation method of lysine methacrylate homopolymer |
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| CN109796549A (en) * | 2016-11-14 | 2019-05-24 | 天津大学 | The preparation method of lysine methacrylate homopolymer |
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