CN103865013A - Preparation method of glucose and temperature-responsive insulin controlled release carrier - Google Patents

Preparation method of glucose and temperature-responsive insulin controlled release carrier Download PDF

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CN103865013A
CN103865013A CN201410084021.6A CN201410084021A CN103865013A CN 103865013 A CN103865013 A CN 103865013A CN 201410084021 A CN201410084021 A CN 201410084021A CN 103865013 A CN103865013 A CN 103865013A
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袁伟忠
沈进
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Tongji University
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Tongji University
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Abstract

The invention relates to a preparation method of a glucose and temperature-responsive insulin controlled release carrier. 1,2-diaminoethane is used as an initial raw material, is protected through di-tert-butyl dicarbonate and then is reacted with acryloyl chloride to prepare 2-aminoethyl acrylamide, and then the 2-aminoethyl acrylamide is reacted with the reaction product 4-chloroformylphenylboronic acid pinacol ester of the 4-carboxybenzeneboronic acid to prepare the monomer N-acrylamide ethyl-4-(tetramethyl-dioxaborolan)-benzamide with glucose responsiveness. The Raft polymerization of the monomer is initiated through trithiocarbonate DDMAT, and then the obtained polymer is used as a macromolecular chain transfer agent to initiate the copolymerization of the MEO2MA and the OEGMA to obtain a block copolymer material PAEB-b-P. The block copolymer has glucose and temperature responsiveness, can control the release of the carried insulin according to the concentration of the glucose in the external environment, has biodegradability, biocompatibility and bioactivity and is widely applied in the fields of medicine controlled release carriers and biological intelligent switches. The preparation method is simple and easy, the raw material can be industrially produced, and the method has great popularization and application value.

Description

The preparation method of a kind of glucose and temperature-responsive Carriers for Protein Drugs
Technical field
The invention belongs to macromolecular material and biomedical engineering field, be specifically related to the preparation method of a kind of glucose and temperature-responsive Carriers for Protein Drugs.
Background technology
In recent years, intelligent macromolecule material relies on its potential application foreground in fields such as medicine control release vehicle, biosensor, biological intelligence switches and has been subject to paying close attention to widely.Intelligent macromolecule be a class to external world the variation of environment make response, and produce the macromolecular material that corresponding physical structure and altered chemical structure are even suddenlyd change.The more intelligent macromolecule of current research mainly contains temperature sensitivity, pH susceptibility and sensitivity of light macromolecular material.Along with to intelligent macromolecule material further investigate, people have found again the intelligent macromolecule of a lot of class susceptibility, such as CO2 susceptibility, brine sensitivity and glucose-sensitive polymer.
Diabetes are one group of metabolic diseases (being divided into I type and II type) take hyperglycemia as feature.Its Etiological is the impaired and hypoinsulinism that causes of beta Cell of islet.So far, for the most effective methods for the treatment of of diabetes be every day multiple subcutaneous injections Regular Insulin help patient and maintain blood sugar at normal level.However; the method of this widespread use still can not be controlled blood sugar concentration well, usually can cause this acute complications of hypoglycemia, when serious, even faint, suffers a shock; and injection also brings very big inconvenience to patient for a long time, also may cause the side effect such as allergy, oedema.According to the statistical information of the World Health Organization (WHO), there is the puzzlement that exceedes 1.5 hundred million people and be subject to diabetes in the whole world in 2004, and to the year two thousand thirty, this numeral will arrive 3.66 hundred million.Therefore, seek more comfortable, more effective methods for the treatment of and just seem necessary.According to the feedback regulation of organism inside, develop a kind of new insulin delivery route, Regular Insulin is wrapped in to certain carrier inside, then be placed in body, this medicine carrying controlled release durg delivery system has susceptibility to the glucose in blood, can respond the burst size of different blood sugar concentrations adjusting Regular Insulin, realizes the function of the intelligent excreting insulin of simulation pancreas islet, this,, by controlling to a great extent diabetics's blood sugar concentration, palliates the agonizing sufferings.
Phenylo boric acid and derivative thereof have electrically charged and two kinds of forms of neutral in the aqueous solution, and these two kinds of forms can realize reversible transformation by following method.Near the pKa place of phenylo boric acid, phenylo boric acid can with form ester with polyhydric compound as glucose, thereby realization is transformed into hydrophilic borate ester from hydrophobic phenylo boric acid.Therefore can utilize phenylo boric acid by its to the recognition function of glucose for restraining oneself formula drug delivery system or regulate some vital movement.Design has the polymer drug of sugared responsiveness and carries material more can make full use of internal milieu variation, discharges medicine easily and safely in the high site of disease selectivity of blood sugar concentration, reaches the object of efficient treatment diabetes.
Utilize MEO 2biocompatibility, biological degradability that MA and OEGMA itself is good, in conjunction with this advanced person's of reversible addition-fracture chain transfer polymerization living polymerisation process, utilization has the prepared macromolecular chain transfer agent of monomer of glucose responding, introduces the hydrophilic segment MEO with temperature-responsive in monomer backbone 2mA and OEGMA, can prepare a kind of amphipathic nature block polymer, this segmented copolymer can self-assembly form micella in the aqueous solution, this micella under physiological condition to external world the glucose in environment there is responsiveness, in the time that glucose concn raises, the sugared responsiveness segment of stratum nucleare can be combined and cause micella to break with glucose, thereby makes inner drug release of wrapping up out, also can accelerate the release of micella internal drug when temperature raises.This will be with a wide range of applications in the treatment in the fields such as biomedicine, nano-medicament carrier especially diabetes.
Summary of the invention
The object of the present invention is to provide the preparation method of a kind of glucose and temperature-responsive Carriers for Protein Drugs.
The object of the invention is that the monomer M EO of temperature-responsive will be there is 2mA and OEGMA are incorporated in the polymkeric substance with glucose responding, make obtained segmented copolymer there is good biocompatibility, biological degradability, biological activity, glucose responding and temperature-responsive simultaneously, and by the stable nanometer micelle that obtains having glucose and temperature-responsive soluble in water this segmented copolymer.Commercial monomer M EO for the present invention 2mA and OEGMA, and there is the monomer of glucose responding by the preparation of the raw material such as commercial quadrol, 4-carboxyl phenylo boric acid, tetramethyl ethylene ketone, adopt reversible addition-fracture chain transfer polymerization method, prepare glucose responding and temperature-responsive block polyarylene block copolymer that a series of monomer ratio are different.
A kind of glucose that the present invention proposes and the preparation method of temperature-responsive Carriers for Protein Drugs, concrete steps are as follows:
(1) quadrol is dissolved in a certain amount of solvent orange 2 A, under the condition stirring, slowly add the solution of the solvent orange 2 A of di-tert-butyl dicarbonic acid ester, time for adding is 5-7h, then at room temperature react 48-60h, obtain product II (N-(2-amino-ethyl) t-butyl formate) finally by filtration, underpressure distillation, precipitation, filtration, extraction and the operation such as dry;
(2) product II N-(2-amino-ethyl) t-butyl formate in step (1) is dissolved in solvent B, adds anhydrous Na after being cooled to 0 ℃ 2cO 3stir after for some time, add wherein the solution of the solvent B that dropwise adds acrylate chloride at 0 ℃, time for adding is 3-5h, then continue at room temperature to react 16-20h, after reaction finishes, obtain product III (N-tertbutyloxycarbonyl-N through operations such as extraction, dry, underpressure distillation 1-acryl diaminoethanes);
(3) the product III in step (2) is dissolved in to a certain amount of solvent B, under stirring, add excessive trifluoroacetic acid (TFA), then at room temperature react 2-4h, reacted rear underpressure distillation and obtained product IV (trifluoroacetate of N-(2-amino-ethyl) acrylamide);
(4) 4-carboxyl phenylo boric acid (CPBA) is slowly joined in the flask that fills solvent B, add again excessive tetramethyl ethylene ketone and molecular sieve, be placed under room temperature and react 48-60h, after filtration, underpressure distillation, washing and the dry product V (4-carboxyl phenylo boric acid is which ester frequently) that obtains;
(5) product V is joined in the flask that fills a certain amount of solvent B, flask connects a drying tube that fills siccative C, add wherein more excessive acyl chlorides D, under stirring, at room temperature react 12-20h, underpressure distillation removes desolventizing and excessive acyl chlorides obtains product VI (4-chloroformyl phenylo boric acid is which ester frequently);
(6) the product IV in step (3) is dissolved in the solvent E being dried, in the time of 0 ℃, add wherein a certain amount of triethylamine, after stirring 1h, dropwise add the solution of the solvent E of product VI in step (5), first under the condition of 0 ℃, stir 2h, then at room temperature react 22h, after reaction finishes, obtain sugared responsiveness monomer VII (N acrylamide base ethyl-4-(tetramethyl--dioxane pentaborane base)-benzamide) through underpressure distillation, acidifying, extraction, washing, dry, recrystallization;
(7) product sugar responsiveness monomer VII, chain-transfer agent F, initiator A IBN in step (6) are joined in the flask that fills solvent G, freezing vacuumizing three times, at N 2or react 24-48h under the protection of Ar, temperature of reaction is 70 ℃.Obtain RAFT macromolecular chain transfer agent PAEBB through precipitation, suction filtration with after being dried;
(8) obtained RAFT macromolecular chain transfer agent PAEBB is dissolved in solvent G, adds monomer M EO 2mA and OEGMA; the concrete add-on of monomer is determined by designed molecular weight and performance thereof; add initiator Diisopropyl azodicarboxylate; system is reacted under argon gas or nitrogen protection; temperature of reaction is 50～100 ℃; react 24～30 hours, and through precipitation, vacuum-drying, obtain target product I PAEBB-b-P (MEO 2mA-co-OEGMA).
In the present invention, described solvent orange 2 A is one or more in Isosorbide-5-Nitrae-dioxane, methylene dichloride, trichloromethane or tetrahydrofuran (THF).
In the present invention, described solvent B is one or more in methylene dichloride, trichloromethane, tetrahydrofuran (THF) or DMF.
In the present invention, described siccative C is anhydrous CaCl 2, anhydrous MgSO 4or one or more in soda-lime.
In the present invention, described acyl chlorides D is one or more in oxalyl chloride, Acetyl Chloride 98Min., chloroacetyl chloride or acrylate chloride.
In the present invention, described solvent E is one or more in tetrahydrofuran (THF), methylene dichloride, trichloromethane or DMF.
In the present invention, described chain-transfer agent F is trithiocarbonate or dithiocarbonates.
In the present invention, described solvent G is DMF, N, N N,N-DIMETHYLACETAMIDE or N, one or more in N diethylformamide.
The invention has the advantages that: raw material sources are extensive monomer M EO used 2mA and OEGMA, and the raw material such as quadrol, 4-carboxyl phenylo boric acid, tetramethyl ethylene ketone, all kinds of SOLVENTS, siccative, chain-transfer agent all can suitability for industrialized production, synthetic method is simple.Synthetic segmented copolymer has glucose responding, temperature-responsive, biocompatibility and biodegradability simultaneously.Multipolymer can be assembled into easily stable nanometer micelle in water.Polymkeric substance minimum critical dissolved each other temperature and glucose responding performance can be by changing monomer M EO in polymkeric substance 2the ratio of the sugared responsiveness monomer of MA, OEGMA and synthesized regulates.Gained stable nanometer micelle has glucose responding, temperature-responsive, biological degradability and biocompatibility simultaneously, aspect the treatment of medicine control release vehicle especially diabetes, the field such as biological intelligence switch, biosensor has a wide range of applications.
Accompanying drawing explanation
Glucose responding prepared by Fig. 1: embodiment 1, the structural representation of temperature-responsive segmented copolymer.
Embodiment
Following examples are to further illustrate of the present invention, rather than limit the scope of the invention.
The magnetic nuclear resonance analyzer for molecular structure (NMR) of this glucose responding, temperature-responsive segmented copolymer and Fourier transform infrared spectrometer (FTIR) are measured.Lower critical solution temperature (LCST) is measured with ultraviolet-visible spectrophotometer with hot platform, LCST be defined as transmittance be down to initial value 50% time corresponding temperature.Assembling gained is transmission electron microscope (TEM) and atomic force microscope (AFM) mensuration for nano-micelle.Nano-micelle particle diameter is measured with dynamic laser light scattering apparatus (DLS).
Embodiment 1
28g quadrol is dissolved in to 1 of 150mL, in 4-dioxane, in advance the di-tert-butyl dicarbonic acid ester of 13.2g is dissolved in to 1 of 150mL, in 4-dioxane, under the condition stirring, slowly join in ethylenediamine solution, time for adding 5h, then at room temperature reacts 48h, obtains product II (N-(2-amino-ethyl) t-butyl formate) through operations such as precipitating, filter, extract and be dry; Take in the methylene dichloride (DCM) that 8.5g product II is dissolved in 100mL, be cooled to the anhydrous Na that adds 5.6g after 0 ℃ 2cO 3stir after for some time, add wherein the DCM solution 80mL that dropwise adds acrylate chloride (6.0g) in the time of 0 ℃, time for adding is 3h, continue at room temperature to react 16h, after reaction finishes, obtain product III (N-tertbutyloxycarbonyl-N through operations such as extraction, dry, underpressure distillation 1-acryl diaminoethanes); Take the DCM that 2.0g product III is dissolved in 10mL, under agitation condition, add the trifluoroacetic acid (TFA) of 30mL, then at room temperature react 2h, reacted rear underpressure distillation and obtain the trifluoroacetate of product IV N-(2-amino-ethyl) acrylamide; The 4-carboxyl phenylo boric acid (CPBA) that takes 4.0g slowly joins in the DCM of 100mL, add again the tetramethyl ethylene ketone of 3.4g and the molecular sieve 4A type of 13.0g, be placed under room temperature and react 48h, after filtration, underpressure distillation, washing and dry product V 4-carboxyl phenylo boric acid any ester frequently that obtains; Take 2.2g product V and join in the DCM flask that fills 35mL, flask connects one and fills the drying tube of soda-lime, then adds wherein oxalyl chloride 5.6g, at room temperature reacts 12h under stirring, and underpressure distillation obtains product VI 4-chloroformyl phenylo boric acid which ester frequently; Take 1.824g product IV and be dissolved in the 15mL tetrahydrofuran (THF) (THF) being dried, 0 ℃ time, add wherein 15mL triethylamine, after stirring 1h, dropwise add the THF solution 15mL being dried of product VI (2.128), first under the condition of 0 ℃, stir 2h, then at room temperature react 22h, after reaction finishes, obtain sugared responsiveness monomer VII (N acrylamide base ethyl-4-(tetramethyl--dioxane pentaborane base)-benzamide) through underpressure distillation, acidifying, extraction, washing, dry, recrystallization;
Take the sugared responsiveness monomer VII of 1g, with after DMF dispersing and dissolving; add 17.8mg dodecyl trithiocarbonate, 4.01mg initiator Diisopropyl azodicarboxylate (AIBN); system is freezing to be vacuumized three times, reacts 24h, 70 ℃ of temperature of reaction under the protection of Ar.Obtain RAFT macromolecular chain transfer agent PAEBB through precipitation, suction filtration with after being dried; Obtained RAFT macromolecular chain transfer agent PAEBB is dissolved in DMF, adds monomer M EO 2mA0.494g and OEGMA0.292g, initiator A IBN4.0mg, system is reacted under argon shield, and temperature of reaction is 50 ℃, reaction times 24h, and through precipitation, vacuum-drying, obtain target product I.
Embodiment 2
25g quadrol is dissolved in the methylene dichloride (DCM) of 100mL, in advance the di-tert-butyl dicarbonic acid ester of 13.0g is dissolved in the DCM of 100mL, slowly join in ethylenediamine solution, time for adding 6h, then at room temperature react 54h, obtain product II (N-(2-amino-ethyl) t-butyl formate) through operations such as precipitating, filter, extract and be dry; Product II N-(2-amino-ethyl) t-butyl formate of 9.0g is dissolved in to the trichloromethane of 80mL, is cooled to the anhydrous Na that adds 5.3g after 0 ℃ 2cO 3stir after for some time, 0 ℃ time, dropwise add wherein the chloroform soln 60mL of acrylate chloride (6.0g), time for adding 4h, continue at room temperature to react 18h, after reaction finishes, obtain product III (N-tertbutyloxycarbonyl-N through operations such as extraction, dry, underpressure distillation 1-acryl diaminoethanes); The product III that takes 1.94g is dissolved in the DCM of 10mL, under stirring, add the trifluoroacetic acid (TFA) of 28mL, then at room temperature react 3h, reacted rear underpressure distillation and obtained product IV (trifluoroacetate of N-(2-amino-ethyl) acrylamide); The 4-carboxyl phenylo boric acid (CPBA) that takes 4.0g slowly joins in the DCM of 100mL, add again the molecular sieve 4A type of 3.5g tetramethyl ethylene ketone and 13.5g, be placed under room temperature and react 54h, after filtration, underpressure distillation, washing and the dry product V (4-carboxyl phenylo boric acid is which ester frequently) that obtains; The product V that takes 2.3g joins in the flask of the DMF that fills 35mL, and flask connects one and fills anhydrous CaCl 2drying tube, add wherein 4.98g oxalyl chloride, under stirring, at room temperature react 15h, underpressure distillation obtains product VI (4-chloroformyl phenylo boric acid frequently which ester); The product IV of 1.596g is dissolved in the 10mL tetrahydrofuran (THF) being dried, add 15mL triethylamine at 0 ℃, after stirring 1.5h, dropwise add the tetrahydrofuran solution 15mL being dried of product VI (1.862g), first under the condition of 0 ℃, stir 2h, then at room temperature react 22h, after reaction finishes, obtain sugared responsiveness monomer VII (N acrylamide base ethyl-4-(tetramethyl--dioxane pentaborane base)-benzamide) through underpressure distillation, acidifying, extraction, washing, dry, recrystallization;
Take the sugared responsiveness monomer VII of 1g, with after N,N-dimethylacetamide dispersing and dissolving; add the dodecyl trithiocarbonate of 17.8mg, the initiator Diisopropyl azodicarboxylate (AIBN) of 4.01mg; system is freezing to be vacuumized three times, under Ar protection, reacts 24h, 70 ℃ of temperature of reaction.Obtain RAFT macromolecular chain transfer agent PAEBB through precipitation, suction filtration with after being dried; Obtained RAFT macromolecular chain transfer agent PAEBB is dissolved in N,N-dimethylacetamide, adds monomer M EO 2mA0.505g and OEGMA0.2336g, initiator A IBN4.0mg, system is reacted under argon shield, and temperature of reaction is 75 ℃, reaction times 26h, and through precipitation, vacuum-drying, obtain target product I.
Embodiment 3
30g quadrol is dissolved in to the trichloromethane of 80mL, in advance the di-tert-butyl dicarbonic acid ester of 14g is dissolved in to the trichloromethane of 120mL, under the condition stirring, slowly join in ethylenediamine solution, time for adding 7h, then at room temperature react 60h, obtain product II (N-(2-amino-ethyl) t-butyl formate) through operations such as precipitating, filter, extract and be dry; Product II N-(2-amino-ethyl) t-butyl formate that takes 9.0g is dissolved in the trichloromethane of 80mL, adds 6.0g anhydrous Na after being cooled to 0 ℃ 2cO 3stir after for some time, in the time of 0 ℃, add wherein the chloroform soln 80mL that dropwise adds acrylate chloride (6.5g), time for adding 5h, continue at room temperature to react 20h, after reaction finishes, obtain product III (N-tertbutyloxycarbonyl-N through operations such as extraction, dry, underpressure distillation 1-acryl diaminoethanes); The product III of 2.15g is dissolved in to the methylene dichloride (DCM) of 10mL, under stirring, add the trifluoroacetic acid (TFA) of 40mL, then at room temperature react 4h, reacted rear underpressure distillation and obtained product IV (trifluoroacetate of N-(2-amino-ethyl) acrylamide); The 4-carboxyl phenylo boric acid (CPBA) that takes 5.0g slowly joins the N of 75mL, in N dimethyl formamide, add again the tetramethyl ethylene ketone of 4.1g and the molecular sieve 4A type of 13.5g, be placed under room temperature and react 60h, after filtration, underpressure distillation, washing and the dry product V (4-carboxyl phenylo boric acid is which ester frequently) that obtains; The product V of 2.47g is joined in the DCM flask that fills 40mL, flask connects one and fills anhydrous MgSO 4drying tube, then add Acetyl Chloride 98Min. 7.85g in flask, under stirring, at room temperature react 18h, underpressure distillation obtains product VI (4-chloroformyl phenylo boric acid is which ester frequently); The product IV of 2.166g is dissolved in to the 20mL tetrahydrofuran solution being dried, in the time of 0 ℃, add wherein the triethylamine of 20mL, after stirring 1h, dropwise add the tetrahydrofuran solution 15mL being dried of product VI (2.527), first under the condition of 0 ℃, stir 2h, then at room temperature react 22h, after reaction finishes, obtain sugared responsiveness monomer VII (N acrylamide base ethyl-4-(tetramethyl--dioxane pentaborane base)-benzamide) through underpressure distillation, acidifying, extraction, washing, dry, recrystallization;
Take the sugared responsiveness monomer VII of 1g, with after N,N-dimethylacetamide dispersing and dissolving; add dodecyl trithiocarbonate 17.8mg, initiator Diisopropyl azodicarboxylate (AIBN) 4.01mg; system is freezing to be vacuumized three times, reacts 24h, 70 ℃ of temperature of reaction under the protection of Ar.Obtain RAFT macromolecular chain transfer agent PAEBB through precipitation, suction filtration with after being dried; Obtained RAFT macromolecular chain transfer agent PAEBB is dissolved in N,N-dimethylacetamide, adds monomer M EO 2mA0.522g and OEGMA0.146g, initiator A IBN4.0mg, system is reacted under argon shield, and temperature of reaction is 80 ℃, reaction times 30h, and through precipitation, vacuum-drying, obtain target product I.
Embodiment 4
35g quadrol is dissolved in Isosorbide-5-Nitrae-dioxane of 120mL, in advance the di-tert-butyl dicarbonic acid ester of 16g is dissolved in to 120mL1,4-dioxane slowly adds it in ethylenediamine solution under the condition stirring, and time for adding 6h, then in room temperature)); Product II N-(2-amino-ethyl) t-butyl formate of 11.23g is dissolved in to the DMF of 90mL, is cooled to the anhydrous Na that adds 7.42g after 0 ℃ 2cO 3stir after for some time, in the time of 0 ℃, add wherein the N that dropwise adds acrylate chloride (7.27g), dinethylformamide solution 80mL, time for adding is 3h, continue at room temperature to react 20h, after reaction finishes, obtain product III (N-tertbutyloxycarbonyl-N through operations such as extraction, dry, underpressure distillation 1-acryl diaminoethanes); The product III of 4.3g is dissolved in to the trichloromethane of 10mL, under stirring, add 60mL trifluoroacetic acid (TFA), then at room temperature react 4h, reacted rear underpressure distillation and obtained product IV (trifluoroacetate of N-(2-amino-ethyl) acrylamide); The 4-carboxyl phenylo boric acid (CPBA) of 6.0g is slowly joined in the tetrahydrofuran (THF) of 100mL, add again the tetramethyl ethylene ketone of 5.1g and the molecular sieve 4A type of 20g, be placed under room temperature and react 55h, after filtration, underpressure distillation, washing and the dry product V (4-carboxyl phenylo boric acid is which ester frequently) that obtains; The product V of 4.4g is joined in the DCM flask that fills 35mL, flask connects a drying tube that fills soda-lime, add wherein Acetyl Chloride 98Min. 14.13g again, at room temperature react 20h under stirring, underpressure distillation obtains product VI (4-chloroformyl phenylo boric acid is which ester frequently); The product IV of 4.56g is dissolved in to the 25mLN being dried, dinethylformamide, in the time of 0 ℃, add wherein the triethylamine of 35mL, after stirring 1h, dropwise add the N being dried of product VI (5.32), dinethylformamide 25mL, first under the condition of 0 ℃, stir 2h, then at room temperature react 22h, after reaction finishes, obtain sugared responsiveness monomer VII (N acrylamide base ethyl-4-(tetramethyl--dioxane pentaborane base)-benzamide) through underpressure distillation, acidifying, extraction, washing, dry, recrystallization;
Take the sugared responsiveness monomer VII of 1g, with after DMF dispersing and dissolving; add dodecyl trithiocarbonate 17.8mg, initiator Diisopropyl azodicarboxylate (AIBN) 4.01mg; system is freezing to be vacuumized three times, under the protection of Ar, reacts 24h, and temperature of reaction is 70 ℃.Obtain RAFT macromolecular chain transfer agent PAEBB through precipitation, suction filtration with after being dried; Obtained RAFT macromolecular chain transfer agent PAEBB is dissolved in DMF, adds monomer M EO 2mA0.549g, initiator A IBN4.0mg, system is reacted under argon shield, and temperature of reaction is 80 ℃, the reaction times 28, and through precipitation, vacuum-drying, obtain target product I.
Embodiment 5
42g quadrol is dissolved in to the N of 125mL, in N dimethyl formamide, in advance the di-tert-butyl dicarbonic acid ester of 19.8g is dissolved in to the N of 100mL, in N dimethyl formamide, under the condition stirring, slowly add in ethylenediamine solution, time for adding 7h, then at room temperature reacts 60h, obtains product II (N-(2-amino-ethyl) t-butyl formate) through operations such as precipitating, filter, extract and be dry; Product II N-(2-amino-ethyl) t-butyl formate of 12.75g is dissolved in the trichloromethane of 80mL, is cooled to the anhydrous Na that adds 8.3g after 0 ℃ 2cO 3stir after for some time, at 0 ℃, add wherein the chloroform soln 80mL that dropwise adds acrylate chloride (9.0g), time for adding is 5h, continue at room temperature to react 20h, after reaction finishes, obtain product III (N-tertbutyloxycarbonyl-N through operations such as extraction, dry, underpressure distillation 1-acryl diaminoethanes); The product III that takes 3.0g is dissolved in the methylene dichloride of 10mL, under stirring, add the trifluoroacetic acid (TFA) of 45mL, then at room temperature react 3h, reacted rear underpressure distillation and obtained product IV (trifluoroacetate of N-(2-amino-ethyl) acrylamide); The 4-carboxyl phenylo boric acid (CPBA) of 6.0g is slowly joined in the tetrahydrofuran (THF) of 100mL, add again the tetramethyl ethylene ketone of 5.1g and the molecular sieve 4A type of 20g, be placed under room temperature and react 60h, after filtration, underpressure distillation, washing and the dry product V (4-carboxyl phenylo boric acid is which ester frequently) that obtains; The product V of 3.3g is joined in the DCM flask that fills 40mL, flask connects one and fills anhydrous CaCl 2drying tube, then add wherein oxalyl chloride 8.4g, under stirring, at room temperature react 18h, underpressure distillation obtains product VI (4-chloroformyl phenylo boric acid is which ester frequently); The product IV of 2.964g is dissolved in the 25mL methylene dichloride (DCM) being dried, in the time of 0 ℃, add wherein the triethylamine of 23mL, after stirring 1h, dropwise add the DCM25mL being dried of product VI (5.32), first under the condition of 0 ℃, stir 2h, then at room temperature react 22h, after reaction finishes, obtain sugared responsiveness monomer VII (N acrylamide base ethyl-4-(tetramethyl--dioxane pentaborane base)-benzamide) through underpressure distillation, acidifying, extraction, washing, dry, recrystallization;
Take the sugared responsiveness monomer VII of 2g, with N, after N-diethylformamide dispersing and dissolving; add dodecyl trithiocarbonate 17.8mg, initiator Diisopropyl azodicarboxylate (AIBN) 4.01mg; system is freezing to be vacuumized three times, under the protection of Ar, reacts 24h, and temperature of reaction is 70 ℃.Obtain RAFT macromolecular chain transfer agent PAEBB through precipitation, suction filtration with after being dried; Obtained RAFT macromolecular chain transfer agent PAEBB is dissolved in to N, in N-diethylformamide, adds monomer M EO 2mA 0.505g, OEGMA0.2336, initiator A IBN4.0mg, system is reacted under argon shield, and temperature of reaction is 90 ℃, reaction times 30h, and through precipitation, vacuum-drying, obtain target product I.

Claims (8)

1. a preparation method for glucose and temperature-responsive Carriers for Protein Drugs, is characterized in that described carrier is PAEBB-b-P (MEO 2mA-co-OEGMA), concrete steps are as follows:
(1) quadrol is dissolved in solvent orange 2 A, under the condition stirring, slowly add the solution of the solvent orange 2 A of di-tert-butyl dicarbonic acid ester, time for adding is 5-7h, then at room temperature react 48-60h, finally by filtration, underpressure distillation, precipitation, filtration, extraction and dry, obtain N-(2-amino-ethyl) t-butyl formate;
(2) N-in step (1) (2-amino-ethyl) t-butyl formate is dissolved in solvent B, adds anhydrous Na after being cooled to 0 ℃ 2cO 3, stir after for some time, at 0 ℃, dropwise add wherein the solution of the solvent B of acrylate chloride, time for adding is 3-5h, then continues at room temperature to react 16-20h, after reaction finishes, through extraction, dry, underpressure distillation, obtains product N-tertbutyloxycarbonyl-N 1-acryl diaminoethanes;
(3) by the N-tertbutyloxycarbonyl-N in step (2) 1-acryl diaminoethanes is dissolved in solvent B, adds excessive trifluoroacetic acid under stirring, then at room temperature reacts 2-4h, has reacted rear underpressure distillation; Obtain (trifluoroacetate of N-(2-amino-ethyl) acrylamide);
(4) 4-carboxyl phenylo boric acid is slowly joined in the flask that fills solvent B, then add excessive tetramethyl ethylene ketone and molecular sieve, be placed under room temperature and react 48-60h, after filtration, underpressure distillation, washing and dry 4-carboxyl phenylo boric acid any ester frequently that obtains;
(5) 4-carboxyl phenylo boric acid step (4) being obtained frequently which ester joins in the flask that fills solvent B, described flask connects a drying tube that fills siccative C, add wherein more excessive acyl chlorides D, under stirring, at room temperature react 12-20h, underpressure distillation, except desolventizing and excessive acyl chlorides, obtains 4-chloroformyl phenylo boric acid which ester frequently;
(6) trifluoroacetate of the N-in step (3) (2-amino-ethyl) acrylamide is dissolved in the solvent E being dried, in the time of 0 ℃, add wherein triethylamine, after stirring 1h, dropwise add the 4-chloroformyl phenylo boric acid solution of the solvent E of any ester frequently in step (5), first under the condition of 0 ℃, stir 2h, then at room temperature react 22h, after reaction finishes, obtain sugared responsiveness monomer N acrylamide base ethyl-4-(tetramethyl--dioxane pentaborane base)-benzamide through underpressure distillation, acidifying, extraction, washing, dry, recrystallization;
(7) product sugar responsiveness monomer N acrylamide base ethyl-4-(tetramethyl--dioxane pentaborane base)-benzamide, chain-transfer agent F, initiator A IBN in step (6) are joined in the flask that fills solvent G, freezing vacuumizing three times, at N 2or react 24-48h under the protection of Ar, temperature of reaction is 70 ℃; Obtain RAFT macromolecular chain transfer agent PAEBB through precipitation, suction filtration with after being dried;
(8) obtained RAFT macromolecular chain transfer agent PAEBB is dissolved in solvent G, adds monomer M EO 2mA and OEGMA; the concrete add-on of monomer is determined by designed molecular weight and performance thereof; add initiator Diisopropyl azodicarboxylate; system is reacted under argon gas or nitrogen protection; temperature of reaction is 50～100 ℃; react 24～30 hours, and through precipitation, vacuum-drying, obtain target product PAEBB-b-P (MEO 2mA-co-OEGMA).
2. the preparation method of a kind of glucose according to claim 1 and temperature-responsive Carriers for Protein Drugs, is characterized in that described solvent orange 2 A is one or more in Isosorbide-5-Nitrae-dioxane, methylene dichloride, trichloromethane or tetrahydrofuran (THF).
3. the preparation method of a kind of glucose according to claim 1 and temperature-responsive Carriers for Protein Drugs, is characterized in that described solvent B is one or more in methylene dichloride, trichloromethane, tetrahydrofuran (THF) or DMF.
4. the preparation method of a kind of glucose according to claim 1 and temperature-responsive Carriers for Protein Drugs, is characterized in that described siccative C is anhydrous CaCl 2, anhydrous MgSO 4or one or more in soda-lime.
5. the preparation method of a kind of glucose according to claim 1 and temperature-responsive Carriers for Protein Drugs, is characterized in that described acyl chlorides D is one or more in oxalyl chloride, Acetyl Chloride 98Min., chloroacetyl chloride or acrylate chloride.
6. the preparation method of a kind of glucose according to claim 1 and temperature-responsive Carriers for Protein Drugs, is characterized in that described solvent E is one or more in tetrahydrofuran (THF), methylene dichloride, trichloromethane or DMF.
7. the preparation method of a kind of glucose according to claim 1 and temperature-responsive Carriers for Protein Drugs, is characterized in that described chain-transfer agent F is trithiocarbonate or dithiocarbonates.
8. the preparation method of a kind of glucose according to claim 1 and temperature-responsive Carriers for Protein Drugs, is characterized in that described solvent G is DMF, N, N N,N-DIMETHYLACETAMIDE or N, one or more in N diethylformamide.
CN201410084021.6A 2014-03-10 2014-03-10 Preparation method of glucose and temperature-responsive insulin controlled release carrier Pending CN103865013A (en)

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