CN101029131A - Branching poly-ethylene and its production - Google Patents
Branching poly-ethylene and its production Download PDFInfo
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- CN101029131A CN101029131A CNA2007100345505A CN200710034550A CN101029131A CN 101029131 A CN101029131 A CN 101029131A CN A2007100345505 A CNA2007100345505 A CN A2007100345505A CN 200710034550 A CN200710034550 A CN 200710034550A CN 101029131 A CN101029131 A CN 101029131A
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Abstract
Production of branched polyglycol consists of four steps: preparing branched polyglycol with glutamic acid of 9-fluorenmethoxy-oxo- protecting amino as central molecule, preparing branched polyglycol with glutamic acid as central molecule, preparing maleimide and preparing the final product. In the structural formula, n is >=1, and m is between 4-500. It can modify protein and polypeptide, decrease biological activity and hindering effect.
Description
Technical field
The present invention relates to a kind of branched polyethylene glycol and preparation method thereof.
Background technology
Transmit the field at current medicine, polyethyleneglycol modified (Pegylation, PEGization, PEGylation) be the important method that a class is widely used, particularly be considered to one of the most successful present technology (J.Clin.Pharmacokinet aspect the clinical application that improves protein and polypeptide drugs, 2000,40:539; J.Adv.Drug Deliv.Rev., 1993,10:91; J.Eur.J.Pharm.Biopharm., 2004,58:185).Yet also there are some problems in actual applications in this technology, shows that mainly pharmaceutical grade protein can cause its biological activity to reduce greatly after PEGization usually.But there are some researches show that recently when adopting a kind of PEG with special construction to modify, can obviously reduce the loss of activity of modifying the back medicine, this PEG with special construction is called as ramiform PEG (Branched PEG).Compare with linear PEG, ramiform PEG not only can keep the biological activity of being modified the back medicine to greatest extent, and its distinctive " class umbellate form " structure more effectively protected protein matter be not hydrolyzed in vivo, can more effectively stop antibody near pharmaceutical grade protein, thereby increase pharmaceutical grade protein circulating half-life in vivo greatly, reduce simultaneously its immunogenicity (J.Bioconjugate Chem., 1995,6:62 in vivo greatly; J.Drug Discovery Today, 2005,10:1451; J.Bioactive Compatible Polym., 1997,12:196; J.Bioconjug.Chem., 2001,12:515; J.Control Release, 1996,40:199).
Research both at home and abroad at present and the ramiform PEG that uses mainly are with the two cladodification PEGs (Lys-(PEG) of Methionin (Lys) as central element
2J.Bioorg.Med.Chem.Lett., 2002,12:177; J.Adv.Drug Deliv.Rev., 2003,55:1261; J.Bioorg.Med.Chem., 2004,12:5031), Lys-(PEG)
2Be as decorating site with the amino on the protein when modifying protein, though by amido modified protein is the most frequently used up to now method, and also be adopted preferred option when protein is carried out PEGization usually, but also there is deficiency in this method, distinct issues are exactly to make and have a large amount of isomer in the product owing to amino more than usually one that can react in the protein molecule, and this mixture of isomers normally is difficult to separation and purification, and, if amino is on the activity of proteins site just, then may cause proteinic inactivation.In order to address this problem, present PEGization technology is developing towards " pointed decoration " direction, and promptly modifier only reacts in specified site.Pointed decoration not only can reduce the generation of isomer in the product significantly, but also can be by being kept its pharmaceutical activity as much as possible by the position of decorating site on the design protein molecule.
It is one of the most effective pointed decoration means (J.Adv.Drug Deliv.Rev., 2002,54:459 in the protein modification chemistry that sulfydryl on the halfcystine is modified; WO9903887; WO9955377), because the common content of halfcystine seldom in natural protein, and can almost a cysteine residues can be imported by any position in the modifying protein molecular sequences in this way with a unessential amino-acid residue in the halfcystine replacement protein molecule by genetic engineering technique.
Research to sulfydryl in the protein chemistry is of long duration, can have with the group of its reaction a lot, wherein maleimide is a kind of preferably, sulfydryl combines by addition reaction with two keys on the maleimide base group, the reaction conditions gentleness can be carried out under neutrality or weakly alkaline (pH (7.5～8.5)) condition.Maleimide has been widely used on the modifying and decorating to protein or polypeptide as the group commonly used of polyethyleneglycol derivative, but up to the present, relevant modifier all is to adopt linear polyethylene glycol, in the branched polyethylene glycol modifier, yet there are no report.
Summary of the invention
The objective of the invention is to overcome above-mentioned the deficiencies in the prior art, provide a kind of and be used for protein and polypeptide drugs sulfydryl pointed decoration, and can reduce the branched polyethylene glycol of the loss of activity of modifying the back medicine and its preparation method greatly.
For addressing the above problem, the present invention by the following technical solutions:
A kind of branched polyethylene glycol, its structure is:
N is 〉=1 integer in the formula, the integer of preferred n=1～18, and m is 4～500 integer.
A kind of method for preparing above-mentioned branched polyethylene glycol comprises the steps:
1) preparation is the branched polyethylene glycol of central element to protect amino L-glutamic acid:
Protecting the amino L-glutamic acid (Fmoc-Glu) and the mol ratio of mono methoxy polyethylene glycol (mPEG) by 9-fluorenylmethyloxycarbonyl in reactor is 1: 2～3 adding Fmoc-Glu and mPEG, described mPEG molecular weight is 200～20000, add 4-Dimethylamino pyridine (DMAP) according to 1%～200% of described Fmoc-Glu mole number, need the ratio of 10～200 liters of solvents to add the analytical pure organic solvent according to 1 mole of Fmoc-Glu, stir under the room temperature and make reactants dissolved; According to described Fmoc-Glu and N, the mol ratio of N '-dicyclohexylcarbodiimide (DCC) is 1: 2～4 adding DCC then, and controlled temperature is-10 ℃～30 ℃, reacts after 10～50 hours, stops temperature control, is stirred to room temperature, continues stirring reaction 1 hour.After removing by filter white precipitate, press 50%～100% of DCC mole number and add oxalic acid, stirring at room was reacted after 1 hour, the filtering reaction thing; Filtrate is rotated evaporation concentration to 1/2～1/10 of original volume under 30 ℃～100 ℃ temperature; naturally after reducing to room temperature; add excessive anhydrous diethyl ether; vigorous stirring; get white precipitate; filter and thorough washing, getting product after the drying is the branched polyethylene glycol (Fmoc-Glu-(mPEG) of central element with the amino L-glutamic acid of Fmoc protection
2).
Described mPEG molecular weight is preferably 750,2000 or 5000.Described organic solvent is methylene dichloride, chloroform, dioxane, ethyl acetate, N, a kind of in N '-dimethyl formamide, methyl-sulphoxide, tetrahydrofuran (THF), the acetone or their mixture; Described control reaction temperature is preferably-5 ℃～5 ℃; The described reaction times is preferably 20-30 hour.
2) step 1 product Fmoc-Glu-(mPEG)
2Removing of amino protecting group
In reactor, restrain step 1 product Fmoc-Glu-(mPEG) according to 1
2The ratio that adds 1 milliliter～5 milliliters of solvents adds Fmoc-Glu-(mPEG)
2With the analytical pure organic solvent, after the stirring and dissolving, add the mass concentration be equivalent to 0.5～5 times of liquor capacity and be the organic solvent solution of 5%～90% piperidines, stirring at room is after 0.5～3 hour, and the rotation evaporation concentration is to 1/2～1/10 of original volume under 30 ℃～100 ℃ temperature, naturally after reducing to room temperature, add excessive anhydrous diethyl ether, vigorous stirring gets white precipitate, filter and thorough washing, getting product after the drying is the branched polyethylene glycol (H of central element with L-glutamic acid
2N-Glu-(mPEG)
2).
Described organic solvent is methylene dichloride, chloroform, dioxane, ethyl acetate, N, a kind of in N '-dimethyl formamide, methyl-sulphoxide, tetrahydrofuran (THF), the acetone or their mixture; The piperidines mass concentration is preferably 30%～70% in the organic solvent solution of described piperidines.
3) preparation dimaleoyl imino lipid acid:
Mol ratio by amino lipid acid of end and alkali in reactor is the amino fatty bronsted lowry acids and bases bronsted lowry of 1: 0.5～1 adding end, adds an amount of solvent, stirs under the room temperature and makes reactants dissolved; Maleic anhydride is dissolved in an amount of analytical pure organic solvent, is 1: 1～2 to add the maleic anhydride solution by the mol ratio of the amino lipid acid of described end and maleic anhydride, and stirring at room was reacted 1 hour; After reaction finishes, extremely acid with the hydrochloric acid regulation system; Filter, filter residue with distilled water wash repeatedly, to filter down liquid be neutral, obtain the white powder intermediate product after the drying; This intermediate product is placed the reactor that thermometer, water trap, prolong are housed; extraordinarily go into the analytical pure organic solvent by 20～100 of intermediate product mole number; heated and stirred makes its dissolving under nitrogen protection; press 10%～200% of intermediate product mole number and add catalyzer; carry out back flow reaction, in water trap, after the anhydrous generation mutually, stop heating; naturally reduce to room temperature, obtain product dimaleoyl imino lipid acid except that after desolvating at rotary evaporation under 80 ℃～160 ℃ temperature.
The structure of the amino lipid acid of described end is:
N is 〉=1 integer in the formula, the integer of preferred n=1～18.
Described alkali is sodium hydroxide, potassium hydroxide; Described solvent is water, methylene dichloride, chloroform, dioxane, ethyl acetate, N, a kind of in N '-dimethyl formamide, methyl-sulphoxide, tetrahydrofuran (THF), the acetone or their mixture; Described organic solvent is benzene,toluene,xylene, acetic anhydride, N, a kind of in N '-dimethyl formamide, the methyl-sulphoxide or their mixture; Described catalyzer is a kind of in triethylamine, tosic acid, sodium-acetate, Potassium ethanoate, the copper sulfate or their mixture.
4) preparation is the branched polyethylene glycol with the maleimide base group of a spacerarm of containing of central element with L-glutamic acid
In reactor set by step 3 the preparation dimaleoyl imino lipid acid: N, N '-dicyclohexylcarbodiimide (DCC): the mol ratio of I-hydroxybenzotriazole (HOBt) is 1: 1: 1 adding dimaleoyl imino lipid acid, N, N '-dicyclohexylcarbodiimide (DCC) and I-hydroxybenzotriazole (HOBt), add an amount of analytical pure organic solvent, stirring at room, after the dissolving of question response thing, 2 preparations is the branched polyethylene glycol of central element with L-glutamic acid set by step: the mol ratio of described dimaleoyl imino lipid acid is that 1: 1～4 to add described be the branched polyethylene glycol of central element with L-glutamic acid, controlled temperature is-10 ℃～30 ℃, react after 10～50 hours, stop temperature control, be stirred to room temperature, continued stirring reaction 1 hour; After reaction finishes, remove by filter white precipitate, then, press 50%～100% of DCC mole number and add oxalic acid, the stirring at room reaction is after 1 hour, and the filtering reaction thing rotates evaporation concentration to 1/2～1/10 of original volume with filtrate under 30 ℃～100 ℃ temperature, naturally after reducing to room temperature, add excessive anhydrous diethyl ether, vigorous stirring gets white precipitate, filter and thorough washing, get product of the present invention after the drying.
Wherein said organic solvent is methylene dichloride, chloroform, dioxane, ethyl acetate, N, a kind of in N '-dimethyl formamide, methyl-sulphoxide, tetrahydrofuran (THF), the acetone or their mixture; Described control reaction temperature is preferably-5 ℃～5 ℃; The described reaction times is preferably 20～30 hours.
Advantage of the present invention is: synthesized a kind of novel polyethyleneglycol derivative with ramiform structure that can realize pointed decoration to protein and polypeptide.This compound can be used as protein and polypeptide drugs modifier, compares with traditional linear polyethylene glycol modifier, and the present invention will reduce greatly by the biological activity of modified medicaments and lose.The present invention adopts L-glutamic acid to connect two polyoxyethylene glycol segments as the centronucleus molecule, and by L-glutamic acid being added protecting group and adopting the method for mixed solvent to solve the problem that L-glutamic acid is insoluble in organic solvent effectively, this structure has not yet to see report.The present invention has introduced maleimide base group in compound molecule, make it become the pointed decoration agent of sulfydryl in a kind of protein and the peptide molecule, thereby can reduce the generation of isomer in the modified outcome significantly, but also can be by being kept its pharmaceutical activity as much as possible by the position of decorating site on the design protein molecule.The present invention has increased a spacerarm when introducing maleimide base group, thereby the generation of steric effect when greatly reducing modifying protein and polypeptide drugs provides guarantee smoothly for modification reaction.
Embodiment
Embodiment 1:
1.Fmoc-Glu-(mPEG
750)
2Preparation
With 3.7 gram Fmoc-Glu, 22.5 gram molecular weights respectively 750 mono methoxy polyethylene glycol (mPEG
750) and 0.9 the gram DMAP place a Kjeldahl flask, add 100 milliliters of analytical pure methylene dichloride and 10 milliliters of analytical pure tetrahydrofuran (THF)s, put into magneton, stir under the room temperature and make reactants dissolved, after 10 minutes, add 8.0 gram DCC, kept under 0 ℃ of condition stirring reaction 24 hours.After reaction finishes, stop temperature control, rise to room temperature naturally, continued stirring reaction 1 hour, remove by filter white precipitate after, add 4.0 gram oxalic acid, stirring at room reaction 1 hour is filtered, with filtrate after rotation evaporation concentration to 20 under 45 ℃ of temperature milliliter, naturally reduce to room temperature, add 200 milliliters of anhydrous diethyl ethers, vigorous stirring gets white precipitate, filter and thorough washing, get product (Fmoc-Glu-(mPEG after the drying
750)
2).
2.Fmoc-Glu-(mPEG
750)
2Removing of amino protecting group
Take by weighing the product Fmoc-Glu-(mPEG in the step 1
750)
220 grams place flask, add 20 milliliters of analytical pure methylene dichloride, after the stirring and dissolving, add massfraction and be 20 milliliters of the dichloromethane solutions of 50% piperidines, stirring at room reaction 1 hour, rotation evaporation concentration to 20 milliliter under 45 ℃ of temperature, naturally after reducing to room temperature, add in 200 milliliters of anhydrous diethyl ethers, vigorous stirring gets white precipitate, filter and thorough washing, get product (H after the drying
2N-Glu-(mPEG
750)
2).
3.6-the preparation of dimaleoyl imino caproic acid (6-MICA)
26 gram 6-aminocaprolc acids (6-ACA) and 7 gram sodium hydroxide mixing are placed a single port flask, add 100 ml distilled waters, stir under the room temperature and make reactants dissolved, continue to stir 30 minutes; Take by weighing 38 gram maleic anhydrides, be dissolved in 100 milliliters of analytical pure ethyl acetate, this maleic anhydride solution is added in the above-mentioned single port flask, stirring at room reaction 1 hour; After reaction finishes, add the hydrochloric acid soln that 200 ml concns are 1N, stir after 15 minutes, filtration, and with distilled water wash repeatedly, liquid is neutral to filtering down, will get intermediate product 6-toxilic acid amide group caproic acid (6-MAICA) after the filter residue and drying;
In a there-necked flask that thermometer, water trap, prolong be housed, add 300 milliliters of analytical pure toluene and 20 milliliters of analytical pure methyl-sulphoxides; under nitrogen protection, add the above-mentioned intermediate product 6-MAICA of 23 grams; heated and stirred; after the dissolving of question response thing, add 26 milliliters of triethylamines, be heated to backflow; reacted 4 hours; stop heating, reduce to room temperature naturally, obtain product 6-MICA except that after desolvating at rotary evaporation under 120 ℃ of temperature.
4. with L-glutamic acid the branched polyethylene glycol (MICA-Glu-(mPEG of containing of central element with the maleimide base group of a spacerarm
750)
2) preparation
6-MICA, 5.8 gram DCC and the 3.8 gram HOBt of the final preparation of 5.9 gram steps 3 are added in the Kjeldahl flask, add 100 milliliters of analytical pure methylene dichloride then, put into magneton and stir, after 10 minutes, add the H of 15 gram step 2 preparations
2N-Glu-(mPEG
750)
2, kept under 0 ℃ of condition stirring reaction 24 hours.After reaction finishes, rise to room temperature naturally, continued stirring reaction 1 hour; After reaction finishes, remove by filter white precipitate, then, add 3.2 gram oxalic acid, stirring at room reaction 1 hour is filtered, and filtrate is rotated evaporation concentration to 25 milliliter under 45 ℃ of temperature, naturally after reducing to room temperature, add 250 milliliters of anhydrous diethyl ethers, vigorous stirring gets white precipitate, filter and thorough washing, get product MICA-Glu-(mPEG after the drying
750)
2
Embodiment 2:
1.Fmoc-Glu-(mPEG
2000)
2Preparation
With 4.1 gram Fmoc-Glu, 60.0 gram molecular weights respectively 2000 mono methoxy polyethylene glycol (mPEG
2000) and 0.8 the gram DMAP place a Kjeldahl flask, add 150 milliliters of analytical pure methylene dichloride and 15 milliliters of analytical pure tetrahydrofuran (THF)s, put into magneton, stir under the room temperature and make reactants dissolved, after 15 minutes, add 6.0 gram DCC, kept under 5 ℃ of conditions stirring reaction 26 hours.After reaction finishes, stop temperature control, rise to room temperature naturally, continued stirring reaction 1 hour.After removing by filter white precipitate, add 3.2 gram oxalic acid, stirring at room reaction 1 hour is filtered, with filtrate after rotation evaporation concentration to 30 under 45 ℃ of temperature milliliter, naturally reduce to room temperature, add 300 milliliters of anhydrous diethyl ethers, vigorous stirring gets white precipitate, filter and thorough washing, get product (Fmoc-Glu-(mPEG after the drying
2000)
2).
2.Fmoc-Glu-(mPEG
2000)
2Removing of amino protecting group
Take by weighing the product Fmoc-Glu-(mPEG of step 1
2000)
245 grams place flask, add 50 milliliters of analytical pure methylene dichloride, after the stirring and dissolving, add massfraction and be 40 milliliters of the dichloromethane solutions of 40% piperidines, stirring at room reaction 2 hours, rotation evaporation concentration to 35 milliliter under 45 ℃ of temperature, naturally after reducing to room temperature, add in 350 milliliters of anhydrous diethyl ethers, vigorous stirring gets white precipitate, filter and thorough washing, get product (H after the drying
2N-Glu-(mPEG
2000)
2).
3.6-the preparation of dimaleoyl imino caproic acid (6-MICA)
26 gram 6-aminocaprolc acids (6-ACA) and 6 gram sodium hydroxide mixing are placed a single port flask, add 80 ml distilled waters, stir under the room temperature and make reactants dissolved, continue to stir 20 minutes; Take by weighing 34 gram maleic anhydrides, be dissolved in 80 milliliters of analytical pure ethyl acetate, this maleic anhydride solution is added in the above-mentioned single port flask, stirring at room reaction 1 hour; After reaction finishes, add the hydrochloric acid soln that 150 ml concns are 1N, stir after 20 minutes, filtration, and with distilled water wash repeatedly, liquid is neutral to filtering down, will get intermediate product 6-toxilic acid amide group caproic acid (6-MAICA) after the filter residue and drying;
In a there-necked flask that thermometer, water trap, prolong be housed, add 250 milliliters of analytical pure toluene and 25 milliliters of analytical pure methyl-sulphoxides; under nitrogen protection, add the above-mentioned intermediate product 6-MAICA of 23 grams; heated and stirred; after the dissolving of question response thing, add 25 milliliters of triethylamines, be heated to backflow; reacted 5 hours; stop heating, reduce to room temperature naturally, obtain product 6-MICA except that after desolvating at rotary evaporation under 120 ℃ of temperature.
4.MICA-Glu-(mPEG
2000)
2Preparation
6-MICA, 4.9 gram DCC and the 3.2 gram HOBt of the final preparation of 5.0 gram steps 3 are added in the Kjeldahl flask, add 150 milliliters of analytical pure methylene dichloride then, put into magneton and stir, after 15 minutes, add the H of 35 gram step 2 preparations
2N-Glu-(mPEG
2000)
2, kept under 5 ℃ of conditions stirring reaction 26 hours.After reaction finishes, rise to room temperature naturally, continued stirring reaction 1 hour; After reaction finishes, remove by filter white precipitate, then, add 2.6 gram oxalic acid, stirring at room reaction 1 hour is filtered, and filtrate is rotated evaporation concentration to 35 milliliter under 45 ℃ of temperature, naturally after reducing to room temperature, add 350 milliliters of anhydrous diethyl ethers, vigorous stirring gets white precipitate, filter and thorough washing, get product MICA-Glu-(mPEG after the drying
2000)
2
Embodiment 3:
1.Fmoc-Glu-(mPEG
5000)
2Preparation
With 4.4 gram Fmoc-Glu, 150.0 gram molecular weights respectively 5000 mono methoxy polyethylene glycol (mPEG
5000) and 0.7 the gram DMAP place a Kjeldahl flask, add 300 milliliters of analytical pure methylene dichloride and 30 milliliters of analytical pure tetrahydrofuran (THF)s, put into magneton, stir under the room temperature and make reactants dissolved, after 20 minutes, add 7.2 gram DCC, kept under-5 ℃ of conditions stirring reaction 30 hours.After reaction finishes, stop temperature control, rise to room temperature naturally, continued stirring reaction 1 hour.After removing by filter white precipitate, add 3.5 gram oxalic acid, stirring at room reaction 1 hour is filtered, with filtrate after rotation evaporation concentration to 50 under 45 ℃ of temperature milliliter, naturally reduce to room temperature, add 500 milliliters of anhydrous diethyl ethers, vigorous stirring gets white precipitate, filter and thorough washing, get product (Fmoc-Glu-(mPEG after the drying
5000)
2).
2.Fmoc-Glu-(mPEG
5000)
2Removing of amino protecting group
Take by weighing the product Fmoc-Glu-(mPEG of step 1
5000)
2100 grams, place flask, add 100 milliliters of analytical pure methylene dichloride, after the stirring and dissolving, add massfraction and be 70 milliliters of the dichloromethane solutions of 60% piperidines, stirring at room reaction 1.5 hours, rotation evaporation concentration to 60 milliliter under 45 ℃ of temperature adds in 600 milliliters of cold diethyl ethers, vigorous stirring, get white precipitate, filter and thorough washing, get product (H after the drying
2N-Glu-(mPEG
5000)
2).
3.6-the preparation of dimaleoyl imino caproic acid (6-MICA)
26 gram 6-aminocaprolc acids (6-ACA) and 6.5 gram sodium hydroxide mixing are placed a single port flask, add 90 ml distilled waters, stir under the room temperature and make reactants dissolved, continue to stir 30 minutes; Take by weighing 36 gram maleic anhydrides, be dissolved in 90 milliliters of analytical pure ethyl acetate, this maleic anhydride solution is added in the above-mentioned single port flask, stirring at room reaction 1 hour; After reaction finishes, add the hydrochloric acid soln that 180 ml concns are 1N, stir after 20 minutes, filtration, and with distilled water wash repeatedly, liquid is neutral to filtering down, will get intermediate product " 6-toxilic acid amide group caproic acid " (6-MAICA) after the filter residue and drying;
In a there-necked flask that thermometer, water trap, prolong be housed, add 280 milliliters of analytical pure toluene and 25 milliliters of analytical pure methyl-sulphoxides; under nitrogen protection, add the above-mentioned intermediate product 6-MAICA of 23 grams; heated and stirred; after the dissolving of question response thing, add 26 milliliters of triethylamines, be heated to backflow; reacted 6 hours; stop heating, reduce to room temperature naturally, obtain product 6-MICA except that after desolvating at rotary evaporation under 120 ℃ of temperature.
4.MICA-Glu-(mPEG
5000)
2Preparation
6-MICA, 3.9 gram DCC and the 2.6 gram HOBt of the final preparation of 4.0 gram steps 3 are added in the Kjeldahl flask, add 200 milliliters of analytical pure methylene dichloride then, put into magneton and stir, after 20 minutes, add the H of 70 gram step 2 preparations
2N-Glu-(mPEG
5000)
2, kept under-5 ℃ of conditions stirring reaction 30 hours.After reaction finishes, rise to room temperature naturally, continued stirring reaction 1 hour; After reaction finishes, remove by filter white precipitate, then, add 2.1 gram oxalic acid, stirring at room reaction 1 hour is filtered, and filtrate is rotated evaporation concentration to 50 milliliter under 45 ℃ of temperature, naturally after reducing to room temperature, add 500 milliliters of anhydrous diethyl ethers, vigorous stirring gets white precipitate, filter and thorough washing, get product MICA-Glu-(mPEG after the drying
5000)
2
Claims (7)
1, a kind of branched polyethylene glycol is characterized in that its structural formula is:
In the formula, n is 〉=1 integer, and m is 4～500 integer.
2, branched polyethylene glycol according to claim 1 is characterized in that n is 1～18 integer.
3, a kind of method for preparing claim 1 or 2 described branched polyethylene glycols is characterized in that may further comprise the steps:
A. protecting the mol ratio of amino L-glutamic acid and mono methoxy polyethylene glycol by 9-fluorenylmethyloxycarbonyl in reactor is amino L-glutamic acid and the mono methoxy polyethylene glycols of 1: 2～3 adding 9-fluorenylmethyloxycarbonyls protections, described mono methoxy polyethylene glycol molecular weight is 200～20000, add the 4-Dimethylamino pyridine according to 1%～200% of the amino L-glutamic acid mole number of described 9-fluorenylmethyloxycarbonyl protection, need the ratio of 10～200 liters of solvents to add the analytical pure organic solvent according to 1 mole of amino L-glutamic acid of 9-fluorenylmethyloxycarbonyl protection, stir under the room temperature and make reactants dissolved; Then according to amino L-glutamic acid and the N of described 9-fluorenylmethyloxycarbonyl protection, the mol ratio of N '-dicyclohexylcarbodiimide is 1: 2～4 adding N, N '-dicyclohexylcarbodiimide, controlled temperature is-10 ℃～30 ℃, react after 10～50 hours, stop temperature control, be stirred to room temperature, continued stirring reaction 1 hour; After removing by filter white precipitate, press N, 50%～100% of N '-dicyclohexylcarbodiimide mole number adds oxalic acid, and stirring at room was reacted after 1 hour, the filtering reaction thing; Filtrate is rotated evaporation concentration to 1/2～1/10 of original volume under 30 ℃～100 ℃ temperature, naturally after reducing to room temperature, add excessive anhydrous diethyl ether, vigorous stirring, get white precipitate, filter and thorough washing, getting product after the drying is the branched polyethylene glycol of central element with the amino L-glutamic acid of 9-fluorenylmethyloxycarbonyl protection;
B. in reactor, be that to add with the amino L-glutamic acid of 9-fluorenylmethyloxycarbonyl protection be the branched polyethylene glycol and the analytical pure organic solvent of central element for ratio that the branched polyethylene glycol of central element adds 1 milliliter～5 milliliters of solvents with the amino L-glutamic acid of 9-fluorenylmethyloxycarbonyl protection according to 1 gram step a product, after the stirring and dissolving, add the mass concentration be equivalent to 0.5～5 times of liquor capacity and be the organic solvent solution of 5%～90% piperidines, after the stirring at room 0.5～3 hour, the rotation evaporation concentration is to 1/2～1/10 of original volume under 30 ℃～100 ℃ temperature, naturally after reducing to room temperature, add excessive anhydrous diethyl ether, vigorous stirring, get white precipitate, filter and thorough washing, getting product after the drying is the branched polyethylene glycol of central element with L-glutamic acid;
C. the mol ratio by amino lipid acid of end and alkali is the amino fatty bronsted lowry acids and bases bronsted lowry of 1: 0.5～1 adding end in reactor, adds an amount of solvent, stirs under the room temperature and makes reactants dissolved; Maleic anhydride is dissolved in an amount of analytical pure organic solvent, is 1: 1～2 to add the maleic anhydride solution by the mol ratio of the amino lipid acid of described end and maleic anhydride, and stirring at room was reacted 1 hour; After reaction finishes, extremely acid with the hydrochloric acid regulation system; Filter, filter residue with distilled water wash repeatedly, to filter down liquid be neutral, obtain the white powder intermediate product after the drying; This intermediate product is placed the reactor that thermometer, water trap, prolong are housed; extraordinarily go into the analytical pure organic solvent by 20～100 of intermediate product mole number; heated and stirred makes its dissolving under nitrogen protection; press 10%～200% of intermediate product mole number and add catalyzer; carry out back flow reaction; in water trap anhydrous generate mutually after; stop heating; naturally reduce to room temperature; obtain product dimaleoyl imino lipid acid at rotary evaporation under 80 ℃～160 ℃ temperature except that after desolvating, the structure of the amino lipid acid of described end is:
In the formula, n is 〉=1 integer;
D. the dimaleoyl imino lipid acid of c preparation: N set by step in reactor, N '-dicyclohexylcarbodiimide: the mol ratio of I-hydroxybenzotriazole is 1: 1: 1 adding dimaleoyl imino lipid acid, N, N '-dicyclohexylcarbodiimide and I-hydroxybenzotriazole, add an amount of analytical pure organic solvent, stirring at room, after the dissolving of question response thing, b preparation is the branched polyethylene glycol of central element with L-glutamic acid set by step: the mol ratio of described dimaleoyl imino lipid acid is that 1: 1～4 to add described be the branched polyethylene glycol of central element with L-glutamic acid, controlled temperature is-10 ℃～30 ℃, react after 10～50 hours, stop temperature control, be stirred to room temperature, continued stirring reaction 1 hour; After reaction finishes, remove by filter white precipitate, then, press N, 50%～100% of N '-dicyclohexylcarbodiimide mole number adds oxalic acid, and the stirring at room reaction is after 1 hour, the filtering reaction thing, filtrate is rotated evaporation concentration to 1/2～1/10 of original volume under 30 ℃～100 ℃ temperature, reduce to room temperature naturally after, add excessive anhydrous diethyl ether, vigorous stirring, get white precipitate, filter and thorough washing, get product of the present invention after the drying.
4, according to the described method for preparing branched polyethylene glycol of claim 3, it is characterized in that: among the described step a, the mono methoxy polyethylene glycol molecular weight is 750,2000 or 5000; Described organic solvent is methylene dichloride, chloroform, dioxane, ethyl acetate, N, a kind of in N '-dimethyl formamide, methyl-sulphoxide, tetrahydrofuran (THF), the acetone or their mixture; Described control reaction temperature is-5 ℃～5 ℃; The described reaction times is 20～30 hours.
5, according to the described method for preparing branched polyethylene glycol of claim 3, it is characterized in that: organic solvent is methylene dichloride, chloroform, dioxane, ethyl acetate, N among the described step b, a kind of in N '-dimethyl formamide, methyl-sulphoxide, tetrahydrofuran (THF), the acetone or their mixture; The piperidines mass concentration is 30%～70% in the organic solvent solution of described piperidines.
6, according to the described method for preparing branched polyethylene glycol of claim 3, it is characterized in that: n is 1～18 integer in the structural formula of the amino lipid acid of described step c middle-end; Alkali is sodium hydroxide or/and potassium hydroxide, and described solvent is water, methylene dichloride, chloroform, dioxane, ethyl acetate, N, a kind of in N '-dimethyl formamide, methyl-sulphoxide, tetrahydrofuran (THF), the acetone or their mixture; Described organic solvent is benzene,toluene,xylene, acetic anhydride, N, a kind of in N '-dimethyl formamide, the methyl-sulphoxide or their mixture; Described catalyzer is a kind of in triethylamine, tosic acid, sodium-acetate, Potassium ethanoate, the copper sulfate or their mixture.
7, according to the described method for preparing branched polyethylene glycol of claim 3, it is characterized in that in the described steps d, organic solvent is methylene dichloride, chloroform, dioxane, ethyl acetate, N, a kind of in N '-dimethyl formamide, methyl-sulphoxide, tetrahydrofuran (THF), the acetone or their mixture; Described control reaction temperature is-5 ℃～5 ℃; The described reaction times is 20～30 hours.
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| CNA2007100345505A CN101029131A (en) | 2007-03-16 | 2007-03-16 | Branching poly-ethylene and its production |
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| CNA2007100345505A CN101029131A (en) | 2007-03-16 | 2007-03-16 | Branching poly-ethylene and its production |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102180807A (en) * | 2011-03-17 | 2011-09-14 | 中国科学院化学研究所 | Amino acid and preparation method thereof |
| CN101724142B (en) * | 2008-10-22 | 2011-12-14 | 中国科学院过程工程研究所 | Poly alkyl ether polymer using thiosulfonate as terminal group and synthesis and application thereof |
| CN103012770A (en) * | 2011-09-24 | 2013-04-03 | 复旦大学 | Polyethylene glycol benzothiazole derivative and preparation method and application thereof |
| WO2018177055A1 (en) * | 2017-03-30 | 2018-10-04 | 北京键凯科技股份有限公司 | Method for preparing y-branched hydrophilic polymer carboxylic acid derivative |
| CN108659227A (en) * | 2017-03-30 | 2018-10-16 | 北京键凯科技股份有限公司 | A kind of preparation method of the hydrophilic polymer carboxylic acid derivates of y-branch |
-
2007
- 2007-03-16 CN CNA2007100345505A patent/CN101029131A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724142B (en) * | 2008-10-22 | 2011-12-14 | 中国科学院过程工程研究所 | Poly alkyl ether polymer using thiosulfonate as terminal group and synthesis and application thereof |
| CN102180807A (en) * | 2011-03-17 | 2011-09-14 | 中国科学院化学研究所 | Amino acid and preparation method thereof |
| CN102180807B (en) * | 2011-03-17 | 2014-07-16 | 中国科学院化学研究所 | Amino acid and preparation method thereof |
| CN103012770A (en) * | 2011-09-24 | 2013-04-03 | 复旦大学 | Polyethylene glycol benzothiazole derivative and preparation method and application thereof |
| CN103012770B (en) * | 2011-09-24 | 2015-03-04 | 复旦大学 | Polyethylene glycol benzothiazole derivative and preparation method and application thereof |
| WO2018177055A1 (en) * | 2017-03-30 | 2018-10-04 | 北京键凯科技股份有限公司 | Method for preparing y-branched hydrophilic polymer carboxylic acid derivative |
| CN108659227A (en) * | 2017-03-30 | 2018-10-16 | 北京键凯科技股份有限公司 | A kind of preparation method of the hydrophilic polymer carboxylic acid derivates of y-branch |
| US11359089B2 (en) | 2017-03-30 | 2022-06-14 | Jenkem Technology Co., Ltd. (Beijing) | Method for preparing Y-branched hydrophilic polymer carboxylic acid derivative |
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