CN101284135A - Mono-modified polyethyleneglycol-insulin complexes and preparation method thereof - Google Patents
Mono-modified polyethyleneglycol-insulin complexes and preparation method thereof Download PDFInfo
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- CN101284135A CN101284135A CNA2008100114789A CN200810011478A CN101284135A CN 101284135 A CN101284135 A CN 101284135A CN A2008100114789 A CNA2008100114789 A CN A2008100114789A CN 200810011478 A CN200810011478 A CN 200810011478A CN 101284135 A CN101284135 A CN 101284135A
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Abstract
The invention relates to the field of bio-medicine. The formula of a polyethylene glycol-modified insulin composite is RNH-X-D, wherein R represents CH3O-(CH2CH2O)nCH2CH2, n is equal to 50-500, X is natural amino acid outside proline of genetic encode or non-natural amino acid of mono-amino mono-carboxyl, and D is B30 amino acid residue-removed natural pork insulin or human insulin. In PEG modification, carboxyl instead that amino is modified, and polyethylene glycol chain is linked to alpha carboxyl on B30 amino acid residue of insulin via amide bond at high yield and high stability. The polyethylene glycol-modified insulin composite has important application prospect in long acting aspect, and substantially is chemically modified insulin. The polyethylene glycol-modified insulin composite has potential long acting function.
Description
One, technical field:
The present invention relates to biomedicine field, particularly relate to the diabetes insulin, its preparation method also is provided in addition.
Two, background technology:
Diabetes are a kind of common multiple metabolic diseases, and sickness rate both domestic and external is all in rising trend.According to incompletely statistics, more than existing diabetics 3,000 ten thousand people in the whole nation, the sickness rate of diabetes is up to 3.2%.Though numerous disease can both cause people's death, diabetes are the tertiary main causes that cause people's death.
Insulin is the specially good effect polypeptide hormone chemical compound of treatment diabetes, does not still have more effective alternative medicine up to now.But, because insulin is a kind of protein, lost activity by the protease hydrolysis in the gastrointestinal tract easily, therefore can only inject use, otherwise administration.For the patient of insulin-dependent, the homergy that earn a bare living, necessary frequent injecting drug use, this brings very big misery and inconvenience just for such patient.
In order to solve to wanting difficulty, delay insulin degraded and drainage in vivo, the novel form of insulin has been carried out broad research both at home and abroad in recent years, the method for chemical modification is one of important subject of preparation insulin novel form.
For the patient of diabetes, effectively Therapeutic Method relates to the application of two kinds of exogenous insulin, Semilente Insulin and long lasting basal insulin that diet uses.
The long-acting mechanism of insulin derivates is divided into three kinds basically at present:
First kind is the aminoacid sequence of transforming the primary structure of insulin, makes it to form insoluble polymer crystalline state, utilizes the slow release in vivo of this insoluble attitude, reaches long-acting;
Second kind is the method aliphatic chain of covalent bond on the side chain of some amino acid residues of insulin with chemical modification, increases the circulation half life with albuminous effect in vivo by the aliphatic chain that connects and reaches long-acting;
The third is exactly to connect one or several polymer substance by chemical method in the functional group of some amino acid residue of insulin, reaches long lasting purpose with the circulation half life that increases insulin.
The representative of taking the insulin preparation of above-mentioned first kind of long-acting mechanism is Ultralente, Lente and half-Lente.But the insoluble meeting that has now shown these durative action preparations causes the inconsistent of dose response, and aspect described time effect uncertain problem, the instability that has caused glycemic control, thereby be easy to generate life-threatening hypoglycemia symptom, therefore still need the strong insulin substitution therapy of soluble long-acting basal insulin to succeed.
The pioneering work (pegylation) of protein being modified with Polyethylene Glycol PEG be the earliest last century late nineteen seventies in the Frank Davis of Rutgers university professor's laboratory, carry out.PEG has the character of a series of uniquenesses.Comprise avirulence, non-immunogenicity and antigenicity; The kidney of very low quality decision is got rid of; Higher flexibility and in water neutralizes some organic solvents very high dissolubility.Correspondingly, PEG-protein has toxicity, immunogenicity and the antigenicity that has reduced, the kidney that has reduced is got rid of speed and protease hydrolysis speed, the dissolubility and the stability that have increased.Be also shown in the improvement of many valuable drug kinetic parameters.
In biomedicine, the people of biotechnology and pharmaceutical industry work is familiar with such a case: the pharmaceutical properties of medicinal polypeptide and the improvement of biological property are closely-related with the covalent bond of this peptide species and PEG.For example, the epitope that can shield polypeptide that combines of PEG and medicine has so just weakened the scavenging action of reticuloendothelial system and by the identification of vivo immuning system, has also reduced the Degradation of proteolytic enzyme simultaneously.PEG conjugation has also increased the apparent size of polypeptide, thereby the filtration that has reduced kidney has also changed simultaneously the bio distribution of polypeptide.The key factor that influences above-mentioned character is: 1) be attached to the number of the PEG chain on the polypeptide, 2) be attached to the molecular weight and the structure of the PEG chain on the polypeptide, 3) yoke of PEG on polypeptide close the site, 4) PEG is attached to chemical method used on the polypeptide.
The Polyethylene Glycol of insulin (PEG) is changed the influence that just has been subjected to above-mentioned four factors.The PEG that uses is attached to the reagent that the method on the polypeptide mainly is parent's electricity at first at present, promptly the hydroxyl of the end of PEG by get married functional group's (as active ester, acyl chlorides etc.) of electricity of chemical method transformation, be beneficial to protein side chain on nucleophilic group (amino, sulfydryl etc.) react.Most protein do not contain the free sulfhydryl groups sulfydryl, and all contain free amino, so the modification of PEG mainly is the amino in the modifying protein.Three free amino are arranged, the N-terminal of A chain, the N-terminal of B chain, and the epsilon-amino of the lysine of B29 in the insulin.Modifying people by the PEG to insulin recognizes, no matter how the PEG chain of macromolecule, what chemical method to be modified at the N-terminal of INSULIN A chain with, the capital causes the activity of insulin to reduce even loses activity fully, reason is that the N-terminal of A chain is very near from the active center of insulin, and the PEG chain of modification can hinder combining of insulin and its receptor; And can't select these three amino with active very strong dressing agent, thereby causing multiple different modifying mixture of products, this is flagrant defective to a medicine.
So remaining just can only be to close the site and PEG is attached to chemical method used on the polypeptide at the yoke on the polypeptide from PEG to seek breakthrough.Someone is by using the N-terminal of chemical method protection A chain, and the epsilon-amino of the lysine of the N-terminal of modification B chain and B29, though can keep the most of active of insulin like this, the separation of single site modified outcome has then caused very big difficulty, has also increased cost to suitability for industrialized production; The somebody causes to a certain extent selective modification by the activity that changes reagent, for example adopts the aldehyde radical derivant of PEG can optionally modify N-terminal amino under low pH, so still will protect the N-terminal of A chain; Present in a word method of modifying can not be avoided the influence of above-mentioned four factors fully, can not obtain gratifying modification result.
Three, summary of the invention:
The objective of the invention is to overcome above-mentioned not enough problem, a kind of mono-modified polyethylene glycol-insulin complex substance is provided, its preparation method also is provided in addition, enzymatic is synthetic, and insulin and being connected of the chemical group that will modify are site-specific and efficiently.
The technical scheme that the present invention is adopted for achieving the above object is: a kind of mono-modified polyethylene glycol-insulin complex substance, and its chemical structure of general formula is RNH-X-D, R is CH in the formula
3O-(CH
2CH
2O) nCH
2CH
2, n=50-500; X is the natural amino acid beyond the proline of genetic coding or the alpha-non-natural amino acid of an amino carboxyl; D is the insulin human of sloughing the natural Iletin II (Lilly) of b30 amino acid residue or sloughing the b30 amino acid residue.
Described X is a threonine.
Be connected for amido link between the carboxyl of described R-NH and X; Between the carboxyl of the amino of X and the C-terminal B29 lysine of D is that amido link is connected.
The precursor of described R-NH is amino Polyethylene Glycol CH
3O-(CH
2CH
2O) nCH
2CH
2NH
2, its molecular weight ranges is 2KD-20KD.
The preparation method of a kind of mono-modified polyethylene glycol-insulin complex substance of the present invention: concrete synthesis step is as follows:
The preparation of the amino Polyethylene Glycol of the first step: with a certain amount of general formula is that (R represents CH to ROH
3O-(CH
2CH
2O) nCH
2CH
2) mono methoxy polyethylene glycol (n=50-500) join in the toluene, heating azeotropic removal of water branch, remaining solution adds the pyridine with the aliphatic alcohol equimolar amounts, back flow reaction also drips the thionyl chloride (SOCl of 2 times of aliphatic alcohol moles
2), reacted 2-6 hour in 80 ℃-100 ℃ again after dropwising, cold filtration is removed crystal, toluene is removed in distilling under reduced pressure then, and the product that obtains is R-Cl, and this material is put into reactor, with a small amount of absolute methanol dissolving, add liquid ammonia then, excess of ammonia and methanol were removed in decompression after still was driven in airtight following 60 ℃ of reactions in 12-24 hour, and obtaining general formula is R-NH
2Amino Polyethylene Glycol, standby;
The preparation of the second step RNH-X: get the amino acid derivativges that a certain amount of amino has been protected with Fmoc, its chemical general formula is Fmoc-(HN)-X-(COOH), be dissolved in dichloromethane CH
2Cl
2With 1 of dimethyl formamide (DMF): 1-1: add standby R-NH in the 6 volume ratio mixed solutions with the amino amino acid derivativges equimolar amounts of having protected with Fmoc
2The carbodiimide (DCC) that adds the amino acid derivativges mole that 2 times of amino have protected with Fmoc again, 0-10 ℃ stirring reaction 12-24 hour, remove by filter by-product DCU then, add excessive piperidines in the solution that obtains, stirring reaction is taken off the Fmoc protecting group, obtain RNH-X, standby, this moment, the carboxyl of X was connected with amino Polyethylene Glycol formation amide, and amino is free state;
The 3rd step was removed the preparation of the insulin intermediate of b30 amino acid residue: add natural Iletin II (Lilly) or insulin human in the ammonium bicarbonate soln of pH8.3, make it dissolving, the Carboxypeptidase A that adds catalytic amount then, effect is 2 hours in 37 ℃ of water-baths, adds sour cessation reaction then, and reactant liquor separates with SephadexG50, lyophilizing, obtain taking off the insulin intermediate of b30 amino acid, its B chain N-terminal is B29-Lys, and is standby;
The preparation of the polyethylene glycol-insulin complex substance that the 4th step is mono-modified: at first a certain amount of standby insulin intermediate of taking off the b30 amino acid residue is used the acetum dissolving of 0.5-2N, get solution 1, then standby RNH-X is dissolved in 1: 1-1: in the EtOH/DMF mixed solution of 6 volume ratios, the mol ratio of raw material insulin intermediate and RNH-X is 1: 10-1: 60, get solution 2, at last solution 1 and 2 is merged and adjusting pH to 6-7, the trypsin that adds catalytic amount, reaction is 12-24 hour in 20-35 ℃ water-bath, can obtain the mono-modified polyethylene glycol-insulin complex substance of end-product of the present invention.
Dripping pyridine afterreaction time that finishes in the preparation of the amino Polyethylene Glycol of the described first step is 4 hours.
Adding the liquid ammonia response time in the preparation of the amino Polyethylene Glycol of the described first step is 18 hours.
The reaction temperature of the middle preparation RNH-X of the preparation of the described second step RNH-X is 4 ℃, and the response time is 18 hours.
The acetum concentration of dissolving the insulin intermediate of taking off the b30 amino acid residue in the preparation of the polyethylene glycol-insulin complex substance that described the 4th step is mono-modified is 1N.
Raw material insulin intermediate and C in the preparation of the polyethylene glycol-insulin complex substance that described the 4th step is mono-modified
nH
2n+1The mol ratio of NH-X is 1: 60.
Solution 1 and 2 merging and adjusting pH value are 6.8 in the preparation of the polyethylene glycol-insulin complex substance that described the 4th step is mono-modified.
The present invention has adopted brand new concept in PEG modification field: the decorations of rebuilding are amino for modifying carboxyl, and polyglycol chain is connected on the α carboxyl of b30 amino acid residue of insulin, and this is connected to amido bond, and high yield is stable.The synthetic a kind of a kind of mono-modified polyethylene glycol-insulin complex substance that the major application prospect is arranged aspect long-acting of the present invention, the essence of this complex is chemically modified insulin.This mono-modified polyethylene glycol-insulin complex substance has potential long-acting function.
Preparation method of the present invention has been used brand-new method of modifying: in the coupling reaction of key, use enzymatic reaction, by enzymatic high efficiency and specificity the B chain C-terminal that is combined in insulin of PEG chain high yield.Specifically, having used the synthetic method of enzymatic under the organic facies will remove the Iletin II (Lilly) of b30 amino acid residue or insulin human intermediate in the present invention is connected with high yield with synthetic aminoacid polyethyleneglycol derivative in advance, form the mono-modified polyethylene glycol-insulin complex substance that a polyglycol chain links to each other with insulin B 30 aminoacid carboxyls, the productive rate of this organic facies enzymatic reaction reaches more than 90% in the present invention, is up to 99%.
When X was alanine, this structural formula represented that a PEG chain is connected to amido link on the carboxyl of b30 amino acid of Iletin II (Lilly).When X is a threonine, on behalf of a PEG chain, this structural formula be connected to amido link on the carboxyl of b30 amino acid of insulin human; No matter initiation material is Iletin II (Lilly) or insulin human, when X is threonine, by method provided by the invention, the insulin human of an aliphatic chain that at last all obtained B chain C-terminal carboxyl modified.Because trypsin catalytic synthetic reaction in organic facies only requires that B29 is a lysine, therefore, no matter X seed amino acid why does not influence the productive rate of last trim.
Four, description of drawings:
Fig. 1 is the electrophoresis behavior contrast figure that the PEG chain is modified insulin and unmodified insulin human.Adopt polyacrylamide nature continuous electrophoresis, gel strength is 10%, and pH8.9 electrode buffer pH is 8.3 in the gel, Kao Masi light blue R250 dyeing.
Fig. 2 is that starting material is the mass spectrum of the RNH-Thr of mPEG-5000, shows that its molecular weight ranges is 3000-5300, and molecular weight ranges changes greatly.
Fig. 3 is the mass spectrum of the complex that forms of above-mentioned RNH-Thr and insulin, and its molecular weight ranges illustrates that at 8000-11000 it is 1: 1 that insulin combines with it, and the synthetic mode of enzymatic has determined RNH-Thr to be connected the c-terminus of the B29 position Lys of D.
Five, the specific embodiment:
Detailed description under doing into below in conjunction with specific embodiment to the present invention, but be not limited to specific embodiment.
Embodiment 1: terminal link molecule amount is the preparation of the insulin-PEG complex of 5000 PEG chain
Step 1: molecular weight is the preparation of 5000 amino Polyethylene Glycol
Getting 20 gram (4 mM) relative molecular weights is 5000 mono methoxy polyethylene glycol, adds and to be equipped with in 500 milliliters of round-bottomed flasks of 300 milliliters of toluene, heats and steams 100 milliliters of toluene to take the moisture in the alcohol out of.Remaining solution adds equimolar pyridine and refluxes, and drips the thionyl chloride (SOCl of 2 times of moles
2), reducing the temperature to 90 ℃ after dropwising and continue reaction 4 hours, cooling removes by filter crystal, pressure reducing and steaming toluene then, residue ether sedimentation, sucking filtration vacuum drying obtain 19 and restrain products, and this is 5000 PEG-Cl for molecular weight.But with this product be dissolved in a small amount of absolute methanol join can be airtight and the reactor of bearing certain pressure in, it is airtight to add liquid ammonia, and 60 ℃ were reacted 24 hours, and opened reactor, the reactant liquor rotary evaporation removes deammoniation and methanol, obtains mean molecule quantity and be 5000 amino Polyethylene Glycol PEG-NH
2Step 2:PEG5000-NH-Thr (NH
2) preparation
The relative molecular weight of getting preparation among the embodiment 1 is 5000 PEG-NH
210 grams (2 mM) are dissolved in CH
2Cl
2In the mixed solution of/DMF (1: 3); the Fmoc-Thr (Fmoc-threonine) that adds the commercially available amido protecting of 0.68 gram (2 mM); add DCC 0.81 gram (4 mM) then, 4 ℃ of following stirring reactions 24 hours remove by filter the DCU precipitation; add an amount of piperidines then and take off the Fmoc protecting group; solution adds the ether sedimentation product, filters washing; vacuum drying obtains PEG5000-NH-Thr (NH
2).
Step 3: the preparation of removing the insulin intermediate of b30 amino acid residue
Getting 100 milligrams of insulin humans (Dongbao of Tonghua company) is dissolved in the 0.1M ammonium bicarbonate soln of 5 milliliters of pH8.3, add 1 milligram of Carboxypeptidase A, above-mentioned solution was 37 ℃ of insulations 2 hours, add the glacial acetic acid cessation reaction then, (110cm 1.6cmID) separates and removes pheron, collects 110 ~ 160 milliliters of component lyophilizing through SephadexG50 with the solution that obtains, the be removed insulin intermediate D of b30 amino acid residue, yield is 97%.
The preparation of step 4:D-Thr-NH-PEG5000 (this is a kind of of claim formula of D-X-HN-PEG)
(1) gets in the step 3 20 milligrams of the insulin intermediate of removing the b30 amino acid residue of preparation, adds 0.15 milliliter of 1N glacial acetic acid and dissolve, obtain solution 1
(2) get the PEG-NH-Thr (NH for preparing among 600 milligrams of embodiment 2 of 3 milliliters of DMF/EtOH (1: 1) mixed liquor dissolving
2), this is a solution 2.
(3) merge solution 1,2, regulate pH6.5, add the trypsin with 1: 200 weight ratio of insulin intermediate again, 30 ℃ of reactions 24 hours with TEA (triethylamine).
(4) after reaction is finished, system is regulated pH3.0 with HAc, the solution that obtains separates with SephadexG50 removes trypsin, the modification component lyophilizing that obtains.
(5) lyophilized products is carried out gel electrophoresis analysis, rp-hplc analysis respectively, the result confirms that obtained RNH-Thr-D, productive rate is more than 95%.Specifically see description of drawings.
Products obtained therefrom is through surveying the result as shown in Figure 1, as seen from Figure 1, the electrophoresis behavior that the PEG chain is modified insulin and unmodified insulin human has notable difference, and this is because in gel, charged particle is moved to positive pole by negative pole by the difference of electric charge, and moving direction is as figure; After the PEG chain has been modified the carboxyl of B chain C-terminal of insulin, the insulin of decorations has lacked a positive charge than the insulin of unmodified, therefore its mobility speed is slower than unmodified insulin, has shown two different district's bands on the electrophoresis film, shown in swimming lane among the figure 1 and swimming lane 2.
Embodiment 2: terminal link molecule amount is the preparation of the insulin-PEG complex of 10000 PEG chain
Step 1: molecular weight is the preparation of 10000 amino Polyethylene Glycol
Getting 20 gram (2 mM) relative molecular weights is 10000 mono methoxy polyethylene glycol, adds and to be equipped with in 500 milliliters of round-bottomed flasks of 300 milliliters of toluene, heats and steams 100 milliliters of toluene to take the moisture in the alcohol out of.Remaining solution adds equimolar pyridine and refluxes, and drips the thionyl chloride (SOCl of 2 times of moles
2), reducing the temperature to 90 ℃ after dropwising and continue reaction 4 hours, cooling removes by filter crystal, pressure reducing and steaming toluene then, residue ether sedimentation, sucking filtration vacuum drying obtain 19 and restrain products, and this is 10000 PEG-Cl for molecular weight.But with this product be dissolved in a small amount of absolute methanol join can be airtight and the reactor of bearing certain pressure in, it is airtight to add liquid ammonia, and 60 ℃ were reacted 24 hours, and opened reactor, the reactant liquor rotary evaporation removes deammoniation and methanol, obtains mean molecule quantity and be 10000 amino Polyethylene Glycol PEG-NH
2
Step 2:PEG10000-NH-Thr (NH
2) preparation
The relative molecular weight of getting preparation among the embodiment 1 is 10000 PEG-NH
210 grams (1 mM) are dissolved in CH
2Cl
2In the mixed solution of/DMF (1: 3); the Fmoc-Thr (Fmoc-threonine) that adds the commercially available amido protecting of 0.68 gram (2 mM); add DCC 0.81 gram (4 mM) then, 8 ℃ of following stirring reactions 24 hours remove by filter the DCU precipitation; add an amount of piperidines then and take off the Fmoc protecting group; solution adds the ether sedimentation product, filters washing; vacuum drying obtains PEG5000-NH-Thr (NH
2).
Step 3: the preparation of removing the insulin intermediate of b30 amino acid residue
Getting 100 milligrams of insulin humans (Dongbao of Tonghua company) is dissolved in the 0.1M ammonium bicarbonate soln of 5 milliliters of pH8.3, add 1 milligram of Carboxypeptidase A, above-mentioned solution was 37 ℃ of insulations 2 hours, add the glacial acetic acid cessation reaction then, (110cm 1.6cmID) separates and removes pheron, collects the lyophilizing of 110-160 milliliter component through SephadexG50 with the solution that obtains, the be removed insulin intermediate D of b30 amino acid residue, yield is 97%.
The preparation of step 4:D-Thr-NH-PEG10000 (this is a kind of of claim formula of D-X-HN-PEG)
(1) gets in the step 3 20 milligrams of the insulin intermediate of removing the b30 amino acid residue of preparation, adds 0.15 milliliter of 1N glacial acetic acid and dissolve, obtain solution 1
(2) get the PEG-NH-Thr (NH for preparing among 600 milligrams of embodiment 2 of 3 milliliters of DMF/EtOH (1: 1) mixed liquor dissolving
2), this is a solution 2.
(3) merge solution 1,2, regulate pH6.8, add the trypsin with 1: 100 weight ratio of insulin intermediate again, 30 ℃ of reactions 24 hours with TEA (triethylamine).
(4) after reaction is finished, system is regulated pH3.0 with HAc, the solution that obtains separates with Sephadex650 removes trypsin, the modification component lyophilizing that obtains.
(5) lyophilized products is carried out gel electrophoresis analysis, rp-hplc analysis respectively, the result confirms that obtained D-X-HN-PEG, productive rate is more than 95%.Specifically see description of drawings.
Embodiment 3: terminal link molecule amount is the preparation of the insulin-PEG complex of 20000 PEG chain
Step 1: molecular weight is the preparation of 20000 amino Polyethylene Glycol
Getting 20 gram (1 mM) relative molecular weights is 20000 mono methoxy polyethylene glycol, adds and to be equipped with in 500 milliliters of round-bottomed flasks of 300 milliliters of toluene, heats and steams 100 milliliters of toluene to take the moisture in the alcohol out of.Remaining solution adds equimolar pyridine and refluxes, and drips the thionyl chloride (SOCl of 2 times of moles
2), reducing the temperature to 90 ℃ after dropwising and continue reaction 4 hours, cooling removes by filter crystal, pressure reducing and steaming toluene then, residue ether sedimentation, sucking filtration vacuum drying obtain 19 and restrain products, and this is 20000 PEG-Cl for molecular weight.But with this product be dissolved in a small amount of absolute methanol join can be airtight and the reactor of bearing certain pressure in, it is airtight to add liquid ammonia, and 60 ℃ were reacted 24 hours, and opened reactor, the reactant liquor rotary evaporation removes deammoniation and methanol, obtains mean molecule quantity and be 20000 amino Polyethylene Glycol PEG-NH
2Step 2:PEG20000-NH-Thr (NH
2) preparation
The relative molecular weight of getting preparation among the embodiment 1 is 20000 PEG-NH
210 grams (0.5 mM) are dissolved in CH
2Cl
2In the mixed solution of/DMF (1: 3); the Fmoc-Thr (Fmoc-threonine) that adds the commercially available amido protecting of 0.68 gram (2 mM); add DCC 0.81 gram (4 mM) then, 10 ℃ of following stirring reactions 24 hours remove by filter the DCU precipitation; add an amount of piperidines then and take off the Fmoc protecting group; solution adds the ether sedimentation product, filters washing; vacuum drying obtains PEG20000-NH-Thr (NH
2).
Step 3: the preparation of removing the insulin intermediate of b30 amino acid residue
Getting 100 milligrams of Iletin II (Lilly) (Xuzhou Wan Bang company) is dissolved in the 0.1M ammonium bicarbonate soln of 5 milliliters of pH8.3, add 1 milligram of Carboxypeptidase A, above-mentioned solution was 37 ℃ of insulations 2 hours, add the glacial acetic acid cessation reaction then, (110cm 1.6cmID) separates and removes pheron, collects 110 ~ 160 milliliters of component lyophilizing through SephadexG50 with the solution that obtains, the be removed insulin intermediate D of b30 amino acid residue, yield is 97%.
The preparation of step 4:D-Thr-NH-PEG20000 (this is a kind of of claim formula of D-X-HN-PEG)
(1) gets in the step 3 20 milligrams of the insulin intermediate of removing the b30 amino acid residue of preparation, adds 0.15 milliliter of 1N glacial acetic acid and dissolve, obtain solution 1
(2) get the RNH-Thr (NH for preparing among 600 milligrams of embodiment 2 of 3 milliliters of DMF/EtOH (1: 1) mixed liquor dissolving
2), this is a solution 2.
(3) merge solution 1,2, regulate pH6.8, add the trypsin with 1: 200 weight ratio of insulin intermediate again, 30 ℃ of reactions 24 hours with TEA (triethylamine).
(4) after reaction is finished, system is regulated pH3.0 with HAc, the solution that obtains separates with SephadexG50 removes trypsin, the modification component lyophilizing that obtains.
(5) lyophilized products is carried out gel electrophoresis analysis, rp-hplc analysis respectively, the result confirms that obtained D-X-HN-PEG, productive rate is more than 95%.
Embodiment 4 terminal link molecule amounts are the preparation of the insulin-PEG complex of 2000 PEG chain
Step 1: molecular weight is the preparation of 2000 amino Polyethylene Glycol
Getting 8 gram (4 mM) relative molecular weights is 2000 mono methoxy polyethylene glycol, adds and to be equipped with in 500 milliliters of round-bottomed flasks of 300 milliliters of toluene, heats and steams 100 milliliters of toluene to take the moisture in the alcohol out of.Remaining solution adds equimolar pyridine and refluxes, and drips the thionyl chloride (SOCl of 2 times of moles
2), reducing the temperature to 90 ℃ after dropwising and continue reaction 4 hours, cooling removes by filter crystal, pressure reducing and steaming toluene then, residue ether sedimentation, sucking filtration vacuum drying obtain 19 and restrain products, and this is 2000 PEG-Cl for molecular weight.But with this product be dissolved in a small amount of absolute methanol join can be airtight and the reactor of bearing certain pressure in, it is airtight to add liquid ammonia, and 60 ℃ were reacted 24 hours, and opened reactor, the reactant liquor rotary evaporation removes deammoniation and methanol, obtains mean molecule quantity and be 2000 amino Polyethylene Glycol PEG-NH
2Step 2:PEG2000-NH-Gly (NH
2) preparation
The relative molecular weight of getting preparation among the embodiment 1 is 2000 PEG-NH
210 grams (2 mM) are dissolved in CH
2Cl
2In the mixed solution of/DMF (1: 3); the Fmoc-Glyr (Fmoc-threonine) that adds the commercially available amido protecting of 0.68 gram (2 mM); add DCC0.81 gram (4 mM) then, 4 ℃ of following stirring reactions 24 hours remove by filter the DCU precipitation; add an amount of piperidines then and take off the Fmoc protecting group; solution adds the ether sedimentation product, filters washing; vacuum drying obtains PEG2000-NH-Gly (NH
2).
Step 3: the preparation of removing the insulin intermediate of b30 amino acid residue
Getting 100 milligrams of insulin humans (Dongbao of Tonghua company) is dissolved in the 0.1M ammonium bicarbonate soln of 5 milliliters of pH8.3, add 1 milligram of Carboxypeptidase A, above-mentioned solution was 37 ℃ of insulations 2 hours, add the glacial acetic acid cessation reaction then, (110cm 1.6cmID) separates and removes pheron, collects 110 ~ 160 milliliters of component lyophilizing through SephadexG50 with the solution that obtains, the be removed insulin intermediate D of b30 amino acid residue, yield is 97%.
The preparation of step 4:D-Gly-NH-PEG2000 (this is a kind of of claim formula of D-X-HN-PEG)
(6) get in the step 3 20 milligrams of the insulin intermediate of removing the b30 amino acid residue of preparation, adds 0.15 milliliter of 1N glacial acetic acid and dissolve, obtain solution 1
(7) get the PEG-NH-Gly (NH for preparing among 600 milligrams of embodiment 2 of 3 milliliters of DMF/EtOH (3: 1) mixed liquor dissolving
2), this is a solution 2.
(8) merge solution 1,2, regulate pH6.5, add the trypsin with 1: 200 weight ratio of insulin intermediate again, 30 ℃ of reactions 24 hours with TEA (triethylamine).
(9) after reaction is finished, system is regulated pH3.0 with HAc, the solution that obtains separates with SephadexG50 removes trypsin, the modification component lyophilizing that obtains.
(10) lyophilized products is carried out gel electrophoresis analysis, rp-hplc analysis respectively, the result confirms that obtained RNH-Gly-D, productive rate is more than 95%.Specifically see description of drawings.
Embodiment 4
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Leu-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 16KD, and aminoacid is leucine, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 5
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Leu-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 14KD, and aminoacid is leucine, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 6
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Phe-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 13KD, and aminoacid is phenylalanine, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 7
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Trp-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 12KD, and aminoacid is tryptophan, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 8
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Ile-D: concrete raw material is the insulin intermediate D that takes off B30, and aliphatic alcohol is C
15H
31OH, aminoacid are isoleucine, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 9
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Ser-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 11KD, and aminoacid is Silk Amino Acids, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 10
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Met-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 9KD, and aminoacid is methionine, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 11
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Cys-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 8KD, and aminoacid is cysteine, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 12
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Glu-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 7KD, and aminoacid is paddy aminoacid, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 13
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Gln-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 6KD, and aminoacid is glutamine, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 14
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Asp-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 4KD, and aminoacid is aspartic acid, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 15
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Asn-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 3KD, and aminoacid is agedoite, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 16
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Arg-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 5KD, and aminoacid is arginine, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 17
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Val-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 10KD, and aminoacid is valine, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 18
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-His-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 20KD, and aminoacid is histidine, according to embodiment 1 described processing step and process conditions preparation.
Embodiment 19
CH
3O-(CH
2CH
2O) nCH
2CH
2The preparation of-Tyr-D: concrete raw material is the insulin intermediate D that takes off B30, CH
3O-(CH
2CH
2O) nCH
2CH
2The molecular weight of OH is 2KD, and aminoacid is tyrosine, according to embodiment 1 described processing step and process conditions preparation.
Claims (11)
1, a kind of mono-modified polyethylene glycol-insulin complex substance, it is characterized in that: its chemical structure of general formula is RNH-X-D, R is CH in the formula
3O-(CH
2CH
2O) nCH
2CH
2, n=50-500; X is the natural amino acid beyond the proline of genetic coding or the alpha-non-natural amino acid of an amino carboxyl; D is the insulin human of sloughing the natural Iletin II (Lilly) of b30 amino acid residue or sloughing the b30 amino acid residue.
2, a kind of mono-modified polyethylene glycol-insulin complex substance according to claim 1, it is characterized in that: X is a threonine.
3, a kind of mono-modified polyethylene glycol-insulin complex substance according to claim 1 is characterized in that: be connected for amido link between the carboxyl of R-NH and X; Between the carboxyl of the amino of X and the C-terminal B29 lysine of D is that amido link is connected.
4, a kind of mono-modified polyethylene glycol-insulin complex substance according to claim 1 is characterized in that: the precursor of R-NH is amino Polyethylene Glycol CH
3O-(CH
2CH
2O) nCH
2CH
2NH
2, its molecular weight ranges is 2KD-20KD.
5, a kind of preparation method of mono-modified polyethylene glycol-insulin complex substance is characterized in that: concrete synthesis step is as follows:
The preparation of the amino Polyethylene Glycol of the first step: with a certain amount of general formula is that (R represents CH to ROH
3O-(CH
2CH
2O) nCH
2CH
2) mono methoxy polyethylene glycol (n=50-500) join in the toluene, heating azeotropic removal of water branch, remaining solution adds the pyridine with the aliphatic alcohol equimolar amounts, back flow reaction also drips the thionyl chloride (SOCl of 2 times of aliphatic alcohol moles
2), reacted 2-6 hour in 80 ℃-100 ℃ again after dropwising, cold filtration is removed crystal, toluene is removed in distilling under reduced pressure then, and the product that obtains is R-Cl, and this material is put into reactor, with a small amount of absolute methanol dissolving, add liquid ammonia then, excess of ammonia and methanol were removed in decompression after still was driven in airtight following 60 ℃ of reactions in 12-24 hour, and obtaining general formula is R-NH
2Amino Polyethylene Glycol, standby;
The preparation of the second step RNH-X: get the amino acid derivativges that a certain amount of amino has been protected with Fmoc, its chemical general formula is Fmoc-(HN)-X-(COOH), be dissolved in dichloromethane CH
2Cl
2With 1 of dimethyl formamide (DMF): 1-1: add standby R-NH in the 6 volume ratio mixed solutions with the amino amino acid derivativges equimolar amounts of having protected with Fmoc
2The carbodiimide (DCC) that adds the amino acid derivativges mole that 2 times of amino have protected with Fmoc again, 0-10 ℃ stirring reaction 12-24 hour, remove by filter by-product DCU then, add excessive piperidines in the solution that obtains, stirring reaction is taken off the Fmoc protecting group, obtain RNH-X, standby, this moment, the carboxyl of X was connected with amino Polyethylene Glycol formation amide, and amino is free state;
The 3rd step was removed the preparation of the insulin intermediate of b30 amino acid residue: add natural Iletin II (Lilly) or insulin human in the ammonium bicarbonate soln of pH8.3, make it dissolving, the Carboxypeptidase A that adds catalytic amount then, effect is 2 hours in 37 ℃ of water-baths, adds sour cessation reaction then, and reactant liquor separates with SephadexG50, lyophilizing, obtain taking off the insulin intermediate of b30 amino acid, its B chain N-terminal is B29-Lys, and is standby;
The preparation of the polyethylene glycol-insulin complex substance that the 4th step is mono-modified: at first a certain amount of standby insulin intermediate of taking off the b30 amino acid residue is used the acetum dissolving of 0.5-2N, get solution 1, then standby RNH-X is dissolved in 1: 1-1: in the EtOH/DMF mixed solution of 6 volume ratios, the mol ratio of raw material insulin intermediate and RNH-X is 1: 10-1: 60, get solution 2, at last solution 1 and 2 is merged and adjusting pH to 6-7, the trypsin that adds catalytic amount, reaction is 12-24 hour in 20-35 ℃ water-bath, can obtain the mono-modified polyethylene glycol-insulin complex substance of end-product of the present invention.
6, the preparation method of a kind of mono-modified polyethylene glycol-insulin complex substance according to claim 5 is characterized in that: dripping pyridine afterreaction time that finishes in the preparation of the amino Polyethylene Glycol of the first step is 4 hours.
7, the preparation method of a kind of mono-modified polyethylene glycol-insulin complex substance according to claim 5 is characterized in that: adding the liquid ammonia response time in the preparation of the amino Polyethylene Glycol of the first step is 18 hours.
8, the preparation method of a kind of mono-modified polyethylene glycol-insulin complex substance according to claim 5 is characterized in that: the reaction temperature of the middle preparation RNH-X of the preparation of the second step RNH-X is 4 ℃, and the response time is 18 hours.
9, the preparation method of a kind of mono-modified polyethylene glycol-insulin complex substance according to claim 5 is characterized in that: to take off the acetum concentration of the insulin intermediate of b30 amino acid residue be 1N in dissolving in the preparation of the polyethylene glycol-insulin complex substance that the 4th step is mono-modified.
10, the preparation method of a kind of mono-modified polyethylene glycol-insulin complex substance according to claim 5 is characterized in that: raw material insulin intermediate and C in the preparation of the polyethylene glycol-insulin complex substance that the 4th step is mono-modified
nH
2n+1The mol ratio of NH-X is 1: 40.
11, the preparation method of a kind of mono-modified polyethylene glycol-insulin complex substance according to claim 5 is characterized in that: to merge and regulate pH value be 6.8 to solution 1 and 2 in the preparation of the polyethylene glycol-insulin complex substance that the 4th step is mono-modified.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102188367A (en) * | 2011-01-05 | 2011-09-21 | 山东新时代药业有限公司 | Insulin glargine injecta and preparation method thereof |
| CN105175514A (en) * | 2015-09-05 | 2015-12-23 | 苏州普罗达生物科技有限公司 | Polyethylene glycol modified insulin-like polypeptide and application thereof |
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2008
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102188367A (en) * | 2011-01-05 | 2011-09-21 | 山东新时代药业有限公司 | Insulin glargine injecta and preparation method thereof |
| CN105175514A (en) * | 2015-09-05 | 2015-12-23 | 苏州普罗达生物科技有限公司 | Polyethylene glycol modified insulin-like polypeptide and application thereof |
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| Publication number | Publication date |
|---|---|
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