CN110591077B - Method for preparing tyrosine oligopeptide and grafted monomethoxy polyethylene glycol through enzyme catalysis - Google Patents
Method for preparing tyrosine oligopeptide and grafted monomethoxy polyethylene glycol through enzyme catalysis Download PDFInfo
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- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 title claims abstract description 63
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 108010038807 Oligopeptides Proteins 0.000 title claims abstract description 43
- 102000015636 Oligopeptides Human genes 0.000 title claims abstract description 43
- 239000002202 Polyethylene glycol Substances 0.000 title claims abstract description 35
- 229920001223 polyethylene glycol Polymers 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 24
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 13
- 238000006555 catalytic reaction Methods 0.000 title claims abstract description 13
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 65
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims abstract description 39
- 238000006243 chemical reaction Methods 0.000 claims abstract description 29
- 239000004365 Protease Substances 0.000 claims abstract description 19
- 108091005804 Peptidases Proteins 0.000 claims abstract description 14
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 14
- 229920001577 copolymer Polymers 0.000 claims abstract description 14
- 229960004441 tyrosine Drugs 0.000 claims description 58
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 33
- 239000011541 reaction mixture Substances 0.000 claims description 28
- RSOCSVWZTHPBBQ-UHFFFAOYSA-N carbonic acid;pyrrolidine-2,5-dione Chemical compound OC(O)=O.O=C1CCC(=O)N1 RSOCSVWZTHPBBQ-UHFFFAOYSA-N 0.000 claims description 26
- 239000002244 precipitate Substances 0.000 claims description 24
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 14
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 14
- 235000019419 proteases Nutrition 0.000 claims description 13
- 239000002904 solvent Substances 0.000 claims description 11
- 238000003828 vacuum filtration Methods 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 10
- 238000000967 suction filtration Methods 0.000 claims description 10
- 229940088598 enzyme Drugs 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 7
- 239000008367 deionised water Substances 0.000 claims description 7
- 229910021641 deionized water Inorganic materials 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- VXYFARNRGZWHTJ-FVGYRXGTSA-N methyl (2s)-2-amino-3-(4-hydroxyphenyl)propanoate;hydrochloride Chemical compound Cl.COC(=O)[C@@H](N)CC1=CC=C(O)C=C1 VXYFARNRGZWHTJ-FVGYRXGTSA-N 0.000 claims description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims description 6
- PFYXSUNOLOJMDX-UHFFFAOYSA-N bis(2,5-dioxopyrrolidin-1-yl) carbonate Chemical compound O=C1CCC(=O)N1OC(=O)ON1C(=O)CCC1=O PFYXSUNOLOJMDX-UHFFFAOYSA-N 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 239000000376 reactant Substances 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 108091005658 Basic proteases Proteins 0.000 claims description 3
- 108090000526 Papain Proteins 0.000 claims description 3
- 235000019834 papain Nutrition 0.000 claims description 3
- 229940055729 papain Drugs 0.000 claims description 3
- 108090000145 Bacillolysin Proteins 0.000 claims description 2
- 108010004032 Bromelains Proteins 0.000 claims description 2
- 108091005507 Neutral proteases Proteins 0.000 claims description 2
- 102000035092 Neutral proteases Human genes 0.000 claims description 2
- 102000004142 Trypsin Human genes 0.000 claims description 2
- 108090000631 Trypsin Proteins 0.000 claims description 2
- 235000019835 bromelain Nutrition 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000012588 trypsin Substances 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 6
- 229920001184 polypeptide Polymers 0.000 abstract description 6
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 6
- 238000003786 synthesis reaction Methods 0.000 abstract description 6
- 150000001413 amino acids Chemical class 0.000 abstract description 4
- 239000003054 catalyst Substances 0.000 abstract description 4
- 238000010511 deprotection reaction Methods 0.000 abstract description 3
- 238000007086 side reaction Methods 0.000 abstract description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 235000019441 ethanol Nutrition 0.000 description 5
- 239000000872 buffer Substances 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 108010033949 polytyrosine Proteins 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical group COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000004973 liquid crystal related substance Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- -1 methyl (-OCH3) Chemical group 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000006320 pegylation Effects 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 1
- DTQVDTLACAAQTR-DYCDLGHISA-N trifluoroacetic acid-d1 Chemical compound [2H]OC(=O)C(F)(F)F DTQVDTLACAAQTR-DYCDLGHISA-N 0.000 description 1
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- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
- C08G65/32—Polymers modified by chemical after-treatment
- C08G65/329—Polymers modified by chemical after-treatment with organic compounds
- C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
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- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
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Abstract
The invention relates to a method for preparing tyrosine oligopeptide and grafted monomethoxy polyethylene glycol by enzyme catalysis, belonging to the technical field of enzyme catalysis. And (2) catalyzing and synthesizing tyrosine oligopeptide by using protease as a catalyst, and grafting monomethoxypolyethylene glycol to the amino terminal of the oligomeric tyrosine oligopeptide to prepare the tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer. The yield reaches 15-55%. The catalyst in the polypeptide synthesis step is enzyme, has the advantages of mild reaction conditions, more environment-friendly property, less side reaction and the like, and avoids the complicated protection and deprotection steps, thereby providing a novel method for preparing the amino acid polypeptide-monomethoxy polyethylene glycol.
Description
Technical Field
The invention relates to a method for preparing tyrosine oligopeptide and grafted monomethoxy polyethylene glycol by enzyme catalysis, belonging to the technical field of enzyme catalysis.
Background
The artificial polypeptide is a polyamino compound formed by taking natural amino acid as a monomer to carry out polymerization reaction through amido bond, and the properties of the artificial polypeptide are similar to those of natural peptide, polyamino acid and protein. The material formed by the artificial polypeptide has good biocompatibility and degradability. The polypeptide or the modified oligomer thereof shows unique structural properties such as self-assembly, liquid crystal behavior and the like, has stronger plasticity, and has great application potential in the fields of biology, medicine and natural high polymer materials. The polytyrosine can be applied to the fields of drug delivery, gene therapy and the like after being modified by pegylation. In addition, each phenolic hydroxyl group of the polytyrosine molecule generates two electrons in the oxidation process, so the polytyrosine molecule is used as an electroactive marker for signal amplification of an electrochemical biosensor.
Examples of the polymerization method of amino acids include a solid phase method, an NCA method and an enzyme catalysis method. Compared with other polymerization methods, the enzyme catalysis method has the advantages of mild reaction conditions, more environment-friendly property, less side reactions and the like, and avoids fussy protection and deprotection steps.
The invention aims to synthesize tyrosine oligopeptide under the catalysis of protease as a catalyst, and then graft monomethoxypolyethylene glycol on the amino terminal of the oligomeric tyrosine oligopeptide to prepare the tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer.
Disclosure of Invention
The invention aims to overcome the defects and provide a method for preparing tyrosine oligopeptide and grafted monomethoxy polyethylene glycol through enzyme catalysis, which has mild reaction conditions and is more environment-friendly.
The technical scheme of the invention is a method for preparing tyrosine oligopeptide and grafted monomethoxy polyethylene glycol by enzyme catalysis, which comprises the following steps: (1) synthesizing tyrosine oligopeptide under the catalysis of protease; (2) preparing monomethoxy polyethylene glycol succinimide carbonate; (3) and (3) grafting the tyrosine oligopeptide with monomethoxy polyethylene glycol to prepare a tyrosine oligopeptide-monomethoxy polyethylene glycol copolymer.
(1) Enzymatic synthesis of tyrosine oligopeptides: adding L-tyrosine methyl ester hydrochloride, dimethyl sulfoxide and protease into a phosphate buffer solution, placing a reaction mixture into a constant-temperature oscillator for reaction, centrifuging after the reaction is finished, taking a precipitate, washing with deionized water and ice ethanol twice respectively, and freeze-drying to obtain a product tyrosine oligopeptide.
The method comprises the following specific steps: adding 0.5-2.0 g of L-tyrosine methyl ester hydrochloride and 2-5U of protease into a disodium hydrogen phosphate-citric acid buffer solution with the volume of 2.0-3.0 mL, the pH value of 5.5-8.0 and the concentration of 0.1-0.2M, and then adding dimethyl sulfoxide accounting for 2.5-25% of the volume of the buffer solution; placing the mixture in a constant temperature oscillator at 200-600 rpm and 30-70 ℃ for reaction for 5-10 hours; and after the reaction is finished, centrifuging the reaction mixture for 3-10 minutes at 5000-10000 rpm, washing the precipitate twice with deionized water and glacial ethanol respectively, and freeze-drying to obtain the tyrosine oligopeptide.
The protease is any one of bromelain, papain, trypsin, neutral protease and alkaline protease.
The phosphate buffer solution is 0.1-0.2M disodium hydrogen phosphate-citric acid buffer solution, and the pH value of the phosphate buffer solution is 5.5-8.0.
The addition amount of the dimethyl sulfoxide is 2.5-25% of that of the phosphate buffer solution according to the volume concentration.
(2) Preparation of monomethoxypolyethylene glycol succinimide carbonate: adding monomethoxy polyethylene glycol, N' -disuccinimidyl carbonate and 4- (dimethylamino) pyridine into anhydrous tetrahydrofuran, and stirring for reaction; and (3) distilling the obtained reactant in vacuum, pouring the distilled reactant into precooled anhydrous ether, carrying out reduced pressure suction filtration, and collecting the precipitate to obtain the monomethoxy polyethylene glycol succinimide carbonate.
Adding 1-2 g of monomethoxy polyethylene glycol, 5-8 g of N, N' -disuccinimidyl carbonate and 3-6 g of 4- (dimethylamino) pyridine into 25mL of anhydrous tetrahydrofuran, and reacting for 2-6 hours at a stirring speed of 200-400 rpm and a temperature of 30-60 ℃; and after the reaction is finished, distilling the reaction mixture at 25-40 ℃ in vacuum to remove the solvent, pouring the solvent into pre-cooled anhydrous ether, and carrying out suction filtration under reduced pressure of 0-10 kPa to collect the precipitate, wherein the obtained precipitate is methoxypolyethylene glycol succinimide carbonate.
The monomethoxypolyethylene glycol has an average molecular weight of 200, 400 or 1000 Da.
(3) Preparation of tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer: adding tyrosine oligopeptide, methoxy polyethylene glycol succinimide carbonate and triethylamine into absolute ethyl alcohol, mixing and reacting under the protection of nitrogen; standing overnight after the reaction is finished, performing vacuum filtration, performing vacuum drying, and then dissolving in acetone; standing again overnight, and carrying out vacuum filtration to obtain the grafted product tyrosine oligopeptide-monomethoxy polyethylene glycol copolymer.
Adding 2-5 g of tyrosine oligopeptide, 2.4-10 g of monooxypolyethylene glycol succinimide carbonate and 12-60 g of triethylamine into 15mL of anhydrous methanol; reacting the reaction mixture for 10-24 h at 30-50 ℃ under the protection of nitrogen; after the reaction is finished, standing the reaction mixture at 0 ℃ overnight, and carrying out vacuum filtration under the reduced pressure of 0-10 kPa; vacuum drying the filtrate at 30-70 ℃ for 6-10 hours, and adding acetone preheated to 30-40 ℃ until the acetone is completely dissolved; and standing the obtained solution at 0 ℃ overnight, and carrying out vacuum filtration under 0-10 kPa to obtain a precipitate which is a tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer.
The weight ratio of the tyrosine oligopeptide, the monomethoxy polyethylene glycol succinimide carbonate and the triethylamine is 1:1.2: 6-1: 2: 12.
The 1HNMR detection is carried out on the tyrosine oligopeptide-monomethoxy polyethylene glycol copolymer by the following method: the 1HNMR spectra of the copolymers were determined at room temperature using an AVANCE III HD-400 MHz NMR spectrometer with CF3COOD as solvent and Tetramethylsilane (TMS) as internal standard.
The invention has the beneficial effects that: the catalyst in the polypeptide synthesis step is enzyme, has the advantages of mild reaction conditions, more environment-friendly property, less side reaction and the like, and avoids the complicated protection and deprotection steps, thereby providing a novel method for preparing the amino acid polypeptide-monomethoxy polyethylene glycol.
Drawings
FIG. 1 is a scheme for detecting a tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer by HNMR in example 11.
Detailed Description
In the following examples, the protease was obtained from Waokay Biotechnology Ltd, the buffer was obtained from Nanchang rain and dew laboratory facilities Ltd, the other reagents were obtained from Chemicals group Ltd or Huaian petrochemical plant of Jiangsu province, and the 4- (dimethylamino) pyridine was obtained from Aradin Biotechnology Ltd.
Example 1
(1) Protease catalyzed synthesis of tyrosine oligopeptides: to 2.0 mL of 0.1M disodium hydrogen phosphate-citric acid buffer pH5.8 was added 0.5 g of L-tyrosine methyl ester hydrochloride, dimethyl sulfoxide and 2U of protease. The volume fraction of dimethyl sulfoxide was 2.5% phosphate buffer. The reaction mixture was placed in a constant temperature shaker (600 rpm) and reacted at 30 ℃ for 10 hours. After the reaction is finished, centrifuging the reaction mixture for 3 minutes at 10000rpm, washing the precipitate twice by deionized water and glacial ethanol respectively, and freeze-drying to obtain the product tyrosine oligopeptide.
(2) Preparation of monomethoxypolyethylene glycol succinimide carbonate: to 25mL of anhydrous tetrahydrofuran were added 2g of monomethoxy polyethylene glycol, 5g of N, N' -disuccinimidyl carbonate, and 3 g of 4- (dimethylamino) pyridine. The average molecular weight of the monomethoxypolyethylene glycol is 200 Da. The reaction mixture was reacted at 30 ℃ for 6 hours under stirring (200 rpm). And after the reaction is finished, distilling the reaction mixture at 40 ℃ in vacuum to remove the solvent, pouring the solvent into precooled anhydrous ether, carrying out suction filtration under reduced pressure of 0-10 kPa, and collecting the precipitate, wherein the obtained precipitate is monomethoxy polyethylene glycol succinimide carbonate.
(3) Preparation of tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer: 2g of tyrosine oligopeptide, methoxypolyethylene glycol succinimide carbonate and triethylamine were added to 15mL of anhydrous methanol. The weight ratio of the tyrosine oligopeptide, the methoxy polyethylene glycol succinimide carbonate and the triethylamine is 1: 2: 12. The reaction mixture was reacted for 10 h at 30 ℃ under nitrogen. After the reaction is finished, standing the reaction mixture at 0 ℃ overnight, and carrying out suction filtration under reduced pressure of 0-10 kPa. After drying the filtrate at 30 ℃ for 6 hours under vacuum, acetone preheated to 40 ℃ was added until all dissolved. Standing the obtained solution at 0 ℃ overnight, and carrying out vacuum filtration under 0-10 kPa to obtain a precipitate which is a tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer with the yield of 15%.
By passing1Detecting the substitution degree of tyrosine oligopeptide-monomethoxy polyethylene glycol copolymer by HNMR method, specifically as shown in figure 1, wherein a is-CH in polyethylene glycol monomethyl ether chain segment3Chemical shift peak of hydrogen; f is a hydrogen chemical shift peak of a terminal methyl (-OCH3) of the tyrosine oligopeptide. From the peak area ratioThe degree of substitution to the tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer was 1.1.
Example 2
(1) Protease catalyzed synthesis of tyrosine oligopeptides: to 2.5 mL of 0.15M disodium hydrogen phosphate-citric acid buffer pH7.0 was added 1 g of L-tyrosine methyl ester hydrochloride, dimethyl sulfoxide and 3.5U of papain. The volume fraction of dimethyl sulfoxide was 15% phosphate buffer. The reaction mixture was placed in a constant temperature shaker (600 rpm) and reacted at 30 ℃ for 8 hours. After the reaction is finished, centrifuging the reaction mixture for 3 minutes at 10000rpm, washing the precipitate twice by deionized water and glacial ethanol respectively, and freeze-drying to obtain the product tyrosine oligopeptide.
(2) Preparation of monomethoxypolyethylene glycol succinimide carbonate: to 25mL of anhydrous tetrahydrofuran were added 1.5g of monomethoxy polyethylene glycol, 6g of N, N' -disuccinimidyl carbonate, and 6g of 4- (dimethylamino) pyridine. The average molecular weight of the monomethoxypolyethylene glycol is 400 Da. The reaction mixture was reacted at 30 ℃ for 6 hours under stirring (200 rpm). And after the reaction is finished, distilling the reaction mixture at 40 ℃ in vacuum to remove the solvent, pouring the solvent into precooled anhydrous ether, carrying out suction filtration under reduced pressure of 0-10 kPa, and collecting the precipitate, wherein the obtained precipitate is monomethoxy polyethylene glycol succinimide carbonate.
(3) Preparation of tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer: 3 g of tyrosine oligopeptide, methoxypolyethylene glycol succinimide carbonate and triethylamine are added into 15ml of anhydrous methanol. The weight ratio of the tyrosine oligopeptide, the methoxypolyethylene glycol succinimide carbonate and the triethylamine is 1:1.5: 6. The reaction mixture was reacted for 16 h at 40 ℃ under nitrogen. After the reaction is finished, standing the reaction mixture at 0 ℃ overnight, and carrying out suction filtration under reduced pressure of 0-10 kPa. After drying the filtrate at 30 ℃ for 6 hours under vacuum, acetone preheated to 40 ℃ was added until all dissolved. Standing the obtained solution at 0 ℃ overnight, and carrying out vacuum filtration under 0-10 kPa to obtain a precipitate which is a tyrosine oligopeptide-monomethoxy polyethylene glycol copolymer with the yield of 30%.
By passing1The substitution degree of the tyrosine oligopeptide-monomethoxy polyethylene glycol copolymer detected by HNMR means is 1.01.
Example 3
(1) Protease catalyzed synthesis of tyrosine oligopeptides: to 2.5 mL of 0.2M, pH 8.0.0 disodium hydrogen phosphate-citric acid buffer was added 1 g of L-tyrosine methyl ester hydrochloride, dimethyl sulfoxide and 3.5U of alkaline protease. The volume fraction of dimethyl sulfoxide was 25% phosphate buffer. The reaction mixture was placed in a constant temperature shaker (600 rpm) and reacted at 70 ℃ for 10 hours. After the reaction is finished, centrifuging the reaction mixture for 3 minutes at 10000rpm, washing the precipitate twice by deionized water and glacial ethanol respectively, and freeze-drying to obtain the product tyrosine oligopeptide.
(2) Preparation of monomethoxypolyethylene glycol succinimide carbonate: to 25mL of anhydrous tetrahydrofuran were added 2g of monomethoxy polyethylene glycol, 8g of N, N' -disuccinimidyl carbonate, and 5g of 4- (dimethylamino) pyridine. The average molecular weight of the monomethoxypolyethylene glycol is 1000 Da. The reaction mixture was reacted at 30 ℃ for 6 hours under stirring (200 rpm). And after the reaction is finished, distilling the reaction mixture at 40 ℃ in vacuum to remove the solvent, pouring the solvent into precooled anhydrous ether, carrying out suction filtration under reduced pressure of 0-10 kPa, and collecting the precipitate, wherein the obtained precipitate is monomethoxy polyethylene glycol succinimide carbonate.
(3) Preparation of tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer: 5g of tyrosine oligopeptide, oxypolyethylene glycol succinimide carbonate and triethylamine are added into 15mL of anhydrous methanol. The weight ratio of the tyrosine oligopeptide, the methoxy polyethylene glycol succinimide carbonate and the triethylamine is 1: 2: 12. The reaction mixture was reacted for 24h at 50 ℃ under nitrogen. After the reaction is finished, standing the reaction mixture at 0 ℃ overnight, and carrying out suction filtration under reduced pressure of 0-10 kPa. After drying the filtrate at 30 ℃ for 6 hours under vacuum, acetone preheated to 40 ℃ was added until all dissolved. Standing the obtained solution at 0 ℃ overnight, and carrying out vacuum filtration under 0-10 kPa to obtain a precipitate which is a tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer, wherein the yield is 55%.
By passing1The substitution degree of the tyrosine oligopeptide-monomethoxy polyethylene glycol copolymer detected by HNMR means is 1.06.
Claims (7)
1. The method for grafting tyrosine oligopeptide by using monomethoxypolyethylene glycol is characterized by comprising the following steps of: adding tyrosine oligopeptide, monomethoxy polyethylene glycol succinimide carbonate and triethylamine into anhydrous methanol, mixing and reacting under the protection of nitrogen; standing overnight after the reaction is finished, performing vacuum filtration, performing vacuum drying, and then dissolving in acetone; standing again overnight, and performing vacuum filtration to obtain a grafted product, namely the tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer;
the tyrosine oligopeptide is synthesized through enzyme catalysis, and the preparation method of the tyrosine oligopeptide comprises the following steps: adding L-tyrosine methyl ester hydrochloride, dimethyl sulfoxide and protease into a phosphate buffer solution, placing a reaction mixture into a constant-temperature oscillator for reaction, centrifuging after the reaction is finished, taking a precipitate, washing with deionized water and ice ethanol twice respectively, and freeze-drying to obtain a product tyrosine oligopeptide.
2. The method for grafting tyrosine oligopeptide by monomethoxypolyethylene glycol according to claim 1, which is characterized by comprising the following steps: adding 2-5 g of tyrosine oligopeptide, 2.4-10 g of monooxypolyethylene glycol succinimide carbonate and 12-60 g of triethylamine into 15mL of anhydrous methanol; reacting the reaction mixture for 10-24 h at 30-50 ℃ under the protection of nitrogen; after the reaction is finished, standing the reaction mixture at 0 ℃ overnight, and carrying out vacuum filtration under the reduced pressure of 0-10 kPa; vacuum drying the filtrate at 30-70 ℃ for 6-10 hours, and adding acetone preheated to 30-40 ℃ until the acetone is completely dissolved; and standing the obtained solution at 0 ℃ overnight, and carrying out vacuum filtration under 0-10 kPa to obtain a precipitate which is a tyrosine oligopeptide-monomethoxypolyethylene glycol copolymer.
3. The method of claim 1, wherein the tyrosine oligopeptide is prepared by the following steps: adding 0.5-2.0 g of L-tyrosine methyl ester hydrochloride and 2-5U of protease into a disodium hydrogen phosphate-citric acid buffer solution with the volume of 2.0-3.0 mL, the pH value of 5.5-8.0 and the concentration of 0.1-0.2M, and then adding dimethyl sulfoxide accounting for 2.5-25% of the volume of the buffer solution; placing the mixture in a constant temperature oscillator at 200-600 rpm and 30-70 ℃ for reaction for 5-10 hours; and after the reaction is finished, centrifuging the reaction mixture for 3-10 minutes at 5000-10000 rpm, washing the precipitate twice with deionized water and glacial ethanol respectively, and freeze-drying to obtain the tyrosine oligopeptide.
4. The method of grafting tyrosine oligopeptide with monomethoxypolyethylene glycol according to claim 1, wherein: the protease is bromelain, papain, trypsin, neutral protease or alkaline protease.
5. The method of claim 1, wherein the monomethoxypolyethylene glycol is grafted with tyrosine oligopeptide, and the monomethoxypolyethylene glycol succinimide carbonate is prepared by the following steps: adding monomethoxy polyethylene glycol, N' -disuccinimidyl carbonate and 4- (dimethylamino) pyridine into anhydrous tetrahydrofuran, and stirring for reaction; and (3) distilling the obtained reactant in vacuum, pouring the distilled reactant into precooled anhydrous ether, carrying out reduced pressure suction filtration, and collecting the precipitate to obtain the monomethoxy polyethylene glycol succinimide carbonate.
6. The method of claim 5, wherein the preparation of monomethoxypolyethylene glycol succinimide carbonate comprises the steps of: adding 1-2 g of monomethoxy polyethylene glycol, 5-8 g of N, N' -disuccinimidyl carbonate and 3-6 g of 4- (dimethylamino) pyridine into 25mL of anhydrous tetrahydrofuran, and reacting for 2-6 hours at a stirring speed of 200-400 rpm and a temperature of 30-60 ℃; and after the reaction is finished, distilling the reaction mixture at 25-40 ℃ in vacuum to remove the solvent, pouring the solvent into pre-cooled anhydrous ether, and carrying out suction filtration under reduced pressure of 0-10 kPa to collect the precipitate, wherein the obtained precipitate is methoxypolyethylene glycol succinimide carbonate.
7. The method of grafting tyrosine oligopeptide with monomethoxypolyethylene glycol according to claim 5, wherein: the monomethoxypolyethylene glycol has an average molecular weight of 200, 400 or 1000 Da.
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