CN114716663B - Method for preparing polyethylene glycol modified lysine - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 31
- 229920001223 polyethylene glycol Polymers 0.000 title abstract description 47
- 239000002202 Polyethylene glycol Substances 0.000 title description 44
- 239000004472 Lysine Substances 0.000 claims abstract description 63
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims abstract description 61
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims abstract description 51
- 239000003960 organic solvent Substances 0.000 claims abstract description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 29
- 238000006243 chemical reaction Methods 0.000 claims description 25
- 239000002904 solvent Substances 0.000 claims description 24
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 239000011541 reaction mixture Substances 0.000 claims description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 4
- 239000007795 chemical reaction product Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 235000018977 lysine Nutrition 0.000 abstract description 55
- 229920001427 mPEG Polymers 0.000 abstract description 12
- 238000002360 preparation method Methods 0.000 abstract description 8
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 abstract description 3
- 150000002669 lysines Chemical class 0.000 abstract description 3
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 31
- 239000012043 crude product Substances 0.000 description 16
- 230000008569 process Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000005227 gel permeation chromatography Methods 0.000 description 6
- 239000003607 modifier Substances 0.000 description 6
- -1 pH adjustment Substances 0.000 description 6
- 239000007810 chemical reaction solvent Substances 0.000 description 5
- 230000035484 reaction time Effects 0.000 description 5
- 150000001408 amides Chemical class 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000012467 final product Substances 0.000 description 3
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
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- 238000001556 precipitation Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 150000001735 carboxylic acids Chemical class 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
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- 235000013922 glutamic acid Nutrition 0.000 description 2
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- 238000005342 ion exchange Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
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- 102000004169 proteins and genes Human genes 0.000 description 2
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- 229940126586 small molecule drug Drugs 0.000 description 2
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- ZJIFDEVVTPEXDL-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) hydrogen carbonate Chemical compound OC(=O)ON1C(=O)CCC1=O ZJIFDEVVTPEXDL-UHFFFAOYSA-N 0.000 description 1
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical class O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000003838 adenosines Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical class [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical class C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- 238000010189 synthetic method Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Abstract
Provided herein are methods of preparing two-arm PEG lysines comprising reacting NHS-activated mPEG carbonate (mPEG-SC) with lysine in the presence of an organic solvent and triethylamine. Compared with the existing preparation process, the preparation method of the two-arm PEG lysine provided by the invention is simple to operate and has higher yield.
Description
Technical Field
The application relates to a method for preparing polyethylene glycol modified lysine, in particular to a method for preparing PEG disubstituted lysine in an organic solvent.
Background
Polyethylene glycol (Polyethylene glycol, PEG) is a linear hydrophilic high molecular compound, and has the advantages of no toxicity, good biocompatibility and low antigenicity. After covalent coupling of PEG to some drug molecules (e.g., proteins, polypeptides, small molecule drugs, liposomes, etc.), these superior properties of PEG can be imparted to the resulting coupled drug molecules. Currently, there are tens of polyethylene glycol modified drugs on the market, including protein drugs such as PEG-modified adenosine dehydrogenase (PEG-ADA), PEG-modified asparaginase, PEG-modified interferon, and small molecule drugs such as PEG-modified camptothecin (PEG-CPT) and PEG-modified doxorubicin.
Polyethylene glycol modification techniques have been developed over several decades and a variety of PEG modifiers have been developed, including multi-arm (branched) PEG modifiers, such as two-arm PEG modifiers. Some advantages of two-arm PEG modifiers over single-arm PEG modifiers may include increased selectivity for modification sites; PEG loading is increased with limited modification sites; effectively preventing cells or other macromolecules (e.g., antibodies) from contacting the modified molecule, thereby increasing half-life and reducing immunogenicity, etc.
The use of lysine to prepare two-arm PEG modifiers has been reported in the literature. U.S. patent application US20130177961 discloses the preparation of two molecule mPEG-conjugated lysines by reacting methoxypolyethylene glycol succinimidyl carbonate (mPEG-SC) with lysine in boric acid buffer pH 8.0 for 24 hours, followed by purification, with a final product yield of around 50%. The reaction process involves the preparation of buffer solution, pH adjustment, acid adjustment and extraction are needed for post-treatment, the reaction time is as long as 24 hours, the yield is low, and the preparation method is difficult to meet the production requirement in practice. Chinese patent application CN105017522 discloses a process for preparing monomethoxy polyethylene glycol coupled glutamic acid comprising stirring a mixture of carboxylated monomethoxy polyethylene glycol, triethylamine, DCC, N-hydroxysuccinimide in methylene chloride overnight at room temperature. The patent application mentions that the product yield can reach 79% -85%. However, when we replace the glutamic acid with lysine, the yield of two-arm PEG lysine is extremely low, less than 10%.
Therefore, there is a need to develop a new method for preparing two-arm PEG lysine.
Disclosure of Invention
In one aspect, provided herein is a method of preparing a two-arm PEG lysine of formula (I), comprising reacting mPEG-SC of formula (II) with lysine in the presence of an organic solvent and triethylamine,
Wherein n in the formula (I) and the formula (II) are the same and are integers of 4 to 500.
In some embodiments, n is an integer between 80 and 150.
In some embodiments, the organic solvent comprises: 1) A solvent a, wherein the solvent a is selected from absolute ethanol, methanol, or DMF; and 2) a solvent B, wherein the solvent B is dichloromethane.
In some embodiments, the volume ratio of solvent a to solvent B is 4:1.
In some embodiments, the lysine, mPEG-SC, and triethylamine are fed in a molar ratio of 1:2.2:4.
In some embodiments, the reaction is carried out at 30 ℃ for about 1 hour.
In some embodiments, the method further comprises adding lysine to the reaction mixture after the reaction has been performed for about 1 hour; wherein the amount of the additional lysine is preferably 1/9 of the initial charge amount of lysine.
In some embodiments, the method comprises: after dissolving lysine with the solvent A, solvent B and mPEG-SC were added, and triethylamine was added after the mPEG-SC was dissolved, followed by reaction for 1h under stirring.
In some embodiments, the method comprises:
1) Dissolving lysine by using the solvent A, adding the solvent B and mPEG-SC, adding triethylamine after the mPEG-SC is dissolved, and then reacting for 1h under stirring; and
2) Lysine was added and the reaction was continued for 1h.
In some embodiments, the method further comprises precipitating a reaction product comprising the two-arm PEG lysine using Methyl Tertiary Butyl Ether (MTBE).
Compared with the existing preparation process, the preparation method of the two-arm PEG lysine provided by the invention is simple to operate and has higher yield.
Drawings
FIG. 1 is a graph showing the results of measuring the lysine content of two-arm PEG in the crude product by GPC. Wherein the abscissa is time (min) and the ordinate is the signal intensity value detected by the differential refractometer detector.
Detailed Description
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
"Two-arm PEG lysine", "PEG disubstituted lysine" or "branched carboxylic acid" are used interchangeably herein to refer to a product of one molecule of lysine linked to two PEG molecules through two amino groups in an amide linkage.
"PEG monosubstituted lysine" is the product of a molecule of lysine linked to a PEG molecule via an amino group via an amide linkage. In the preparation methods provided herein, it may be considered as an impurity molecule in the reaction product.
"Polyethylene glycol modified lysine" refers to a compound comprising a PEG moiety and a lysine moiety in the molecule, and may include the two-arm PEG lysines described above and/or PEG monosubstituted lysines.
Herein, references to polyethylene glycol (PEG) may include chemical derivatives thereof, for example, polyethylene glycol monomethyl ether (mPEG). The size of the PEG molecules (e.g., mPEG) used is not essentially limited, e.g., the average molecular weight may be in the range of 200 to 20000Da (e.g., 200, 400, 600, 800, 1000, 1500, 2000, 4000, 5000, 6000, 8000, 10000, 20000Da, etc.), and may comprise approximately 4-500 monomer molecules. In other words, the number n of CH 2CH2 O repeat units in a PEG molecule can be any integer between 4 and 500, for example, 5, 10, 20, 50, 100, 150, 200, 300, 400, 500, etc. Molecular weights or n values exceeding the above ranges are also possible.
"Crude product" refers to the product obtained after completion of the reaction by precipitation of MTBE alone, without further purification by subsequent ion exchange columns.
The method for preparing two-arm PEG lysine provided herein can be represented by the following reaction scheme:
NHS activated mPEG carbonate reacts with two amino groups in lysine in the presence of triethylamine, resulting in an m-PEG disubstituted lysine (also referred to herein as a "branched carboxylic acid") in which two mPEG molecules are bound to one lysine molecule via an amide linkage. The branched carboxylic acids obtained carry carboxyl functions and can be used for chemical modification of proteins or other small molecules after activation. It is noted that the number n of CH 2CH2 O repeat units of different arms may be different in a particular two-arm PEG lysine molecule prepared. This is because the starting materials used are typically not homogeneous molecules of mPEG carbonate activated by NHS. Thus, n as used herein may be considered as the number average (typically an integer value) of monomers in a population of PEG molecules. Those skilled in the art will recognize that such non-uniformities do not affect the practice of the methods provided herein,
The present invention is based, at least in part, on the unexpected discovery by the inventors that the purity and reaction yield of branched carboxylic acids in the reaction crude product can be significantly improved by varying the reaction solvent combination, wherein the absolute ethanol and methylene chloride mixed solvent is optimal. In addition, the inventors have unexpectedly found that the reaction yield can be further improved by changing the feeding manner.
The invention is further illustrated by the following specific examples.
Example 1 reaction solvent selection
Taking mPEG-SC with molecular weight of 5K as a raw material, and the feeding mole ratio is lysine: mPEG-SC: triethylamine = 1:2.2:4. 13.3mg of lysine (0.091 mmol) was weighed by analytical balance and added to a 25mL round bottom flask, placed in a stirrer, 16mL of reaction solvent A was added, dissolved by stirring in an oil bath at 32℃and 4mL of Dichloromethane (DCM) and 1g of mPEG-SC (0.2 mmol) were added, 50.5. Mu.L of triethylamine (0.36 mmol) was added after stirring and dissolution, stirred and reacted at 30℃for 1 hour, distilled off under reduced pressure, the solvent was concentrated off, precipitated with 20mL of methyl tert-butyl ether (MTBE), filtered and dried under vacuum to obtain a crude product containing branched carboxylic acid.
In addition to the target branched carboxylic acid, there may be mPEG monosubstituted lysines (i.e., only one amino group in the lysine molecule is bound to mPEG via an amide linkage) as well as unreacted starting materials in the crude product. Theoretically, the higher the branched carboxylic acid content in the reaction crude product, the higher the yield.
The mPEG coupling on lysine can be detected by Gel Permeation Chromatography (GPC). Specifically, gel chromatographic columns and phosphate buffer with the pH of 7.0 can be adopted, different peaks can appear according to different retention time of substances after loading, and the branched carboxylic acid content can be obtained according to the ratio of peak areas (normalization method). An example of the GPC detection results is shown in fig. 1. In the figure, a peak of 10K molecular weight is shown at about 14min, and a peak of 5K molecular weight is shown at about 16 min. The component with the molecular weight of 10K is the target product branched carboxylic acid, and the component with the molecular weight of 5K comprises PEG monosubstituted lysine and unreacted raw materials. The peak ratio of 10K was 90% by area percent, and the branched carboxylic acid content was considered to be 90% (wt).
The inventors conducted extensive screening of the reaction solvent A, and found that most solvents are unsuitable for the reaction, and that the objective product can be obtained in a high yield by using only a few solvents. These solvents and the reaction result data are shown in table 1 below.
TABLE 1 reaction solvent A and branched carboxylic acid content results obtained
Conclusion: the reaction effect is best by using a mixed system of absolute ethyl alcohol and DCM.
Example 2 optimization of feed mode
The inventors studied the manner of reaction addition in order to improve the yield.
2.1 Increasing lysine feeding ratio
In theory, increasing the lysine feed ratio makes it possible to increase the branched carboxylic acid content in the crude product. The inventors therefore first conducted experiments to increase the lysine feed ratio.
Except that the feed molar ratio was changed to lysine: mPEG-SC: triethylamine=1:2:4, i.e. lysine charge 14.7mg (0.1 mmol), was carried out in the same manner as in example 1. The branched carboxylic acid content of the obtained crude product was 82.3% by detection. The reaction time was prolonged by 1h, and the branched carboxylic acid content in the obtained crude product was 82.6%.
Further improving the lysine feeding proportion or prolonging the reaction time, and the content of the branched carboxylic acid in the crude product is not further increased.
2.2 Increasing the branched Carboxylic acid content by supplementing lysine
The inventors have unexpectedly found that by supplementing lysine, the branched carboxylic acid content in the crude product is facilitated to be increased.
Taking mPEG-SC with molecular weight of 5K as a raw material, and the feeding mole ratio is lysine: SC: triethylamine = 1:2.2:4. 13.3m g lysine (0.091 mmol) was weighed by analytical balance and added to a 25mL round bottom flask, placed in a stirrer, 16mL absolute ethanol was added, dissolved in an oil bath at 32℃with stirring, 4mL DCM and 1g mPEG-SC (0.2 mmol) were added, 50.5. Mu.L triethylamine (0.36 mmol) was added after stirring and dissolution, stirred at 30℃for 1 hour, and the branched carboxylic acid content in the reaction mixture was detected by sampling (sample A). 1.4mg of lysine (0.01 mmol) was added to the reaction mixture, the reaction was continued for 1 hour, and the reaction mixture was sampled (sample B) to detect the branched carboxylic acid content. 1.4mg of lysine (0.01 mmol) was further added thereto, the reaction was continued for 1 hour, and a sample (sample C) was taken to examine the branched carboxylic acid content in the reaction mixture. The addition of lysine and sampling were repeated until the branched carboxylic acid content no longer increased.
Experimental results: the branched carboxylic acid content in the samples was measured using the method described in example 1. The branched carboxylic acid content in sample A was 80.5%, the branched carboxylic acid content in sample B was 90.4%, and the branched carboxylic acid content in sample C was 90.5%. And the subsequent addition of lysine is continued, and the content of branched carboxylic acid is not increased.
Conclusion: in order to improve the yield of the target product, one lysine feeding can be added in the reaction process, and the addition of the lysine feeding amount, the addition of the lysine for more times or the extension of the reaction time in one feeding has no obvious effect.
Example 3 amplification experiments
Taking mPEG-SC with molecular weight of 5K as a raw material, and the feeding mole ratio is lysine: SC: triethylamine = 1:2.2:4. 266mg of lysine (1.82 mmol) is weighed by an analytical balance, added into a 1000mL round bottom flask, placed into a stirrer, 320mL of absolute ethyl alcohol is added, stirred and dissolved in an oil bath at 32 ℃, 80mL of DCM and 20g of mPEG-SC (4 mmol) are added, 1mL of triethylamine (7.2 mmol) is added after stirring and dissolving, the reaction is carried out for 1h under 30 ℃ with stirring, 29mg of lysine (0.2 mmol) is added again, the reaction is continued for 1h, the solvent is evaporated under reduced pressure, 400mL of MTBE is used for precipitation, filtration and vacuum drying are carried out, and 18.9g of crude product is obtained, the content of branched carboxylic acid is detected to be 90.6%, and the yield is 94.5%. Then 18.9g of the crude product was dissolved in 2L of purified water, loaded onto a DEAE ion exchange column, eluted with purified water for one column volume, eluted with 315us/cm sodium chloride solution, the eluate was collected, ph=3 was adjusted with HCl, extracted with DCM, dried over anhydrous sodium sulfate, filtered, concentrated by spin-evaporation under reduced pressure to remove the solvent, precipitated with 400ml of MTBE, filtered, dried under vacuum to finally yield 14.2g of branched carboxylic acid with yield=75%. The branched carboxylic acid content in the final product was found to be almost 100% by GPC. The molecular weight of the final product was determined by GPC to be about 10832.
Branched carboxylic acid NMR Data (DMSO): 1.2 to 1.4ppm (6H, 3,4,5 methylene of lysine); 1.6ppm (2H, 6 methylene of lysine); 3.14ppm (s, 3H, mPEG terminal methoxy groups); 3.49ppm (s, mPEG backbone methylene); 4.05ppm (t, 2H, -CH 2, -OCO-; 7.18ppm (t, 1H, -NH-lysine); 7.19ppm (d, 1H, -NH-lysine).
Advantages of the synthetic methods provided herein compared to the methods disclosed in U.S. patent application US20130177961 include, but are not limited to, the following:
1. In the reaction system, the process of the patent document needs to use buffer solution, needs to be configured, needs to adjust pH=8 again, has complex operation and poor control, and has simple and easy operation of the system of the prior process;
2. the reaction time of the process in the patent document is too long, the time is saved in the existing process, and the efficiency is high;
3. in the step of aftertreatment, as the process of the patent document is a water phase, the steps of acid adjustment, extraction and the like are involved, the time is consumed, the complexity is high, the steps cannot be involved in the existing process, the MTBE precipitation is mainly adopted, the operation is simple, the time is saved, and the yield is improved;
4. Although the subsequent purification steps are substantially consistent, the methods provided herein can increase the branched carboxylic acid content of the crude product from about 60% to about 90% of the patent literature process, on the branched carboxylic acid content of the crude product.
In general, the methods provided herein significantly improve branched carboxylic acid content and yield in the crude product by optimizing reaction conditions and dosing regime.
Claims (6)
1. A process for preparing a two-arm PEG lysine of formula (I) comprising reacting mPEG-SC of formula (II) with lysine in the presence of an organic solvent and triethylamine,
Wherein n in the formula (I) and the formula (II) are the same and are integers of 4 to 500,
The organic solvent includes: 1) A solvent a, wherein the solvent a is selected from absolute ethanol, methanol, or DMF; and 2) a solvent B, wherein the solvent B is methylene dichloride, and the volume ratio of the solvent A to the solvent B is 4:1,
The method comprises the following steps: after dissolving lysine with the solvent a, the solvent B and the mPEG-SC were added, and triethylamine was added after the mPEG-SC was dissolved, followed by reaction at 30 ℃ for 1h with stirring.
2. The method of claim 1, wherein n is an integer between 80 and 150.
3. The method of claim 1 or 2, wherein the lysine, mPEG-SC, and triethylamine are fed in a molar ratio of 1:2.2:4.
4. The method of claim 1 or 2, further comprising adding lysine to the reaction mixture after 1h of the reaction; wherein the amount of the added lysine is 1/9 of the initial feeding amount of the lysine.
5. The method of claim 4, further comprising continuing the reaction for 1h after the lysine is added.
6. The method of claim 1 or 2, further comprising precipitating a reaction product comprising the two-arm PEG lysine using methyl tert-butyl ether.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210337793.0A CN114716663B (en) | 2022-03-31 | Method for preparing polyethylene glycol modified lysine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210337793.0A CN114716663B (en) | 2022-03-31 | Method for preparing polyethylene glycol modified lysine |
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| Publication Number | Publication Date |
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| CN114716663A CN114716663A (en) | 2022-07-08 |
| CN114716663B true CN114716663B (en) | 2024-07-02 |
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Non-Patent Citations (1)
| Title |
|---|
| A Practical Synthesis of N -Monomethoxypoly(ethylene glycol)carbonyl-L-lysine Hydrochloride: A Precursor to Chiral and Mixed Branched Pegylated Reagents;Susan D. Van Arnum;Macromolecules;第908-912页 * |
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