Background and overview[1][2]
With the rapid development of biotechnology, more and more protein and peptide drugs are being used in the prevention and treatment of diseases. Although protein and peptide drugs have specific action sites and precise efficacy, they have shortcomings such as low solubility, poor stability, short half-life, and immunogenicity, which limit their administration routes and efficacy. Polyethylene glycol (PEG) modification can solve the above problems well. PEG is a pH-neutral, non-toxic, highly water-soluble hydrophilic polymer polymerized from epoxy ethylene monomer. In the site-directed modification of sulfhydryl groups of protein and peptide drugs, the most commonly used activated PEG is maleimide polyethylene glycol monomethyl ether (mPEG-MAL). In order to avoid cross-linking and agglomeration during the modification process, monomethoxypolyethylene glycol (mPEG-OH) is usually used as the synthetic raw material of the modifier.
Preparation[1]
1) Preparation of mPEG-OTs: Weigh 5g (1mmol) of mPEG5000-OH and place it in an eggplant-shaped bottle, add 20ml of methylene chloride to dissolve, then add 0.86ml of triethylamine (6mmol) and 1.14g of p-toluenesulfon Acid chloride (6mmol), stirred at room temperature for 24h. During the reaction, use TLC (methanol: dichloromethane = 1:6) to detect the reaction progress. When the raw material point disappears, indicating that the reaction is complete, wash three times with 0.1% sodium citrate solution, saturated sodium chloride solution, and anhydrous sulfuric acid. Sodium drying.
The solvent was removed by rotary evaporation under reduced pressure, anhydrous ether was added to precipitate the solid, and the product was filtered with suction. The product was a white solid, which was dried in a desiccator. 4.85g of product was obtained, with a yield of 97%. 1.3.2 Synthesis of mPEG-NH2: Dissolve 4.5gmPEG5000-OTs (0.9mmol) in 15mlN, N-dimethylformamide, add 1g phthalimide potassium salt (5.4mmol), and nitrogen at 120°C Protection reaction 4h. The solvent was removed by rotary evaporation under reduced pressure at 90°C, the residue was dissolved in 35 ml of absolute ethanol, 3.5 ml of hydrazine hydrate was added, and the reaction was heated to reflux at 90°C for 4 hours. The solvent was removed by rotary evaporation under reduced pressure at 60°C, and the product was dissolved in methylene chloride. Insoluble matter was filtered off, washed three times with pure water and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. TLC spot board. The solvent was evaporated under reduced pressure, anhydrous ether was added to precipitate the solid, and the product was filtered with suction. The product was a white powdery solid, which was dried in a desiccator. 4.2g of product was obtained, with a yield of 93%.
2) Synthesis of maleimide polyethylene glycol monomethyl ether: Dissolve 3gmPEG5000-NH2 (0.6mmol) in 30ml dioxane, and mix with 0.24g under the condition of magnetic stirring at 80℃ Maleic anhydride (2.4 mmol) was reacted for 30 min. After the reaction is completed, dioxane is removed by rotary evaporation at 55°C, and the ether is settled and dried. The obtained solid was dissolved in 30 ml of acetic anhydride, 1 g of anhydrous sodium acetate was added, and the reaction was stirred at 100°C for 45 min. Filter out the solids in the reaction solution, add 10 times the volume of acetic anhydride in pure water to the reaction solution, and then extract the water layer three times with dichloromethane. After washing the dichloromethane layer three times with saturated sodium chloride solution, add anhydrous sodium sulfate. dry. TLC detection. The solvent was evaporated under reduced pressure, the ether was settled, and suction filtered to obtain 2.3 g of light brown solid with a yield of 76%. 2 Results During the reaction, TLC detection was used to determine the presence of the product and the extent of the reaction. mPEG-OH dots develop color with iodine, mPEG-OTs dots develop color with iodine and 254nm UV, mPEG-NH2 ninhydrin develops color, and maleimide polyethylene glycol monomethyl ether dots develop UV color.
The final product was analyzed by RP-HPLC, the detection wavelength was 215nm, gradient elution A: 0.05% TFA/H2O, B: 0.05% TFA/70% CH3CN/H2O, the flow rate was 1ml/min. mPEG5000-MAL retention time is 29.273min. React maleimide polyethylene glycol monomethyl ether and cysteine-linked polypeptide in equivalent amounts of 1:3, 1:1, and 3:1 respectively. Use RP-HPLC to monitor the reaction progress and observe the maleic acid residues. The generation changes of the iminopolyethylene glycol monomethyl ether peak, polypeptide peak and new substance peak are consistent with the theoretical values, confirming its activity.
Main reference materials
[1] Research on the synthesis of maleimide-based polyethylene glycol monomethyl ether
