Editing Synthesis Chitosan Folic Acid

[[Image:Folic_acid_PEG_Chitosan.jpg]]
[[Image:Chitosan_folic_acid_symthesis.jpg|560px]]


'''Folate-NHS-ester:&nbsp;'''folic acid (300 mg, 0.67 mmol) was dissolved in 12 mL of dry dimethylsulfoxide to which 93 mg (0.45 mmol) of '''dicyclohexylcarbodiimide (DCC)''' and 77 mg (0.67 mmol) of '''N-hydroxysuccinimide (NHS) '''were added. The mixture was stirred at room temperature for 6 hours. The white precipitate (a side product of the reaction) was removed by filtration. The filtrate was added to 100 mL of 30% acetone in diethyl ether. The resulting yellow precipitate (folate-NHS-ester) was collected by filtration and washed with acetone and ether. It was used immediately in the next step of synthesis. The yield was 250 mg (83%). 

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'''Folate-PEG-COOH:''' '''NH2-PEG-COOH '''(450 mg, 0.13 mmol, molecular weight 3400 Da, Shearwater Polymers, Huntsville, AL) was dissolved in 100 mL of dry pyridine. The folate-NHS-ester (approximately 250 mg, 0.55 mmol) was added to the solution and the reaction mixture was stirred overnight in the dark. Upon completion of the reaction, the pyridine was evaporated in vacuo and the remaining solid was dissolved in 20 mL of deionized water. The product was purified by dialysis against pH 7.4 phosphate buffer (10 mM) for one day and against deionized water for two days. The yield was 450 mg (76%). 

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'''Chitosan-PEG-folate''': Chitosan (200 mg, 1.0 mmol amino groups) was dissolved in 20 mL of 2% acetic acid aqueous solution. To this solution, folate-PEG-COOH (40 mg, 10 μmol in 5 mL of dimethylsulfoxide and preactivated by reacting with '''[3-(-dimethylamino)propyl]carbodiimide (EDC''', 2.5 mg, 15 μmol) and '''NHS''' (5.8 mg, 25 μmol) for 4 hours) was added dropwise. The reaction was continued in the dark for 20 hours with constant stirring. The reaction mixture was then dialyzed using a membrane (molecular weight cutoff, 12–14 kDa) against deionized water for 3 days. The chitosan-PEG-folate conjugate was recovered by freeze-drying. The yield was 210 mg. 

[[Category:Paul]] [[Category:Transfection]]