Editing Synthesis of PEI Cho (section)

== Protocol 2<br>  ==

Synthesis of Branched PEI-Chol. 

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[[Image:Branch PEI Chol synthesis.png]] 

Stage I. <br>1. Dissolve Benzylchloroformate (1.76 g, 1.5 mL, 10.36 mmol) in methylene chloride (5 mL) and place in a three-neck flask under nitrogen atmosphere. 

2. Dissolve imidazole (1.4 g, 20.6 mmol) in methylene chloride (20 mL) and place in an addition funnel. 

3. Cool three-neck flask to 0 °C using ice and add imidazole solution gradually over 30 min. 

4. Stir the mixture at room temperature for 2 h. 

5. Add methylene chloride (25 mL) and citric acid (10%; 25 mL). 

6. Separate layers and wash the organic fraction with citric acid (10%; 25 mL). 

7. Dry organic component over magnesium sulfate and concentrate in vacuo. 

8. Dry the residue under high vacuum for 2 h at ambient temperature. 

Stage II. 

9. Add DMAP (35 mg) and methylene chloride (25 mL) to the product of Stage I 

10. Cool the mixture to 0 °C under nitrogen atmosphere. 

11. Add PEI (MW 1800; 1.7 g, âŒ10 mmol PEI repeat unit) in methylene chloride (25<br>mL) gradually over 30 min. 

12. Stir for 18 h at ambient temperature. 

13. Concentrate in vacuo and thoroughly wash three times with ethyl ether. 

Stage III. 

14. Dissolve cholesteryl chloroformate (600 mg, 1.34 mmol) in methylene chloride (10 mL) 

15. Add dropwise quickly with the product of Stage II (PEI-CBz). 

16. Add triethylamine (1 mL) in methylene chloride (10 mL) cooled to under -10 °C by ice-salt bath, under nitrogen atmosphere, under stirring 

17. Stir the reaction mixture for 6 h at 0 °C and for another 6 h at ambient temperature. 

18. Concentrate the product in vacuo at ambient temperature. 

19. Wash three times with ethyl ether. 

20. Remove residual cholesteryl chloroformate with ethyl ether. 

<br>Stage IV. 

21. Dissolve the product of stage III (Chol-PEI-CBz) in 25 mL of acetic acid 

22. Add 50 mg of 10% palladium on activated carbon (Pd/C). 

23. Purge the solution with nitrogen. 

24. Stir under hydrogen at atmospheric pressure for âŒ8 h. 

25. Filter the reaction mixture 

26. Wash with 200 mL of ethyl acetate/acetic acid (9/1 v/v). 

27. Concentrate in vacuo to give a residue. 

28. Dissolve the crude product in 20 mL of 1 N NaOH. 

29. Precipitate with excessive acetone below 0 °C. 

30. Wash three times with ethyl ether.<br> 

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