Editing Synthesis of CBA-PolyAmines (section)

== Older Protocol (simple)  ==

'''Sigma'''<br> 

Hyperbranched poly(ethylenimine) (bPEI, 25 kDa)<br> 

tert-Butyl-N-(6-aminohexyl)carbamate (N-Boc-1,6-diaminohexane, N-Boc-DAH)<br> 

trifluoroacetic acid (TFA)<br> 

triisopropylsilane<br> 

triisobutylsilane<br> 

N,N-diisopropylethylamine (DIPEA)<br> 

piperidine<br> 

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)<br> 

chloroquine diphosphate salt<br> 

trypan blue solution (0.4%)<br> 

DL-buthionine-sulfoxamine<br> 

'''PolySciences''': N,N′-cystaminebisacrylamide (CBA)&nbsp;<br> 

Novabiochem: 2-(1H-benzotriazole-1-yl) 1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)&nbsp;<br> 

'''Anaspec''': Fmoc-L-Arg(pbf)-OH <br> 

<br> 

'''Procedure:'''<br> 

Dissolve equimolar amounts of N,N′-cystaminebisacrylamide (CBA) and corresponding polyamines,N-Boc-DAH, linear polyethylenimine (LPEI, MW 473), triethylenetetramine (TETA) and spermine (SPE)&nbsp;in a minimal volume of methanol containing 10% water, MeOH/H2O solution (9:1, v/v). 

Stire in&nbsp;tightly-closed vials under argon overnight at 60°C (or in the dark under a nitrogen atmosphere). (Michael reaction) 

Add an additional amount of polyamine&nbsp;(20%) (or 10% mole of N-Boc-DAH), dissolved in 1 mL of methanol to viscous solutions 

Let the reaction&nbsp;continue for another 3 h at 65°C&nbsp; to consume unreacted acrylamide functional groups 

Precipitation with diethyl ether. Disolve the pellet in water and adjust with 1M HCl to pH 5&nbsp; to obtain homogeneous solutions 

If N-Boc is used, add N-Boc removal reagent solution (TFA: triisobutylsilane: H2O=95:2.5:2.5, v/v) at ice bath temperature for 30 min 

Precipitation with diethyl ether, dissolve in ultrapure water<br> 

Dialyze (MWCO 1,000 Da) several times&nbsp;against deionized water. 

The polymeric products, pLPEI, pTETA and pSPE, can be&nbsp;concentrated in vacuo and lyophilized to yield sticky solid. 

The typical yields varied from 40 to 75%. 

<br> 

Add 4 equivalents of Fmoc-Arg(pbf)-OH and HBTU, and 8 equivalents of DIPEA in DMF at room temperature 

let react for 2 days.&nbsp; 

Add excess of diethyl ether&nbsp; to reaction mixture to precipitate and remove unreacted reagents, remove once 

Add&nbsp; an equal volume of piperidine solution (30% in DMF) at room temperature for 30 min to remove the Fmoc groups of grafted Fmoc-Arg(pbt)-OH. 

After two additional precipitations with ether, add deprotection reagent solution (TFA: triisopropylsilane: H2O=95:2.5:2.5, v/v) to the precipitates to deprotect the pbf groups of arginine residues at room temperature for 30 min. 

Do another precipitation with ether 

Dialyse the final product, poly(CBA-DAH-R)&nbsp; against ultra-pure water overnight 

lyophilize.<br> 

<br> 

<br> 

[[Image:CBA polyamine synthesis.png]] 

[[Image:Cytotoxicity CBA polyamine.png]] 

[[Image:Transfection efficiency CBA polyamine.png]] 

<br> 

[[Image:Abp synthesis.png|700px|Abp synthesis.png]] 

[[Image:ABP cell viability.png|500px|ABP cell viability.png]] 

[[Image:ABP transfection efficiency.png|500px|ABP transfection efficiency.png]] 

[[Image:ABP transfection efficiency 1.png|500px|ABP transfection efficiency 1.png]] 

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