Paul.xu at 16:00, 30 July 2014

2014-07-30T16:00:19Z

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== Chemicals ==

Spermine (SP, Sigma, S3256-5G, $194.50), 5GM Alfa Aesar, $138 

N,N′-bis(3-aminopropyl)-1,3-propanediamine (APPD, Sigma, 404810-5G, $90.40) 

'''N,N′-bis(3-aminopropyl)-ethylenediamine''' ('''APED''', Sigma: 239399-100ML, '''$29.30,'''  Acros Organics) 

N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD, Sigma, 333131-5G, $173.00) 

triethylenetetramine (TETA, Sigma 90460-10ML, $145.00) 

N,N′-cystaminebisacrylamide (CBA, PolySciences, 5 g $143.00) 

branced polyethylenimine (bPEI, M w =25kDa, Sigma) 

ethylenediamine (EDA, Sigma, 03550-250ML $63.50) 

2-acetyldimedone (Dde-OH , EMD Chemicals, 102575-756, Chem Impex International, 5 g, $67.90) 

hydroxylamine hydrochloride (NH2OH·HCl, Sigma, 255580-100G, $58.00) imidazole (Sigma) 

N-methyl-2-pyrrolidinone (1-Methyl-2-pyrrolidinone , NMP, Sigma, M79603-1L, $87.70) 

== Synthesis ==

Dissolve SP (0.202g, 1 mmol) and Dde-OH (0.419 g, 2.3 mmol)  in 1 mL MeOH, stirring at room temperature for 24 h to let Dde group specifically protect primary amine groups in spermine. 

The next day, add CBA (0.260g, 1 mmol) into above system in 1.5 mL of MeOH/diH 2 O (9/1 v/v). 

Polymerize at 60° C in the dark under nitrogen atmosphere for 3-4 days. 

Add 10% mol excess EDA  to consume any unreacted acrylamide groups, stirring at 60°C for at least additional 2 h. 

Precipitate polymer in 40 mL anhydrous diethyl ether to get the intermediate polymer poly(CBA-SP-Dde). 

Remove the Dde groups in the deprotection mixture of NH2OH·HCl/Imidazole/NMP/DMF by stirring at room temperature for 4 h. 

'''To make deprotection mixture: ''' 

1.25 g (1.80 mmol) of NH2OH·HCl 

0.918 g (1.35 mmol) of imidazole is suspended in 5 mL of NMP. 

Sonicate the mixture until complete dissolution. 

Just before reaction, dilute 5 volume of this solution  with 1 volume of DMF to make the deprotection mixture. 

After deprotection, purify the crude product by dialysis (MWCO = 1000) against MilliQ water for 24 h 

Dry by lypholization to obtain poly(CBA-SP) as solid gel. 

 

[[Image:Polyamine CBA synthesis.png|600px|Polyamine CBA synthesis.png]] 

[[Image:Polyamine CBA cytotoxicity.png|600px|Polyamine CBA cytotoxicity.png]] 

[[Image:Polyamine CBA TR efficiency.png]] 

poly(CBA-TETA) and poly(CBA-AEPD)is the best for uptake 

but poly(CBA-SP), poly(CBA-APPD) and poly(CBA-APED) have higher transfection efficiency 

for cost + efficiency: use poly(CBA-APED) 

 

== Older Protocol (simple) ==

'''Sigma''' 

Hyperbranched poly(ethylenimine) (bPEI, 25 kDa) 

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

trifluoroacetic acid (TFA) 

triisopropylsilane 

triisobutylsilane 

N,N-diisopropylethylamine (DIPEA) 

piperidine 

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

chloroquine diphosphate salt 

trypan blue solution (0.4%) 

DL-buthionine-sulfoxamine 

'''PolySciences''': N,N′-cystaminebisacrylamide (CBA)  

Novabiochem: 2-(1H-benzotriazole-1-yl) 1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)  

'''Anaspec''': Fmoc-L-Arg(pbf)-OH 

 

'''Procedure:''' 

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

Stire in 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 (20%) (or 10% mole of N-Boc-DAH), dissolved in 1 mL of methanol to viscous solutions

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

Precipitation with diethyl ether. Disolve the pellet in water and adjust with 1M HCl to pH 5  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 

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

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

The typical yields varied from 40 to 75%.

 

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. 

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

Add  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)  against ultra-pure water overnight

lyophilize. 

 

 

[[Image:CBA polyamine synthesis.png]]

[[Image:Cytotoxicity CBA polyamine.png]]

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

 

[[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]]

Synthesis of CBA-PolyAmines - Revision history

Paul.xu at 16:00, 30 July 2014