Synthesis of TEPA CBA

Diethylenetriamine, abbreviated dien or DETA, (H2N-CH2CH2-NH-CH2CH2-NH2, an analog of diethylene glycol)
Triethylenetetramine, abbreviated trien or TETA, (H2N-CH2CH2-NH-CH2CH2-NH-CH2CH2-NH2)
Tetraethylenepentamine, abbreviated TEPA, (H2N-CH2CH2-NH-CH2CH2-NH-CH2CH2-NH-CH2CH2-NH2),
Pentaethylenehexamine, abbreviated PEHA, (H2N-CH2CH2-NH-CH2CH2-NH-CH2CH2-NH-CH2CH2-NH-CH2CH2-NH2), up to polyethylene amine. Similarly piperazine is an analogue of dioxane.

Syntheis of Reducible TEPA-CBA copolymer

1. Tetraethylenepentamine (2.2 mmol) +  bisacrylate CBA (0.58 mg, 2.2 mmol)   in 2.2 ml 10 % aqueous methanol at 45 °C for 3~4 days.

2, Add excess amount of TEPA, 20 mol% excess and react for additional 3 days

3, Purify by ultrafiltration (MWCO 1,000)

4, Freeze dry

5, Analysis: 1H-NMR  and gel permeation chromatography (GPC), MW:3,350.

6, Transfection:

Dissolve in 20 mM HEPES buffered saline

1.5:1 (polymer:DNA) in PBS or serum free medium, complex: 15~30 min

Serum free medium for 3~4 hr

then add complete serum

TEPA-CBA has the best transfection efficiency

Reducible TATA by Michael’s-type addition

Reaction: ethylene amine monomer (TETA, DETA, or EDA) and CBA in 10% aqueous MeOH at 50 °C in the dark for a period of 18 h to 2 days under an argon atmosphere.

1, Add  CBA (0.8 g, 3.07 mmol) to TETA (449 mg, 3.07 mmol) in 10% methanol (2.8 mL)

2, The polymerization ((50 °C) until stirring of the magnetic bar becomes difficult due to the high viscosity of the reaction mixture (16 h).

3, Add excess TETA (∌50 mg) in MeOH to consume any remaining acrylamide functional groups, stir for an additional 2 h.

4, Add 10 ml ultrapure deionized water (ddH2O), adjust pH to 5, isolate polymer product by ultrafiltration (MWCO 1000).

5, Freeze dry

6, DNA:polymer=1:3, incubate 15~30 min

7, Serum-free medium for 4 hours, then replace with complete medium

Tetraethylenepentamine (TEPA)

Ethylenediamine (EDA)

Triethylenetetramine (TETA)

Diethylenetriamine (DETA)