Diquaternary ammonium salts constitute a new class of reagent for mediating
transfection of DNA in mammalian cell lines. N,N'-dioleyl-N,N,N',N'-tetram
ethyl-1,2-ethanediamine (TmedEce), N,N' dioleyl-N,N,N',N'-tetramethyl-1,3-p
ropanediamine (PropEce), N,N'-dioleyl-N,N,N',N'-tetramethyl-1,6-hexanediami
ne (HexEce), and their corresponding N,N'-dicetyl saturated analogues (Tmed
Ace, PropAce and HexAce) have all been synthesized and characterized. They
were prepared via a bis-Menshutkin reaction of the corresponding tetramethy
ldiamine with 2.2 M equiv of a long-chain alkyl halide (saturated or unsatu
rated). The reaction was run in anhydrous acetonitrile for ca. 3 days at 60
degreesC, which produced the diquaternary ammonium halides in good to near
ly quantitative yields for most derivatives. DNA transfection comparable to
commercially available reagents such as Lipofectin, Lipofectace, Lipofecta
mine, and O-ethyldioleoylphosphatidylcholinium triflate has been achieved i
n vitro with these new reagents. There was no need to use a colipid for eff
ective transfection, but serum did significantly inhibit transfection. The
saturated and the unsaturated derivatives differed with respect to hydratio
n behavior. The saturated derivatives appeared to retain a lamellar-type cr
ystalline array structure upon hydration, whereas the unsaturated versions
formed micelles and/or liposomes, depending on the ionic strength: HexEce w
as micellar in both water and saline; PropEce was micellar in water but lam
ellar in saline; and TmedEce was lamellar in both. Despite these different
hydration patterns, all of these unsaturated derivatives formed productive
transfection complexes with DNA. Varying the distance between the quaternar
y sites affected transfection efficacy in the order HexAce > TmedAce = Prop
Ace for the saturated derivatives and in the order PropEce = HexEce > TmedE
ce, with a smaller spread, for the unsaturated derivatives.