Huang, Z., Li, W., MacKay, J. A., and Szoka, F. C.
Supplementary Material
Cell Viability
Cytotoxicity of TCL
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
4/DOPE
3/DOPE
DOTAP/DOPE
6/DOPE
0.5
1
2
4
8
16
32
64 128
Charge ratio (+/-)
Cytotoxicity of TCL lipoplexes on cultured CV-1 cells. TCL/DOPE and DOTAP/DOPE
were complexed with 0.1 g per well of β-galactosidase plasmid DNA at charge ratios
(+/-) ranging from 0.5/1 to 128/1. The viability of cells exposed for 4 h was tested and
compared with the untreated cells.
Synthesis of TCL
1
H NMR (400 MHz) spectra were recorded on a Varian 400 MHz instrument. Chemical
shifts are expressed as parts per million using tetramethylsilane as internal standard.
J
values are in Hertz. Mass spectra were obtained at the Mass Spectrometry Facility,
University of California at San Francisco. Elemental analyses were performed by M-H-W
Laboratories (Phoenix, AZ). TLC analyses were performed on 0.25-mm silica gel plates.
Unless noted otherwise, the ratios describing the composition of solvent mixtures
1
Huang, Z., Li, W., MacKay, J. A., and Szoka, F. C.
represent relative volumes.
2-Pyridinyl 3-cholesteryl disulfide (1) Thiocholesterol (0.402 g, 1 mmol) and 2,
2-pyridyl disulfide (0.244 g, 1.1 mmol) were dissolved in 5 mL chloroform under argon.
The reaction mixture was stirred at room temperature for 48 h. Solvent was then removed
by rotary evaporation under reduced pressure. The residue was washed with 20 mL
methanol and filtered. The filter cake was used directly for next step reaction. High purity
product was obtained by silica gel flash column chromatography (hexane to hexane/ethyl
acetate 20/1). Rf = 0.87, (hexane/ethyl acetate 2/1). White solid (0.45g, 88% yield) was
obtained. 1H NMR (CDCl3),  0.67 (3H, s); 0.85-1.65 (33H, m); 1.78-2.01 (5H, m); 2.36
(2H, d, J = 8.4); 2.79 (1H, m); 5.35 (1H, d, J = 4.4); 7.06 (1H, m ); 7.63 (1H, m ); 7.77
(1H, d, J = 8.0); 8.44 (1H, d, J = 4.0). ESI-MS: m/z 512.7 (M + H+). EI-HRMS for
C32H50NS2: found 512.3376, calcd 512.3384.
N,N-Dimethylaminoethyl 3-cholesteryl disulfide (2) To a solution of 1 (3.07 g, 6
mmol)
in
CHCl3
(60
mL)
and
triethylamine
(10
mL)
was
added
N,N-dimethylaminoethylthiol hydrochloride (4.23 g, 30 mmol). The reaction mixture was
stirred at room temperature for 24 h. The solution was washed with 2% aqueous KOH
(50 mL) and 50 mL brine consecutively. The oil layer was dried over anhydrous sodium
sulfate, filtered and evaporated. The crude product was purified by silica gel flash column
chromatography (hexane/ethyl acetate 3/1 to 2/1). Rf = 0.14, (hexane/ethyl acetate 2/1).
Colorless viscous liquid (2.40 g, 81.4% yield) was obtained. 1H NMR (CDCl3),  0.68
(3H, s); 0.85-1.62 (33H, m); 1.78-2.03 (5H, m); 2.28 (6H, s); 2.36 (2H, d, J = 6.8);
2
Huang, Z., Li, W., MacKay, J. A., and Szoka, F. C.
2.60-2.70 (3H, m); 2.82 (2H, t, J = 6.0); 5.37 (1H, d, J = 4.8). MALDI-MS: m/z 506.1 (M
+ H+).
3-Cholesteryldithioethyl dimethyl amine hydrochloride (3) To a solution of 2 (0.33
g, 0.65 mmol) in hot methanol (30 mL) was added hydrochloric acid (3 mL, 1.2 M). The
solution obtained was then evaporated to dryness under vacuum. The residue was
recrystallized from methanol (5 mL), dried over vacuum. White crystal (0.32 g, 90.3%
yield) was obtained. 1H NMR (CDCl3),  0.68 (3H, s); 0.85-1.63 (33H, m); 1.79-2.05
(5H, m); 2.34 (2H, d, J = 4.0); 2.71-2.79 (1H, m); 2.85 (6H, s); 3.15 (2H, t, J = 5.2 ); 3.33
(2H, t, J = 6.0); 5.39 (1H, d, J = 4). MALDI-MS: m/z 506.4 (C31H56ClNS2+). Anal. Calcd
for C31H56ClNS2: C, 68.65; H, 10.41; N, 2.58. Found: C, 68.80; H, 10.48; N, 2.65.
3-Cholesteryldithioethyl trimethyl ammonium iodide (4) To a solution of 2 (0.15 g,
0.3 mmol) in methylene chloride (6 mL) was added methyl iodide (0.1 mL, excessive).
The mixture was stirred under argon at room temperature for at least 2 h. Analysis on
TLC indicated the completion of the reaction. Solvent and excessive methyl iodide was
removed by rotary evaporation under reduced pressure at low temperature. White crystal
(0.18 g, 95% yield) was obtained. Rf = 0.5, (CHCl3/MeOH/NH4OH, 65/25/4). 1H NMR
(CDCl3),  0.68 (3H, s); 0.85-1.63 (33H, m); 1.79-2.05 (5H, m); 2.34 (2H, d, J = 4.4 Hz);
2.71-2.79 (1H, m); 3.11 (2H, t, J = 7.2 Hz); 3.55 (9H, s); 3.90 (2H, t, J = 7.2 Hz); 5.40
(1H, d, J = 4.8 Hz). MALDI-MS: m/z 520.4 (C32H58NS2+). Anal. Calcd for C32H58INS2:
C, 59.33; H, 9.02; N, 2.16. Found: C, 59.53; H, 9.16; N, 2.27.
3
Huang, Z., Li, W., MacKay, J. A., and Szoka, F. C.
(2-tert-Butoxycarbonylamino-ethyl)-dimethyl-(2-(3-cholesteryldithioethyl))
ammonium bromide (5) To a solution of 2 (1.01 g, 2 mmol) in methylene chloride (5
mL) was added 2-tert-butoxycarbonylamino-ethyl bromide (0.64 g, 2.87 mmol). The
mixture was stirred under argon at room temperature for 48 h. Then, the solvent was
removed. Diethyl ether (8 mL) was added to the viscous residue. A white precipitate was
collected and rinsed with diethyl ether twice. White solid (0.73 g, 50% yield) was
obtained. Rf = 0.65, (CHCl3/MeOH/NH4OH, 65/25/4). 1H NMR (CDCl3),  0.68 (3H, s);
0.85-1.63 (42H, m); 1.79-2.05 (5H, m); 2.34 (2H, d, J = 4.4 ); 2.71-2.79 (1H, m); 3.09
(2H, t, J = 7.2 ); 3.43 (6H, s); 3.76 (4H, m); 3.90 (2H, t, J = 6.0); 5.40 (1H, d, J = 4.8);
6.05 (1H, br). MALDI-MS: m/z 649.2 (C38H69N2O2S2+). Anal. Calcd for C38H69BrN2O2S2:
C, 62.52; H, 9.53; N, 3.84. Found: C, 62.63; H, 9.77; N, 3.92.
(2-Amino-ethyl)-dimethyl-(2-(3-cholesteryldithioethyl))
ammonium
bromide
hydrochloride (6) To a solution of 5 (0.65 g, 0.89 mmol) in hot methanol (15 mL) was
added conc. hydrochloride acid (1mL). The mixture was stirred at room temperature for 1
h. The white precipitate was recrystallized from MeOH/conc. HCl (100/5). White crystal
(0.52 g, 88% yield) was obtained. Rf = 0.33, (CHCl3/MeOH/NH4OH, 65/25/4). 1H NMR
(CDCl3-CD3OD, 1/1),  0.68 (3H, s); 0.85-1.63 (33H, m); 1.79-2.05 (5H, m); 2.34 (2H, d,
J = 6); 2.71-2.79 (1H, m); 3.09 (2H, t, J = 7.2); 3.30 (6H, s); 3.42 (2H, t, J = 7.2 ); 3.79
(2H, t, J = 7.2); 5.40 (1H, d, J = 4.8). ESI-MS: m/z 549.4 (C33H61N2S2+). Anal. Calcd for
C33H62BrClN2S2: C, 59.48; H, 9.38; N, 4.20. Found: C, 59.58; H, 9.48; N, 4.15.
4