(
247 mg, 0.5 mmol) dissolved in dry MeOH (2 mL) was added to
27.4, 27.7, 28.3, 28.9, 30.7, 31.8, 32.5, 36.2, 36.7, 37.4, 38.0, 39.2,
39.9, 43.6, 49.4, 56.5, 57.9, 61.1, 62.8, 64.2, 65.7, 80.2, 121.9, 139.7.
the N,N-dimethylamine solution. The pressure tube containing
the reaction mixture inside was screw-capped and heated at 80 C
◦
+2
∑
ESI-MS: 522.4 (M /2). Anal. (C69
H
124Br
2
N
2
O
4
2H O) Calcd. C,
2
for 48 h. Then it was cooled and the solvent was removed by
rotary evaporation. Crude reaction mixture was then crystallized
repeatedly from a mixture of MeOH and ethyl acetate to afford
a white solid (H-M) in 81% of yield 236 mg, (0.41 mmol).
66.75; H, 10.39; N, 2.26; found. C, 66.49; H, 10.12; N, 2.37.
◦
-1
Lipid HG-6. Mp: 223–226 C. IR (neat) (cm ): 3442, 2944,
1
2
870, 1460, 1347, 1276 and 1077. H-NMR (CDCl
3
, 300 MHz):
d 0.67 (s, 6H), 0.85–2.32 (m, 90H), 3.16–3.24 (m, 2H), 3.36 (br s,
◦
1
Mp: 164–168 C. H-NMR (CDCl
3
, 300 MHz): d 0.67 (s, 3H),
6
4
2
3
1
H), 3.6–3.69 (br m, 12H), 3.82–3.92 (br m, 8H), 5.34 (d, 2H, J =
0
6
7
3
5
-
.85–2.35 (m, 41H), 3.2 (m, 1H), 3.67 (s, 6H), 3.82–3.92 (m,
13
.5 Hz). C-NMR (CDCl , 75 MHz): d 11.6, 18.6, 19.7, 21.2, 22.5,
3
1
3
H), 4.15 (s, 2H), 5.36 (d, 1H, J = 4.5 Hz). C-NMR (CDCl ,
3
2.9, 23.7, 27.6, 27.8, 28.4, 28.7, 31.5, 31.9, 32.5, 36.2, 36.5, 37.3,
5 MHz): d 11.6, 19.1, 20.7, 22.7, 23.2, 24.7, 27.3, 27.7, 28.2, 29.6,
8.0, 39.3, 39.6, 43.3, 49.6, 56.0, 57.4, 61.3, 62.7, 64.3, 65.4, 79.9,
0.4, 31.9, 35.8, 36.1, 37.2, 37.8, 39.7, 39.9, 44.0, 50.8, 52.5, 56.5,
+2
22.4, 139.9. ESI-MS: 529.4 (M /2). Anal. (C70
H
126Br
2
N
2
O )
4
+
8.3, 64.7, 65.4, 65.8, 67.7 80.6, 122.2, 140.1. ESI-MS: 503.1 (M
Calcd. C, 68.94; H, 10.41; N, 2.30; found. C, 68.69; H, 10.23;
N, 2.44.
-
∑
Br ). Anal. (C33
H
60
O
2
NBr 0.5H
2
O) Calcd. C, 66.98; H, 10.39;
N, 2.37; found. C, 67.28; H, 10.45; N, 2.06.
◦
-1
Lipid HG-12. Mp: 230–233 C. IR (neat) (cm ): 3432, 2945,
General method for the synthesis of Gemini Lipids. A solution
of cholest-5-en-3b-oxyethan-N-methyl-N-2-hydroxyethylamine
1
2
0
6
4
2
3
1
872, 1454, 1353, 1282 and 1079. H-NMR (CDCl , 300 MHz): d
3
.67 (s, 6H), 0.85–2.30 (m, 102H), 3.18–3.26 (m, 2H), 3.32 (br s,
(
5) (0.2 mmol) and an appropriate a,w-dibromoalkane
H), 3.64–3.72 (br m, 12H), 3.84–3.9 (br m, 8H), 5.38 (d, 2H, J =
(
0.07 mmol) in dry MeOH-EtOAc (4 mL, v/v: 1/1) was
1
3
.5 Hz). C-NMR (CDCl , 75 MHz): d 11.6, 18.8, 19.6, 21.6, 22.7,
3
refluxed over a period of 48–72 h in a screw-top pressure tube,
until TLC indicated complete disappearance of the dibromide.
After that, each reaction mixture was cooled and the solvent was
evaporated to furnish a crude solid. The solid was repeatedly
washed with ethyl acetate to remove any of the unreacted
cholest-5-en-3b-oxyethan-N-methyl-N-2-hydroxyethyl amine (5)
and the residue was finally subjected to repeated crystallizations
from a mixture of MeOH and ethyl acetate. This furnished a
white solid in each case and the product yields ranged from ca.
3.1, 23.8, 27.0, 27.4, 28.4, 28.8, 30.7, 31.8, 32.3, 36.4, 36.7, 37.4,
8.5, 39.4, 39.7, 43.5, 49.6, 56.5, 57.9, 61.9, 62.4, 64.7, 65.5, 79.7,
+2
22.7, 139.4. ESI-MS: 571.5 (M /2). Anal. (C76
H
138Br
2
N
2
4
O )
Calcd. C, 70.02; H, 10.67; N, 2.15; found. C, 69.82; H, 10.43;
N, 2.36.
Liposome preparation. Cationic liposomes were prepared ei-
ther from monomeric or gemini lipid alone or from its mixture with
DOPE at specified ratios. For the mixed liposome preparation,
individual solution of lipids in chloroform was taken in different
molar ratios of DOPE and gemini lipids in Wheaton glass
vials. For pure lipid vesicle preparation, only a given lipid was
dissolved in chloroform and taken in Wheaton glass vials. After
mixing the organic solvent was evaporated by rotating the vials
4
0% to 50%. The purity of each lipid was ascertained from TLC;
the R values ranged from 0.2 to 0.3 in (10 : 1) CHCl /MeOH. All
the new gemini lipids were fully characterized by melting point,
f
3
1
13
IR, H-NMR, C-NMR, mass spectrometry, and CHN analysis.
Pertinent spectroscopic and analytical data are given below.
under N stream, which afforded a thin layer of a lipid or lipid
2
◦
-1
Lipid HG-3. Mp: 202–204 C. IR (neat) (cm ): 3434, 2926,
mixtures on the bottom wall of the Wheaton glass vials. Traces
of organic solvents were removed under high vacuum condition.
1
2
858, 1469, 1355, 1274 and 1078. H-NMR (CDCl
d 0.68 (s, 6H), 0.85–2.36 (m, 84H), 3.35 (br s, 8H), 3.69 (br s,
2H) 3.88 (br s, 8H), 5.3 (d, 2H, J = 4.5 Hz). C-NMR (CDCl ,
5 MHz): d 11.7, 18.4, 19.1, 21.3, 22.4, 22.8, 23.9, 27.3, 27.5, 28.0,
8.6, 31.1, 31.5, 32.5, 36.0, 36.6, 37.0, 38.1, 39.5, 39.7, 43.1, 49.8,
6.1, 57.5, 61.6, 62.8, 64.5, 65.1, 79.4, 122.6, 139.9. ESI-MS: 508.4
3
, 300 MHz):
◦
The resulting dry thin lipid layer was hydrated at 4 C for 10–
1
3
1
7
2
5
(
2
3
1
2 h with freshly autoclaved water (Milli-Q). Buffered water was
added in such a way that the final lipid concentration in the
-
1
suspension was maintained at 0.5 mg ml . To assume optimal
hydration, the lipid layer was subjected to freeze-thaw cycles from
+2
M /2). Anal. (C67
H
120Br
2
N
2
O
4
) Calcd. C, 68.30; H, 10.27; N,
◦
◦
0
(
C to 60 C several times to furnish multi-lamellar vesicles
.38; found. C, 68.34; H, 9.95; N, 2.49.
◦
MLVs). Subsequent bath sonication of MLVs at 60 C for
◦
-1
Lipid HG-4. Mp: 211–215 C. IR (neat) (cm ): 3436, 2937,
20 min then afforded unilamellar liposomes. Cationic lipid or
lipid-DOPE suspensions were prepared and kept under sterile
condition. Formulations were found to be stable when stored at
1
2
859, 1461, 1346, 1281 and 1087. H-NMR (CDCl , 300 MHz):
d 0.67 (s, 6H), 0.85–2.33 (m, 86H), 3.28 (m, 2H), 3.36 (br s, 6H),
.76 (br s, 12H) 3.87–3.96 (br m, 8H), 5.34 (d, 2H, J = 4.5 Hz).
C-NMR (CDCl , 75 MHz): d 11.5, 18.6, 19.3, 21.6, 22.3, 22.9,
3.5, 27.7, 27.9, 28.02, 28.7, 31.0, 31.7, 32.3, 36.3, 36.5, 37.2, 38.4,
9.3, 39.8, 43.4, 50.1, 56.5, 57.2, 61.3, 62.5, 64.7, 65.3, 79.9, 122.9,
39.3. ESI-MS: 515.4 (M /2). Anal. (C68
Calcd. C, 67.03; H, 10.34; N, 2.30; found. C, 66.87; H, 10.41; N,
3
◦
3
4 C.
1
3
3
Plasmid DNA. Plasmid pEGFP-c3 (Clontech, USA) which
encodes for an enhanced green fluorescence protein (GFP) under
a CMV promoter was employed for transfection studies. It was
amplified in Escherichia coli (DH10a). Plasmid pEGFP-c3 has
anti kanamycin gene which allows transformed E. coli to grow
selectively in kanamycin containing bacterial culture medium
2
3
1
+2
∑
H
122Br
2
N
2
O
4
1.5H O)
2
2
.21.
◦
-1
-1
Lipid HG-5. Mp: 219–224 C. IR (neat) (cm ): 3429, 2936,
(conc. 10 mg ml ). Amplified pEGFP-c3 was extracted and
1
2
0
3
869, 1456, 1361, 1283 and 1080. H-NMR (CDCl
.67 (s, 6H), 0.85–2.3 (m, 88H), 3.24 (m, 2H), 3.34 (br s, 6H), 3.69–
.77 (br m, 12H) 3.86–3.95 (br m, 8H), 5.32 (d, 2H, J = 4.5 Hz). C-
, 75 MHz): d 11.9, 18.7, 19.1, 21.8, 22.2, 22.5, 24.1,
3
, 300 MHz): d
purified using Qiagen Midi Prep Plasmid Purification protocol
(Qiagen, Germany). Purity of the plasmid was checked by
electrophoresis on 1.0% agarose gel. Concentration of DNA
was estimated spectroscopically by measuring the absorption
1
3
NMR (CDCl
3
4
610 | Org. Biomol. Chem., 2011, 9, 4600–4613
This journal is © The Royal Society of Chemistry 2011