1
366
Pierrat et al.
(
3.25 g, 11.3 mmol) in anhydrous CH Cl (50 mL) was treated
from DOPC and 10b in CHCl (5 mL). TLC R 0.26
2
2
3
f
1
with oxalyl chloride (1.10 mL, 12.5 mmol) at 0°C under an
argon atmosphere. After 30 min, the reaction mixture was
allowed to warm to room temperature and was stirred for an
additional period of 1 h. Volatiles were removed under re-
duced pressure, and the residue was coevaporated twice with
anhydrous toluene. The expected acyl chloride derivative was
recovered in quantitative yield and used in the next step
without further purification. To dodecyl 4-chloro-4-oxobuta-
noate (275 mg, 0.90 mmol) were successively added parafor-
maldehyde (27 mg, 0.90 mmol) and anhydrous ZnCl2
(CH Cl /MeOH 90:10). H-NMR (400 MHz, CDCl ) δ
2
2
3
6.41 (m, 1H), 5.35–5.19 (m, 5H), 4.53 (br s, 2H), 4.28–4.11
(m, 6H), 3.50 (s, 8H), 2.31–2.26 (m, 6H), 1.97 (m, 8H),
3
1.58–1.51 (m, 9H), 1.26–1.17 (m, 56H), 0.84 (t, JHH=
1
3
6.7 Hz, 9H). C-NMR (100 MHz, CDCl ) δ 173.5,
3
173.1, 172.1, 130.2, 129.8, 91.7, 91.7, 91.6, 91.5, 69.6,
69.5, 69.4, 66.7, 66.6, 65.6, 65.5, 62.1, 62.0, 61.8, 54.7,
34.3, 34.2, 32.1, 29.9, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3,
3
1
29.2, 27.4, 27.3, 25.0, 24.6, 22.8, 21.4, 14.3. P-NMR
(162 MHz, CDCl ) δ –5.62, –5.72, –5.76, –5.84. ESI-MS
3
(2.5 mg, 2%mol.). The reaction mixture was heated at 35°C
(m/z): calc. for C H NO P 1012.79, found 1012.7.
5
8
111
10
under an argon atmosphere for 18 h. After cooling to room
temperature, the residue was purified by flash column chro-
1,2-Dioleoyl-sn-glycero-3-[(hexanoyloxy)methyl]phosphocho-
line chloride (3). This compound (mixture of two diaster-
eomers, 100 mg, 28%) was obtained as a waxy solid from
DOPC and 10c in CHCl (5 mL). TLC R 0.20
matography over silica gel (PE/Et O 80:20) to yield com-
2
pound 12 (150 mg, 50%) as a clear oil. TLC R 0.42
f
1
(
2
PE/Et O 80:20). H-NMR (400 MHz, CDCl ) δ 5.69 (s,
H), 4.07 (t, J =6.9 Hz, 2H), 2.70–2.62 (m, 4H), 1.61–
.58 (m, 2H), 1.28–1.24 (m, 18H), 0.86 (t, JHH=6.7 Hz, 3H).
C-NMR (100 MHz, CDCl ) δ 172.1, 170.7, 68.9, 65.4,
2
3
3
f
3
1
(CH Cl /MeOH 90:10). H-NMR (400 MHz, CDCl ) δ
HH
2
2
3
3
1
5.67–5.57 (m, 2H), 5.33–5.22 (m, 5H), 4.57 (br s, 2H), 4.34–
4.13 (m, 6H), 3.49 (s, 9H), 2.40–2.27 (m, 6H), 1.93 (m, 8H),
1
3
3
3
3
2
2.1, 29.8 (2C), 29.7 (2C), 29.6, 29.5, 29.2, 28.9, 28.8, 26.1,
2.9, 14.3.
1.65–1.53 (m, 6H), 1.27–1.24 (m, 44H), 0.85 (t, JHH=6.6 Hz,
13
9H). C-NMR (100 MHz, CDCl ) δ 173.5; 173.0, 172.3,
3
1
30.2, 129.8, 83.1, 69.5, 69.4, 66.7, 66.6, 65.6, 65.3, 62.4,
62.3, 62.2, 61.6, 57.7, 34.3, 34.2, 34.0, 32.1, 31.3, 29.9, 29.7,
9.5, 29.4, 29.3, 29.2, 17.4, 27.3, 25.0, 24.3, 22.9, 22.4, 14.3,
General Procedure for the Preparation of Cationic Lipids
2
31
To a solution of DOPC in anhydrous CHCl was added a
14.0. P-NMR (162 MHz, CDCl ) δ –3.77, –3.94. ESI-MS
3
3
solution of the freshly prepared electrophiles 10a-e, 11a-c
(m/z): calc. for C H NO P 914.69, found 914.6.
51
97
10
or 12 (8 eq.) in CHCl . The resulting reaction mixture was
3
refluxed for 18 h under an argon atmosphere. Then it was
cooled down at room temperature and concentrated under
reduced pressure. Analytically pure compounds were
obtained after purification by flash chromatography over
silica gel (CH Cl /MeOH 98:2 to 90:10).
1,2-Dioleoyl-sn-glycero-3-[1-(hexanoyloxy)ethyl]phosphocho-
line chloride (4). This compound (mixture of four diaster-
eomers, 50.6 mg, 21%) was obtained as a waxy solid from
DOPC and 10d in CHCl (4 mL). TLC R 0.23
3
f
1
(CH Cl /MeOH 90:10). H-NMR (400 MHz, CDCl ) δ
2
2
2
2
3
6.43–6.37 (m, 1H), 5.34–5.27 (m, 4H), 5.21 (br s, 1H), 4.54
1
,2-Dioleoyl-sn-glycero-3-[(dodecanoyloxy)methyl]phospho-
(br s, 2H), 4.30–4.13 (m, 6H), 3.48 (s, 9H), 2.32 (m, 4H), 1.99
(m, 8H), 1.56 (m, 9H), 1.31–1.24 (m, 44H), 0.86 (m, 9H). C-
1
3
choline chloride (1). This compound (mixture of two diaster-
eomers, 110 mg, 28%) was obtained as a waxy solid from
NMR (100 MHz, CDCl ) δ 173.5, 173.1, 172.5, 172.2, 130.3,
3
DOPC and 10a in CHCl3 (5 mL). TLC R 0.20
129.9, 91.8, 69.4, 66.3, 65.8, 62.5, 61.8, 61.6, 55.0, 50.1, 34.4,
34.3, 34.2, 32.1, 31.4, 29.9, 29.7, 29.4, 29.3, 29.2, 27.4, 27.3,
25.1, 24.4, 22.9, 22.4, 21.5, 14.3, 14.1. P-NMR (162 MHz,
f
1
(
CH Cl /MeOH 90:10). H-NMR (400 MHz, CDCl ) δ
2
2
3
3
1
5
.67–5.56 (m, 2H), 5.36–5.28 (m, 4H), 5.23 (br s, 1H), 4.57
(
br s, 2H), 4.32–4.11 (m, 6H), 3.49 (s, 9H), 2.39–2.27 (m,
CDCl ) –5.67, –5.78, –5.84, –5.93. ESI-MS (m/z): calc. for
3
6
0
1
6
2
2
–
9
H), 1.97 (m, 8H), 1.62–1.57 (m, 6H), 1.27–1.23 (m, 56H),
C H NO P 928.70, found 928.7.
5
2
99
10
3
13
.85 (t, JHH=6.6 Hz, 9H). C-NMR (100 MHz, CDCl ) δ
3
73.4, 173.0, 172.5, 130.2, 129.8, 91.7, 69.4, 69.3, 66.2,
5.7, 65.6, 62.3, 62.2, 61.6, 54.7, 34.3, 34.2, 34.1, 32.1,
9.9, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 27.4, 27.3,
1,2-Dioleoyl-sn-glycero-3-[(propionyloxy)methyl]phosphocho-
line chloride (5). This compound (mixture of two diaster-
eomers, 103 mg, 29%) was obtained as a waxy solid from
DOPC and 10e in CHCl (6 mL). TLC R 0.17
3
1
5.0, 24.7, 22.8, 21.4, 14.3. P-NMR (162 MHz, CDCl ) δ
3
3
f
1
3.94, –4.01. ESI-MS (m/z): calc. for C H NO P
(CH Cl /MeOH 90:10). H-NMR (400 MHz, CDCl ) δ
5
7
109
10
2
2
3
98.78, found 998.7.
5.66–5.62 (m, 2H), 5.32–5.29 (m, 4H), 5.23 (br s, 1H), 4.60
3
(
br s, 2H), 4.30–4.14 (m, 6H), 3.48 (s, 9H), 2.41 (q, JHH=
3
1
,2-Dioleoyl-sn-glycero-3-[1-(dodecanoyloxy)ethyl]phospho-
7.5 Hz, 2H), 2.29 (q, J =7.6 Hz, 4H), 1.99–1.95 (m, 8H),
HH
3
choline chloride (2). This compound (mixture of four dia-
stereomers, 803 mg, 38%) was obtained as a waxy solid
1.57–1.54 (m, 4H), 1.31–1.11 (m, 40H), 1.13 (t, JHH=
3
13
7.37 Hz, 3H), 0.83 (t, J =6.9 Hz, 6H). C-NMR
HH