S. Lin et al. / Chemistry and Physics of Lipids 86 (1997) 171–181
175
MHz) (CDCl3/CD3OD (2:1)) d 174.24 (C1%),
173.87 (C1¦), 70.64 (d, 3JCP=7.8 Hz, sn-2-C),
66.74 (br s, b-C), 63.96 (d, JCP=5.1 Hz, sn-3-C),
62.95 (sn-1-C), 59.36 (d, JCP=4.8 Hz, a-C), 54.37
(N-Me3), 34.51 and 34.36 (C2%, C2¦), 32.21 (C14%,
C14¦), 29.40–30.00 (C4%-C13% and C4¦-C13¦),
25.20 and 25.15 (C3%, C3¦), 22.94 (C15%, C15¦),
14.21 (C16%, C16¦); mass spectrum m/z 734 (MH+);
HRMS calcd. for C40H81NO8P (MH+) m/z
734.5700, found m/z 734.5704. Anal. Calcd. for
C40H80NO8P·H2O: C, 63.88; H, 10.99; N, 1.86.
Found: C, 63.80; H, 11.14; N, 1.79.
((D3C)2SO) d 7.05–7.25 (m, 8 H, Ar), 6.90–7.00
(m, 8 H, Ar), 6.75–6.85 (m, 4 H, Ar), 3.70–3.85
1
(m, 2 H, C1H2), 3.35 (dm, 2 H, JHC=140 Hz,
C2H2), 3.07 (s, 9 H, N(CH3)3).
2.11. 1,2-Di-O-palmitoyl-sn-glycero-3-
phosphocholine (6)
A mixture of 180 mg (0.263 mmol) of 1,2-di-O-
palmitoyl-sn-glycero-3-phosphatidic acid (5) and
95 mg (0.78 mmol) of dimethylaminopyridine
(DMAP) was dried by repeated evaporation of
anhydrous pyridine (3×10 ml) according to the
literature report (Kingsley and Feigenson, 1979).
The residue was then dissolved in 10 ml of
pyridine and warmed to 40°C (bT) for 30 min
until fully dissolved. To this mixture was added
314 mg (1.04 mmol) of 2,4,6-triisopropylbenzene-
sulfonyl chloride (TPS) (freshly recrystallized be-
fore use from n-pentane containing 1% thionyl
chloride) followed by 110 mg (0.260 mmol) of
choline tetraphenylborate (4) and the reaction
mixture was stirred at 35°C (bT) for 4 h. The
excess TPS was decomposed by the addition of 1
ml of H2O, and the solvent was evaporated in
6acuo to dryness. Chromatography with silica gel
(elution was begun with CHCl3/MeOH/H2O
(60:30:0) and the solvent polarity was increased
stepwise to CHCl3/MeOH/H2O (60:30:4)), evapo-
ration, followed by microfiltration (0.5-mm Teflon
membrane) of a CH2Cl2 solution of the product
then gave 152 mg (0.202 mmol, 77% yield) of
1,2-di-O-palmitoyl-sn-glycero-3-phosphocholine
(6) (Robles and Van Den Berg, 1969; Gupta et al.,
1977; Patel et al., 1979; Hermetter and Paltauf,
1981; Singh, 1990) as a white solid: TLC (CHCl3/
MeOH/H2O (60:30:4)) Rf 0.20; 1H NMR (500
MHz) (CDCl3/CD3OD (2:1)) d 5.24 (m, 1 H,
sn-2-CH), 4.42 (dd, 1 H, J=12, 2.8 Hz, sn-1-
CHa), 4.26 (m, 2 H, a-CH2), 4.16 (dd, 1 H,
2.12. 1, 2 -Di - O - palmitoyl - sn - glycero - 3 -
phospho[h-13C]choline (6a)
1,2-Di-O-palmitoyl-sn-glycero-3-phospho[a-13C]
choline (6a) was prepared from 1.00 g (1.46
mmol) of 1,2-di-O-palmitoyl-sn-glycero-3-phos-
phatidic acid (5), 400 mg (3.27 mmol) of DMAP,
1.32 g (4.36 mmol) of TPS, and 1.20 g (2.83
mmol) of [1-13C]choline tetraphenylborate (4a)
following the procedure described for unlabeled 6
to give 820 mg (1.09 mmol, 75% yield) of 6a as a
white solid: TLC (CHCl3/MeOH/H2O (60:30:4))
Rf 0.20; 1H NMR (500 MHz) (CDCl3/CD3OD
(2:1)) d 5.24 (m, 1 H, sn-2-CH), 4.42 (dd, 1 H,
1
J=12, 2.8 Hz, sn-1-CHa), 4.26 (dm, 2 H, JHC
=
145 Hz, a-CH2), 4.16 (dd, 1 H, J=12, 7.0 Hz,
sn-1-CHb), 4.01 (dd, 2 H, JHH=3JHP=6 Hz,
sn-3-CH2), 3.62 (m, 2 H, b-CH2), 3.23 (s, 9 H,
N(CH3)3), 2.33 and 2.32 (t, 2 H, J=8.1 Hz, and
t, 2 H, J=8.1 Hz; C2%H2 and C2¦H2), 1.61 (m, 4
H, C3%H2, C3¦H2), 1.27 (br s, 48 H, C4%H2-
C15%H2, C4¦H2-C15¦H2), 0.89 (t, 6 H, J=6.8 Hz,
C16%H3, C16¦H3); 13C NMR (125 MHz) (CDCl3/
CD3OD (2:1)) d 174.27 (C1%), 173.89 (C1¦), 70.61
3
1
(d, JCP=7.6 Hz, sn-2-C), 66.73 (d, JCC=39 Hz,
b-C), 64.03 (d, JCP=5.5 Hz, sn-3-C), 62.93 (sn-1-
C), 59.42 (d, JCP=4 Hz, a-C), 54.39 (N-Me3),
34.51 and 34.36 (C2%, C2¦), 32.20 (C14%, C14¦),
29.40–30.00 (C4%–C13% and C4¦–C13¦), 25.20 and
25.15 (C3%, C3¦), 22.94 (C15%, C15¦), 14.21 (C16%,
C16¦); mass spectrum m/z 735 (MH+); HRMS
J=12, 7.0 Hz, sn-1-CHb), 4.01 (dd, 2 H, JHH
=
3JHP=6 Hz, sn-3-CH2), 3.62 (m, 2 H, b-CH2),
3.23 (s, 9 H, N(CH3)3), 2.33 and 2.32 (t, 2 H,
J=8.1 Hz, and t, 2 H, J=8.1 Hz; C2%H2 and
C2¦H2), 1.61 (m, 4 H, C3%H2, C3¦H2), 1.27 (br s,
48 H, C4%H2–C15%H2, C4¦H2–C15¦H2), 0.89 (t, 6
H, J=6.8 Hz, C16%H3, C16¦H3); 13C NMR (125
calcd. for C CH81NO8P (MH+) m/z 735.5733,
13
39
found m/z 735.5728. Anal. Calcd. for