J Am Oil Chem Soc
Japan), and (S)-(?)-1-(1-naphthyl)ethyl isocyanate (99 %;
optical purity, 96 %) and (R)-(-)-1-(1-naphthyl)ethyl iso-
cyanate (98 %; optical purity, 95 %) from Aldrich (Sigma-
Aldrich Japan, Tokyo, Japan). Dry toluene, dry pyridine,
and N,N0-dicyclohexylcarbodiimide (DCC) were obtained
from Kanto Chemical (Tokyo, Japan). 4-Dimethylamino-
pyridine (4-DMAP) was purchased from Merck (Darms-
tadt, Germany). Palmitic acid (16:0; free form),
eicosapentaenoic acid (20:5; ethyl ester), and docosahex-
aenoic acid (22:6; ethyl ester) were obtained with purities
higher than 99 %. Tetracosahexaenoic acid (24:6) and
hexacosaheptaenoic acid (26:7) were concentrated from
total fatty acids of roughscale sole in the form of methyl
esters [15]. The ethyl and methyl esters were used after
saponification.
(10:3, v/v; 2 mL), 4-DMAP (4 mg), and (S)- or (R)-1-(1-
naphthyl)ethyl isocyanate (2b,c; 0.12 mmol = 23.7 mg),
and held at 50 °C overnight in the dark. After 1-propanol
(10 lL) was added to stop the reaction (with standing
over 10 min), solvents were removed under a stream
of nitrogen. The products were dissolved in 1 mL of
chloroform.
Preparation of Fatty Acid Adducts of Glycerol
Urethane Derivatives
MAG and DAG Urethane Derivatives (10–15)
To a 1-mL reaction vial containing isomers of glycerol
mono-urethane derivatives (3–5; 0.015 lmol = 4.3–4.5 lg)
or di-urethane derivatives (6–8; 0.03 lmol = 14.6–15.3 lg)
were added fatty acid (9; 0.033 lmol = 10 lg for di-ure-
thanes and 0.165 lmol = 50 lg for mono-urethanes), a
solution of 4-DMAP (0.03 lmol = 3.7 lg) in carbon tetra-
chloride (0.2 mL), and a solution of DCC (1.1 molar equiv-
alent of fatty acids) in carbon tetrachloride (10 lL). After the
mixture had been stirred at room temperature for 2 h, 1-pro-
panol(10 lL)wasaddedtostopthereaction. Thesolutionwas
filtered through a small cotton-wool plug. The solvents were
removed in a stream of nitrogen. The products were dissolved
in 0.1 mL of chloroform.
Enantiomeric 1- and 3-palmitoyl-sn-glycerols (sn-1- and
sn-3-16:0-MAG) were synthesized by addition of 16:0 to
(R)-(-)-2,3- and (S)-(?)-1,2-O-isopropylidene-sn-glycerols
(optical purity, [98 %; Tokyo Chemical Industry, Tokyo,
Japan), respectively, followed by acidification using triflu-
oroacetic acid [9, 10]. 1,2-Dipalmitoyl-sn-glycerol (sn-1,2-
16:0-DAG) and 1,2(2,3)-dipalmitoyl-rac-glycerol (rac-
1,2(2,3)-16:0-DAG) were obtained from Sigma (Sigma-
Aldrich Japan). Their positional isomers, sn-2-16:0-MAG
and sn-1,3-16:0-DAG, were isolated using boric acid TLC
from the products of partial degradation of tripalmitoyl-
glycerol by the reaction with ethylmagnesium bromide [2].
HPLC
Partial Carbamoylation of Glycerol with Isocyanates
Silicic acid HPLC separation of the glycerol urethane
derivatives (3–8) was carried out with a Hitachi L-6200
pump (Hitachi, Tokyo, Japan), a Shimadzu CTO-10ASvp
column oven (Shimadzu, Kyoto, Japan), a Jasco 875-UV/
VIS detector (Jasco, Tokyo, Japan), and a Shimadzu
The glassware articles used in this step were preliminarily
dried over a flame and cooled in a desiccator to room
temperature.
Glycerol Mono- and Di-3,5-DNPU Derivatives (3a–8a)
C-R6A integrator.
A
LiChrospher Si 60 column
(25 cm 9 4.6 mm id, 5 lm particles; Merck) was used at
25 °C eluting with HPLC-grade hexane (A) and hexane/
dichloromethane/ethanol (40:12:3, v/v/v) (B) as the mobile
phase at a flow rate of 1 mL/min. Elution starting with A/B
(75:25) (0 min) was gradually changed to A/B (0:100) over
30 min and then held at B for 20 min. UV detection was at
254 and 280 nm for the 3,5-DNPU and 1-(1-naphthyl)ethyl
urethane derivatives, respectively.
To a 10-mL screw-capped test tube were added dry glyc-
erol (1; 0.12 mmol = 11.0 mg), dry toluene/dry pyridine
(10:3, v/v; 1 mL), and 3,5-dinitrophenyl isocyanate (2a;
0.06 mmol = 12.5 mg). The solid block of isocyanate was
crushed after its addition to the reaction mixture to prevent
it from being exposed to atmospheric moisture. The test
tube was allowed to stand at room temperature for 1 h in
the dark. After 1-propanol (10 lL) was added to stop the
reaction (with standing for 10 min), the solvents were
removed in a stream of nitrogen. The products were dis-
solved in 1 mL of ethanol.
Silicic acid HPLC analysis of MAG and DAG urethane
derivatives (10–15) was carried out with a Shimadzu LC-
6A pump, a Hitachi L-4200 UV/VIS detector, and a
Shimadzu C-R3A integrator. A column of Inertsil SIL-
100A (25 cm 9 4.0 mm id, 5 lm particles; GL Sciences,
Tokyo, Japan) was used at 30 °C with hexane/2-propanol
in 97:3 (v/v) and 99:1 (v/v) compositions as the mobile
phase for MAG and DAG 3,5-DNPU derivatives,
respectively. For the analyses of MAG and DAG 1-(1-
naphthyl)ethyl urethanes, hexane/2-propanol in 97:3 (v/v)
Glycerol Mono- and Di-(S)- or (R)-1-(1-naphthyl)ethyl
Urethane Derivatives (3b,c–8b,c)
To a 10-mL screw-capped test tube were added dry glyc-
erol (1; 0.06 mmol = 5.5 mg), dry toluene/dry pyridine
123