Preparation of stereospecifically deuterated palmitates
JCD 19 Hz, upfield a-deuterium isotope shift (0.4 ppm)), 29.07
(C4, upfield b-deuterium isotope shift (0.1 ppm)) 24.95, 22.71,
14.13.
Methyl (4R)-[4-2H1]-palmitate ((4R)-[4-2H1]-1). A solution of
(S)-5-hydroxy-1-heptadecene (906 mg, 3.57 mmol) in dry pyridine
(7.5 mL) was treated with TsCl (1.41 g, 7.14 mmol) at 0 C. The
◦
Methyl (5S)-[5-2H1]-palmitate ((5S)-[5-2H1]-1). From (R)-6-
hydroxy-1-heptadecene. Obtained as a white solid. The spectral
data of the title compound was identical to that of the corre-
sponding (R)-enantiomer.
solution was stirred for 1 h at 0 ◦C and left at 4 ◦C (3 days).
The precipitated pyridinium hydrochloride salt was dissolved by
adding H2O (25 mL) to the reaction mixture and the aqueous
layer extracted with Et2O (3 × 30 mL). The combined organics
were washed with sat. CuSO4 (4 × 15 mL), H2O (2 × 15 mL),
5% NaHCO3 (2 × 15 mL), sat NaCl (1 × 15 mL), dried and
concentrated in vacuo to give the tosylate (1.29 g) as a viscous oil.
1H NMR d 7.79 (d, J 9.0 Hz, 2H), 7.33 (d, J 9.0 Hz, 2H), 5.70
(m, 1H), 4.98 (dm, J 5.1 Hz, 1H), 4.92 (m, 1H), 4.57 (p, J 6.0 Hz,
1H), 2.44 (s, 3H), 1.94–2.06 (m, 2H), 1.61–1.73 (m, 2H), 1.15–
1.33 (m, 22H), 0.88 (t, J 6.6 Hz, 3H). The tosylate intermediate
(1.14 g, ca. 2.8 mmol) was dissolved in dry ether (5 mL) and
lithium aluminium deuteride (372 mg, 8.8 mmol) was added; the
reaction mixture was stirred for 5 h at rt under N2. The reaction
was quenched with H2O (12 mL) and 6 M HCl (21 mL) and
then extracted with Et2O (3 × 30 mL). The combined ethereal
layers were washed with H2O (1 × 15 mL), dried and concentrated
in vacuo to give the crude monodeuterated 1-heptadecene (686
Desaturase assay. Reactions of acyl-ACP derivatives with D4
desaturase were carried out at room temperature. Each reaction
mixture consisted of ivy D4 desaturase dimer (2.4 nmol), dithio-
threitol (400 nmol), bovine liver catalase (16 nmol), Anabaena
vegetative ferredoxin (4.7 nmol), maize root NADPH:ferredoxin
reductase (0.3 nmol), acyl-ACP (10.6 nmol) in a total volume of
660 lL of buffer. The reaction was initiated by the addition of
NADPH (0.7 lmol) in buffer (32 lL) and allowed to continue
for 30 min. The reaction was terminated with the addition of
toluene (1 mL) and the thioester linkage was transesterifed to
give the corresponding methyl ester with freshly prepared 0.5 M
NaOMe at 55 ◦C for 30 min. The residue was acidified with acetic
acid (100 lL) and extracted with hexane (2 × 2 mL). The phases
were separated by centrifugation and the combined organics were
evaporated under a steady stream of N2 and the residue was diluted
with 100 lL of hexane for analysis by GC-MS.
1
mg) as a low melting solid: H NMR d 5.81 (ddt, J 18, 10.5,
7.5 Hz, 1H), 4.99 (dm, J 18 Hz, 1H), 4.92 (dm, J 9 Hz, 1H),
2.04 (dt, J 9.0, 6.0 Hz, 2H), 1.26 (br s, 25 H), 0.88 (t, J 6.0, 3H);
MS (EI, 70 eV) m/z 239 (M+), 211 (M+ − 28). A portion of the
monodeuteroheptadec-1-enyl intermediate so obtained (289 mg,
ca. 1.26 mmol) was oxidized with aqueous KMnO4 (800 mg,
5 mmol in 19 mL) containing hexadecyltributyl phosphonium
bromide (11 mg) for 5 h at rt with vigorous stirring. The product
mixture was treated with sodium sulfite (1.5 g) in 15 mL 4 M HCl
and then extracted with hexanes (3 × 30 mL) followed by washing
with H2O, (1 × 20 mL) and sat. NaCl (1 × 20 mL) to give the
crude carboxylic acid (319 mg) after drying and evaporation of the
organic layer. A portion of this material (232 mg) was subsequently
methylated by BF3/MeOH and purified by flash chromatography
(2.5% EtOAc/hexanes) to give the title compound as a white solid
(125 mg, 0.46 mmole, 44% overall yield from (S)-5-hydroxy-1-
heptadecene). Rf 0.18 (2.5% EtOAc in hexanes). 1H NMR d 3.66
(s, 3H), 2.30 (t, J 7.4, 2H), 1.61 (m, 2H,), 1.26 (br s, 23H), 0.88
(t, J 6.7, 3H); 13C NMR d 174.37, 51.44, 34.11, 31.94, 29.71,
29.71, 29.71, 29.67, 29.67, 29.62, 29.45, 29.38, 28.77 (C4, t, JCD
19 Hz, upfield a-deuterium isotope shift21 (0.41 ppm)), 29.18 (C5,
upfield b-deuterium isotope shift (0.1 ppm)) 24.88 (C-3, upfield
b-deuterium isotope shift (0.1 ppm)), 22.71, 14.14; MS (EI, 70 eV)
m/z 271 (M+), 240 (M+ − CH3O).
Acknowledgements
This work was supported by an NSERC URS (to A. T.),
N.S.E.R.C. Grant OGP-2528 (to P. H. B.); by the Office of
Basic Energy Sciences of the United States Department of Energy
(to J. S.).
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Methyl (4S)-[4-2H1]-palmitate ((4S)-[4-2H1]-1). From (R)-5-
hydroxy-1-heptadecene. Obtained as a white solid. The spectral
data of the title compound was identical to that of corresponding
(R)-enantiomer.
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Methyl (5R)-[5-2H1]-palmitate ((5R)-[5-2H1]-1). From (S)-6-
hydroxy-1-heptadecene. Obtained as a white solid. The spectral
data of the title compound were similar to those reported for the
isotopomer above except for: 13C NMR d 174.36, 51.44 34.13,
31.94, 29.70, 29.70, 29.70, 29.69, 29.67, 29.67, 29.38 ((C6, upfield
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1274 | Org. Biomol. Chem., 2007, 5, 1270–1275
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