J = 1.8, H-2), 7.44 (1H, dd, J = 8.5, 1.8, H-6), 7.73 (1H, d, J =
15.8, H-b). 13C-NMR (CDCl3, d): 21.0, 21.1 (2 OCOCH3), 118.8
(C-a), 123.4 (C-2), 124.4 (C-5), 127.1 (C-6), 133.3 (C-1), 142.9
(C-3), 144.3 (C-4), 145.5 (C-b), 168.3, 168.5 (2 OCOCH3), 171.7
(CO2H).
Oxidation by NaIO4. To 3 mL of a freshly prepared 10 mM
solution of phenol in a pH 7.4 phosphate–NaCl buffer was added
12.8 mg of NaIO4. The mixture was placed at 25 ◦C under
stirring and analyzed by HPLC–MS every hour.
Inhibition of linoleic acid peroxidation. A freshly prepared
2.55 mM solution of linoleic acid (2 mL) in a pH 7.4 phosphate
buffer containing 0.1 M SDS were placed at 37 ◦C in the
spectrometer cell. At time zero, 25 lL of a freshly prepared
80 mM solution of AAPH in the same buffer was added,
followed ca. 15 min later by 25 lL of an antioxidant solu-
tion in MeOH. The experiments were repeated with different
phenol concentrations (1 mM and lower). The initial level
of hydroperoxides (molar absorption coefficient at 234 nm =
26 100 M−1 cm−1)28 was below 2% in all experiments. The
uninhibited and inhibited peroxidation rates were calculated
from the slope of the absorbance at 234 nm vs time lines before
and after antioxidant addition using fixed time intervals. Each
experiment was run in triplicate. Standard deviations were lower
than 10%.
n-Hexadecyl-3,4-diacetoxycinnamate (4). Compound
3
(334 mg, 1.26 mmol) was dissolved in a minimum of dry
CH2Cl2. DMF (8 mL) and oxalyl chloride (167 mL) were then
added and the mixture was stirred at room temperature for
3 h. The solvent was removed under a reduced pressure and
the resulting syrupy residue was dissolved in dry toluene and
evaporated to dryness. The resulting solid was dissolved in
CH2Cl2–pyridine (1 : 1) and 1-hexadecanol (1.1 equiv.) and
a catalytic amount of DMAP were added. The solution was
stirred overnight at room temperature. After evaporation to
dryness, the mixture was purified by column chromatography
on silica gel with 2 : 8 EtOAc–hexane as eluent. The product
was crystallized from ether–heptane to afford pure compound
1
4 as a white powder (yield 60%). H-NMR (CDCl3, d): 0.88
(3H, t, J = 6.4, CH3), 1.27–1.58 (m, 26H, 13CH2), 1.70 (2H, m,
CH2), 2.32, 2.33 (6H, s, 2OAc), 4.21 (2H, t, J = 6.7, CH2), 6.40
(1H, d, J = 16.0, H-a), 7.24 (1H, d, J = 8.3, H-5), 7.39 (1H,
d, J = 2.0, H-2), 7.42 (1H, dd, J = 8.3 and J = 2.0, H-6), 7.63
(1H, d, J = 16.0, H-b). 13C-NMR (CDCl3, d): 14.5 (CH3), 21.0
(2 OCOCH3), 23.1, 26.1, 26.4, 28.2, 29.1, 29.7; 29.8; 29.9; 30.0,
30.1, 32.3, 33.2, 65.3 (CH2), 119.9, 123.1, 124.3, 126.7 (C-a,
C-2, C-5, C-6), 133.8 (C-1), 142.8, 143.0, 143.8 (C-3, C-4, C-b),
Data analysis. Molecular modeling was performed with
Hyperchem (Autodesk, Sausalito, USA). The Scientist program
(MicroMath, Salt Lake City, USA) was used for all curve-fitting
and simulation procedures.
Abbreviations. DPPH:
2,2
diphenyl-1-picrylhydrazyl;
AAPH: 2,2ꢂ-azo-bis (2-methylpropionamidine) dihydrochloride;
SDS: sodium dodecylsulfate.
=
167.1, 168.3, 168.4 (2 OCOCH3, OCOCH CH).
n-Hexadecyl-3,4-dihydroxycinnamate or n-hexadecylcaffeate
(5). To a solution of compound 4 (0.45 g, 0.922 mmol) in
MeOH–CH2Cl2 (1 : 1) was added a catalytic amount of K2CO3
and the mixture was stirred for 5 h at room temperature under
N2. After removal of the solvent under a reduced pressure,
the residue was dissolved in EtOAc and the organic layer
washed twice with water, dried over anhydrous Na2SO4 and
concentrated. The residue was purified by recrystallization in
ether–heptane, yielding compound 5 as a white solid (yield 56%).
1H-NMR (CDCl3, d): 0.90 (3H, t, J = 6.4, CH3), 1.21–1.59 (m,
26H, 13CH2), 1.71 (2H, m, CH2); 4.20 (2H, t, J = 6.7, CH2), 6.28
(1H, d, J = 15.9, H-a), 6.88 (1H, d, J = 8.2, H-5), 7.04 (1H, dd,
J = 8.2 and J = 2.0, H-6), 7.10 (1H, d, J = 2.0, H-2), 7.58 (1H,
d, J = 15.9, H-b). 13C-NMR (CDCl3, d, tentative assignment
according to the literature27): 14.4 (CH3), 23.1, 26.4, 29.2, 29.4,
29.5, 29.6, 29.7, 29.9, 30.0, 30.1, 32.3, 65.2 (CH2) 114.9 (C-2),
116.0 (C-5), 116.4 (C-a), 122.8 (C-6), 128.2 (C-1), 144.2 (C-3),
Acknowledgements
M. Roche is grateful to the General Council of the PACA region
and to INRA for financial support.
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Antioxidant tests
Reduction of the DPPH radical. To 2 mL of a freshly
prepared 0.2 mM solution of DPPH in MeOH (molar ab-
sorption coefficient at 515 nm = 11 240 M−1 cm−1 assuming
a purity of 95%) placed in the spectrometer cell was added
20 lL of a freshly prepared 2.5 mM solution of antioxidant
in MeOH. The reaction was monitored at 25 ◦C over 250–600 s.
Each experiment was repeated six times. Standard deviations
were lower than 5%.
AAPH-induced oxidation. To 5 mL of a freshly prepared
1 mM solution of phenol in a pH 7.4 phosphate–NaCl buffer
was added 70 mg of AAPH. The mixture was placed at 37 C
under stirring and analyzed by HPLC–MS every hour.
◦
Oxidation by (KSO3)2NO. To 4.5 mL of a freshly prepared
1 mM solution of phenol in a pH 7.4 phosphate–NaCl buffer
was added 0.5 mL of a freshly prepared 20 mM solution of
(KSO3)2NO. The mixture was placed at 25 ◦C under stirring
and analyzed by HPLC–MS every 30 min.
18 F. A. M. Silva, F. Borges, C. Guimara˜es, J. L. F. C. Lima, C. Matos
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O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 2 3 – 4 3 0
4 2 9