Vitamin B6-Derived Aminopyridinol Antioxidants
FULL PAPER
period lengths (t) were determined by the intersection between the re-
gression lines to the inhibited and the uninhibited traces. Initiation rates,
Ri, were determined in preliminary experiments by the inhibitor method
(100 mL), if applicable, or additional PBS (100 mL) in case aqueous anti-
oxidant was not used. Samples were vortexed for 2 min. Compound 17
(2.12 mmol) in MeOH (50 mL) was added, and milky mixtures were vor-
texed for an additional 20 s and incubated at 378C for 120 min (single an-
tioxidant) or 210 min (two antioxidants).
by using PMC as reference antioxidant: Ri =2ACTHNUTRGNE[UNG PMC]/t. The absolute rate
constant for inhibition kinh was obtained by Equation (6),[33] where kp is
[2a]
the propagation rate constant, which in the case of styrene is 41mꢀ1 sꢀ1
.
To monitor disappearance of antioxidants, aliquots were withdrawn every
15 min over at least 1 h. In case of aqueous antioxidants, aliquots
(100 mL)
were
vortexed
(20 s)
with
BHT/PPh3/MeOH
kp½styreneꢄ
ð6Þ
ꢀD½O2ꢄt ¼
lnð1 ꢀ t=tÞ
(0.25 mg:0.30 mg:400 mL) and an internal standard in MeOH (50 mL).
Samples were centrifuged (2 min at 10000 rpm), supernatants (300 mL)
were withdrawn, and solvents were blown off under a stream of argon.
Residues were dissolved in aqueous MeOH (70%, 70 mL, for 11) or
aqueous MeOH (50%, 100 mL, for uric acid), and mixtures were placed
in the autosampler at 58C prior to sample injection on HPLC.
kinh
Theoretical calculations: All calculations were carried out by using the
B3LYP density functional[34] and CBSB7 basis set[35] as implemented in
the Gaussian 03 suite of programs,[36] compiled to run on Sun Microsys-
tems SunFire 25000 or Enterprise M9000 servers with UltraSPARC-IV+
or Sparc64 VII CPUs, respectively. Briefly, geometries were first opti-
mized and then vibrational frequencies calculated to characterize the sta-
tionary points as either minima or transition states on the surface. Fol-
lowing thermochemical corrections to 298 K, the enthalpies of activation
were calculated relative to the separated reactants, and given relative to
that calculated for the reaction of 2 and methylperoxyl as shown in
Figure 1.
For a-TOH analysis, an aliquot (200 mL) was vortexed (20 s) with BHT/
PPh3/MeOH (0.50 mg:0.60 mg:700 mL) containing an internal standard in
MeOH (100 mL). Highly lipophilic compounds were extracted with
hexane (2ꢂ1 mL), organic layers were combined and dried over Na2SO4.
Solvents were blown off under a stream of argon, residues were dissolved
in hexanes (60 mL) and placed in the autosampler at 58C prior to sample
injection on HPLC.
Analyses of MeLin-OHs were performed as for oxidations initiated by
AIPH. Antioxidant consumption profiles were plotted on charts: [antiox-
idant] versus time, whereas the lag-phase duration periods and Rox,inh
values were extracted from [PLPC-OOHs] versus time.
Stability of antioxidants in aerated solutions: Antioxidant solutions
(1.00 mmol) in benzene (10 mL) were placed in a sand bath at 378C. Ali-
quots (400 mL) were withdrawn every 20 min over 3 h (3a) or every 8 h
over 48 h (2, 11, 12, and 14) and added to an ice-cold mixture of BHT
and P
G
Partition of antioxidants between PLPC (MLV) and PBS: Antioxidants
(52.2 nmol) in MeOH (100 mL) were added to PLPC (20 mg) in chloro-
form (800 mL). Solvents were blown off under a stream of argon to form
thin films. Degassed PBS (2.00 mL) was added to those films, mixtures
were vortex (2 min) and further degassed for 5 min with bubbling argon.
Vials containing milky suspensions were then carefully closed and incu-
bated for 2 h at 378C. The aqueous phase was separated from lipid aggre-
gates by centrifugation (3ꢂ5 min at 3500 rpm) in Amicon centrifugal
filter units (10 kDa cut off). Filtrates obtained after the initial 5 min of
centrifugation were discarded, an aliquot of the remaining supernatants
(400 mL) was vortexed with an internal standard in MeOH (400 mL) and
mixtures were placed in the HPLC autosampler at 58C prior to sample
injection on HPLC. The extent of partitioning of an antioxidant into lipo-
somes was assigned by comparison of [antioxidant]aqueous found in samples
incubated with and without PLPC and otherwise identically following the
above procedure.
were added in benzene (80 mL), mixtures were vortexed for 30 s and the
solvent was blown off under a stream of argon. The residue was dissolved
in MeOH/H2O (65%, 150 mL) and placed in the autosampler at 58C
prior to sample injection on HPLC. For aqueous mixtures, aliquots were
drawn every hour over eight hours (2, 11, 12, and 14) or every 15 min
over two hours (3a). Compounds were incubated in PBS (pH 7.4, 50 mm)
and a mixture of BHT and PACTHNUTRGNE(UGN OMe)3 (2.0 mmol each) was added in
MeOH (500 mL). Internal standards were also added in MeOH. Informa-
tion about stability was extracted from plots of [antioxidant] versus time.
Oxidation of antioxidants in homogenous solutions: Experiments were
conducted similar to the stability measurements, except antioxidants
were incubated in the presence of
MeOAMVN (in benzene) or AIPH (in PBS), and aliquots were with-
drawn every 5 min over 20 min (2, 3a, 11, 12, and 14) for benzene solu-
tions, whereas aliquots from the PBS solutions were taken every 15 min
over 90 min (2, 11, 12, and 14) or every 5 min over 25 min (3a). The half-
life times for antioxidants in the presence of radical initiators were ex-
tracted from plots of [antioxidant] versus time. Stoichiometric factors
were calculated as shown in the Supporting Information.
Reproducibility of the reported results: All experiments described were
repeated at least three times and an average value (stoichiometric factor,
rate of lipid peroxidation, rate of hydrogen atom donor autoxidation, per-
cent distribution in aqueous and lipid phase) was reported for each mea-
surement. In homogeneous systems, the repeated values were within a
10% margin of the reported value. In heterogeneous systems, the highest
standard deviation was less than 13%. Stability tests in aerated solvents
were also repeated at least three times consistent with all other results.
Oxidation of PLPC (MLV) in PBS in the presence of AIPH: Antioxi-
dants (26.6 nmol) in methanol (100 mL) were added to PLPC (20 mg) in
chloroform (800 mL). Solvents were blown off under a stream of argon to
form thin films. PBS (1.90 mL) was added to those films followed by an
addition of AIPH (1.06 mmol) in PBS (100 mL). Samples were vortexed
for 2 min and incubated at 378C for 4 h. Every 30 min, aliquots (200 mL)
were drawn, vortexed (20 s) with an ice-cold BHT/PPh3/MeOH
(1.0 mg:1.2 mg:1 mL), then vortexed (20 s) with KOH/MeOH (0.50m,
1 mL) and left for 15 min at RT. After that time, mixtures were vortexed
(20 s) with aqueous NH4Cl (5%, 1 mL) extracted with hexane (2ꢂ1 mL).
Organic layers were combined and dried over Na2SO4, and solvents were
blown off under a stream of argon. The residual material was dissolved
in cinnamyl alcohol/hexane (12.5 mm, 100 mL) and placed in the HPLC
autosampler at 58C prior to sample injection on HPLC. The lag-phase
duration periods were extracted from plots of [PLPC-OOHs] versus
time. Data points (at least four per reaction) obtained at early stages of
PLPC oxidation were fit to linear equations from which Rox,inh values
were calculated.
Acknowledgements
We thank Dr. Maikel Wijtmans for helpful discussions. This work was
supported by the National Science Foundation, the Natural Sciences and
Engineering Research Council of Canada and by a Research Foundation
Grant funded by the Korean Government (MOEHRD, Basic Research
Promotion Fund, KRF-2008-331-E00460).
Carew, J. C. Khoo, J. L. Witztum, New Engl. J. Med. 1989, 320, 915;
d) P. Holveoet, D. Collen, FASEB J. 1994, 8, 1279; e) N. A. Porter,
Oxidation of PLPC (MLV) in PBS in the presence of 17: a-Tocopherol
(13.3 nmol) in MeOH (100 mL) was added (if applicable) to PLPC
(20 mg) in chloroform (800 mL). Solvents were blown off under a stream
of argon to form thin films. PBS (1.85 mL) was added to those films fol-
lowed by an addition of an aqueous antioxidant (13.3 nmol) in PBS
Chem. Eur. J. 2010, 16, 14106 – 14114
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
14113