Synthesis of α-tocohexaenol (α-T6) a fluorescent, oxidatively sensitive polyene analogue of α-tocopherol

Article abstract of DOI:10.1016/j.bmc.2009.11.051

A polyunsaturated analogue of α-tocopherol was synthesized that is both fluorescent and sensitive to peroxidative chemistry that occurs in phospholipid membranes. α-Tocohexaenol 1, [(S)-2,5,7,8-tetramethyl-2-((1E/Z,3E,5E,7E,9E)-4,8,12-trimethyltrideca-1,3,5,7,9,11-hexaenyl)chroman-6-ol, α-T6] was prepared by condensing a known triene fragment triphenyl-(2,6-dimethyl-octa-2,4,6-trienoic acid methyl ester)-phosphonium bromide with a protected chromanol aldehyde, (2S)-6-{[tert-butyl(dimethyl)silyl]oxy}-2,5,7,8-tetra-methyl-3,4-dihydro-2H-chromene-2-carbaldehyde. The full side chain was then completed with isopentyl(tri-n-butyl)phosphonium bromide to give 1. The geometry of the C1′-C2′ alkene appears to be Z (cis) although the coupling constants of the olefinic protons are intermediate between values normally assigned to E and Z-isomers. In ethanol, α-T6 has a maximum absorption at 368 nm with an absorption coefficient of 45,000 M^-1 cm^-1, and displays a maximum fluorescence emission at 523 nm. The susceptibility of α-T6 to peroxidative chemistry was dependent on the concentration of azo-initiators of lipid oxidation in acetonitrile solution as well as in phospholipid vesicles. A loss of fluorescence at 520 nm was observed when α-T6 (vesicles or α-T6-lipid mixtures) was exposed to peroxidative conditions, and this loss mirrored the production of conjugated dienes and trienes during the peroxidation of bulk phospholipids. Addition of natural α-tocopherol during the AMVN induced oxidation of 4 μM α-T6 and 0.5 mg/ml soybean PC induced a characteristic lag phase, after which the fluorescence of α-T6 began to lessen. Thus, α-T6 may be a useful reporter not only of tocopherol location in cells, but also of the extent of peroxidative events.

Full text of DOI:10.1016/j.bmc.2009.11.051

Bioorganic & Medicinal Chemistry 18 (2010) 777–786
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of
a-tocohexaenol (
a-T6) a fluorescent, oxidatively sensitive
polyene analogue of
a-tocopherol
Yongsheng Wang a,b, Candace Panagabko b, Jeffrey Atkinson b,
*
a Toronto Research Chemicals, Inc., 2 Brisbane Rd., North York, ON, Canada M3J 2J8
b Department of Chemistry and Centre for Biotechnology, Brock University, St. Catharines, ON, Canada L2S 3A1
a r t i c l e i n f o
a b s t r a c t
Article history:
A polyunsaturated analogue of
peroxidative chemistry that occurs in phospholipid membranes.
a
-tocopherol was synthesized that is both fluorescent and sensitive to
-Tocohexaenol 1, [(S)-2,5,7,8-tetra-
methyl-2-((1E/Z,3E,5E,7E,9E)-4,8,12-trimethyltrideca-1,3,5,7,9,11-hexaenyl)chroman-6-ol, -T6] was
prepared by condensing known triene fragment triphenyl-(2,6-dimethyl-octa-2,4,6-trienoic acid
Received 27 October 2009
Revised 20 November 2009
Accepted 21 November 2009
Available online 27 November 2009
a
a
a
methyl ester)-phosphonium bromide with a protected chromanol aldehyde, (2S)-6-{[tert-butyl(di-
methyl)silyl]oxy}-2,5,7,8-tetra-methyl-3,4-dihydro-2H-chromene-2-carbaldehyde. The full side chain
was then completed with isopentyl(tri-n-butyl)phosphonium bromide to give 1. The geometry of the
C10ꢀC20 alkene appears to be Z (cis) although the coupling constants of the olefinic protons are interme-
Keywords:
Vitamin E
a-Tocopherol
diate between values normally assigned to E and Z-isomers. In ethanol, a-T6 has a maximum absorption
Fluorescent membrane probe
Lipid peroxidation
at 368 nm with an absorption coefficient of 45,000 Mꢀ1 cmꢀ1, and displays a maximum fluorescence
emission at 523 nm. The susceptibility of -T6 to peroxidative chemistry was dependent on the concen-
tration of azo-initiators of lipid oxidation in acetonitrile solution as well as in phospholipid vesicles. A
loss of fluorescence at 520 nm was observed when -T6 (vesicles or -T6-lipid mixtures) was exposed
to peroxidative conditions, and this loss mirrored the production of conjugated dienes and trienes during
the peroxidation of bulk phospholipids. Addition of natural -tocopherol during the AMVN induced oxi-
-T6 and 0.5 mg/ml soybean PC induced a characteristic lag phase, after which the fluo-
-T6 began to lessen. Thus, -T6 may be a useful reporter not only of tocopherol location in
a
a
a
a
dation of 4
rescence of
lM a
a
a
cells, but also of the extent of peroxidative events.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
nol (
a
-T6) (Fig. 1), keeps much of the structure of
a
a
-tocopherol:
-tocopherol,
it has the same chromanol head and the skeleton of
The absorption and distribution of the lipophilic antioxidant
and the same (R)-stereochemistry at C-2. Furthermore, the inclu-
vitamin,
a-tocopherol, has been studied for many years by using
sion of six conjugated double bonds in the side chain will make
mass spectrometry to follow deuterium containing synthetic toc-
opherols.1–9 [For reviews see Refs. 10–13.] However, such isotopi-
cally labeled compounds are less useful at the cellular level
because identification of the labeled tocopherol and its metabolites
would normally require extraction from tissue samples, and this
precludes any time and spatial resolution of vitamin E movement
in a cell. It is advantageous, therefore, to use fluorescent molecular
probes that report cellular locations directly at low concentrations.
As vitamin E is only weakly fluorescent, we have prepared a series
it fluorescent. Using the Fieser–Kuhn rules,
have an absorption maximum of 366 nm and we predicted that
the fluorescence emission of -T6 would be >500 nm based on
its similarity to a reported fluorescent didehydrofarnesol ana-
logue22 a pentaene that fluoresces at 465 nm. Compared with
tocopherol, which has an absorption maximum of 292 nm and
emission maximum of 325 nm, the fluorescent properties of -T6
a-T6 is calculated to
a
a
-
a
will make it a useful fluorescent reporter not interfered with by
endogenous cellular chromophores.
of fluorescent analogues of
a
-tocopherol14 one of which (NBD-
a-
A similar molecule named FeAOX-6 has been made and charac-
terized by the group of Palozza et al.21,23,24 FeAOX-6 is missing one
double bond at C90 and the lack of full conjugation leads to a short-
ening of the wavelength of absorption (to ꢁ300 nm), which is not
tocopherol) has been successfully used in the study of intracellular
trafficking of vitamin E.15–20
We envisioned that a new fluorescent analogue of
could be designed and synthesized that would be less bulky and
less polar than the NBD moiety. This new molecule, -tocohexae-
a-tocopherol
that different from natural a-tocopherol. Also, the lesser degree of
a
conjugation was insufficient for our intended use as a fluorophore.
Polyenes such as retinoids25 and carotenoids26 are susceptible
to oxidation. Indeed, both retinoic acid27 and b-carotene28–30 have
been studied as inhibitors of lipid peroxidation and they have been
* Corresponding author. Tel.: +1 905 6885550 3967; fax: +1 904 682 9020.
E-mail address: jatkin@brocku.ca (J. Atkinson).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.051

778
Y. Wang et al. / Bioorg. Med. Chem. 18 (2010) 777–786
We planned to convert alcohol 5 to the bromide 4 and eventu-
ally the phosphonium salt 3, which would be coupled with Trolox
aldehyde 214,33 to provide
-T6, 1. We followed reported synthetic
HO
HO
O
N
H
N
a
N
methods for preparation of pentaenol 522,34 with minor modifica-
tions, the most important of which was the substitution of LiHMDS
for n-BuLi during the synthesis of the intermediate 8-(tert-butyl-
dimethylsilanoxy)-2,6-dimethylocta-2,4,6-trienoic acid methyl es-
ter (compound 7 in their numbering22) which we found to give
higher yields.
O
O
O
NBD-α-tocopherol
NO2
H
Unfortunately, all our efforts to transform the alcohol 5 to the
corresponding phosphonium salt 3 failed. Treatment of 5 with bro-
minating reagents, such as HBr, PBr3, or CBr4 and PPh3, followed by
reaction with PPh3 or PBu3, did not provide the desired phospho-
nium salt. Treatment of alcohol 5 with triphenylphosphine hydro-
bromide proceeded in only 10% yield despite the results of
Furuhata et al.35 who reported a one-pot Wittig reaction with a
similar conjugated allylic alcohol as starting material. Reacting
alcohol 5 with triphenylphosphine hydrobromide followed by add-
ing aldehyde 2 and KOH in methanol, did not produce an olefin
product, only aldehyde 2 was recovered. Alcohol 5 was presumably
lost to polymerization to form unrecoverable products. In an at-
tempt to circumvent this failure we tried to prepare a phospho-
nium salt from the bromochromanol 8, however, 8 did not react
with triphenylphosphine, even when refluxed in toluene for 5 days
(Scheme 2), presumably due to the sterically congested nature of
this neopentylic position.
FeAOX-6
H
HO
1«
9«
5«
α-Tocohexaenol (α-T6), 1
(S)-2,5,7,8-tetramethyl-2-((1E/Z,3E,5E,7E,9E)-4,8,12-trimethyltrideca-
1,3,5,7,9,11-hexaenyl)chroman-6-ol
Figure 1. Structure of the fluorescent tocopherol derivative NBD-
FeAOX-621 and the
-tocohexaenol ( -T6) prepared in this work.
a ,
-tocopherol14
a
a
shown to be capable of reacting with lipid hydroperoxides. If the
fully conjugated side chain of -T6 is susceptible to oxidation in
a manner similar to carotenoids31 then oxidation should destroy
the -conjugation and thus the fluorescence intensity. We hoped
that the fluorescence signal of -T6 would act as an oxidatively
sensitive membrane probe whose structure would mimic the
membrane location and behavior of
-tocopherol32, but whose
fluorescent signal would diminish when local oxidative stress
overwhelmed antioxidant defenses. In effect, -T6 would act as a
fluorescent-reporting tocopherol until it was consumed by oxida-
tive events. This would represent a unique manner of visualizing
not only the location of tocopherol within a cell, but also poten-
tially the differential loss of tocopherol-specific fluorescence in
those parts of the cell where oxidative events are greatest. Admit-
a
p
a
1.2. Synthesis of a-T6, 1 (C14 + C10 + C5 strategy)
a
Our final and successful approach involved the coupling of alde-
hyde 2 with a smaller phosphonium salt 12 that was available from
an intermediate we had already prepared in the synthesis of 5, fol-
lowed by side chain extension (Scheme 3).
Deprotection of silyl ether 10 gave alcohol 11. The conversion of
alcohol 11 to bromide 12 gave only fair yields (ꢁ40%) using hydro-
bromic acid solution, PBr3, or CBr4/PPh3. However, good yields
(88%) of 12 were obtained using triphenylphosphine hydrobro-
mide. Treatment of 12 with LiHMDS and coupling to aldehyde 2
gave ester 13. Reduction of ester 13 with DIBAL-H at 0 °C afforded
14 (70%), which was oxidized by active MnO2 to yield aldehyde 15.
A final olefination using isopentyl(tri-n-butyl)phoshphonium bro-
mide 17 provided the silyl protected phenol 16 that, following
a
tedly, the polyene tail of
than -tocopherol so that it may not have the exact same proper-
ties when embedded in a membrane.
a-tocohexaenol will make it more rigid
a
1.1. Synthesis of a-tocohexaenol (a-T6), 1
deprotection, gave the target a-T6, 1 (Fig. 2).
Our synthetic strategy first pursued a convergent synthesis
(chromanol + C15 fragment) based on our past use of a chroman
aldehyde derived from the available starting material Trolox and
a pentaene phosphonium salt which would form the final double
bond in a Wittig reaction. Scheme 1 illustrates this approach.
1.3. Establishment of the alkene geometry
The geometry of the double bonds in intermediate 10 were con-
firmed as trans based on the comparison to published spectra.22
Only the alkenes at C10ꢀC20 and C90ꢀC100 were created after the
use of intermediate 10. Palozza et al. report the chemical shift of
HO
the proton on C10 in FeAOX-6 (similar to
a-T6 but lacking the al-
O
α-Tocohexaenol, 1
RO
RO
RO
CBr4/PPh3
DIBAL-H, 0 oC
OH
O
TBDMSO
Br
O
O
CO2Me
+
(Bu)3P
7
O
CHO
6
3
2
RO
Br
Br
X
PPh3
O
R
8
4 R = Br
5 R = OH
9
R = TBDMS
Scheme 1.
Scheme 2. Attempted synthesis of protected chromanol phosphonium salt.

Y. Wang et al. / Bioorg. Med. Chem. 18 (2010) 777–786
779
PPh3.HBr
TBAF
65%
HO
CO2Me
11
RO
CO2Me
88%
10
R = TBDMS
Br
LHMDS, -78 o
aldehyde 2
C
RO
H
DIBAL-H, 0 o
70%
CO2Me
C
(Ph)3P
CO2Me
12
O
R = TBDMS
13
RO
n-BuLi,17
RO
H
-78 oC
MnO2
TBAF
H
OH
O
O
CHO
87%
14
15
RO
HO
H
H
O
O
1
16
Br
(n-Bu3)P,
toluene, reflux
Br
(n-Bu)3P
91 %
17
Scheme 3.
Figure 2. HPLC chromatogram of a-T6. The column was a Synergi 4 lm Hydro-RP (C18), 150 ꢂ 4.6 mm (Phenomenx) running on a Waters 626 pump with 990 photodiode
array with detection at 525 nm. The solvent system was 100% methanol at 1.0 ml/min.
kene between C90 and C100) as d 6.82 with a coupling constant (J-
with J-values of 10.7 and 17.5 Hz. We conclude, therefore, that the
C10–C20 double bond in the
-T6 we prepared likely has the cis-
geometry. The structures of C10-cis and C10-trans
-T6 were inves-
tigated by molecular mechanics minimization using Spartan
(Wavefunction, Inc.). When the alkenyl -systems were kept in
the same plane, the distance between the C2 of the pyran ring
value) of 12 Hz to H-C20. The analogous C10 proton in tetraene alde-
a
hyde 15 was a doublet at d 6.89 (J = 12.1 Hz) and in
d 6.90 (J = 12.3 Hz). The proton at C20 is not easily discernable in
the 1D spectrum of -T6 due to the multiplicity of signals between
d 6.6ꢀ6.2, but the 2D COSY of -T6 clearly shows a correlation be-
a
-T6 is found at
a
a
p
a
tween the C10 signal at d 6.90 and a doublet centered at d 6.37 with
J = 12.3 Hz. This value of the coupling constant is intermediate be-
tween those normally expected for cis- or trans-alkenes: J3 H–H
coupling of 8ꢀ12 Hz for cis and 14ꢀ17 Hz for trans. By way of
comparison, our previous preparation of the alkenes 18
(Scheme 4)14,33,36 used a similar triphenylphosphonium salt and
clearly produced a cis-alkene with a J-value for an overlapped dou-
blet of doublets at d 5.3 of ꢁ6 Hz. Similar monoalkenes have been
made previously using triphenylphosphonium salts,37–39 and in
one case40 (Scheme 4) the product was reported to be a 14:1 mix-
ture of cis:trans, but only coupling constants for the trans isomer
were discernible: the C10 proton at d 5.79 was a doublet of doublets
and C120 was reduced from 14.84 Å in the all-trans-
a
-T6 to
13.82 Å in the C10ꢀC20 cis-
a-T6. No attempt was made to photoiso-
merize this bond.
1.4. Fluorescence characterization of
a-T6
The absorbance spectrum of -T6 includes several maxima. In
a
ethanol these occur at 349, 368, and 390 nm. The maximum
absorption at 368 nm in ethanol shifts slightly to 367 nm in hexane
(Fig. 3). The absorption coefficients at 368 nm are ꢁ45,000 and
24,000 Mꢀ1 cmꢀ1 in ethanol and hexane, respectively. The maxi-
mum fluorescence emission is at 523 nm in EtOH and 520 nm in

780
Y. Wang et al. / Bioorg. Med. Chem. 18 (2010) 777–786
RO
RO
H
H
base
+
OH
(Bu)3P
Br
OH
O
CHO
O
n
n
18
2 R=TBDMS
5.00, 1H, ddd, J = 1.4, 10.7, 17.3 Hz
(Compound 28 in Nozawa et al. Ref. 40)
5.79, 1H, dd J =10.7 & 17.3 Hz
BnO
H
O
H
Scheme 4.
0.45
0.4
0.35
0.3
in EtOH
in hexane
0.25
0.2
0.15
0.1
0.05
0
300
350
400
450
Wavelength (nm)
Figure 3. Absorption spectrum of 9.2 lM a-T6 in ethanol and hexane.
hexane (Fig. 4). Fluorescence intensity in ethanol–water solutions
decreases with increasing water content and is almost absent be-
low 10% (vol/vol) ethanol concentrations.
The rate of fluorescence loss at 520 nm was dependent on the
concentration of AMVN and was inhibited by the addition of
10 lM a-tocopherol, exhibiting the typical lag phase seen with
a
-T6 was designed to be fluorescent and yet oxidatively sensi-
tive. Since the structure has been kept as similar to -tocopherol
as possible, the expectation is that -T6 will mimic -tocopherol’s
location and orientation in phospholipid bilayers. If -T6 is ex-
these sorts of assays.56–59 Importantly,
solution when no initiator is present.
a-T6 is stable in aerated
a
a
a
Niki has shown that, in benzene, b-carotene is 32 times less
reactive than -tocopherol towards peroxyl radicals generated
from AMVN in the presence of methyl linoleate.29 However, when
-tocopherol and b-carotene are combined in the same soybean PC
a
a
posed to conditions that initiate and support polyunsaturated
phospholipid peroxidation, one should be able to observe a loss
of fluorescence that parallels peroxidative chemistry. Several other
fluorescent probes are available that accomplish the same goal,
such as cis-parinaric acid41–49 and BODIPY-(581/591),48,50–55 but
these are global lipid sensors and may not share the same cellular
a
liposomal membrane both are consumed at the same rate. These
authors noted that, ‘. . .b-carotene can be as or even more effective
than
membrane.’
This is thought to be due to the position of a-tocopherol in the
a-tocopherol toward lipophilic radicals present in the
membrane location and dynamics as
a-tocopherol.
To get a preliminary sense of whether
a
-T6 meets these criteria
membrane where the phenol hydroxyl is near the head group re-
gion and the isoprenoid side chain is anchored in the core of the
membrane.32 b-Carotene has no similar polar functional group
and is thus likely buried in the bilayer core.60 Thus, when hydro-
phobic AMVN is the radical initiator in solution, peroxyl radicals
we measured the loss of fluorescence of this compound in both or-
ganic solution and phospholipid vesicles when subjected to various
common initiators of lipid peroxidation. Figure 5 shows a repre-
sentative time course of fluorescence traces when 4 lM a-T6 was
combined with 0.5 mg/ml soy PC and 1.25 mM 2,20-azobis(2,4-
dimethylvaleronitrile) (AMVN) in acetonitrile at 37 °C. The fluores-
cence maxima at 520 nm slowly decayed over several hours, while
the maxima at 485 nm initially rose slightly and then dropped.
Figure 6 shows fluorescence decay curves observed when acetoni-
have easy access to the phenolic hydrogen of a-tocopherol and this
spares the b-carotene. In a membrane the reaction is slowed by the
greater difficulty of the peroxyl radical reaching the tocopherol
phenol, that is, oriented away from the center of the bilayer.
In the case of
a-T6 the structural elements of both a-tocopherol
trile solutions of a-T6, soy PC, and AMVN were followed over time.
(phenol) and b-carotene (polyene) reside in the same molecule. We

Y. Wang et al. / Bioorg. Med. Chem. 18 (2010) 777–786
781
1.1
1.2
1
Ethanol
DOPC
Hexane
1.0
0 mM AMVN
0.8
0.6
0.4
0.2
0
1. 25 mM AMVN
2. 5 mM AMVN + α-toc
2. 5 mM AMVN
0.9
0.8
0.7
0.6
0
100
200
300
400
Time (min)
425
475
525
575
Wavelength (nm)
Figure 6. AMVN induced oxidation of 3.0
l
M
a
-T6 and 0.5 mg/ml soy PC in the
presence and absence of 10 -tocopherol in acetonitrile at 37 °C. a
lM
a
-T6 was
monitored as the ratio of fluorescence at 520 nm/485 nm (kex 368 nm) since over
the course of oxidation the 520 nm emission decreases and the 485 nm emission
increases slightly before decreasing.
Figure 4. Fluorescence spectra of 6.0 lM a-T6 in ethanol, 100 nm DOPC unilamel-
lar vesicles, and hexane. The excitation wavelength was at 368 nm. The fluores-
cence intensities are normalized to the maximal fluorescence at 523 nm in ethanol.
ene chain where it disrupts conjugation and destroys
fluorescence. When
threefold excess over
a
a
-tocopherol is present in approximately
-T6 there appears to be some sparing of
1.05
0.95
a-T6 which delays the loss of fluorescence. This would also occur
if the polyene tail of a-T6 did not react as quickly with peroxyl rad-
icals as does b-carotene—a reasonable assumption given the great-
er degree of conjugation of b-carotene. Should -T6 be used as a
probe to detect peroxidative events in cells it will be easy to dose
-T6 in concentrations equivalent to or more than endogenous
tocopherol thus eliminating any sparing effect.
Figure 7 confirms that -T6 is susceptible to oxidative loss of
t =0 min
0.85
0.75
0.65
0.55
0.45
0.35
0.25
0.15
a
a
a-
18
25
a
fluorescence and that this occurs simultaneously with lipid perox-
idation and the production of conjugated dienes as monitored at
40
234 nm. Importantly, the absorption of
creased over time, mirroring the loss of fluorescence, showing that
-T6 was being consumed. That -T6 is present in the bilayer of
a-T6 at 368 nm also de-
a
a
85
125
vesicles is strongly suggested by the half-lives for fluorescence loss
being more sensitive to the concentration of the hydrophobic
AMVN than the more polar ABAP (Fig. 8). At dilute concentrations
220
280
315
of either initiator (0.5 mM) the half-life of
a-T6 was approximately
the same, but rates of -T6 disappearance rapidly diverged as the
a
1080
amount of each initiator was increased.
Other commonly used initiators of lipid peroxidation also pro-
475
495
515
535
555
moted loss of
50 M CuSO4, 250
loss of fluorescence was 83 min (data not shown). The half-life of
-T6 in egg PC vesicles when exposed to 125 M Fe(ClO4)2 and
1.0 mM H2O2 was 280 min. Treatment of lipid samples with
1 mM hydrogen peroxide or 100 M inorganic superoxide (as
KO2) did not decrease -T6 fluorescence.
Titration of a solution of TTP with aliquots of
a
-T6. When
a-T6 in egg PC vesicles was treated with
Wavelength (nm)
l
l
M ascorbate and 1.5 mM H2O2, the half-life for
Figure 5. Fluorescence decay of
a-T6 (4.0 lM) combined with 0.5 mg/ml soy PC
a
l
and 1.25 mM AMVN in acetonitrile at 37 °C. Fluorescent spectra were normalized
based on the maximal fluorescence at 520 nm (F0) at the beginning of the reaction
(t = 0 min). Spectra were recorded at various intervals throughout the reaction
where spectra representative of the progressive changes in the fluorescence as
oxidation proceeds are shown in the figure.
l
a
a-T6 provided the
binding curve shown in Figure 9. A dissociation constant, Kd, was
obtained by fitting to a one-site binding model and was found to
be 540 35 nM. We attempted to illustrate the specificity of this
therefore expect that it is able to act as a hydrogen atom donating
antioxidant like
to the polyene like b-carotene. The lack of a substantial lag phase in
fluorescence loss when only -T6 is present suggests that the phe-
noxyl radical formed by H-atom donation delocalizes to the poly-
a
-tocopherol as well as have peroxyl radicals add
binding by first equilibrating
cence signal, followed by sequential addition of increasing
amounts of -tocopherol as competitive ligand. Unfortunately,
the fivefold excess of -T6 used to assure high occupancy on TTP
a-T6 and TTP to a constant fluores-
a
a
a

782
2.40
Y. Wang et al. / Bioorg. Med. Chem. 18 (2010) 777–786
120
A
ABAP-LUV
2.00
1.60
1.20
0.80
0.40
0.00
AMVN-LUV
100
80
60
40
20
0
234 nm (+ α-T6)
234 nm (- α-T6)
270 nm (+ α-T6)
270 nm (- α-T6)
AMVN-PC/CH3CN
0
2
4
6
8
10
12
0
50
100
150
200
250
300
Initiator concen. (mM)
Time
Figure 8. Changes in the observed half-lives of a-T6 fluorescence when oxidations
0.18
0.16
0.14
0.12
0.1
were performed with water soluble (ABAP) and lipid soluble (AMVN) initiators in
100 nM large unilamellar vesicles (LUV) composed of egg PC or in solutions of soy
PC in acetonitrile at 37 °C.
B
368 nm (- α-T6)
368 nm (+ α-T6)
16000
14000
12000
10000
8000
6000
Kd = 540 35 nM
4000
2000
0
0.08
0.06
0.0
2.5
5.0
7.5
10.0
[α-T6] μM
Figure 9. Titration of 0.2
l
M
TTP with
a-T6. Values have been corrected for
0
50
100
150
Time (min)
200
250
300
fluorescence measured when
a-T6 was added at the same concentrations to buffer
solution containing no TTP. See Section 2 for full details.
Figure 7. UV absorbance changes during AMVN induced oxidation of 4 lM a-T6
and 0.5 mg/ml soybean PC by 2.5 mM AMVN in acetonitrile at 37 °C. (A) Lipid
oxidation was monitored by the absorbance of conjugated dienes at 234 and
night, and then cooled in desiccators before use. Cooling baths
were prepared with acetone/liquid nitrogen for ꢀ78 °C and
ꢀ60 °C, and ice/water for 0 °C. Air or moisture sensitive reactions
were performed under N2. THF (tetrahydrofuran) was distilled
with sodium/benzophenone immediately before use, benzene
was distilled over sodium metal, dichloromethane was distilled
over CaH2, methanol was distilled from Mg/iodine. n-Butyllithium
was titrated with diphenylacetic acid or N-benzylbenzamide to a
yellow or a blue endpoint, respectively, immediately before use.
Reagent grade solvents were used for extractions. Distilled water
was used for all aqueous workups.
270 nm for dienes and trienes, respectively. (B) Oxidation of
the loss of absorbance at 368 nm.
a-T6 was followed by
supported an increase in fluorescence when tocopherol was added,
presumably due to interaction of the two hydrophobic compounds
in free solution.
Thus, the polyene tocopherol analogue a-T6 is sensitive to con-
ditions of polyunsaturated lipid peroxidation and its consumption
over time can be followed by the loss of fluorescence. In future
work we plan to explore the utility of
of peroxidative events.
a-T6 as a cellular marker
Analytical thin-layer chromatography (TLC) was performed on
Merck 0.25 mm pre-coated Silica Gel 60 Å F-254 aluminum plates
and visualized under UV light, or stained in 5% KMnO4 solution.
Column chromatography was carried out on silica gel (200–300
mesh) purchased from Aldrich.
2. Experimental
2.1. General
1H NMR and 13C NMR spectra were measured on a Bruker Ad-
vance DPX-300 digital FT NMR spectrometer (300 and 75 MHz,
respectively) in CDCl3 with residual chloroform as internal
All reagents were purchased from Sigma–Aldrich Chemical Co.,
Oakville, Ontario. All glassware was dried in an oven of 90 °C over-

Y. Wang et al. / Bioorg. Med. Chem. 18 (2010) 777–786
783
reference (7.281 ppm for 1H and 77.0 ppm for 13C) unless otherwise
2.2.3. Triphenyl-(2,6-dimethyl-octa-2,4,6-trienoic acid methyl
noted. Chemical shifts are reported as d values and coupling con-
stants (J) are reported in hertz (Hz). The following abbreviations
are used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
andbr(broad). Massspectra(MS)wererecordedonaCarloErba/Kra-
tosGC/MSConcept1S doublefocusingmassspectrometer interfaced
to a Kratos DART acquisition system and a Sun SPARC workstation.
Samples were introduced througha direct inlet system. Ions are gen-
erated using electron impact (EI) or Fast Atom Bombardment (FAB)
and were reported in m/z values. Fluorescence was recorded on a
Photon Technologies International QuantaMaster Model QM-2001
ester)-phosphonium bromide (12)
Br
Ph3P
CO2Me
The mixture of alcohol 11 (312 mg, 1.59 mmol) and triphenyl-
phosphine hydrobromide (657 mg, 1.91 mmol) in DCM and ethyl
ether (10 ml, 3:2) was stirred for 4 h at room temperature under
N2. After the solvent was removed, the residue was washed with
hexane (5 ꢂ 10 ml), and dried under vacuum to give 865 mg of
12 as an orange foam. This was used directly in next step without
further purification. Mp 122–124 °C.
L-format, equipped with double-grating monochromators,
150 W xenon lamp, and Felix 32 software.
a
1H NMR d 7.9–7.6 (m, 15H), 7.18 (d, J = 9.6 Hz, 1H, @CH), 6.36
(m, 2H, @CH), 5.58 (virtual quartet, J = 7.5 Hz, 1H, @CH), 4.86 (dd,
J1 = 8.1 Hz, J2 = 16.2 Hz, 2H, P+–CH2), 3.67 (s, 3H, OCH3), 1.89 (s,
3H, CH3), 1.46 (d, J = 3.6 Hz, 3H, CH3).
2.2. Synthesis
2.2.1. 8-(tert-Butyldimethylsilanyloxy-2,6-dimethyl-octa-2,4,6-
trienoic acid methyl ester (10)
Mass spectra [FAB] m/z 441 (MꢀBr), 100%), 262 (38.3%), 55 (58%).
HRMS calcd: 441.19834, found: 441.20047.
2.2.4. (S)-6-(tert-Butyldimethylsilanyloxy)-2,5,7,8-tetramethyl-
chroman-2(4,8-dimethyl-1,3,5,7-tetraenoic acid methyl ester) (13)
TBDMSO
CO2Me
To a solution of a 3:2 mixture of (E)-methyl 4-(diethoxyphos-
phoryl)-2-methylbut-2-enoate and (Z)-methyl 2-((diethoxyphos-
phoryl)methyl)but-2-enoate61 (6.75 g, 27 mmol) in dry THF
(100 ml) was added LiHMDS (27 ml, 1 M in THF) at 0 °C. After stir-
ring for 30 min, 4-(tert-butyldimethylsilanyloxy)-2-methyl-but-2-
enal34 (1.89 g, 8.83 mmol) in THF (80 ml) was added dropwise.
After 3 h, the reaction was quenched by water and extracted with
ethyl acetate (4 ꢂ 50 ml). The combined organic extracts were
dried over anhydrous MgSO4 and concentrated. Purification on
SiO2 (ethyl acetate/hexane 1:9) afforded 2.36 g (86%) of 10 as a
light yellow oil. TLC Rf = 0.4 (hexane/ethyl acetate = 9:1).
1H NMR d 7.28 (d, J = 8.2 Hz, 1H HC@C), 6.53 (d, J = 15.3 Hz, 1H,
CH@CH), 6.44 (m, 1H, CH@CH), 5.78 (t, J = 6.2 Hz, 1H, CH@), 4.38
(d, J = 6.2 Hz, 2H O–CH2), 3.78 (s, 3H, OCH3), 2.74 (s, 3H, CH3),
1.99 (s, 3H, CH3), 0.93 (s, 9H), 0.10 (s, 6H).
TBDMSO
CO2Me
O
To a solution of phosphonium bromide 12 (410 mg, 0.79 mmol)
in THF (10 ml) was added LiHMDS (0.27 ml, 0.27 mmol) at 0 °C un-
der N2. The resulting red colored solution was stirred for 30 min,
then cooled to –78 °C. The Trolox aldehyde 233 (92 mg, 0.26 mmol)
in THF (2 ml) was added via syringe. The mixture was allowed to
warm up to room temperature and stirred overnight. Water was
then added, followed by extraction with ethyl acetate
(4 ꢂ 20 ml). The combined organic extracts was dried over anhy-
drous MgSO4 and concentrated. Purification on SiO2 (DCM/hexane
3:1) afforded 55.6 mg of 13 (40%) as a light yellow oil. TLC Rf = 0.42
(DCM/hexane = 3:1).
13C NMR d 168.94, 143.7, 138.84, 135.92, 134.05, 126.24,
122.71, 60.39, 51.79, 25.94, 25.84, 18.6, 12.79, 12.61, ꢀ5.14, ꢀ5.29.
Mass spectra [EI+] m/z 310 (M+, 6.2%), 253 (46.9%), 221 (49.1%),
178 (58.9%), 147 (71.5%), 119 (73.9%), 73 (100%).
1H NMR d 7.32 (d, J = 12 Hz, 1H, @CH), 7.06 (d, J = 12 Hz, 1H,
@CH), 6.60–6.48 (m, 2H, @CH), 6.34 (t, J = 12 Hz, 1H, @CH), 5.59
(d, J = 12 Hz, 1H, @CH), 3.80 (s, 3H, CH3), 2.60 (m, 1H, CH2), 2.19
(s, 3H, Ar-CH3), 2.11 (s, 3H, Ar-CH3), 2.05 (s, 3H, Ar-CH3), 2.01 (s,
3H, CH3), 1.88 (s, 3H, CH3), 1.56 (s, 3H, CH3), 1.05 (s, 9H, 3CH3),
0.13 (s, 6H, 2CH3). Protons from the chroman C3–CH2 are partially
obscured by the multiple CH3 signals.
HRMS calcd: 310.19642, found: 310.19487.
2.2.2. 8-Hydroxy-2,6-dimethyl-octa-2,4,6-trienoic acid methyl
ester (11)
13C NMR d 144.47, 139.04, 137.25, 135.89, 130.73, 126.09,
125.69, 123.60, 123.25, 117.68, 51.81, 33.73, 27.51, 26.10, 21.24,
18.60, 14.37, 13.43, 12.84, 12.44, 11.88, ꢀ3.30.
Mass spectra [EI+] m/z 510 (M+, 23.9%), 378 (23.1%), 319
(58.4%), 279 (42.3%), 73 (100%).
HO
CO2Me
HRMS calcd: 510.31645, found: 510.31621.
To a solution of compound 10 (2.23 g, 7.19 mmol) in THF
(250 ml) was added TBAF (33 ml, 1 M in THF) under N2. After 4 h
the reaction was quenched with water followed by extraction with
ethyl acetate (4 ꢂ 50 ml). The combined organic extracts was dried
over anhydrous MgSO4 and concentrated. Purification on SiO2
(hexane/ethyl acetate 1:1) afforded 962 mg (68%) of pale yellow
crystal. Mp 58–60 °C. TLC Rf = 0.27 (hexane/ethyl acetate = 9:1).
1H NMR d 7.28 (m, 1H, @CH), 6.6–6.45 (m, 2H, @CH), 5.84 (t,
J = 6 Hz, 1H, @CH), 4.35 (d, J = 6.6 Hz, 2H, CH2), 3.78 (s, 3H, CH3),
2.00 (s, 3H, CH3), 1.87 (s, 3H, CH3).
2.2.5. (2Z,4E,6E,8E)-9-(6-(tert-Butyldimethylsilanyloxy)-(S)-
2,5,7,8-tetramethylchroman-2-yl)-2,6-dimethylnona-2,4,6,8-
tetraen-1-ol) (14)
TBDMSO
OH
O
13C NMR d 168.62, 143.29, 138.63, 135.92, 134.12, 126.74,
123.45, 59.51, 51.84.
To a solution of ester 13 (270 mg, 0.53 mmol) in THF was added
DIBAL-H (1.6 ml, 1 M in DCM) at 0 °C. The resulting solution was
stirred for 2 h under N2. The reaction was quenched with methanol
Mass spectra [EI+] m/z 196 (53.3%), 119 (91.7%), 107 (96.3%), 91
(100%), 41 (59.1%).

784
Y. Wang et al. / Bioorg. Med. Chem. 18 (2010) 777–786
and water, followed by extraction with ethyl acetate (4 ꢂ 20 ml).
The combined organic extracts were dried over anhydrous MgSO4
and concentrated. Purification on SiO2 (ethyl acetate/hexane 1:3)
afforded 180 mg (71%) of 14 as a clear oil. TLC Rf = 0.33 (hexane/
ethyl acetate = 3:1).
was cooled to ꢀ78 °C. The mixture was allowed to warm up to
room temperature and stirred overnight. Water was added fol-
lowed by extraction with ethyl ether (4 ꢂ 15 ml). The combined or-
ganic extracts was dried over anhydrous MgSO4 and concentrated.
Purification on SiO2 (ethyl acetate/hexane 1:9) afforded 79.3 mg
(88%) of 16 as an orange oil. TLC Rf = 0.67 (hexane/ethyl
acetate = 9:1).
1H NMR d 6.91 (d, J = 12 Hz, 1H, @CH), 6.49–6.28 (m, 3H, @CH),
6.18 (d, J = 10.8 Hz, 1H, @CH), 5.47 (d, J = 12 Hz, 1H, @CH), 4.13 (m,
2H, OH–CH2), 2.60 (m, 2H, CH2), 2.18 (s, 3H, CH3), 2.10 (s, 3H, CH3),
2.05 (s, 3H, CH3), 1.88–1.85 (m, 6H, 2CH3), 1.55 (s, 3H, CH3), 1.05 (s,
9H, 3CH3), 0.13 (s, 6H, 2CH3). Protons from the chroman C3–CH2
are partially obscurred by the multiple CH3 signals at ꢁ2.1–2.0.
13C NMR d 136.30, 135.29, 127.66, 127.40, 125.92, 125.76,
122.57, 76.58, 69.40, 68.67, 63.18, 33.71, 27.83, 27.47, 27.05,
29.09, 21.26, 21.06, 20.44, 20.37, 18.60, 14.32, 12.36, 12.01,
ꢀ3.32, ꢀ3.37.
1H NMR d 6.90 (d, J = 12 Hz, 1H, @CH), 6.60–6.18 (m, 6H, @CH),
5.97 (d, J = 10.8 Hz, 1H, @CH), 5.48 (d, J = 12 Hz, 1H, @CH), 2.60 (m,
2H, CH2), 2.20 (s, 3H, CH3), 2.12 (s, 3H, CH3), 2.06 (s, 3H, CH3), 1.87–
1.83 (m, 14H, CH2 and 4 ꢂ CH3), 1.57 (s, 3H, CH3), 1.06 (s, 9H,
3CH3), 0.14 (s, 6H, 2CH3).
13C NMR d ppm 145.88, 144.37, 137.70, 136.74, 136.01, 135.74,
135.19, 134.98, 131.28, 127.43, 126.06, 125.10, 124.80, 123.54,
122.61, 117.79, 75.96, 33.74, 27.52, 26.29, 26.11, 21.30, 18.61,
18.58, 14.37, 13.43, 12.90, 12.37, 11.98, ꢀ3.30.
Mass spectra [EI+] m/z 482 (M+, 4%), 86 (81%), 84 (100%).
HRMS calcd: 482.32162, found: 482.30629.
Mass spectra [EI+] m/z 532 (M+, 9.5%), 319 (39.3%), 279 (30.5%),
221 (24.9%), 91 (44.5%), 73 (100%), 57 (50.2%), 43 (83.8%).
HRMS calcd: 532.37366, found: 532.37321.
2.2.6. (2Z,4E,6E,8E)-9-(6-(tert-Butyldimethylsilanyloxy)-(S)-
2,5,7,8-tetramethylchroman-2-yl)-2,6-dimethylnona-2,4,6,8-
tetraenal) (15)
2.2.8. Tri-n-butyl-(3-methyl-but-2-enyl)-phosphonium
bromide (17)62
TBDMSO
P(n-Bu)3
CHO
O
A mixture of tri-n-butylphosphine (4.25 ml, 17.0 mmol) and 1-
bromo-3-methyl-2-butene (2.0 ml, 17.3 mmol) in toluene (30 ml)
was heated at reflux for 5 h. After the toluene was removed under
reduced pressure, hexane (60 ml) was added and the solution was
heated at reflux for 1 h. After cooling to room temperature, the so-
lid that formed was filtered and dried under vacuum to give a
white crystal (5.48 g, 91%). Mp 61–63 °C.
A mixture of alcohol 14 (180 mg, 0.38 mmol), MnO2 (600 mg,
6.9 mmol) and Na2CO3 (200 mg, 1.89 mmol) in DCM (15 ml) was
stirred for 3 h at room temperature. After filtration and concentra-
tion, the residue was purified on SiO2 (ethyl acetate/hexane 1:3) to
give 69 mg (39%) of 15 as a yellow oil. TLC Rf = 0.58 (hexane/ethyl
acetate = 3:1).
1H NMR d 4.95 (m, @CH), 3.28 (q, J = 7.8 Hz, 2H), 2.28–2.38 (m,
6H), 1.68–1.71 (m, 6H), 1.36–1.55 (m, 12H), 0.83–0.89 (m, 9H).
13C NMR d 109.08, 108.96, 25.9, 24.00, 23.80, 23.74, 23.68,
19.19, 18.57.
1H NMR d 9.49 (s, 1H, CHO), 7.16 (d, J = 12 Hz, 1H, @CH), 6.97 (d,
J = 9.6 Hz, 1H, @CH), 6.72–6.65 (m, 2H, @CH), 6.36 (t, J = 12 Hz, 1H,
@CH), 5.62 (d, J = 12 Hz, 1H, @CH), 2.60 (t, J = 6.6 Hz, 2H, CH2), 2.20
(s, 3H, CH3), 2.11 (s, 3H, CH3), 2.06 (s, 3H, CH3), 1.98–1.85 (m, 8H,
CH2 and 2 ꢂ CH3), 1.56 (s, 3H, CH3), 1.05 (s, 9H, 3CH3), 0.13 (s, 6H,
2CH3).
Mass spectra [FAB] m/z 271 (M+ꢀBr, 100%).
13C NMR d ppm 194.68, 149.25, 146.41, 138.25, 137.02, 135.73,
132.41, 126.10, 125.55, 123.70, 122.85, 122.36, 117.68, 76.09,
33.72, 27.48, 26.10, 21.23, 18.60, 14.39, 13.44, 12.45, 11.87, 9.64,
ꢀ3.31.
2.2.9. (S)-6-Hydroxy-2,5,7,8-tetramethyl-2-((1Z,3E,5E,7E,9E)-
4,8,12-tetramethyl-trideca-1,3,5,7,9,11-hexaenyl)chromane (1)
HO
Mass spectra [EI+] m/z 480 (M+, 21.6%), 221 (27.9%), 149
(39.7%), 95 (55.2%), 73 (100%).
HRMS calcd: 480.30597, found: 480.30605.
O
2.2.7. tert-Butyldimethylsilanyloxy ((S)-2,5,7,8-tetramethyl-2-
((1Z,3E,5E,7E,9E)-4,8,12-tetramethyltrideca-1,3,5,7,9,11-
hexaenyl)chroman-6-yoxyl)silane (16)
To a solution of compound 16 (50 mg, 0.09 mmol) in THF (5 ml)
was added TBAF (0.2 ml, 1 M in THF) at room temperature under
N2. After stirring for 2 h, the reaction was quenched with water
and extracted with ethyl acetate (4 ꢂ 10 ml). The combined organ-
ic extracts were dried over anhydrous MgSO4 and concentrated.
Purification on SiO2 (ethyl acetate/hexane 1:3) afforded 14.3 mg
(38%) of compound 1 as an orange oil.
TBDMSO
O
TLC Rf = 0.45 (hexane/ethyl acetate = 3:1).
1H NMR d 6.90 (d, J = 12 Hz, 1H, @CH), 6.63–6.21 (m, 6H, @CH),
5.95 (d, J = 10.8 Hz, 1H, @CH), 5.47 (d, J = 12 Hz, 1H, @CH), 4.19 (b,
1H), 2.65 (m, 2H, CH2), 2.22 (s, 3H, CH3), 2.17 (s, 3H, CH3), 2.11 (s,
3H, CH3), 1.98–1.85 (m, 14H, 1CH2 and 3CH3), 1.57 (s, 3H, CH3).
13C NMR d 145.47, 144.84, 137.62, 136.92, 136.10, 135.81,
134.94, 131.21, 127.25, 126.33, 126.03, 125.19, 124.84, 121.13,
To a solution of tri-n-butyl(3-methylbut-2-enyl)phosphonium
bromide 17 (160 mg, 0.46 mmol) in THF (12 ml) was added n-BuLi
(0.33 ml, 1.39 M in hexane) at 0 °C. The resulting pale yellow solu-
tion was stirred for 30 min under N2. The aldehyde 15 (81.7 mg,
0.17 mmol) in THF (2 ml) was added after the reaction mixture

Y. Wang et al. / Bioorg. Med. Chem. 18 (2010) 777–786
785
118.52, 117.65, 75.89, 34.66, 33.74, 31.58, 27.59, 26.27, 25.27,
21.16, 12.88, 12.23, 12.16, 12.00, 11.29.
Mass spectra [EI+] m/z 418 (M+, 1.6%), 205 (3.8%), 149 (13.6%),
75 (100%).
in Prism Graphpad 4.0. The data was analyzed using an exponential
one phase decay model for derivation of the rate constants and
half-lives for each condition of oxidizing agent. Plateau values
were taken as the maximal oxidation from the 520/485 nm ratio
of the spectra obtained after incubation of each sample overnight
at room temperature.
HRMS calcd: 418.28718, found: 418.28669.
2.3. Controlled oxidation of 1
2.3.4. Fluorescence titration of TTP with a-T6
Fluorescence spectra were recorded on a Photon Technology
International (PTI) QuantaMaster 2000 fluorimeter using Felix 32
software. All solvents were degassed prior to use. Oxidation trials
Binding of a-T6 to the tocopherol transfer protein (TTP) was
investigated using methods previously reported.14 Briefly, the
baseline fluorescence from 515 to 535 nm (kex = 366 nm) of
of
a
-tocohexaenol were conducted with
a
-tocohexaenol incorpo-
0.2
50 mM Tris, 1 mM EDTA, pH 7.4) in a quartz cuvette. Subsequently,
an aliquot of -T6 was added from an ethanolic stock solution and
equilibrated on a rotating mixer for 15 min at room temperature in
the dark prior to measurement of fluorescence intensity. Further
lM a-TTP in SET buffer (250 mM sucrose, 100 mM KCl,
rated into dioleoyl phosphatidylcholine (DOPC), and egg yolk phos-
phatidylcholine (EYPC) large unilamellar vesicles, and with
soybean PC dispersions in acetonitrile. All preparations consisted
a
of approximately 1 mol % a-tocohexaenol per mol lipid. The oxidiz-
ing agents tested included (1) potassium superoxide, KO2, (2) iron
perchlorate with hydrogen peroxide, H2O2, (3) copper sulfate,
ascorbate and H2O2 and the radical initiators (4) AMVN, and (5)
ABAP. All oxidizing agents were prepared fresh before use as
additions of
-T6 while never exceeding 1% ethanol in the sample. Control
titrations with buffer only (no TTP) were conducted as above.
The fluorescence at 520 nm at each -T6 concentration was plotted
a-T6 continued up to a final concentration of 17 lM
a
a
approximately 1000-fold stock solutions. Oxidation of
hexaenol was examined in DOPC and egg yolk vesicles by
a
-toco-
after subtracting the fluorescence of the buffer control. The data
was fit using a one-site saturation model by non-linear regression
in Prism GraphPad 4.0. Each titration curve was performed in
duplicate in three independent experiments.
100
150
l
l
M KO2, 125 lM FeClO4/1.0 mM H2O2, 150 lM CuSO4/
M ascorbate/1.5 mM H2O2 and 1 mM H2O2 in addition to 4–
10 mM 2,20-azobis-(2-amidinopropane) dihydrochloride (ABAP)
and 0.2–4 mM 2,20-azobis-(2,4-dimethylvaleronitrile) (AMVN).
References and notes
AMVN was also used to induce oxidation of
bean PC dispersions in acetonitrile at a range of 0.2–8 mM.
a-tocohexaenol soy-
1. Traber, M. G.; Ingold, K. U.; Burton, G. W.; Kayden, H. J. Lipids 1988, 23, 791.
2. Traber, M. G.; Burton, G. W.; Ingold, K. U.; Kayden, H. J. J. Lipid Res. 1990, 31,
675.
Assays were initiated by adding an appropriate volume of oxi-
dizing agent stock solution to a quartz cuvette containing the
tocohexaenol liposomes in 10 mM phosphate 0.1 M NaCl pH 7.4
buffer or -tocohexaenol: soy PC in acetonitrile. The solution was
mixed and the fluorescence spectrum was recorded using an exci-
tation wavelength of 368 nm and the UV absorbance at 215, 234,
270, and 368 nm were measured. Subsequently, the sample was
incubated at 37 °C in the dark and measured at various intervals
over 2–5 h time spans.
a
-
3. Traber, M. G.; Rudel, L. L.; Burton, G. W.; Hughes, L.; Ingold, K. U.; Kayden, H. J. J.
Lipid Res. 1990, 31, 687.
4. Traber, M. G.; Burton, G. W.; Hughes, L.; Ingold, K. U.; Hidaka, H.; Malloy, M.;
Kane, J.; Hyams, J.; Kayden, H. J. J. Lipid Res. 1992, 33, 1171.
5. Traber, M. G.; Rader, D.; Acuff, R. V.; Brewer, H. B., Jr.; Kayden, H. J.
Atherosclerosis 1994, 108, 27.
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Clin. Nutr. 1994, 60, 397.
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J. Am. J. Clin. Nutr. 1998, 68, 847.
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Traber, M. G. Free Radical Biol. Med. 2005, 38, 857.
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Commun. 1990, 11, 99.
11. Kaempf-Rotzoll, D. E.; Traber, M. G.; Arai, H. Curr. Opin. Lipidol. 2003, 14, 249.
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13. Lodge, J. K. J. Plant Physiol. 2005, 162, 790.
a
2.3.1. Oxidation of lipid vesicles
Hundred-nanometer vesicles were prepared from chloroform
stock solutions of DOPC, EYPC, and soybean PC from Avanti Lipids
using standard methods that we have reported previously.20
a
-T6
was added from an ethanolic stock for integration into liposomal
membranes. Due to the light sensitivity of -T6, all manipulations
a
14. Nava, P.; Cecchini, M.; Chirico, S.; Gordon, H.; Morley, S.; Manor, D.; Atkinson, J.
Bioorg. Med. Chem. 2006, 14, 3721.
were performed shielding the samples from direct irradiation
using opaque glassware in dim lighting. Briefly, aliquots of the
phospholipid stocks with or without added a-T6 were evaporated
under N2 and dried for 1.5 h under vacuum. The dried films were
re-suspended in 10 mM phosphate, 0.1 M NaCl pH 7.4 and vor-
texed for full dispersion of the phospholipids into solution. Unila-
mellar vesicles were prepared from the suspension by extruding
through 100 nm polycarbonate membranes. These liposome stock
15. Morley, S.; Panagabko, C.; Shineman, D.; Mani, B.; Stocker, A.; Atkinson, J.;
Manor, D. Biochemistry 2004, 43, 4143.
16. Qian, J. H.; Morley, S.; Wilson, K.; Nava, P.; Atkinson, J.; Manor, D. J. Lipid Res.
2005, 46, 2072.
17. Morley, S.; Cross, V.; Cecchini, M.; Nava, P.; Atkinson, J.; Manor, D. Biochemistry
2006, 45, 1075.
18. Qian, J.; Atkinson, J.; Manor, D. Biochemistry 2006, 45, 8236.
19. Morley, S.; Cecchini, M.; Zhang, W.; Virgulti, A.; Noy, N.; Atkinson, J.; Manor, D.
J. Biol. Chem. 2008, 283, 17797.
20. Zhang, W. X.; Frahm, G.; Morley, S.; Manor, D.; Atkinson, J. Lipids 2009, 44, 631.
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