Active oxygen chemistry within the liposomal bilayer: Part III: Locating Vitamin E, ubiquinol and ubiquinone and their derivatives in the lipid bilayer

Article abstract of DOI:10.1016/j.chemphyslip.2004.04.007

We have previously shown that the location and orientation of compounds intercalated within the lipid bilayer can be qualitatively determined using an NMR chemical shift-polarity correlation. We describe herein the results of our application of this method to analogs of Vitamin E, ubiquinol and ubiquinone. The results indicate that tocopherol - and presumably the corresponding tocopheroxyl radical - reside adjacent to the interface, and can, therefore, abstract a hydrogen atom from ascorbic acid. On the other hand, the decaprenyl substituted ubiquinol and ubiquinone lie substantially deeper within the lipid membrane. Yet, contrary to the prevailing literature, their location is far from being the same. Ubiquinone-10 is situated above the long-chain fatty acid slab . Ubiquinol-10 dwells well within the lipid slab, presumably out of striking range of Vitamin C. Nevertheless, ubiquinol can act as an antioxidant by reducing C- or O-centered lipid radicals or by recycling the lipid-resident tocopheroxyl radical.

Full text of DOI:10.1016/j.chemphyslip.2004.04.007

Chemistry and Physics of Lipids 131 (2004) 107–121
Active oxygen chemistry within the liposomal bilayer
Part III: Locating Vitamin E, ubiquinol and ubiquinone
and their derivatives in the lipid bilayer
Michal Afria, Benjamin Ehrenbergb, Yeshayahu Talmond, Judith Schmidtd,
Yael Cohena, Aryeh A. Frimera,c,∗
a
Department of Chemistry, Bar-Ilan University, Ramat Gan 52900, Israel
Department of Physics, Bar-Ilan University, Ramat Gan 52900, Israel
b
c
The Ethel and David Resnick Chair in Active Oxygen Chemistry, Ramat Gan 52900, Israel
d
Department of Chemical Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel
Received 6 January 2004; received in revised form 2 April 2004; accepted 13 April 2004
Available online 5 June 2004
Abstract
We have previously shown that the location and orientation of compounds intercalated within the lipid bilayer can be quali-
tatively determined using an NMR chemical shift-polarity correlation. We describe herein the results of our application of this
method to analogs of Vitamin E, ubiquinol and ubiquinone. The results indicate that tocopherol—and presumably the corre-
sponding tocopheroxyl radical—reside adjacent to the interface, and can, therefore, abstract a hydrogen atom from ascorbic acid.
On the other hand, the decaprenyl substituted ubiquinol and ubiquinone lie substantially deeper within the lipid membrane. Yet,
contrary to the prevailing literature, their location is far from being the same. Ubiquinone-10 is situated above the long-chain
fatty acid “slab”. Ubiquinol-10 dwells well within the lipid slab, presumably out of “striking range” of Vitamin C. Nevertheless,
ubiquinol can act as an antioxidant by reducing C- or O-centered lipid radicals or by recycling the lipid-resident tocopheroxyl
radical.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Liposomes; NMR; ␣-Tocopherol; Tocopheroxyl radical; Ubiquinol; Ubiquinone; Vitamin C; Lipid bilayer; Membranes
1. Introduction
relatively small molecule natural antioxidants, which
protect membrane lipids from oxidative damage. The
tocopherol molecule consists of two functional do-
mains: a C16 hydrocarbon chain—which is responsi-
ble for the lipophilicity of the molecule and its proper
location within the membrane bilayer (Sebrel and
Harris, 1972; Horwitt, 1993; Sharma and Buettner,
1993; Veris, 1994); and a chromanol head group—
which is responsible for the antioxidant activity. In
Vitamin E (tocopherol, TocH, 1a; see Scheme 1),
ubiquinol-10 (reduced coenzyme Q10, CoQH2-10,
2a) and Vitamin C (ascorbic acid) are examples of
∗
Corresponding author. Tel.: +972-3-5318610;
fax: +972-3-5351250.
E-mail address: frimea@mail.biu.ac.il (A.A. Frimer).
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2004.04.007

108
M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 107–121
confirm the high efficiency of Vitamin E as an antiox-
OR2
4
Me
5
4
idant. This is consistent with the proposed recycling
of the resulting tocopheroxyl radical (Toc•) back to
TocH, either by ascorbic acid and/or ubiquinol (Beyer,
1994; Packer et al., 1979; Noguchi and Niki, 1998;
Bisby and Parker, 1995; Podmore et al., 1998). The
mechanistic details of such cycling are clouded by the
fact that the relative locations of the lipid-active an-
tioxidants (TocH and ubiquinol) with respect to the
interface are still unknown. In particular, how does
the water-residing ascorbic acid undergo interaction
with the highly lipophilic TocH and/or ubiquinol? Var-
ious studies have attempted to resolve this issue and
a variety of models have been suggested. Following
Fukuzawa et al. (1993), Fig. 1 shows three basic mod-
els which have been proposed.
MeO
MeO
Me
R1
R1O
Me
3
5
4a
8a
6
7
3
Me
R2
6
2
2
O
1
8
OR2
Me
Vitamin E derivatives
1a: R1 = H, R2 = C16H33
1b: R1 = Ac, R2 = C16H33
1c: R1 = H, R2 = CH3
1d: R1 = Ac, R2 = CH3
Ubiquinol derivatives
2a: R1 = Decaprenyl, R2 = H
2b: R1 = H, R2 = H
2c: R1 = Decaprenyl, R2 = Ac
2d: R1 = H, R2 = Ac
1e: R1 = PhCO, R2 = CH3
O
OH
MeO
MeO
Me
R
3
2
5
6
O
O
OH
4
1
HO
OH
(1) According to the first proposal (Fig. 1A), toco-
pherol behaves much like a kite. The TocH tail
is well anchored within the bilayer; nevertheless,
the chromanol moiety floats upwards near the
water–lipid interface placing the phenolic oxy-
gen in easy access of Vitamin C (Niki, 1989;
Srivastava et al., 1983; Perly et al., 1985; Ekiel
et al., 1988). The localization of the tocopherol
radical near the interface has also been discussed
(Buettner, 1993).
O
Vitamin C
Ubiquinone derivatives
3a: R = Decaprenyl
3b: R=H
b
h
j
d
c
f
d
a
g
CH2
Decaprenyl =
i
e
e
8
g'
c'
i'
Scheme 1. Natural antioxidant molecules and derivatives.
(2) The second model (Fig. 1B) situates the chro-
manol OH-group somewhat deeper within the
membrane, ca. 10 Å from the interface. Here it
interacts with the carbonyl group of an acyl es-
ter of the phospholipid (Fragata and Bellemare,
1980; Villalain et al., 1986; Gómez-Fernández
et al., 1989). Nevertheless, vertical oscillations
within the membrane allow the tocopheroxyl rad-
ical (Toc•) to be within “striking distance” of the
cycling ascorbic acid.
(3) The last school (Fig. 1C) argues that the chro-
manol is located deep in the hydrophobic region
(fatty acid chain “slab”) of the membrane bilayer
(Kagan and Quinn, 1988; Bisby and Ahmed,
1989; Fukuzawa et al., 1992; Fukuzawa et al.,
1997; Urano et al., 1993). The mechanism of
recycling remains to be resolved.
the latter capacity, the chromanol moiety functions
as a chain-breaking antioxidant terminating radical
chains by donating a hydrogen atom (Buettner, 1993).
Ubiquinol-10 (CoQH2-10), too, is comprised of a
polar (hydroquinone) head group and a long tail of ten
isoprenyl units (dubbed the decaprenyl chain). This
hydroquinone functions much the same way as a radi-
cal chain-breaking antioxidant (Poderoso et al., 1999;
Constantinnescu et al., 1994) either by reducing C-
or O-centered lipid radicals or by reducing the toco-
pheroxyl radical (Toc•) back to tocopherol (TocH).
Ubiquinol-10 undergoes very facile oxidation, and it
is its more stable quinone precursor, ubiquinone-10
(coenzyme Q10, CoQ-10, 3a), which is readily avail-
able in biological systems. Indeed, there are even some
biological studies which have used the commercially
available ubiquinone as a stand in for ubiquinol, seem-
ingly unaware that it is only the latter that is the true
antioxidant.
The discussion in the literature regarding the loca-
tion of ubiquinone and ubiquinol within the cell mem-
brane treats these very different compounds as one and
the same. As shown in Fig. 2 (using ubiquinone-10
The standard one-electron reduction potentials for
ascobate, tocopherol and ubiquinol (Buettner, 1993)

M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 107–121
109
(A)
O
(B)
(C)
O
OH
H
OH
OH
H
OH
H
OH
OH
O
O
O
O
O
O
O
N
O
O
N
O
O
H
O
H
O
H
O
H
O
O
O
O
O
O
O
N
O
O
O
P
P
P
CH CH2
CH CH2
CH CH2
CH2
CH2
CH2
O
O
O
O
O
O
O
O
O
H
O
O
O
H
O
O
O
O
Fig. 1. Schematic models of the location of Vitamin E within the lipid bilayer.
as the intercalant), here, too, there are three basic
positions. (1) The first (Fig. 2A) places the quinone
and hydroquinone rings near the water–lipid interface
(Samori et al., 1992; Lenaz, 1988). (2) The second
school (Fig. 2B) argues that, to the contrary, the rings
are located in the hydrophobic slab area of the mem-
brane (Metz et al., 1995; Alonso et al., 1981; Kingsley
and Feigenson, 1981; Aranda and Gomez-Fernandez,
1985; Aranda et al., 1986; Ondarroa and Quinn, 1986).
(3) Finally, it has been suggested that the quinone and
hydroquinone rings are actually sandwiched between
the layers of the phospholipid bilayer (i.e., at the end of
the fatty acid chains) (Fig. 2C), perhaps even as head
to head aggregates (Fig. 2D) (Katsikas and Quinn,
1981; Gomez-Fernández et al., 1999). According to
this model, the head group oscillates between the two
bilayer surfaces, thus remaining at all times within a
hydrophobic environment (Fato et al., 1986).
et al., 2002), we have shown that it is possible to
qualitatively determine the location and orientation
of compounds intercalated within the lipid bilayer
by using an NMR chemical shift-polarity correla-
tion. Specifically, a generally good correlation exists
between the 13C NMR chemical shifts (δ) of the var-
ious carbons of a given compound and the polarity
of the solvent in which the spectrum is measured
(Maciel and Ruben, 1963; Ueji and Makamaura, 1976;
Menger et al., 1978, 1988; Menger, 1979; Janzen
et al., 1989). The solvent polarity can be evaluated by
Reichardt’s ET(30) parameter (Reichardt, 1990, 1994)
or other polarity parameters reported in the literature.
The solvents examined generally ranged in polarity
from benzene [ET(30) = 34.5 kcal/mol] to methanol
[ET(30) = 55.5 kcal/mol]. Substrates containing a
polar function with an electron-deficient carbon (e.g.,
conjugated carbonyl systems) are preferred for this
technique since the increase in the NMR chemi-
cal shift (ꢀδ) with solvent polarity is particularly
dramatic.
In the present paper, we will try to clear up some
of the above confusion. In previous work (Strul et al.,
1994; Frimer et al., 1996; Weitman et al., 2001; Afri

110
M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 107–121
Fig. 2. Schematic models of the location of ubiquinone within the lipid bilayer.
Once a correlation graph for the various carbon
static fields from the time-averaged structure, as well
as from water penetration and perhaps partial exit of
the surfactants. Given this caveat, we nevertheless
believe that this 13C NMR chemical-shift/polarity cor-
relation technique is a useful tool for approximating
the location of substrates within lipid bilayers.
nuclei in a substrate is prepared, the substrate can be
intercalated within the liposomal phospholipid bilayer
and its 13C chemical shift data measured. Using the
correlation graph, this liposomal 13C chemical shift
data can be correlated with a corresponding polarity
value (ET). However, a gradient of solvent polarity
is expected within the liposomal bilayer, increasing
as one goes from deep within the lipid bilayer (ap-
proaching the polarity of hexane with an ET(30) of
30.9 kcal/mol) out towards the aqueous phase (ap-
proaching the polarity of pure water with an ET(30)
of 63.1 kcal/mol). Thus, the polarity data obtained via
the correlation graph gives us a qualitative measure of
the distance of the various carbons within a substrate
from the interface. This will then allow us to correlate
a substrate’s average location/orientation within the
bilayer with its reactivity.
We should also note that the aforementioned 13C
NMR techniques enable us to determine the polarity
of the micro-environment of the substrate carbons and
thereby qualitatively determine the substrates distance
from the interface. It is this which allows us to cor-
relate a substrate’s location/orientation within the bi-
layer with its reactivity. There is, however, no clear
linear correlation between the polarity felt by a sub-
strate and its quantitative distance from the interface
in Angstroms. We have begun to synthesize a series of
“chemical rulers” that will allow us to determine just
this relationship at different depths within the phos-
pholipid bilayer. This work, however, is beyond the
scope of this paper. Our results and inferences must,
perforce, remain qualitative and not quantitative.
We describe herein the results of our application
of this NMR method to determine the qualitative
We use the word “average” advisedly. The timescale
of molecular motion is faster than that of the NMR
experiment; hence, the values we obtain by the above
method are actually average locations. The NMR
chemical shift data will reflect polarity due to electro-

M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 107–121
111
location and orientation of five analogs of Vitamin
E (1a–1e), four of ubiquinol (2a–2d), and two of
ubiquinone (3a–3b). The results indicate that to-
copherol lies adjacent to the interface (in-between
the suggestions shown in Fig. 1A and B), and can
therefore abstract a hydrogen from ascorbic acid.
Ubiquinol-10 and its quinone analog lie well within
the lipid bilayer substantially deeper than tocopherol
and presumably out of “striking range” of Vitamin
C. Nevertheless, ubiquinol can act as an antioxidant
by reducing C- or O-centered lipid radicals or by
recycling the tocopheroxyl radical.
transmission electron microscopy) was performed on
tocopherol-intercalated liposomal solutions, which
underwent ultra-fast vitrification with liquid ethane at
its freezing point (Nilsson et al., 2000). The samples
were then studied in a Philips CM120 transmission
electron microscope at approximately −175 ◦C, in an
Oxford Instruments CT3500 cryo-specimen holder.
Images were digitally recorded by a Gatan MultiScan
791 CCD camera.
Acetyl chloride, acetyl-1-13C chloride, acetic-1-13C
anhydride, benzoyl chloride, pyridine, n-hexane,
sodium dithionite (sodium hydrosulfite) and the
deuterated solvents were obtained from Aldrich,
Milwaukee, WI. Dimyristoylphosphatidylcholine
(DMPC), 2,2,5,7,8-pentamethyl-6-chromanol, (+)-␣-
tocopherol (Vitamin E), (+)-␣-tocopherol acetate
(Vitamin E acetate), coenzyme Q10 and 2,3-dime-
thoxy-5-methyl-1,4-benzoquinone were purchased
from Sigma, St. Louis, MO. Acetic anhydride was
obtained from Bio Lab, Jerusalem, Israel. KH2PO4
and KOH were used in the preparation of a 0.1 M
phosphate buffer solution (pH 7.8 and containing
10−4 M EDTA); the latter was utilized, in turn, to
prepare all aqueous solutions, unless otherwise spec-
ified. The general procedure for the preparation of
DMPC liposomal suspensions for NMR studies, as
previously described (Afri et al., 2002), was utilized,
with the following modifications. For the Vitamin E
derivatives 1a–1e, the substrate concentration was
0.025 M and the substrate to lipid molar ratio was
1:5; the NMR analysis was carried out at 31 ◦C. For
the ubiquinols 2c–2d and ubiquinones 3a–3b, the
substrate concentration was 0.050 M and the substrate
to lipid ratio was 1:5; the NMR analysis was carried
out at 45 ◦C (Weitman et al., 2001). To verify that
the substrates did not undergo autoxidation during
the course of lengthy NMR measurements, selected
samples were sealed under argon and the NMR ex-
periment repeated. No changes were observed.
2. Materials and methods
2.1. General
EI and CI Mass spectra were run on a GC/MS
Finnigan-4021 (San Jose, CA, USA). High resolution
mass spectra were run on a VG-Fison AutoSpecE high
resolution spectrometer (Wythenshawe, Manchester,
UK). Analytical thin layer chromatography (TLC)
was performed using Merck silica gel microcards
(Darmstadt, Germany). Preparative chromatography
was performed using Merck silica gel F254 cards.
The NMR spectra were recorded on a Bruker DPX
300 or Bruker DMX 600 Fourier transform spectrom-
eter (Bruker, Rheinstetten, Germany). For 1D NMR
spectra we used a QNP probe. All 2D experiments
(COSY, HMQC, HMBC and NOESY) were run us-
ing the programs from the Bruker software library.
NMR spectra were generally taken at 25 1 ◦C. The
dimyristoylphosphatidylcholine (DMPC) vesicle so-
lutions, however, were run at 45 ◦C, above the phase
transition temperature (TC) of DMPC (Zachariasse
et al., 1981). Below this temperature the mobility of
the intercalated molecules is low and the resulting
NMR absorptions are very broad. Raising the tem-
perature sharpens the peaks but does not seem to
affect the chemical shifts. The NMR spectra were
generally recorded while locked on the deuterium
signals of the respective solvent. The chemical shifts
were measured relative to internal tetramethylsilane
(TMS), except in the case of the aqueous vesicle
solutions in which we calibrated the spectrum ac-
cording to the trimethylammonium peak at 54.6 ppm.
Cryo-TEM (Talmon, 1999) (cryogenic temperature
2.2. Substrates—preparation and spectral data
A rather complete spectral analysis of chromanols
1a–1e appears in the literature (Baker and Myers,
1991; Witkowski et al., 2000). Ubiquinones 3a–3b
(Vanliemt et al., 1994) are also known. Regarding
ubiquinol 2d, while the compound is known (Ohkawa
and Nagai, 1997), some of the spectral data are lack-

112
M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 107–121
ing and, hence, the complete data are given below. The
numbering of the carbons in the spectral data below is
as shown in Scheme 1. To aid the reader in comparing
hydroquinones 2 with quinones 3, we have kept the
numbering of the corresponding carbons in these two
compounds the same (see Scheme 1 and Table 3).
(CDCl3) δ 6.48 (1H, s, H6), 3.91 (3H, s, OMe on C3),
3.88 (3H, s, OMe on C2), 2.17 (3H, s, Me on C5);
13C NMR (CDCl3) δ 141.55 (C1), 140.38 (C4), 139.04
(C3), 137.17 (C2), 119.36 (C5), 111.30 (C6), 60.82 and
60.71 (OMe), 15.32 (Me); MS (EI) 184 (M+, 62%),
169 (M+-CH3, 52%).
2.2.1. 2,2,5,7,8-Pentamethylchroman-6-yl acetate
(1d)
2.2.5. 1,4-Diacetoxy-2,3-dimethoxy-5-methyl-6-
decaprenylbenzene (ubiquinol-10 diacetate, 2c)
Diacetoxyubiquinol 2c was synthesized, according
to the procedure previously described for the prepa-
ration of 2d (Obol’nikova et al., 1976), by refluxing
ubiquinol-10 with acetic anhydride for 2 h under N2.
The desired diacetate was precipitated from acetic
anhydride at 0 ◦C MP 44 ◦C. An analog enriched
with 13C at the ester carbonyls was prepared using
acetic-1-13C anhydride, and diluted with three parts of
the 12C compound. NOESY was particularly helpful
This acetoxy derivative of chromanol 1c was pre-
pared with acetyl chloride following the general
procedure for the esterification of cromanols as pre-
viously described (Wheeler, 1963). An analog en-
riched with 13C at the ester carbonyl was prepared
using acetyl-1-13C chloride, and diluted with the 12C
compound in a 1:1 ratio. MS (CI, 70 eV) m/z 263
(MH+, 100%), 221 (MH+-Ac, 39%); HRMS calcd.
(C16H22O3, M+) 262.1569, obsd. 262.1562.
1
in confirming the NMR peak assignments. H NMR
(CDCl3) δ 5.12 (8H, m, Hf), 5.08 (1H, m, Hh), 4.98
2.2.2. 2,2,5,7,8-Pentamethylchroman-6-yl benzoate
(1e)
ꢀ
ꢀ
(1H, t, J = 6 Hz, Hb), 3.83 (6H, s, H3 and H2 ), 3.20
This benzoyloxy derivative of chromanol 1c was
prepared with benzoyl chloride following the general
procedure for the esterification of cromanols as pre-
viously described (Wheeler, 1963). MP 104 ◦C; MS
(CI, 70 eV) m/z 324 (M+, 100%), 219 (M+-C6H5CO,
59%); HRMS calcd. (C21H24O3, M+) 324.1725, obsd.
324.1709.
ꢀꢀ
ꢀꢀ
ꢀ
(2H, d, J = 6 Hz, Ha), 2.34 (3H, s, H4 or H1 ), 2.31
ꢀꢀ
ꢀꢀ
(3H, s, H1 or H4 ), 2.06 (21H, m, He and H5 ), 1.98
ꢀ
(18H, m, Hd), 1.73 (3H, s, Hc ), 1.68 (6H, s, Hj and
Hi ), 1.60 (24H, m, Hg ); 13C NMR (CDCl3) δ 168.92
ꢀ
ꢀ
ꢀ
ꢀ
and 168.78 (C1 and C4 ), 143.38 and 143.20 (C3 and
C2), 140.71 (C4), 140.43 (C1), 135.75 (Cc), 134.83
(Cg), 131.13 (Ci), 128.31 (C6), 124.35 (C5), 124.21
ꢀ
ꢀ
(Cf), 123.91 (Ch), 121.18 (Cb), 60.58 (C2 and C3 ),
2.2.3. Ubiquinol-10 (2a)
39.68–39.54 (Cd), 26.54 (Ce), 26.19 (Ca), 25.63 (Cj),
20.40 (C4 or C1 ), 20.36 (C1 or C4 ), 16.17 (Cc ),
15.95 (Cg ), 12.07 (C5 ); MS (FAB), 947.1 (M–H,
0.6%), 865.8 (M–C6H10, 1.33%), 239.3 (100%);
HRMS (DCI) calcd. (C63H97O6, MH+) 949.7285,
obsd. 949.7258.
The reduction of ubiquinone 3a was carried out fol-
lowing the literature procedure (Mukai et al., 1990).
1H NMR (CDCl3) δ 5.31 (2H, broad-s, OH), 5.12 (8H,
m, Hf), 5.10 (1H, t, J = 6.5 Hz, Hb), 3.88 (6H, s,
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
H3 and H2 ), 3.33 (2H, d, J = 6.5 Hz, Ha), 2.14 (3H,
ꢀ
s, H5 ), 2.06 (18H, t, J = 8 Hz, He), 1.98 (18H, t,
ꢀ
J = 8 Hz, Hd), 1.77 (3H, s, Hc ), 1.68 (3H, s, Hj),
1.59 (27H, m, Hi and Hg ); 13C NMR (CDCl3) δ
140.11 (C4), 139.86 (C1), 136.90 and 136.81 (C2 and
C3), 135.30 (Cc), 135.27–134.90 (Cg), 131.26 (Ci),
124.41–123.85 (Cf), 122.29 (Cb), 122.02 (C6), 117.98
2.2.6. 1,4-Diacetoxy-2,3-dimethoxy-5-methylbenzene
(ubiquinol-0 diacetate, 2d)
ꢀ
ꢀ
Diacetoxyubiquinol 2d was obtained as previously
described (Obol’nikova et al., 1976) by refluxing
ubiquinol with acetic anhydride for two hours under
N2. Distillation of the excess anhydride yielded the
desired diacetate. An analog enriched with 13C at the
ester carbonyls was prepared using acetic-1-13C an-
hydride, and diluted with three parts of the 12C com-
ꢀ
ꢀ
(C5), 60.80 and 60.74 (C2 and C3 ), 39.75–39.734
(Cd), 26.92 (Cj), 26.77–26.53 (Ce), 25.28 (Ca), 16.25
ꢀ
ꢀ
ꢀ
ꢀ
(Cc ), 16.04 (Ci ), 16.03 (Cg ), 11.95 (C5 ); MS (FAB),
865.4 (MH+, 69%).
1
2.2.4. Ubiquinol-0 (2b)
The reduction of ubiquinone 3b was carried out as
previously described (Mukai et al., 1990). H NMR
pound. H NMR (CDCl3) δ 6.67 (1H, d, J = 0.6 Hz,
ꢀ ꢀ
H6), 3.85 (3H, s, H3 ), 3.83 (3H, s, H2 ), 2.34 (3H,
1
ꢀꢀ
ꢀꢀ
s, H4 ), 2.31 (3H, s, H1 ), 2.11 (3H, d, J = 0.6 Hz,

M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 107–121
113
13
ꢀ
ꢀ
ꢀ
H5 ); C NMR (CDCl3) δ 169.12 (C1 ), 168.71 (C4 ),
145.72 (C3), 143.48 (C2), 141.48 (C4), 140.74 (C1),
ꢀ
ꢀ
126.11 (C5), 118.44 (C6), 60.76 (C2 and C3 ), 20.62
ꢀꢀ
ꢀꢀ
ꢀ
(C1 ), 20.38 (C4 ), 15.73 (C5 ).
3. Results and discussion
3.1. Preliminary comments regarding the substrate
to lipid ratio
One crucial point needs to be elucidated be-
fore we discuss the data. The common lore in the
field of liposome research is that the amount of
“intercalant”—substrate incorporated within liposo-
mal bilayers—should not rise above 5 mol% lest its
high concentration affect the integrity of the physical
properties of the liposome itself. In the NMR exper-
iments described below, however, spectral detection
is nigh impossible if the intercalant concentration is
much below 17 mol%. It was, therefore, imperative
to confirm the formation of liposomes under those
conditions, and to examine the shape and size of the
liposomes.
Fig. 3. Cryo-TEM image of “empty” DMPC liposomes (the black
areas are part of the support film).
3.2. Location of Vitamin E analogs within liposomal
bilayer
As we pointed out in our introductory comments,
there is substantial confusion in the literature as to the
exact location of Vitamin E and its analogs within the
The method of choice for studying our systems
was cryogenic temperature transmission electron mi-
croscopy (Cryo-TEM) (Talmon, 1999; Nilsson et al.,
2000). The main advantage of this technique is that
the ultra-fast vitrification with liquid ethane retains
the internal structure of the liposomal bilayer. We pre-
pared both “empty” dimyristoylphosphatidylcholine
(DMPC) liposomes (with lipid concentration of
0.125 M), and those containing Vitamin E acetate (1b)
as our model intercalant in a concentration of 0.025 M
(the same conditions used in the NMR technique). In
the case of the former, when we dispersed the lipid
by sonication, we observed the formation of a variety
of liposomes, both multilamellar and unilamellar (see
Fig. 3). The average width of the bilayers was found
to be 45 Å and their diameter ranged from 1300 to
2000 Å. Inclusion of 1b within the liposomal bilayer
(see Fig. 4) led to the formation of smaller liposomes
with an average diameter of 200–500 Å. These results
are preliminary, and the exact influence of the inter-
calant concentration is presently under investigation.
There remains a second issue, namely the biological
relevance of our results, which we will deal with in
Section 3.4.
Fig. 4. Cryo-TEM image of DMPC liposomes containing 17 mol%
Vitamin E acetate (1b) (the black area is part of the support film).

114
M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 107–121
lipid bilayer. We hoped that, by applying the aforemen-
tioned chemical shift-polarity correlation NMR tech-
nique to chromanols 1a–1e, we would be able to shed
light on this problem. While tocopherol (1a), toco-
pherol acetate (1b), and chromanol (1c) are commer-
cially available (Sigma), acetoxychromanol (1d) and
its benzoyloxy analog 1e were readily prepared by re-
acting chromanol 1c with acetyl or benzoyl chloride,
respectively.
there is a large ꢀδ (2.1–4.5 ppm units) at ring carbons
C-5, C-7, C-8 and in chromanol 1c, C-4a, as well. On
the other hand, for the corresponding ester derivatives,
1b, 1d and 1e, the ꢀδ are significant (1.8–4.5 ppm
units) at carbons C-2, C-4a and C-5, carbonyl C-6a,
and—in the benzoate derivate 1e—at the carbon para
to the ester carbonyl. The discrepancies between these
systems somewhat complicate their comparison and
analysis.
In order to prepare the necessary correlation graph,
we measured the 13C NMR of compounds 1a–1e in
four different deuterated solvents of varying polar-
ities: carbon tetrachloride, chloroform, ethanol and
methanol. As expected, as one proceeds from the least
polar (CCl4) to the most polar solvent (CD3OD), the
difference in chemical shifts (ꢀδ) increase with in-
creasing solvent polarity; nevertheless, theses changes
are not significant (i.e., ꢀδ is not >1.8) for all carbons.
This result is well precedented and has much to do
with how solvent polarity affects the electronic distri-
bution on the various carbons. However, for reasons
not as yet clear to us, this ꢀδ is significant for differing
carbons in the various analogs. For chromanols with
To implement the NMR technique, we prepared
graphs of 13C NMR chemical shift vs. polarity of
the solvent (using Reichardt’s ET(30) parameter
(Reichardt, 1990, 1994). The correlation coefficient
(r2) for the hydroxy analogs (0.83–0.99) was a bit
better than that of the ester derivatives (0.75–0.87).
In the next stage, we incorporated the various sub-
strates within the liposomal phospholipid bilayer and
measured the 13C NMR chemical shifts of the key
carbons listed above. We note that the peaks of the
liposome do not overlap those of interest in the in-
tercalated substrates. From the correlation graphs,
we calculated the ET(30) of the various carbons. To
exemplify this process, we show the detailed results
for Vitamin E acetate 1b, which appear in Table 1
=
a free 6-hydroxy group (1, R H), namely 1a and 1c,
Table 1
13C NMR shifts (ppm) for Vitamin E acetate (1b) in pure solvents and intercalated within DMPC liposomes
Solvent
ET(30)a
C-2
C-4a
C-5
C-6a
CCl4
CDCl3
EtOD
MeOD
DMPC liposomesb
(ET(30) calc.)c
32.5
39.1
51.9
55.5
74.33
75.06
75.50
76.11
116.31
117.35
117.96
118.71
124.33
124.88
125.54
126.14
166.97
169.69
170.43
171.47
74.96 (40.4)
117.49 (43.7)
125.84 (53.4)
170.27 (48.5)
Correlation
0.93
0.93
0.97
0.86
coefficient (r2)
Line equation
δ = 0.067 × ET
+ 72.25
δ = 0.091 × ET
+ 113.51
δ = 0.072 × ET
+ 122.01
δ = 0.166 × ET
+ 162.23
a
kcal/mol at 25 ◦C.
b
Spectra were normally measured at 25 1 ◦C. However, because the phase transition temperature of DMPC is ca. 22 ◦C and in order
to improve the mobility of the long-chained analogs, the liposomal spectra were measured at 45 ◦C.
c
From Plate 1. Error in the calculated ET is 1 kcal/mol.

M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 107–121
115
180
160
140
C-6a (r2=0.86)
MeOD
Liposome
EtOD
CDCl3
CCl4
C-5 (r2=0.97)
C-4a (r2=0.93)
120
80
C-2 (r2=0.93)
30
35
40
45
50
55
60
ET(30) (kcal/mol)
Plate 1. Correlation graph for Vitamin E acetate (1b).
and Plate 1. Table 2 summarizes the important data
of the calculated ET(30) for the key carbons of the
liposome-intercalated analogs 1a–1e.
is a saturated lipid and, hence, no lipid autoxidation
should be observed. To verify that the substrates did
not undergo autoxidation under these conditions, se-
lected samples were sealed under argon and the NMR
experiment repeated. No changes were observed.
We note that some of the NMR measurements were,
at times, quite lengthy—up to 48 h at 45 ◦C. DMPC
Table 2
Calculated ET(30) for Vitamin E analogs 1a–1e
C-pa
ꢀET (30)adjb(adjacent carbons)
=
Compound
C-6a (C O)
C-5
C-7
C-8
C-4a
C-2
Vitamin E (1a)
Acetate 1b
Chromanol 1c
Acetate 1d
48.0
53.4
49.5
50.3
44.9
48.1
43.8
–
–
–
–
4.3 (7–8)
9.7 (5–4a)
7.5 (5–4a)
5.7 (5–4a)
3.5 (5–4a)
48.5
43.7
42.0
44.6
41.4
40.4
48.6
43.2
46.9
41.1
42.7
42.6
Benzoate 1e
46.4
a
The para carbon on the benzoate ester.
The difference in ET(30) value of adjacent carbons.
b

116
M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 107–121
Regarding the calculated ET(30) data for Vitamin
E analogs 1a–1e (Table 2), we need to examine two
parameters.
deshielding of carbons C-5 and C-7 (for rea-
sons we do not yet understand, the ester car-
bonyl carbons C-6a in these acetates lie in a less
polar environment than the corresponding C-5
carbons).
(1) The relative ET(30) value of the various carbons,
which gives us a qualitative measure of the relative
depth of the molecules within the liposome.
(4) As noted above, the C-5 and C-7 carbons in Vita-
min E (1a) are essentially parallel and experience
the same polarity of ET(30) = 48. We have no
evidence whether or not there is a linear corre-
lation between depth and ET, though we have
been attempting to develop “chemical rulers” to
elucidate this question. Nevertheless, given that
ꢀET(30)adj is 4.3 and assuming that this quantity
remains essentially unchanged as we rise to the
interface, we should now be able to approximate
the location of the free C-6 hydroxyl group hy-
drogen. There are three bond lengths from C-5
to the C-6 hydroxyl group hydrogen: one vertical
(4.3 units) and two at an angle of 30◦ (2 sin 30◦
× 4.3 = 4.3 units). This places the C-6 hydroxyl
hydrogen (or the corresponding oxy-radical or-
bital) at an ET(30) value of ca. 56.6 [ET(30)
of pure water is 63.1] clearly in striking range
of the interface (as pictorially represented in
Scheme 2). This is consistent with a positioning
of Vitamin E below model A of Fig. 1—perhaps
approaching model B, but permitting facile recy-
cling of tocopheroxyl radical by water-resident
Vitamin C.
(2) The difference in ET(30) value of adjacent car-
bons [ꢀET(30)adj] in the various molecules. This
quantity supplies information as to the orientation
of the molecule within the liposomal bilayer. If the
molecule lies horizontally, parallel to the interface,
we would expect the ꢀET(30)adj for the various
adjacent carbons of the molecule to approach zero.
As the molecule becomes more vertical (along the
C-6/C-8a axis) lying perpendicular to the inter-
face, the ꢀET(30) is expected to become maximal.
From the numbers in Table 2, we get the following
picture.
(1) The data indicates that with respect to relative
depths within the liposome, the five analogs di-
vide themselves into three groups. Preliminary
results (Afri and Frimer, unpublished results) indi-
cate that the ester carbonyls of the DMPC, which
comprises the liposomes and which presumably
lie just below the hydrophilic phosphatidylcholine
head groups (Abrams and London, 1993), are
located at an ET(30) value of ca. 51–52. Thus,
C-5 of Vitamin E acetate (1b), with a calculated
ET(30) of 53, is not far from the interface. Vitamin
E (1a), chromanyl 1c and its corresponding ac-
etate 1d lie somewhat deeper; the carbons C-5 and
C-7 experience essentially the same polarity with
an ET(30) of 48-50 (vide infra). The chromanyl
benzoate 1e lies deepest with an ET(30) value
of 45.
OH
OH
O
O
O
O
Water Phase
Lipid Phase
H
H
ET = 63
ET = 53
O
(2) Based on the values for ꢀET(30)adj, its clear
that Vitamin E acetate (1b) with a value of 9.7
lies vertically within the liposome. Chromanol
1c with a ꢀET(30)adj of 7.5 is close to verti-
cal, while the remaining analogs lie at various
angles.
ET = 50
ET = 44
O
ET = 34
(3) The C-5 carbons in acetate analogs 1b and 1d are
more polar than the free hydroxyl analogs 1a and
1c. This is consistent with the suggestion that the
ester carbonyl enhances the electron-withdrawing
induction of the adjacent oxygen group and the
Scheme 2. Pictorial representation of the location and orientation of
the tocopheroxyl radical within the liposomal bilayer with respect
to Vitamin C.

M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 107–121
117
3.3. Location of ubiquinol (2) and ubiquinone (3)
derivatives within the liposomal bilayer
(3b). The highly lipophilic character of the former lim-
its it solubility to acetone, chloroform and tert-butanol.
Interestingly, the chemical shifts of the carbonyls of
CoQ-10 are relatively insensitive to changes in sol-
vents polarity (ꢀδ = 0.82–0.85 ppm); hence, only car-
bons C-2, C-3, C-5 and C-6 (with ꢀδ > 1.2 ppm) can
be used to determine the molecules location. A dif-
ferent story was observed in the case of CoQ-0. The
latter dissolves well even in pure water, but here, only
carbons C-1, C-4 and C-5 had large ꢀδ values. In both
systems, r2 = 0.80–0.97.
As mentioned in the Introduction, ubiquinol (2) is
an important natural antioxidant, whose activity in-
volves its oxidation through the semiquinone to the
corresponding ubiquinone (3). We could not imple-
ment our NMR technique directly on ubiquinols 2a
and 2b, because the chemical shifts (ꢀδ) of the var-
ious carbons in these diol derivatives are essentially
insensitive to solvent polarity. Because of our expe-
rience in locating the esters of Vitamin E within the
liposome, we decided to also synthesize and examine
the ubiquinol esters.
The next step was to intercalate compounds 2c, 2d.
3a and 3b within the liposomal bilayer and measure
their 13C NMR spectra. Table 3 summarizes the cor-
responding calculated ET(30) for the various carbons
of these four molecules.
Unlike the 6-protio derivative diacetate ubiquinol-0
(2d), ubiquinol-10 diacetate (2c) proved quite insolu-
ble in highly polar protic solvents such as ethanol or
methanol. As a result, in the latter case we were limited
to the deuterated aprotic solvents carbon tetrachloride,
benzene, chloroform and acetone. In both system, the
chemical shifts of the two carbonyls C-1ꢀ and C-4ꢀ
are reliably (r2 = 0.84–0.87) sensitive to solvent po-
larity; carbon C-6, on the other hand, is only mildly
sensitive to polarity and the correlation coefficient is
marginal (r2 = 0.60) in the case of 2c and unreliable
(r2 = 0.36) in the case of 2d.
The results indicate that each of the four molecules
lies essentially parallel to the interface but in vari-
ous polarity regions. As expected, the long-chained
decaprenyl substituted analogs ubiquinol-10 diacetate
(2c) and CoQ-10 (3a) are pulled substantially deeper
into the bilayer (ET(30) ≈ 34.5 and 39.0, respectively)
than the corresponding short derivatives, ubiquinol-0
diacetate (2d), and CoQ-0 (3a), which lie in a shal-
lower region of the bilayer (ET(30) ≈ 49.5 and 51.0,
respectively).
We turn now to two commercially available mem-
bers of the ubiquinone family, CoQ-10 (3a) and CoQ-0
Several observations need to be made at this junc-
ture. Firstly, since ubiquinone-10 (3a) lies at an
Table 3
Calculated ET(30) for ubiquinol-10 diacetate (2c), ubiquinol-0 diacetate (2d), ubiquinone-10 (CoQ-10, 3a) and ubiquinone-0 (CoQ-0, 3b)
Compound
C-1ꢀ
C-4ꢀ
C-1
C-2
C-3
C-4
C-5
C-6
Ubiquinol-10 diacetate (2c)
Ubiquinol-0 diacetate (2d)
Ubiquinone-10 (CoQ-10, 3a)
Ubiquinone-0 (CoQ-0, 3b)
34.3
49.5
34.5
49.3
34.9
40.1
40.4
38.0
52.0
38.5
51.9
50.3

118
M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 107–121
ET of ca. 39, the tail is presumably embedded in
the slab with the head-group just above, but distant
from the lipid–water interface. Thus in the case of
ubiquinone-10, it is likely that this compound lies in
between the models represented in Fig. 2A and B.
Comparing now ubiquinol diesters 2c and 2d with
the corresponding ubiquinones 3a and b, we see that
the esters lie 1.5–4.5 ET units deeper into the bilayer.
Thus, ubiquinol-10 diacetate (2c) lies at an ET of ca.
34.5 (the same ET as non-polar benzene); hence, it is
presumably situated within the fatty acid slab.
The question now becomes the relative position of
ubiquinol-10 (2a). Previous research has suggested
that the latter lies above the corresponding quinone
(3a) (Kingsley and Feigenson, 1981; Salgado et al.,
1993; Aranda et al., 1986; Gomez-Fernández et al.,
1999). However, as observed in the case of the Vi-
tamin E derivatives (Table 2: 1a versus 1b, and 1c
versus 1d), monoacetylation of the chromanols causes
the resulting esters to be less lipophilic by several ET
units. Without clear-cut evidence, similar extrapola-
tion from the di-esters 2c and 2d to the di-ols 2a and
2b is, however, highly questionable. We can only def-
initely conclude, therefore, that ubiquinol-10 2a lies
deep within the lipid bilayer. We can make no defi-
nite statement, though, as to whether it is situated near
ubiquinol-10 diacetate (2c) within the fatty acid slab
at an ET of ca. 34.5, or higher up near ubiquinone-10
(3a). We can, nevertheless, rule out the suggestion that
the head-group of ubiquinol 2a lies near the interface
(analogous to model A of Fig. 2).
Based on the above data, the relative location and
orientation of tocopherol (1a), ubiquinol-diacetate (2c)
and ubiquinone (3a) within the liposomal bilayer can
pictorially represented in Scheme 3.
3.4. Biological relevance of data
As noted above (Section 3.1), in order to facili-
tate NMR measurements it was necessary to use an
overall substrate concentration of 0.025–0.050 M and
a substrate to lipid ratio of 1:5 (17 mol%). While
we demonstrated that liposomes are indeed formed
under these conditions, the question remains as to
the biological relevance of the data obtained for such
liposomes—in which the substrate concentration is
much above 5 mol%. After all, such a high ratio might
affect the physical properties of the liposome and,
hence, the location and orientation of the intercalated
substrates within the lipid bilayer.
To this end, we synthesized cromanol acetate (1d),
ubiquinone-0 diacetate (2d) and ubiquinone-10 di-
acetate (2c), which were enriched with 13C in the
ester carbonyl. These were then incorporated within
DMPC liposomes in a substrate to lipid ratio of 1:20
(4.75 mol%) and an overall substrate concentration
of 0.01 M. In each case, the calculated ET(30) val-
ues obtained for the labeled carbons were exactly
Water Phase
ET = 63
H
Lipid Phase
O
ET = 53
ET = 50
ET = 44
O
MeO
O
OMe
ET = 40
O
Me
MeO
OMe
ET = 34
Ac
Ac
O
O
Me
3a
1a
2c
Scheme 3. Pictorial representation of the relative location and orientation of tocopherol (1a), ubiquinol-diacetate (2c) and ubiquinone (3a)
within the liposomal bilayer.

M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 107–121
119
the same as those previously determined for the un-
labeled analogs. These experiments clearly indicate
that high substrate to lipid ratio of 1:5 has little ef-
fect on the location of intercalants within the lipid
bilayer. Our results are then indeed biologically
relevant.
water–lipid interface, but it cannot distinguish, how-
ever, between models B, C or D.
Acknowledgements
We should note, however, that even at a concentra-
tion of 4.75 mol% phase separation might take place
with domains of heterogeneous lipid/tocopherol com-
position resulting (Villalain et al., 1986; Micol et al.,
1990). Under these conditions, 13C NMR may reflect
average signals from ␣-tocopherol molecules located
at different positions in the membrane. Resolution of
this possibility is beyond the scope of this paper and
is presently under investigation.
We would like to acknowledge the kind and gener-
ous support of The Israel Science Foundation (Grants
Nos. 588/98 and 327/02) and The Ethel and David
Resnick Chair in Active Oxygen Chemistry. This pa-
per is based in part on the Ph.D. Thesis of Michal Afri
at Bar-Ilan University (Afri, 2001).
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