Interaction of ferulic acid derivatives with human erythrocytes monitored by pulse field gradient NMR diffusion and NMR relaxation studies

Article abstract of DOI:10.1016/j.chemphyslip.2004.12.005

Ferulic acid (Fer), a natural anti-oxidant and chemo-protector, is able to suppress experimental carcinogenesis in the forestomach, lungs, skin, tongue and colon. Several Fer derivatives have been suggested as promising candidates for cancer prevention, being the biological activity related also to the capacity of partitioning between aqueous and lipid phases. In the present work, pulsed field gradient (PFG) NMR diffusion measurement and NMR relaxation rates have been adopted for investigating the interaction of three Fer derivatives (Fer-C11, Fer-C12 and Fer-C13) with human erythrocytes. Binding to the erythrocyte membrane has been shown for all derivatives, which displayed a similar interaction mode such that the aromatic moiety and the terminal part of the alkyl chain were the most affected. Quantitative analysis of the diffusion coefficients was used to show that Fer-C12 and Fer-C13 display higher affinity for the cell membrane when compared with Fer-C11. These findings agree with the higher anti-oxidant activity of the two derivatives.

Full text of DOI:10.1016/j.chemphyslip.2004.12.005

Chemistry and Physics of Lipids 134 (2005) 109–117
Interaction of ferulic acid derivatives with human
erythrocytes monitored by pulse field gradient NMR
diffusion and NMR relaxation studies
Cecilia Anselmia, Francesca Bernardib,
Marisanna Centinia, Elena Gaggellib, Nicola Gaggellib,
Daniela Valensinb, Gianni Valensinb,∗
a
Department of Pharmaceutical and Technological Science, University of Siena, Via della Diana 2, 53100 Siena, Italy
b
Department of Chemistry, University of Siena, Via Aldo Moro, 53100 Siena, Italy
Received 14 June 2004; received in revised form 20 December 2004; accepted 22 December 2004
Abstract
Ferulic acid (Fer), a natural anti-oxidant and chemo-protector, is able to suppress experimental carcinogenesis in the forestom-
ach, lungs, skin, tongue and colon. Several Fer derivatives have been suggested as promising candidates for cancer prevention,
being the biological activity related also to the capacity of partitioning between aqueous and lipid phases. In the present work,
pulsed field gradient (PFG) NMR diffusion measurement and NMR relaxation rates have been adopted for investigating the inter-
action of three Fer derivatives (Fer-C11, Fer-C12 and Fer-C13) with human erythrocytes. Binding to the erythrocyte membrane
has been shown for all derivatives, which displayed a similar interaction mode such that the aromatic moiety and the terminal
part of the alkyl chain were the most affected. Quantitative analysis of the diffusion coefficients was used to show that Fer-C12
and Fer-C13 display higher affinity for the cell membrane when compared with Fer-C11. These findings agree with the higher
anti-oxidant activity of the two derivatives.
© 2005 Published by Elsevier Ireland Ltd.
Keywords: Ferulic acid derivatives; PFG NMR; Relaxation; Erythrocytes
1. Introduction
Ferulic acid (3-hydroxy-4-methoxycinnamic acid)
is a ubiquitous compound in nature, found especially
in wheat, corn, rice, tomatoes, spinach, cabbage and as-
paragus. Together with its alkyl esters it (Fig. 1) elicits
∗
Corresponding author. Tel.: +39 0577 234231;
fax: +39 0577 234233.
E-mail address: valensin@unisi.it (G. Valensin).
0009-3084/$ – see front matter © 2005 Published by Elsevier Ireland Ltd.
doi:10.1016/j.chemphyslip.2004.12.005

110
C. Anselmi et al. / Chemistry and Physics of Lipids 134 (2005) 109–117
parameters, also the alkyl chain length plays a critical
role; in fact the anti-oxidant activity decreases from
tridecyl ferulates onward.
Moreover, the evaluation of the protective effect of
ferulicacidandsomen-alkylderivativesagainsthemol-
ysis of an erythrocyte suspension induced by free rad-
ical promoter CuOOH showed that the n-dodecyl fer-
ulate is more active than the n-octyl and n-hexadecyl
compounds (Anselmi et al., 2004b).
Fig. 1.
The interaction of undecyl, dodecyl and tridecyl
ferulates (Fig. 1) with human erythrocytes are here
presented with the aim of ascertaining whether the
anti-oxidant activity correlates with the mode of such
interaction. An improvement in delineating the anti-
oxidant potential is also expected since the last is often
tested by measuring the protection offered by drugs
against hemolysis induced by hydroxyl or peroxyl rad-
icals. Among the several methods developed to inves-
tigate drug-membrane interactions, the rapid and pre-
cise evaluation of self-diffusion coefficients, allowed
by pulse field gradient (PFG) NMR (Canet, 1997), was
believedtoprovidequitesuitableapproach. Infact, dur-
ing the so-called diffusion delay time (vide infra), the
drug rapidly exchanges between the membrane bound
state(s) and the bulk solution such that the measured
diffusion coefficient maintains memory of both envi-
ronments in a weighted average way. As a matter of
fact, the PFG NMR technique has successfully been
applied to drug-macromolecule associations (Canet,
1997; Atkinson et al., 2003; Lin et al., 1997; Fielding,
2000; Waldeck et al., 1997; Price et al., 2002; Bleicher
et al., 1998) and to molecular aggregation in solution
(Mansfield et al., 1998, 1999; Viel et al., 2002; Yao et
al., 2000; Price et al., 1999). A further advantage arises
from measuring NMR relaxation rates in the same sam-
ple, thus yielding independent and complementary in-
formation on the system under study.
interesting anti-oxidant properties (Graf, 1992; Rice-
Evans et al., 1996; Niki, 1997; Kikuzaki et al., 2002;
Trombino et al., 2004), that are potentially useful not
only to prevent lipid oxidation in food but, also, to
prevent free-radical-induced diseases such as cancer
and atherosclerosis or aging caused by oxidative tis-
sue degeneration (Niki, 1997; Kikuzaki et al., 2002;
Anselmi et al., 2001; Balasubashini et al., 2004). The
anti-oxidant properties rely on the radical scavenging
activity (Niki, 1997) and, as a consequence, ferulic acid
is by far the most effective among hydroxycinnamic
acids, due to the resonance stabilization of the phe-
noxy radical by an extended side-chain coniugation.
Different group at the end of the carbon side chain are
also able to affect anti-oxidant behavior (Nenadis et
al., 2003). In particular esterification of ferulic acids
with medium chain alcohols (Niki, 1997; Kikuzaki et
al., 2002) determines a further increase in activity and
suggests that also preferential localization and relative
partitioning ability between aqueous and lipophilic in-
terfaces may be related to the anti-oxidant potential.
The anti-oxidant properties of a series of n-alkyl fer-
ulates of natural source or originating from chemical
modification of naturally occurring compounds were
recently investigated (Anselmi et al., 2004a) in order to
define (i) the optimum chain length of the molecule for
anchoring to the membrane binding sites; (ii) the orien-
tation of the phenoxy group with respect to the mem-
brane bilayer; and (iii) the effect of side-chain folding
upon the orientation or positioning at the membrane.
It came out that the dodecyl and the tridecyl ferulates
were the most active among the even and odd mem-
bers derivatives, respectively. It was, in fact, stressed
that the three dimensional arrangement relative to the
membrane bilayer plays a key role in dictating the anti-
oxidant ability of n-alkyl ferulates and furnishes a rea-
sonable explanation of the mechanism of action. More-
over it was found out that, besides the conformational
2. Materials and methods
The ferulic derivatives were synthesized according
to standard procedure by reaction of ferulic acid (of
natural origin) (Taniguchi et al., 1999) with the cor-
responding alkanol in the presence of an acid catalyst
(Taniguchi et al., 1998). In a three-necked flask, 0.1 mol
of ferulic acid, and 0.1 mol of alkanol were dissolved in
200 ml of toluene and 1.0 g of p-toluensulphonic acid

C. Anselmi et al. / Chemistry and Physics of Lipids 134 (2005) 109–117
111
was added. The reaction mixture was stirred at 80 ◦C
for 16 h.
gitudinal storage periods (Wu et al., 1995; Gibbs and
Johnson, 1991; Dingley et al., 1995; Johnson, 1999).
The gradient strength was incremented (with an ini-
tial value of 0.86 G cm−1 and a step size of 2.65 G cm−1
for 2 ms), while the separations of the field gradients
and the total echo time were kept constant. A series
of 16 spectra, with a number of scans ranging from
16 to 32, was recorded in 2D mode for each measure-
ment, with a recycle time of 10 s between scans. The
diffusion values were calculated by regression anal-
ysis of the signal decay leading to errors not larger
than 2–5%. The strength of the B0 field gradient was
calibrated by measuring the self-diffusion coefficient
of the residual HDO signal in a 100% D2O sample
at 298 K (Callaghan et al., 1983; Price, 1998). The
measurement of the DMSO diffusion coefficient was
used to determine the hydrodynamic radius of DMSO
at 0.148 nm using a viscosity for DMSO at 298 K of
2.00 × 10−3 kg m−1 s−1. A diffusion experiment was
performed on a sample of TMS in DMSO at 298 K in
order to evaluate its hydrodynamic radius r by the ratio
(Longsworth, 1960):
After cooling, the mixture was washed with a satu-
rated aqueous solution of sodium hydrogen carbonate.
The organic layer was dried over anhydrous sodium
sulfate. The solvent was fully evaporated off from the
organic layer under reduced pressure. The crude prod-
ucts were purified on silica gel column and identified
by TLC, UV and NMR analysis.
FerulicacidwassuppliedbyTsunoRiceFineChem-
icals (Wakayama, Japan); toluene (analytical grade),
p-toluensulfonic acid, sodium hydrogen carbonate, an-
hydrous sodium sulfate, 1-undecanol, 1-dodecanol and
1-tridecanol were purchased from Aldrich (Milan,
Italy).
Human erythrocytes of group A were collected from
healthy volunteers, suspended in anti-coagulant solu-
tion (heparin and EDTA), and centrifuged at 1000 × g
for 5 min. The cells were washed five times with
Tris–HCl buffer (172 mM, pH 7.6) and centrifuged at
1600 × g for 10 min. The buffy coat of white cells was
removed carefully after each wash. The erythrocytes
were resuspended at 10% hematocrit in 10 mM phos-
phate buffered saline (PBS, pH 7.4 containing 137 mM
NaCl and 2.7 mM KCl) and stored at 4 ◦C for 3 days
maximum.
rDMSODDMSO
rTMS
=
= 0.150 nm
(1)
DTMS
Such value was then used to measure the viscos-
ity of the solution containing the ferulic derivatives by
measuring the diffusion of TMS (vide infra).
1H NMR spectra were obtained at 14.1 T with a
Bruker Avance 600 Spectrometer operating at con-
trolled temperature ( 0.2 K) and using a 5 mm triple
resonance probe with gradients along x, y, z direc-
tions. Chemical shifts were referenced to external
tetramethylsilane (TMS). COSY, TOCSY, NOESY and
ROESY spectra were obtained by using standard pulse
sequences. TOCSY experiments were acquired with
a total spin-locking time of 75 ms using a MLEV-17
mixing sequence. NOESY and ROESY spectra were
acquired with a mixing time ranging between 100 and
350 ms. Spin lattice relaxation rates were measured
with inversion recovery pulse sequences. The same se-
quencewasalsousedtomeasurethesingle-selectivere-
laxation rates by means of suitably shaped ␲-pulses in-
stead of the usual non-selective ␲-pulse. All rates were
calculated by regression analysis of the initial recovery
curves of longitudinal magnetization components lead-
ing to errors not larger than 3%. The diffusion coef-
ficients were measured at 298 K by a PFG longitudinal
eddy-current delay (LED), pulse sequence with bipolar
gradients incorporating spoil gradients during both lon-
3. Results and discussion
With the exception of a different intensity of the
merged methylene resonances, the NMR spectra of the
three derivatives were identical (Table 1). Also NOESY
and ROESY spectra (not shown) were identical, show-
ing only trivial dipolar connectivities consistent with
the absence of stabilized conformations.
The extremely low water solubility of ferulic acid
esters led to the choice of DMSO as a solvent in view
of an experimental protocol requiring addition of red
blood cells to the NMR sample. It is, in fact, known
that DMSO is used for cell cryopreservation (Rowley,
1992). The integrity of cells was anyway continuously
checked by microscopy inspection (data not shown).
Several NMR methods have been suggested for in-
vestigating ligands interacting at macromolecular sites.
All methods rely on changes induced by slowing down

112
C. Anselmi et al. / Chemistry and Physics of Lipids 134 (2005) 109–117
Table 1
1H NMR chemical shifts (600 MHz) (δ, ppm), diffusion coefficients (D × 1010, m2 s−1) for the three ferulic acid esters 21 mM free and in
presence of erythrocytes 10% in volume, in DMSO-d6 at T = 298 K
δ (ppm)
D (m2 s−1
Free
)
1a
1b
1c
+RBCs
1a
1a
1b
1c
1b
1c
H1
H2
OH
H5
OMe
H7
H8
H9
H10
H11
(CH2)n
CH3
7.10
6.78
9.62
7.31
3.81
6.46
7.52
4.11
1.62
1.34
1.24
0.85
7.10
6.78
9.62
7.31
3.81
6.46
7.52
4.11
1.62
1.34
1.24
0.85
7.10
6.78
9.62
7.31
3.81
6.46
7.52
4.11
1.62
1.34
1.24
0.85
2.73
2.79
nd
2.72
2.72
nd
2.78
2.77
nd
1.88
1.86
nd
1.86
nd
1.86
1.88
nd
1.82
1.85
nd
1.73
1.73
nd
1.67
nd
1.71
1.69
nd
1.70
1.70
nd
1.67
1.69
nd
1.66
nd
1.65
1.65
nd
1.67
1.70
nd
2.72
2.75
2.79
2.77
2.77
2.66
2.62
nd
2.74
2.74
2.71
2.72
2.74
2.67
2.69
nd
2.79
2.79
2.76
2.77
2.79
2.77
2.79
nd
2.76
2.73
2.81
1.78
1.68
1.64
nd: not determined.
of the reorientational dynamics of the ligand when go-
ing from the free to the bound environment. From this
point of view, the PFG NMR method of measuring
changes in the diffusion rates of the ligand has been
shown to provide a very good piece of information on
small molecules interacting with biological or model
membranes (Waldeck et al., 1997, 1995; Price et al.,
2002; Guy et al., 1986; Price and Kuchel, 1990; Lennon
et al., 1994).
As shown in Table 1, the diffusion coefficients mea-
sured by PFG NMR on different resonances of all the
ferulic acid esters 21 mM in DMSO-d6 are, within the
experimental error limit, consistent with a unique dif-
fusion behavior of the three molecules. As also shown
in Table 1, the addition of aliquots of the erythro-
cytes aqueous suspension yields slower diffusion coef-
ficients. Such change is directly related to the volume
of added RBC and reaches a ca. 40% decrease upon ad-
dition of 60 ␮l (Table 1). Quite interestingly the three
derivatives are diversely influenced by the presence of
erythrocytes, being 1b and 1c much more affected than
1a.
The relatively slow diffusion features of the free fer-
ulic compounds are apparently due to the viscosity of
the solvent (η = 2.00 × 10−3 kg m−1 s−1 as compared
to η = 8.94 × 10−4 kg m−1 s−1 of water at the same
temperature) rather than to aggregation. In fact, dilut-
ing the sample (Table 2) was not accompanied by faster
diffusion coefficients or changes in chemical shifts.
As already stated, the effect of the viscosity change
due to water was independently checked by measuring
the diffusion coefficient of the 1b derivative (averaged
over all protons), DMSO, H2O and TMS upon addi-
tion of 60 ␮l of water to three DMSO solutions having
different amounts of dissolved 1b (Table 2).
The following experimental protocol was therefore
used:
a. The diffusion coefficients of the ferulic acid deriva-
tives were measured in DMSO solution at different
concentrationinordertoascertaineventualaggrega-
tion phenomena and provide the basis of subsequent
comparison.
b. The same diffusion coefficients were then measured
upontheadditionofvariablevolumesoftheaqueous
erythrocyte suspension to the NMR samples.
c. Since addition of water to DMSO yields unpre-
dictable effects on the solution viscosity, that, in
turn, strongly affects the NMR features, the diffu-
sion coefficients of the free ligand had to be some-
how normalized before attempting any comparison
with the effects induced by the presence of cells.
As a consequence, diffusion coefficients were also
measured after addition of variable volumes of wa-
ter alone to the three derivatives NMR samples.
It is apparent that addition of water leads to substan-
tial slowing of diffusion features of all molecules in so-
lution consistently with a sizeable increase in solvent

C. Anselmi et al. / Chemistry and Physics of Lipids 134 (2005) 109–117
113
Table 2
Diffusion coefficients (D × 1010, m2 s−1) and the η × D product for the Fer-C12 (1b) derivative at different concentrations in DMSO-d6 at
T = 298 K
D (m2 s−1) × 1010
η × D (kg s−2) × 1012
21 mM
9 mM
4.5 mM
21 mM
9 mM
4.5 mM
No water
1b
DMSO
H2O
2.72
7.30
9.70
7.20
2.88
7.54
9.79
7.42
2.86
7.54
9.92
7.42
0.55
1.47
1.96
1.45
0.56
1.48
1.92
1.45
0.56
1.47
1.94
1.45
TMS
+60 ␮l H2O
1b
DMSO
H2O
2.13
5.58
7.38
5.51
2.17
5.66
7.40
5.55
2.05
5.51
7.34
5.44
0.56
1.47
1.95
1.45
0.57
1.48
1.94
1.45
0.55
1.48
1.97
1.46
TMS
The viscosity η (kg m−1 s−1) was calculated in absence of water: 2.02 × 10−3 (21 mM), 1.96 × 10−3 (9 mM), 1.96 × 10−3 (4.5 mM), and in
presence of 60 ␮l of water: 2.64 × 10−3 (21 mM), 2.62 × 10−3 (9 mM), 2.68 × 10−3 (4.5 mM).
viscosity. Such change is well documented in the liter-
ature, where it is reported that the presence of H2O as a
cosolvent yields a further increase in the viscosity that
reachesamaximumatχDMSO ca. 0.36asaconsequence
of well-defined intermolecular interactions (Mizuno et
hydrodynamic radius of the molecule, here assumed of
spherical shape (Stilbs and Lindman, 1981).
The use of TMS as internal diffusion reference al-
lows to take into account the variation in solvent prop-
erties while comparing solutions differing for solute
concentration or solvent composition, making it possi-
ble to correct the measured diffusion values for any
change in the viscosity (Cabrita and Berger, 2001).
The analysis of the η × D product (Table 2) is an easy
way to understand if a change in diffusion values is
due to viscosity or not: if that product, measured in
different conditions, has the same value, then the de-
crease of diffusion coefficient depends only on viscos-
ity.
´
al., 2000; Kirchner and Reiher, 2002; Catalan et al.,
2001). Since H-bonding of 1b with one or the other
solvent may contribute to the viscosity, while TMS is
not expected to be involved in H-bonding, the diffusion
coefficient of TMS was used to calculate the medium
viscosity, as reported in the caption of Table 2, by using
the equation (Rice-Evans et al., 1996):
kBT
D =
(2)
6πηrH
It follows that the effects of adding water to DMSO
solutions of the three ferulic acid derivatives are com-
pletely accounted for by the change in viscosity and
wherekB = 1.380658 × 10−23 J K−1 istheBoltzmann’s
constant, η is the viscosity of the medium and rH is the
Table 3
Diffusion coefficients (D × 1010, m2 s−1) and the η × D product for the three derivatives (1a, 1b, 1c) 21 mM in DMSO-d6 at T = 298 K
D (m2 s−1) × 1010
η × D (kg m s−2) × 1012
1a
1b
1c
1a
1b
1c
Free
TMS
Fer
7.24
2.74
7.20
2.72
7.46
2.78
1.45
0.55
1.45
0.56
1.45
0.54
+60 ␮l RBCs
TMS
Fer
5.11
1.85
5.11
1.70
5.00
1.67
1.46
0.53
1.46
0.48
1.45
0.49
Fer: ferulic derivatives. The viscosity η (kg m−1 s−1) of the solution was calculated in the free samples: 2.01 × 10−3 (1a), 2.02 × 10−3 (1b),
1.95 × 10−3 (1c), and in presence of 60 ␮l of RBCs: 2.85 × 10−3 (1a), 2.85 × 10−3 (1b), 2.91 × 10−3 (1c).

114
C. Anselmi et al. / Chemistry and Physics of Lipids 134 (2005) 109–117
Table 4
preferential solvation can be ruled out, at least at the
measured solvent composition.
Molar fraction of the three ferulic derivatives
pf
pb
If, with this in mind, we reconsider the effect of ery-
throcytes upon the observed diffusion coefficients of
the three ferulic acid derivatives it is now evident that,
while the η × D values of TMS are not changed, those
of 1a, 1b, and 1c (again averaged over all proton reso-
nances) are somehow and differentially slowed down,
being, again, 1b and 1c more affected than 1a (Table 3).
When a molecule self-aggregates or binds a macro-
molecule site, and binding involves rapid exchange,−o1n
1a
1b
1c
0.96
0.88
0.90
0.04
0.12
0.10
Errors in calculated fractions are in the range 4–10%.
As already stated, we considered the product η × D
instead of the diffusion value in order to take the vis-
cosity changes into account. Equation (Trombino et al.,
2004) has been therefore considered:
thetimescaleofNMRdiffusionexperiment(∆ ꢀ koff
,
where ∆ is the separation between the leading edges
of the gradient pulses and koff is the kinetic constant
for the dissociation process), then the estimated dif-
fusion coefficient is a weighted average of diffusion
coefficients of free and bound states, being the weight-
ing factors the corresponding occupational probabili-
ties (Stilbs and Lindman, 1981, 1982; Stokkeland and
Stilbs, 1985).
(η × D)obs = pf(η × D)f
(5)
From such equation we could calculate the values
of pf and pb of all three derivatives (Table 4), thus esti-
mating the strength of the interaction. It is now evident
that 1b and 1c have almost the same affinity for the
erythrocyte membrane, whereas the association of 1a
is weaker by a factor of ca. 3.
Such trend correlates with the different anti-oxidant
activity of the three ferulates being the 1b and 1c com-
pounds more active than 1a: the concentration able to
inhibit the lipid peroxidation process in rat liver micro-
somal membranes (IC50 (␮M)) was for undecyl deriva-
tives two or three times larger than that for the other
two compounds (Anselmi et al., 2004a).
Ligand binding studies are commonly analyzed us-
ing the simplest two-site model in which the ligand can
bind to any of the n identical independent binding sites
on a macromolecule (Luo et al., 1999). In this case
the observed ligand diffusion coefficient, Dobs, is the
population-weighted average of the free, Df, and the
bound, Db, values (Price et al., 2002):
The independent measurements of spin-lattice re-
laxation rates provided further support to the observed
binding interaction and also allowed some inferences
ontheinteractionmode. Suchratesweremeasuredafter
selectiveexcitationofprotonresonances, sinceitiswell
ascertained that the Risel parameter is a very sensitive
tool for detecting binding of relatively small ligands
to macromolecular binding sites (Fielding, 2000; Luo
et al., 1999; Valensin et al., 1982; Delfini et al., 2000;
Rossi et al., 1999; Berti et al., 2000). The methoxy
group could be of primary importance to map the in-
teraction between the cells and ferulic acid derivatives
but unfortunately this signal is almost masked by the
broad water resonance in the presence of erythrocytes.
Nevertheless significant information comes from the
analysisofrelaxationeffectsoftheotherresonancesbe-
longing to the aromatic ring. When plotting the normal-
ized proton relaxation enhancement ꢀRsiel/Risfel, where
Dobs = pfDf + pbDb
(3)
Such approach suitably probes relatively weak in-
teraction only; the exchange rate between the two en-
vironments must, in fact, be fast enough to provide
significant modulation. Eq. (3) can be exploited by
considering that (i) Df is likely much larger than Db
and (ii) Db can be approximated by the diffusion co-
efficient of the macromolecule which is not likely to
be affected by ligand binding (Fielding, 2000). In the
case of erythrocytes, the Db value is expectedly very
small (the lateral diffusion is very slow with a limit
value of 10−12 m2 s−1) (Eisinger and Scarlata, 1987;
Kuba et al., 2002); in addition, also pb is small, being
the ligand concentration exceedingly larger than the
likely concentration of cellular binding sites. In such
conditions the term pbDb can be neglected and Eq. (3)
becomes:
ꢀRsiel is the measured change in selective relaxation
sel
− Rsifel) and Rsifel is the corresponding rate
Dobs = pfDf
(4)
rate (= R
iobs

C. Anselmi et al. / Chemistry and Physics of Lipids 134 (2005) 109–117
115
Fig. 2. Normalized selective relaxation enhancements of protons of the three ferulic acid derivatives 21 mM in DMSO at T = 298 K in the
presence of 60 ␮l of RBCs.
in the free state, the diverse affinity of the three deriva-
tives can be immediately viewed (Fig. 2). Moreover
some inferences on the binding mode can be laid based
on the following. An increase in Rsiel may arise from
either a slowing down of molecular motions or an in-
crease in the number of 1H–1H dipole–dipole interac-
tions contributing to relaxation or both. From this point
of view, the data in Fig. 2 indicates that:
The observed findings suggest that PFG NMR dif-
fusion measurements provide a suitable tool for delin-
eating the interaction between whole cellular double
bi-layer and such that (i) some quantitative aspects of
binding can be inferred; (ii) structure-activity corre-
lations can be attempted; and (iii) inferences on the
interaction modes can be made.
Acknowledgements
(1) 1a, 1b and 1c bind in the same way, though with
different affinities;
The financial supports by COFIN 2001 is gratefully
acknowledged. We also thank Dr. Antonio Vivi (NMR
Center, Siena) for his skilful technical assistance in
NMR diffusion spectroscopy and for helpful discus-
sion.
(2) the strongest effect experienced by aromatic or
olefinic protons suggests either location close to a
relatively more extended proton-rich environment
or a more pronounced slowing down of internal
motions. By the same way, the alkyl side chain,
which most likely is embedded in the membrane
double layer (thus determining the relative bind-
ing affinity), can be hypothesized to retain higher
degrees of internal flexibility.
Appendix A. Supplementary data
Supplementary data associated with this ar-
ticle can be found, in the online version, at
doi:10.1016/j.chemphyslip.2004.12.005.
TheobservedeffectsontheethylenicprotonsH7, H8
and on aromatic proton H1 and H2 support the previous
evidence that the most effective anti-oxidants are char-
acterized by main mean folded conformations, where
the n-alkyl chain bends towards the ethylenic protons
which are in turn coplanar to the aromatic moieties
(Anselmi et al., 2004a).
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