Synthesis and physico-chemical characterization of bolaamphiphiles derived from alkenyl d-xylosides

Article abstract of DOI:10.1039/c1nj20158a

The two step synthesis of a new series of bolaamphiphiles derived from alkenyl d-xylosides is described. Yields between 57 and 90% have been reached. Aggregation behavior and membrane interaction properties of a particular sugar-bolaform composed of two xylose heads connected by an ether link to a hydrophobic chain comprising 18 carbon atoms and an insaturation were studied. Its CAC value (～0.05 mM) is low compared to those of conventional surfactants. Thermodynamic analysis has revealed that the aggregation phenomenon is mainly driven by an entropy process. The compound is able to interact with DPPC monolayers, in particular if their surface pressure is ≥15 mN m ^-1. Our results highlight the potential of this bolaamphiphile as surfactant and membrane interacting agent.

Full text of DOI:10.1039/c1nj20158a

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PAPER
Synthesis and physico-chemical characterization of bolaamphiphiles
derived from alkenyl D-xylosideswz
a
b
b
a
´
Magali Deleu,y Celine Damez,y Sylvain Gatard, Katherine Nott,
Michel Paquota and Sandrine Bouquillon*b
Received (in Montpellier, France) 24th February 2011, Accepted 7th June 2011
DOI: 10.1039/c1nj20158a
The two step synthesis of a new series of bolaamphiphiles derived from alkenyl D-xylosides is
described. Yields between 57 and 90% have been reached. Aggregation behavior and membrane
interaction properties of a particular sugar-bolaform composed of two xylose heads connected by
an ether link to a hydrophobic chain comprising 18 carbon atoms and an insaturation were
studied. Its CAC value (B0.05 mM) is low compared to those of conventional surfactants.
Thermodynamic analysis has revealed that the aggregation phenomenon is mainly driven by an
entropy process. The compound is able to interact with DPPC monolayers, in particular if their
surface pressure is Z 15 mN mꢀ1. Our results highlight the potential of this bolaamphiphile as
surfactant and membrane interacting agent.
To our knowledge, the synthesis of sugar-based bolaforms
Introduction
mainly involves hexoses or disaccharides.7–15 Pentose-based
bolaforms have received less attention. Different symmetric
sugar-based bolaamphiphiles have been synthesized. Their
monosaccharidic glycosylated heads have a linear form7 or a
pyranose/furanose shape4d,8–10 and are connected by ether
bonds,11 amide bonds7,8 or acetalic functions9e to a hydro-
phobic spacer having a variable length. This lipophilic spacer
can also be a simple or a double hydrocarbon chain.4d,10,12
Few dissymmetric bolaforms containing a sugar based head
Sugar-based surfactants are compounds of interest for food,
cosmetic and pharmaceutical applications because of their natural
origin, their high biodegradability and their biocompatibility.
Our previous studies have shown the possibility of
synthesizing pentosides having an octadienyl chain, with a
double bond at the terminal position, from the Pd-catalyzed
telomerisation of butadiene with pentoses.11-O-Alkenoyl-D-
xylose and alkenyl D-xylosides were shown to exhibit interesting
surface active properties.2
have been described in the literature. Goue
´
th et al.9b as well as
For many years, bolaform-type surfactants have received
increasing attention because of their original properties. Their
general structure consists of two polar heads connected by a
hydrophobic spacer. In an aqueous environment, they have
been reported to organize into aggregates of various morpho-
logies depending on the nature of the polar head, and the
length and flexibility of the spacer.3 Potential applications of
these compounds as encapsulation or vectorization agents
have been reported.4 Among bolaamphiphiles, sugar-based
ones offer the advantages of providing the required hydro-
solubility to the aggregates and reducing cytotoxicity.5
Prata et al.13 have prepared dissymmetric bolaamphiphilic
compounds comprising two distinct saccharidic moieties.
Other studies have described the synthesis of bolaforms with
a sugar head and acidic,14 or ammonium15 hydrophilic heads.
Other sugar-based bolaforms with two chains have also been
prepared.3b,c,4,6
Sugar bolaamphiphiles have been reported to form ultrathin
monolayer membranes and to be able to insert into lipid
membranes.12 Mixture of some bolaforms with conventional
lipids leads to membranes with a higher stability against
chemical and thermal degradation.16a In contrast, some mono-
meric and polymeric bolaamphiphiles can disrupt membranes.16b
In this paper, we first describe the synthesis of xylose-based
bolaform surfactants. The novel symmetric bolaforms com-
prise two xylose polar heads connected by an ether or an ester
link to a hydrocarbon segment with an insaturation and
increasing chain lengths. In the second part, the aggregation
behavior and the membrane interaction properties of a sugar-
bolaform (20aa) composed by two xylose heads connected by
an ether link to a hydrophobic chain comprising 18 carbon
atoms and an insaturation are investigated.
a
´
Unite de Chimie biologie industrielle, Gembloux Agro-Bio Tech,
Universite de Liege, 2, Passage des Deportes, B-5030 Gembloux,
´
`
´
´
Belgium
´
b
´
´
Unite Mixte de Recherche ‘‘Reactions Selectives et Applications’’,
´
CNRS - Universite de Reims Champagne-Ardenne, Boıte n1 44,
B.P. 1039, F-51687 Reims, France.
E-mail: sandrine.bouquillon@univ-reims.fr;
ˆ
Fax: +33 (0) 3-26-91-31-66; Tel: +33 (0) 3-26-91-89-73
w Dedicated to Prof. Didier Astruc on the occasion of his 65th birthday.
z Electronic supplementary information (ESI) available. See DOI:
10.1039/c1nj20158a
y These authors have equally contributed to this work.
c
2258 New J. Chem., 2011, 35, 2258–2266
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Table 1 Metathesis reactions on xylosides: Z and Z/E isomers ratio
depending on the catalyst addition conditions
Results and discussion
Synthesis
Entry
Catalyst addition conditions
Xyloside
Z (%)
Z/Ea
The metathesis of acetylated xylosides having an octadienyl
chain1 was performed in the presence of Grubbs I catalyst to
obtain six precursors of anomeric pure bolaform surfactants,
1aa, 2aa, 3aa and 1bb, 2bb, 3bb with a spacer comprising,
respectively, 10, 18 and 20 carbon atoms (Scheme 1). The
synthesis isolated yields were comprised between 57 and 90%.
Metathesis reactions were undertaken under classical
conditions in dichloromethane at 40 1C for 24 h. The
precursor of bolaform 1bb resulting from the metathesis of
hex-50-enyl(2,3,4-tri-O-acetyl)-b-D-xylopyranoside was isolated
with a yield of 74% with a single addition of the catalyst.
Optimization of the metathesis reaction was carried out on the
1aa compound by varying the mode of addition of the catalyst
and its concentration. The compound 1aa resulting from the
metathesis of hex-50-enyl(2,3,4-tri-O-acetyl)-a-D-xylopyranoside
was obtained with a yield of 90% and of 87% by adding the
catalyst in one shot or by two successive additions of catalyst
respectively (Table 1, entries 3 and 2). These conditions were
adopted for the synthesis of 2bb, 3bb and 3aa and the yields
obtained vary from 69% to 80%. For the preparation of
compound 2aa only, the catalyst was added 3 times with a
metathesis yield reaching 69%. The aa and bb derivatives
react in a relatively similar way. However the metathesis is not
stereoselective for the configuration of the double bond
because both Z and E isomers are obtained and are not
separable by chromatography on silica gel. NMR analysis
showed that isomer E was by far the most abundant in all
instances and was used to determine the ratio Z/E.
1
2
3
4
5
6
7
Fastb
Fastc
Slowd
Slowd
Faste
Slowf
Slowd
1bb
1aa
74
87
90
69
69
76
80
22/78
26/74
25/75
19/81
15/85
18/82
16/84
2bb
2aa
3bb
3aa
a
b
Determined by 1H NMR. Addition by means of a transfer cannula.
c
Addition by means of a syringe taddition = 2 ꢁ 40 s (t = 0 and t = 12 h).
d
Addition in one shot by means of a syringe taddition = 4 h (Flow (Q) =
e
0.7 mL hꢀ1). Addition by means of a syringe taddition = 3 ꢁ 40 s
f
(every 2 h). Addition by means of a syringe taddition = 2 h (Q =
0.7 mL hꢀ1).
undec-100-enoyl(2,3,4-tri-O-acetyl)-b-D-xylopyranoside and undec-
100-enoyl(2,3,4-tri-O-benzyl)-D-xylopyranoside was also studied.
The metathesis of undec-100-enoyl(2,3,4-tri-O-acetyl)-b-D-
xylopyranoside was performed in the presence of Grubbs I
catalyst in dichloromethane (Scheme 4). The 10,200-bis-eicos-
100-enedioyl(2,3,4-tri-O-acetyl)-b-D-xylopyranoside 4bb was
isolated with 67% yield.
For the deprotection, a mixture of triethylamine/methanol/
water (1: 8 : 1) known to selectively deprotect the acetylated
ester derivatives of hexoses17 was applied to compound 4bb
(Scheme 5). In these conditions a mixture of free D-xylose and
eicos-10-enedioate was obtained but not the expected bolaform.
Protection of the xylose hydroxyl groups by benzylic groups
was then considered. Since no optimal conditions have
been found to separate the a and b anomers of undec-100-
enoyl(2,3,4-tri-O-benzyl)-D-xylopyranoside by chromato-
graphy on silica gel, the metathesis was carried out on the
mixture of anomers (Scheme 6, Table 2).
The acetylated precursors of the ether bolaforms were then
deprotected in an almost quantitative way in the presence of
sodium methanolate to give the desired bolaforms (Scheme 2).
All compounds were obtained with a NMR purity higher than
95% after extraction.
A single addition of the catalyst results in a yield not
exceeding 46% (Table 2, entries 1 and 2). On the other hand,
multiple additions of the catalyst yield 81% of 10,200-bis-eicos-
100-enedioyl(2,3,4-tri-O-benzyl)-D-xylopyranoside 5 (Table 2,
entries 3 and 4).
The metathesis was also carried out with an unprotected
xyloside, the hex-50-enyl-a-D-xylopyranoside, to check the
possibility of directly obtaining the bolaform (Scheme 3). This
allowed the isolation of 10,100-bis-dec-50-enyl-a-D-xylopyranoside
10aa with a yield of 75%.
To obtain the corresponding ester bolaform, the benzylic
groups were hydrogenated. The different conditions employed
are described in Scheme 7 and in Table 3.
As 1-O-alkenoyl-D-xylose has been shown to have very
interesting surface active properties,2 the metathesis of
Scheme 1 Homodimerization reactions of xylosides having an octadienyl chain.
c
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Scheme 2 A. Deprotection step of acetylated precursors of the bolaforms. B. Structure of the ether bolaform synthesized. The percentage
indicated below each structure corresponds to the yields after isolation.
Scheme 3 Metathesis from hex-50-enyl-a-D-xylopyranoside.
Scheme 4 Metathesis on undec-100-enoyl(2,3,4-tri-O-acetyl)-b-D-xylopyranoside.
Scheme 5 Deprotection reaction of 10,200-bis-eicos-100-enedioyl(2,3,4-tri-O-acetyl)-b-D-xylopyranoside 4bb.
Using Pd/C, 5 or 10%, no hydrogenation of the compound
is observed (Table 3, entries 1–4). The deprotection of the
benzylic groups takes place only when using Pd(OH)2/C (20%)
in CH2Cl2/MeOH (1 : 20).18 However, the mass spectrometry
showed that 10,200-bis-eicosanedioyl-1-D-xylopyranoside was
only obtained in the form of a mixture with partially
c
2260 New J. Chem., 2011, 35, 2258–2266
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Scheme 6 Metathesis on undec-100-enoyl(2,3,4-tri-O-benzyl)-D-xylopyranoside.
Table 2 Metathesis reactions on undec-100-enoyl(2,3,4-tri-O-benzyl)-
Table 3 Hydrogenolysis reactions on 10,200-bis-eicos-100-enedioyl-
(2,3,4-tri-O-benzyl)-D-xylopyranosidea
D-xylopyranosidea
Catalyst addition
conditions
Reaction
time/h
Entry Catalyst
Solvent
t/h Conv. (%)
Entry
Z (%)
E/Zb
1
2
3
4
5
Pd/C (10%)
Janssen
Pd/C (5%)
Engelhart 5011
EtOH 99%
48
72
48
48
—
—
—
—
1
2
3
4
Fastc
Fastc
Fastd
Faste
3
14
24
51
36
46
72
81
78/22
EtOH/Et2O (1/4)
EtOH 99%
EtOH 95%
Pd(OH)2/C (20%) CH2Cl2/MeOH (1/20) 24 98%
a
a
Ester (100 mg, 017 mmol), Grubbs catalyst (14.1 mg, 0.017 mmol,
b
5 (100 mg, 0.087 mmol), H2 (1 atm.), RT.
0.1 eq.) CH2Cl2 (7 mL), 40 1C. Determined by 1H NMR. Addition
by means of a transfer cannula. Addition by means of a syringe
c
d
below 0.1 mM. As the solubility of 20aa in water is higher than
1 mM, the break indicates a saturation of the interface leading
to a spontaneous formation of self-assemblies in the bulk. The
intersection between the two linear fittings of the curve defines
the critical aggregation concentration (CAC) and the corres-
ponding surface tension (gCAC). At 25 1C, the CAC of 20aa
is 0.052 mM and gCAC is B44.0 mN mꢀ1. The dynamic light
scattering measurement of a 100 mM solution of 20aa shows
a single population at 227 ꢂ 70 nm suggesting the presence
of vesicles rather than micelles. However, symmetrical glucose
bolaamphiphiles with a 12 or 14 carbon atoms hydrophobic
chain have been shown to form helical crystalline nano-
fibers and no monolayer spherical vesicles.19c Further
investigations using electron microscopy, small angle X-ray
scattering and wide angle X-ray scattering20 are necessary to
determine the precise morphology of the 20aa supramolecular
aggregates.
e
taddition = 2 ꢁ 60 s (t = 0 and t = 12 h). Addition by means of a
syringe taddition = 2 ꢁ 60 s (t = 0, t = 9 h and t = 18 h).
deprotected compounds. Purification by chromatography on
silica gel was fruitless.
Physico-chemical study
The potential of these sugar-based bolaforms as surfactants
and membrane interacting agents has been studied by
analysing the surface and the membrane interaction properties
of the 20aa compound composed of two xylose heads
connected by an ether link to a hydrophobic chain comprising
18 carbon atoms and an insaturation. Further examination of
the relationships between the structure of these bolaamphi-
philes and their physico-chemical properties will be performed
in a future work. Indeed, the self-assembly properties of sugar-
based bolaforms can be influenced by the stereochemistry of
the glycosidic linkage and the length and the nature of the
hydrocarbon chain (insaturated or not).3c,19
In the present study, the aggregation behaviour of 20aa in
water was investigated by surface tension measurements and
isothermal titration calorimetry (ITC). The mixing behaviour
of 20aa with a dipalmitoylphosphatidylcholine (DPPC) mono-
layer taken as a simplified model membrane is analysed using
surface pressure–area compression isotherms. Interaction of
the bolaform with DPPC is also assessed by its ability to
penetrate into a DPPC monolayer.
Aggregation behaviour. The aggregation behaviour of 20aa
was first analysed by tensiometry measurements. The plot
(Fig. 1) of the equilibrium surface tension as a function of
20aa concentration in the aqueous medium shows a break
Fig. 1 Equilibrium surface tension (geq) as a function of 20aa
concentration (c) (logarithmic scale). Measurements were performed
in milli-Q water at room temperature (25 ꢂ 2 1C).
Scheme 7 Hydrogenation of 10,200-bis-eicos-100-enedioyl(2,3,4-tri-O-benzyl)-D-xylopyranoside 5.
c
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In comparison with the critical micellar concentration
(CMC) of conventional surfactants such as SDS, CTAB,
CHAB, triton X-10021 and alkyl glucoside,22 20aa CAC value
is very low. It reflects its high capacity to aggregate.
The absence of repulsive electrostatic forces between the
neighbouring polar heads, as is the case for SDS and CTAB,
favours such aggregation.
When the interface is saturated, the minimal area occupied
by the molecule at the interface, a, can be determined by the
equation:
Fig. 2 Molar heat of 20aa disaggregation (dhi/dn) versus the concen-
tration of 20aa (c) in the cell.
a = 1/NaGm
where Na is Avogadro’s number and Gm is the maximal excess
concentration of the surfactant at the surface calculated by the
Gibbs equation:
determined by the first derivative of dhi/dn curve (see the inset)
corresponds to the CAC. The value obtained is 0.053 mM and
is in perfect accordance with the tensiometry measurements.
The step between the two plateaux of the sigmoid gives the
ꢀ
ꢁ
1
dg
2:303 RT d log c
Gm ¼ ꢀ
enthalpy of disaggregation, equal in magnitude but opposite in
sign to the aggregation enthalpy (DHw
-
ag).26 Under our
where R is the gas constant (8.314 J molꢀ1
K
ꢀ1), T is the
experimental conditions, DHw is ꢀ0.33 kJ molꢀ1
The standard free energy of aggregate formation and the
.
-
ag
absolute temperature and dg/d log c is the slope of the curve of
geq as a function of the surfactant aqueous concentration
(log c) at concentrations below the CAC.
aggregation entropy can be calculated by the equation:
ꢀ
ꢁ
Under our conditions, the calculated a for 20aa is 81 A2.
This value is almost twice the area occupied by an n-alkyl
glycoside surfactant with one single glucose or mannose as
polar head and a lipid chain between C8 and C12.23 This
suggests that bolaform molecules form a loop structure at the
interface with both xylose heads close to each other and
immersed in water as suggested for other bolaform types.24
Unsurprisingly, a is lower than the one developed by hexose-
based bolaforms at the air–water interface.12 The configura-
CAC
DG0ag ¼ RT ln
¼ DH ꢀ TDS
55:5
where R, the gas constant, is 8.314 J molꢀ1 Kꢀ1
.
In our case, DGa0g is ꢀ34.1 kJ molꢀ1 and TDS is 33.8 kJ molꢀ1
.
At 25 1C, the aggregation of 20aa is spontaneous (DG o 0),
exothermic (DH o 0) and generates a large positive change of
the system entropy (DS > 0) indicating a mainly entropy-
driven process.
1
tion of the double bond of the spacer is not unique and H
The large gain in entropy is caused by a reduction of the
hydrophobic area that is exposed to water when aggregates are
formed. The low contribution of the enthalpy to the Gibbs
energy is not common for sugar-based surfactants for the
temperature range used.26 For octyl glucoside, changes in head
group hydration between the monomeric and the micellar state
of the surfactant have been assumed to add a constant
enthalpic term. Our divergent result could be explained by a
different hydration phenomenon of the xylose molecule
(between the monomer and the aggregated state) compara-
tively to the glucose one, implying a distinct aggregation
process. Indeed, the hydration of the sugar residue is
known to influence the supramolecular arrangement of
bolaamphiphiles15 and according to Gray et al.27 the aqueous
solubility of xylose monomer is much higher than the glucose
unit. Further investigations using spectroscopic methods,28 for
example, are necessary to elucidate this phenomenon.
NMR showed that the E isomer was by far the most abundant
(Z/E ratio of 15/85). This should favor the extended shape of
the spacer. However, according to the calculated a for 20aa,
the presence of the insaturation in a long (18 carbon atoms)
chain does not impair its flexibility, allowing it to adopt a bent
conformation at the interface. Further investigations on xylose
bolaamphiphiles with the corresponding saturated chain
and/or with a shorter hydrophobic spacer could confirm this
hypothesis. The geometry of the 20aa within the aggregate
cannot be deduced from our results. In the case of vesicle
formation, freeze fracture electron microscopy could be
helpful for determining it.15 Compression isotherms at an
air–water interface of a Langmuir trough can also provide
information about the conformation of bolaforms at an
interface.25
Thermodynamic parameters associated with aggregate forma-
tion were determined by ITC. Injections of small volumes
of a concentrated solution of 20aa into milli-Q water in the
measurement cell are accompanied by an endothermic heat of
reaction. After subtracting the heat related to the dilution of
the drop, integration of each peak and normalization as a
function of the number of surfactant moles injected, the molar
heat of disaggregation (dhi/dn) is obtained. As the injections
are performed, dhi/dn decreases because the concentration of
20aa in the cell increases limiting the disaggregation pheno-
menon and finally reaches zero (Fig. 2). The inflection point
Interaction with lipid monolayers. Compression isotherms of
mixed 20aa–DPPC monolayers in different proportions were
established in order to check the existence of interactions
between these two compounds (Fig. 3).
At low molar ratios of bolaform (0.10 r Xb r 0.50), the
isotherms of the mixed films lie between those of the pure
components but they display a different overall shape. At
Xb = 0.25 and 0.5, the characteristic transition observed in
the DPPC isotherm is absent. Moreover, the maximal surface
c
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Fig. 5 Surface pressure increase as a function of the time elapsed
after injection of increasing amounts of bolaform 20aa into the
subphase (milli-Q water at 25 1C). The indicated concentrations are
the final ones reached in the subphase.
Fig. 3 Surface pressure–area isotherms of pure 20aa and DPPC
monolayers and of mixed 20aa–DPPC monolayers at different
bolaform molar ratios (Xb). The isotherms were recorded at 25 1C
with milli-Q water as subphase. Duplicate experiments using separately
prepared solutions gave similar results.
DPPC monolayer was studied and results compared to the
adsorption of the bolaform at a clean air–water interface.
Adsorption of 20aa at a clean interface (Fig. 5) is concen-
tration dependent as is usually observed for conventional
surfactants.31 At 0.42 mM in the subphase, no effect on the
surface pressure is observed. At higher concentrations, a
significant increase of surface pressure is detected after a delay
(B600 s) which is not influenced by the concentration of 20aa
in the subphase. Nonetheless this concentration has an effect
on the adsorption kinetics reflected by the slope of the linear
part of the curve and on the maximal increase of surface
pressure. The higher the concentration of bolaform in the
subphase is, the steeper the slope is and the greater the
equilibrium surface pressure is.
pressure is higher than the one reached with pure DPPC.
These observations are in favour of the existence of inter-
actions between the bolaform and DPPC molecules. In these
proportions, these interactions have a stabilizing effect on the
mixed monolayer, especially at high compression states.
Compression isotherms of mixed films with a higher bolaform
molar ratio (Xb = 0.75 and 0.90) are on the left hand side of
the pure bolaform curve. Globally, molecules in these mixed
films occupy a lower area than those of the pure compounds.
It can arise from a particular arrangement of both molecules
or from a destabilizing effect leading to a partial displacement
of one or both compounds from the interface.
For two P (10 and 20 mN mꢀ1), the plot of the molecular
area versus Xb is shown in Fig. 4. The mixed monolayers
Xb = 0.5 and 0.90 exhibit a molecular area significantly higher
and lower, respectively, than those obeying the additivity rule
(dashed line). According to Maget-Dana,29 a deviation from
the additivity rule means that specific interactions (i.e. excess
interaction) exist between the two compounds. A positive
deviation, such as observed at Xb = 0.5, suggests that one
of the compound, at least, forms bidimensional aggregates
at the interface as already observed for other surfactant/
phospholipid mixed monolayers.30 Negative deviation observed
at Xb = 0.90 could result from formation of a complex
between both molecules or from a partial displacement of
the bolaform 20aa from the interface.
In the presence of a DPPC monolayer at the interface, the
adsorption profile of the bolaform is modified (Fig. 6). For a
DPPC monolayer initially compressed between 10 and
30 mN mꢀ1, the injection of bolaform molecules into the
subphase (5 mM in the subphase) gives rise to a surface
pressure increase giving evidence of bolaform penetration into
the DPPC monolayer. The delay before surface pressure
increase is shorter (100–250 s) than in the absence of DPPC.
This suggests that DPPC molecules exert an attractive effect
on the bolaform adsorption at the interface.
The maximal surface pressure increase depends on the initial
surface pressure of the DPPC monolayer (Fig. 7).
At 15 mN mꢀ1 and above, the variation is linear. Up to
31 mN mꢀ1, the increase is higher than in the absence of
DPPC. According to Maget-Dana,29 this means that specific
interactions exist between the two compounds. The intersection
To further investigate the possible interactions between the
bolaform molecules and DPPC, bolaform penetration into a
Fig. 6 Surface pressure increase as a function of the time elapsed
after injection of bolaform 20aa (final concentration of 5 mM) into the
subphase (milli-Q water at 25 1C) under a DPPC monolayer initially
compressed at different surface pressures (between 10 and 30 mN mꢀ1).
Fig. 4 Mean molecular area at a surface pressure of 10 (E) and
20 (J) mN mꢀ1 versus 20aa molar ratio of 20aa/DPPC mixtures (Xb).
The dashed line represents the additivity rule values.
c
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hydrophobic nature between the aliphatic spacer of the
bolaform and the hydrocarbon chains of phospholipids could
lead to a stabilization of the bolaform molecules at the
interface. Thanks to its membrane interaction properties,
20aa bolaform can be suggested as a potential component
for the formulation of drug delivery systems.
Researches on the influence of the spacer length, of the
insaturation and of the stereochemistry of the ether linkage of
xylose-based bolaamphiphiles on their supramolecular
organization and interaction properties with membrane mod-
els are in progress.
Fig. 7 Maximal surface pressure increase observed after the injection
of bolaform 20aa as a function of the initial surface pressure of the
DPPC monolayer. The white dot on the ordinate axis corresponds to
the equilibrium surface pressure increase reached after adsorption of
20aa at a clean interface.
Experimental section
Syntheses
of the regression line with the x-axis determines the exclusion
surface pressure (Pex), i.e. the maximal DPPC monolayer
surface pressure permitting bolaform molecules penetration.
Under our conditions, Pex is 48.6 mN mꢀ1. This is much
higher than values observed for membrane active compounds
such as mellitin, d-lysin, paclitaxel and fengycin.32 It suggests
that bolaform 20aa is able to penetrate into biological
membrane in vivo, for which P is estimated to be between 31
All reagents were commercially available and used as received.
Solvents were dried and distilled under argon before use
(CH2Cl2 over CaCl2 and diethyl ether, THF over sodium/
1
benzophenone) and stored over molecular sieves. H and 13C
NMR spectra were recorded on an AC 250 Bruker in CDCl3,
MeOD or acetone-d6 with TMS as reference for 1H spectra
and CDCl3 (d 77.0), MeOD (d 49.9) or acetone-d6 (d 30.6)
for 13C spectra. The infrared spectra were recorded with
Spectrafile IRt Plus MIDAC. C, H and N analyses were
performed on a Perkin Elmer 2400 CHN equipment. GC was
recorded on a Hewlett-Packard HP-6890 gas chromatograph,
fitted with DB-1 capillary column (25 m, 0.32 mm), a flame
ionisation detector and HP-3395 integrator; chromatography
was carried out on SDS Silica 60 (40–63 mm), Art 2050044
(flash-chromatography) or Silica 60 F254 (TLC plates).
All experiments (MS and HRMS) were obtained on a
hybrid tandem quadrupole/time-of-flight (Q-TOF) instrument,
equipped with a pneumatically assisted electrospray (Z-spray)
ion source (Micromass, Manchester, UK) operated in positive
mode. The electrospray potential was set to 3 kV in positive
ion mode (flow of injection 5 mL minꢀ1) and the extraction
cone voltage was usually varied between 30 and 90 V.
The detailed synthesis conditions for each compound are
given in the ESI.z
and 34 mN mꢀ1 33
Further investigation under more realistic
.
conditions must be carried out to confirm this hypothesis.
Surprisingly, an initial surface pressure of 10 mN mꢀ1 is
unfavourable for bolaform insertion. The surface pressure
increase is even lower than in the absence of DPPC. In other
words, a lateral pressure threshold is necessary to allow
bolaform to be inserted within the DPPC monolayer. To our
knowledge, this biphasic aspect of the DP–Pi plot has only
been observed for charged peptides29,34 for which electrostatic
interactions play a crucial role in their interaction with the
phospholipid layer. In our case, electrostatic forces cannot
explain the results as the bolaform is uncharged. An optimal
arrangement between 20aa and DPPC at a higher lateral
pressure could be at the origin of the bolaform stabilization
at the interface.
The purity of the compound 20aa was checked by RP-
HPLC-ELSD (see below for the conditions) and was >97%.
Conclusion
A novel series of bolaamphiphiles consisting of two xylose
heads connected by an ester or an ether link to a hydrophobic
spacer with increasing chain length and an insaturation were
synthesized with satisfactory yields by sequential protection/
metathesis/deprotection or by direct metathesis.
Determination of 20aa aqueous solubility
The 20aa concentrations in the aqueous medium were checked
by external calibration by RP-HPLC-ELSD on an Agilent
Technologies 1200 series HPLC. The column, a Zorbax XDB
C18 (3.5 mm, 4.6 ꢁ 150 mm, Agilent), was thermostated at
30 1C. The flow rate was 0.8 mL minꢀ1 and a linear gradient of
milli-Q water and ACN starting at 30% ACN and increasing
to 100% ACN in 5 min was used. Then, 100% ACN was
maintained for 5 min. The ELSD parameters were 40 1C and
3.5 bars N2.
The interfacial behaviour and membrane interaction
properties of the compound 20aa with a hydrophobic chain
comprising 18 carbon atoms and connected by an ether link to
both xylose heads with an a anomeric form were analyzed.
The important self-assembly properties of the xylose-based
bolaform 20aa highlight its high potential as a surface-active
agent. The aggregation is mainly driven by an entropy process.
The area occupied by the molecule at the interface suggests
that it adopts a loop structure. Its interaction with membrane
lipids favours its adsorption and stabilization at the interface.
This interaction occurs if the density of phospholipids is high
enough to exert an optimal lateral pressure. Interaction of a
Molecular aggregation in water
By tensiometry. Equilibrium surface tension was measured
with the tensiometer Tensimat N3 (Prolabo) equipped with a
platinum Wilhelmy plate at room temperature (25 ꢂ 2 1C).
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The bolaform 20aa was dissolved in dimethylsulfoxide
(DMSO) and the concentrated organic solution was diluted
with milli-Q water (final DMSO concentration of 1% (v/v)).
All measurements were repeated twice with two distinct
bolaform solutions.
interface. These devices were placed at two fixed positions on
the trough to ensure a reproducible injection process. Further-
more, during the entire duration of the experiment, the sub-
phase was stirred using two cylindrical micromagnetic rods
(8 ꢁ 1.5 mm2) and two electronic stirrer heads located beneath
the trough (model 300, Rank Brothers, Bottisham, U.K.). An
autoreversing mode with slow acceleration and a stirring speed
of 100 rpm was selected. After the injection of bolaform, the
increase in surface pressure was recorded.
By dynamic light scattering. Particle size distribution of a
100 mM 20aa aqueous dispersion was measured using a Delsat
Nano (Beckman Coulter, Brea, CA, USA). The bolaform 20aa
was dissolved in DMSO and the concentrated organic solution
was diluted with milli-Q water (final DMSO concentration
of 1% (v/v)).
For the penetration experiments, DPPC monolayers were
prepared with the same LB system as above. The defined
initial surface pressure of these monolayers was obtained by
spreading a precise volume of a DPPC 1 mM solution
prepared in dichloromethane/methanol (9 : 1 v/v). As soon as
the initial surface pressure was stabilized (B20 min),
bolaform, solubilized in DMSO, was injected (20 mL) into
the subphase (milli-Q water) to a final concentration of
5 ꢁ 10ꢀ6 M using the same injection setup as described above.
After the injection of bolaform, the increase in surface
pressure was recorded.
By isothermal titration calorimetry. The thermodynamic
parameters associated with the aggregation were determined
by isothermal titration calorimetry performed on a VP-ITC
Microcalorimeter (Microcal, Northampton USA).
Aliquots of 4 mL of a 1.2 mM bolaform 20aa solution were
injected into the calorimeter cell (Vcell = 1.4565 mL) containing
milli-Q water with 1% (v/v) of DMSO at constant time
intervals (300 s). The cell was maintained very accurately
at 25 1C.
Pure DMSO injections into the subphase did not modify the
initial surface pressure of the DPPC monolayers within the
time span of the penetration experiments.
The bolaform 20aa solutions in milli-Q water were prepared
as for the experiments with the tensiometer.
The solution in the sample cell was stirred at a speed of
305 rpm. The reference cell was filled with milli-Q water. Prior
Acknowledgements
to each analysis, all solutions were degassed using
sonicator bath.
a
This work was supported by the ‘‘Contrat d’objectifs’’ in
‘‘Europol’Agro’’ framework (Glycoval program). We are
grateful to the public authorities of Champagne-Ardenne for
a fellowship to C. D. and material funds. The authors thank
Julien Ertel and Alexandre Schandeler for their technical
assistance. M. D. thanks the F.R.S.-F.N.R.S. (National Funds
for Scientific Research, Belgium) for her research associate
position. K. N. thanks the Superzym ARC grant, financed by
the French Community of Belgium.
The heats related to the fall of the drop were determined by
injecting at constant time interval 4 ml of water with 1% (v/v)
of DMSO into the measuring cell containing water with 1%
(v/v) of DMSO. These values were subtracted from the
observed heats for determining the effective heats. All
measurements were repeated twice with two distinct bolaform
solutions. Raw data were processed by software Origin 7
(Originlab, Northampton, USA).
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