Chiral recognition in aggregates formed by chiral bola-amphiphiles

Article abstract of DOI:10.1016/j.tetasy.2010.07.002

The aggregates of three new isomeric chiral bola-amphiphiles have been taken into consideration as models for investigating chiral recognition in biological membranes. The recognition capabilities of the aggregates were explored following (by CD and HPLC

Full text of DOI:10.1016/j.tetasy.2010.07.002

Tetrahedron: Asymmetry 21 (2010) 2117–2123
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Chiral recognition in aggregates formed by chiral bola-amphiphiles
Francesca Ceccacci a, Pietro di Profio b,c, Luisa Giansanti d,c, Stefano Levi Mortera e, Giovanna Mancini d,c,
,
*
Alessandro Sorrenti d,c, Claudio Villani e
a Dipartimento di Chimica, Universita‘ degli Studi di Roma ‘La Sapienza’, P. le A. Moro 5, I-00185 Roma, Italy
b Dipartimento di Chimica, Universita‘ di Perugia, Via Elce di Sotto, 06123 Perugia, Italy
c Centro di Eccellenza Materiali Innovativi Nanostrutturati per Applicazioni Chimiche, Fisiche e Biomediche, Universitá di Perugia, Via Elce di Sotto, 06123 Perugia, Italy
d CNR, Istituto di Metodologie Chimiche-Sezione Meccanismi di Reazione, Dipartimento di Chimica, Universita‘ degli Studi di Roma ‘La Sapienza’, P.le Aldo Moro 5, 00185 Roma, Italy
e Dipartimento di Chimica e Tecnologie del Farmaco, Universita‘ degli Studi di Roma ‘La Sapienza’, P. le A. Moro 5, I-00185 Roma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The aggregates of three new isomeric chiral bola-amphiphiles have been taken into consideration as
models for investigating chiral recognition in biological membranes. The recognition capabilities of the
aggregates were explored following (by CD and HPLC experiments) the shift in the equilibrium between
the inter-converting enantiomers of a biphenylic derivative and bilirubin, used as markers of chirality.
The chiral recognition experiments were performed under different pH and concentration conditions.
The results were compared with those relative to the aggregates formed by an equimolar mixture of single
head/single chain amphiphiles that mimic the structure of the bola surfactant devoid of a covalent link.
Ó 2010 Published by Elsevier Ltd.
Received 31 May 2010
Accepted 1 July 2010
Available online 31 July 2010
1. Introduction
ences being the molecular structure of their lipids. In archaebacte-
ria, the lipid molecules contain isoprenoid hydrocarbon chains
Chiral homogeneity is one of the main features of Nature both
at the molecular and the macroscopic levels. Most of the processes
that are essential to life are unequivocally governed by chiral inter-
instead of the linear chain of other lipids that are ether-, instead
of ester-, linked to the sn-2 and sn-3 positions (instead of sn-1
and sn-2) of a glycerol moiety. Some lipids found in thermoacido-
philic archaebacteria have a fourth peculiarity as both ends of the
hydrocarbon chains are linked to the polar head groups (glycerol or
nonitol).1 Lipids featuring two polar head groups linked to both ends
of a hydrocarbon chain are known as bolaform amphiphiles2 (or
bola-amphiphiles or bola-lipids) as they resemble the ‘bolas’, the
weapons used by the South American gauchos. The peculiar phase
behavior of bola-lipids has suggested that the ‘bola’ structure could
be the evolutionary response to the frequent and extensive temper-
ature fluctuations faced by thermoacidophilic archeabacteria.1
Although natural bola-amphiphiles are chiral, to the best of our
knowledge, aggregates formed by either natural or synthetic chiral
bola-amphiphiles have not been taken into consideration as mod-
els for investigating chiral recognition phenomena in the biological
membrane.
actions, as all metabolic pathways are linked to
L-aminoacids, to D-
sugars, to the right-handed helix of proteins, and to the right-
a
handed helix of nucleic acids; therefore there is an interest in
studying the non-covalent interactions responsible for the organi-
zation deriving from chiral recognition in biological membranes
that are in charge of most life processes. The central role of biolog-
ical membranes in the cell life suggests their possible involvement
either in the breaking of symmetry or in the amplification process
of a first enantiomeric imbalance. It is possible that the organiza-
tion of a primitive bio-membrane had been involved in the enan-
tiomeric selection that yielded the present homochirality of
Nature. The amphiphilic components of primitive bio-membranes
were very different from the components of current bio-mem-
branes and a trace of the actual nature of primitive lipids might
be found in the membranes of archaebacteria, microorganisms that
are able to live in extreme habitats, hostile to all other forms of
life.1 In fact, the prefix ‘Archae’, meaning ‘Ancient’, is due to the fact
that these organisms are considered living fossils and that they
probably resemble the first forms of life on Earth. Archaebacteria
differ in many ways from other bacteria, with one of the differ-
Herein we report on the chiral recognition experiments carried
out in aggregates formed by chiral synthetic bola-amphiphiles for
investigating the effect of the ‘bola’ structure on chiral recognition.
In particular, we describe deracemization experiments on racemic
2-carboxy-2’-dodecyloxy-6-nitrobiphenyl, 1, and bilirubin IXa, BR,
carried out in aggregates formed by the three isomeric bola-
amphiphiles 2 and, for comparison, in aggregates formed by an
equimolar mixture of the single head/single chain amphiphiles 3
and 4 (Chart 1) that mimic the structure of the bola surfactant
devoid of a covalent link. Both biphenylic derivative 1 and bilirubin
* Corresponding author.
E-mail address: giovanna.mancini@uniroma1.it (G. Mancini).
0957-4166/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.tetasy.2010.07.002

2118
F. Ceccacci et al. / Tetrahedron: Asymmetry 21 (2010) 2117–2123
)
N
H
-O2C
O(CH
O(CH2)12
HN
H
O(CH2)12
HN
OC12H25
NO2
-O2C
H
H
-O2C
H
O(CH12 12
HN
-O2C
)
)
O(CH
H
N
CO2H
O(CH2)12
HN
-O2C
-O2C
H
H
1
2c
2b
2a
CO2H
CO2H
O(CH2)12 HN
-O2C
-
HN
HN
CO2
NH
NH
NH
12H25
H
H
C
O
O
4
3
BR
Chart 1.
were exploited previously as markers of the chirality of micellar
aggregates3 because within these systems the stereochemical
information can be transferred from the chiral aggregates to the
marker, inducing an imbalance in the 1:1 equilibrium ratio of the
inter-converting enantiomers. Such an imbalance (deracemization)
is detectable by circular dichroism3 and, depending on the rota-
tional barrier and experimental conditions, by HPLC3a,c,d,e and
NMR3a,d,g, experiments.
Here, the deracemization of both racemic mixtures was investi-
gated by circular dichroism (CD) experiments. The relatively high
rotational barrier of 13c (21 kcal/mol vs 17 kcal/mol of bilirubin4)
also allowed us to ascertain the extent of its deracemization by
HPLC experiments on a chiral stationary phase.
2.2. Aggregation behavior
The size distribution of the aggregates formed in water by bola-
amphiphiles 2 and by a 1:1 mixture of surfactants 3 and 4 was
evaluated by Dynamic Laser Light Scattering (DLLS) measure-
ments. The aggregation properties of the various surfactants are,
as expected, very different. Surfactant 2a forms vesicles in all pH
conditions, as indicated by DLLS experiments showing the pres-
ence of a broad population whose number distribution is of 30–
40 nm diameter whereas 2b gives micellar aggregates regardless
of the pH conditions. Actually, as reported in Table 1, in the case
of 2b we observed three peaks in all pH conditions, one relative
to micellar aggregates (3–6 nm), and two relative to larger aggre-
gates (probably vesicles). However, the micellar population was
largely the most abundant.
2. Results and discussion
Due to the strong insolubility of compound 2c, it was not possi-
ble to study its aggregating properties by DLLS. The DLLS experi-
ments relative to the mixture of 3 and 4 did not give statistically
acceptable results.
2.1. Preparation of amphiphiles
All amphiphiles have been prepared by reductive amination of
The Critical Micellar Concentration (cmc) of 2b at 50 °C at the
spontaneous pH was measured by fluorescence experiments, fol-
lowing the pyrene 1:3 ratio method.5 The estimated cmc is
3.1 ꢀ 10ꢁ5 M and the gradual increase in the I3/I1 ratio suggests
the formation of premicellar aggregates.5a,5c
the proper aldehyde with L-proline. For the preparation of 2 and
4 (Scheme 1), the aldehyde was obtained by oxidation with IBX
of the corresponding alcohol which in turn was prepared by alkyl-
ation of the commercially available diphenols and phenol with 12-
bromo-dodecan-1-ol.
O(CH2)12
HN
O(CH2)12OH
OH
O(CH2)11CHO
a
b
c
H
-O2C
R
R
R
R
2a ortho
6a ortho R=-O(CH2)12OH
6b meta R=-O(CH2)12OH
6c para R=-O(CH2)12OH
6d R=H
6a ortho R=-O(CH2)11CHO
6b meta R=-O(CH2)11CHO
6c para R=-O(CH2)11CHO
6d R=H
5a ortho R=OH
5b meta R=OH
5c para R=OH
5d R=H
2b meta
2c para
4 R=H
R=
O(CH2)12 HN
-O2C
H
Scheme 1. Reagents and conditions: (a) 12-bromododecan-1-ol, K2CO3, CH3CN, reflux; (b) IBX, DMSO, THF, rt; (c) H2, Pd(C) 10%, THF, MeOH,
L
-proline.

F. Ceccacci et al. / Tetrahedron: Asymmetry 21 (2010) 2117–2123
2119
Table 1
Diameters (D) and percentage scattered intensities obtained in the DLLS experiments
on aqueous 25 mM solutions of 2b at 55 °C under different pH conditions
pH
Peak 1
% si (% n)a
Peak 2
% si (% n)a
Peak 3
% si (% n)a
D (nm)
D (nm)
D (nm)
1
7
14
3.2
3.9
6.1
1.9 (97)
0.5 (98)
11.6 (99)
9.3
17.5
28.0
23 (3.2)
42 (1.9)
17.5 (<0.1)
112.6
120.6
160.5
75 (0.1)
57 (0.1)
70.9 (<0.1)
a
% si: percentage of light scattered by the respective population; % n: number
percent of the respective population as calculated according the inverse laplace
transform procedure used.
2.3. Chiral recognition experiments
The chiral recognition experiments on biphenylic derivative 1
Figure 2. CD spectra of 5.0 mM 1 in aggregates of 3 (25 mM) and 4 (25 mM) at
spontaneous pH (solid line), pH 13 (dashed line) and pH 1 (dotted line). The molar
ellipticity has to be considered as apparent molar ellipticity because it is calculated
from the analytical concentration of 1.
were carried out at different concentrations of
1 (5.0 mM,
0.30 mM, and 0.010 mM) in the presence of aggregates of both
25 mM bola-amphiphiles 2 and the mixture of 25 mM 3 and
25 mM 4 (in order to keep constant the concentration of the polar
head groups) and under different pH conditions, namely pH 1, pH
13, and spontaneous pH (ꢂ6.5). The temperature of the experi-
ments was varied according to the solubility of the surfactants un-
der the various experimental conditions and each sample was set
in order to obtain clear solutions (see Experimental).
All CD spectra of 5.0 mM 1 in aggregates of 2 showed a band
centered at 330 nm; the band is negative under acidic and sponta-
neous pH conditions, and positive under alkaline pH conditions, as
shown in Figure 1 for surfactant 2a.
induced by the aggregates as already observed in the aggregates of
3.3d Furthermore, HPLC also allowed us to investigate samples un-
der concentration conditions not suitable for CD experiments due
to the low concentrations of 1. The changes in the stereochemical
bias upon pH variations were reversible both in the presence of
aggregates of 2 and in the presence of the aggregates formed by
the mixture of 3 and 4, as observed previously in aggregates
formed only by 3.3d These results demonstrate, as suggested previ-
ously,3d that in these systems the deracemization process is con-
trolled by the charge features of the biphenylic derivative and of
the surfactants, hence by the different organization of the aggre-
gates, which can change dramatically in response to extreme pH
variation, leading to a reversal of the equilibrium of the inter-con-
verting enantiomers.
The enantiomeric excesses (ees) measured by HPLC in the
experiments relative to 2a and to the mixture of 3 and 4 are re-
ported in Table 2. The ee of 1 induced by the aggregate changes
with the concentration of 1, with the nature of the amphiphile
and with the pH of the medium. At the highest value of concentra-
tion of 1, the aggregates formed by 2–4 induce only modest ees on
1. Interestingly, under these conditions the ees induced by bola-
amphiphiles 2, under all pH values, are higher than those induced
by the mixture of 3 and 4.
Table 2
Enantiomeric excesses of 1 as a result of deracemization due to the chiral aggregates
formed by surfactants 2a and by a mixture of surfactants 3 and 4 (the sign in brackets
refers to the sign of the CD band of the excess enantiomer)
Figure 1. CD spectra of 5.0 mM 1 in aggregates of 2a (25 mM) at spontaneous pH
(solid line), pH 13 (dashed line) and pH 1 (dotted line). The molar ellipticity has to
be considered as apparent molar ellipticity because it is calculated from the
analytical concentration of 1.
Surfactant
[HCl] = 0.10 M Spontaneous [NaOH] = 0.20 M
pH
[1] = 5.0 mM
[2a] = 25 mM (ꢁ) 2%
[3] = 25 mM (ꢁ) 1%
+
(ꢁ) 11%
(ꢁ) 3%
(+) 3%
(+) 1%
The CD spectra of 1 in the presence of the aggregates formed by
the three different bola-amphiphiles showed bands characterized
by the same shape and intensity, although the experiments were
carried out at different temperatures. The CD experiments of 1 in
the presence of the equimolar mixture of 3 and 4 showed bands
characterized by the same sign as those observed in the presence
of surfactants 2, however, under neutral and alkaline conditions,
the spectra showed a less intense band which shifted to higher
and lower wavelength, respectively (Fig. 2). The presence of a band
in the CD spectra of such systems could be due to deracemization,
induced CD effect, or superimposition of both phenomena. Derace-
mization was ascertained and measured by HPLC on a chiral sta-
tionary phase, confirming that the CD sign reversals following
extreme pH changes were due to a switch in the excess enantiomer
[4] = 25 mM
[1] = 0.30 mM [2a] = 25 mM (ꢁ) 7%
(ꢁ) 22%
(ꢁ) 20%
(+) 6%
(+) 4%
[3] = 25 mM (ꢁ) 7%
+
[4] = 25 mM
[1] = 0.010 mM [2a] = 25 mM (ꢁ) 20%
(ꢁ) 40%
(ꢁ) 40%
(+) 20%
(+) 20%
[3] = 25 mM (ꢁ) 20%
+
[4] = 25 mM
At low concentrations of 1, when the biphenylic derivative is
highly diluted in the chiral aggregates, the ees are larger, in all

2120
F. Ceccacci et al. / Tetrahedron: Asymmetry 21 (2010) 2117–2123
aggregates and under all pH conditions. However, in these concen-
trations the observed ee values of 1 are comparable or identical in
the aggregates formed either by bola or by the conventional surfac-
tants. It is remarkable that the extent of deracemization obtained
at the spontaneous pH is the highest (ee = 40%) we have ever ob-
served in this kind of system.
The different wavelengths of the maximum featured by the CD
bands obtained under neutral and alkaline conditions in the mix-
ture of surfactants 3 and 4 could be ascribed to a dependence of
the optical features of 1 on its state of charge. If this were the case,
the spectra obtained would be the evidence of an interfacial posi-
tion of 1, therefore exposed to the bulk. However, we verified with
a sample of enantiopure 1, isolated by HPLC on a chiral stationary
phase,3c,6 that the wavelength of the maximum of the CD band is
centered at 330 nm, independent from the pH conditions. There-
fore, the variations observed in Figure 2 should indicate a different
dihedral angle between the two aromatic rings of 1 induced by a
specific change of the morphology of the aggregate according to
pH conditions.
A comparison on the recognition capabilities of the aggregates
formed by bola-amphiphiles 2 (25 mM) and of the aggregates
formed by an equimolar mixture of 3 (25 mM) and 4 (25 mM)
was also carried out by using bilirubin as a marker of chirality.
The experiments were carried out by CD on samples at different
concentrations of BR (0.10 mM, 0.010 mM, and 0.0010 mM) under
different pH conditions, namely pH 1, pH 9 (at higher pH BR is not
stable7), and at spontaneous pH, at the same temperature at which
the analogous experiments concerning the biphenylic derivative
were performed. All the CD spectra of BR in the presence of the
aggregates formed by the bola-amphiphiles showed a conserva-
tive-positive bisignate band whose intensity and wavelength de-
pended on the pH conditions, as shown in Figure 3 for 2a, and on
the regiochemistry of the bola-amphiphile (Figs. 4–6), although
being independent from the concentration of bilirubin.
Figure 4. CD spectra of 0.010 mM BR in aggregates of 2a (solid line), 2b (dotted),
and 2c (dashed line) at spontaneous pH ([2] = 25 mM). The molar ellipticity has to
be considered as apparent molar ellipticity because it is calculated from the
analytical concentration of BR.
The results of the deracemization experiments in the aggregates
formed by the equimolar mixture of surfactants 3 and 4 are re-
ported in Figure 7 and concern only the experiment carried out
at spontaneous pH, since under the other pH conditions (pH 9
and pH 1), we obtained very noisy spectra. The CD spectrum re-
ported in Figure 7 shows a positive bisignate band similar to those
observed in Figure 4, although more intense. The presence of a
bisignate band in such systems, lacking additional chromophores
that absorb in the same region of the spectrum as bilirubin, can
be reasonably attributed to the deracemization of bilirubin.8 Due
Figure 5. CD spectra of 0.10 mM BR in aggregates of 2a (solid line), 2b (dotted line),
and 2c (dashed line) at pH
9 ([2] = 25 mM). The molar ellipticity has to be
considered as apparent molar ellipticity because it is calculated from the analytical
concentration of BR.
Figure 3. CD spectra of 0.10 mM BR in aggregates of 25 mM 2a at spontaneous pH
(dashed line), pH 9 (solid line), and pH 1(dotted line). The molar ellipticity has to be
considered as apparent molar ellipticity because it is calculated from the analytical
concentration of BR.
Figure 6. CD spectra of 0.10 mM BR in aggregates of 2a (solid line), 2b (dotted line),
and 2c (dashed line) at pH
1 ([2] = 25 mM). The molar ellipticity has to be
considered as apparent molar ellipticity because it is calculated from the analytical
concentration of BR.

F. Ceccacci et al. / Tetrahedron: Asymmetry 21 (2010) 2117–2123
2121
deracemization confirm that small amounts of solute do not per-
turb the organization of the aggregates. All of the results obtained
confirm the hypothesis we suggested previously3d,h,k that in poly-
molecular aggregates, the recognition process is due to chiral cav-
ities that result from a complex texture of specific interactions
controlling their shape and location, and that the perturbation of
these cavities reduces the recognition capabilities.
3. Experimental
3.1. General
Chemicals (Sigma–Aldrich) were of the highest grade available
and were used without further purification. Solvents employed in
the spectroscopic studies were of spectroscopic grade and used as
received. TLC: Merck Silica Gel 60 F254. PTLC: Silica Gel 60 F254
2 mm. CC: Merck Silica Gel 60, 70–230 mesh ASTM. 1H NMR spectra
were recorded on a Bruker AC 300 operating at 300.13 and
75.47 MHz for 1H and 13C, respectively, equipped with a sample tube
thermostating apparatus. Signals were referenced with respect to
TMS (d = 0.000 ppm). CD spectra were recorded on a Jasco spectro-
polarimeter J-715. HPLC determinations of enantiomeric excess
were performed as previously described3c,d on a quinine t-butylcar-
bammate stationary phase9—250 ꢀ 4.6 mm I.D.—injecting directly
the aqueous samples (eluent: methanol/acetonitrile = 60/40 +
ammonium acetate 8.4 mM + acetic acid 42 mM; 283 K; flow rate
1.5 mL/min). HPLC isolation of the enantiomers of 1 was carried
out on a Chiralpak AD column (250 ꢀ 4.6 mm I.D), cooled at 277 K
(water/ice bath), as previously reported.3c,6 The collected fractions
of the pure enantiomers were stored at 253 K until used.
Figure 7. CD spectrum of 0.10 mM BR in aggregates formed by 25 mM 3 and
25 mM 4 at spontaneous pH. The molar ellipticity has to be considered as apparent
molar ellipticity because it is calculated from the analytical concentration of BR.
to the very low barrier of interconversion, in the case of BR, it was
not possible to measure the extent of its deracemization by HPLC.
However, despite the absence of a quantitative CD for the pure dia-
stereomeric complex bilirubin enantiomer/aggregate, we could
estimate the extent of deracemization of ꢂ10% in the experiments
of BR in aggregates of 2, and of 15% in those of BR in aggregates of 3
and 4 on the basis of the reported data, relative to a computed CD
spectrum of bilirubin.8b
The results reported in Table 2 indicate that the bola-amphi-
philes and the mixture of ‘conventional’ amphiphiles showed sim-
ilar recognition capabilities when the biphenylic derivative 1 was
used at very low concentrations. On the other hand, at high con-
centrations of 1 all the aggregates showed reduced recognition
capabilities; however, those of bola aggregates were higher than
those of ‘conventional’ aggregates, independent of the regiochem-
istry of the bola monomer and therefore of the morphology of the
aggregates. These observations support the view that a high con-
centration of solute perturbs the structure of the aggregates and
that, because of a preorganization due to the covalent bond joining
remotely two head groups, the structure of the bola aggregates is
perturbed to a minor extent.
3.2. Determination of aggregate size by dynamic laser light
scattering
Light scattering measurements were made on ca. 1 mL samples
in 6 mm diameter Pyrex glass culture tubes, protected from dust
by Parafilm caps. The cylindrical glass sample tube was fixed at
the center of a toluene-filled fluorimeter cuvette to provide refrac-
tive index matching against stray light reflections. The cuvette was
housed in a black-anodized-aluminum cell block, whose tempera-
ture was regulated by a Peltier thermoelectric element. The light
source was a Coherent Innova 70-3 argon-ion laser operating at
488 nm. Light scattered at 90° was collected from approximately
one coherence area and imaged onto the slit of a photomultiplier
tube (Products for Research, Inc.). A64-channel Nicomp model
370 computing autocorrelator was used to calculate and display
the diffusion coefficient, D, and the associated derived parameters
from the cumulant analysis10, and nonlinear least-squares (NLLS)
fits (inverse Laplace transform, ILT,11 method) to the intensity-
autocorrelation function. All the measurements showed reasonable
goodness-of-fit values. The hydrodynamic radii, Rh, were estimated
by applying the Stokes–Einstein relation (for stick-boundary
conditions):12
Due to the high value of
e of BR, all the experiments involving
this chiral marker were carried out at a high [surfactant]/[BR] ratio
under conditions where we can assume that the aggregate organi-
zation is not perturbed by the marker. Based on the results relative
to the deracemization of the biphenylic derivative, we could have
expected the bola and ‘conventional’ aggregates to show similar
recognition capabilities, actually, in this case the ‘conventional’
aggregates showed slightly better recognition capabilities.
It is noteworthy that the recognition capabilities of the isomeric
bola aggregates were actually equivalent toward the biphenylic
derivative, whereas they were different toward bilirubin, particu-
larly under acidic conditions, where the highest extent of derace-
mization was observed in the aggregates formed by surfactants
2b and 2c (in the latter case at 70 °C), respectively. This result dem-
onstrates that chiral recognition cannot be ascribed to the interac-
tion of the solute with the head group of a monomeric unit within
the aggregate, but to the interaction with the entire aggregate or
with a region of the aggregate.
D ¼ kT=6pgRh
where
g is the viscosity of the solution, which can be approximated
to that of water.
3.3. Determination of the critical micelle concentration by
fluorescence experiments
The hypothesis that the bola structure of primordial lipids
might have a role in the development of the chiral homogeneity
of biomolecules is nicely supported by the experiments of derace-
mization of 1 at different [surfactant]/[1] ratios, showing that the
bola aggregates are perturbed to a minor extent by the inclusion
of a high amount of probe. On the other hand, the results of BR
Fluorescence experiments were performed on the Horiba Jobin
Yvon spectrofluoremeter Fluoromax-4, equipped with a thermo-
stating apparatus. Samples were prepared by drying 80
ethanol solution of 4.0 M pyrene in a glass tube by a nitrogen flux
and adding aqueous solutions (3 mL) of 2b at concentrations be-
lL of an
l

2122
F. Ceccacci et al. / Tetrahedron: Asymmetry 21 (2010) 2117–2123
tween 1.0 ꢀ 10ꢁ6 M and 1.0 ꢀ 10ꢁ2 M to obtain a 1.1 ꢀ 10ꢁ7 M fi-
nal concentration of pyrene. The solutions were left to incubate
at 50 °C under stirring for 12 h. Emission spectra of the solutions
were acquired in the range 350–450 nm (kexc = 335 nm).
113.99; 69.10; 43.78; 29.46; 29.41; 29.29; 29.23; 29.05; 25.92;
21.95.
3.5.3. (2S)-1-[12-(2-{12-[(2S)-2-Carboxypyrrolidine-1-yl]dodecyl-
oxy}phenoxy)dodecyl]pyrrolidine-2-carboxylic acid 2a
3.4. Sample preparation for recognition experiments
To a solution of 0.40 g (3.5 mmol) of L-proline in 18 mL of
CH3OH, a solution of 0.47 g (0.99 mmol) of 7a in 14 mL of THF
was slowly added under stirring. After removal of oxygen by flux-
ing through in the solution, 40 mg of 10% Pd(C) was added. The
mixture was saturated with hydrogen and kept under stirring at
room temperature till the complete disappearance of the starting
material (ꢂ5 days, TLC, CHCl3/CH3OH/H2O = 64:34:2).
Samples of 5.0 mM 1 in aqueous surfactants were prepared by
adding 0.70 mL of H2O (or aqueous 0.10 M HCl or 0.20 M NaOH)
to the proper amounts of surfactant and biphenylic derivative.
The solutions were sonicated and gently heated to obtain clear
solutions.
Samples at a lower concentration of 1 were prepared by the eth-
anol injection method, adding a proper volume of a concentrated
ethanolic solution of the biphenylic derivative to the aqueous solu-
tion of the surfactant.
All samples of 1 were incubated overnight prior to the measure-
ment. The chosen temperature for the recognition experiments
was such as to allow the obtainment of clear solutions. For 2a
the temperature was 50 °C for all pH values; for 2b 50 °C at the
spontaneous pH, 45 °C under alkaline pH, and 30 °C under acidic
pH; for 2c temperature was 70 °C; for the mixture of 3 and 4
57 °C at the spontaneous pH, 37 °C under alkaline pH, and 45 °C
under acidic pH.
Samples containing BR were prepared by adding an appropriate
volume of a concentrated DMSO solution of the probe to the aque-
ous solution of the surfactant at the spontaneous pH, at acid pH
(0.10 M HCl), and at basic pH (H3BO3/NaOH/HBr).
The solution was then filtered under suction on a Celite Pad by
means of a gooch funnel and the residue washed with hot EtOH. The
crude product, obtained after removal of the solvent from the fil-
trate, was purified by chromatography on silica gel (CHCl3/CH3OH/
H2O = 64:34:2), followed by crystallization from t-BuOMe, yielding
0.62 g (92% yield) of 2a as white crystals. (Calcd for C40H68N2O6: C,
71.39; H, 10.12; N, 4.17. Found: C, 71.27; H, 10.31; N, 4.01). 1H
NMR (CD3OD): 7.05–6.83 (4H, m, Ar); 3.951 (4H, t, ArOCH2–,
J = 6.4 Hz); 3.89–3.70 (4H, m, d,
a); 3.29–2.95 (6H, m, –CH2N, d);
2.48–2.31 (2H, m, b); 2.20–1.58 (14H, m, b,
c, ArOCH2CH2–, –
CH2CH2N); 1.56–1.24 (32H, m, –(CH2)8–). 13C NMR (CD3OD):
173.71; 150.60; 122.41; 115.63; 70.61; 70.39; 56.68; 55.98; 30.89;
30.80; 30.64; 30.41; 30.32; 27.76; 27.36; 27.13; 24.33.
3.5.4. 12-[3-(12-Hydroxy-dodecyloxy)-phenoxy]-dodecan-1-ol
6b
Compound 6b, a white solid, was obtained from resorcinol 5b in
90% yield, following the same procedure described for 6a. 1H NMR
(CDCl3): 7.135 (1H, t, Ar, J = 8.1 Hz); 6.52–6.42 (3H, m, Ar); 3.914
(4H, t, ArOCH2–, J = 6.6 Hz); 3.628 (4H, t, –CH2OH, J = 6.6 Hz);
1.82–1.68 (4H, m, ArOCH2CH2–); 1.64–1.21 (36H, m, –(CH2)9–).
13C NMR (CDCl3): 160.38; 129.76; 106.62; 101.42; 67.98; 63.13;
32.83; 29.58; 29.44; 29.40; 29.29; 26.07; 25.76.
3.5. Preparation of surfactants and probes
Bilirubin IXa was purchased from Fluka. Biphenylic derivative
13j and surfactant 33d,13 were prepared as previously described.
o-Iodoxybenzoic acid (IBX) was prepared following a procedure de-
scribed in the literature.14
3.5.1. 12-[2-(12-Hydroxy-dodecyloxy)-phenoxy]-dodecan-1-ol
6a
3.5.5. 12-[3-(11-Formyl-undecyloxy)-phenoxy]-dodecanal 7b
Compound 7b, a white solid, was obtained from 6b in 85% yield
(column chromatography, CHCl3), following the same procedure
described for 7a. 1H NMR (CDCl3): 9.750 (2H, m, –CHO,
J = 1.8 Hz); 7.133 (1H, t, Ar, J = 7.8 Hz); 6.53–6.40 (3H, m, Ar);
3.910 (4H,t, ArOCH2–, J = 6.6 Hz); 2.406 (4H, dt, –CH2CHO,
J = 1.8 Hz, J = 7.3 Hz); 1.751 (4H, q, ArOCH2CH2–, J = 6.6 Hz); 1.74–
1.55 (4H, m, –CH2CH2CHO); 1.50–1.18 (28H, m, –(CH2)7–). 13C
NMR (CDCl3): 203.04; 160.39; 129.76; 106.62; 101.42; 67.96;
43.94; 29.55; 29.50; 29.41; 29.38; 29.35; 29.29; 29.17; 26.06;
22.10.
To 0.50 g (4.5 mmol) of catechol 5a in 80 mL of CH3CN, 2.7 g
(10 mmol) of 12-bromododecan-1-ol and 4.2 g (30 mmol) of anhy-
drous K2CO3 were added. The resulting mixture was kept at reflux
for four days, monitoring the reaction by TLC (Et2O/hexane = 8:2).
The hot mixture was then filtered and the residue was washed
thoroughly with hot CH3CN. The precipitate that separated from
the organic solution upon cooling was filtered and crystallized
from CH3CN, yielding 2.1 g of the product as white crystals (96%
yield). 1H NMR (CDCl3): 6.873 (4H, s, Ar); 3.975 (4H, t, ArOCH2–,
J = 6.6 Hz); 3.625 (4H, t, –CH2OH, J = 6.6 Hz); 1.87–1.73 (4H, m, Ar-
OCH2CH2–); 1.65–1.21 (36H, m, –(CH2)9–). 13C NMR (CDCl3):
149.22; 121.01; 114.09; 69.27; 63.10; 32.83; 29.64; 29.47; 29.35;
26.07; 25.77.
3.5.6. (2S)-1-[12-(3-{12-[(2S)-2-Carboxypyrrolidine-1-yl]dodecyl-
oxy}phenoxy)dodecyl]pyrrolidine-2-carboxylic acid 2b
Compound 2b, a white solid, was obtained from 7b in 97% yield,
following the same procedure described for 2a (TLC, CC: CHCl3/
CH3OH/H2O = 64:34:2, crystallization from t-BuOMe. Calcd for
3.5.2. 12-[2-(11-formyl-undecyloxy)-phenoxy]-dodecanal 7a
To 1.8 g (6.4 mmol) of IBX in 7 mL of a 3/4 ratio DMSO/THF solu-
tion, 1.1 g (2.3 mmol) of 6a was added and the resulting solution
was left under stirring for 12 h (TLC, THF/hexane = 3:7). The yellow
solid that precipitated after the addition of 20 mL of water to the
reaction solution was filtered under suction and washed thor-
oughly with water. The solid was then washed with CHCl3,
and removal of the solvent from the organic layer obtained yielded
0.98 g (2.1 mmol, 90% yield) of the pure product as a pale yellow
solid. The product was used as such without further purification.
1H NMR (CDCl3): 9.753 (2H, t, –CHO, J = 1.7 Hz); 6.874 (4H, s, Ar);
3.975 (4H, t, ArOCH2–, J = 6.7 Hz); 2.401 (4H, dt, –CH2CHO,
J = 1.7 Hz, J = 7.3 Hz); 1.93–1.68 (4H, m, ArOCH2CH2–); 1.62–1.15
(32H, m, –(CH2)8–). 13C NMR (CDCl3): 202.73; 149.09; 120.89;
C
40H68N2O6: C, 71.39; H, 10.12; N, 4.17. Found: C, 71.19; H,
10.37; N, 3.92). 1H NMR (CD3OD): 7.106 (1H, t, Ar, J = 8.1 Hz);
6.50–6.36 (3H, m, Ar); 3.911 (4H, t, –CH2OAr, J = 6.4 Hz); 3.85–
3.58 (4H, m,
a, d); 3.26–2.98 (6H, m, –CH2N, d); 2.50–2.32 (2H,
m, b); 2.20–1.55 (14H, m, b,
c
, –CH2CH2OAr, –CH2CH2N); 1.50–
1.20 (32H, m, –(CH2)8–). 13C NMR (CD3OD): 173.69; 161.69;
130.84; 107.66; 102.46; 70.58; 68.86; 56.59; 55.83; 30.72; 30.70;
30.68; 30.55; 30.43; 30.30; 30.26; 27.71; 27.20; 27.05; 24.27.
3.5.7. 12-[4-(12-Hydroxy-dodecyloxy)-phenoxy]-dodecan-1-ol
6c
Compound 6c, a pale pink solid, was obtained from hydroqui-
none 5c in 95% yield, following the same procedure described for

F. Ceccacci et al. / Tetrahedron: Asymmetry 21 (2010) 2117–2123
2123
6a. 1H NMR (CDCl3): 6.804 (4H, s, Ar); 3.881 (4H, t, ArOCH2–,
J = 6.5 Hz); 3.627 (4H, t, –CH2OH, J = 6.5 Hz); 1.80–1.64 (4H, m, Ar-
OCH2CH2–); 1.62–1.15 (36H, m, –(CH2)9–). 13C NMR (CDCl3):
153.20; 115.40; 68.67; 63.13; 32.83; 29.58; 29.41; 26.07; 25.76.
3.22–3.06 (1H, m, CH2N), 3.02–2.86 (1H, m, CH2N), 2.86–2.66
(1H, m, d), 2.40–2.05 (2H, m, b), 2.04–1.86 (2H, m, c), 1.82–1.60
(4H, m, ArOCH2CH2, CH2CH2N), 1.48–1.08 (16H, m, –(CH2)8). 13C
NMR (CDCl3): 170.20, 159.05, 129.37, 120.40, 114.41, 69.88,
67.78, 55.82, 54.89, 29.53, 29.49, 29.44, 29.38, 29.26, 29.17,
26.76, 26.04, 25.89, 23.59.
3.5.8. 12-[4-(11-Formyl-undecyloxy)-phenoxy]-dodecanal 7c
Compound 7c, a white solid, was obtained from 6c in 70% yield
after purification (column chromatography, CHCl3), and following
the same procedure described for 7a. 1H NMR (CDCl3): 9.745 (2H,
t, –CHO, J = 1.7 Hz); 6.801 (4H, s, Ar); 3.878 (4H, t, ArOCH2–,
J = 6.6 Hz); 2.406 (4H, dt, –CH2CHO, J = 7.3 Hz, J = 1.7 Hz); 1.84–
1.51 (8H, m, ArOCH2CH2, –CH2CH2CHO), 1.50–1.18 (28H, m, –
(CH2)7–). 13C NMR (CDCl3): 202.96; 153.14; 136.53; 115.32;
68.58; 43.88; 29.50; 29.46; 29.37; 29.31; 29.28; 29.23; 29.11;
26.01; 22.04.
Acknowledgments
We acknowledge the financial support from FIRB, Research pro-
gram: Ricerca e Sviluppo del Farmaco (CHEM-PROFARMA-NET),
Grant no. RBPR05NWWC 003 and from Dipartimento di Progettaz-
ione Molecolare. This work was carried out in the frame of CM0703
‘Systems Chemistry’ COST Action.
References
3.5.9. (2S)-1-[12-(4-{12-[(2S)-2-Carboxypyrrolidine-1-yl]dodecyl-
oxy}phenoxy)dodecyl]pyrrolidine-2-carboxylic acid 2c
1. (a) De Rosa, M.; Gambacorta, A.; Gliozzi, A. Microbiol. Rev. 1986, 50, 70–80; (b)
De Rosa, M.; Gambacorta, A. Prog. Lipid Res. 1988, 27, 153–175; (c) Gambacorta,
A.; Gliozzi, A.; De Rosa, M. World J. Microbiol. Biotechnol. 1995, 11, 115–131; (d)
Gliozzi, A.; Relini, A.; Lee Gau Chong, P. J. Membr. Sci. 2002, 206, 131–147.
2. (a) Mc Kenzie, D. C.; Bunton, C. A.; Nicoli, D. F.; Savelli, G. J. Phys. Chem. 1987, 91,
5709–5713; (b) Fuhrhop, J. H.; Mathieu, J. Chem. Commun. 1983, 144–145; (c)
Fuhrhop, J. H.; Wang, T. Chem. Rev. 2004, 104, 2901–2937; (d) Meister, A.;
Blume, A. Curr. Opin. Colloid Interface Sci. 2007, 12, 138–147; (e) Yan, Y.; Lu, T.;
Huang, J. J. Colloid Interface Sci. 2009, 337, 1–10. and reference cited therein.
3. (a) Alzalamira, A.; Ceccacci, F.; Monti, D.; Levi Mortera, S.; Mancini, G.; Sorrenti,
A.; Venanzi, M.; Villani, C. Tetrahedron: Asymmetry 2007, 18, 1868–1876; (b)
Borocci, S.; Ceccacci, F.; Galantini, L.; Mancini, G.; Monti, D.; Scipioni, A.;
Venanzi, M. Chirality 2003, 15, 441–447; (c) Ceccacci, F.; Diociaiuti, M.;
Galantini, L.; Mancini, G.; Mencarelli, P.; Scipioni, A.; Villani, C. Org. Lett. 2004,
6, 1565–1568; (d) Ceccacci, F.; Mancini, G.; Sferrazza, A.; Villani, C. J. Am. Chem.
Soc. 2005, 127, 13762–13763; (e) Ceccacci, F.; Cruciani, O.; Diociaiuti, M.;
Galantini, L.; Formisano, G.; Lindner, W.; Mancini, G.; Villani, C. Tetrahedron:
Asymmetry 2006, 17, 1603–1608; (f) Ceccacci, F.; Giansanti, L.; Levi Mortera, S.;
Mancini, G.; Sorrenti, A.; Villani, C. Bioorg. Chem. 2008, 36, 252–254; (g)
Ceccacci, F.; Giansanti, L.; Mancini, G.; Mencarelli, P.; Sorrenti, A. New J. Chem.
2007, 31, 86–92; (h) Bombelli, C.; Borocci, S.; Lupi, F.; Mancini, G.; Mannina, L.;
Segre, A. L.; Viel, S. J. Am. Chem. Soc. 2004, 126, 13354–13362; (i) Andreani, R.;
Bombelli, C.; Borocci, S.; Lah, J.; Mancini, G.; Mencarelli, P.; Vesnaver, G.;
Villani, C. Tetrahedron: Asymmetry 2004, 15, 987–994; (j) Bella, J.; Borocci, S.;
Mancini, G. Langmuir 1999, 15, 8025–8031; (k) Bombelli, C.; Bernardini, C.;
Elemento, G.; Mancini, G.; Sorrenti, A.; Villani, C. J. Am. Chem. Soc. 2008, 130,
2732–2733.
Compound 2c, a pale yellow solid, was obtained from 7c in 90%
yield, following the same procedure described for 2a (TLC, CC:
CHCl3/CH3OH/H2O = 64:34:2, crystallization from t-BuOMe, Calcd
for C40H68N2O6: C, 71.39; H, 10.12; N, 4.17. Found: C, 71.10; H,
10.40; N, 3.88). 1H NMR (CD3OD): 6.791 (4H, s, Ar); 3.90–3.65
(8H, m, –CH2OAr,
a, d); 3.27–2.94 (6H, m, –CH2N, d); 2.47–2.31
(2H, m, b); 2.17–1.82 (6H, m, b,
c
); 1.78–1.58 (8H, m, –CH2CH2OAr,
–CH2CH2N); 1.50–1.21 (32H, m, –(CH2)8–). 13C NMR (CD3OD):
173.72; 154.63; 116.45; 70.65; 69.61; 56.70; 55.95; 30.70; 30.65;
30.62; 30.53; 30.29; 30.25; 27.66; 27.18; 27.01; 24.36.
3.5.10. 12-Phenoxydodecan-1-ol 6d
Compound 6d, a white solid, was obtained from phenol 5d in
92% yield, following the same procedure described for 6a. 1H
NMR (CDCl3): 7.35–7.20 (2H, m, Ar), 6.95–6.80 (3H, m, Ar), 3.943
(2H, t, ArOCH2, J = 6.6 Hz), 3.630 (2H, t, CH2OH, J = 6.6 Hz), 1.90–
1.68 (2H, m, ArOCH2CH2), 1.62–1.20 (18H, m, –(CH2)9). 13C NMR
(CDCl3): 159.11, 129.38, 120.44, 114.48, 67.86, 63.05, 32.80,
29.56, 29.43, 29.40, 29.29, 26.06, 25.74.
3.5.11. 12-Phenoxydodecanal 7d
4. Navon, G.; Frank, S.; Kaplan, D. J. Chem. Soc., Perkin Trans. 2 1984, 1145–
1149.
5. (a) Aguiar, J.; Carpena, P.; Molina-Bolívar, J. A.; Carnero Ruiz, C. J. Colloid
Interface Sci. 2003, 258, 116–122; (b) Kalyanasundaram, K.; Thomas, J. K. J. Am.
Chem. Soc. 1977, 9, 2039–2044; (c) Ananthapadmanabhan, K. P.; Goddard, E. D.;
Turro, N. J.; Kuo, P. L. Langmuir 1985, 1, 352–355.
6. Ceccacci, F.; Mancini, G.; Mencarelli, P.; Villani, C. Tetrahedron: Asymmetry
2003, 14, 3117–3122.
7. McDonagh, A. F.. In The Porphyrin; Dolphin, D., Ed.; Academic Press: New York,
1979; Vol. 6, pp 293–491. Chapter 5.
Compound 7d, a white solid, was obtained from phenol 6d in
87% yield, following the same procedure described for 7a. 1H
NMR (CDCl3): 9.753 (1H, t, CHO, J = 1.8 Hz), 7.34–7.15 (2H, m,
Ar), 7.05–6.80 (3H, m, Ar), 3.939 (2H, t, ArOCH2, J = 6.6 Hz), 2.410
(2H, dt, CH2OH, J = 1.8 Hz, J = 7.3 Hz), 1.84–1.70 (2H, m, Ar-
OCH2CH2), 1.70–1.50 (2H, m, CH2CH2CHO), 150–1.20 (14H, m, –
(CH2)7). 13C NMR (CDCl3): 203.00, 159.12, 129.40, 120.44, 114.48,
67.84, 43.92, 29.53, 29.50, 29.40, 29.35, 29.29, 29.16, 26.06, 22.09.
8. (a) Lightner, D. A.; Gawromski, J. K.; Gawromska, K. J. Am. Chem. Soc. 1985, 107,
2456–2461; (b) Lightner, D. A.; Gawromski, J. K.; Wijekoon, W. M. D. J. Am.
Chem. Soc. 1987, 109, 6354–6362.
3.5.12. (2S)-1-(12-Phenoxydodecyl)-pyrrolidine-2-carboxylic
acid 4
9. Tobler, E.; Lammerhofer, M.; Lindner, W.; Mancini, G. Chirality 2001, 13, 641–
647.
10. Koppel, D. E. J. Chem. Phys. 1972, 57, 4814–4820.
11. Provencher, S.; Hendrix, J.; De Maeyer, L.; Paulussen, N. J. Chem. Phys. 1978, 69,
4273–4276.
12. Berne, B. J.; Pecora, R. Dynamic Light Scattering with Applications to Chemistry,
Biology and Physics; Wiley: New York, 1976.
13. Hébrant, M.; Burgoss, P.; Assfeld, X.; Joly, J.-P. J. Chem. Soc., Perkin Trans. 2 2001,
998–1004.
14. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155–4156.
Compound 4, a pale yellow solid, was obtained from 7d in 90%
yield, following the same procedure described for 2a (TLC, CC:
CHCl3/CH3OH/H2O = 64:34:2; crystallization from MeOH/t-BuOMe.
Calcd for C23H37NO3: C, 73.56; H, 9.93; N, 3.73. Found: C, 73.45; H,
10.01; N, 3.65). 1H NMR (CDCl3): 7.28–7.16 (2H, m, Ar), 6.92–6.78
(3H, m, Ar), 4.04–3.80 (3H, om, ArOCH2, d), 3.72–3.60 (1H, m,
a),