Calix[4]arene-cholic acid conjugates: A new class of efficient synthetic ionophores

Article abstract of DOI:10.1039/b415908j

The synthesis of a new class of amphiphilic calix[4]arene-based ionophores, relying on direct reductive amination as a key step, and the evaluation of their H⁺and Na⁺transporting properties is described. The Royal Society of Chemistr

Full text of DOI:10.1039/b415908j

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Calix[4]arene-cholic acid conjugates: a new class of efficient synthetic
ionophores{
Nakia Maulucci,a Francesco De Riccardis,*a Cinzia Barbara Botta,a Agostino Casapullo,b Elena Cressina,c
Massimo Fregonese,c Paolo Tecilla*c and Irene Izzo*a
Received (in Cambridge, UK) 14th October 2004, Accepted 3rd December 2004
First published as an Advance Article on the web 21st January 2005
DOI: 10.1039/b415908j
according to Scheme 1 in five steps and 37% overall yield.{
The tetraaldehyde 9 (Fig. 2) was synthesized through Cs2CO3
tetraalkylation (known to induce 1,3-alternate calix[4]arene
conformation) from commercially available calix[4]arene and
2-methoxyethyl p-toluensulfonate.7 High yield tetraformylation8
furnished the expected, stable, tetraaldehyde (61% overall yield).
Direct reductive amination of 9, in the presence of a slight excess
(0.5 eq. per functional group) of amine 8 and the mild reducing
agent sodium triacetoxyborohydride (NaBH(OAc)3)9 afforded in
good yield (78%) the tetraaminated 1. To evaluate the effect of the
steroid substituents in the H+ and Na+ transporting essays, two
related compounds (2 and 3) were also prepared. K2CO3-mediated
C-3 selective hydrolysis gave the C-12 tetracetylated 2 (92%).
Full deprotection was obtained with methanolic KOH and gave 3
(89%).
The synthesis of a new class of amphiphilic calix[4]arene-based
ionophores, relying on direct reductive amination as a key step,
and the evaluation of their H+ and Na+ transporting properties
is described.
The control of membrane permeability is of paramount impor-
tance for all living organisms1 and, since the publication of the
first successful model, reported by Tabushi and co-workers in
1982,2 synthetic chemists have designed and constructed a variety
of chemically diverse and effective low molecular weight ion
conductors.
A recent survey from Matile3 shows that among non-peptide
systems, the rigid steroid scaffold is gaining more attention:
monomeric and dimeric steroids self-assemble into barrel-like
supermolecules and exert powerful ionophoric activities. However,
in this context, only one example is known where a sterol-based
ionophore forms a membrane spanning, tunnel-like, unimolecular
channel.4
The synthesis of the calix[4]arene cone analogues 4 (63% yield)
and 5 (98%) was realized in a similar manner starting from the
tetraalkylated aldehyde 10 (Fig. 2). This was obtained through
Unimolecular ion channels are considered a particularly
attractive model to study, in that most of the biochemical
processes that characterize life (signal transduction, muscle
contraction, proton-coupled ATP-synthesis, and so on), depend
on it. In cells they can be found in high molecular weight
transmembrane proteins (‘‘pore helix’’).1
With the idea to synthesize new steroid-based transmembrane
unimolecular ionophores, we designed a new class of compounds
incorporating the versatile calix[4]arene platform, an ideal
molecular scaffold offering easy protocols of derivatization,
different preorganized structures and showing ion affinity.5
In this paper, we describe the synthesis and the H+ and Na+
transporting activity of five conformationally restricted calix[4]-
arene-3a,12a-dihydroxy-24-amino-cholane conjugates synthesized
in both the 1,3-alternate and the cone conformations in order to
understand the influence of the overall length of compounds on
the ion transporting properties. Preliminary molecular mechanics
Fig. 1 Calix[4]arene-cholic acid conjugates 1–5.
˚
studies indicated a 35.0 ¡ 2.0 A length for derivatives 1–3, and a
˚
25.0 ¡ 2.0 A length for derivatives 4–5, in their fully extended
conformation (Fig. 1).6
The synthetic strategy chosen to assemble the four amine units
with the 1,3 alternate calix[4]arene scaffold, relied on the reductive
amination reaction. To this end the steroid amine 8 was prepared
{ Electronic supplementary information (ESI) available: full synthetic and
analytical details, and results of permeability studies on liposomes. See
http://www.rsc.org/suppdata/cc/b4/b415908j/
*iizzo@unisa.it (Irene Izzo)
tecilla@dsch.univ.trieste.it (Paolo Tecilla)
Scheme 1 Reagents and conditions: a. Ac2O, Py, DMAP, CH2Cl2; b.
BH3?THF, THF, 0 uC; c. TsCl, Py, CH2Cl2; d. NaN3, DMF, 60 uC; e. H2,
PtO2, EtOH (37%, 5 steps).
1354 | Chem. Commun., 2005, 1354–1356
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Fig. 3 Plot of kobs for the transport of H+ (left panel) and Na+ (right
panel) as a function of mol% of 2 ($) and 5 (#).
Fig. 2 Calix[4]arene scaffolds.
NaH mediated alkylation10 of calix[4]arene and subsequent
formylation.8
are very small indicating that in these systems there is not a clear
preference toward free or acetylated hydroxyl moieties.
The ionophoric properties of compounds 1–5 were investigated
in large unilamellar vesicles (100 nm diameter) with a 95 : 5 egg
phosphatidylcholine (PC) and egg phosphatidylglycerol (PG) lipid
composition. Ultrafiltration experiments and UV-Vis analysis of
the water solution, free of vesicles, showed that more than 95% of
the calixarene derivatives were bound to the aggregate. The ability
of the ionophores to mediate a pH or Na+ gradient collapse was
measured using two different essays based, respectively, on the
response of the intravesicular pH-sensitive pyranine fluorophore,11
and on a 23Na+ NMR experiment.12 Kinetic experiments were run
at 25 uC and in both processes we found first order kinetic profiles.
The fitting of the data13 allows one to obtain the apparent first
order rate constants (kobs, min21) for the transport of H+ or Na+
across the membrane. The data obtained at a fixed 1% molar ratio
of calixarene derivatives with respect to the total concentration of
lipid are reported in Table 1.
Fig. 3 reports the dependence of kobs versus the concentration of
2 and, for comparison, of 5. Increasing the concentration of
ionophore the rate of transport of H+ and Na+ increases linearly
and the same linear dependence is observed with the other
calixarene derivatives. This linear dependence of transport rate on
ionophore concentration, taken together with the higher activity of
the 1,3-alternate derivatives characterized by a length that roughly
matches the thickness of the hydrophobic core of the membrane,
is in full accord with a unimolecolar transmembrane active
structure.3 On the contrary, the cone derivatives are too short to
span the membrane and the formation of an active structure
probably requires the assembly of more than one molecule.15 This
process is clearly less favoured with respect to a unimolecular pore
formation and, therefore, the cone derivatives are less active than
the 1,3 alternate isomers.
In conclusion the first example of artificial ionophores
presenting calix[4]arene-cholic acid substructures is reported.16
These compounds seem to act through a unimolecolar mechanism;
this result, together with the different activity of compounds 2 and
5, suggest that the overall length plays a critical role in the ion
transporting properties. Studies to better evaluate the mechanism
of action, the ion selectivity, and towards the synthesis of more
efficient ionophores, using different steroidal scaffolds, are in
progress.
Inspection of Table 1 shows that all the calixarene derivatives
are effective in promoting the transport of both ions although to a
different extent. In particular, compounds 1–3, characterized by
the 1,3-alternate conformation, are clearly more active than
compounds 4, 5 which display the cone conformation. As a
matter of fact, the activity of compounds 1–3 are comparable in
the case of Na+ or higher in the case of H+ than that of
amphotericin B, a natural occurring ionophore which is often
taken as a reference compound.14 The tetracetylated derivative 2 is
the most active both in the transport of H+ and Na+ while an
inverse selectivity is observed between 1 and 3 with the first more
active than the second in the transport of H+ and slightly less
active in that of the Na+ ion. However, these activity differences
Financial support for this research has been provided by the
Ministry of Instruction, University and Research (MIUR contract
2002031238 and MIUR contract 2003039581_004) and by the
Universita` degli Studi di Salerno. The authors are indebted to Prof.
Placido Neri and Dr Marco Martino for helpful discussion.
Nakia Maulucci,a Francesco De Riccardis,*a Cinzia Barbara Botta,a
Agostino Casapullo,b Elena Cressina,c Massimo Fregonese,c
Paolo Tecilla*c and Irene Izzo*a
Table 1 Observed rate constants (min21) for the entry of H+ (kH) and
Na+ (kNa) into 95 : 5 egg PC/PG vesicles in the presence of the different
ionophores (1% respect to the total concentration of lipids)a
aUniversity of Salerno, Department of Chemistry, Via S. Allende,
I-84081 Baronissi (SA), Italy. E-mail: iizzo@unisa.it;
Fax: +39 089 965296; Tel: +39 089965230
Ionophore
kH
kNa
bUniversity of Salerno, Department of Pharmaceutical Science, Via
Ponte Don Melillo, I-84084 Fisciano (SA), Italy
None
1
2
3
4
0.043
0.90
1.06
0.47
0.14
0.13
0.14
3.3 6 1025b
1.21 6 1023
1.96 6 1023
1.92 6 1023
6.01 6 1024
6.53 6 1024
2.67 6 1023c
cUniversity of Trieste, Department of Chemical Sciences, Via L.
Giorgieri, I-34127 Trieste, Italy. E-mail: tecilla@dsch.univ.trieste.it;
Fax: +39 040 5583903; Tel: +39 040 5583925
5
Amphotericin B
Notes and references
a
The total concentration of lipid was 0.17 mM and 10 mM in the
{ Compounds 1–10 have been fully characterized (1H and 13C NMR,
HRESI-MS) for details see ESI.{ Selected data and procedure for 1: Direct
reductive amination. General procedure: To a solution of amine 8 (0.332 g,
H+ and Na+ experiments, respectively. T = 25 uC. Estimated from
initial rate. Ref. 13.
b
c
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0.720 mmol) and tetraaldehyde 9 (0.091 g, 0.120 mmol) in 1,2-
dichloroethane (2.5 ml) NaBH(OAc)3 (0.152 g, 0.720 mmol) and AcOH
(30 ml, 0.480 mmol) were added. The reaction mixture was stirred at r.t.
overnight, quenched with 1 N NaOH solution and extracted with
methylene chloride. The combined organic phases were washed with brine,
dried (MgSO4) and concentrated in vacuo. The crude residue was flash-
chromatographed (5–25% of methanol in dichloromethane to give 1
(0.240 g, 78%) as an amorphous solid. 1: Rf: 0.38 (20% methanol in
dichloromethane); [a]D: 70 (c = 1.0, CHCl3); 1H NMR (400 MHz, TCDE,
110 uC) d 0.60 (3 H, s, CH3-18), 0.72 (3 H, bd, CH3-21), 0.78 (3 H, s, CH3-
19), 1.84 (3 H, s, COCH3), 2.72 (2 H, CH2CH2NH), 3.30 (3 H, s, OCH3),
3.55–3.97 (8 H, m, CH2OCH3, ArOCH2, ArCH2Ar, HNCH2Ar, 4.53 (1 H,
m, H-3), 4.91 (1 H, bs, H-12), 7.13 (2 H, s, ArH); 13C NMR (400 MHz,
TCDE, 110 uC) d 10.7, 16.2, 19.2, 19.3, 20.9, 21.1, 21.7, 23.9, 24.2, 24.9,
25.2, 25.6, 30.7, 31.5, 32.3, 32.9, 33.1, 33.2, 33.7, 34.2, 40.3, 43.6, 45.4, 46.5,
47.8, 48.0, 56.6, 69.6, 70.5,72.4, 74.1, 121.4, 130.4 (62), 132.1 (62), 155.8,
167.9, 168.2. HRESI-MS (m/z): calcd. for C156H236N4O24 [M + 2H]2+
1275.8763; found 1275.8676.
6 The thickness of a phosphatidylcholine/phosphatidylglicerol membrane
˚
bilayer model is around 30–35 A.
7 W. Verboom, S. Datta, Z. Asfari, S. Harkema and D. N. Reinhaudt,
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10440–10441 and references cited therein.
13 The pseudo-first order rate constants were obtained by non-linear
regression analysis of the fluorescence or the Na+ NMR peak areas vs.
time data and the fit error on the rate constant was always less than 1%.
14 F. De Riccardis, M. Di Filippo, D. Garrisi, I. Izzo, F. Mancin,
L. Pasquato, P. Scrimin and P. Tecilla, Chem. Commun., 2002,
3066–3067.
15 The formation of pores by the assembly of hydrophobic calix[4]arene
amide has been reported: V. Sidorov, F. W. Kotch, G. Abdrakhmanova,
R. Mizani, J. C. Fettinger and J. T. Davis, J. Am. Chem. Soc., 2002, 124,
2267–2278.
16 A ionophore based on a resorcin[4]arene-cholic acid ether assembly has
been reported. In this case, however, only the cone-like rccc isomer was
prepared. N. Yoshino, A. Satake and Y. Kobuke, Angew. Chem. Int.
Ed., 2001, 40, 457–459.
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4601–4604.
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1356 | Chem. Commun., 2005, 1354–1356
This journal is ß The Royal Society of Chemistry 2005