Molecular recognition of zwitterions: Enhanced binding and selective recognition of miltefosine by a squaramide-based host

Article abstract of DOI:10.1002/asia.201200881

The importance of a synergic combination between electrostatic and hydrogen-bonding interactions has been demonstrated. A self-complementary amidosquarate-squaramide host for miltefosine has been disclosed. In DMSO, this rather structurally simple host exhibits high affinity and enhanced selectivity for miltefosine owing to the goal-keeping effect of the amidosquarate unit. Copyright

Full text of DOI:10.1002/asia.201200881

COMMUNICATION
DOI: 10.1002/asia.201200881
Molecular Recognition of Zwitterions: Enhanced Binding and Selective
Recognition of Miltefosine by a Squaramide-Based Host
Carlos Lꢀpez, Elena Sanna, Lucas Carreras, Manel Vega, Carmen Rotger, and
Antoni Costa*[a]
It is widely accepted that recognition of zwitterionic mole-
cules is challenging. Systems qualified for this function are
of interest as molecular sensors[1] and in fundamental re-
search at the biological level. In this respect, control over re-
organization and recognition events that take place on the
cell membrane is an area in which zwitterionic receptors can
play an important role.[2] However, compared to the large
number of receptors for cations or anions, receptors for
zwitterions are scarce because of the simultaneous presence
of highly solvated negative and positive charged functional
groups on the same molecule.[3] In addition, these receptors
tend to aggregate to some extent because of self-comple-
mentarity. Hence, new receptors with high affinity and selec-
tivity for zwitterions need to be designed and synthesized.
Such receptors could be useful as simplified models for in-
vestigating the mechanisms of the interactions between
small-molecule ligands and zwitterionic molecular compo-
nents of cells such as phospholipids and peptides.
cluding halides[7] and oxoanions.[8] We have previously re-
ported that conformational transitions promoted by recogni-
tion events allow squaramide-based hosts to adapt their geo-
metries, thereby satisfying the requirements for efficient
ion-pair recognition.[9] All these features are incorporated
here to recognize MI, a zwitterionic compound of biological
interest (Scheme 1).
In the supramolecular area, rigid preorganized hosts have
been favored candidates. However, rigidity and preorganiza-
tion are not mandatory. In fact, an enhanced host–guest
binding affinity with conformationally mobile hosts has
been recently demonstrated, owing to the cooperative syner-
gism existing between the guest binding and conformational
changes of the host.[4] Inspired, in part, by biological ideas
and continuing with our ongoing interest in selective recog-
nition from flexible hosts, herein we report a squaramide-
based host that features high-affinity and selectivity in the
presence of a biologically relevant guest such as miltefosine
(hexadecylphosphocholine) (MI), a membrane-active syn-
thetic ether lipid analogue that is very effective for the treat-
ment of human visceral leishmaniasis.[5]
Scheme 1. Synthesis of the amidosquarate receptor 4.
Here, we use the dynamic dimerization and conformation-
al reorganization of a flexible squaramide host that is syner-
gically combined with cooperative hydrogen-bonding inter-
actions for the improved recognition of MI. In our ap-
proach, we introduce for the first time a charged amidosqua-
rate unit as the binding site for the cationic part of the zwit-
terion and a neutral squaramide unit, located at a precise
distance, which acts as the anion binding site. This combina-
tion of the two different squaramide units shows an effective
way to enhance the affinity and selectivity for a zwitterionic
guest using a relatively simple host.[10]
Squaramides are rigid amide-type compounds that have
a dual binding mode capable of recognizing quaternary am-
monium cations[6] or, alternatively, a variety of anions in-
The amidosquaric acid precursor 1 was prepared by direct
condensation of 4-aminobenzylamine with squaric acid in
water. The success of this highly chemoselective transforma-
tion arises from the in situ protonation of the benzylic
amine group. Host 4, the sodium salt, was prepared first by
sequential condensation of diethyl squarate with p-nitroani-
line,[12] and then with amidosquaric acid 2. The occurrence
of the charged amidosquarate site enables 4 to spontaneous-
ly self-assemble with concomitant formation of hydrogen
[a] C. Lꢀpez, E. Sanna, L. Carreras, M. Vega, Prof. C. Rotger,
Prof. A. Costa
Departament de Quꢁmica
Universitat de les Illes Balears
Ctra. Valldemossa, km 7.5, 07122 Palma de Mallorca, Illes Balears
(Spain)
Fax : (+34)971173426
E-mail: antoni.costa@uib.es
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201200881.
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Antoni Costa et al.
bonded head-to-tail pairs or even oligomeric aggregates
CArH···O hydrogen bonding. At the same time, protons Hg
and Hh experienced small, but significant, upfield shifts; this
suggests a slight magnetically-shielded environment. On the
other hand, the sharp resonances of the amidosquarate NHc
proton as well as those of Hd and Hg remained essentially
unchanged as expected for non-interacting hydrogen atoms.
Data from 1H NMR titrations were analyzed by using
a 1:1 (4·MI) and 2:1 (42·MI) binding model, including the
self-association constants already calculated: 42 (280mÀ1)
and 43 (90mÀ2) to evaluate the stepwise association constants
K11 =3.6(1)ꢃ103 mÀ1 and K21 =2.7(6)ꢃ103 mÀ2 for 4·MI and
42·MI complexes, respectively. All other attempts with differ-
ent binding models failed to attain convergence.
(Figure 1). For this reason, the aggregation state of 4 was in-
1
itially investigated by H NMR spectroscopy. With increas-
ing concentration of 4 in [D6]DMSO, the three NH protons
The complexation of 4 with MI at millimolar concentra-
tions shows the dominant formation of 1:1 complexes (Fig-
ure 2v), although at low concentration of miltefosine (<5ꢃ
10À4 m), 2:1 complexes as well as dimers of 4 are observed.
In accordance, rotational frame nuclear Overhauser effect
Figure 1. Concentration-dependence of the 1H NMR chemical shifts of
the significant protons of
4 in [D6]DMSO at ambient temperature
(298 K) for a range between 3.0ꢃ10À4 and 1.5ꢃ10À2 m. The symbols are
experimental data points. Best fit curves were calculated by simultaneous
nonlinear regression of experimental data with the HypNMR program.[12]
shifted downfield and several aromatic resonances experi-
enced small but significant upfield (Hg) or downfield (He,
Hf) shifts, thus indicating the formation of an aggregated
species. Global analysis of chemical shift data with the
HypNMR program[12] yielded dimerization and trimerization
constants Kdim =285(2)mÀ1 and Ktrim =90(2)mÀ2 that corre-
spond to 42 and 43, respectively. In spite of repulsion, owing
to a similar negative charge, the observed chemical shifts
and the calculated constants indicate that a dynamic equilib-
rium between monomeric, dimeric and, in a smaller propor-
tion, trimeric species (<8% at 10À3 m) exists.[13] In accord-
ance with this result, molecular modeling studies (B3LYP/6-
31G*) on 42 suggest an antiparallel packing motif with
C2 symmetry, where the ArHe proton participates in hydro-
gen-bonding interactions to help stabilize the dimer
(Figure 1).[14]
Molecular modeling also indicated that the squaramide-
amidosquarate host 4 was well suited for simultaneously
binding to the tetralkylammonium and phosphate groups of
MI with the amidosquarate and squaramide units of 4, re-
spectively. Thus, the mode of binding of amidosquarate 4 to
1
MI was investigated by H NMR spectroscopy. Titration of
Figure 2. i) Changes in the 1H NMR spectra of
4
(7.38ꢃ10À4 m) in
MI with a solution of 4 in [D6]DMSO, produced large paral-
lel downfield shifts of the NHa and NHb protons (>
1.0 ppm), thereby showing that the phosphate group of MI
is interacting through a hydrogen bond with the two NHs of
the squaramide unit of 4. The aromatic proton Hc was also
shifted downfield, thus indicating its participation in
[D6]DMSO at 298 K, in the presence of 6 equivalents of miltefosine. ii–
iv) Plots of complexation-induced changes of selected protons of 4 (7.38ꢃ
10À4 m) as function of increasing amounts of MI. Symbols are experimen-
tal data points. Solid lines are best fit curves obtained by simultaneous
nonlinear regression of five resonances, NHa, NHb, ArHe, ArHg, and
CHh. v) Simulated distribution profiles of the five participating species:
4·MI, 42·MI, 4, 42, and 43 (<0.6%, not visible at this scale).
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Antoni Costa et al.
spectroscopy (ROESY) experiments revealed intermolecu-
lar contacts between the NH and aromatic protons of 4 as
well as several key hydrogen atoms of MI, especially those
close to the phosphodiester moiety. The 1:1 complex is also
clearly seen in negative-ion mode MALDI-TOF mass spec-
trometry: a signal at m/z=840.393 is assigned to [4·MI]À.
Furthermore, diffusion-ordered spectroscopy (DOSY) ex-
periments also provide evidence of the formation of a multi-
component system (see the Supporting Information).
Figure 3. Change in the 1H NMR spectra of
[D6]DMSO at 298 K in the presence of 8 equivalents of MI.
4
(1.03ꢃ10À3 m) in
The affinity of the host for MI cannot be improved by in-
creasing the number of amidosquarate units from one to
two within the same host. Titrations of model compounds 5
and 6 with MI and analysis of the corresponding complexa-
tion-induced shifts (CIS) gave values of K11 =5.8(1)ꢃ102 mÀ1
and K11 =6.6(1)ꢃ102 mÀ1 for 5·MI and 6·MI, respectively.
Both values are roughly five times lower than that obtained
for 4·MI. The magnitude of the association constant for
5·MI, lacking the amidosquarate unit, is in the range of
those previously reported.[11] However, that of 6·MI is lower
than expected owing to mutual repulsion between the two
amidosquarate units. Therefore, host 4 features an optimal
balance of interacting groups and a favorable conformation
to attain strong binding of MI. The self-complementary
character of 4, also helps to enhance the selectivity in the
presence of potentially competitive charged guests. For ex-
ample, titration of 4 with tetramethylammonium bromide or
iodide or with sodium bis(p-nitrophenyl)phosphate did not
result in any CIS shifts. However, the strongly interacting
tetraethylammonium chloride (TEACl) yielded K11 =
Although host 4 is selective in the presence of ion pairs
and non-matching zwitterions, there are still several limita-
tions. The more significant of which refers to the absence of
structural elements to differentiate between zwitterions that
have the same charged groups but different alkyl ester resi-
dues on the phosphate. To illustrate this point, phosphatidyl-
choline (PC) that has a zwitterionic part identical to that of
MI is an ideal substrate. Titration of 4 with PC, under the
same experimental conditions described above for MI, af-
forded a K11 =3.6(1)ꢃ103 mÀ1 for 4·PC. This is the same
value found for 4·MI, and reflects the limits in the selectivity
of 4.
In summary, a high affinity and selective host incorporat-
ing two different squaramide units for recognition of milte-
fosine has been synthesized. Compared to existing hosts for
zwitterions such as those combining calixarenes or crown
ethers for recognition of the cationic portion, here we intro-
duce an unprecedented amidosquarate unit, which, in con-
junction with a squaramide for binding the anionic portion,
results in a straightforward and selective host for MI and re-
lated lipids in dimethyl sulfoxide (DMSO). The results indi-
cate that host 4 could be useful for investigating the interac-
tions of phospholipids with biologically relevant ligands.
1.3(1)ꢃ102 mÀ1,
5·
(TEACl), K11 =2.8(1)ꢃ102 mÀ1.
a lower value than that measured for
ACHTUNGTRENNUNG
Experimental Section
Experimental procedures including synthesis, spectral characterization, ti-
tration data, and NMR spectroscopy experiments can be found in the
Supporting Information.
Thus, the role of the amidosquarate unit is not only to en-
Acknowledgements
hance binding, but also to be the goalkeeper of 4 to avoid
or at least to minimize the binding of potentially competi-
tive guests. To illustrate this point, we investigated the bind-
ing of 2-(triethylammonio)ethane sulfonate (TEAES), a sul-
fonated zwitterion, with 4. Here, the extended intercharge
distance of TEAES (4.3 ꢄ)[15] is shorter than that of miltefo-
sine (5.0 ꢄ), and the lower basicity of the sulfonate anion
compared to the phosphate results in a completely different
mode of interaction with 4 (Figure 3).
We thank CONSOLIDER-Ingenio 2010 (project CSD2010-0065), the
MICINN of Spain (project CTQ2011-27512/BQU, FEDER funds) and
the “Direcciꢀ General de Recerca, Desenvolupament Tecnolꢅgic i Inno-
vaciꢀ del Govern Balear” (CAIB; project 23/2011, FEDER funds) for fi-
nancial support. E.S. thanks the CAIB for a predoctoral fellowship.
Keywords: host–guest systems
· ion pairs · molecular
recognition · squaramide · zwitterions
Upon addition of a large excess of TEAES to 4, only the
NHc and Hg protons shifted slightly, while the others re-
mained essentially unchanged. This experiment demon-
strates that TEAES does not interact with the squaramide
core but only marginally with the amidosquarate moiety.
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Received: September 21, 2012
Published online: November 5, 2012
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