Tuning chloride binding, encapsulation, and transport by peripheral substitution of pseudopeptidic tripodal small cages

Article abstract of DOI:10.1002/chem.201202182

A highly efficient synthesis of small pseudopeptidic cages from simple precursors has been achieved by the triple S_N2 reaction between tripodal tris(amido amines) and several 1,3,5-tris(bromomethyl)benzene electrophiles. The success of the macrobicyclization strongly depends on the central triamine scaffold, which dictates the correct preorganization of the intermediates. The chloride binding properties of the protonated pseudopeptidic cages have been studied in the solid state (by X-ray diffraction) as well as in solution (by NMR spectroscopy and ESI-MS) and in the gas phase (by collision-induced dissociation (CID)-MS). The crystal structure of the HCl salts of several cages show a chloride partially or completely caged within the cavity of the macrobicycle. Both the amino acid side chain and the substitution at the aromatic tripodal ring have an effect on the chloride binding ability. The cages derived from the 1,3,5-benzene moiety show low affinity, whereas the triple substitution in the ring (either with Me or Et) increases the chloride binding by one order of magnitude. Besides, the cages derived from aliphatic amino acids display a stronger interaction than those derived from phenylalanine. The basis for the different mode of binding depending on the receptor structure is proposed according to the structural data (X-ray and NMR spectroscopy). Finally, the transport of the chloride anion through lipid bilayers has been studied for selected cages. Despite the important differences in the chloride binding, the transport properties are better correlated with the lipophilicity of the molecules. Therefore, the pseudopeptidic cages sharing the same binding motif for chloride rendered very different interaction and transport properties depending on the peripheral substitution. Caging of chloride: A family of pseudopeptidic molecular cages has been synthesized. They bind chloride in the solid state (X-ray), in solution (ESI-MS and NMR spectroscopy), and in the gas phase (collision-induced dissociation (CID)-MS). Moreover, some of them are able to transport chloride ions through lipid bilayers. The chloride binding, encapsulation, and transport abilities of the cages strongly depend on the peripheral substitution, such as the residues on the tripodal aromatic cap or the amino acid side chain (see figure). Copyright

Full text of DOI:10.1002/chem.201202182

DOI: 10.1002/chem.201202182
Tuning Chloride Binding, Encapsulation, and Transport by Peripheral
Substitution of Pseudopeptidic Tripodal Small Cages
Inꢀs Martꢁ,[a] Jenifer Rubio,[a] Michael Bolte,[b] M. Isabel Burguete,[a] Cristian Vicent,[c]
Roberto Quesada,[d] Ignacio Alfonso,*[e] and Santiago V. Luis*[a]
Abstract: A highly efficient synthesis
of small pseudopeptidic cages from
simple precursors has been achieved by
the triple SN2 reaction between tripo-
dal tris(amido amines) and several
1,3,5-tris(bromomethyl)benzene elec-
trophiles. The success of the macrobi-
cyclization strongly depends on the
central triamine scaffold, which dic-
tates the correct preorganization of the
intermediates. The chloride binding
properties of the protonated pseudo-
peptidic cages have been studied in the
solid state (by X-ray diffraction) as
well as in solution (by NMR spectro-
scopy and ESI-MS) and in the gas
phase (by collision-induced dissociation
(CID)-MS). The crystal structure of
the HCl salts of several cages show a
chloride partially or completely caged
within the cavity of the macrobicycle.
Both the amino acid side chain and the
substitution at the aromatic tripodal
ring have an effect on the chloride
binding ability. The cages derived from
the 1,3,5-benzene moiety show low af-
finity, whereas the triple substitution in
the ring (either with Me or Et) increas-
es the chloride binding by one order of
magnitude. Besides, the cages derived
from aliphatic amino acids display a
stronger interaction than those derived
from phenylalanine. The basis for the
different mode of binding depending
on the receptor structure is proposed
according to the structural data (X-ray
and NMR spectroscopy). Finally, the
transport of the chloride anion through
lipid bilayers has been studied for se-
lected cages. Despite the important dif-
ferences in the chloride binding, the
transport properties are better correlat-
ed with the lipophilicity of the mole-
cules. Therefore, the pseudopeptidic
cages sharing the same binding motif
for chloride rendered very different in-
teraction and transport properties de-
pending on the peripheral substitution.
Keywords: cage compounds · chlor-
ide binding · pseudopeptides · su-
pramolecular chemistry · transport
Introduction
[a] I. Martꢀ, Dr. J. Rubio, Prof. Dr. M. I. Burguete, Prof. Dr. S. V. Luis
Departamento de Quꢀmica Inorgꢁnica y Orgꢁnica
Universitat Jaume I
The study of macrocycles and cage-like structures derived
from amino acids (peptides and pseudopeptides) is an
emerging research topic, closely connected with synthetic
chemistry,[1] natural products,[2] material science,[3] and bio-
logical chemistry.[4] The geometrical constraints produced by
the cyclic frame are extremely useful to implement a rigid
conformation within a small peptidic sequence.[5] This has al-
lowed the chemists to design structural scaffolds with inter-
esting biological activities.[6] Moreover, this type of com-
pounds has been used as receptors for some species of inter-
est, either ions (cations[7] and anions[8]) or small neutral mol-
ecules.[9] Among these structures, those defining a three-di-
mensional inner space are especially attractive for
recognition purposes, because they usually lead to stronger
interaction and higher selectivity towards a given comple-
mentary guest. Thus, the study of three-dimensional cavities
as molecular containers is a hot topic nowadays.[10] In this
context, macrobicyclic pseudopeptides defining a cage-like
architecture[11] have become very appealing as synthetic tar-
gets for molecular recognition purposes[12] and biomimetic
studies.[13] However, their preparation is very often challeng-
ing, requiring multi-step synthetic pathways with low yields
and tedious purification steps. In this regard, the key step is
usually the reaction leading to the macrobicyclic structure,
Avda. Sos Baynat, s/n, 12071 Castellꢂn (Spain)
Fax : (+34)964728214
E-mail: luiss@uji.es
[b] Dr. M. Bolte
Institut fꢃr Anorganische Chemie
J.-W.-Goethe-Universitꢄt
Max-von-Laue-Str.7
60438 Frankfurt/Main (Germany)
[c] Dr. C. Vicent
Serveis Centrals d’Instrumentaciꢂ Cientꢀfica
Universitat Jaume I
Avda. Sos Baynat, s/n
12071 Castellꢂn (Spain)
[d] Dr. R. Quesada
Departamento de Quꢀmica, Facultad de Ciencias
Universidad de Burgos
09001 Burgos (Spain)
[e] Dr. I. Alfonso
Departamento de Quꢀmica Biolꢂgica y Modelizaciꢂn Molecular
Instituto de Quꢀmica Avanzada de CataluÇa (IQAC-CSIC)
Jordi Girona, 18-26
08034 Barcelona (Spain)
Fax : (+34)932045904
E-mail: ignacio.alfonso@iqac.csic.es
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201202182.
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FULL PAPER
which very often leads to an untreatable mixture of cyclic
and open-chain oligomers.[14] Some interesting approaches
have been developed to improve the efficiency of the cycli-
zation processes, mainly based on the implementation of a
defined preorganization, which would stabilize the correct
pre-cyclic conformation in the transition state of the reac-
tion.[15] Then, a faster and a more selective formation of the
intended cyclic structure can be achieved. This preorganiza-
tion can be obtained by using geometrically rigid building
blocks[16] or after tuning the protecting groups[17] or the sol-
vent,[18] by using some additional non-covalent interac-
tions[19] or by taking advantage of designed templates, either
kinetically or thermodynamically.[20]
On the other hand, chloride is the most abundant anion
in biological systems; therefore, it is an interesting target for
synthetic receptors. Despite the structural simplicity of
chloride anion, its physicochemical properties make its se-
lective and efficient molecular recognition very challenging,
especially in highly competitive media, like aqueous solu-
tion.[21] The control of chloride transport and maintenance
of appropriate chloride concentrations is fundamental for
the function of living organisms. Defective or altered func-
tioning of the natural transport mechanism is associated
with serious diseases termed channelopathies, such as cystic
fibrosis.[22] Recently, the development of synthetic small
molecules designed for the transport of chloride through
lipid bilayers has received an increasing attention.[23] These
compounds can facilitate chloride transport in living cells
and display biological activity.[24] A number of these mole-
cules have been shown to promote apoptosis in cancer cells
as a result of their anionophoric activity.[25] On the other
hand, the design and preparation of new transporters dis-
playing low toxicity is also needed. In this regard, pseudo-
peptidic cages are very appealing targets, because it is rela-
tively simple to modulate their physicochemical properties
by the variation of the side chains from different amino
acids. Moreover, the preparation of simple transporters with
systematic structural changes would allow the understanding
of the transport process at the fundamental level in order to
propose some structure–activity relationships.
amino acidic units. Compounds of this type have recently
shown interesting properties in nanotechnology and for the
preparation of new biomaterials.[27] These tripodal pseudo-
peptides can be used to synthesize cage-like structures by
the reaction with a suitable tripodal complementary moiety
(grey circle in Scheme 1). Molecular models (CPK) show
that the inner cavity of these cages is suitable for hosting a
chloride anion.
Here we report on the simple preparation of tripodal
pseudopeptides and on their macrobicyclization reaction
toward the corresponding pseudopeptidic cages. Our studies
allowed us to understand the structural factors affecting the
macrobicyclization reaction and to define the scope and lim-
itations of the proposed synthetic procedure. Moreover, the
ability of the cages to bind chloride has been studied in gas,
solution and solid states. Finally, the chloride transport
through lipid bilayers as a model for cell membranes was
also studied for selected cages.
Results and Discussion
Design and synthesis of the tripodal cages: Our design of
the corresponding tripodal pseudopeptides is quite simple
and straightforward. As the central scaffold, we used three
different triamines, having three-fold symmetry (Scheme 2),
Within our ongoing research project devoted to the syn-
thesis and the study of the properties of new pseudopeptidic
compounds,[26] we envisioned the preparation of three-di-
mensional cage-like structures (Scheme 1). A simple and
reasonable proposal to reach this aim should be the prepara-
tion of pseudopeptidic tris(amido amines) based on a tripo-
dal central scaffold (black circle in Scheme 1) and three
Scheme 2. Synthesis of the tripodal pseudopeptidic tris(amido amines).
DME=dimethoxyethane.
which are either commercially available (Tren, 1a) or easily
accessible from simple starting materials[28] (TMBn and
TEBn, 1b and 1c, respectively). These have been described
forming part of different receptors, where a preorganized
concave conformation is proposed.[29] This geometrical dis-
position would favor the use of the corresponding tripodal
tris(amido amines) for the closure of a macrobicyclic archi-
tecture, leading to the preparation of small pseudopeptidic
molecular cages. The coupling of the triamines (1a–c) with
the Z-protected (Z=benzyloxycarbonyl) N-hydroxysuccin-
AHCTUNGTRENNUNG
Scheme 1. Tripodal pseudopeptides.
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I. Alfonso, S. V. Luis et al.
Table 1. Synthesis of the tripodal pseudopeptides 3a–h.
Table 2. Synthesis of the pseudopeptidic tripodal cages.
Entry
Triamine
X
R1 (Aaa)
2 ([%])[a]
3 ([%])[a]
Entry
3
Scaffold
R1
Aaa
4 (R2)
Cage ([%])[a]
1
2
3
4
5
6
7
8
1a (Tren)
1a (Tren)
1a (Tren)
1a (Tren)
1b (TMBn)
1b (TMBn)
1c (TEBn)
1c (TEBn)
–
–
–
–
Me
Me
Et
Et
Bn[b] (Phe)
iPr (Val)
iBu (Leu)
sBu (Ile)
Bn (Phe)
iPr (Val)
Bn (Phe)
iPr (Val)
2a (85)
2b (87)
2c (95)
2d (96)
2e (94)
2 f (71)
2g (81)
2h (82)
3a (74)
3b (72)
3c (84)
3d (62)
3e (71)
3 f (76)
3g (76)
3h (71)
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3a
3b
3c
3d
3a
3b
3c
3d
3e
3 f
3g
3h
Tren
Tren
Tren
Tren
Tren
Tren
Tren
Tren
Tren
Tren
Tren
Tren
TMBn
TMBn
TEBn
TEBn
Bn
Phe
Val
Leu
Ile
Phe
Val
Leu
Ile
Phe
Val
Leu
Ile
Phe
Val
Phe
Val
4a (H)
4a (H)
4a (H)
4a (H)
5a (38)
5b (43)
5c (20)
5d (34)
5e (39)
5 f (47)
5g (25)
5h (53)
5i (33)
5j (49)
5k (37)
5l (42)
iPr
iBu
sBu
Bn
iPr
iBu
sBu
Bn
iPr
iBu
sBu
Bn
iPr
Bn
4b (Me)
4b (Me)
4b (Me)
4b (Me)
4c (Et)
4c (Et)
4c (Et)
4c (Et)
4b (Me)
4b (Me)
4b (Me)
4b (Me)
9
[a] Yields of the isolated products are given in parenthesis. [b] Bn=
benzyl.
10
11
12
13
14
15
16
[b]
–
–
–
[b]
ventional deprotection of the carbamates 2a–h in acidic
medium produced the intended tripodal pseudopeptides 3a–
h in good overall yields. The synthetic procedures can be
easily carried out in relatively large scale, because none of
the steps required a chromatographic purification, and the
final products can be isolated as analytically pure solids by
precipitation and washing procedures. No significant differ-
ences in the reactivity of the systems due to the central scaf-
fold or to the amino acid side chains were observed, being
the different final yields ascribed to a slight difference in the
solubility of the compounds.
We also used these tripodal pseudopeptides for the con-
struction of small pseudopeptidic cages. To this aim, we per-
formed the triple SN2 reaction between the corresponding
tris(amido amine) and the symmetric triply electrophilic ar-
omatic bromides 4a–c (Scheme 3). Different substitution
[b]
[b]
iPr
–
[a] Yields of the isolated products after chromatographic purification.
[b] A complicated mixture was obtained as observed by 1H NMR spec-
troscopy and ultra-performance liquid chromatography (UPLC)-MS
analysis of the crude reaction mixture.
involved, which is necessary for the isolation of analytically
pure materials. This purification process is usually the limit-
ing step for the preparation of pseudopeptidic-related com-
pounds. Anyway, 1H NMR spectroscopy and UPLC-MS
analysis of the crude reaction mixture suggested that the re-
action is very selective towards the formation of the cage
(see the Supporting Information). Within the Tren deriva-
tives, the yields were better for the pseudopeptides derived
from Val and Ile although, once again, the differences are
more related to the purification process than to the reactivi-
ty of the compounds. No important differences were found
for the substitution pattern in compounds 4a–c, suggesting a
low effect of the steric hindrance in the reaction.
Unfortunately, the reactions performed with the tripodal
amido amines bearing an aromatic central scaffold (3e–h,
Table 2, entries 13–16) led, in all cases, to a more complex
1
mixture of compounds (as observed by TLC, H NMR spec-
troscopy, and UPLC-MS). The accurate UPLC-MS analysis
of the crude reaction mixtures allowed us to identify the in-
tended [1+1] cage, accompanied with a large amount of the
bigger [2+2] cage, along with other open-chain oligomeric
minor products (see the Supporting Information). In most
of the examples, these two cages were the major products
and in a very similar proportion as inferred from the UPLC
traces. After many attempts, we were unable to isolate both
cage compounds as pure products, even by using careful and
exhaustive preparative HPLC techniques. Thus, we conclud-
ed that the macrobicyclization reaction is much more effi-
cient in the case of the Tren derivatives 3a–d than for the
compounds having a central aromatic scaffold (TMBn or
TEBn, 3e–h). These results seemed quite intriguing, because
similar aromatic scaffolds have been used to preorganize tri-
podal moieties for the preparation of concave receptors.
Thus, a more in-depth study was performed in order to find
a reasonable explanation for the different behavior. First of
Scheme 3. Macrobicyclization reaction for the synthesis of the pseudo-
peptidic cages 5a–l.
patterns in the electrophiles were assayed (R2 =H, Me, Et)
for studying the steric effect on the formation of the cages.
The reaction was performed in acetonitrile, which was
heated to reflux, by using an excess of anhydrous potassium
carbonate as a base. Moreover, we used tetrabutylammoni-
um bromide as an additive (0.5 mol with respect to com-
pounds 3a–h). This highly soluble salt can act as a phase-
transfer catalyst and the anionic bromide also serves as a
stabilizing species of the transition states, as we have recent-
ly observed in the formation of pseudopeptidic macrocy-
cles.[30] The obtained results are displayed in Table 2.
For the Tren derivatives, very good yields of the isolated
products were obtained (Table 2, entries 1–12), considering
that three substitution reactions are performed in one pot
and that a careful chromatographic purification process is
1
all, H NMR spectra (500 MHz, CD3CN) obtained for repre-
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Pseudopeptidic Tripodal Small Cages
FULL PAPER
sentative precursors (3b, 3 f, and 3h, R1 =iPr) showed that
the starting tris(amido amine) is able to weakly interact with
the bromide anion present in the reaction mixture (added as
a catalyst and also formed during the reaction process). The
tripodal pseudopeptides set hydrogen bonding between the
amide and amino NH hydrogen atoms and the Br anion,
and molecular modeling suggested that these interactions
would produce a concave conformation with the three
amino nitrogen atoms in a similarly close proximity (see Fig-
ures S1–S4 in the Supporting Information). Therefore, we
concluded that no important differences in the preorganiza-
tion of the precursors should be expected. However, we
must consider that the key step to render either the [1+1]
or the [2+2] cage is the third SN2 reaction in a hypothetical
macrocyclic intermediate, as depicted in Scheme 4. From
podal aromatic spacer, thus explaining the observed differ-
ences in the corresponding reactivity.
All the isolated pure cages were unambiguously character-
ized by spectroscopic techniques, showing the expected C3
symmetry by NMR spectroscopy and the formation of the
[1+1] macrobicycle by mass spectrometry. Besides, a defini-
tive proof for the cage structure was obtained by X-ray dif-
fraction analysis (see below).
Crystal structures of the chloride complexes: We obtained
crystals suitable for X-ray diffraction with several cages as
the corresponding tetrahydrochloric acid salts, obtained by
slow evaporation of a methanolic solution of the given com-
pound in the presence of a slight excess of concentrated
aqueous hydrochloric acid. Thus, we were able to get suit-
AHCTUNGTRENNUNG
able crystals for all the Phe derivatives, that is, 5a (R2 =H),
5e (R2 =Me), and 5i (R2 =Et); and for two of the Val deriv-
atives, that is, 5 f (R2 =Me) and 5j (R2 =Et). The compari-
son between the observed structures illustrates the effect of
the amino acid nature (R1) and of the substitution at the tri-
podal aromatic ring (R2). All the crystals showed the pres-
ence of four chloride anions per cage unit, confirming the
protonation of the four amino groups of the receptors. The
cages crystallized with solvent (water or methanol) mole-
cules, being in most cases disordered. For the crystals show-
ing two geometries per asymmetric unit (5a, 5 f, and 5j),
very little differences (mainly in the disposition of the
amino acid side chains) were observed by the corresponding
superposition of the structures (see Figures S32, S36, and
S38 in the Supporting Information). Thus, a unique geome-
try per crystal structure will be discussed. Remarkable dif-
ferences between different receptors were observed attend-
ing to the cage–chloride contacts and to the cage geometry
(see Figures 1 and 2). By comparing the structures obtained
for the Phe cages, a very intriguing trend was observed
(Figure 1). The cages bearing a Me (5e) or Et (5i) residue
at R2 showed a tight inclusion complex with chloride (Fig-
ure 1B and C). Thus, one chloride anion settles in the
center of the inner cavity of the cages, stabilized by four
electrostatic hydrogen-bonding interactions with all the am-
monium groups of the cages (at a Cl···HN distance of 2.1 ꢆ
for the tertiary nitrogen atom and 2.6–2.9 ꢆ for the secon-
dary nitrogen atoms), defining the anion coordination with
tetrahedral geometry. Besides, the included chloride is posi-
tioned on top of the centroid of the tripodal aromatic ring
(at a distance of 3.09 ꢆ) suggesting the participation of an
anion–p interaction.[31]
Scheme 4. Key step for the competition between the inter-/intramolecular
SN2 reactions.
this intermediate, an effective intramolecular reaction would
exclusively produce the intended [1+1] cage, whereas the
competing intermolecular reaction would give access to the
larger [2+2] cage. According to the difference observed in
the outcome of the reactions depending on the central scaf-
fold of the tripodal pseudopeptide, we hypothesized that a
rational explanation for our observations could be obtained
by studying the corresponding macrocyclic intermediate in
selected representative cases. Because the nucleophilic sub-
stitution is an irreversible reaction, the spatial proximity be-
tween the reacting centers in the intermediate (namely the
distance between the amino nitrogen atom and the methyl-
À
Interestingly, the amide NH groups of 5e and 5i are
pointing outwards and are hydrogen bonded to crystal water
molecules, and do not interact with any of the outer chloride
ions. Thus, the main interaction of the included chloride is
through electrostatic hydrogen bonds with the ammonium
nitrogen atoms. However, a very different situation was ob-
tained for compound 5a, lacking substitution at the tripodal
aromatic ring (R2 =H, Figure 1A). For this molecule, the
crystal structure showed a less symmetric cage without any
encapsulated chloride (Figure 1A). The inner cavity of the
ene bromide (dACHTUNGTRENNUNG(N C) in Scheme 4) must be fundamental
regarding the competition between the intra- and intermo-
lecular reactions. The closer are the two reacting atoms, the
more efficient should be the intramolecular reaction, where-
as a long distance would increase the feasibility of the inter-
molecular process. Theoretical calculations on representa-
tive intermediates (see Figure S7 in the Supporting Informa-
À
tion) showed that dACHTUNGTRENNUNG(N C) was significantly larger for the tri-
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I. Alfonso, S. V. Luis et al.
Figure 2. Side and upper view of the crystal structures of the correspond-
ing tetrahydrochloric acid salts of A) 5 f and B) 5j. Only the closest
chloride ions are shown as a CPK model (30% of van der Waals radius),
whereas the additional chloride ions, solvent molecules, and non-polar
hydrogen atoms have been omitted for clarity. Hydrogen bonds are
shown as dashed black lines.
to the cavity of the receptors. One of these chloride ions is
partially included in the inner cavity of the molecule,
namely at the entrance of the cage. This Cl(1) interacts
through three ammonium NH···Cl hydrogen bonds with two
secondary nitrogen atoms and the tertiary N atom (at
Cl···HN distances of 2.2–2.4 ꢆ). The second chloride Cl(2) is
hydrogen bonded through one ammonium (Cl···HN dis-
AHCTUNGTREGtNNUN ances ca. 2.4–2.5 ꢆ) and two amide NH groups (Cl···HN
Figure 1. Side and upper view of the crystal structures of the correspond-
ing tetrahydrochloric acid salts of A) 5a, B) 5e, and C) 5i. Only the clos-
est chloride is shown as a CPK model (30% of van der Waals radius),
whereas the additional chloride atoms, solvent molecules, and non-polar
hydrogen atoms have been omitted for clarity. Hydrogen bonds are
shown as dashed black lines, whereas possible anion–p contacts are
shown as dashed gray lines.
distances ca. 2.3–2.4 ꢆ), being at the outer face of the cage.
Also in these Val cages, an intramolecular weak hydrogen
bond was observed between the C=O group and the amide
NH group of two arms of the capsule. These results under-
score remarkable differences in the mode of binding of the
chloride anions due to the aliphatic/aromatic side chain. The
Phe cages (with R2 =Me or Et) showed the inclusion of one
chloride ion mainly interacting through ammonium NH
bonds, whereas the corresponding Val cages (with R2 =Me
or Et) displayed a more complex binding implicating ammo-
nium and amide groups and two chloride ions per cage.
Thus, the nature of the side chain could change the mode of
chloride recognition, at least from the information gathered
in their corresponding crystal structures.
cage is highly reduced by the collapse of one of the arms,
which sets an intramolecular hydrogen bond between an
amide carbonyl group and the amide NH group of the
closer arm (see Figure 1A). As a consequence, the closest
chloride is bound out of the cavity, interacting through both
ammonium and amide NH···Cl hydrogen bonds (Cl···HN
distances of 2.2–2.3 ꢆ, Figure 1A). The other three chloride
ions are found to interact with ammonium groups and water
molecules, one of them being also located close to one of
the cage windows (see Figure S31 in the Supporting Infor-
mation). Thus, surprisingly, the substitution on the aromatic
tripodal ring improves the ability of the cages for including
the chloride within the inner cavity, at least as observed in
the solid state.
On the other hand, the compounds from aliphatic amino
acids showed a remarkably different structure in the solid
state, though the conformation was very similar between the
Val cages bearing either Me (5 f) or Et (5j) at R2 (Figure 2,
for a superposition of both cages, see Figure S39 in the Sup-
porting Information). Two chloride atoms were found close
Chloride binding in solution and in the gas phase
ESI-MS experiments: The chloride recognition abilities of
selected hosts were also investigated by ESI-MS-based com-
petition experiments.[32] To this aim, we used the cages de-
rived from Phe (5a, 5e, and 5i) and Val (5b, 5 f, and 5j) in
order to screen the effect of the substitution at both R1 (aro-
matic vs. aliphatic) and R2 (H, Me, Et). A compound de-
rived from Leu (5k) was also added to the study for the
comparison of different aliphatic side chains. The ESI mass
spectra of solutions of an equimolar mixture of the studied
receptors in the absence of HCl and in the presence of a 30-
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Pseudopeptidic Tripodal Small Cages
FULL PAPER
fold excess of HCl in CH2Cl2/CH3OH (7:3) showed impor-
tant differences (Figure S8 in the Supporting Information).
In the absence of added HCl, the ESI mass spectrum re-
vealed the dominant presence of a singly charged [M+H]+
species and a minor doubly charged [M+2H]2+ peak (where
M stands for the mass of the corresponding cages). When a
30-fold excess of HCl was added, the doubly charged species
[M+2H]2+ became dominant with respect to the singly
charged peaks, indicating that the protonation of the amine
groups has occurred as expected after the addition of HCl.
Moreover, the corresponding 1:1 host–chloride adducts of
the general formula [M+2H+Cl]+ were clearly identified
on the basis of both accurate m/z determinations as well as
their characteristic isotopic pattern, and remarkably their in-
tensity was strongly dependent on the identity of the host.
This is illustrated in Figure 3 where the expanded region in
the m/z=500–850 range of the ESI mass spectrum of the
equimolar mixture of the cages in the presence of a 30-fold
excess of HCl is presented.
displaying Val and Leu residues, more intense signals are
observed. This suggests that the receptors from aliphatic
amino acids seem to bind the anion more efficiently. We
have already observed that the Phe pseudopeptidic macro-
cycles usually displayed a reduced ability to participate in
non-covalent interactions with a given substrate or upon
self-assembly,[34] and we reasoned this trend as a shielding
effect of the aromatic ring of the side chain, which precludes
the close amide and ammonium groups from establishing
hydrogen-bonding interactions. On the basis of the ESI-MS
competition results we propose host 5k (Leu derivative with
R2 =Et) as the most efficient chloride receptor.
Energy-resolved collision-induced dissociation (CID) ex-
periments were also carried out on the formed
[M+2H+Cl]+ cations. In general, the expelled fragment in
all cases is HCl in the collision energy range investigated, to
yield the [M+H]+ singly charged free receptor. Breakdown
profiles for each host–guest couple are represented in Fig-
ure 4A and illustrative CID spectra at increasing collision
energy (CE) for the [5k+2H+Cl]+ cation in Figure 4B. The
differences between the gas dissociation of the complexes
were not very large, although a higher stability of those
formed with the cages bearing Et at R2 was apparent. Also
in this case, the best receptor was 5k, like in the competition
experiments.
NMR experiments: The interaction between the pseudopep-
tidic cages and chloride anions in solution was also studied
by NMR spectroscopic titration. To this aim, the addition of
chloride (as the tetrabutylammonium salt, TBACl) to a solu-
tion of the cages was monitored by 1H NMR (500 MHz,
303 K) spectroscopy. Interestingly, when the experiments
were performed with the receptors as the free amine, a very
weak interaction was observed. However, when performing
the experiment with the protonated cages (either as the
HClO4 or the trifluoroacetic acid (TFA) salts) a strong bind-
ing was observed by the perturbation of the chemical shifts
of several 1H NMR signals upon the addition of TBACl.
After trying different experimental conditions, we obtained
suitable NMR spectra (reasonably well-defined signals for
the accurate quantitative analysis) by titrating the corre-
sponding tetra-TFA salts of the cages in aqueous acetonitrile
solution (CD3CN/H2O 95:5) with increasing amounts of
TBACl (see the Supporting Information for details). Several
signals of the cages were affected by the presence of the
chloride anion, mainly those flanking the secondary amino
nitrogen atoms corresponding to the benzylic positions of
the tripodal aromatic ring (labeled as HB/HB’) and the
proton at the chiral center (the one at the a carbon of the
amino acid, labeled as HA), as well as the amide NH proton
signal (which was visible because we used non-deuterated
water). Other representative signals from the Tren moiety
also changed upon chloride addition, though severe broad-
ening and important overlapping precluded their accurate
analysis. A single set of signals was observable during the ti-
Figure 3. ESI mass spectrum in the m/z=500–850 range of an equimolar
mixture of the selected cages (5a, 5b, 5e, 5 f, 5i, 5j, and 5k) in the pres-
ence of a 30-fold excess of HCl.
Two clear trends in the chloride selectivity can be seen
along the series, as judged by ESI mass spectrometry. First
of all, except for M=5a and 5b, the species [M+2H+Cl]+
are detected. We reasonably take this as an experimental
evidence for a weaker binding of chloride by the hosts 5a
and 5b, thus anticipating the importance of the alkyl sub-
stituents in the tripodal aromatic ring to promote chloride
fixation in the capsule. We hypothesize that this observed
selectivity does not rely on electronic anion–p interactions,
because an enhanced stability of the Cl–p interaction is ex-
pected for the more electron-poor non-substituted phenyl
groups present in 5a and 5b.[33] However, this observation in
the ESI-MS competition experiments parallels the behavior
observed in solid state for the Phe derivatives, where 5a
(R2 =H) showed a less efficient geometry for the interaction
with the closest chloride within the crystal cell. Another in-
teresting trend is observed when comparing the correspond-
ing cages bearing aliphatic versus aromatic side chains.
Thus, whereas for M=5e and 5i, that is, cages that feature
Phe residues, [M+2H+Cl]+ cations are barely detected in
the ESI mass spectrum, for M=5 f, 5j, and 5k, that is, cages
1
tration experiments, implying fast exchange on the H NMR
timescale. We performed the titrations with all the Phe de-
Chem. Eur. J. 2012, 18, 16728 – 16741
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16733

I. Alfonso, S. V. Luis et al.
appeared as an AB quartet in all the cases and their corre-
sponding anisochrony increased in the presence of chloride.
This suggests that the formation of chloride complexes pro-
duces a more rigid cage, with a more different chemical en-
vironment for these protons. For the illustration of the
effect of the substitution at the tripodal aromatic ring, the
chloride-induced chemical shifts for 5a (R2 =H) and 5e
(R2 =Me) are shown in Figure 5. For a fair comparison, the
Figure 4. A) Breakdown profiles of mass-selected [M+2H+Cl]+ com-
plexes at increasing center of the mass collision energies (ꢇ =5e (Phe,
!
&
^
Me), & =5i (Phe, Et), =5 f (Val, Me), =5j (Val, Et), and =5k
(Leu, Et)) and B) illustrative CID spectra of mass-selected
[5k+2H+Cl]+ at m/z=720.5 at increasing CElab from 1, 3, 6, 9, and
12 eV (from the bottom to the top).
Figure 5. Effect of the substitution on R2 on the chloride binding proper-
ties illustrated by the plot of the chloride-induced chemical shifts of sev-
2
eral signals of cages A) 5a (R2 =H, ) and B) 5e (R =Me, ). NMR ti-
^
^
trations were performed at 500 MHz, 303 K, and in CD3CN/H2O 95:5 (ꢇ
rivatives, that is, 5a, 5e, and 5i, to show the effect of differ-
ent substitution at the tripodal aromatic ring (R2). Addition-
ally, the effect of the amino acid side chain (R1) was studied
by measuring all the cages with R2 =Et, that is, 5i (Phe), 5j
(Val), 5k (Leu), and 5l (Ile). As a general trend, most of
the signals move downfield during the first points of the ti-
tration, up to the addition of one to two equivalents of
chloride. Then, some signals continue moving downfield
upon further addition, whereas others started to move up-
field. This behavior suggests the formation of a strong 1:1
cage–chloride complex accompanied with complexes with
higher chloride stoichiometry (see below). The methylene
protons at the benzylic position of the tripodal aromatic ring
(HB/HB’) showed a very interesting trend. These protons
!
&
=NH, & =HB, =HB’, =HA).
same scales were used for both representations. The plots
clearly show a stronger interaction of chloride with the Me
derivative (see below for a quantitative analysis). Thus, no
signs of saturation were observed for 5a after four equiva-
lents of the anion were added, whereas most of the signals
of 5e clearly showed saturation after addition of less than
two equivalents of chloride. Noteworthy, both the signals
from the benzylic protons (HB/HB’) and the signal for the
methyl groups directly attached to the aromatic ring in 5e
reached saturation at approximately 1.2 equivalents of chlor-
ide, suggesting that these groups are directly involved in the
16734
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Chem. Eur. J. 2012, 18, 16728 – 16741

Pseudopeptidic Tripodal Small Cages
FULL PAPER
that, the simultaneous non-
linear regression fit of all the
available signals was per-
formed (see the Supporting In-
formation for details). Com-
plexes with different stoichi-
ometry were detected by fit-
ting the titration data. Howev-
er, in all cases the 1:1 complex
was more stable, suggesting the
preference for the interaction
with one chloride ion. For a
suitable comparison of supra-
molecular complexes of differ-
ent stoichiometry, we used the
BC050 parameter that gives an
idea of the intrinsic chloride
affinity, independently on the
stoichiometry of the consid-
ered complexes.[36] Because we
observed multinuclear com-
plexes in the titration agent
(chloride), the BC050 parameter
is here defined as the concen-
tration of chloride necessary to
bind 50% of the corresponding
cage, starting from the free
species and considering all the
possible complexes formed.
For simple 1:1 complexes, the
BC050 parameter is equal to the
Figure 6. Effect of the nature of the amino acid side chain on the binding of chloride, illustrated by the plot of
the chloride-induced chemical shifts of several signals of A) 5i (Phe), B) 5j (Val), C) 5k (Leu), and D) 5l
&
(Ile). NMR titrations were performed at 500 MHz, 303 K, and in CD3CN/H2O 95:5 (ꢇ =NH, & =HB, =HB’,
!
=HA).
binding of the first chloride anion. These data are in perfect
agreement with the results observed in the solid state, that
is, the formation of the cage–chloride 1:1 supramolecular
complex.
dissociation constant of the corresponding supramolecular
complex and, therefore, the lower is the BC050 value, the
stronger is the host–guest interaction. The obtained results
are shown in Table 3, whereas the titration experiments and
For the study of the effect of the nature of the amino acid
side chain, similar plots of the chloride-induced chemical
shifts are shown in Figure 6 for the cages bearing an ethyl
moiety at R2. A remarkable difference between the aromat-
ic and aliphatic amino acids was observed. The Leu cage
seems to be the one forming the most stable complexes with
chloride. In the aliphatic cages, a very interesting behavior
was observed for the chemical shift of the amide NH
proton. In all cases, the signal moves downfield until reach-
ing a maximum at approximately 1.2 equivalents of chloride
and, after that, a marked upfield shift is evident. This sug-
gests that in the case of the aliphatic cages, the amide NH
proton is directly implicated in the binding of the first chlor-
ide anion and that the formation of the complexes of higher
stoichiometry further affects the chemical environment of
the amide group, probably due to a conformational change.
These experimental observations in solution are in a very
good agreement with the data obtained in the solid state by
X-ray diffraction of the corresponding chloride complexes
of Phe and Val cages (see above).
1
Table 3. Chloride-binding data in solution, obtained by H NMR spectro-
scopic titration (500 MHz, 5% aqueous CD3CN, 303 K) of the corre-
sponding TFA salts of the cages with TBACl.
Entry Cage R1 (Aaa)
R2
Logb[a] (cage/Cl)
BC050 [mm]
1
2
5a
5e
Bn (Phe)
Bn (Phe)
H
(2.35Æ0.03) (1:1)[b] (4467Æ309)
Me (4.01Æ0.08) (1:1)[b]
G
E
3
4
5
5i
Bn (Phe)
Et
Et
(3.37Æ0.09) (1:1)[b]
ACHTUNGTRENNUNG
A
5j
5k
iPr (Val)
(3.68Æ0.07) (1:1)[b]
(5.86Æ0.09) (1:2)[c]
(4.35Æ0.11) (1:1)[b]
ACHTUNGTRENNUNG
iBu (Leu) Et
A
N
8
5l
sBu (Ile)
Et
(3.87Æ0.08) (1:1)[b]
A
(5.9Æ0.1) (1:2)[c]
[a] Obtained by simultaneous non-linear regression fitting of all the avail-
able 1H NMR signals. [b] We defined b (1:1) as the formation constant
for the 1:1 cage/Cl complex in mÀ1. [c] In this case b (1:2) is the cumula-
tive formation constant for the corresponding 1:2 cage/Cl complex in mÀ2
.
[d] In this case b (1:3) is the cumulative formation constant for the 1:3
cage/Cl complex in mÀ3. Despite the weak binding of the third chloride
ion, it was necessary for an accurate fitting of the data.
For the quantitative analysis of the data, we followed the
fitting procedure proposed by Roelens and co-workers.[35] To
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16735

I. Alfonso, S. V. Luis et al.
the corresponding fittings are given in the Supporting Infor-
mation. Interestingly, the substitution at the tripodal aro-
matic ring produced an increase in the strength of the chlor-
ide binding by more than one order of magnitude from
BC050 =4 to 0.1 mm for R2 =H or Me, respectively. The large
increase of the chloride binding, which is influenced by the
R2 substituent, is in very good agreement with the observa-
tions in the solid state by single-crystal X-ray diffraction and
in solution by ESI-MS competition experiments. As previ-
ously anticipated, this difference cannot be ascribed to en-
hanced anion–p interactions. We reasoned that the substitu-
tion at R2 could produce an efficient desolvation of the
nearby benzylic ammonium groups, thus leaving the corre-
+
sponding NH2 sites more accessible for the binding to the
anion. A slight decrease in the chloride binding was ob-
served by going from Me to Et substitution among the Phe
cages, which can be ascribed to an increased steric hindrance
in the Et derivative. Stronger interactions are obtained by
changing from an aromatic to an aliphatic amino acid side
chain, as also observed in the competition ESI-MS experi-
ments and for other pseudopeptidic macrocyclic hosts.[34] Be-
sides, we consistently observed the formation of complexes
of 1:2 stoichiometry, as well as chloride binding through the
participation of the amide NH protons, which is also in
agreement with the mode of binding observed in the solid
state by X-ray diffraction for the Val cages. The cage show-
ing the strongest interaction (with BC050 =44 mm) was 5k,
which is in perfect accordance with the competition ESI-MS
and CID experiments. Among the cages bearing aliphatic
side chains, the one from Leu lacks substitution at the b
carbon atom of the amino acid, which could decrease the
steric hindrance for the binding, thus explaining the im-
proved chloride recognition of 5k versus 5l and 5j.
Figure 7. 35Cl NMR spectra (48.97 MHz, CD3OD/D2O 1:1) of A) HCl at
303 K, B) 5i·4HCl at 297 K, and C) 5i·4HCl at 333 K. In C) the experi-
mental spectrum was de-convoluted into two bands (black traces), being
the corresponding addition simulated spectrum (gray trace) and the dif-
ference between the simulated and the experimental spectra (light gray
trace) also shown. The numerical results of the fitting are shown in the
inset.
servations, we estimated the chloride in/out exchange within
the low milliseconds scale.
The chloride recognition ability of cage 5i was also stud-
ied by 35Cl NMR spectroscopy, in order to know whether
the inclusion complex observed in the solid state was re-
tained in solution.[37] Thus, the 35Cl NMR spectrum of
5i·4HCl was acquired in a highly competitive solvent
(CD3OD/D2O 1:1), in addition to the 35Cl NMR spectrum of
HCl in the same medium as a reference (Figure 7). The
spectrum of 5i·4HCl showed an almost undetectable very
broad signal at room temperature (Figure 7B), which slight-
ly sharpened at higher temperature (333 K, Figure 7C). The
very sharp peak at d=3.29 ppm observed for HCl in this
medium (Figure 7A) suggested that the Cl nuclei in the
sample of 5i·4HCl are involved in a dynamic exchange
process within a non-symmetrical environment.[38] Interest-
ingly, the signal observed at 333 K was non-symmetrical,
and the accurate de-convolution by line-shape fitting ren-
dered two broad 35Cl NMR peaks at d=17.83 and
10.86 ppm in a relative intensity of approximately 1:3, re-
spectively. These data suggest that the 1:1 chloride inclusion
complex is also present in solution and that the chloride in/
out exchange occurs at an intermediate rate on the
Chloride transport through lipid bilayers: The potential of
these compounds to facilitate the transmembrane transport
of chloride in model phospholipid bilayers was explored by
using
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) liposomes. Chloride efflux from chloride-loaded
vesicles was monitored over time by using a chloride-selec-
tive electrode. The liposome suspension was placed in an
isotonic, chloride-free medium and the compounds studied
were added as a solution in DMSO. Addition of a detergent
at the end of the experiment lysed the vesicles and provided
a final chloride concentration value corresponding to 100%
chloride release. The results obtained are shown in Figure 8.
Compound 5i was found to be the most active chloride car-
rier in these assays, promoting around 50% chloride release.
Compounds 5e, 5k, and 5l promoted similar chloride efflux
accounting for approximately 35%, and limited chloride
efflux was detected in the case of compound 5a. These re-
sults showed that in compounds 5a, 5e, and 5i, which are
derived from Phe, the transport efficiency increased signifi-
cantly as the substitution of the tripodal aromatic ring
changed from hydrogen (5a) to methyl (5e) and ethyl (5i)
groups. The fact that 5a was found to be the less active de-
rivative of these compounds is in good agreement with the
1
35Cl NMR timescale, but fast on the H NMR timescale as
observed in the titration experiments. Considering the reso-
nance frequencies of both nuclei and our experimental ob-
16736
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Chem. Eur. J. 2012, 18, 16728 – 16741

Pseudopeptidic Tripodal Small Cages
FULL PAPER
ous to the closure of the macrobicycle. The binding of chlor-
ide ions by the protonated forms of the pseudopeptidic
cages has been studied in the solid state (by X-ray diffrac-
tion), in solution (by NMR spectroscopy and competition
ESI-MS) and in the gas phase (by tandem MS CID), show-
ing trends, which are in very good agreement. Despite the
fact that the binding pockets of the different cages are es-
sentially identical (same size, shape, and potential binding
sites), the chloride recognition abilities are strongly affected
by the peripheral substitution, such as the residues at the ar-
omatic tripodal ring (R2) and the nature of the amino acid
side chain (R1). The substitution at R2 is favoring the inclu-
sion of the chloride ion (as observed by X-ray diffraction)
and is improving the binding in solution (as observed by
competition ESI-MS and NMR spectroscopy) by more than
one order of magnitude. This is very surprising because the
more substituted aromatic ring should display reduced
anion–p interactions, but can be explained with a less effi-
cient solvation of the ammonium groups in the more substi-
tuted cages. On the other hand, the cages built from aliphat-
ic amino acids interact more strongly with the chloride than
the cages derived from Phe. Interestingly, the aliphatic/aro-
matic substitution seems to change the mode of the binding
to chloride, as inferred from the NMR spectroscopic and X-
ray data. Thus, the Phe cages are more prone to completely
encapsulate the chloride ion by electrostatic ammonium
NH···ClÀ hydrogen bonds, whereas the aliphatic cages bind
two chloride ions through ammonium and amide NH
groups, leading to a partial encapsulation. An important
counterintuitive conclusion can be extracted by comparing
the results of the Phe and Val cages bearing an Et group at
R2: the chloride inclusion within the cage structure does not
necessarily imply a more efficient binding. Thus, the Phe
cages (with Me or Et at R2) fully encapsulate the anion but
the interaction is stronger with the receptors built from ali-
phatic amino acids. The ability of some of these cages to
transport chloride through lipid bilayers has also been dem-
Figure 8. Chloride efflux promoted by 25 mm (5 mol% carrier-to-lipid
concentration) of the indicated compounds in unilamellar POPC vesicles.
The vesicles were loaded with 489 mm NaCl buffered at pH 7.0 with
10 mm phosphate dispersed in 489 mm NaNO3 buffered at pH 7.0. Each
~
&
*
trace represents the average of three trials ( =5i, =5k, =5l, & =5e,
^
*
=5a, =DMSO).
results observed in both the solid state and in solution.
Thus, compound 5a did not encapsulate chloride in the solid
state and the chloride binding affinity was an order of mag-
nitude lower than that of the other derivatives. In order to
further rationalize these results we also studied the lipophi-
licity of these compounds. This parameter has been shown
to greatly influence the transmembrane transport activity of
other ionophores.[39] Average logP values were calculated by
using computational methods.[40] Moreover, an experimental
measure of the lipophilicity was obtained by reverse-phase
HPLC.[41] The results indicated that an increase in the lipo-
philicity of the compounds resulted in an increase of the
transport activity (Table S1 in the Supporting Information),
with the most lipophilic derivative (5i) being the most active
transporter and the most hydrophilic derivative (5a) only
displaying limited activity as chloride transporter. The simi-
lar values of both logP and the retention time for com-
pound 5e, 5k, and 5l were in agreement with the results ob-
served in the liposome assays.
onstrated. Once again, remarkACTHNUGTRNEUNGable differences were ob-
served in the chloride transport rates of the different cages,
which are better correlated to the corresponding lipophilici-
ty than to the chloride binding. Therefore, we have demon-
strated that slight changes in the structure of these pseudo-
peptidic cages produce important differences in the corre-
sponding chloride affinity and transport ability, even if they
have apparently the same binding pocket. The behavior of
these minimalistic systems parallels the situation in Nature,
where far structural mutations in natural receptors can enor-
mously affect their function, and even produce pathological
disorders. Our deep study by using small and simple mole-
cules could serve for the understanding of more complicated
systems, as well as a platform for the further design of im-
proved chloride receptors and transporters based on pseudo-
peptidic molecules.
Conclusion
We described herein the efficient and selective synthesis of
a new family of small pseudopeptidic cages, through a triple
nucleophilic substitution and from simple tripodal precur-
sors. The success of the reaction highly depends on the
structure of the central scaffold of the tris(amido amine)
precursor. The pseudopeptides derived from tris(2-amino-
ACHTUNGTRENNUNGethyl)amine (Tren) led to the [1+1] cages in very good
yields and complete selectivity, whereas those bearing a cen-
tral aromatic scaffold produced complex mixtures, mainly
containing the [1+1] and the [2+2] cages. The obtained re-
sults have been rationalized in terms of the preorganization
of the corresponding monomacrocyclic intermediate, previ-
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16737

I. Alfonso, S. V. Luis et al.
were analyzed and ordered attending to their relative energy. Their popu-
lation was also calculated by using a Boltzmann distribution with the
same software.
Experimental Section
General: Reagents and solvents were purchased from commercial suppli-
ers (Aldrich, Fluka, or Merck) and were used without further purifica-
tion.
General procedure for the preparation of the cages
Synthesis of 5a: N-N’-N’’-tris(N-l-phenylalanine)-Tren (3a) (761 mg,
1.295 mmol), anhydrous K2CO3 (1.789 g, 12.95 mmol), tetrabutylammoni-
um bromide (208.64 mg, 0.648 mmol), and 1,3,5-tris(bromomethyl)-ben-
zene (476.39 mg, 1.295 mmol) were placed in a flask containing dry
CH3CN (500 mL) and the mixture was heated to reflux for 12 h under an
argon atmosphere. The reaction mixture was filtered and the solvent
evaporated under reduced pressure. The crude product was dissolved in
CHCl3 (50 mL) and extracted with aqueous NaOH 0.01m (3ꢇ50 mL).
The organic phase was dried over anhydrous MgSO4 and the solvent was
evaporated under reduced pressure. The product was purified by silica
flash chromatography by using MeOH/CH2Cl2 (1:40) as eluent. Com-
pound 5a was obtained in 38% (345.34 mg, 0.492 mmol) yield. M.p. 94–
968C; [a]2D5 =À78.5 (c=0.01, CHCl3); 1H NMR (500 MHz, CDCl3): d=
1.83 (s, 1H), 2.24 (t, J=6.2 Hz, 2H), 2.79 (dd, J=9.9, 14.0 Hz, 1H), 2.99
(q, J=6.0 Hz, 2H), 3.33 (dd, J=4.0, 14.1 Hz, 1H), 3.43 (d, J=14.5 Hz,
1H), 3.50 (dd, J=4.1, 9.9 Hz, 1H), 3.76 (d, J=14.5 Hz, 1H), 7.01 (s, 1H),
7.07 (t, J=5.6 Hz, 1H), 7.22–7.25 (m, 1H), 7.28–7.38 ppm (m,
4H);13C NMR (126 MHz, CDCl3): d=37.9, 38.8, 51.9, 54.6, 64.5, 125.5,
126.8, 128.8, 129.1, 138.00, 141.0, 173.8 ppm; IR (ATR): 3318, 2930, 2830,
NMR spectroscopy: The NMR experiments were carried out on a Varian
INOVA 500 spectrometer (500 MHz for 1H, 125 MHz for 13C, and
49 MHz for 35Cl). Chemical shifts are reported in [ppm] by using TMS as
a reference. Titrations experiments followed by 1H NMR spectroscopy
were performed in CD3CN/H2O 95:5 at 303 K and data fitting was car-
ried out with HypNmr 2008 version 4.0.71 software (see the Supporting
Information for details).
Mass spectrometry: A Q-TOF Premier mass spectrometer with an elec-
trospray source (Waters, Manchester) operating in the V-mode was used.
The drying gas as well as the cone gas was nitrogen at a flow of 400 LhÀ1
and 60 LhÀ1, respectively. The temperature of the source block was set to
1208C and the desolvation temperature was set to 1508C. A capillary
voltage of 3.5 kV was used in the positive scan mode. For characteriza-
tion purposes, methanol sample solutions were infused through a syringe
pump directly connected to the ESI source at a flow rate of 10 mLminÀ1
and the cone voltage was set to 20 V. Mass calibration was performed by
using a mixture of 0.05m NaOH and 10% formic acid (50:50) from m/z=
50–1250. For the accurate mass measurements, a solution of leucine enke-
phalin (m/z=556.2771) was introduced through the lock spray needle at
a flow rate of 30 mLminÀ1. For competition experiments, to an equimolar
mixture of the receptors (1ꢇ10À3 m in CH2Cl2/CH3OH 7:3) a 30-fold
excess of HCl was added. The resulting mixture was diluted to a final
concentration of 1ꢇ10À5 m and the ESI mass spectra were recorded. Col-
lision-induced dissociation (CID) experiments were performed by mass-
selecting the monoisotopic peak of interest with Q1 (isolation width
1 Da), interacted with argon in the T-wave collision cell while analyzing
the ionic fragments with the TOF analyzer. The collision energy was sys-
tematically stepped in the Elab =1–12 eV range. For a qualitative analysis
of the energy-dependent CID experiments, the laboratory collision ener-
1653, 1509, 1451 cmÀ1
;
HRMS (ESI-TOF)+ calcd for C42H51N7O3
[M+H]+: 702.4132; found 702.4134; elemental anal. calcd for
C42H51N7O3·H2O: C 70.07, H 7.42, N 13.62; found: C 69.7, H 7.1, N 13.3.
Preparation of the phospholipid vesicles: A solution of 1-palmitoyl-2-
oleoyl-sn-glycero-3-phosphocholine (POPC) (Sigma–Aldrich) (chloro-
form, 20 mgmLÀ1) was evaporated and the obtained lipid film was dried
under high vacuum for at least 2 h. The lipid film was rehydrated by ad-
dition of a solution of sodium chloride (489 mm, 10 mm phosphate buffer,
pH 7.0) followed by careful vortexing. The obtained lipid suspension was
then subjected to nine freeze-thaw cycles and twenty-nine extrusions
through a 200 nm polycarbonate Nucleopore membrane by using a Lipo-
soFast Basic extruder (Avestin, Inc.). The resulting unilamellar vesicles
were dialyzed against a solution of NaNO3 (489 mm, and 10 mm phos-
phate buffer, pH 7.0) to remove unencapsulated chloride.
gies were converted to the center-of-mass frame, ECM =m/ACHTGNUETRN(UNNG m+M)Elab,
where m and M stand for the masses of the collision gas and the ionic
species, respectively. For the breakdown profile representations, the
signal intensities were obtained from the average of 80 scans and measur-
ing of the area of the fragmentation peaks. These graphs were represent-
ed by taking into account the relative abundance of the precursor and
the product peaks of each compound (Iprecursorion/[Iprecursorion+Iproduction])
against ECM. We selected the value of the collision energy required for
50% reduction of the precursor ion (E1/2) as a semi-quantitative measure
of the intrinsic gas-phase stability of the studied non-covalent complexes.
Ion selective electrode (ISE) transport assays: Unilamellar POPC vesi-
cles (200 nm mean diameter) containing an encapsulated solution of
NaCl (489 mm) and phosphate buffer (10 mm, pH 7.0) were suspended in
a solution containing NaNO3 (489 mm) and phosphate buffer (10 mm,
pH 7.0) for a final lipid concentration of 0.5 mm and a total volume of
5 mL. A solution of the carrier molecule in DMSO (12.5 mL, 10 mm) was
added and the chloride release from the vesicles was monitored by using
a sympHony chloride-selective electrode. At the end of the experiment
the vesicles were lysed with a detergent (triton-X 10% dispersion in
water, 60 mL) to release all chloride ions. This final reading value was
considered to represent 100% release and used as such.
Infrared spectroscopy: FTIR spectra were acquired in a JASCO 6200
equipment having a MIRacle Single Reflection ATR Diamond/ZnSe ac-
cessory.
X-ray crystallographic analysis: Data were collected on a STOE IPDS II
two-circle diffractometer with graphite-monochromated MoKa radiation.
Empirical absorption corrections were performed by using the
MULABS[42] option in PLATON.[43] The structures were solved by direct
methods by using the program SHELXS[44] and refined against F2 with
full-matrix least-squares techniques by using the program SHELXL-97.[44]
Hydrogen atoms were geometrically positioned and refined by using a
riding model. Hydrogen atoms of water and methanol molecules could
not be unequivocally located and were omitted from the refinement. The
absolute configuration of all molecules was determined by refining the
Flack parameter. In compound 5 f, three isopropyl groups are disordered
over two positions. CCDC-883493 (5 f), 883494 (5a), 883495 (5j), 883496
(5i), and 883497 (5e) contain the supplementary crystallographic data for
this paper. These data can be obtained free of charge from The Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_re-
quest/cif.
Acknowledgements
Financial support from the Spanish Ministerio de Ciencia e Innovaciꢂn
(MICINN) and the Ministerio de Economꢀa
y
Competitividad
(MINECO) is gratefully acknowledged (CTQ2009-14366-C02), as well as
the Consejerꢀa de Educaciꢂn de la Junta de Castilla y Leꢂn (project
BU337A12–2). I.M. (FPI), J.R. (FPU), and R.Q. (Ramꢂn y Cajal) also
thank the MICINN/MINECO for personal financial support. We thank
Prof. Stefano Roelens for providing us with the BC50 calculator program.
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16738
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