Pseudopeptidic cages as receptors for N-protected dipeptides

Article abstract of DOI:10.1021/jo500629d

The molecular recognition of short peptides is a challenge in supramolecular chemistry, and the use of peptide-like cage receptors represents a promising approach. Here we report the synthesis and characterization of a diverse family of pseudopeptidic macrobicycles, as well as their binding abilities toward N-protected dipeptides using a combination of different techniques (NMR, ESI-MS, and fluorescence spectroscopy). The cage hosts were assayed for dipeptide binding using competition ESI-MS experiments as high-throughput screening to obtain general trends for the recognition phenomena. Selected hosts were additionally studied by NMR spectroscopy ( ¹H NMR titration and diffusion-ordered spectroscopy experiments) in different solvents. The results unambiguously demonstrated the formation of the [cage·dipeptide] supramolecular complexes and rendered quantitative information about the strength of the interaction (K_ass). The structural variables within the pseudopeptidic cage framework that produced a stronger and more selective recognition were thus identified. The cages showed a remarkable selectivity for N-protected dipeptides with an aromatic amino acid at the carboxylic terminus, which prompted us to propose a mode of binding based on polar and nonpolar noncovalent interactions. Accordingly, we faced the molecular recognition of a target dipeptide (Ac-EY-OH) mimicking a biologically relevant sequence by NMR and fluorescence spectroscopy in highly competitive media.

Full text of DOI:10.1021/jo500629d

Article
pubs.acs.org/joc
Pseudopeptidic Cages as Receptors for N‑Protected Dipeptides
Enrico Faggi,† Alejandra Moure,† Michael Bolte,‡ Cristian Vicent,§ Santiago V. Luis,*,∥
and Ignacio Alfonso*,†
†Departamento de Química Biolog
Girona 18-26, E-08034 Barcelona, Spain
́ ́
ica y Modelizacion Molecular, Instituto de Química Avanzada de Cataluna (IQAC-CSIC), Jordi
̃
‡Institut fur Anorganische Chemie, J.-W.-Goethe-Universitat, Max-von-Laue-Strasse 7, D-60438 Frankfurt/Main, Germany
̈
̈
§Serveis Centrals d’Instrumentacio
́
Científica and ∥Departamento de Química Inorgan
́
ica y Organ
́
ica, Universitat Jaume I, Avda. Sos
́
Baynat, s/n, E-12071 Castellon, Spain
S
* Supporting Information
ABSTRACT: The molecular recognition of short peptides is a
challenge in supramolecular chemistry, and the use of peptide-
like cage receptors represents a promising approach. Here we
report the synthesis and characterization of a diverse family of
pseudopeptidic macrobicycles, as well as their binding abilities
toward N-protected dipeptides using a combination of
different techniques (NMR, ESI-MS, and fluorescence spec-
troscopy). The cage hosts were assayed for dipeptide binding
using competition ESI-MS experiments as high-throughput
screening to obtain general trends for the recognition
phenomena. Selected hosts were additionally studied by
NMR spectroscopy (1H NMR titration and diffusion-ordered
spectroscopy experiments) in different solvents. The results unambiguously demonstrated the formation of the [cage·dipeptide]
supramolecular complexes and rendered quantitative information about the strength of the interaction (Kass). The structural
variables within the pseudopeptidic cage framework that produced a stronger and more selective recognition were thus identified.
The cages showed a remarkable selectivity for N-protected dipeptides with an aromatic amino acid at the carboxylic terminus,
which prompted us to propose a mode of binding based on polar and nonpolar noncovalent interactions. Accordingly, we faced
the molecular recognition of a target dipeptide (Ac-EY-OH) mimicking a biologically relevant sequence by NMR and
fluorescence spectroscopy in highly competitive media.
molecular recognition of short peptides is still a challenge in
supramolecular chemistry.8 The external intervention on these
interactions could serve for the design of therapeutic or
diagnostic tools.9 The use of synthetic receptors for dipeptides
is a fundamental approach to the problem, since the relative
simplicity of the systems could allow the full understanding of
the rules controlling the process.10
During the past decade, we have designed and prepared
different pseudopeptidic macrocycles with interesting supra-
molecular properties.11 They were shown to be efficient
receptors for different species with positive or negative
charge.12 More interestingly, they are hosts for N-protected
amino acids and dipeptides.13 However, the strength and
selectivity of the binding are moderate, and in most cases, the
process has been studied in noncompetitive solvents. More
recently, we have increased the complexity of the pseudopepti-
dic receptors by adding a third arm defining a cage structure.14
This second generation of pseudopeptidic receptors is able to
partially or completely include the guest even in more
INTRODUCTION
■
Macrocyclic structures derived from amino acids are attractive
molecules with a formidable potential in the molecular
recognition field.1 The different functionalities of the side
chains could implement a large molecular diversity and,
concomitantly, many potential noncovalent binding sites and
different chemical properties (such as charge or polarity).
Moreover, the peptide bond also represents a structural and
interactional functional group.2 This combination has allowed
the use of pseudopeptidic macrocycles for the recognition of
different species.3 Regarding that, the addition of another
dimension to the binding motif could improve the recognition
abilities, both by increasing the strength of the interaction and
by improving the selectivity of the process.4 In this context,
molecular cages have recently emerged as interesting entities
for the binding of specific guests.5 Accordingly, the macro-
bicyclic compounds containing amino acid moieties can be
considered as pseudopeptidic cages with an inner cavity
surrounded by functional groups defining a binding pocket
for potential substrates.6 On the other hand, although some
important biological processes are closely related to the
noncovalent interaction with specific peptide sequences,7 the
Received: March 18, 2014
Published: April 21, 2014
© 2014 American Chemical Society
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a
Scheme 1. (A) Synthesis of Pseudopeptidic Cages and (B) Chemical Structure of the Monomacrocycle Analogue 6
a
Conditions: (1) equilibrium at rt either in the absence or in the presence of 5·TBA; (2) BH3·py; (3) hydrolysis.
competitive media, leading to interesting functional molecules
with potential biological applications.15 Following our work in
this field, here we aimed to prepare larger pseudopeptidic cages
able to recognize more elaborate substrates such as dipeptides.
We envisioned that the three-dimensional structure of the cages
should display more efficient recognition of these challenging
substrates through a well-defined network of noncovalent
bonds. Besides, the understanding of the intimate interaction
would allow the better design of improved binders with
potential applications in different fields such as sensing,
catalysis, and biomedicine.
the precise [3 + 2] hexaimine cage. Previous studies showed
that the process is mainly controlled by the preorganization of
the bis(amidoamine) precursor in a U-shaped conformation.
Thus, for pseudopeptidic diamines containing a rigid scaffold
(such as trans-cyclohexane-1,2-diamine, chx) with a match
combination of the stereocenters ((R,R)-diamine and (S)-
amino acids), the formation of the correct imine intermediate is
highly favored.18 For the more flexible aliphatic linear spacers,
the formation of the macrocyclic oligoimine is not so favorable,
and the use of the suitable anionic template improves the
outcome of the reaction.19 Taking into account these data, we
envisioned the preparation of different pseudopeptidic cages
with a representative molecular diversity. As expected, the use
of the chx spacer induced the formation of the cages with good
yields considering that six simultaneous C−N bonds in two
steps were formed (Scheme 1, Table 1). We obtained cages
4a−c bearing aliphatic (4a, entry 1), aromatic (4b, entry 2), or
polar (4c, entry 3) residues, which allowed us to map a wide
range of chemical properties (such as solubility or potential
noncovalent interactions). The formation of the hexaimine
intermediate in the case of 3a was additionally studied by NMR
and molecular modeling.16 Most remarkably, the corresponding
pseudopeptidic cage derived from serine was obtained in an
excellent overall yield (entry 3), calculated from 1c and
including the reductive amination reaction, hydrolysis with the
tBu deprotection, and a final reversed-phase chromatographic
purification necessary to obtain the pure compound.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of the Pseudopeptidic
Cages. Our proposal for the preparation of the intended cages
was based on a multicomponent [3 + 2] reductive amination
condensation between a bipodal pseudopeptidic bis-
(amidoamine) (1a−g) and a simple aromatic trialdehyde (2)
(Scheme 1).16 This approach allows a high degree of
modularity in the pseudopeptidic moiety, which will control
the size, flexibility, polarity, and binding sites of the final cage.
Thus, we faced the preparation of a representative family of
receptors (4a−g) bearing aliphatic, aromatic, or polar side
chains and displaying different conformational properties
through the use of different aliphatic spacers in the
pseudopeptidic bis(amidoamine). The overall process com-
prises the initial formation of the corresponding hexaimine
intermediate (3) by a dynamic covalent chemistry method-
ology17 followed by the in situ reduction of the imine bonds.
Therefore, the success of the reaction highly depends on the
ability of the system to self-correct, leading to the formation of
When using a simple linear aliphatic spacer in the
pseudopeptidic moiety, the reaction also led to the formation
of the cage, though in a lower yield and selectivity. Actually, the
HPLC trace of the crude reaction showed the presence of other
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Table 1. Synthesis of Pseudopeptidic Molecular Cages
a
entry
1a−g
spacer
R
template
cage (yield, %)
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1d
1e
1f
chx
chx
chx
et
iPr
4a (47)
4b (30)
Bn
b
b
CH2OtBu
iPr
4c (59)
4d (15)
4d (24)
4e (24)
et
iPr
5a·TBA
5a·TBA
5a·TBA
5a·TBA
et
Bn
c
et
CH2OtBu
iPr
4f (11)
1g
pr
4g (13)
a
b
c
Values in parentheses correspond to the overall isolated yields.
During the reaction procedure, the tBu protecting group was cleaved.
The final hydrolysis was performed at less acidic pH to avoid tBu
elimination, and thus, the isolated cage retained the CH2OtBu side
chain.
undesired side products. Interestingly, the addition of the
suitable anionic template (5·TBA) improved the yield of the
reaction (compare entries 4 and 5 in Table 1) and, more
importantly, rendered a more selective process toward cage 4d.
The template 5·TBA promoted the formation of the correct
hexaimine intermediate (3d) from the dynamic covalent
mixture of pseudopeptidic imines. A detailed study of this
intermediate using NMR (including diffusion-ordered spec-
troscopy (DOSY) and rotating frame Overhauser effect
spectroscopy (ROESY)), ESI-MS, CD, and molecular modeling
allowed us to propose the formation of a supramolecular
complex where the tricarboxylate 5 is included within the cavity
of the hexaimine cage 3d, stabilized by six amide−carboxylate
H-bonds.16 The formation of this complex increased the rate of
formation and the thermodynamic stability of the cage, finally
leading to an improvement of the overall process. The template
methodology was also applied to the synthesis of cages bearing
different side chains (4e−f) or a longer aliphatic spacer (4g)
with reasonably good overall yields (Table 1).
Figure 1. X-ray diffraction analysis of the crystal structure of 4a·
6HClO4: (A) molecular cage structure without perchlorate anions and
crystallization water molecules, (B) a different view of the cage with
the four perchlorate anions (in a CPK representation) in close contact.
The nonpolar H atoms have been omitted for clarity.
inserted into the macrocyclic cavity, connected by a complex
network of hydrogen bonds, implicating both the ammonium
and amide NH protons. This observation suggested that these
cages would be suitable hosts for the binding of anionic entities,
where the amino amide moieties would serve as H-bonding
donor sites.
Molecular Recognition of N-Protected Dipeptides.
Although most of the pseudopeptidic cages were purified and
isolated as salts of trifluoracetic acid, they were transformed
into their corresponding free amino bases by an ion-exchange
resin prior to their use for the binding experiments (see the
Supporting Information for details).
ESI-MS Experiments. Motivated primarily by the potential
binding abilities of the pseudopeptidic cages 4a−4g, we faced
first the use of a convenient experimental approach for high-
throughput screening of different guests. Soft ionization mass
spectrometric techniques are among the most versatile and
widespread for this purpose as exemplified in high-throughput
investigations on drug discovery.20 This is mainly due to the
ability of soft ionization MS methods to gently transfer
noncovalent complexes from the solution to the gas phase, and
excellent reviews in the context of molecular recognition can be
found elsewhere.21 In particular, competition ESI-MS methods
have been extensively used to estimate binding affinities (both
qualitatively and quantitatively) in protein−ligand complexes
that proved to correlate well with the binding constants
determined by solution methods.22 The molecular recognition
properties of smaller synthetic receptors can also be addressed
by ESI-MS-based methods,23 for example, in the study of
alkylammonium,24 anion,25 or amino acid26 complexation.
In competition ESI-MS experiments, the ESI mass spectrum
of a selected host is investigated in the presence of a set of
potential guests (or a selected guest in the presence of a group
of hosts), so that identification and quantification of different
The final structures of all the cages were confirmed by the
full spectroscopic characterization of the compounds (see the
1
Supporting Information). The H and 13C NMR data showed
the expected D3 symmetry of the systems, while the high-
resolution MS spectra confirmed the identity of the macro-
bicyclic architecture. Moreover, we were able to grow crystals of
4a (chx; R = iPr) as the corresponding perchlorate salt, suitable
for X-ray diffraction studies. The macrobicyclic molecule
appeared fully protonated, and thus, six perchlorate anions
were found per cage unit, accompanied by several water
molecules. The cagelike architecture was fully confirmed in the
solid state (Figure 1A), although the average D3 symmetry
found in solution by NMR was broken in the crystal structure.
The three cyclohexane rings showed a perfect chair
conformation with the amide substitutions in a trans-
diequatorial conformation, setting the amide NH anti to the
CH of the chiral centers of the cyclohexane. The isopropyl side
chains are set in a pseudoequatorial conformation, pointing out
of the macrobicyclic cavity. Besides, each hydrogen atom
attached to the chiral center of the amino acid moiety is syn to
the closest amide NH. Overall, all these conformational
preferences are in good agreement with the characteristic
behavior found in related macrocycles and also with the results
obtained by molecular modeling of the imine precursor.
Among the six perchlorate anions present in the asymmetric
unit, four of them are in closer contact with the macrobicyclic
structure (Figure 1B). These perchlorate anions are partially
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host−guest complexes are simultaneously measured. In the
present work, we used competition ESI-MS for the initial
screening of the binding abilities of the synthetic pseudopepti-
dic cages toward N-protected dipeptides. In the first round of
experiments, we selected four representative cages to gauge
how the structure of each host modulates the binding process.
These cages bear (1) the rigid cyclohexane moiety (chx) with
aliphatic (4a; R = iPr) and polar (4c; R = CH2OH) side chains
or (2) a more flexible linear spacer of different lengths (Et/Pr)
and from different amino acids (4e, ethylene spacer with R =
Bn; 4g, propylene spacer with R = iPr). Besides, to determine
the effect of the macrobicyclic structure, an open-chain tripodal
derivative was included in the study as a control and revealed a
lesser extent of adduct formation in agreement with lower
binding affinities (data not shown). As the substrates, we
considered three N-(benzyloxy)carbonyl (Z) dipeptides
derived from alanine and phenylalanine in different sequences,
namely, Z-AA-OH, Z-AF-OH, and Z-FF-OH. The competition
ESI-MS experiments were carried out as follows: To a stock
solution containing an equimolar (5 × 10−5 M) mixture of the
4a, 4c, 4e, and 4g receptors in CH3CN/MeOH (2:1) in the
presence of 0.5% trifluoroacetic acid were added equimolar
amounts of each dipeptide in separated experiments, and the
resulting solutions were analyzed by positive ESI-MS. We
observed the formation of the supramolecular complexes
between every cage and every substrate, all of them showing
a 1:1 cage:dipeptide stoichiometry. These noncovalent species
were observed in their respective ESI mass spectra mainly as
doubly ([M + 2H]2+) charged species accompanied by singly
charged ([M + H]+) species, though to a lesser extent. We took
the ratio between the peak intensities of every 1:1 complex (in
all charge states) with respect to the peak intensities of the free
host (in all charge states) as an indication of the relative
binding affinities between the 4a, 4c, 4e, and 4g hosts and each
N-protected dipeptide. The obtained results are plotted in
Figure 2.
4g cages, we concluded that the rigidity imposed by the
cyclohexane moiety is beneficial for the molecular recognition
of the dipeptides. Overall, the full analysis of the ESI-MS data
rendered several general trends: (1) the rigidity of the
cyclohexane moiety within the cage architecture improves the
binding and (2) the side chains of the receptors are not
innocent in the recognition phenomena: Ser and Val improve
the interaction, while Phe reduces it. Interestingly, the
combination of the cyclohexane spacer with the Val side
chain had proved to be the best receptor in the case of the
macrocyclic family (compound 6 in Scheme 1B).13b
Focusing on the most efficient receptor 4c, we faced a second
round of experiments directed to find out the binding abilities
of 4c toward a wider scope of dipeptide sequences.
Accordingly, additional systematic variations in the sequence
of the dipeptide substrates were studied (entries 8 and 10−15
in Table 3). The complex formation ability of 4c with the guest
ions was investigated by competition ESI-MS on equimolecular
solutions of 4c and a mixture of all guests. Incidentally, two
pairs of guests were isobaric, namely, Z-AF-OH/Z-FA-OH and
Z-AE-OH/Ac-EF-OH, so two separate experiments containing
the nonoverlapped guests were conducted. Figure 3 illustrates
the competitive ESI mass spectrum of an equimolecular
solution of host 4c and a mixture of Z-AA-OH, Ac-EY-OH,
Ac-EF-OH, Z-FA-OH, Z-AY-OH, Z-AW-OH, and Z-FF-OH.
Each N-protected dipeptide formed noncovalent complexes
with the 4c host with a 1:1 stoichiometry as judged by ESI-MS.
We made the assumption that the ion efficiencies for the 1:1
adducts and the free hosts are similar. This essentially means
that the relative intensities in the ESI mass spectrum directly
reflect the relative concentrations in the injected solution. We
are aware that this only holds when the presence of the
substrate does not affect the ionization efficiency in the 1:1
complex, which is dominated by the contribution of the
receptor. Closely related competition ESI-MS experiments were
described by Jorgensen and Heck22a for a series of vancomycin
group antibiotics and bacterial cell wall peptide analogues, for
which association constants could be quantitatively extracted
directly from equimolar solutions of a given host and different
guests. Using this approach (see details in Table S3 in the
Supporting Information), we have estimated the association
binding constants of the 4c host and the series of N-protected
dipeptides. Estimated Kass values derived from ESI-MS
competition experiments are collected in Table 2 for receptor
4c in CH3CN/CH3OH (2:1) mixtures. In general, we observed
a good correlation between the Kass values derived from ESI-
MS and those values determined by strictly solution-based
methods such as NMR (see below). Notice that both
techniques (i) gave comparable absolute Kass values, (ii)
suggest moderate selectivity for N-Ac dipeptides, and (iii)
clearly identify the Ac-EF-OH and Ac-EY-OH sequences as the
most specific for the 4c host.28
Figure 2. Relative intensities of the complexed/free receptor observed
by ESI-MS competition experiments.
1H NMR and DOSY Experiments. We also aimed to tackle
the interaction between the pseudopeptidic cages and the
dipeptides by NMR, which provides more precise information
about the binding process. Initially, we decided to study the
complex between 4a and Z-AF-OH, since the structurally
related system was deeply characterized in the case of the
macrocyclic hosts (such as compound 6 depicted in Scheme
1B, which interacts with Z-AF-OH dipeptide). Thus, we
acquired the 1H NMR spectrum (500 MHz, CDCl3, 298 K) of
a 1:1 host−guest mixture, and we compared it with those of the
partners alone (Figure 4). Several signals changed their
We can extract very important information from the
comparison of the obtained data (Figure 2). First, the results
suggested that the best receptor should be 4c, since it showed
invariably the highest proportion of bound species. We
hypothesized that this cage could establish a higher number
of H-bond interactions, implicating the OH groups of the Ser
side chains. On the other hand, cage 4e was shown to be the
less efficient host, probably due to a more flexible structure
(from the ethylene spacer) and to the presence of benzyl side
chains, which disfavor the participation of the amino amide
moieties in noncovalent interactions.27 From the results for 4a/
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Figure 3. Positive ESI mass spectrum of equimolar (5 × 10 −5 M) solutions of the host 4c and selected N-protected dipeptides using 2:1 CH3CN/
CH3OH mixtures.
1
Table 2. Binding Constants (Kass, M−1) Obtained by H NMR Titration (500 MHz, 298 K) and ESI-MS Competition
Experiments
a
b
c
entry
host
substrate
solvent
Kass (M−1
)
Kass (M−1
)
ΔG (kJ·mol−1
)
d
1
2
6
Z-AF-OH
Z-AF-OH
Z-AF-OH
Z-FF-OH
Z-AA-OH
Z-FA-OH
Z-AF-OH
Z-AF-OH
Z-AF-OH
Z-FF-OH
Z-AA-OH
Z-FA-OH
Z-AY-OH
Z-AW-OH
Z-AE-OH
Ac-EF-OH
Ac-EF-OH
Ac-EY-OH
Ac-EY-OH
Ac-EY-OH
CDCl3/CD3OH (100:1)
CDCl3/CD3OH (100:1)
CD3CN/CD3OH (100:15)
CD3CN/CD3OH (100:15)
CD3CN/CD3OH (100:15)
CD3CN/CD3OH (100:15)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
CD3CN/CD3OH (2:1)
161
e
−12.6
−17.0
−13.0
−12.7
−11.7
−9.4
−11.9
−13.2
−10.4
−13.4
−12.6
−12.3
−12.8
−12.7
−13.5
−9.9
4a
4a
4a
4a
4a
4a
4c
4e
4c
4c
4c
4c
4c
4c
4a
4c
4a
4c
4e
955
190
166
112
44
e
3
e
4
e
5
e
6
e
7
123
209
66
e
8
220
e
9
10
11
12
13
14
15
16
17
18
19
20
224
162
141
178
166
229
55
240
206
214
215
212
234
e
324
69
501
e
−14.3
−10.5
−15.0
−8.9
417
36
414
e
a
b
1
Obtained by H NMR titration, estimated error ≤15% (see the Supporting Information for details). Obtained by ESI-MS, estimated error ≤7%
(see the Supporting Information for details). Calculated from the NMR-based binding constant. Taken from ref 13b. Not measured by ESI-MS.
c
d
e
chemical shifts, supporting the interaction between them. All
the amide and carbamate NH signals moved downfield,
supporting the establishment of strong H-bonding interactions.
Besides, the proton attached to the α carbon of the Phe residue
of the dipeptide (Fα in Figure 4) moved upfield, while the
benzylic and CαH signals of the cage (d and c protons,
respectively) moved downfield. These observations are
consistent with a proton transfer from the carboxylic acid of
the substrate to the secondary amine of the receptor, leading to
the formation of an ion pair. An interesting behavior was also
observed for the aromatic signals of the Phe side chain of the
dipeptide. The protons in the meta and para positions (F3 and
F4 in Figure 4) moved upfield, while the ortho hydrogens (F2)
practically did not alter. Besides, these signals appeared as a
broad band within the complex, which suggests a restricted
motion of the Phe side chain. On the contrary, the aromatic
signals sourcing from the Z protecting group were less affected
by the interaction. These observations highlight the importance
of the aromatic residue in the carboxylic terminus and suggest a
close interaction with the aromatic rings of the receptor.29
The self-diffusion rate obtained by DOSY is also a useful
parameter for the characterization of supramolecular species in
solution.30 The formation of stable intermolecular complexes is
accompanied by an increase of the apparent size of the
molecules with the concomitant reduction of the self-diffusion
rate (D). In the case of macrocyclic receptor 6 with Z-AF-OH,
we also observed a change of the diffusion properties of both
the host and guest upon binding, which could be used to
confirm the formation of the corresponding complex (Table 3,
entries 1−3).13b The DOSY spectra of the macrobicyclic
receptor 4a showed a self-diffusion rate very similar to that
found for the [6 + Z-AF-OH] complex, in good agreement with
their similar molecular sizes (entry 4 in Table 3). Moreover, in
the DOSY spectrum of a sample with an equimolecular mixture
of 4a and Z-AF-OH, all the signals diffused with the same rate,
demonstrating the presence of a unique supramolecular species
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Figure 5. (A) Superposed DOSY (500 MHz, CDCl3, 298 K) spectra
for 4a, Z-AF-OH, and an equimolecular mixture of both. (B)
Molecular size (molar mass or molecular volume) versus the self-
diffusion rate. CPK models for (C) cage 4a and (D) the complex of 4a
with Z-AF-OH. For clarity, the carbon atoms of the dipeptide
substrate are represented in dark green.
1
Figure 4. H NMR (500 MHz, CDCl3, 298 K) spectra of 4a, Z-AF-
OH, and an equimolecular mixture of both (with arbitrary numbering/
lettering of the signals).
in solution (entry 5 in Table 3, Figure 5A). Thus, also in this
case, both the cage and the substrate increased their apparent
molecular size in solution upon binding (Table 3 and Figure
5A). Very remarkably, the data displayed in Table 3 indicate the
internal consistency of the DOSY experiments performed with
these systems, where a good correlation (R2 > 0.99) between
the molecular sizes and the DOSY-measured self-diffusion rates
was obtained (Figure 5B).
mixtures that would avoid the complication of the analysis of
the data due to dipeptide self-aggregation.13b The value of the
binding constant between 4a and Z-AF-OH was markedly
reduced in the CD3CN/CD3OH (100:15) mixture (entry 3,
Table 2), reflecting the importance of the intermolecular H-
bonding interactions for the stability of the supramolecular
complex. We studied the substrate sequence selectivity of 4a by
measuring the corresponding N-protected dipeptides with Ala
and Phe residues (entries 3−5), and we obtained the same
stability order as that observed by ESI-MS (Z-AF-OH > Z-FF-
OH > Z-AA-OH). Even more strikingly, the scrambled
sequence Z-FA-OH (entry 6) showed a much weaker binding
to 4a (ΔΔG = 3.6 kJ mol−1), meaning that the presence of an
aromatic residue at the C-terminus modulates the selectivity
toward dipeptides. To explain these results, we hypothesized
the inclusion of this aromatic residue within the inner cavity of
the host. Thus, a tentative proposal for the main interactions
between the pseudopeptidic cage and the dipeptide is shown in
Figure 6, where the combined action of electrostatic, H-
bonding, and hydrophobic contacts would explain the observed
selectivity.
NMR Titration Experiments. The quantitative measurement
of the interaction between 4a and Z-AF-OH was also
1
performed by H NMR titration experiments in the same
solvent as that used for the structurally related monomacrocycle
6. The simultaneous nonlinear fitting of the variations of
different NMR signals (both from the host and from the guest)
rendered an association constant of 955 M−1 (Table 2, entry 2),
which is nearly 1 order of magnitude higher than that found for
the monomacrocyclic derivative (entry 1 in Table 2), thus
supporting the beneficial effect of the cage architecture for the
binding of dipeptides.
Several additional cage−N-protected dipeptide host−guest
interactions were also analyzed by 1H NMR titration
procedures. The somehow stronger binding allowed us to
study the interaction in more polar and competitive solvent
Table 3. Self-Diffusion Rates Measured by DOSY Experiments (500 MHz, CDCl3, 298 K) with Different Pseudopeptide−
Dipeptide Host−Guest Systems
a
entry
sample
Z-AF-OH
molar mass (g·mol−1
)
volume (Å3)
D (10−6 cm2·s−1
)
b
1
2
3
4
5
370.4
829.2
379.6
914.1
7.6 0.2
b
6
6.0 0.2
b
[6 + Z-AF-OH]
4a
1202.6
1165.7
1536.1
1305.3
1273.8
1645.8
4.9 0.3
4.86 0.15
4.17 0.20
[4a + Z-AF-OH]
a
b
CPK volume calculated with Spartan ‘06. Taken from ref 13b.
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17 in Table 2; ΔΔG = 4.4 kJ mol−1), strongly supporting our
hypothesis for the explanation of the observed trends.
Taking into account the binding behavior with the studied
cages, we decided to investigate the replacement of phenyl-
alanine by tyrosine in the C-terminus of the dipeptide. The
obtained sequence (Ac-EY-OH) bears an electron-rich
aromatic residue with additional H-bonding properties and
would allow the binding phenomena to be monitored with a
different technique, taking advantage of the fluorescence
1
emission of the phenol group (see the next section). The H
NMR titration experiments with the three cages and the Ac-EY-
OH dipeptide showed a much stronger complexation with 4c
(see entries 18−20 in Table 2; ΔΔG = 4.5−6.1 kJ mol−1),
which again reinforces our general model for the interaction.
Besides, by comparing the binding of 4c with both the Ac-EF-
OH and Ac-EY-OH dipeptides (entries 17 and 19,
respectively), we concluded that the phenolic OH group
must be implicated in an additional stabilizing (ΔΔG = 0.7 kJ
mol−1) interaction with the host. Interestingly, in spite of the
much weaker interaction of these dipeptides with 4a, a very
similar difference was obtained due to the presence of the
phenolic OH (entries 16 and 18; ΔΔG = 0.6 kJ mol−1).
Fluorescence Spectroscopy Experiments. As anticipated
and due to the presence of the Tyr residue, the Ac-EY-OH
dipeptide shows a strong fluorescence emission band with a
Figure 6. Schematic representation of the tentative proposed model
for the interaction between a pseudopeptidic cage and an N-Z-
protected dipeptide bearing an aromatic residue at the carboxylic
terminus.
For the comparison between different receptors, we had to
use a slightly more polar mixture of solvents (2:1 CD3CN/
CD3OH) due to solubility issues of 4c. In this case, the
comparison of the binding constants between three different
cages and the Z-AF-OH dipeptide (entries 7−9 in Table 2)
rendered the stability order 4c > 4a > 4e. These results are fully
consistent with the data obtained by ESI-MS competition
experiments and underscore the importance of the overall
flexibility and of the nature of the side chains in the molecular
recognition properties of the pseudopeptidic cages. The
combination of a rigid cage structure with polar side chains
produced a more efficient host.
maximum at 304 nm upon excitation at 275 nm (1.5 × 10−4
M
in a 2:1 CH3CN/CH3OH mixture). The addition of increasing
amounts of the host 4c produced a quenching of the emission
(Figure 7). The decrease of the emission intensity at 304 nm
Focusing on the best receptor 4c, several systematic
variations in the sequence of the dipeptide substrates were
studied (entries 8 and 10−15). Also for this host, the presence
of an aromatic residue at the C-terminus favored the interaction
(for instance, compare entries 8 and 12 for scrambled
sequences), although the selectivity was lower than that with
4a (Val derivative). This effect could be due to the coexistence
of structurally different supramolecular complexes in solution,
where the aromatic ring of the Z protecting group would
displace the aromatic side chain of the amino acid from the host
cavity. This possibility could be present for all the hosts, but for
4a, an additional hydrophobic interaction between the Z group
and the iPr side chain (Figure 6) would stabilize the outer
disposition of the N-protecting group. In the case of 4c, the Ser
side chains form a highly polar surrounding periphery, favoring
the competition between the two aromatic rings (side chain
and Z) for the host cavity. Since the Z group is present in all
the dipeptides, the observed overall effect is a decreased
selectivity in 4c. Little difference was observed by comparing
the nature of the aromatic amino acid residue at the C-terminus
(entries 13 and 14), which also points to the possible
competition of the aromatic group of the carbamate.
Interestingly, the introduction of an additional carboxylic acid
at the C-terminus (entry 15) had a stabilizing effect comparable
to that of an aromatic ring, suggesting the possibility of
implementing both residues in a cooperative fashion. Following
this rationale, we envisioned that a dipeptide bearing an
aromatic residue at the C-terminus and a polar carboxylic
residue at the N-terminus should display much stronger
binding with 4c than with 4a. Besides, to improve the
selectivity, the N-protecting group was replaced by acetyl,
which lacks the aromatic ring and is also a more realistic model
of a peptide bond. Very gratifyingly, the binding constant of Ac-
EF-OH was much higher with 4c than with 4a (entries 16 and
Figure 7. Emission fluorescence spectra of Ac-EY-OH (1.5 × 10−4
M
in 2:1 CH3CN/CH3OH) at increasing concentrations ((0−9) × 10−4
M) of the receptor 4c. Inset: relative fluorescence quenching (I/Io) at
304 nm versus the receptor concentration (mM) for 4a, 4c, and 4e.
was accompanied by a slight increase of the emission at ∼400
nm, with an isosbestic point at 360 nm that can be taken as
proof of the formation of a new species in solution. The
analysis of the quenching process with the Stern−Volmer
equation (see the Supporting Information) indicated that both
dynamic and static quenching mechanisms are present in the
system. This fact additionally supports the formation of a
supramolecular complex between 4c and Ac-EY-OH in the
ground state (static quenching). Moreover, this static
quenching suggests the participation of the phenol residue of
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the Tyr moiety in the binding with 4c. The binding abilities of
the different cages were also studied by fluorescence spectros-
copy. The plot of the decrease of the fluorescence intensity at
304 nm versus the cage concentration for the three hosts 4a, 4c,
and 4e (Figure 7, inset) confirmed the data obtained by NMR
titrations, showing the stability order 4c > 4a > 4e.
Model for the [4c + Ac-EY-OH] Supramolecular Complex.
Taking into account all the data obtained in our study, we also
aimed to propose a reasonable binding mode for the interaction
between the receptor 4c and the dipeptide Ac-EY-OH. We
selected this supramolecular complex for several reasons. This
was the most stable species analyzed by NMR titration (and
ESI-MS) in the most competitive solvents considered in our
study. Moreover, this motif can represent a model for one of
the target sequences for kinases, with important biological
implications.31 Several general considerations were taken into
account for building such a model. First, the main interactions
are due to the electrostatic H-bonds between the carboxylates
of the dipeptide and the amino amide moieties (as generally
observed by the NMR induced chemical shifts in the titration
experiments). These anion binding motives resemble the
contacts observed between the cage and the perchlorate anions
in the crystal structure of the 4a·6HClO4 salt (Figure 1B). In
the case of 4c, also the OH from the side chains could form H-
bonds with the corresponding anions, as suggested from the
stronger interaction observed with this Ser derivative. More-
over, our binding studies clearly showed a remarkable
preference for aromatic residues at the C-terminus, implying
the participation of the aromatic ring in the binding
phenomena. This is also supported by the quenching of the
tyrosine side chain fluorescence upon the addition of the cages
(Figure 7). A proposal for the [4c + Ac-EY-OH] supra-
molecular complex, obtained by molecular mechanics calcu-
lations, is shown in Figure 8. The carboxylate at the C-terminus
and that of the glutamic side chain set several H-bonds with
two of the amino amide moieties of the receptor (with the
amino groups partially protonated in the model). Additional H-
bonds with the corresponding OH of the side chains are
possible. The tyrosine side chain of the dipeptide is included
within the cage cavity, showing aryl stacking interactions with
the aromatic rings of the receptor. Moreover, the phenolic OH
is implicated in additional H-bonding interactions (both as a
donor and as an acceptor, Figure 8) with the third amino amide
moiety of the cage. This specific interaction could explain the
observed quenching of the fluorescence emission of the
substrate, since this H-bond pattern would increase the
electronic density on the aryl ring (by incipient abstraction of
the OH proton). Experimental support for this binding mode
was also obtained by ROESY experiments (500 MHz, D2O, 288
K) of a 6 mM sample of 4c in the presence of a 5-fold excess of
Ac-EY-OH. A relatively weak but measurable NOE was
observed between the aromatic proton of the cage and the
proton ortho to the phenol hydroxyl of the tyrosine moiety,
supporting the existence of the proposed complex (Figure 8B).
Although we cannot discard the participation of other binding
modes in solution, the proposed complex fully agrees with all
the experimental data and draws a plausible picture of the
recognition process.
Figure 8. (A) Two views of the proposed model for the structure of
the supramolecular complex formed by the receptor 4c (stick) and the
dipeptide Ac-EY-OH (CPK). (B) Schematic representation of the
complex with the corresponding substrate in blue and section of the
2D ROESY spectrum showing the intermolecular contacts as a red
double-headed arrow in the structure.
allows the implementation of large molecular diversity
regarding the size, flexibility, polarity, and potential interaction
sites on the molecular cages. The molecular recognition of the
obtained cages toward N-protected dipeptides has been deeply
studied using a combination of different techniques. The
competition ESI-MS experiments allowed us to screen the
binding abilities for the determination of the most efficient
receptors. Moreover, for the best host, we were able to obtain
the quantitative assessment of the binding interactions with a
family of dipeptides by mass spectrometry. The ESI-MS-
obtained binding constants are in very good agreement with
those obtained by NMR. In general, ESI mass spectrometric
techniques are very restrictive regarding the systems for which
absolute binding constants can be estimated directly on the
basis of the relative intensities of the complex and the free
receptor. In fact, it is important to emphasize that noncovalent
binding based on electrostatic interactions is reinforced in the
absence of solvent whereas hydrophobic interactions become
less important. In this context, it is crucial to validate whether
the observed complexes truly reflect the specific interactions
that are present in solution. If intensity ratios truly reflect the
ratio of the concentrations in solution, as demonstrated in the
present work, ESI-MS can be regarded as a powerful alternative
method for high-throughput screening of recognition processes.
For selected host−guests pairs, a detailed study of the
supramolecular complexes in solution has been performed by
CONCLUSIONS
■
The efficient synthesis of large pseudopeptidic cages has been
accomplished by a [3 + 2] multicomponent reductive
amination reaction. The synthetic strategy is very robust and
1
NMR, including H NMR titrations and DOSY and ROESY
studies. Thus, the quantitative binding constants were
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J = 12.6 Hz, 6H, CH2NH), 2.86 (d, J = 6.8 Hz, 6H, CH), 2.06 (d, J =
9.5 Hz, 6H, CH2), 1.85 (m, 6H, CH), 1.78 (d, J = 6.9 Hz, 6H, CH2),
1.45−1.25 (m, 12H, CH2), 0.99 (t, J = 7.3 Hz, 36H, CH3). 13C NMR
(100 MHz, MeOD-d4): δ = 176.6 (CO), 140.8 (CAr), 127.8 (CHAr),
96.4 (CH), 69.8 (CH), 53.7 (CH2NH), 34.1 (CH), 33.0 (CH2), 25.8
(CH2), 19.9 (CH3), 19.7 (CH3). HRMS (m/z) (M + 1): calcd for
C66H108N12O6, 1165.8594; found, 1165.8665.
measured, and a reliable picture of the mode of binding can be
proposed. More interestingly, the wide structural diversity
accessible for the host molecules permitted the study of the
molecular recognition process in media with different polarities,
ranging from nonpolar organic solvents to very competitive
media. We extracted several general trends in the structural
parameters of the receptors: the rigid cagelike structure leads to
more efficient hosts, and the side chains can participate in the
binding. Overall, the data rendered the cage containing the
cyclohexane spacer and the serine amino acid (4c) as the most
efficient receptor. With respect to the dipeptide substrate
structure, we observed some selectivity for those bearing an
aromatic residue at the C-terminus and an acetyl group at the
N-terminus. This trend allowed us to study a biologically
interesting sequence (EY) as a model substrate by NMR and
fluorescence spectroscopy. Both techniques showed excellent
agreement regarding the host efficiencies. The strongest
supramolecular complex was qualitatively characterized in
water by ROESY experiments. These results seemed promising
for the future applications of related systems in biological
processes since the EY dipeptide is a target sequence for
kinases, which are phosphorylating enzymes with key
implications for cell regulation and metabolism. However, in
this regard, more efficient receptors able to operate in water at
neutral pH must be designed and developed. Studies in these
directions are under way and will hopefully render optimized
artificial receptors with true biological applications. Moreover,
our findings have a fundamental relevance since we were able to
build an efficient and selective binding motif from simple and
readily accessible pseudopeptidic cages, where further structural
modifications can be carried out.
4b was isolated as a trifluoroacetate salt. Yield: 48 mg, 30%; RP-
1
HPLC (gradient from 10% to 60% CH3CN). H NMR (500 MHz,
CDCl3: MeOD-d4): δ = 7.23−7.00 (30H, CHAr), 6.45 (6H, CHAr),
3.50 (m, 6H, CH), 3.28 (d, J = 12.2 Hz, 6H, CH2NH), 3.12 (d, J =
12.2 Hz, 6H, CH2NH), 3.25 (m, 6H, CH), 2.86 (dd, J = 13.1 and 5.5
Hz, 6H, CH2), 2.75 (dd, J = 13.1 and 9.4 Hz, 6H, CH2), 1.51 (d, J =
7.9 Hz, 6H, CH2), 1.45 (d, J = 12.6 Hz, 6H, CH2), 1.07 (t, J = 9.6 Hz,
6H, CH2), 0.70 (m, 6H, CH2). 13C NMR (100 MHz, MeOD-d4): δ =
174.1 (CO), 138.2 (6CAr), 137.2 (6CAr), 129.3 (12CHAr), 128.5
(12CHAr), 127.9 (6CHAr), 126.8 (6CHAr), 63.1 (CH), 52.2 (CH),
51.5 (CH2NH), 39.5 (CH2Ar), 32.0 (CH2), 24.5 (CH2). HRMS (m/
z) (M + 1): calcd for C90H108N12O6, 1453.8594; found, 1453.8539.
4c was isolated as a trifluoroacetate salt. Yield: 86.7 mg, 59%; RP-
1
HPLC (gradient from 10% to 50% CH3CN). H NMR (500 MHz,
MeOD-d4): δ = 7.04 (6H, CHAr), 3.88 (dd, J = 10.8 and 5.2 Hz, 6H,
CH2OH), 3.82 (dd, J = 10.8 and 5.2 Hz, 6H, CH2OH), 3.79 (m, 6H,
CH), 3.73 (d, J = 13.1 Hz, 6H, CH2NH), 3.53 (d, J = 13.1 Hz, 6H,
CH2NH), 3.23 (t, J = 5.2 Hz, 6H, CH), 2.16 (d, J = 12.2 Hz, 6H,
CH2), 1.80 (d, J = 7.3 Hz, 6H, CH2), 1.48−1.27 (m, 12H, CH2). 13C
NMR (100 MHz, MeOD-d4): δ = 174.9 (CO), 140.2 (CAr), 125.4
(CHAr), 65.0 (CH), 63.1 (CH2OH), 54.2 (CH), 53.1 (CH), 49.8
(CH2NH), 33.3 (CH2), 25.9 (CH2). HRMS (m/z) (M + 1): calcd for
C54H84N12O12, 1093.6406; found, 1093.6425.
General Procedure for the Anion-Templated Macrocycliza-
tion Reaction (Synthesis of 4d−h). Pseudopeptidic bis-
(amidoamine) 1d (68 mg, 0.26 mmol) was dissolved in 8 mL of
CHCl3/CH3OH (9:1) inside a flask under nitrogen. Benzene-1,3,5-
tricarboxylate 5·TBA (82 mg, 0.09 mmol) was dissolved in 8 mL of
CHCl3/CH3OH (9:1) and added to the solution of 1d. Benzene-1,3,5-
tricarbaldehyde (2) (29 mg, 0.17 mol) was dissolved in 8 mL of
CHCl3/CH3OH (9:1) and added to the solution mixture of 1d + 5·
TBA. The mixture was stirred overnight, then a large excess of BH3·py
complex (330 μL of a 8 M solution of BH3·py, 2.6 mmol) was carefully
added, and the mixture was allowed to react for 24 h. before being
hydrolyzed (concd HCl, to acidity) and evaporated to dryness. The
residue obtained was dissolved in water and basified with 1 N NaOH.
The product was extracted with CH2Cl2. The combined organic layers
were dried (MgSO4), and the solvents were evaporated in vacuum.
The product was purified using reversed-phase flash chromatography,
yielding 4d as a trifluoroacetate salt. Yield: 12 mg, 24%; RP-HPLC
(gradient from 5% to 30% CH3CN). 1H NMR (400 MHz, MeOD-d4):
δ = 7.64 (s, 6H, CHAr), 4.29 (d, J = 12.8 Hz, 6H, CH2NH), 4.14 (d, J
= 12.8 Hz, 6H, CH2NH), 3.64 (d, J = 5.9 Hz, 6H, CH), 2.98 (m, 6H,
CH2), 2.84 (m, 6H, CH2), 2.15 (m, 6H, CH), 0.97 (d, J = 6.8 Hz,
18H, CH3), 0.89 (d, J = 6.8 Hz, 18H, CH3). 13C NMR (100 MHz,
MeOD-d4): δ = 169.2 (CO), 163.0 (c, J = 34.9 Hz, COTFA), 136.4
(CHAr), 133.9 (CAr), 67.7 (CH), 51.9 (CH2NH), 40.0 (CH2), 31.5
(CH), 19.3 (CH3), 18.4 (CH3). HRMS (m/z) (M + 1): calcd for
C54H90N12O6,1003.7188; found, 1003.7195.
EXPERIMENTAL SECTION
■
General Experimental Methods. Reagents and solvents were
purchased from commercial suppliers and were used without further
purification. Compounds 1a−g were prepared as previously
described.16,18,19 Preparative reversed-phase purifications were per-
formed on an MPLC instrument with a C18 column as the stationary
phase and CH3CN/water with 0.1% TFA as the mobile phase. The
cages obtained as TFA salts were transformed into the free-base
amines using an ion-exchange resin prior to the binding studies.
Analytical RP-HPLC was performed using a C8 (15 × 0.46 cm, 5 μm)
column. CH3CN/H2O mixtures containing 0.1% TFA at 1 mL/min
were used as the mobile phase, and the monitoring wavelength was set
at 220 and 254 nm. The NMR spectra were performed on
1
spectrometers operating at 500 or 400 MHz for H NMR and 125
or 100 MHz for 13C NMR. The chemical shifts are reported in parts
per million using tetramethylsilane (TMS) as a reference. High-
resolution mass spectrometry (HRMS) was performed on a UPLC
system coupled with an orthogonal acceleration time-of-flight (oa-
TOF) analyzer equipped with an electrospray ionization source.
General Procedure for the Macrocyclization Reaction with
the Preorganized Precursors (Synthesis of 4a−c). A solution of
33.5 mg of benzene-1,3,5-tricarbaldehyde (0.21 mmol) in 15 mL of
CH3OH was added to a solution of 1a (96.8 mg, 0.31 mmol) in 15 mL
of CH3OH. The mixture was stirred overnight. Then 390 μL of an 8 M
solution of BH3·py complex (3.1 mmol) was carefully added, and the
mixture was allowed to react for 16 h before being hydrolyzed (concd
HCl, to acidity) and evaporated to dryness. The residue obtained was
dissolved in water and basified with 1 N NaOH, and the product was
extracted with CH2Cl2. The combined organic layers were dried over
MgSO4, and the solvents were evaporated in vacuum. The product was
purified using reversed-phase flash chromatography, yielding 4a as a
trifluoroacetate salt. Yield: 89 mg, 47%; RP-HPLC (gradient from 10%
1
4e was isolated as a trifluoroacetate salt. Yield: 24 mg, 24%. H
NMR (400 MHz, MeOD-d4): δ = 7.69 (s, 6H, CHAr), 7.37−7.22
(18H, CHAr), 7.15 (12H, CHAr), 4.34 (d, J = 12.9 Hz, 6H, CH2NH),
4.20 (d, J = 12.9 Hz, 6H, CH2NH), 4.08 (dd, J = 10.2 and 5.1 Hz, 6H,
CH), 3.24 (dd, J = 12.9 and 5.1 Hz, 6H, CH2), 3.02 (dd, J = 12.9 and
10.2 Hz,, 6H, CH2), 2.61 (m, 6H, CH2), 2.34 (m, 6H, CH2). 13C
NMR (100 MHz, MeOD-d4): δ = 169.2 (CO), 162.9 (c, J = 34.9 Hz,
COTFA), 135.6 (CAr), 135.2 (CHAr), 134.3 (CAr), 130.4 (12C, CHAr),
129.9 (12C, CHAr), 128.8 (6C, CHAr), 63.6 (CH), 51.3 (CH2NH),
39.5 (CH2), 38.2 (CH2). HRMS (m/z) (M + 1): calcd for
C78H90N12O6, 1291. 7188; found, 1291.7241.
In the case of 4f, the reaction mixture was hydrolyzed with satd aq
1
to 50% CH3CN). H NMR (400 MHz, MeOD-d4): δ = 6.75 (s, 6H,
NH4Cl. The product was isolated as a trifluoroacetate salt. Yield: 8.1
1
CHAr), 3.81 (m, 6H, CH), 3.47 (d, J = 12.6 Hz, 6H, CH2NH), 3.16 (d,
mg, 11%. H NMR (400 MHz, MeOD-d4): δ = 7.10 (6H, CHAr),
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3.64−3.48 (24H, 12 × CH2OtBu + 12 × CH2NH), 3.44−3.30 (m,
12H, CH2), 3.26 (dd, J = 6.4 and 4.8 Hz, 6H, CH), 1.19 (s, 54H,
CH3). 13C NMR (500 MHz, MeOD-d4): δ = 175.5 (CO), 140.9 (CAr),
127.7 (CHAr), 74.6 (C), 64.1 (CH), 63.5 (CH2OtBu), 53.1 (CH2NH),
40.1 (CH2), 27.9 (CH3). HRMS (m/z) (M + 1): calcd for
C66H114N12O12, 1267.8750; found, 1267.8799.
In the case of 4g, the product was purified by preparative reversed-
phase chromatography and then by flash chromatography on silica gel
using CHCl3 as the eluent, slowly increasing the polarity with MeOH
and several drops of aqueous ammonia. The product was isolated as
the free amine. Yield: 13 mg, 13%. 1H NMR (400 MHz,
CDCl3:MeOD-d4): δ = 7.22 (s, 6H, CHAr), 3.69 (d, J = 13.3 Hz,
6H, CH2NH), 3.66 (d, J = 13.3 Hz, 6H, CH2NH), 3.13 (m, 12H,
CH2), 2.86 (d, J = 5.9 Hz, 6H, CH), 1.92 (m, 6H, CH), 1.57 (m, 6H,
CH2), 0.95 (d, J = 3.1 Hz, 18H, CH3), 0.93 (d, J = 3.9 Hz, 18H, CH3).
13C NMR (100 MHz, CDCl3/MeOD-d4): δ = 175.8 (6 × CO), 140.7
(6 × CAr), 128.0 (6 × CHAr), 68.8 (6 × CH), 53.3 (6 × CH2NH),
36.8 (6 × CH2), 32.2 (6 × CH), 30.2 (3 × CH2), 19.7 (6 × CH3),
19.0 (6 × CH3). HRMS (m/z) (M + 1): calcd for C57H96N12O6,
1045.7656; found, 1045.7727.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Further experimental details, characterization data, X-ray
diffraction data, ESI-MS determination of Kass, NMR titration
procedures and data, fluorescence spectra, additional NMR
spectra, and crystal structure data in CIF format. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*Fax: +34-964728214. E-mail: luiss@uji.es.
*Fax: +34-932045904. E-mail: ignacio.alfonso@iqac.csic.es.
■
(5) (a) Henley, P. D.; Kilburn, J. D. Chem. Commun. 1999, 1335−
1336. (b) Fujita, D.; Suzuki, K.; Sato, S.; Yagi-Utsumi, M.; Yamaguchi,
Y.; Mizuno, N.; Kumasaka, T.; Tanaka, M.; Noda, M.; Uchiyama, S.;
Kato, K.; Fujita, M. Nat. Commun. 2012, 3, No. 1093. (c) Tashiro, S.;
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(6) (a) Soo Yoon, S.; Still, W. C. Angew. Chem., Int. Ed. Engl. 1994,
33, 2458−2460. (b) Hong, J.-I.; Namgoong, S. K.; Bernardi, A.; Still,
W. C. J. Am. Chem. Soc. 1991, 113, 5111−5112. (c) Fiehn, T.;
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Am. Chem. Soc. 2006, 128, 11206−11210. (e) Wareham, R. S.;
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This work was supported by the Spanish Ministry of Economy
and Competitiveness (MINECO; CTQ2012-38543-C03 proj-
ect). E.F. thanks CSIC and the European Social Fund for
personal financial support (JAE-doc program). We thank SCIC
of the Universitat Jaume I for providing us with mass
́
spectrometric facilities and Dr. Yolanda Perez for her helpful
assistance with the acquisition of specific NMR experiments.
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