Synthesis and conformational studies of peptido-squaramide foldable modules: A new class of turn-mimetic compounds

Article abstract of DOI:10.1039/c2ob06715c

The β-turn unit is one of the most important secondary structure elements in proteins. The access to new conformationally controlled foldable modules can afford compounds with interesting bioactivities. Here, we describe a new family of peptido-squaramide foldable modules based on the considerable potential of the squaramide unit as a hydrogen bond donor and acceptor as well as the low rotational barrier of the C-N bond. The conformational analysis by NMR of these modules in chloroform and acetonitrile solution shows that a disecondary squaramide with the 4-aminobutyric acid in one of its substituents can mimic the β-turn structure driven by the formation of an intramolecular hydrogen bonded ten-membered ring. This structure, although flexible, has been successfully combined with dipeptide chains to induce the formation of a hairpin-like structure driven by the formation of several cross-strand intramolecular hydrogen bonds.

Full text of DOI:10.1039/c2ob06715c

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Organic &
Biomolecular
Chemistry
Cite this: Org. Biomol. Chem., 2012, 10, 1914
www.rsc.org/obc
PAPER
Synthesis and conformational studies of peptido-squaramide foldable
modules: a new class of turn-mimetic compounds†
Luis Martínez, Angel Sampedro, Elena Sanna, Antoni Costa* and Carmen Rotger*
Received 10th October 2011, Accepted 8th December 2011
DOI: 10.1039/c2ob06715c
The β-turn unit is one of the most important secondary structure elements in proteins. The access to new
conformationally controlled foldable modules can afford compounds with interesting bioactivities. Here,
we describe a new family of peptido-squaramide foldable modules based on the considerable potential of
the squaramide unit as a hydrogen bond donor and acceptor as well as the low rotational barrier of the
C–N bond. The conformational analysis by NMR of these modules in chloroform and acetonitrile
solution shows that a disecondary squaramide with the 4-aminobutyric acid in one of its substituents can
mimic the β-turn structure driven by the formation of an intramolecular hydrogen bonded ten-membered
ring. This structure, although flexible, has been successfully combined with dipeptide chains to induce the
formation of a hairpin-like structure driven by the formation of several cross-strand intramolecular
hydrogen bonds.
of small molecules that mimic the β-turn structure when the bio-
Introduction
logical activity of many peptides was related with a turn contain-
ing conformation.7 This can be achieved through two different
Proteins and peptides exert their biological activity through
approaches: the use of covalent bonds8 to introduce the confor-
small regions of well-defined secondary structures such as
α-helix, β-turn and β-sheet1 which can be mimicked by means of
mational constrains that stabilise the turn geometry, and the
several different foldable modules.2 Unfortunately, small and
induction of the folded state of linear molecules through the for-
mation of intramolecular hydrogen bonds.5,9 In the latter, when
medium size peptides and peptidomimetics are often unstruc-
tured in solution and rarely possess adequate function. Therefore,
the access to new conformationally controlled foldable modules3
is of great interest because they can be incorporated into peptide-
like structures to afford compounds with compelling bioactivities
and better pharmacokinetic properties than peptides.4 Thus,
hybrid sequences are acquiring increasing importance in the
design of biologically active peptides,5 because of the enhanced
stability of these modified backbones to proteolytic cleavage. In
this sense, unnatural scaffolds designed to reproduce structural
protein features while retaining the property to bear the chemical
diversity of the amino acid side chains have a potential appli-
cation in drug discovery.6
the conformational control is needed, one useful strategy is the
incorporation of proline residues to stabilise the folded confor-
mation due to its β-turn propensity.10
Alternatively, other foldable modules of a different nature can
be used as proline substitutes. In this context, for example, β, γ,
and δ-amino acids have proved to encourage turn formation11
and a combination of several non-natural residues of an aliphatic
or aromatic nature are well suited to serve as turn mimetics or
β-hairpin initiators.
Many successful aliphatic folding modules contain a saturated
carbon chain separating amide or urea groups. The squaramide
scaffold, as well as the urea group, share with the amide linkage
a number of interesting features, such as planarity, polarity, and
hydrogen bonding capability12 and represent an interesting
surrogate.
The β-turn unit is one of the most important secondary struc-
ture elements in proteins. The initial interest on the studies of its
conformational properties increased and focused on the synthesis
Like amides, and due to the restricted rotation about the C–N
bond, double secondary squaramides may exist, in principle, as a
mixture of anti/syn conformers (Fig. 1). The extended anti/anti
conformer allows the simultaneous participation of the two NH
groups in the formation of hydrogen bonding interactions.
However the syn/anti states lead to folded structures. On the
other hand, the syn/syn conformer is not significantly populated
due to the mutual steric hindrance of the substituents. It has been
estimated that the rotation about the C–N bond for a secondary
Departament de Química, Universitat de les Illes Balears, Crta de
Valldemosa km 7.5, Palma, Spain. E-mail: carmen.rotger@uib.es;
Fax: +34 971 173426; Tel: +34 971 259576
†Electronic supplementary information (ESI) available: Detailed exper-
imental procedures and the characterisation data of new compounds. The
1H and 13C NMR spectra for compounds 1–4 and 9–15. Stacked plots
1
of H RMN spectra obtained in concentration and temperature variation
experiments for compounds 1–3. Selected fragments of NOESY spec-
trum of 4. See DOI: 10.1039/c2ob06715c
1914 | Org. Biomol. Chem., 2012, 10, 1914–1921
This journal is © The Royal Society of Chemistry 2012

View Article Online
Results and discussions
The conformational preferences of the new squaramide foldable
modules were investigated on model compounds to identify
those that may preferentially adopt adequate folding patterns.
Thus, compounds 1–3 (Chart 1) were designed for this
purpose. In them, the squaramide moiety is linked to two differ-
ent substituents, an n-butyl chain which is common to the three
compounds, and a dipeptide formed by one of the three amino
acids of different length, namely glycine, β-alanine and γ-amino-
butyric acid (GABA) respectively, linked to the natural amino
acid L-phenylalanine. To verify the propensity of compounds
1–3 to induce folded structures, we investigated their capability
to form intramolecular hydrogen bonds between the oxygen of
the carbonyl group of the amino acid and the squaramide NH
proton of the n-butyl substituent.
Compounds 1–3 were synthesised according to conventional
diethyl squarate and amine condensation reactions (Scheme 1).
In short, diethyl squarate was condensed with one equivalent of
n-buthylamine, as in a previously reported method,13 to obtain
the n-butylsquaramide ethyl ester 5. Subsequently, 5 was separ-
ately condensed with the sodium salt of glycine, β-alanine
and γ-aminobutyric acid (GABA) in ethanol to afford the three
disquaramide compounds 6–8. Finally, the terminal carboxylic
groups of 6–8 were coupled with L-phenylalanine methyl
ester using O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HBTU) as a coupling reagent, affording
the three model compounds 1–3.
We investigate the formation of well-defined folded structures
in solution using a combination of NMR spectroscopic tech-
niques. From our previous work on squaramide-based foldable
modules13 and due to the conformational properties and the
hydrogen bonding propensity of disecondary squaramides, we
expect to have in solution a mixture of syn/anti conformers that
Fig. 1 Top: representation of the conformational equilibrium of di-
secondary squaramides. Bottom: hydrogen bonding options for the new
squaramide-based turn fragment stabilised by intramolecular hydrogen
bonding interaction.
squaramide has energetically accessible barriers to rotation
(∼ 63 kJ mol−1), slightly lower than the barrier to rotation
reported for tertiary squaramides (∼ 71 kJ mol−1) and are com-
parable to what is found for carbamates (∼ 65 kJ mol−1). Indeed,
the C–N bond of a secondary squaramide also compares well
with the C–N bond of proline derivatives. Secondary squara-
mides are easy to synthesise from squaric esters and amines. The
combination of these two features convert to disecondary squara-
mides in a very interesting foldable module.
In this sense, we have described a squaramide-based foldable
module that contains a donor atom located at the δ position of
the alkyl chain of a disecondary squaramide.13 The folding capa-
bility of this module is based on the low rotational barrier of the
squaramide unit and driven by the intramolecular hydrogen bond
formation of a nine-membered-ring. Oligosquaramide14 com-
pounds based on this module nicely fold in a hairpin-like struc-
ture and have been successfully used as preorganised linear
precursors in macrocyclisation reactions in protic solvents.
In order to enrich the category of squaramide foldable
modules and test their ability to promote hairpin structures, we
describe here the synthesis and the conformational analysis of a
new family of squaramide-based foldable modules that combine
two motifs, a disecondary squaramide moiety and α-, β- or
γ-amino acid residues (Fig. 1). The syn/anti conformers of these
squaramide compounds driven by the formation of an intramole-
cular hydrogen bond between the carbonyl group of the amino
acid residue and the squaramide NH proton of the neighbour
substituent, result in the formation of eight-, nine-, and ten-
membered-rings respectively. Moreover, the new squaramide-
based turn module can be easily combined with peptide chains
to build β-hairpin-like molecular structures mimicking the fea-
tures of two antiparallel strands connected by a short loop
segment. Consequently, these small turn-forming fragments
could be a valuable tool pursuing bioactive ligand conformations
in rational drug design.
Chart 1 Structure of squaramides 1–3.
Scheme 1 Synthesis of squaramide model compounds 1–3.
Org. Biomol. Chem., 2012, 10, 1914–1921 | 1915
This journal is © The Royal Society of Chemistry 2012

View Article Online
can also aggregate to give higher order species depending on the
concentration and solvent properties. All these different species
in equilibrium can be characterised by analysing the effect on
the NMR chemical shifts produced by variations in temperature,
and concentration. Assignment of the stereochemistry of the
species is based on the evidence that signals of syn-N-substitu-
ents are shifted downfield relative to those of anti, due to the
paramagnetic influence of the squaramide carbonyl group. As in
amides, the NMR chemical shift values for the squaramide NH
protons are very sensitive to hydrogen bond formation. However,
for small molecules like 1–3, equilibration among non-hydrogen
bonded and hydrogen-bonded states is usually rapid on the
NMR time scale. Therefore, the observed NH values reflect a
population-weighted average. Low temperatures allow slowing
down of the interconversion processes among the different
species in equilibrium. The interpretation of δ NH data in terms
of intramolecular hydrogen bonding requires then, that the
studied molecule be sufficiently dilute to prevent hydrogen
(Δδ = 0.3 ppm) under the same conditions. The expected chemi-
cal shift value for non-hydrogen-bonded NH amide protons at
298 K in CDCl3 ranges from 5.8 to 6.4 ppm.15 Therefore, the
observed data suggest that the amide group is, to some extent,
intramolecularly hydrogen bonded. To further investigate this
assumption, we performed a variable temperature experiment.
1
The H NMR spectrum of 1 (2 mM) in CDCl3 is temperature
dependent (313–237 K) with large temperature coefficients
(Δδ/ΔT = −11 ppb K−1) for the three NH protons of the mol-
ecule. This, together with the broadness observed for all the
signals at 237 K, is indicative of the existence of a dynamic equi-
librium among different species even at low temperature.16 At
237 K, the signal NH (c) at 8.5 ppm is also clearly shifted
downfield with respect to the two squaramide protons observed
at 7.8 and 7.9 ppm.
Moreover, the NMR data obtained from these experiments
evidence that the two squaramide signals as well as the two
α-methylene protons CH2 (e) and CH2 (d) of the two squaramide
N-substituents show quite similar chemical shifts and behaviour,
independently of the concentration and temperature investigated.
Therefore, we assumed that the squaramide moiety prefers the
extended anti/anti rather than the syn/anti conformation which is
adequate to induce the sought folded structure. In the anti/anti
conformation, the two squaramide NH groups are able to estab-
lish intermolecular hydrogen bonds. We expect that the aggre-
gates formed in chloroform are mainly dimers due to the high
solubility observed for compound 1 in organic non-polar sol-
vents. The chemical shift displacements experienced by the two
squaramide NH protons upon concentration are consistent with a
homodimerisation process and fit17 very well to a 1 : 1 dimerisa-
tion model, obtaining a dimerisation constant of Kdim = 260 85
M−1 at 298 K.
1
bond-mediated aggregation. All the H NMR spectra could be
unequivocally assigned performing 2D NMR COSY and
TOCSY experiments.
Conformational studies of 1
First, we studied the conformational properties of 1. The 1H
NMR spectrum of a diluted solution of 1 (0.5 mM, 298 K) in a
non-polar solvent like chloroform presents broad signals for the
three NH protons of the molecule. Unexpectedly, the amide NH
(c) proton is shifted downfield (δ ≈ 7.2 ppm) with respect to the
two squaramide signals NH (a) and NH (b), which show similar
chemical shifts at 6.4 ppm and 6.6 ppm respectively. These two
NH protons are also shifted downfield relative to the usual
region for non-hydrogen-bonded squaramide protons (δ ∼
6.0–6.2 ppm), (Fig. 2).
Additionally to this process, the behaviour observed by NMR
for proton NH (c) upon concentration and temperature can be
explained supposing that the N-Gly-Phe-OMe substituent of 1
folds, establishing an intramolecular hydrogen bond between the
amide proton NH (c) and the nearest squaramide carbonyl group,
inducing the formation of an eight-membered ring (Fig. 3).
Similar conformations to this have been observed in the crystal
structure of some proline squaramide derivates.18 Moreover, the
1
The H NMR spectrum of 1 in CDCl3 (1–100 mM) at 298 K
is concentration dependent. The two squaramide signals NH (a)
and NH (b) show relatively large and almost identical chemical
shift changes upon dilution (Δδ = 0.8 ppm), which indicates
the formation of intermolecular hydrogen bonds upon concen-
tration. However, the amide signal NH (c) exhibits little change
Fig. 2 Assigned 1H NMR spectrum of 1 (0.5 mM) in CDCl3 at 298 K.
1916 | Org. Biomol. Chem., 2012, 10, 1914–1921
Fig. 3 Conformational equilibrium and homodimerisation of 1.
This journal is © The Royal Society of Chemistry 2012

View Article Online
proposed structure of the folded homodimer shown in Fig. 3 is
consistent with those found for squaramide-related compounds
described earlier in crystallographic structures of the N-carba-
moyl squaramide dimer.19
of dimer, and consequently, additional intermolecular hydrogen
bonds.
Taking all this together, the data obtained for compound 1
support the existence in solution of different species in equili-
brium, with a significant percentage of the folded conformer that
leads to the formation of an intramolecular hydrogen-bonded
eight-membered ring between the amide proton NH (c) and the
nearest squaramide carbonyl group.
To further investigate this assumption, we studied the confor-
mational properties of compound 1 in acetonitrile. In this
solvent, the hydrogen bonding interactions are less favoured than
in chloroform, therefore hydrogen bond-mediated aggregation
can be diminished and meanwhile the intramolecular hydrogen
bonds are still favoured due its tightness.20 In acetonitrile, at
298 K the chemical shift observed for NH (c) is 6.9 ppm. Even
though the expected value for free amide protons in acetonitrile
is approximately 6.1–6.3 ppm,20 in this case, the downfield shift
observed for this proton should be attributed to the paramagnetic
effect of the carbonyl of the methylester group and not to the for-
mation of hydrogen bond interactions.21 On the other hand, the
squaramide protons NH (a) and NH (b) appear at 6.2 ppm which
is the usual spectral region for non-hydrogen-bonded squaramide
protons. The monomeric state of 1 in acetonitrile was corrobo-
rated with a variable temperature experiment (313–237 K). The
three NH signals showed small temperature coefficients (Δδ/ΔT
= −3 ppb K−1) which can be interpreted in terms of these groups
being either involved in intramolecular hydrogen bonds or fully
solvated by the solvent.14–22 Unlike in chloroform, at 237 K the
1H NMR spectrum of 1 show sharp signals and some new low
intensity signals can be observed. These new signals assigned to
minor syn/anti conformations of the squaramide substituents.
These results indicate that, in acetonitrile, the prevalent
species is the monomeric anti/anti conformer, in equilibrium
with small amounts of unfolded syn/anti conformers.
We carried out a theoretical conformational analysis of com-
pound 1. Energies of the conformers were evaluated using the
RB3LYP/6-311G* method. From all the structures generated, the
two conformations of lowest energy correspond with the two
conformers A and B that form the intramolecular hydrogen-
bonded eight-membered rings. These structures are shown in
Fig. 4 and indicate the tendency of compound 1 to form intramo-
lecular hydrogen-bonded conformers. The two conformers A
and B have almost identical relative energies. Conformer A can
easily homodimerise retaining the folded conformation, estab-
lishing four additional hydrogen bonds. On the other hand, the
alternative folded structure B prevents the formation of this kind
Conformational studies of 2
In contrast to compound 1, squaramide 2 showed low solubility
in chloroform and prevented us running concentration-dependent
studies in this solvent. At 298 K, a solution of 2 (1 mM) in
CDCl3 show two separated signals of different intensity for the
three NH protons of the molecule, one at 6.1 ppm assigned to
the squaramide NH (a) proton and the other at 6.4 ppm assigned
to the squaramide NH (b) and the amide NH (c) protons (Fig. 5).
From these data we assumed that neither of these protons
experience much hydrogen bonding interactions in these con-
ditions because the values are consistent with the expected ones
for free NH amide and squaramide protons in chloroform. We
also observed that the signal of the α-methylene protons CH2 (e)
of the β-alanine substituent splits into two peaks at 3.8 and
3.9 ppm, respectively, due to the presence of syn/anti conformers
or a restricted rotation along the N–Csqu bond of this substituent.
To further analyse the conformational properties of 2 we ran a
variable temperature experiment. The chemical shifts of these
signals are temperature dependent (237–313 K). The two squara-
mide protons NH (a) and NH (b) and the amide NH (c) proton
considerably shift downfield upon decreasing temperature and
show almost identical chemical shifts at any temperature investi-
gated. The large temperature coefficients (15 ppb K−1) obtained
for all of them indicate that the three NH protons are involved in
the formation of hydrogen bonding interactions.16
Taken together, these results suggest that the squaramide ring
prevails in an anti/anti conformation, discarding the formation
of any folded conformation. As a consequence, compound 2
exhibits a large tendency to form intermolecular hydrogen-
Fig. 4 Calculated conformations of 1. The relative free energies (kcal
mol−1) were calculated at the RB3LYP/6-311G*level in the gas phase.
The N–H⋯O hydrogen bonds are indicated.
Fig. 5 Assigned 1H NMR spectrum of squaramide 2 (1 mM) in CDCl3
at 298 K.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 1914–1921 | 1917

View Article Online
Fig. 6 Representation of two possible head-to-tail squaramide hydro-
gen-bonded aggregates of compound 2.
bonded aggregates, in which the amide group is also involved.
We can assume then, that compound 2 tends to form small–
medium sized self-assembled structures.
In them, the molecules are organised in a head-to-tail fashion
as disecondary squaramides usually assemble, either in solution
or in solid phase, due to their double donor–acceptor hydrogen
bonding capability23 (Fig. 6).
Fig. 7 Assigned 1H NMR spectrum of squaramide 3 (2 mM) in CDCl3
at 298 K.
This trend is the cause of the observed low solubility of many
disecondary squaramides in non-competitive solvents, as is the
case of squaramide 2. Besides, in compound 2, the tendency to
form this kind of aggregate is strengthened by the presence of
the amide linkage in the squaramide N-dipeptide substituent.
Contrary to what happens in compound 1, the NH (3) proton of
the usually more stable trans isomer of the amide bond is
oriented in the same direction as the two anti/anti squaramide
NH protons. As a result, compound 2 can be tightly self-
assembled through the formation of additional hydrogen bonds,
instead of the formation of any folded conformer.
Conformational studies of 3
Finally, the conformational properties of the model compound 3
were also investigated. Unlike the previous studied compounds 1
1
and 2, the H NMR spectrum of 3 in CDCl3 (2 mM, 298 K)
shows significant evidence of the existence of a folded confor-
mation of compatible features with the formation of a ten-mem-
bered ring induced by the formation of an intramolecular
hydrogen bond (Fig. 7). The two NH squaramide protons NH (a)
and NH (b) show quite different chemical shifts at 7.2 and
6.4 ppm respectively, as well as the α-methylene protons CH2
(d) and CH2 (e) at 3.7 and 3.4 ppm respectively. This can be
attributed to a syn/anti conformation of the squaramide unit. The
downfield shift observed for protons NH (b) and CH2 (d) is due
to the paramagnetic shielding of the squaramide carbonyl
groups. Thus, the notable downfield shift observed for the squar-
amide NH (a) proton at 7.3 ppm can be attributed to the for-
mation of the intramolecular hydrogen bond responsible for the
formation of the turn. In this case, the chemical shift shown by
the amide proton NH (c) at 6.3 ppm corresponds with the non-
hydrogen-bonded state, and supports the formation of the
expected ten-membered ring folded structure. To corroborate this
evidence, the concentration dependence (1–100 mM) of the
1H NMR spectrum of 3 in CDCl3 was analysed.
Fig. 8 Concentration dependence of 3 (2 mM) at 237 K in CDCl3.
case, the chemical shift data observed also perfectly fit in a 1 : 1
dimerisation model, obtaining a dimerisation constant of Kd = 50
7 M−1, which is one order of magnitude lower than the dimeri-
sation constant calculated for 1. Therefore, the homodimers are
in equilibrium with the monomeric folded structure that predo-
minates at low concentrations. This could be corroborated when
a 2 mM solution of 3 in chloroform was cooled to 237 K
(Fig. 8). At this temperature, the signals corresponding to the
NH protons split, indicating almost the existence of two different
species in equilibrium in a 3 : 1 ratio. The majority species has
signals at 8.4 and 6.3 ppm that were assigned to NH (a) and NH
(c) respectively; meanwhile the corresponding NH (b) could be
located at approximately 7.1 ppm shielded by the aromatic
signals of the phenylalanine residue. From this data we can
assume that the squaramide is in a syn/anti conformation and the
The three NH signals show a downfield chemical shift upon
concentration, suggesting the formation of aggregates. In this
1918 | Org. Biomol. Chem., 2012, 10, 1914–1921
This journal is © The Royal Society of Chemistry 2012

View Article Online
amide group NH (c) is not hydrogen bonded. The large
downfield chemical shift exhibited by the NH (a) proton is
indicative of the formation of an intramolecular hydrogen bond.
All these results confirm the existence of the ten-membered ring
folded structure. On the other hand, the second group of signals
observed in the spectrum contains three peaks of very close
chemical shift at 7.8, 7.6, and 7.5 ppm and were assigned to the
protons labelled NH (c), NH (b), and NH (a) respectively. These
chemical shift values are in agreement with intermolecular
hydrogen-bonded aggregated structures like those observed at
298 K.
The aggregated nature of these structures was further
confirmed with a concentration dependence experiment. Thus,
the 1H NMR spectrum of 3 changes upon concentration at
237 K. The relative intensity of the signals assigned to intermo-
lecular hydrogen-bonded species increases upon concentration
with respect to those assigned to the folded conformer (Fig. 8).
We can conclude that in diluted solutions of 3, the monomeric
intramolecular hydrogen-bonded structure that forms a ten-mem-
bered ring prevails over the aggregates but upon concentration,
those are the prevalent species. This fact was confirmed in aceto-
nitrile, where the presence of aggregates can be avoided. In these
conditions, the intramolecular hydrogen-bonded structure could
also be detected at 298 K24
of this polypeptide-like compound were also conveniently func-
tionalised by means of the formation of an amide and carbamate
linkage, respectively, in order to increase the cross-strand hydro-
gen bonding capability.
The squaramide tetrapeptide compound 4 was synthesised as
shown in Scheme 2 and according to known procedures to
obtain secondary squaramides and peptides. Briefly, the Boc-
mono-protected ethylene diamine was coupled to L-Leu-Cbz
assisted by the coupling reagent HBTU to give compound 12. In
parallel and under the same conditions, Gly-Boc and phenylala-
nineciclohexylamide were also coupled to give the dipeptide 14.
Subsequently, the amino protecting group of 14 was cleaved
under acidic conditions and coupled to the amino Boc-protected
γ-aminobutyric acid, affording the tripeptide 15. Finally, the
amino protecting groups of 12 and 15 were cleaved under acidic
conditions to give the respective free amines. These were con-
densed in two steps with the squaramide moiety. First, the free
amine of 12 was condensed in diethyl ether to obtain the squara-
mide ethyl ester 13, which was further functionalised by conden-
sation with the free amine of 15 in a 50 : 50 v : v mixture of
ethanol–aqueous borax buffer (pH = 9) to give the new squara-
mide tetrapeptide model compound 4.
Finally, the conformational properties of 4 were analysed. Due
to the noticeable tendency of this compound to form aggregates
in chloroform, 4 was studied in acetonitrile at 298 K. The 2D
NMR (TOCSY, COSY) experiments performed (1mM, 298 K)
At 244 K we observed the presence of unfolded conformers in
equilibrium with the intramolecular hydrogen-bonded folded
structure.
The conformational analysis performed for the three model
compounds 1–3 evidenced that compound 3 presents the ade-
quate features of a turn-forming module and is a good candidate
to mimic natural β-turns that can be incorporated into hybrid
structures.
1
allowed us to fully assign all the signals on the H NMR spec-
trum.25 In these conditions, the signals of the structural fragment
ethylenediamine-Leu-Cbz split in two, suggesting the existence
of different conformers in solution.26
Synthesis and conformational analysis of the squaramide-
tetrapeptide 4
Once we had characterised the optimum new squaramide-based
turn module consisting of a secondary squaramide scaffold com-
bined with the γ-amino butyric acid in one of its substituents, we
investigated the capability of this module to organise more
complex structures. Therefore, the new loop structure was incor-
porated into the squaramide tetrapeptide 4 with the aim to obtain
a minimal model of a β-hairpin structure (Chart 2). We assumed
that the attachment of several natural amino acids, namely Gly,
Phe and Leu and the linker ethylene diamine, to the basic fold-
able structure could lead to the formation of cooperative favour-
able interactions in the flanking regions of the loop segment,
driven by the formation of two additional intramolecular hydro-
gen bonds in an antiparallel β-sheet fashion. This arrangement
could overcome the observed flexibility of the turn segment
in compound 3. The carboxylic and amino terminal groups
Scheme 2 Synthesis of 4. (a) DIPEA, HBTU, DMF; (b) TFA,
CH2Cl2; (c) diethylsquarate, DIPEA, EtOH-Et2O; (d) DIPEA, HBTU,
DMF, 4-aminobutyric acid; (e) EtOH-Borax (pH = 9) 50 : 50 v : v.
Chart 2 Structure of the squaramide-tetrapeptide 4.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 1914–1921 | 1919

View Article Online
Fig. 9 Top: Folded conformations of 4 showing H atom labels and the corresponding NOE interactions observed in acetonitrile (1 mM, 298 K).
Cross peaks represented by red arrows support the existence of the turn. The cross peaks represented by the blue arrows support the evidence of an
equilibrium between conformers in solution. Bottom: Minimised structure of two conformers of 4 at RB3LYP/6-311G*level in the gas phase. The
N–H⋯O hydrogen bonds are indicated. Non-acidic hydrogen atoms and bulky substituents have been omitted for clarity.
First of all, we observed that the squaramide NH protons
named NH (a) and NH (b) show chemical shifts at 7.14 and
6.76 ppm, respectively, and are in agreement with a syn/anti con-
formation of the squaramide unit, where the NH (a) is intramole-
cularly hydrogen-bonded to the carbonyl group from the GABA
fragment, and the NH (b) proton experiences the deshielding of
the squaramide carbonyl group. This assumption is corroborated
with the chemical shift of the signals corresponding to CH2 ( j)
and CH2 (h) at 3.58 and 3.77 ppm respectively. These data
suggest the formation of an intramolecularly hydrogen-bonded
ring system that features the turn architecture that we have
characterised for the model compound 3. The analysis of the
chemical shift observed for the rest of the NH groups of the mol-
ecule indicates that they are also involved, to some extent, in
hydrogen bonding interactions.27 It is noteworthy that the NH
(d) splits in two at 7.22 and 6.96 ppm, suggesting the existence
of two conformations for this group participating in hydrogen
bonding interactions in one of them.
To fully characterise the conformational state of 4, we per-
formed 2D-NOESY studies. We found several cross peaks
between both chains supporting the existence of folding (see
Supporting Information†). As shown in Fig. 9, we observed
strong nOe signals between the amide protons NH (a) and the
methylenic protons CH2 (k) and CH2 (l) of the GABA fragment,
which are consistent with the turn conformation. However, the
corresponding signal of NH (d) at 7.22 also gives strong nOe
signals with the methylenic protons CH2 (k) and CH2 (l). These
data, together with the downfield shift observed for NH (d),
suggest that this group is also involved in the turn fragment by
means of a hydrogen bonding interaction with the carbonyl
group of the GABA residue. In this scenario, the ethylenedi-
amine linker acts as a bi-dentate ligand and the turn is driven by
the formation of two hydrogen bonding interactions. However,
some nOe signals detected suggest the presence of a slightly
different conformation of the molecule. For example, the cross-
peaks observed, such as the one between the methylene protons
CH2 (i) and CH2 (k) of both squaramide N-substituents and the
carbamate NH (f) and the CHα (m) of the phenylalanine
residue. These proximities are reasonably explained, considering
that the ethylenediamine fragment adopts an extended confor-
mation in which the corresponding NH (d) at 6.96 ppm faces
outside of the folded molecule.
Minimised structures at RB3LYP/6-311G* level in the gas
phase of 4 featuring both proposed loops show a good parallel
alignment of the strands linked to them, favouring the cross-
strand hydrogen bonding interactions that were detected by
NOESY experiments.28 The analysis of the 1- and 2D NMR
spectra of 4 indicates that the two conformations of the loop are
mostly equally populated.
Hence, the data obtained from this experiment support the
existence of the hairpin-like folded conformation and show that
the new turn-inducing module, due its conformational properties,
is able to organise peptidomimetic structures in a hairpin
fashion.
Conclusions
We have developed and characterised a new turn module based
on the combination of a disecondary squaramide with the 4-ami-
nobutyric acid moieties that form a 10-membered ring induced
by the formation of intramolecular hydrogen bonds. Confor-
mational studies based on 1H NMR data for compound 3
showed that the new mimetic β-turn module is quite flexible.
1920 | Org. Biomol. Chem., 2012, 10, 1914–1921
This journal is © The Royal Society of Chemistry 2012

View Article Online
S. Lavielle, P. Bertus, J. Szymoniak, O. Lequin and P. Karoyan, Chem-
BioChem, 2011, 12, 1039–1042.
Nevertheless, this system has been successfully combined with
dipeptide chains to induce the formation of a hairpin-like struc-
ture driven by the formation of several cross-strand intramolecu-
lar hydrogen bonds. The conformational flexibility of this new
loop is still considerable and leads to two slightly different con-
formations. These results indicate that this kind of hybrid struc-
ture that constitutes a new family of foldamers are good
candidates to be considered, for example, in the development of
conformational switches, or in drug design. Further work to
fully control the conformational properties of this new module is
under process in our laboratory.
11 D. Yang, X.-W. Chang, D.-W. Zhang, Z.-F. Jiang, K. S. Song,
Y.-H. Zhang, N.-Y. Zhu, L.-H. Weng and M.-Q. Chen, J. Org. Chem.,
2010, 75, 4796–4805; S. Chatterjee, R. S. Roy and P. Balaram, J. R. Soc.
Interface, 2007, 4, 587–606; R. Rai, P. G. Vasudev, K. Ananda,
S. Raghothama, N. Shamala, I. L. Karle and P. Balaram, Chem.–Eur. J.,
2007, 13, 5917–5926.
12 M. N. Piña, B. Soberats, C. Rotger, P. Balester, P. M. Deyà and A. Costa,
New J. Chem., 2008, 32, 1919–1923; R. Prohens, M. C. Rotger, M.
N. Piña, P. M. Deyà, J. Morey, P. Ballester and A. Costa, Tetrahedron
Lett., 2001, 42, 4933–4936; S. Tomas, R. Prohens, M. Vega,
M. C. Rotger, P. M. Deyà, P. Balester and A. Costa, J. Org. Chem., 1996,
61, 9394–9401.
13 M. C. Rotger, M. N. Piña, A. Frontera, G. Martorell, P. Ballester,
P. M. Deyà and A. Costa, J. Org. Chem., 2004, 69, 2302–2308.
14 C. Rotger, M. N. Piña, M. Vega, P. Ballester and A. Costa, Angew.
Chem., Int. Ed., 2006, 45, 6844–6848.
Acknowledgements
15 J. D. Fisk, D. R. Powell and S. H. Gellman, J. Am. Chem. Soc., 2000,
122, 5443–5447.
This research is supported by the Ministry of Science and Inno-
vation (MICINN) of Spain grant no. CTQ2008-00841/BQU and
MICINN Consolider-Ingenio grant no. CSD2010-00065. L. M is
grateful to the Ministry of Science and Innovation (MICINN) of
Spain for a fellowship.
16 L. Cipolla, C. Airoldi, D. Bini, M. Gregori, F. Marcelo, J. Jiménez-
Barbero and F. Nicotra, Eur. J. Org. Chem., 2011, 128–136.
17 The binding constants were calculated using HypNMR (Protonic Soft-
ware, version 2008). C. Frassineti, S. Ghelli, P. Gans, A. Sabatini,
M. S. Moruzzi and A. Vacca, Anal. Biochem., 1995, 231, 374;
C. Frassineti, L. Alderighi, P. Gans, A. Sabatini, A. Vacca and S. Ghelli,
Anal. Bioanal. Chem., 2003, 376, 1041.
18 T. Kolev, R. W. Seidel, H. Mayer-Figge, M. Spiteller, W. S. Sheldrick and
B. B. Koleva, Spectrochim. Acta, Part A, 2009, 72, 502–509.
19 A. P. Davis, S. M. Draper, G. Dunne and P. Ashton, Chem. Commun.,
1999, 2265–2266.
Notes and references
1 J. S. Nowick, E. M. Smith and M. Pairish, Chem. Soc. Rev., 1996, 25,
401–415.
2 C. M. Goodman, S. Choi, S. Shandler and W. F. DeGrado, Nat. Chem.
Biol., 2007, 3, 252–262; D. Yang, Y.-H. Zhang, B. Li, D.-W. Zhang,
J. C.-Y. Chan, N.-Y. Zhu, S.-W. Luo and Y.-D. Wu, J. Am. Chem. Soc.,
2004, 126, 6956–6966.
3 K. D. Stigers, M. J. Soth and J. S. Nowick, Curr. Opin. Chem. Biol.,
1999, 3, 714–723.
20 S. H. Gellman, G. P. Dado, C.-B. Liang and B. R. Adam, J. Am. Chem.
Soc., 1991, 113, 1164–1173.
21 The chemical shift observed for the amide proton of the dipeptide Boc-
GABA-Phe-OMe in acetonitrile (1mM, 298 K) is 6.9 ppm. The chemical
shift of this signal in chloroform (2 mM, 298 K) is 6.4 ppm.
22 E. S. Stevens, N. Sugawara, G. M. Bonora and C. Toniolo, J. Am. Chem.
Soc., 1980, 102, 7048–7050.
23 C. Rotger, B. Soberats, D. Quiñonero, A. Frontera, P. Ballester, J. Benet-
Buchholz, P. M. Deyà and A. Costa, Eur. J. Org. Chem., 2008, 1864–
1868.
24 The chemical shifts observed for the NH groups of 3 at 298 K in aceto-
nitrile are 6.9, 6.5 and 6.2 ppm corresponding to the hydrogens labelled
as b, a, and c, respectively. The CH2 g and d also show different chemical
shifts at 3.4 and 3.6 respectively.
25 The low solubility of 4 either in chloroform or acetonitrile prevented us
from running concentration- or temperature-dependent experiments by
NMR techniques.
4 S. J. Hershberger, S.-G. Lee and J. Chmielewski, Curr. Top. Med. Chem.,
2007, 7, 928–942.
5 J. M. Beierle, W. S. Horne, J. H. van Maarseveen, B. Waser, J. C. Reubi
and M. R. Ghadir, Angew. Chem., Int. Ed., 2009, 48, 4725–4729.
6 K. Suat Kee and J. D. S. Seetharama, Curr. Pharm. Des., 2003, 9, 1209–
1224; A. Giannis and T. Kolter, Angew. Chem., Int. Ed. Engl., 1993, 32,
1244–1267.
7 J. D. A. Tyndall, B. Pfeiffer, G. Abbenante and D. P. Fairlie, Chem. Rev.,
2005, 105, 793–826.
8 A. Pinsker, J. Einsiedel, S. Hearterich, R. Waibel and P. Gmeiner, Org.
Lett., 2011, 13, 3502–3505; R. F. Hirschmann, K. C. Nocolau,
A. R. Angeles, J. S. Chen and A. B. Smith III, Acc. Chem. Res., 2009,
42, 1511–1520; L. Lomlim, J. Einsiedel, F. W. Heinemann, K. Meyer and
P. Gmeiner, J. Org. Chem., 2008, 73, 3608–3611; D. Blomberg, P. Kreye,
C. Fowler, K. Brickmann and J. Kihlberg, Org. Biomol. Chem., 2006, 4,
416–423.
26 The split signals observed for this structural fragment are not only due to
the quite probably also occurring interconversion equilibrium E vs. Z of
the carbamate group. The carbamate is located at the end of the molecule
and can freely rotate without provoking the splitting of the ethylendi-
amine NH (d) signal.
9 F. Chen, N.-Y. Zhu and D. Yang, J. Am. Chem. Soc., 2004, 126, 15980–
15981; C. Gennari, B. Salom, D. Potenza, C. Longari, E. Fioravanzo,
O. Carugo and N. Sardone, Chem.–Eur. J., 1996, 2, 644–655.
10 C. Mothes, M. Larregola, J. Quancard, N. Goasdoué, S. Lavielle,
G. Chassaing, O. Lequin and P. Karoyan, ChemBioChem, 2010, 11, 55–
58; J. D. Fisk, D. R. Powell and S. H. Gellman, J. Am. Chem. Soc., 2000,
122, 5443–5447; K. Guitot, M. Larregola, T. K. Pradhan, J.-L. Vasse,
27 The chemical shifts observed for the NH groups of compound 4 are: NH
(a) at 7.14 ppm; NH (b) at 6.76 ppm; NH (c) at 7.07 ppm; NH (d) at 7.22
and 6.96 ppm; NH (e) 6.83 and 6.15 ppm; NH (g) 6.68 ppm.
28 The minimised structures shown here were calculated to illustrate the
experimental evidence of the existence of the two conformations
observed by 2D RMN experiments. No further assumptions were made
from them.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 1914–1921 | 1921