Hydrogen Bonded Squaramide-Based Foldable Module Induces Both β- And α-Turns in Hairpin Structures of α-Peptides in Water

Article abstract of DOI:10.1021/acs.orglett.5b01268

A novel tertiary squaramido-based reverse-turn module SQ is reported, and its conformational properties are evaluated. This module is easily incorporated into a α-peptide sequence by conventional solid-phase peptide synthesis. The structure characterizati

Full text of DOI:10.1021/acs.orglett.5b01268

Letter
pubs.acs.org/OrgLett
Hydrogen Bonded Squaramide-Based Foldable Module Induces Both
β- and α‑Turns in Hairpin Structures of α‑Peptides in Water
†
‡
†
§,∥
Luís Martínez, Gabriel Martorell, Angel Sampedro, Pablo Ballester, Antoni Costa,†
́
and Carmen Rotger*,†
‡
†Departament de Química and Seveis Cientificotec
Mallorca, Spain
̀
nics, Universitat de les Illes Balears, Ctra. Valldemossa, km 7.5, 07122 Palma de
§Institut of Chemical Research of Catalonia (ICIQ), Avda. Països Catalans 16, 43007 Tarragona, Spain
∥Catalan Institution of Research and Advanced Studies (ICREA), Passeig Lluís Compays 23, 08010 Barcelona, Spain
S
* Supporting Information
ABSTRACT: A novel tertiary squaramido-based reverse-turn
module SQ is reported, and its conformational properties are
evaluated. This module is easily incorporated into a α-peptide
sequence by conventional solid-phase peptide synthesis. The
structure characterization of the hybrid squaramido-peptide 4
is described, showing that the turn segment induces the
formation of hairpin structures in water through the formation of both αSQ- and βSQ-turns.
he hairpin structural motif, with two strands of α-peptides
rotational barrier of the squaramide unit and the hydrogen-
Tconnected by a reverse turn forming an antiparallel β-sheet, bonding capabilities of the squaramide framework. These,
combined with the presence of hydrogen-bonding donor
atoms in one of the squaramide substituents, induce folding
driven by the formation of an intramolecular hydrogen bond.12
Our attempts in the field have led us to preorganize linear
oligosquaramide compounds in protic solvents to yield cyclic
oligosquaramidic compounds with interesting antitumor proper-
ties.13 Recently, we have described a squaramide-based module
that can be easily combined with peptide strands and induce
folding in organic solvents, although the conformational
flexibility of the turn moiety is still considerable.14
Herein, we present the design, synthesis, and conformational
studies of the squaramide-based scaffold SQ1a. The minimalist
loop 1a, formally an amino acid, is constructed from diethyl
squarate and (2-aminoethyl)methylamine to give a tertiary
squaramido-ethyl ester (Supporting Information). This module
can be directly coupled with both the amino and the carboxylic
acid groups of natural or non-natural amino acids to obtain
peptidomimetic compounds, Figure 1.
Unlike secondary squaramides, tertiary squaramides such as
N,N-dialkylsquaramides show little preference for any con-
formation in solution and exist as a mixture of Z,Z and E,Z
conformers due to their lower rotational barrier energy (∼17 kcal
mol−1) for the squaramide C−N bond.15 Therefore, the turn
module 1a was envisioned to adopt a high population of the E,Z
conformation when conveniently functionalized driven by the
formation of an intramolecular hydrogen bond and two
additional C−H··O hydrogen bonds due the coplanar disposi-
tion of the squaramide substituents, Figure 2.16
is widespread among proteins and crucial to many biomolecular
recognition events such as protein−protein and protein−nucleic
acid interactions.1 Reverse turns are classified by the number of
amino acid residues involved in the turn segment. For instance,
β-turns are formed by four amino acid residues and feature 10-
membered hydrogen-bonded rings. Frequently observed in
nature, peptides with this secondary structure display many
biologically relevant properties such as antimicrobial and
antiviral activities, among others.2 α-Turns are larger structures
formed by five amino acid residues and an ensemble of 13-
membered hydrogen-bonded rings that usually are internally
stabilized by tight smaller turns such as β-turns. Although the α-
turn motif is rarely observed in peptides or proteins, it can be
found in enzyme active sites, metal-binding domains,3 and
bioactive natural3a,4 or non-natural cyclopeptides5and also plays
a key role in relevant molecular recognition processes such as cell
proliferation signaling6 or reducing the HIV infection7 capability.
Therefore, numerous efforts have been made to design and
synthesize non-natural reverse β-turn scaffolds that could induce
autonomous folding of peptide or peptidomimetic strands with
the aim of developing more selective and potent therapeutic
agents,8 new catalysts,9 preorganized linear peptides,10 etc. The
continuous attempts made to develop modules that display α-
turns in peptidomimetic compounds have afforded a limited
number of successful examples.11 However, it is still a challenge
to overcome the drawbacks of the conformational flexibility of
small peptides in water.
As part of our program aimed at studying the conformational
properties of the squaramide compounds,12 we have studied the
folding capability of disecondary squaramide-based turn mimetic
modules. Our folding segments rely on the relatively low
Received: April 30, 2015
Published: June 2, 2015
© 2015 American Chemical Society
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Letter
driven by an intramolecular hydrogen bond mimicking natural β-
turns (Supporting Information).
Single crystals obtained from 2 by slow evaporation of AcOEt
yield the solid-state structure, revealing the folded arrangement
of the turn framework in the crystals. X-ray data show the
formation of the intramolecular hydrogen bonded 10-membered
ring observed by NMR (N−H··O = 1.931 Å), and as in solution,
the amide NHe is engaged in an intermolecular hydrogen bond
(N−H··O = 2.015 Å) with one of the two squaramide carbonyls
of a neighbor molecule, building hydrogen-bonded ribbons of
folded molecules. Examination of the torsional angles reveals that
the turn segment is almost planar as shown in Figure 3. Thus, the
Figure 1. (Top) Schematic representation of typical β-turn and α-turn
internally stabilized by a β-turn. (Bottom) Structure of the squaramide-
based minimalist loop SQ (1a, 1b, and 1c) and the model compounds 2
and 3.
Figure 3. (Left) ORTEP front and side views of 2. (Right) Crystal
packing of 2 showing the intermolecular association of the folded
squarmide molecules. All hydrogen bonds are represented by dotted
lines. Some hydrogen atoms have been removed for clarity.
Figure 2. Representation of the conformational equilibrium of the
squaramide-based turn module observed for 2. Selected nonsequential
NOEs observed for the E,Z conformation in chloroform are shown.
coplanar orientation of the N-methyl and N−CH2 groups is
favored by the formation of two additional C−H··O hydrogen
bonds. Thus, distances C−Hf··O = 2.513 Å and C−Hb··O =
2.459 Å are 0.207 and 0.261 Å shorter than the sum of the van der
Waals atoms radii, respectively.16 As a result, the turn segment is
arranged in a well-defined conformation suited to direct the
formation of hairpin structures.
As a step forward in our study, we synthesized compound 3 to
evaluate the suitability of the new loop segment to be
incorporated in peptidomimetic structures. The conformational
studies were performed in methanol, hydrogen-bonding
competitive media, by NMR.
Compound 3 was synthesized from 1b, first by a conventional
squaramide condensation reaction with the amine group of the
dipeptide H2N-Gly-Phe-CONHtBu and followed by the cleavage
of the Boc protecting group and the amide coupling reaction with
the carboxylic group of the dipeptide AcNH-Ala-Ala-CO2H
(Supporting Information). Thus, upon folding, 3 may form two
additional interstrand hydrogen bonds to stabilize the secondary
structure.
At 233 K, solution-state NMR studies of 3 run in methanol
show the existence of two equally populated conformers. These
species were unambiguously assigned to the folded 3A (E,Z) and
unfolded 3B (Z,Z) structures, respectively, Figure 4.
Selected nonsequential NOEs observed for 3 are shown in
Figure 4. Thus, consistent with a Z,Z conformation, a strong
NOE signal between the proton NHa′ and the N−(CH3)b′ was
observed, revealing the proximity of these groups in the unfolded
structure 3B. Alternatively, and as observed in 2, the folded
conformer 3A shows a strong NOE signal between the NHa and
the methylene group (CH2)c pointing out the E,Z conformation
of the squaramide module SQ. In addition, the folded structure
shows several characteristic interstrand NOEs observed in
hairpin-like structures.18 These contacts are indicative of the
To evaluate the new loop design, we synthesized and studied
the conformational properties of the model compounds 2 and 3
as a previous step to the incorporation of the novel turn sequence
into a peptide strand, Figure 1.
Thus, in order to promote the formation of the intramolecular
hydrogen-bonding interaction within the loop scaffold, com-
pound 1b was condensed with n-butylamine followed by the
cleavage of the amino protecting group Boc and coupling with
acetyl chloride to afford compound 2 (Supporting Information).
We ran NMR studies in chloroform to investigate the solution-
state conformation of 2. At 298 K, the squaramide proton NHa
has little concentration dependence (Δδ = 0.34 ppm) in the 1−
25 mM range. However, the amide proton NHe suffers an
important downfield shift (Δδ = 0.80 ppm) at the same
concentration interval, suggesting that NHe forms intermolec-
ular hydrogen bonds upon concentration while NHa is shielded
from others molecules. Below the coalescence temperature of
258 K, two different species are clearly observed in a 9:1 ratio and
assigned to the folded and unfolded conformers, respectively.
The addition of increasing amounts of DMSO up to 10% to a 1
mM solution of 2 in CDCl3 induces the downfield chemical shift
of the NHa (Δδ = 0.08) and NHe (Δδ = 1.5 ppm), respectively,
confirming that unlike NHe, NHa is shielded from the solvent
(Supporting Information).
NOESY studies of a 1 mM solution of 2 in CHCl3 run at 244 K
also evidence the folded state of 2 showing strong crosspeak
signals between the nonadjacent protons NHa and the
methylene groups (CH2)c and (CH2)d from the opposite
substituent, Figure 2. Conformational studies performed in
acetonitrile for 2, in which self-aggregation is unlikely, show the
same results as in chloroform (Supporting Information). Thus,
the E,Z conformer induces the formation of a 10-membered ring
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At this temperature, compound 4 shows sharp and spread
signals. Two main conformers 4A and 4B of equal proportion
were clearly identified in both samples and conveniently assigned
by NMR experiments (TOCSY, NOESY, and ROESY), Figure 5.
Chemical shifts analysis reveals that pH does not significantly
affect the turn module conformational behavior, since significant
differences were observed only for the amino acids Val and Leu
located at the carboxyl terminus (Supporting Information).
Interestingly, the main differences in the chemical shifts
between the two conformers are observed for the NH groups
located at the turn segment and nearby. Thus, NHa is shifted
upfield (Δδ = −0.25 ppm) in 4B when compared with 4A. In
contrast, the NH (Phe) and NH (Thr) signals are shifted
downfield (Δδ = 0.55 ppm) in 4B, which is indicative of their
involvement in the formation in of stronger hydrogen bonding
interactions than in 4A. As anticipated, the 2D NMR (TOCSY
and ROESY) studies of 4 run at pH 2.5 and 278 K show the
characteristic contacts between nonadjacent residues, indicating
the formation of a hairpin structure in both conformers. The
detected critical cross-strand NOEs are represented in Figure 6.
Figure 4. Nonsequential NOEs observed for 3 in methanol and
representation of the assigned structures for conformers 3A (E,Z) and
3B (Z,Z).
antiparallel alignment of the two peptide strands. As a
consequence, the juxtaposition of the residue side chains favors
noncovalent interactions that contribute to the hairpin
stabilization in the highly hydrogen bonding competitive media.
Motivated by this result, we investigated the conformational
behavior of our turn module in water by its incorporation into a
decapeptide strand using a conventional peptide solid-phase
synthesis. For this purpose, we designed the hybrid peptidos-
quaramide compound 4.
As shown in Figure 5, the structure follows the peptide
sequence H2N-Glu-Trp-Arg-Tyr-Thr-Xxx-Gly-Phe-Lys-Val-
Figure 5. Turn module 1c was conveniently modified to obtain the
Fmoc-protected squaramido amino acid 5 as a suitable synthetic module
for the solid-phase synthesis of the hybrid peptido-squaramide
compound 4. Turn module structure is highlighted in red.
Leu-CO2H, where Xxx represents the squaramide-based turn
module. Compound 4 was designed to fold, induced by the turn
module, giving an antiparallel hairpin structure that can hold four
additional interstrand hydrogen bonds. Inspired in natural
hairpin structures, we conveniently distributed amino acids
with hydrophobic side chains (Phe/Tyr and Trp/Val) to build a
hydrophobic cluster that could stabilize the folded structure in
water through interstrand side chain−side chain interactions.
Furthermore, the peptide sequence was completed with amino
acids with basic groups (Arg, Lys) that at physiological or acid pH
are positively charged to enhance water solubility and avoid self-
aggregation.17
In peptide solid-phase synthesis, amino acid coupling reactions
usually are performed with the aid of coupling reagents using
DMF as a solvent. The Fmoc-protected compound 1c was
condensed with the amino acid glycine in a water-buffered (BBS,
borate buffered saline)−ethanol−mixture at pH 9.5 to give
compound 5 in a 94% yield. Then, the vinylogous amino acid 5
was successfully incorporated into the peptide sequence by Fmoc
solid-phase chemistry using the 2-chlorotrityl alcohol resin (1
mmol/g).18 Squaramido-peptide 4 was obtained as the
corresponding TFA salt in 95% of purity.
Figure 6. Relevant nonadjacent NOEs observed in the NOESY
experiments of 4 (5 mM) in H2O (5% D2O) at pH 2.60 (red arrows) and
pH 5.25 (blue arrows). Representation of the α- and β-turn moieties
observed in the folded structure. Amino acid side chains have been
removed for clarity.
In 4A, NOE cross-peaks could be observed between NHa and
(CH2)c and (CH2)d, confirming the formation of the
squaramido-based reverse turn observed in compounds 2 and
3. The antiparallel β-sheet structure of the peptide chains is
reflected by backbone−backbone contacts between NH(Thr)--
CHα(Phe) and CHα(Tyr)--CHα(Phe) and interstand contacts
CHβ (Tyr)--CHα(Phe). Further evidence of the hairpin
structure of conformer 4A was found at pH 5.2 with the
interstrand contacts NH(Thr)--CHβ(Phe), NH(Leu)-Trp side
chain and CHα(Leu)--Trp side chain.
Conformer 4B also shows interstrand NOE cross-peaks within
the turn segment and along the peptide sequence. Interestingly,
at pH 2.5 the corresponding NH(Phe) gives NOE contacts with
the NH(Thr) and the squaramidic NHa signals showing their
proximity. This, together with the downfield shift observed for
NH(Phe) and NH(Thr) in 4B, indicates that these two NH are
The secondary structure of the squaramido-peptide 4 was
studied by NMR in H2O at pH 2.5 and 5.2 and at 278 K.
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involved in a different hydrogen bonding pattern within the turn.
This situation is consistent with the formation of a bidentate
hydrogen bonding interaction of the Thr carbonyl group with
NHa and NH(Phe). This alternative folded structure forms an
unusual 13-membered ring that mimics natural occurring α-turns
with maintenance of the antiparallel strand registry. In this case as
in nature, the αSQ-turn is internally stabilized by a βSQ-turn,
Figure 6. The formation of the αSQ-turn is favored by the
presence of glycine in the turn segment that allows fulfilling the
geometry due its lack of bulky side chain.19
This supposition was confirmed by the addition of up to 70%
of D2O to a 5 mM sample of 4 at pH 2.6. As anticipated, we
observed the fast disappearance of nonintramolecular hydrogen
bonded amides oriented toward the solvent such as the free NH
groups located at the N-terminal peptide strand of the two
proposed folded structures 4A and 4B (Supporting Informa-
tion). The rest of NH amide protons and the squaramide NHa
remain unchangeable up to 4−10 h as expected for intra-
molecular hydrogen bonded signals. In compound 4, the
equilibrium between the two proposed conformers slows the
exchange rate of all NH signals located at the C-terminal strand
because those that do not participate in the intramolecular
hydrogen bonds in conformer 4A are bound in conformer 4B
and vice versa. Moreover, in spite of that secondary squaramides
are more acidic than secondary amides, NHa shows an exchange
rate comparable to amides which is indicative of the formation of
the expected intramolecular hydrogen bond within the turn
module. However, NHa shows a faster exchange rate in 4B than
4A as a consequence of the weaker hydrogen bond formed in this
conformer as the chemical shift analysis reveals.
Further evidence of the highly populated hairpin structure of 4
in water was obtained from the circular dichroism spectrum of a
0.5 mM sample in this solvent (Supporting Information). The
CD shows the characteristic intense exciton-coupled bands at
212 and 228 nm, indicating interaction between the aromatic
chromophores as occurs in aromatic rich peptides folded in a
hairpin structure.20
In conclusion, we have designed the efficient N-methylated
squaramide based turn module 1a that induces folding in small
peptidomimetic structures. This module was easily incorporated
into a α-peptide sequence by conventional solid-phase peptide
synthesis obtaining the squaramide-decapeptide 4. This hybrid
compound folds in water, giving both αSQ- and βSQ-turns
through a conformational equilibrium. Taking advantage of its
dynamic conformational properties, this squaramide-based turn
module could become a key scaffold to design bioactive
peptidomimetic molecules.
ACKNOWLEDGMENTS
■
This work was supported by the Ministry of Economy and
Competitiveness (Grant Nos. CTQ2014-57393-C2-1-P and
Consolider-Ingenio CSD2010-00065) and CAIB (Grant No.
23/2011, FEDER funds). We thank Dr M. Royo for synthesizing
compound 4 at the Combinatorial Chemistry Unit of the Parc
́
Cientific of Barcelona.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed synthetic procedures and characterization data for the
new compounds, NMR conformational studies, and CD
spectrum of 4. The Supporting Information is available free of
charge on the ACS Publications website at DOI: 10.1021/
acs.orglett.5b01268.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: carmen.rotger@uib.es.
Notes
The authors declare no competing financial interest.
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