The Role of N-Methyl Squaramides in a Hydrogen-Bonding Strategy to Fold Peptidomimetic Compounds

Article abstract of DOI:10.1002/chem.201803930

Small peptides and peptomimetic compounds are valuable tools to probe and study biological systems. Small synthetic peptide analogues adopt a given secondary structure driven by structural modules that organize the compound architecture. Among them, β- an

Full text of DOI:10.1002/chem.201803930

A Journal of
Accepted Article
Title: The role of N-Methyl squaramides in a hydrogen bonding strategy
to fold peptidomimetic compounds
Authors: Carmen Rotger, Luís Martínez-Crespo, Eduardo C. Escudero-
Adán, and Antonio Costa
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The role of N-Methyl squaramides in a hydrogen bonding strategy
to fold peptidomimetic compounds
a
b
a
a
Luís Martínez-Crespo , Eduardo C. Escudero-Adán , Antonio Costa , and Carmen Rotger*
Abstract: Small peptides and peptomimetic compounds are valuable
tools to probe and study biological systems. Small synthetic peptide
analogs adopt a given secondary structure driven by structural
modules that organize the compound architecture. Among them, β-
and α-turn mimetics are widely used. We have developed SQ4 and
SQ5 squaramido-based turn modules that combine tertiary and
secondary squaramide bonds in their structure to control their
conformational properties. We have evaluated the efficacy of this
combination to promote folding in peptide-like compounds to obtain
parallel and antiparallel-hairpin model compounds in hydrogen
bonding competitive media. Crystallographic structures of model
compounds and the conformational studies performed by NMR on the
squaramido-peptides evaluated confirm that secondary-tertiary
squaramides are more prone to adopt the E,Z conformation than di-
secondary squaramides, and consequently are more suitable to gain
conformational control over foldable peptidomimetic compounds
synthetic analogs and enhance peptide stability against
peptidases.[11]
Among secondary protein structures, β-turns have drawn much
attention due to their crucial role in the bioactive peptide
conformation,[12] the formation of β-sheets and protein-protein
interactions.[13] In this context, β-turn mimics have been used to
develop new drugs[14][2] and β-sheet models, as well as to improve
the yield of the macrocyclization step in the synthesis of
cyclopeptidomimetic compounds.[9] Alternatively, α-turns are
unusual secondary structures in proteins that, ensemble 13-
membered hydrogen-bonded rings stabilized by internal
hydrogen bonds that correspond to tighter structures as β-turns.
Although they have a unique role in relevant bioactive peptides,
there are just a few examples of synthetic modules mimicking α-
turns because of the difficulty of reproducing its features in
synthetic modules.[
15][16]
Therefore, the access to modules that
could mimic the formation of α- and β-turns in bioactive
peptidomimetic compounds is of significant interest. Many
different scaffolds mimic β-turn fragments. However, most of them,
based on cyclic or polycyclic structures, substitute the hydrogen
Introduction
The secondary structure of proteins and peptides are crucial to
their role in a broad array of fundamental biological processes.
Protein misfolding is involved in the etiology of neurodegenerative
disorders, including Parkinson, Alzheimer, and prionic diseases,
among others.[1] However, structural and conformational studies
of proteins can be challenging because of their complexity and
limited availability. As an alternative, small-sized peptides are
valuable and fruitful models to study complex systems since they
have simple architectures and are accessible via chemical
synthesis.[2][3] Peptides are also natural candidates to develop
bioactive compounds.[4–6] However, small peptides rarely retain
their parent conformation and have poor pharmacokinetic
properties. The use of peptidomimetics with engineered structural
elements,[7][8] as macrocyclic scaffolds[9] and turn modules,[10]
becomes fundamental to control the secondary structure of the
covalent bond.[17]
bond found in natural peptides by
a .
Nevertheless, there are fewer examples of hydrogen bond driven
turn modules that fold efficiently linear peptomimetic structures,
since there are limited organic building blocks that provide the
required conformational control, such as modified amino
acids,[18][19][20]lactams,[21][22] ureas[10][23] and thioureas,[24] among
others. Thus, the development of these type of modules is still a
challenging issue.
In the past, we have reported several studies about the
conformational properties of squaramide compounds.[25]
Squaramides are well known for their hydrogen bonding capability
and their use as anion and cation recognition moieties.[
26][27][28]
In
secondary squaramides, the nitrogen atoms are coplanar with the
2
cyclobutene ring showing an (sp ) hybridization as in amides.
2
Accordingly, the length of the C(sp )-N bond of 1.32 Å is shorter
3
compared with a single C(sp )-N bond of 1.46 Å. The hindered
2
−1
rotation around the C (sp )-N bond of 63 kJ mol measured for
secondary squaramides is slightly lower to the values measured
[
[
a]
b]
Dr. L. Martínez-Crespo, Dr. A. Costa, Dr. C. Rotger
Department of Chemistry
University of Balearic Islands
Cra Valldemossa km 7.5, 07122 Palma, Spain
E-mail: carmen.rotger@uib.es
Dr. E. C. Escudero-Adán
-1
for secondary amides (63-96 kJ mol ) and leads to the
observation of Z,Z, and E,Z conformers at room temperature.[
29]
Alternatively, N,N-dialkylureas have a rotational barrier of ∼ 46
-1
kJmol , distinctly lower than squaramides. In this realm,
squaramides are superior to amides and ureas to promote
controlled folding of linear compounds. Furthermore, the
resistance to the hydrolysis observed for squaramide compounds
in the pH range of 3-10 at 37 °C converts to the squaramide unit
in a suitable candidate to incorporate into bioactive compounds
Institute of Chemical Research of Catalonia (ICIQ)
Av. Països Catalans 16 – 43007 Tarragona (Spain)
Supporting information for this article is given via a link at the end of
the document.
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Figure 2. Structure of the peptidomimetic compounds 12 and 13 folded as an
antiparallel and parallel hairpin-like structure, respectively containing the SQ5
module, highlighted in purple.
Here in, we present the conformational studies performed to
investigate the behavior of the secondary-tertiary squaramides as
turn modules in comparison with the analogous di-secondary
squaramide turn modules SQ1 and SQ2, studied previously.
These studies revealed that α-, and β amino acids when attached
to the squaramide ring do not induce significant folding of the
structures, and the best performance was observed when we
used -aminobutiric acid, a natural amino acid as a linker as
shown in SQ2 (Figure1) mimicking the 10 membered ring found
in natural β-turns. Therefore, we designed SQ4 and SQ5, two new
tertiary-secondary squaramide-based turn modules that bear an
alkyl chain decorated at the  position with an acceptor group, an
amine, and a carbonyl group respectively, to induce folding
through the formation of an intramolecular hydrogen bond.
Figure 1. a) Di-secondary squaramides: head-to-tail self-assembly (left) and
Z,Z and E,Z conformers in equilibrium (right) b) Structures of the squaramides
1-6 containing respectively the turn modules SQ1, SQ2, SQ3 SQ4, and SQ5.
Turn modules are highlighted in green (SQ1 and SQ4), purple (SQ2 and SQ5)
and orange (SQ3).
as turnable modules.[30] In our first approach, we used di-
secondary squaramides to develop the turn modules SQ1 and
SQ2, Figure 1a.[25][31] The formation of an intramolecular hydrogen
bond between a squaramide NH and an acceptor group located
at  position of the other N-substituent, stabilizes the E,Z
conformation in solution, forming respectively nine- and ten-
membered rings, as shown in Figure 1b.
For this purpose, we have synthetized the minimalist molecules 2,
3, 5, and 6 that contain modules SQ4 or SQ5 and analyzed the
role of the squaramide N-methyl substituent in the folding process.
Additionally, to gain insight into the versatility of the folding
module SQ5 we have synthetized the peptidomimetic compounds
12 and 13 and have evaluated its role, folding these two
Module SQ1 have been used to fold oligosquaramide strands as
pre-organized precursors in the synthesis of variable-size
[
32]
macrocyclic oligosquaramides. Additionally, we inserted SQ2
in a short peptide sequence that mainly folds into a β-hairpin
structure in polar organic solvents.[31] Despite these achievements,
compounds in a antiparallel and parallel hairpin fashion,
respectively in MeOH, a hydrogen bond competitive solvent
(Figure 2).
di-secondary squaramide-based turn modules SQ1 and SQ2 still
retain some conformational flexibility that prevents fully folding
control (Figure 1b).
To gain conformational control, we designed a new turn module
based on a secondary-tertiary squaramide unit.[33] Tertiary Results and Discussion
-
squaramides have a slightly higher rotational barrier ∼71 kJ mol
1
than secondary squaramides.[34] However, we hypothesized that
the E,Z conformation would prevail as a result of a favorable
balance between the formation of stabilizing intramolecular
interactions and the impossibility to stabilize the Z,Z conformation
commonly observed in bis-di-secondary squaramides through the
arrangement of head-to-tail aggregates (Figure 1a). Thus, we
The synthesis of compounds 2-3 and 5-6 is straightforward
through the sequential condensation of diethyl squarate with
amino compounds. Squaramide 2 and squaramido-ester 7 were
prepared as stated elsewhere.[
25][35]
Squaramido ester 9 was
prepared by the condensation of diethyl squarate with N-
acetylethylenediamine in mixture Et O-EtOH 10%.
a
2
designed
and
successfully
incorporated
a
tertiary
Squaramides 3, 5 and 6 were obtained as shown in Scheme 1.
Shortly, squaramido ester 7 was condensed with N-Boc-N,N’-
dimethyl-1,3-diaminopropane in MeOH to give the bis-
squaramide 8. Finally, the protecting group of 8 was cleaved by
treatment with DCM-TFA 10% to obtain squaramide 3. Di-
squaramides 5 and 6 share in their structure one of the
substituents, dipeptide 10 , that was obtained by coupling N-Boc-
N-methyl-γ-aminobutyric acid with phenylalanine methyl ester in
squaramidoester compound into a decapeptide sequence by
conventional solid-phase peptide synthesis. The resulting
squaramido-peptide compound bearing the SQ3 motif adopted
folded hairpin structures in water, through the formation of both α-
and β-turns. These results pointed out the suitability of secondary-
tertiary squaramides to build more efficient folding scaffolds
(
Figure 1b).[33]
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DMF
and
using
O-(Benzotriazole-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate (HBTU) as the coupling
Scheme 2: Synthesis of peptido-squaramide 12. Reagents and conditions: (a)
Scheme 1. Synthesis of squaramides 3, 5 and 6. Reagents and conditions (a)
DIPEA, Et
2h, r.t.
2 2 2
O-EtOH 7:3 v:v; (b) MeOH-Borax, pH 9, 1:1 v:v, (c) CH Cl -TFA 10%,
EtOH, (b) DCM-TFA 10%, (c) Et
DIPEA, MeOH
2
O-EtOH 9:1, (d) DIPEA, HBTU, DMF, (e)
Scheme 3: Synthesis of peptido-squaramide 13. Reagents and conditions: (a)
NaOEt, EtOH; (b) EtOH-BBS (borate buffer solution, pH 9.5) 50%; (c) HBTU,
2 2
DIPEA, DMF, (d) CH Cl -TFA 10%, 2h, r.t.
reagent. The free amine of 10 was condensed with the
squaramido esters 7 and 9 in MeOH to afford squaramides 5 and
6, respectively.
Synthesis of 12 and 13 was made by condensation of short
peptide sequences with diethyl squarate or the corresponding
squaramidoester. Briefly, the dipeptides used in the synthesis of
12 and 13 were prepared using standard peptide coupling
conditions (HBTU, DIPEA, DMF). Amino groups were protected
with the tert-butoxy carbonyl group and cleaved under acidic
conditions when needed. Synthesis of the squaramido-peptide 12
was performed by the subsequent condensation of compound
First, we studied the conformational properties of the minimalist
1
4b and 17b with diethylsquarate, (Scheme 2). Thus, 14a was
deprotected (DCM-TFA 10%) and condensed with one equivalent
of diethyl squarate in a mixture of Et O-EtOH 7:3 v:v, obtaining
compounds 2-3 and 5-6 that contain modules SQ4, and SQ5
respectively, by NMR spectroscopy in CDCl
3
and polar aprotic
2
solvents, such as DMSO-d
The H NMR spectrum of 2 (1 mM in CDCl
6
. or acetonitrile.
1
monosquaramide-ester 15. Then, after the deprotection of
tripeptide 17a the resulting amine 17b was condensed with 15 in
a mixture of MeOH-Borax buffer at pH 9 1:1 v:v, yielding
compound 12.
To obtain peptidomimetic 13, diethylsquarate was condensed
with one equivalent of N-methyl-γ-aminobutyric acid in EtOH in
the presence of NaOEt to give the squaramide-ester 18 (Scheme
3
at 298 K) shows that
the chemical shift of the squaramide NHa signal at 8.35 ppm is
shifted downfield when if compared with the expected value for
unbound squaramide NH groups in chloroform, which is ~5.5
ppm.[25] A variable concentration experiment (1-100 mM) showed
that the chemical shift of NHa, as well as the rest of the other
signals of the spectrum, are not concentration dependent,
indicating the absence of aggregated species in solution in this
range of concentration.[36] Additionally, when decreasing the
temperature (237-298 K), the NHa signal shifts downfield. The
calculated temperature coefficient for this peak is  (NH)/T = -
0.011 ppm/K. This value is in agreement with those obtained for
NH centers implicated in inter- or intramolecular hydrogen bonds
that strengthen at low temperature. The stepwise addition of
3). First, this unit was condensed with 19b in a mixture of EtOH-
BBS (borate buffer solution, pH 9.5) to give di-squaramide 20, and
then, coupled with 16b using HBTU as coupling reagent in DMF,
to yield compound 13. The synthetic route followed to obtain 13
demonstrated that the turn module SQ5 is compatible with
standard reaction conditions in peptide synthesis.
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DMSO-d
of 2 in CDCl
6
, a hydrogen bonding competitive solvent, to a solution
to 10% v/v does not significantly affect the chemical
(C-H··O = 2.498 Å) and (CH
showing shorter distances than the sum of the Van der Waals
atoms radii (2.72 Å), as previously reported.[33]
2
)g (C-H··O = 2.574 Å) groups,
3
Figure 3. a) Representation of the E, Z conformer of 2 and selected non-
sequential NOEs observed for this compound in chloroform. b) Crystallographic
structure of 2 showing the intramolecular interactions (NH···N and CH···O) that
Figure 4. a) Representation of the E,
Z conformer of 3 and selected
nonsequential NOEs observed for this conformation in chloroform. b)
Crystallographic structure of 3 showing the intramolecular interactions (NH···N
and CH···O) that stabilize the E, Z conformation. c) and d) Crystal packing of 3
showing the intermolecular association of the folded squaramide molecules. All
hydrogen bonds are represented by dotted lines.
stabilize the E,
Z conformation. c) Crystal packing of 2 showing the
intermolecular association of the folded squaramide molecules. Dotted lines
represent the hydrogen bonds.
shift of the NHa proton, confirming that this center is isolated from
the solvent due to the formation of an intramolecular hydrogen
bond. Since we did not appreciate signal splitting at low
temperatures, we assumed that the E,Z-folded conformer is the
In the crystal, the folded molecules are arranged in columns. As
seen in solution, the participation of the NH in the intramolecular
hydrogen bond that folds the structure, and the lack of a second
NH squaramide bond on the molecule prevent the formation of
only significant species in solution (Supporting Information S1-S2). the typical head-to-tail hydrogen-bonded aggregates, found in the
A NOESY experiment run at 258 K showed the spatial proximity
of the NHa with the methylene (CH )c and (CH )e groups. This
together with the four cross-peaks detected between (CH )f and
the butyl chain prove that 1 is in the E,Z conformation, and in a
folded state. Furthermore, a 15N-HMBC experiment at 237 K in
CDCl3, revealed a strong cross-peak that connects NHa with the
tertiary amine nitrogen, showing the strength of the hydrogen
bond that connects these two centers (Supporting Information S3)
crystal structures of di-secondary squaramide compounds.
Therefore, compound 2 adopts a folded structure either in solution
or in the solid state and it is not affected by hydrogen bonding of
competitive solvents. Conversely, compound 1, the di-secondary
squaramide analog, stands in solution as a mixture of conformers
(Z, Z, and E, Z) in equilibrium. These folded and unfolded species
aggregate upon concentration.
2
2
3
We observed the same effect when analyzed the conformational
properties of squaramide 3, where the hydrogen bond acceptor
group is a secondary amine. We performed the conformational
Accordingly, the NOESY experiment run at 298 K in DMSO-d
highly hydrogen bonding competitive solvent, showed analogous
cross-peaks to those observed in CDCl (Supporting information
6
, a
1
3
study as was done for squaramide 2. Consequently, the H NMR
S4), proving that, in this solvent, the folded structure is also the
main conformer in solution. This experiment demostrates the
robustness of the intramolecular hydrogen bond and the
conformational stability of the turn module
3
spectra in CDCl at 298 K showed that the NHa at 8.59 ppm was
shifted 0.24 ppm downfield compared with the corresponding
NHa of squaramide 2. As occurred for 2, this signal showed no
concentration dependence, and the calculated temperature
coefficient for NHa (NH)/T = -0.011 ppm/K is indicative of a
strong temperature dependence. We corroborated the fold state
2 2
Single crystals obtained from a mixture CH Cl -pentane afforded
the solid-state structure of 2, revealing its folded arrangement.
The X-ray data show the formation of the intramolecular hydrogen
bonded nine-membered ring observed by NMR (N−H··N = 2.100
Å), (Figure 3b).
of 3 observing the interstrand cross-peaks such as NHa--(CH
2
)c,
NHa--(CH )f and (CH )f--(CH )g, which are a clear indication of its
3
3
2
folded state in solution (Figure 4a), (Supporting information S8a,
S8b-S9).
Additionally, we observed two stabilizing C−H··O intramolecular
3
hydrogen bonds between the squaramide carbonyls and (CH )b
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Single crystals of 3 were obtained from a CH
2
Cl
2
-pentane mixture.
bonds; while NHa is shielded from other molecules through the
formation of an intramolecular hydrogen bond that is slightly
strengthened as the temperature decreases (Supporting
Information). The NOESY experiment run on 5 (10 mM in CDCl ,
3
237 K) confirmed the E, Z conformation and the folded state of 5,
The analysis of the X-ray diffraction spectroscopy data revealed
the formation as in solution, of a nine-membered ring folded
structure, stabilized by the intramolecular hydrogen bonds
N−Ha··N (2.086 Å), and the C-Hb··O (2.442 Å) and C-Hg··O
(
2.540 Å) interactions, (Figure 4b). In this case, the N-Ha··N
showing strong cross-peak signals between NHa and the
distance is 0.014 Å shorter in compound 3 than in 2, an indication
of the formation of a stronger intramolecular hydrogen bond, as
seen in solution, where the minor steric interactions in secondary
amine 3, favor the formation of the intramolecular hydrogen bond.
The subtle structural difference between 2 and 3 provokes an
entirely different packing of the folded molecules in the crystal
leading to the formation of ribbons, but yet avoiding the formation
of head-to-tail hydrogen bonds (Figure 4c and 4d, Supporting
information).
2 2
methylene groups (CH )c and (CH )d (Figure 5a).
Conformational studies performed for squaramide 5 (5 mM) in
acetonitrile, where intermolecular and weak intramolecular
hydrogen bonds are disfavored, also prove the prevalence of the
E, Z folded conformer (93%), that coexists in solution with a 7 %
of the Z, Z unfolded form, (Supporting information S11-S15).
The turn structure observed for 5 in solution was also identified in
the solid state from the RX analysis of crystals obtained from a
2 2
CH Cl -pentane mixture, showing the intramolecular hydrogen
bond NH···N (1.895 Å), forming a ten-membered ring. Once again,
we observed the additionally stabilizing C-Hb··O (2.440 Å) and C-
Hj··O (2.499 Å) interactions, (Figure 5b).
In stark contrast to these results, our previously reported studies
indicate that di-secondary squaramide 4, exists in acetonitrile
solution as a mixture of Z, Z and E, Z forms in approximately equal
proportion, reflecting clearly the dominant folding tendency in
secondary-tertiary squaramides that contain the SQ5 module. As
occurred for compound 2 and 3, we observed the predominant
formation of the folded structure of 5, either in solution and the
solid state. This result evidences the superior capability of SQ5 to
induce folding compared to SQ2.
To explore the scope of the tertiary- secondary squaramide unit
as turn module, we investigated if module SQ5 could favor the
formation of α-turns in peptidomimetic sequences when combined
with a suitable linker. For this purpose, we designed compound 6
that combines SQ5 with the diethylene amine segment to form a
13-membered α-turn-like ring (Figure 6a).
Figure 5. (a) Folded chemical structure of compound 5. (b) Crystallographic
structure of 5 showing the intramolecular interactions (NH···O and CH···O) that
stabilize the E, Z conformation. (c) Crystal structures of hydrogen-bonded folded
structures of 5 forming ribbons through the crystal.
Compounds 5 and 6 contain in their structure the turn module
named SQ5. In this case, folding is driven by the formation of an
intramolecular hydrogen bond between a squaramide NH and an
amide carbonyl group located at the
position of the other
squaramide substituent. This interaction yields a ten-membered
hydrogen bonding cycle that mimics natural β-turns.
Once again, we studied the conformational properties of 5 in
1
CDCl
3
by H NMR. In CDCl
3
at 298 K, the squaramide proton NHa
appears at 7.56 ppm, fairly deshielded compared to the value
expected for unbound squaramide NH. In agreement with the
formation of an intramolecular hydrogen bond, NHa has little
concentration dependence (Δ = 0.054 ppm) in the 1-25 mM
range. However, the amide proton NHe suffers a moderate
downfield shift (Δ = 0.295 ppm) at the same concentration
interval. The calculated (NH)/T coefficients for NHa and NHe
in the range of 313-237 K are -0.005 and -0.003 ppm/K,
respectively. The data indicate that NHe is exposed to the solvent
and bonds upon concentration through intermolecular hydrogen
Figure 6. a) Folded structure of 6 showing the formation of the α- and β-turns
b) chemical shift variation observed for the signals ♦ NHa, ▲ NHf and ■ NHi of
a 2.5 mM solution of 6 in CDCl
3
upon the addition of different amounts of DMSO-
d
6
up to 13 %.
3
Conformational analysis of 6 in CDCl showed that the NHa
proton is concentration non-dependent in the 1-50 mM range, in
contrast with NHf and NHi, that are shifted 0.50 and 0.26 ppm
downfield, respectively. The temperature coefficients  (NH)/T
3
calculated from a 2.5 mM solution of 6 in CDCl for signals NHa,
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Figure 6. Representation of the conformational equilibrium and selected non-sequential NOEs observed for squaramides (a) 12 and (b) 13 in CD
3
OH. Color code:
NOEs corresponding to common signals 12A-12B (red), 12A (purple), 12B (blue) and 12C (green), both 13A and 13C (red) and the unfolded 13B (green)
NHf and NHi are accordingly -0.007, -0.002 and -0.008 ppm/K,
Supporting Information S17a and S17b). These experiments
As a step forward in this study, the turn module SQ5 was inserted
in β-hairpin mimetics 12 and 13, to evaluate its ability to induce
the formation of antiparallel and parallel hairpins, respectively
(Figure 2). These two short hairpin models were designed to
establish three intramolecular hydrogen bonds, and in both
examples, the extremes of the chains have been functionalized
with aliphatic residues to gain solubility in organic solvents.
In compound 12, module SQ5 was combined with the linker
ethylenediamine to revert the directionality of the short peptide
chain and obtain an antiparallel hairpin structure. Meanwhile, in
compound 13 SQ5 was directly combined with the terminal amino
(
revel that the proton NHa is shielded from the solvent through the
formation of an intramolecular hydrogen bond. Similarly, the NHi
proton, with little concentration dependence and similar
temperature coefficient that NHa, is also intramolecular hydrogen
bonded and shielded from the solvent, although in lesser extent
as can form intermolecular interactions upon concentration.
6
When we added increasing amounts of DMSO-d up to 13 % to a
2
.5 mM solution of 6 in CDCl , we observed the downfield shift of
3
NHf (Δ = 1.60 ppm) and NHi (Δ = 0.41 ppm) signals which is an
indication that NHf is exposed to the solvent while NHi is partially
shielded from it. Conversely, NHa is shifted upfield (Δ = -0.30
ppm) as a result of the weakening of the external intramolecular
hydrogen bond, but still mainly shielded from the solvent. Thus,
the conformational analysis of compound 6 in chloroform proves
the formation of α-turn-like structure stabilized by an internal β-
turn, and as occurs in nature, the outer ring is held by a weaker
hydrogen bond than the internal β-turn. This assumption was
group of the dipeptide L-Ala-L-Ala to organize
arrangement of the peptidomimetic strands.
a parallel
We studied the conformational properties of 12 and 13 in
methanol, highly competitive hydrogen bonding solvent.
a
Variable temperature experiments showed that at 231 and 233 K
respectively, the equilibrium between species is slow on the NMR
1
time scale and the H NMR spectrum of both compounds show
sharp and well-resolved signals. We could unequivocally assign
the peaks of the different species in the equilibrium and perform
the structural studies at 600 mHz by 2D NMR COSY, TOCSY and
NOESY experiments. For compound 12 (5 mM, CD OH, 231 K),
3
we identified three monomeric species in equal proportion, named
3
confirmed when we studied compound 6 in CD CN (2.5 mM)
where we could only observe the formation of the ten-membered
ring (Supporting information S18). Therefore, the turn module
SQ5 can induce both α- and β-turns and is a suitable structure to
develop new α-turn mimetics.
12A, 12B, and 12C, respectively, since a dilution experiment run
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in the range of concentration 1-10 mM at 231 K discarded the
existence of aggregates. Noteworthy, the chemical shifts of
species 12A and 12B only differ in those corresponding to the
3
Comparably, the signals of 13 split (5 mM, CD OH, 233 K) and
we detected and fully assigned three set of peaks corresponding
to three species named 13A, 13B, and 13C, respectively. The
main differences regarding the chemical shifts are between 13A
and 13B. Thus, the NHa proton of 13A at 8.48 ppm is 0.49 ppm
shifted downfield compared to the same signal in 13B at 7.99 ppm.
dipeptide
indistinguishable the rest of the molecule. Thus, in both 12A and
2B, the signal NHa at 8.49 ppm and (CH )b at 3.38 ppm are
segment
Leu-Phe-NH-cyclohexyl,
being
1
3
deshielded compared with the chemical shift of the corresponding
signals of 12C that appear at 8.13 and 3.18 ppm, respectively.
Based on the analysis of the chemical shifts, we assumed that in
species 12A and 12B the squaramide moiety is in an E, Z
conformation. The downfield shifts observed for NHa and (CH )b
3
signals are in agreement with the formation of the intramolecular
hydrogen bond involving the NHa proton and as a consequence
2
At this temperature, the two (CH )c protons in 13 become
anisochronous. In 13A, these protons located at 3.42, and 3.34
ppm are 0.61, and 0.30 ppm shifted upfield respectively if
compared with the corresponding signals in 13B found at 4.03
and 3.64 ppm. These features indicate that in conformer 13A, the
squaramide is in the E,Z conformation favoring the formation of
the intramolecular hydrogen bond between NHa and the amide
carbonyl from the other squaramide substituent. Accordingly, in
3
the localization of the (CH )b group under the paramagnetic
shielding of the neighbor squaramide carbonyl.
2
the E,Z conformation the (CH )c protons are not exposed to the
Conversely, in species 12C the squaramide module adopts a Z,
Z conformation that leads to an unfolded structure. To confirm this
assumption, we run a NOESY experiment from which we
observed fifteen NOEs between nonadjacent residues (Figure 6a)
whose signals correspond to the conformers 12A and 12B. For
example we detected NOEs that originate from the common
paramagnetic effect of the neighbor squaramide carbonyl
explaining the upfield shift of the signals. Conversely, the
chemical shifts found for the corresponding 13B signals suggest
that in this species, the squaramide is in the Z,Z conformation, as
2
locates the (CH )c signals under the influence of the neighbor
squaramide carbonyl and hence undergo a downfield chemical
shift. In 13A, NHq, and NHl are also shifted downfield respect of
the same signals in 13B, in agreement with the formation of an
intramolecular hydrogen bonded parallel hairpin structure.
Conformer 13C has the same chemical shift pattern than 13A,
revealing that it is also a folded structure. However, the lower
downfield shift observed for the corresponding NHa and NHq is
indicative of the formation of weaker intramolecular hydrogen
bonds.
signals of 12A and 12B, as those that connect (CH
protons (CH )g, CHj, (CH )k and (CH )m located at the opposite
)o—NHv between the two
2
)e with the
2
2
3
squaramide substituent and (CH
terminal groups
3
Furthermore, we detected for 12A several additional interstrand
NOEs such as CHp--(CH )g, CHt--(CH )g, (CH )g--NHs,
CH )g—Ar and NHn--(CH )r. For 12B we also detected the
additional interstrant crosspeaks CHj--NHf, (CH )g--Ar, (CH )o—
Ar, and (CH )m--Ar confirming the antiparallel disposition of the
2
2
2
(
2
3
2
3
3
2 2 2
The nonadjacent NOEs (CH )c--NHa, (CH )d--NHa and (CH )e--
peptide chains in both species.
NHa observed in methanol confirm the E, Z folded conformation
The chemical shifts of NHs and NHn are also in agreement with
of the species 13A and 13C. Also, the interstrand cross peaks
the hydrogen-bonding pattern of
differences detected for the Leu-Phe-NHcyclohexyl segment in
2A and 12B may be a consequence of slightly different
conformations of this fragment within the folded structure. On the
other hand, a strong NOE cross peak between NHa and (CH )b
a
hairpin structure. The
(CH
induces the molecule folding in a parallel hairpin-like structure. On
the other hand, a strong NHa--(CH )b cross peak confirms the Z,
Z unfolded conformation of 13B. This conformer represents the
40 % of the compound in the solution (Figure 6b).
3 3
)h--NHq and (CH )h--CHn observed for 13A prove that SQ5
1
3
3
protons of 12C confirms the Z, Z conformation of the squaramide
moiety and the unfolding nature of 12C. Therefore, in methanol
and at 231 K, 66% of molecules of 12 adopt an antiparallel
hairpin-like structure. Compound 12 combines turn module SQ5
with the flexible linker ethylenediamine to promote an antiparallel
hairpin. Unlike in compound 6, no evidence exists of any
conformer of squaramido-peptide 12 showing the α-turn ring. In
this case, we must point out that the formation of the α-turn
through a weak hydrogen bonding interaction would lead to a
The relative disposition of faced hydrogen-bonding donor and
acceptor groups in a parallel hairpin structure is not as optimum
as in the antiparallel one. We believe that the folding observed
here is a promising result and we hope that the design of larger
structures with more hydrogen-bonding groups and additional
stabilizing interactions between the side chains of the two peptide
residues will lead to obtaining more efficient parallel hairpin
mimetics.
folded structure with a minor number of inter-strand hydrogen Conclusions
bonding interactions, which are fundamental to stabilize the
folded structure in hydrogen bonding competitive solvents like
methanol. Nevertheless, the SQ5 module remarkably improves
the folding capability of short peptide-squaramide compounds if
compared with the SQ2 performance that leads to more
conformational free structures, obtaining when incorporated in
short peptide sequences similar to compound 12, an useless
mixture of unfolded and folded species even in a non-hydrogen
bonded competitive solvent as acetonitrile.[31]
We have developed two new squaramide-based turn modules
SQ4 and SQ5. Conformational studies performed with the model
compounds 2, 3, 5, 6, 12 and 13 that include these turn modules,
confirm that secondary-tertiary squaramides are more prone to
adopt the E, Z conformation than di-secondary squaramides, and
consequently are more suitable to gain conformational control
over foldable peptidomimetic compounds in hydrogen bond
competitive solvents as methanol.
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Chemistry - A European Journal
10.1002/chem.201803930
FULL PAPER
NMR experiments performed in solution have evidenced that the
folding of the structures is induced by the formation of NH-X (X=
N, O) intramolecular hydrogen bonds. Moreover, X-Ray analysis
of the crystal structures of secondary-tertiary squaramides 2, 3
(CDCl
3
) : 5.85 (1H, br), 3.74 (2H, q, J = 6.6Hz), 3.47 (2H, br), 3.29 (3H, s), 2.88
(3H, s), 1.89 (2H, q, J = 7.2Hz), 1.63 (2H, q, J = 7.5Hz), 1.47 (9H, s), 1.41 (2H,
q, J = 7.5Hz), 0.95 (3H, q, J = 7.5Hz) ppm. 13C NMR (DMSO-d
) : 183.9, 183.8,
6
169.5, 169.0, 156.8, 80.2, 54.5, 48.66, 47.2, 45.9, 36.5, 34.0, 24.1, 20.2, 14.2
+
and
5
had shown the formation of an additional CH
3
-O
ppm. HRMS (MALDI-TOF): m/z calc. for C18
H
31
N
3 4
O
Na [M+Na] 376.4462, exp.
intramolecular interaction. This interaction, nonexistent in di-
secondary squaramides E,Z conformation, contributes to the
stabilization of the turn conformation either in solution and solid
state. The formation of a thirteen-membered hydrogen bonded
cycle has also been observed in compound 6, mimicking the α-
turns found in proteins. In addition, and as a further step towards
foldable peptidomimetic structures, the turn module SQ5 was
successfully incorporated into short peptide strands to induce
their folding in protic solvents, obtaining both parallel and
antiparallel hairpin structures. In conclusion, the N-methyl-
squaramides studied here are suitable folding units to incorporate
into linear peptide o pertidomimetic sequences due their excellent
conformational properties and straightforward incorporation by
direct condensation reactions.
376.4470.
3-(butylamino)-4-(methyl(3-(methylamino)propyl)amino)cyclobut-3-ene-
1,2-dione, 3. 8 (100 mg, 0.28 mmol) was stirred for 2 hours at r.t in a mixture of
CH
stream at r.t. The crude was dissolved in NaOH 1M (5 mL) and the solution was
extracted with CH Cl (6  6 mL). The organic phases were combined, dried
(MgSO ) and concentrated. The solid was dissolved in CH Cl (3 mL) and
2 2
Cl -TFA 10 % (10 mL). Then, the solvent was removed under an argon
2
2
4
2
2
pentane (10 mL) was added to obtain a precipitate. The solid was filtered and
the remaining solution was concentrated under reduced pressure to afford 3 (10
mg, 0.04 mmol) as a white solid. Yield 14 %. m.p 208 ºC; 1H NMR (CDCl
3
) :
8.63 (1H, br), 3.67 (2H, q, J = 6.6Hz), 2.27 (2H, br), 2.63 (2H, t, J = 5.4Hz), 2.41
(3H, s), 1.74 (2H, q, J = 5.4Hz), 1.57 (2H, q, J = 7.8Hz), 1.38 (2H, q, J = 9Hz),
0.94 (2H, t, J = 7.2Hz) ppm. 13C NMR (DMSO-d
67.7, 55.4, 48.8, 46.9, 44.8, 38.6, 36.5, 36.3, 34.0, 29.5, 26.7, 20.2, 14.2 ppm.
HRMS (ESI): m/z calc. for C13
[M+H]+ 254.1869, exp. 254.1874.
) : 183.6, 182.3, 169.2, 168.8,
6
1
Experimental Section
24 3 2
H N O
All reagents and solvents were commercially available and were used without
further purification unless otherwise stated. All reactions sensitive to air or
moisture were carried out under argon atmosphere in dry freshly distilled
4-((tert-butoxycarbonyl)(methyl)amino)butanoic
(Methylamino)butyric acid hydrochloride was dissolved in a mixture of NaOH
1M-dioxane 1:1 v:v (20 mL) and cooled to 6 C. To this solution, Boc O (1.421
acid
.
4-
solvents under anhydrous conditions. DMSO-d
6
and CDCl
3
(99.8 %D) were
2
1
stored on molecular sieves (3 Å). H and 13C NMR spectra were recorded at 600
g, 6.5 mmol) in dioxane (6 mL) was added dropwise and then the mixture was
stirred at r.t. for 15 hours. The crude was filtered and the liquid phase was
and 300 MHz at 23 ºC unless otherwise specified. Chemicals shifts are reported
as parts per million (δ) referenced to the residual deuterium lock solvents. The
coupling constants values J are given in Hz. NMR peak assignments were
performed by COSY, TOCSY, HSQC, HMBC, and NOESY experiments. HRMS
data were obtained on a MicroMass Autospec 3000 double focusing magnetic
sector mass spectrometer operating at 3000 m/z with a cone voltage of 4V
4
concentrated under reduced pressure. The residue was dissolved in KHSO 1M
(20 mL) and product was extracted in AcOEt (2  20 mL). The organic phase
was washed with brine (20 mL), dried, and the solvent was evaporated to afford
N-Boc-N-methyl--aminobutyric acid (680 mg, 3.12 mmol) as a pale yellow oil.
1
Yield 96 %. H NMR (CDCl
3
) : 3.29 (2H, br), 2.85 (3H, s), 2.36 (2H, t, J = 7.2Hz),
) : 178.7, 156.4,
Na
1.85 (2H, m, J = 6.9Hz), 1.46 (9H, s) ppm. 13C NMR (CDCl
(
HRMS-ESI), and on a Bruker Autoflex (MALDI−TOF) with 2,5-dihydroxibenzoic
3
acid (DHB) as matrix.
80.2, 48.3, 34.6, 31.4, 28.8, 23.3 ppm. HRMS (ESI): m/z calc. for C10
H19NO
4
[M+Na]+ 240.1212, exp. 240.1211.
Concentration dependence experiments were run by recording the spectra of
the studied compounds at different concentration in the range 1-100 mM.
Temperature coefficients were determined by recording the spectra of 1-10 mM
solution of the compounds at different temperatures varying between 318-237
K.
(N)-methyl
2-(4-((tert-butoxycarbonyl)(methyl)amino)butanamido)-3-
phenylpropionate, 10. 10 was synthesized according to the general procedure
aminoacid coupling reactions from N-Boc-N-methyl--aminobutyric acid (120
mg, 0.55 mmol) and Phe-OMe·HCl (178 mg, 0.99 mmol) in DMF (15 mL). The
General procedure for amino acid coupling reactions
solvent was removed and the crude was dissolved in CH
with HCl 3M (2  20 mL) and brine (20 mL), dried (MgSO
Et O (10 mL) was added to the viscous oil, and the mixture was stirred for 10
2
Cl
2
(20 mL), washed
The amino acids with the free carboxylic acid and free amine were dissolved in
dry DMF with DIPEA (8 equivalents). To this solution, HBTU (3 equivalents) in
DMF was added under inert atmosfera. The reaction mixture was covered witha
tin foil and stirred for 15 hours at room temperature. The crude was purified as
stated below for each compound.
4
) and concentrated.
2
min. Then, the solvent was decanted and this procedure was repeated three
times. The combined organic phases were concentrated to afford 10 (202 mg,
0.53 mmol) as a pale brown oil. Yield 97 %. 1H NMR (CDCl
br), 3.73 (1H, s), 3.16 (4H, m, J = 5.7Hz), 2.82 (3H, s), 2.13 (2H, m, J = 6.9Hz),
1.79 (2H, m, J = 6.9Hz), 1.46 (9H, s) ppm. 13C NMR (CDCl
) : 172.7, 156.8,
136.8, 129.7, 128.9, 127.5, 79.9, 53.7, 52.7, 47.6, 38.2, 34.6, 33.6, 28.9, 24.2
ppm. HRMS (MALDI-TOF): m/z calc. for C20 K: 401.2052, exp. 401.2033.
3
) : 7.28, 4.84 (1H,
tert-butyl
(3-((2-(butylamino)-3,4-dioxocyclobut-1-en-1-
3
yl)(methyl)amino)propyl)(methyl)carbamate, 8. A solution of 7 (283 mg, 1.40
mmol) in EtOH (6 mL) was added to another solution of N-(tert-
butyl)oxycarbonyl-N,N’-trimethyl-1,3-diaminopropane (276 mg, 1.40 mmol) in
EtOH (5 mL). The mixture was stirred for 20 hours at room temperature. Then
the solvent was removed under reduced pressure and the residue was
30 2 5
H N O
(N)-methyl
yl)(methyl)amino)butanamido)-3-phenylpropanoate, 5. A solution of 10 (68
mg, 0.18 mmol) in a mixture of CH Cl -TFA 10 % (10 mL) was stirred for 2 hours
2-(4-((2-(butylamino)-3,4-dioxocyclobut-1-en-1-
dissolved in CH
2  15 mL) and NaCl sat. (15 mL), dried on MgSO
under reduced pressure and the resulting solid was washed with pentane and
O to afford 8 (321 mg, 0.91 mmol) as a white solid. Yield 65 %. 1H NMR
Et
2
Cl
2
(20 mL). The resulting solution was washed with HCl 0.1M
2
2
(
4
. The solvent was removed
at r.t. and the solvent was removed under an argon stream , obtaining the TFA-
salt of 11. This salt was mixed with DIPEA (0.09 mL, 0.9 mmol) and to this
solution, 7 (39 mg, 0.20 mmol) in MeOH (5 mL) was added. The mixture was
2
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Chemistry - A European Journal
10.1002/chem.201803930
FULL PAPER
stirred for 15 hours at r.t. The solvent was removed under reduced pressure and
41.9, 40.9, 40.6, 34.4, 28.8, 25.2, 23.6, 23.2, 22.6 ppm. HRMS (MALDI-TOF):
the crude was dissolved in CH
and brine (10 mL), dried (MgSO
by flash column chromatography (Al
as a white solid. Yield 58 %. m.p 137 ºC; 1H NMR (CDCl
2
Cl
) and concentrated. The product was purified
O3; AcOEt) to afford 5 (25 mg, 0.09 mmol)
) : (CDCl ) : 7.55
1H, s), 7.10 (2H, d, J = 5.7Hz), 6.03 (1H, d, J = 5.7Hz), 4.84 (1H, q, J = 6.9Hz),
2
(10 mL), washed with HCl 3M (2  10 mL)
29 3 4
m/z calc. for C15H N O
Na [M+Na]+ 328.2056, exp. 328.2066.
4
2
(S)-2-acetamido-N-(2-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)-
4-methylpentanamide, 15: A solution of 14 (160 mg, 0.51 mmol) in CH Cl
TFA 10 % (10 mL) was stirred for 2 hours at r.t. and the solvent was removed
under an argon stream at r.t. The resulting TFA-salt was dissolved in Et O:EtOH
2:1 v:v (18 mL) and DIPEA (0.408 mL, 4.08 mmol). This solution was added
dropwise to another solution of diethyl squarate (129 mg, 0.76 mmol) in Et O (2
mL) and the reaction mixture was stirred for 48 hours at r.t. The solvent was
removed under reduced pressure and the crude was washed with Et O (4  20
mL) to afford 15 (130 mg, 0.38 mmol) as a white solid. Yield 75 %. m.p 151 ºC;
1H NMR (CDCl
) : 4.75 (2H, br), 4.29 (1H, br), 3.58 (2H, br), 3.36 (2H, m, J =
7.8Hz), 2.02 (3H, s), 1.63 (3H, m), 1.47 (3H, t, J = 6.3Hz), 1.47 (6H, dd, J =
6.6Hz) ppm. 13C NMR (CDCl
) : 190.1, 183.1, 177.3, 174.1,173.2, 170.7, 69.6,
44.6, 44.2, 34.4, 25.2, 23.6, 23.2, 22.6, 16.5 ppm. HRMS (MALDI-TOF): m/z
3
3
2
2
-
(
3.78 (5H, s), 3.37 (3H, s), 3.12 (4H, m, J = 6.6Hz), 2.29 (2H, q, J = 7.8Hz), 1.89
2
(
2H, m, J = 5.1Hz), 1.64 (2H, m, J = 7.8Hz), 1.42 (2H, m, J = 7.8Hz), 0.96 (3H,
t, J = 7.2Hz) ppm. 13C NMR (CDCl
) : 183.2, 173.0, 172.2, 168.2, 136.4, 129.6,
29.0, 128.9, 127.6, 54.0, 52.9, 51.9, 44.7, 38.2, 37.7, 33.5, 31.1, 24.2, 20.1,
4.2 ppm. HRMS (ESI): m/z calc. for C23 Na: 452.2161, exp. 452.2162.
3
2
1
1
H
31
N
O
3 5
2
N-(2-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)acetamide, 9.
solution of diethyl squarate (233 mg, 1.37 mmol) in Et O (20 mL) was added to
a stirred solution of N-(2-amino)ethylacetamide (116 mg, 1.14 mmol) in Et O-
EtOH 21:4 v:v (15 mL). The mixture was stirred for 12 hours at r. t. The crude
was filtered and the solid was washed with Et O to obtain part of the final product.
The solution was concentrated under reduced pressure and the residue was
dissolved in EtOH (0.5 mL) and to this solution, Et O (10 mL) was added to
obtain a precipitate which was filtered to afford the rest of the product. Altogether,
30 mg (0.57 mmol) of 9 were obtained as a white solid. Yield 50 %. m.p 136
ºC; 1H NMR (CDCl
) : 6.87 (1H, s), 6.19 (1H, s), 4.76 (2H, br), 3.78 (1H, br),
.61 (1H, br), 3.50 (2H, br), 2.00 (3H, s), 1.47 (3H, t, J = 6.6Hz) ppm. 13C NMR
A
3
2
2
3
2
25 3 5
calc. for C16H N O
Na [M+Na]+ 362.1692, exp. 362.1702.
2
tert-butyl
((S)-1-(((S)-1-(cyclohexylamino)-1-oxo-3-phenylpropan-2-
yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate, 16: 16 was synthesized
according to the general procedure for amino acid coupling, using N-Boc-L-
valine (265 mg, 1.22 mmol) and phenylalanine-cyclohexylamide (300 mg, 1.22
1
3
3
mmol) in DMF (10 mL). After, CH
and the resulting solution was washed with HCl 1M (3  50 mL), H
and brine (50 mL), dried over MgSO and concentrated. The solid obtained was
2
Cl
2
(50 mL) was added to the reaction mixture,
(
DMSO-d
6
) : 190.1, 183.1, 177.3, 173.9, 173.5, 170.2, 69.6, 44.6, 44.2, 23.4,
2
O (50 mL)
+
1
6.5 ppm. HRMS (MALDI-TOF): m/z calc. for C10
H
14
N
2 4
O K [M+K] 265.059, exp.
4
265.058.
crushed in Et
2
O (20 mL) and filtered to afford 16 (475 mg, 1.07 mmol) as a white
1
solid. Yield 88 %. m.p 205 ºC con descomposición; H NMR (CDCl
3
) : 6.48 (1H,
(
S)-methyl 2-(4-((2-((2-acetamidoethyl)amino)-3,4-dioxocyclobut-1-en-1-
yl)(methyl)amino)butanamido)-3-phenylpropanoate, 6. 10 (200 mg, 0.53
mmol) in CH Cl -TFA 10 % (10 mL) was stirred for 2 hours at r.t. and the solvent
was removed under an argon stream at r.t. To this TFA-salt 11, a solution of 9
120 mg, 0.53 mmol) in MeOH (9 mL) and DIPEA (0.477 mL, 4.77 mmol) was
added. The mixture was stirred for 96 hours at r.t. The solvent was removed
under reduced pressure and the crude was washed with Et O (3  2 ml) until
the oil became a solid. The solid was filtered and dissolved in CH Cl (2 ml).
br), 5.74 (1H, br), 4.83 (1H, br), 4.57 (1H, q, J = 6.3Hz), 3.89 (1H, br), 3.67 (1H,
m, J = 7.8Hz), 3.19 (1H, m, J = 7.8Hz), 2.99 (1H, m, J = 7.8 Hz), 2.18 (1H, m, J
= 5.7Hz), 1.77 (2H, m), 1.58 (4H, m), 1.42 (9H, s), 1.26 (4H, m), 1.05 (2H, m),
2
2
0.93 (3.3H, d, J = 6.9Hz), 0.82 (2.7H, d, J = 6.9Hz) ppm. 13C NMR (CDCl
3
) :
(
172.5, 162.9, 156.5, 79.8, 70.2, 50.1, 49.2, 37.9, 37.4, 37.0, 36.6, 28.8, 28.3,
+
16.3 ppm. HRMS (MALDI-TOF): m/z calc. for C25
H
39
N
3 4
O
K [M+K] 484.258, exp.
2
484.257.
2
2
Hexane was added to obtain two phases and was let to stand until a solid was
tert-butyl
(4-(((S)-1-(((S)-1-(cyclohexylamino)-1-oxo-3-phenylpropan-2-
obtained to afford 6 (90 mg, 0.19 mmol) as a white solid. Yield 35 %. m.p 105
yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-
1
ºC; H NMR (CDCl
3
) : 7.76 (1H, br), 7.11 (2H, d, J = 7.5Hz), 7.67 (1H, br), 6.23
oxobutyl)(methyl)carbamate, 17: A solution of 16 (150 mg, 0.34 mmol) in a
(1H, d, J = 7.5Hz), 4.86 (1H, q, J = 6.6Hz), 3.78 (5H, m), 3.48 (2H, t, J = 5.4Hz),
2 2
mixture of CH Cl -TFA 10% (10 mL) was stirred for 2 hours at r.t., and then the
3
6
1
4
.39 (3H, s), 3.16 (3H, m, J = 5.7Hz), 3.09 (2H, m, J = 6.6Hz), 2.30 (2H, q, J =
solvent was removed under an argon stream at r.t. The TFA-salt was combined
with N-Boc-N-methyl--aminobutiric acid (74 mg, 0.34 mmol) in DMF (10 mL)
following the general procedure described for aminoacdi coupling. The solvent
.6Hz), 1.91 (2H, m), 1.59 (3H, br) ppm. 13C NMR (CDCl
3
) : 183.4, 182.7, 172.9,
72.4, 171.8, 168.7, 167.8, 136.3, 129.6, 129.1, 127.6, 53.9, 53.0, 52.0, 44.2,
1.0, 38.2, 37.8, 31.6, 24.1, 23.5 ppm. HRMS (ESI): m/z calc. for
was removed and the crude was dissolved in CH
2
Cl
2
(25 mL), washed with HCl
and the solvend was
C
46
H
60
N
8
O
12Na [2M+Na]+ 939.4228, exp. 939.4243.
3M (2  25 mL) and brine (25 mL), dried over MgSO
4
evaporated under reduced presure. The solid obtained was crushed in acetone
(
S)-tert-butyl (2-(2-acetamido-4-methylpentanamido)ethyl)carbamate, 14:
4 was synthesized according to the general procedure for aminoacid coupling
from N-Boc-ethylendiamine (477 mg, 2.89 mmol) and N-acetyl-leucine (500 mg,
.89 mmol) in DMF (20 mL). The solvent was removed and the crude was
dissolved in CH Cl (60 mL), washed with HCl 0.3M (4  60 mL), H O (2  60
mL) and brine (60 mL), dried over MgSO and concentrated. The product was
dissolved in Et O (60 mL) and the 90 % of the solvent was removed under
(20 mL) and filtered to afford 17 (78 mg, 0.14 mmol) as a white solid. Yield 42 %.
1
1
m.p 210 ºC (decomposes); H NMR (CDCl
3
) : 6.97 (1H, d, J = 7.5Hz), 6.87 (1H,
br), 6.25 (1H, br), 4.70 (1H, q, J = 5.4Hz), 3.68 (1H, br), 3.38 (1H, br), 3.20 (1H,
br), 3.03 (2H, m), 2.84 (3H, s), 2.19 (3H, m), 1.77 (2H, br), 1.60 (4H, m), 1.48
(9H, s), 1.25 (4H, m), 1.07 (2H, m), 0.84 (6H, dd, J = 7.5Hz) ppm. 13C NMR
2
2
2
2
4
(CDCl
54.6, 48.9, 47.2, 37.8, 34.5, 33.2, 33.0, 30.1, 28.9, 25.9, 25.4, 24.1 ppm. HRMS
(ESI): m/z calc. for C30
Na [M+Na]+ 567.3522, exp. 567.3513.
3
) : 174.3, 171.6, 170.0, 157.3, 137.9, 129.7, 128.8, 127.0, 80.4, 60.7,
2
reduced pressure to the formation of a solid. The solid was filtered from the
48 4 5
H N O
remaining solvent to afford 14 (170 mg, 0.54 mmol) as a white solid. Yield 19 %.
m.p 135 ºC; 1H NMR (CDCl
) : 6.67 (1H, br), 5.88 (1H, br), 4.92 (1H, br), 4.42
1H, m, J = 5.4Hz), 3.48 (2H, br), 3.28 (2H, br), 2.03 (3H, s), 1.57 (2H, m), 1.46
11H, br), 0.96 (6H, s) ppm. 13C NMR (CDCl
) : 173.2, 170.7, 157.0, 80.2, 52.3,
(S)-2-acetamido-N-(2-((2-((4-(((S)-1-(((S)-1-(cyclohexylamino)-1-oxo-3-
phenylpropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-
3
(
(
3
oxobutyl)(methyl)amino)-3,4
dioxocyclobut-1-en-1-yl)amino)ethyl)-4-
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Chemistry - A European Journal
10.1002/chem.201803930
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methylpentanamide, 12: A solution of 17 (80 mg, 0.15 mmol) in CH
2
Cl
2
-TFA
solid was formed and the solution was filtered to afford 19 (209 mg, 0.66 mmol)
1
1
0% (10 mL) was stirred for 2 hours at r.t., then the solvent was removed under
an argon stream at r.t. The TFA-salt was dissolved in MeOH (5 mL) and DIPEA
0.135 mL, 1.35 mmol). The resulting solution was added to another solution of
as a white solid. Yield 53 %. m.p 138 ºC; H NMR (CDCl
3
) : 6.59 (1H, br), 5.94
(1H, s), 4.93 (1H, br), 4.31 (1H, t, J = 7.2Hz), 4.11 (1H, br), 1.44 (9H, s), 1.33
(15H, s) ppm. 13C NMR (CDCl
) : 172.9, 171.5, 80.4, 51.7, 50.9, 49.7, 29.1,
(
3
29 3 4
H N O
Na [M+Na]+
1
5 (50 mg, 0.15 mmol) in MeOH (2 mL) and the reaction mixture was stirred for
28.7, 18.9, 18.6 ppm. HRMS (MALDI-TOF): m/z calc. for C15
338.204, exp. 338.206.
8
days. The solvent was removed under reduced pressure and the crude was
crushed in water, filtered and dried to afford 12 (81 mg, 0.11 mmol) as a white
solid. Yield 73 %. m.p 188 ºC con descomposición; 1H NMR (DMSO-d
): 8.03
1H, br), 7.91 (3H, br), 7.60 (2H, br), 7.17 (5H, br), 4.44 (1H, q, J = 5.7Hz), 4.19
6
4-((2-(((S)-1-(((S)-1-(tert-butylamino)-1-oxopropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-3,4-dioxocyclobut-1-en-1-
(
(
(
(
1H, q, J = 7.2Hz), 4.04 (1H, br), 3.57 (2H, br), 3.14 (2H, br), 2.89 (1H, br), 2.79
1H, d, J = 8.7Hz), 2.16 (2H, br), 1.78 (5H, br), 1.56 (6H, br), 1.35 (2H, br), 1.05
6H, br), 0.81 (3H, d, J = 6.3Hz), 0.77 (3H, d, J = 6.6Hz), 0.71 (6H, d, J = 5.7Hz)
yl)(methyl)amino)butanoic acid, 20: A solution of 19 (189 mg, 0.60 mmol) in
a mixture of CH
solvent was removed under an argon stream at r.t. The TFA-salt was dissolved
in H O (1 mL) the solution was basified to pH 9 with NaOH 1M and borate buffer
2 2
Cl -TFA 10% (10 mL) was stirred for 2 hours at r.t. Then, the
ppm. 13C NMR (DMSO-d
): 183.3, 182.7, 173.5, 173.0, 171.6, 170.4, 170.0,
68.5, 168.1, 138.6, 130.0, 128.8, 127.1, 59.1, 54.6, 52.0, 48.3, 43.9, 41.9, 41.9,
8.6, 37.1, 33.1, 33.0, 32.2, 31.1, 26.1, 25.3, 25.1, 23.8, 23.4, 22.5, 20.0, 19.0
Na [M+Na]+ 760.4374, exp.
6
2
1
3
solution (BBS) at pH 9.5 (6 mL) was added. This solution was added to a
solution of 18 (145 mg, 0.60 mmol) in EtOH (7 mL) and the reaction mixture was
stirred for 15 hours at r.t. The EtOH of the mixture was removed under reduced
ppm. HRMS (ESI): m/z calc. for
39 59 7 7
C H N O
7
60.4375.
pressure and H
aqueous solution was acidified with HCl 37% to pH 1 and was extracted with
AcOEt (2 20 mL). The organic phases were dried over MgSO and
concentrated. Finally, the product was washed in Et O and filtered to afford 20
(123 mg, 0.30 mmol) as a white solid. Yield 50 %. m.p 147 ºC; H NMR (DMSO-
) : 12.15 (1H, br), 7.92 (1H, d, J = 7.5Hz), 7.57 (1H, d, J = 8.1Hz), 7.29 (1H,
2
O (10 mL) was added to the remaining solution. The resulting
4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)(methyl)amino)butanoic acid, 18.

4
EtONa (62 mg, 0.98 mmol) was added to a solution of N-methyl--aminobutiric
acid (150 mg, 0.98 mmol) in Et O (15 ml) and the mixture was stirred for 30 min.
after adding DIPEA (0.490 mL, 4.90 mmol) the resulting solution was added
dropwise to a stirred solution of diethyl squarate (167 mg, 0.98 mmol) in Et O (5
mL). The reaction mixture was stirred for 48 hours. The solvent was removed
under reduced pressure and the crude was washed with Et O (2  50 mL) and
dissolved in H O (20 mL). The resulting aqueous solution was acidified with HCl
7% to pH 1 and was extracted with CH Cl (3  25 mL). The organic phases
were dried over Na SO and concentrated to afford 18 (115 mg, 0.48 mmol) as
a colorless oil. Yield 49 %. 1H NMR (CDCl
) : 4.76 (2H, q, J = 7.2Hz), 3.74
1.1H, t, J = 6.9Hz), 3.47 (2H, q, J = 6.9Hz), 3.34 (1.3H, s), 3.16 (1.7H, s), 2.43
2H, m, J = 3.6Hz), 1.98 (2H, m, J = 6.9Hz), 1.451 (3H, t, J = 6.9Hz) ppm. 13C
) : 189.2, 183.4, 183.2, 169.0, 168.4, 63.8, 58.6, 53.2, 46.7,
5.5, 25.4, 19.1, 7.7 ppm. HRMS (ESI): m/z calc. for C11
Na [M+Na]+
64.0848, exp. 264.0856.
2
1
2
d
6
2
s), 4.82 (1H, m, J = 7.8Hz), 4.17 (1H, q, J = 7.2Hz), 3.18 (2H, s), 2.23 (3H, br),
1.77 (2H, t, J = 6.6Hz), 1.33 (3H, d, J = 6.9Hz), 1.20 (9H, s), 1.14 (3H, d, J =
7.2Hz) ppm. 13C NMR (DMSO-d
167.3, 53.2, 51.3, 50.9, 49.5, 31.2, 29.3, 23.8, 19.9, 19.3 ppm. HRMS (MALDI-
TOF): m/z calc. for C19
[M+H]+ 441.224, exp. 411.226.
2
6
) : 183.8, 182.8, 177.0, 172.3, 172.0, 168.9,
2
3
2
2
31 4 6
H N O
2
4
3
(S)-2-(4-((2-(((S)-1-(((S)-1-(tert-butylamino)-1-oxopropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-3,4-dioxocyclobut-1-en-1-
(
(
yl)(methyl)amino)butanamido)-N-((S)-1-(cyclohexylamino)-1-oxo-3-
phenylpropan-2-yl)-3-methylbutanamide, 13. A solution of 16 (127 mg, 0.29
NMR (DMSO-d
6
2
2
H
15NO
5
2 2
mmol) in CH Cl -TFA 10% (10 mL) was stirred for 2 hours at r.t., and then the
solvent was removed under an argon stream at r.t. The TFA-salt was combined
with 20 (90 mg, 0.22 mmol) in DMF (10ml) following the general procedure
(
S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid, N-
Boc-L-Ala-L-Ala:. L-Ala-L-Ala (250 mg, 1.56 mmol) was dissolved in a mixture
of NaOH 1M:dioxane 1:1 v:v (12 mL) and cooled to 0 C. A solution of Boc
852 mg, 3.90 mmol) in dioxane (6 mL) was added dropwise and then the
described for amino acid coupling. Then, CH
reaction mixture, and the resulting solution was washed with HCl 1M (2  25
mL) and brine (25 mL), dried over MgSO and the solvent was removed. The
solid obtained was crushed in Et O, filtered and precipitated from a CH Cl
2 2
Cl (30 mL) was added to the
2
O
4
(
2
2
2
-
mixture was stirred at r.t. for 15 hours. A solid was formed and it was filtered
from the solution. The dioxane was removed under reduced pressure and the
hexane mixture following the two-phase method. Finally, the solid was crushed
in water at pH 5 (4 X 10 mL) to afford 13 (75 mg, 0.10 mmol) as a white solid.
crude was dissolved in KHSO
4
1M (20 mL). The product was extracted in AcOEt
Yield 46 %. m.p 160 ºC; 1H NMR (DMSO-d
6
) : 8.52 (1H, d, J = 8.4Hz), 7.94
(
3  30 mL) and the organic phase was washed with brine (20 mL), dried over
(2H, br), 7.33 (1H, d, J = 7.5Hz), 7.23 (7H, s), 4.83 (0.5H, m), 4.57 (1H, br), 4.44
(0.5H, m), 4.17 (0.5H, m), 4.04 (0.5H, br), 3.65 (1H, t, J = 7.2Hz), 2.81 (8H, br),
2.18 (2H, br), 1.98 (2H, q, J = 6.6Hz), 1.62 (7H, br), 1.35 (2H, d, J = 6.9Hz), 1.20
(9H, s), 1.12 (3H, br), 0.92 (3H, d, J = 6.6Hz), 0.85 (3H, d, J = 6.6Hz), 0.70 (3H,
MgSO
4
and concentrated to afford N-Boc-L-Ala-L-Ala (372 mg, 1.43 mmol) as
1
a white solid. Yield 92 %. m.p 109 ºC; H NMR (CDCl
3
) : 6.89 (1H, d, J = 6.9Hz),
5
.15 (1H, br), 5.56 (1H, t, J = 6.9Hz), 4.22 (1H, br), 1.44 (12H, s), 3.55 (3H, d, J
=
6.9Hz) ppm. 13C NMR (CDCl
3
) : 176.0, 173.4, 156.2, 67.5, 50.4, 48.6, 28.7,
d, J = 6.6Hz) ppm. 13C NMR (DMSO-d
6
) : 183.9, 182.6, 172.9, 172.2, 172.1,
2
7.8, 18.6, 18.4 ppm. HRMS (MALDI-TOF): m/z calc. for C11
H
20
N
O
2 5
K [M+K]+
171.5, 170.4, 168.5, 167.4, 138.6, 130.0, 128.8, 127.0, 59.0, 54.6, 53.2, 50.9,
49.5, 48.3, 38.7, 33.1, 33.0, 31.1, 29.3, 26.1, 25.3, 24.4, 20.0, 19.9, 19.3, 19.0
299.101, exp. 299.100.
59 7 7
H N O
Na [M+Na]+ 760.437, exp.
ppm. HRMS (MALDI-TOF): m/z calc. for C39
760.439.
tert-butyl
((S)-1-(((S)-1-(tert-butylamino)-1-oxopropan-2-yl)amino)-1-
oxopropan-2-yl)carbamate, 19. 19 was synthesized according to the general
procedure for amino acid coupling using N-Boc-L-Ala-L-Ala (325 mg, 1.25
Crystallographic Data
mmol) and tert-butylamine (365 mg, 4.99 mmol) in DMF (10 mL). After, CH
2
Cl
2
(50 mL) was added to the reaction mixture, and the resulting solution was
CCDC −1859732, CCDC-1859737, and CDCC-1859738 contain
the supplementary crystallographic data for this paper. These
washed with HCl 1M (4  30 mL), H
2
O (50 mL) and brine (50 mL), dried over
data
can
be
obtained
free
of
or
charge
via
MgSO . The solvent was removed and Et O (30 mL) was added to the crude. A
4 2
www.ccdc.cam.ac.uk/data_request/cif,
by emailing
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FULL PAPER
data_request@ccdc.cam.ac.uk, or by contacting The Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2
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Chemistry - A European Journal
10.1002/chem.201803930
FULL PAPER
FULL PAPER
N-Methylation of squaramides
promotes the conformational control of
peptidomimetic-based squaramides.
The combination of tertiary and
secondary squaramide bonds in their
structure, efficiently folds peptide-like
compounds to obtain parallel and
antiparallel-hairpin analogs driven by
hydrogen bonds and C-H···O
Luís Martínez-Crespo, Antoni Costa,
Carmen Rotger*
Page No. – Page No.
The role of N-Methyl squaramides in a
hydrogen bonding strategy to fold
peptidomimetic compounds
interations in hydrogen bonding
competitive media.
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