New self-assembled supramolecular hydrogels based on dehydropeptides

Article abstract of DOI:10.1039/c5tb00501a

Supramolecular hydrogels rely on small molecules that self-assemble in water as a result of the cooperative effect of several relatively weak intermolecular interactions. Peptide-based low molecular weight hydrogelators have attracted enormous interest ow

Full text of DOI:10.1039/c5tb00501a

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Journal Name
RSCPublishing
ARTICLE
New selfꢀassembled supramolecular hydrogels based
on dehydropeptides
Cite this: DOI: 10.1039/x0xx00000x
H. Vilaça,a G. Pereira,a T. G. Castro,a B. F. Hermenegildo,b J. Shi,c T. Q. Faria,d N.
Micaêlo,a R. M. M. Brito,d B. Xu,c E. M. S. Castanheira,b J. A. Martins,*a P. M. T.
Ferreira*a
Received 00th January 2012,
Accepted 00th January 2012
Supramolecular hydrogels rely on small molecules that selfꢀassemble in water as a result of
the cooperative effect of several relatively weak intermolecular interactions. Peptideꢀbased
low molecular weight hydrogelators have attracted enormous interest owing to the simplicity
of small molecules combined with the versatility and biocompatibility of peptides. In this
DOI: 10.1039/x0xx00000x
www.rsc.org/
work, naproxen, a well known nonꢀsteroidal antiꢀinflammatory drug, was
Nꢀconjugated with
various dehydrodipeptides to give aromatic peptide amphiphiles that resist proteolysis.
Molecular dynamic simulations were used to obtain insight into the underlying molecular
mechanism of selfꢀassembly and to rationalize the design of this type of hydrogelators. The
results obtained were in excellent agreement with the experimental observations. Only
dehydrodipeptides having at least one aromatic amino acid gave hydrogels. The
characterization of the hydrogels was carried out using transmission electron microscopy
(TEM), circular dichroism (CD), fluorescence spectroscopy and also rheological assays.
In this work a multidisciplinary approach that combines molecular
dynamic simulations with experimental results was devised for
Introduction
The preparation of biomaterials, such as hydrogels, using a “bottomꢀ
up” approach is based on molecular selfꢀassembly through nonꢀ
covalent interactions such as hydrogen bonding, Van der Waals
forces and πꢀπ and electrostatic interactions. Small peptides with
bulky aromatic moieties can selfꢀassemble into nanostructures that
interweave giving threeꢀdimensional (3D) networks that entrap water
giving biocompatible and biodegradable hydrogels. These
biomaterials have a wide range of applications, from drug delivery to
tissue engineering and regenerative medicine.1ꢀ8 One limitation of
peptide based hydrogelators is their susceptibility to enzymatic
hydrolysis, which shortens their in vivo lifetime and narrows the
scope of their application. One of the strategies used to circumvent
this limitation is to introduce nonꢀproteinogenic amino acids into
peptide hydrogelators.9ꢀ13 Recently Xu et al. described the synthesis
of new hydrogelators based on dipeptides containing unnatural Dꢀ
amino acids Nꢀcapped with naproxen.14 The Dꢀamino acids in the
conjugates not only increased the proteolytic stability of the
hydrogelators but also enhanced their selectivity for inhibiting
cyclooxygenaseꢀ2 (COXꢀ2). The same authors also prepared and
studied other peptides Nꢀconjugated with other NSAID drugs,
namely ibuprofen and flurbiprofen.15
developing new efficient dehydropeptide hydrogelators. Five
dehydrodipeptides Nꢀconjugated with naproxen were prepared and
studied. These compounds were designed taking into consideration
several factors: dehydroamino acids16ꢀ21 are known to increase the
resistance of peptides against proteolytic enzymes, the naproxenꢀ
capped hydrogelators (and hydrogels) are likely to retain the NSAID
properties of naproxen;14,15 the naphthalene moiety of naproxen is
prone to engage in intermolecular πꢀπ stacking interactions as
described for other peptide hydrogels functionalised with
naphthalene moieties.12,14,15,22
The goal of this work was to understand the selfꢀassembly behaviour
of aromatic dehydrodipeptide amphiphiles and to create a rational
basis for the design of new dehydropeptide hydrogelators. Molecular
dynamic simulations, together with characterization assays [circular
dichroism (CD), fluorescence spectroscopy, transmission electron
microscopy (TEM) and rheometry], evidenced the propensity of
dehydrodipeptides containing an aromatic amino acid and Nꢀ
conjugated to
a polyaromatic moiety to selfꢀassemble into
nanostructures that give hydrogels. Furthermore this new class of
hydrogelators revealed to resist proteolytic degradation.
This journal is © The Royal Society of Chemistry 2013
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VJieowuArrtnicalelONnlainme e
ARTICLE
DOI: 10.1039/C5TB00501A
The stereochemistry of compounds 2ꢀ5 was confirmed by
Nuclear Overhauser (NOE) difference experiments by
irradiating the αꢀNH proton of the dehydroamino acid residue
Results and discussion
and observing NOE enhancements in the signals of the
βꢀ
Synthesis
methyl or
β
ꢀphenyl protons. All 1H NMR spectra of compounds
Five new dehydrodipeptides
N
ꢀprotected with naproxen (Npx)
tert
ꢀhydroxydipeptides. The strategy deployed
involved a dehydration reaction followed by cleavage of the
tertꢀbutoxycarbonyl group (Boc), reaction with )ꢀ(+)ꢀ
5aꢀe in dimethylsulfoxide (DMSOꢀd6) show one doblet and two
singlets between 8.11 pm and 12.67 ppm due to the NH and
CO2H protons. The βꢀCH proton of the dehydroamino acid
residues appears in the aromatic region in the case of
dehydrophenylalanine and as a quartet near 6.5 ppm for
dehydroaminobutyric acid. In the 13C NMR spectra of these
compounds the signals assigned to the βꢀcarbon atoms of the
dehydroamino acid residues appear in a narrow zone of high
chemical shift between 131.81 ppm and 133.14 ppm. This is
were prepared from the corresponding methyl esters of
butoxycarbonylꢀ
N
ꢀ
ꢀ
β
(
S
naproxen chloride and alkaline hydrolysis of the methyl esters
(Scheme 1). The dehydroamino acids used were
dehydrophenylalanine (ꢁPhe) and dehydroaminobutyric acid
(ꢁAbu). This synthetic methodology was chosen to avoid
racemization issues concerning the naproxen moiety. The
diprotected dipeptides having a ꢀhydroxyamino acid (Scheme
1, 1aꢀe) were dehydrated in good to high yields by treatment
with tertꢀbutyldicarbonate (Boc2O) and 4ꢀ
N’
N,Cꢀ
due to deprotection resulting from conjugation of the
double bond with the carbonyl group.
α,βꢀ
β
dimethylaminopyridine (DMAP) followed by
N,
N
,
N’
,
ꢀ
Molecular Dynamic Simulations
tetramethylguanidine (TMG)17 (Scheme 1, 2aꢀe). The Boc
Molecular dynamic simulations (MD) were carried out for all
dehydrodipeptides prepared (5aꢀe) to examine the process
spontaneous of selfꢀassembly. The peptides were placed in
cubic boxes of 4.5 x 4.5 x 4.5 mm solvated with the SPC water
model.23 The average number of clusters observed for each
peptide is presented in Table 1. This analysis was carried out by
calculating the number of peptides that are clustered using a
cutꢀoff of 1.4 nm for each simulation frame and averaged over
the last 20 ns sampling time. The average number of
intermolecular hydrogen bonds was calculated and averaged
group was removed with trifluoroacetic acid (TFA) (Scheme 1,
3aꢀe) and the
Nꢀdeprotected dehydrodipeptides were conjugated
with ( )ꢀ(+)ꢀnaproxen (Scheme 1, 4aꢀe). Finally, the methyl
S
esters were removed by treatment with a solution of NaOH (1
M) affording compounds 5aꢀe in good yields (Scheme 1).
R2 OH
R2
O
1) Boc2O, DMAP
2) TMG
O
H
H
N
OMe
N
OMe
Boc
N
H
Boc
N
H
R1
TFA
O
2
R1
O
1
R2
O
over the same time interval. The number of
πꢀstacking
+H3N
OMe
N
H
interactions was divided by the number of peptides present in
the simulation box and normalized by the number of simulated
frames (last 20 ns). This average percentage of πꢀπ interaction
R1
O
3
Cl
O
Et3N
H3CO
is read as the number of
solution. Different types of
considered were: sandwich (S), parallel displaced (PD) or Tꢀ
shaped (T). Experimental and theoretical data24 were used to
define the cutꢀoff distances and angles between aromatic groups
π
ꢀstacking / number of peptides in
ꢀstacking interactions were
R2
R2
O
H
N
O
π
H
N
OMe
NaOH
H3CO
OH
N
H
N
H
O
R1
O
O
R1
O
H3CO
4
5
R1 = CH2Ph, R2 = Ph, a; R1 = CH2Ph, R2 = CH3, b;
R1 = CH(CH3)2, R2 = Ph, c; R1 = CH3, R2 = Ph, d;
R
1 = CH3, R2 = CH3, e.
.
that characterize these types of
intermolecular ꢀstacking interactions analyzed were those
between naproxen groups, naproxen and the aromatic moieties
of phenylalanine (Phe) and dehydrophenylalanine ( Phe) and
between the phenyl groups of the two aromatic amino acid
residues. All of these types of ꢀstacking interactions could be
πꢀstacking interactions. The
π
Scheme 1. Synthesis of dehydrodipeptides
N
ꢀconjugated to
ꢀꢁPheꢀOH, 5a; Npxꢀ ꢀPheꢀ
ꢀꢁPheꢀOH, 5c Npxꢀ ꢀAlaꢀ
ꢀꢁAbuꢀOH, 5e
naproxen (Npx): Npxꢀ
L
ꢀPheꢀ
Z
L
L
Zꢀ
Zꢀ
ꢀ
ꢁAbuꢀOH, 5b
;
Npxꢀ
L
ꢀValꢀ
Z
;
ꢁPheꢀOH, 5d; Npxꢀ
L
ꢀAlaꢀ
Z
π
found solely for peptide 5a, the other peptides show only some
of these types of interactions. In the case of peptide 5e the only
π
ꢀstacking interactions observed are those between the
naproxen moieties.
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5TBA00R5T0I1CALE
Table 1. Average number of clusters, hydrogen bonds and percentage of intra/intermolecular
each system. The total number of
and T, are shown. Standard deviation for the first two analyses is shown in parenthesis.
π
ꢀstacking interactions observed for
π
ꢀstacking interactions and also the individual contribution of each
π
ꢀstacking geometry, S, PD
NpxꢀPheꢀ
5a
ꢀ
PheꢀOH,
NpxꢀPheꢀ
5b
ꢀ
AbuꢀOH, NpxꢀValꢀ
ꢀ
PheꢀOH,
NpxꢀAlaꢀ
5d
ꢀ
PheꢀOH,
NpxꢀAlaꢀ
5e
ꢀ
AbuꢀOH,
5c
Average
clusters
Average
number
number
of
of
3.2 (±0.10)
3.8 (±0.04)
2.4 (±0.09)
4.7 (±0.04)
2.8 (±0.08)
3.5 (±0.04)
3.8 (±0.10)
3.2 (±0.05)
4.5 (±0.09)
3.7 (±0.04)
hydrogen bonds
8.5 (S: 5.1; PD: 2.0; T: 6.5 (S: 4.3; PD: 1.1; T: 7.0 (S: 4.5; PD: 1.5; T: 6.3 (S: 4.4; PD: 1.5; T:
11.3 (S: 7.7; PD: 2.3;
T: 1.3)
NpxꢀNpx
NpxꢀPhe
NpxꢀꢀPhe
PheꢀPhe
1.4)
1.1)
1.0)
0.4)
3.6 (S: 1.9; PD: 0.7; T: 4.5 (S: 2.5; PD: 1.1; T:
―
―
―
―
1.0)
1.7 (S: 0.8; PD: 0.4; T:
0.5)
0.9)
―
2.7 (S: 1.3; PD: 0.5; T: 2.0 (S: 0.9; PD: 0.4; T:
Percentage of
0.9)
0.7)
π
stacking
0.5 (S: 0.1; PD: 0.1; T: 0.6 (S: 0.2; PD: 0.1; T:
interactionsa
―
―
―
0.3
0.3)
ꢀPheꢀꢀPhe
PheꢀꢀPhe
Total
0.4 (S: 0.1; T: 0.3)
―
0.1 (T: 0.1)
0.2 (PD: 0.1; T: 0.1)
―
0.8 (S: 0.1; PD: 0.2; T:
0.5)
―
―
―
―
15.5
11.6
9.8
8.5
11.3
a Sandwich (S): R ≤ 4.5 Å and θ ≤ 15° or θ ≥ 165°; Parallel Displaced (PD): R ≤ 4.0 Å and 15° < θ < 30° or 150° < θ < 165°; Tꢀshaped (T): R ≤ 3.5
Å and θ ≤ 10°.
geometric center of each cluster. Figures were obtained with
Pymol.25
Figure 1 shows the clustering arrangement of a given frame
taken from the simulation trajectory of 5aꢀe
.
Visual analysis of the clustering behaviour within each frame
clearly shows selfꢀaggregation of peptides 5aꢀd, and, poor
aggregation of peptide 5e. Hydrogelators 5b and 5c show the
lowest numbers of clusters (Figure 1, Table 1), which means
higher extent of aggregation. The number of intermolecular
hydrogen bonds is very low and comparable for all systems
(Table 1) suggesting that this type of molecular interaction does
not explain the aggregation properties observed for peptides 5aꢀ
d
. The aggregation phenomenon seems to be better explained
by the formation of intermolecular ꢀstacking interactions
between the naproxen moiety and the aromatic amino acids.
Figure 2 shows the representative ꢀstacking interactions
observed for compound 5b
π
π
.
Figure 1. Snapshot of the MDS of peptides 5aꢀe after
equilibration. The water molecules have been omitted for better
viewing of the peptides. The spheres in cyan represent the
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conclude that the aggregation of this type of peptides is
dominated by interactions between the ꢀaromatic
π
ꢀ
π
N
component and other aromatic amino acid moieties. Aromatic
amino acid residues in addition to the Nꢀaromatic capping
group are required for peptide aggregation (as seen for 5e).
Hydrogelation
Dehydrodipeptides 5aꢀe were tested as hydrogelators in order to
validate this methodology for the rational design of efficient
hydrogelators. Gelation was triggered via pH change and/or
heating and subsequent cooling. Compounds 5a and 5b were
solubilized in PBS buffer at 60 ºC and gelation occurred upon
cooling to room temperature (Figure 3). Compounds 5c and 5d
were dissolved in water with addition of NaOH (1 M) and
gelation occurred by pH adjustment with HCl (1 M) (Figure 3).
The results showed that gelation of compounds 5aꢀd occurs at
low critical gelation concentrations (CGC), between 0.4 wt%
and 0.8 wt% and gelꢀsol phase transition pH (pHgs) between 5
and 8 (Table 2).
Figure 2. Illustration of representative πꢀstacking interactions
observed for hydrogelator 5b during the last 20 ns of the
simulation. (A) Intermolecular interaction between the
naproxen moieties, (B) Intermolecular interaction between Npx
and Phe, (C) Intermolecular interaction between Phe groups,
(D) Intra and intermolecular interactions, (E) Multiple
intermolecular interactions and (F) Tꢀshaped interaction
between Phe and Npx.
C
N
H
N
H
O
O
O
Naproxen
in all five dehydrodipeptides studied. PD
significant (Figure 2a). It is interesting to note that naproxen
stacking alone cannot explain the aggregation phenomenon.
Although peptide 5e presents a high percentage of naproxen
π
ꢀstacking, preferentially as the S type mode, occurs
C
P
h
O H
2
π
ꢀstacking is also
π
ꢀ
2
π
ꢀ
stacking interactions, the theoretical simulations suggest that
this peptide is not able to selfꢀaggregate (Figure 1). This leads
to the conclusion that the sole intermolecular interaction
between naproxen groups is not sufficient to promote peptide
aggregation. On the other hand, the interactions between
naproxen and other aromatic groups seem to be responsible for
the aggregation of the peptides 5aꢀd. Analyzing the systems of
peptides 5a or 5b suggests that the selfꢀaggregation
phenomenon is determined by the intermolecular interaction
between naproxen and the phenyl group of the aromatic amino
acid (Figure 2b). The system containing peptide 5a also
suggests that the presence of two aromatic amino acid residues
does not shows any additive effect on cluster formation, since
the 5a system shows less selfꢀaggregation than 5b. In fact,
combining two aromatic residues such as phenylalanine and
dehydrophenylalanine seems to have a detrimental effect on
H3
P
h
N
H
C
N
O
H O
Figure 3. Optical images of hydrogels 5aꢀd.
Table 2. CGC and gelꢀsol phase transition pH of compounds
5aꢀd
Compound
selfꢀaggregation. The systems containing peptides 5c and 5d
,
.
indicate that selfꢀaggregation in dehydrodipeptides with a
single aromatic residue in the Cꢀterminal position is less
effective. Systems containing peptides 5a, 5c and 5d also
CGC
[wt%]
pHgs
demonstrate that the dehydrophenylalanine residue does not
establish significant intermolecular interactions with itself,
instead, this amino acid seems to interact preferentially with
naproxen. Furthermore, although 5c and 5d are structurally
similar, 5c shows a higher propensity to form clusters. This
could result from the presence of valine that makes peptide 5c
less polar than peptide 5d. From these results it is possible to
Npxꢀ
Npxꢀ
Npxꢀ
L
L
L
ꢀPheꢀ
Z
ꢀꢁPheꢀOH, 5a
ꢀꢁAbuꢀOH, 5b
ꢀꢁPheꢀOH, 5c
0.4
0.4
0.6
8.0
6.0
8.0
ꢀPheꢀ
Z
ꢀValꢀ
Z
4 | J. Name., 2012, 00, 1-3
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NpxꢀLꢀAlaꢀZꢀꢁPheꢀOH, 5d
0.8
5.0
As predicted by the molecular dynamic simulations, compound
5e (NpxꢀLꢀAlaꢀ ꢀꢁAbuꢀOH), lacking an aromatic amino acid
residue, failed to give a hydrogel in all conditions tested. The
dehydropeptides with a capped ꢀterminal phenylalanine
Z
N
residue (5a and 5b) display lower CGC compared to peptides
5c and 5d, bearing an alkyl Nꢀterminal amino acid (Val or
Ala). These experimental results are in excellent agreement
with those obtained by the molecular dynamic simulations. This
means that the molecular modelling methodology presented
here might be a valuable new toll for the design of efficacious
peptide hydrogelators..
Comparing the experimental conditions for hydrogelation of
dehydrodipeptide
5a
with
the
dipeptide
phenylalanylphenylalanine
Nꢀprotected with naproxen (0.4 wt%
and pH 8,0 vs 0.8 wt% and pH 7, respectivelly),14 it is possible
Figure 4. Fluorescence spectra of compounds 5a and 5b (2×10ꢀ6
M) at different pH values (λexc= 290 nm). Insets: Variation of the
maximum fluorescence intensity and intensity ratio I2/I1 with pH.
to conclude that the presence of the
α,βꢀdouble bond decreases
the CGC value and increases the gelꢀsol phase transition pH.
Hydrogelators 5a and 5b, containing different dehydroamino
acids, dehydrophenylalanine and dehydroaminobutyric acid,
respectively, were selected for further characterization, namely
fluorescence studies, Circular Dichroism (CD) studies and
proteolitic stability assays, to get insight into the effect of the
structure of the dehydroaminoacid on the selfꢀassembly and
gelation processes and proteolitic stability. The properties of
hydrogelator 5a can be directly compared to its “natural”
For both compounds 5a and 5b, the maximum emission
intensity rises with pH, with a tendency to stabilize for pH
values above 5 for compound 5a and 6 for compound 5b
.
Considering the ratio between the aggregate band and naproxen
monomer band, I2/I1, a different behaviour is observed: the
maximum value of I2/I1 occurs at pH 3 for both compounds,
near the pKa value of the peptide terminal carboxylic acid group
(pKa∼
3)27. For both compounds, a minimum is observed at pH
dipeptide analogue phenylalanylphenylalanine Nꢀprotected with
Naproxen.14Moreover hydrogelator 5a (bearing two aromatic
amino acid residues) was expected to display fluorescence and
CD spectra more insightful towards the selfꢀassembly and
5 with stabilization observed thereafter. A slight rise in I2/I1
ratio is observed at pH 8 for compound 5a and pH 6 for
compound 5b, identified as the pH gelation values. The pH at
which a gel is formed is highly dependent on the molecular
structure of the hydrogelator and correlates with the apparent
pKa of the peptide.28 The extent of deprotontion of the
carboxylic acid group on the hydrogelators is pHꢀdependent
and determines their hydrophilicity. Accordingly, compound
5b, bearing only one aromatic amino acid, is more hydrophilic
than 5a (bearing two aromatic amino acid residues ) thus
requiring lower pH (pH= 6) for gelation comparing to 5a (pH
8.0)
Excitation spectra provide relevant information about the nature
of the aggregate emission band (Figure 5). It can be observed
that upon collecting emission in the naproxen band (360 nm) or
in the aggregate band (450 nm), the spectra completely modify
showing different excited species, and not dynamic exciplexes
formed at the excited state.
gelation processes than 5c and
aromatic amino acid residue). Rheological and Transmission
Electron Microscopy (TEM) characterisation was carried out
5d (containing only one
with gels 5aꢀd (5e failed to produce a hydrogel).
Photophysical Studies
The critical aggregation concentration (CAC) as well as the
influence of pH in the aggregation of peptides 5a and 5b were
studied by fluorescence spectroscopy. Figure 4 shows the
influence of pH in the fluorescence properties of molecules 5a
and 5b. The fluorescence emission of peptides 5a and 5b is
clearly dominated by the emission of the naproxen moiety,
λ
max=353 nm (λexc= 290 nm), similar to the results reported for
naproxen in methanol and water.26 However, it is possible to
observe a second fluorescence band, with maximum emission
near 440 nm. This band is ascribed to the formation of an
emissive dimmer between naproxen and the phenylalanine
residues. At the excitation wavelength used (
phenylalanine is not electronically excited.
λexc= 290 nm),
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at lower concentrations, which may indicate the formation of
intermolecular aggregates that play an important role in gel
formation.
These results show that fluorescence spectroscopy is a good
methodology to estimate the critical gelation concentration and
to get insight into the intramolecular/intermolecular interactions
between the aromatic moieties of these molecules.
Hydrogel Characterization
The CD spectra of hydrogelators 5a and 5b and of the dipeptide
Npxꢀphenylalanylphenylalanine (NpxꢀLꢀPheꢀLꢀPheꢀOH) were
recorded in order to get insight into the peptide secondary
structure (Figure 7).
Figure 5. Excitation spectra (λem=360 nm and λem=450 nm) of
compounds 5a and 5b (2×10ꢀ6 m) at selected representative pH
values. Insets: Normalized spectra at the peak of minimum energy.
Figure 6 shows the dependence of fluorescence emission of
compounds 5a and 5b with concentration.
Figure 6. Fluorescence spectra of compounds 5a (pH 8) and 5b
(pH 6) at several concentrations (λexc= 290 nm). Insets:
Variation with concentration of the maximum emission
wavelength of the first band and intensity ratio I2/I1.
It can be seen that the ratio of intensities of the aggregate and
monomer bands, I2/I1 (insets of Figure 6), is almost constant for
concentrations below 0.4 wt%, but increases sharply for higher
concentrations. At this concentration, a clear change in the
naproxen maximum emission wavelength is also detected, with
a red shift that tends to stabilize at higher concentrations (inset
of Figure 6, left). These results point clearly to hydrogel
formation at 0.4 wt% for compound 5a. For compound 5b, the
ratio I2/I1 follows the same trend (presenting lower values) with
a noticeable rise above 0.4 wt%, as for compound 5a. The red
shift in naproxen emission with increasing hydrogelator
concentration is smaller than that observed for compound 5a.
From these two indicators, a clear change in behaviour is
detected above 0.4 wt%, pointing to hydrogel formation (inset
of Figure 6, right). Above 0.4 wt% concentration, the
aggregate emission band is clearly higher than what is observed
6 | J. Name., 2012, 00, 1-3
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Figure 7. CD spectra of Npxꢀ
L
ꢀPheꢀ
L
ꢀPheꢀOH (A), NpxꢀPheꢀ
ꢀ
PheꢀOH 5a (B) and NpxꢀPheꢀꢀAbuꢀOH 5b (C).
Figure 8. TEM images of hydrogels obtained from
dehydrodipeptides (A) 5a; (B) 5b; (C) 5c; (D) 5d (scale bar of 100
nm).
The three peptides show similar CD spectra, displaying Cotton
effects only in the far UV wavelength (190ꢀ260 nm). The CD
spectra of NpxꢀLꢀPheꢀLꢀPheꢀOH (Figure 7A) at low
temperatures exhibit bands around 196 nm (positive peak), 220
nm (broad negative peak) and 235 nm (positive Cotton effect).
Hydrogelator 5a selfꢀassembles into nonꢀuniform nanofibers
displaying different widths: minimum width of 9 nm and a
maximum width of 18 nm, similar to those shown by Npxꢀ
Lꢀ
Pheꢀ
L
ꢀPheꢀOH.14 Hydrogel 5b exhibit long nanofibers, that
The signals at 196 nm and 220 nm indicate a
βꢀsheet like
arrangement of the peptide backbone, corresponding to πꢀπ
*
entangle to form a network, with an average width of 10 nm.
The nanofibers of hydrogelator 5c are short (length between
170ꢀ750 nm) and nonꢀuniform displaying a minimum width of
12 nm and maximum width of 16 nm. Hydrogel 5d comprises
long and entangled nanofibers with widths ranging between 8
and 16 nm.
and nꢀπ* transitions, respectively.29ꢀ34 The CD spectra of
compounds 5a and 5b (Figure 7B and C) at low temperatures
are similar to those obtained for Npxꢀ
suggests the same type of intermolecular interactions and
sheet like structure. However, in Npxꢀ ꢀPheꢀ ꢀPheꢀOH the
LꢀPheꢀLꢀPheꢀOH. This
β
ꢀ
L
L
strongest bands are the ones resulting from the peptide
backbone (195 nm and 220 nm), while in dehydrodipeptides 5a
and 5b, the most intense bands originate from the naphthalene
interactions (220 nm and 235 nm). This indicates that for the
dehydrodipeptide hydrogelators the naphthalene interactions
are more important than the peptide backbone arrangement.
The variation of the CD spectra of these three compounds with
temperature shows a progressive loss of structure up to 40 °C.
For higher temperatures, the absence of CD signals suggests
high mobility of the peptide backbone and the lack of an
organized structure.31 Cooling the peptide solutions leads to
gelation, shown by the enhancement of the CD signals31 and
the blue shift of the λmax of the bands. This indicates gradual
transition from an isotropic solution to an anisotropic
The rheological data obtained with hydrogels 5aꢀd are
presented in Table 3.
Table 3. Rheological properties of hydrogels formed by
naproxenꢀdehydropeptides 5aꢀd
.
Hydrogel [a] Dynamic strain sweep
Dynamic frequency sweep
G’max [Pa] G’’max [Pa] Critical strain
[%]
G’[b] [Pa]
G’’[b] [Pa]
5a
5b
5c
5d
1.6x103
8.1x102
5.9x102
9.8x102
2.2x102
92.7
5.0
1.6
0.3
8.0
1.7x103
7.1x102
6.6x102
8.0x102
2.2x102
79.3
1.1x102
1.0x102
89.1
environment in the gel state.35 In the case of Npxꢀ
LꢀPheꢀLꢀPheꢀ
1.17x102
OH, structure forming is abrupt and occurs at temperatures
below 40 °C, where the signal strength increases rapidly as the
temperature decreases. For compound 5a the appearance of
organized structures starts at a slightly higher temperature
(around 60 ºC), suggesting that the dehydrophenylalanine
residue increases the propensity for hydrogelation at higher
temperatures. The hydrogelator 5b showed a behaviour similar
to that observed for NpxꢀLꢀPheꢀLꢀPheꢀOH. According to these
results it is possible to conclude that the dehydrophenylalanine
residue increases the hydrogel thermal stability.
[a] The concentration of the hydrogel is 0.4 wt% for compounds 5a and 5b, 0.6
wt% for compound 5c and 0.8 wt% for compound 5d. [b] The value is taken at
6.32 rad sꢀ1.
All hydrogels presented a storage modulus (G’) significantly
higher than their loss modulus (G’’) and independent from
frequency, which indicates a viscoelastic behaviour. The
hydrogels of compounds 5d and 5a present the greater storage
modulus and critical strains (8.0 % and 5.0 %, respectively),
suggesting a more resilient network in these two hydrogels.
Comparison between the critical strains of hydrogels of Npxꢀ
Pheꢀ ꢀꢁPheꢀOH (5a), Npxꢀ ꢀPheꢀ
ꢀPheꢀOH (0.62 %)15 and
Npxꢀ ꢀPheꢀ ꢀdouble
ꢀPheꢀOH (1.0 %)14 shows that the
Lꢀ
Morphologycal analysis of the new hydrogels based on
dehydrodipeptides was carried out using transmission electron
microscopy (TEM). The TEM images of hydrogels obtained
from compounds 5aꢀd are shown in Figure 8.
Z
D
L
L
D
α,β
bond in 5a increases the resistance of this gel to external force.
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 7

Journal of Materials Chemistry B
Page 8 of 14
View Article Online
ARTICLE
Journal Name
DOI: 10.1039/C5TB00501A
Enzymatic and Toxicity Assays
biocompatibility of the hydrogels as cell culture media will be
evaluated in the near future.
The stability of the new dehydrodipeptide hydrogelators 5a and
5b, against proteolytic degradation with
α
ꢀchymotrypsin was
ꢀChymotrypsin
compared to Npxꢀ ꢀPheꢀ ꢀPheꢀOH (Figure 9). α
L
L
was chosen for its ability to preferentially cleave peptide amide
bonds where the carboxyl side of the amide bond is an aromatic
amino acid. Thus, the peptide bond between the Phe residues on
120
100
the control substrate Npxꢀ
bond between the Phe residue and the dehydroamino acid
residue in peptides 5a (Npxꢀ ꢀPhe ꢀꢁPheꢀOH) and 5b (Npxꢀ
ꢀPhe ꢀꢁAbuꢀOH) are the likely clevage sites for
chymotripsin. Dehydropeptides 5c (Npxꢀ ꢀVal ꢀꢁPheꢀOH
and 5d (Npxꢀ ꢀAla ꢀꢁPheꢀOH lacking an aromatic amino
LꢀPheꢀLꢀPheꢀOH and the peptide
L
ꢀZ
Buffer 1% (v/v)
Buffer 2% (v/v)
Buffer 10% (v/v)
80
L
ꢀZ
L
ꢀ
Z
)
60
40
20
0
L
ꢀ
Z
)
acid residue in position P1 are not likelly to be recognized by
chymotrypsin as substrates. The results show that while the
control substrate Npxꢀ
proteolytic degradation, the dehydrodipeptides 5a and 5b are
completely stable when treated with ꢀchymotrypsin for 80 hrs
(Figure 9). The capping ꢀterminal amide bond of naproxen on
LꢀPheꢀLꢀPheꢀOH undergoes fast
α
[ꢁM]
-
-
-
50
100
500
N
Buffer only
Npx-Ala-
ꢀPhe-OH
peptides 5a and 5b was also found stable towards
chymotripsinꢀcatalysed hydrolysis.
Replacement of the
substrate by a dehydroamino acid rends that peptide bond
resistant to hydrolysis.
Cꢀtermimal Phe residue on the control
Figure 10. Cell viability of adult human skin fibroblasts after
incubation for 48 h with 50 ꢂM, 100 ꢂM or 500 ꢂM of NpxꢀLꢀAlaꢀZꢀ
ꢁPheꢀOH (5d), as compared with buffer controls. No significant
differences were observed (P>0.05).
Experimental
Melting points (ºC) were determined in a Gallenkamp apparatus
and are uncorrected. 1H and 13C NMR spectra were recorded on
a Bruker Avance III at 400 and 100.6 MHz, respectively, or in a
1
Varian Unity Plus 300 at 300 and 75.4 MHz, respectively. H–
1H spin–spin decoupling and DEPT θ 45º were used. HMQC
and HMBC were used to attribute some signals. Chemical shifts
(δ) are given in parts per million (ppm) and coupling constants
(J) in hertz (Hz). High resolution mass spectrometry (HRMS)
data were recorded by the mass spectrometry service of the
University of Vigo, Spain. Elemental analysis was performed
Figure 9. Evaluation of the proteolitic stability of hydrogelators
on
a LECO CHNS 932 elemental analyzer. Column
Npxꢀ
Pheꢀ
7.4, 25 ºC).for 80 hours.
LꢀPheꢀ
LꢀPheꢀOH; Npxꢀ
LꢀPheꢀ
ZꢀꢁPheꢀOH, 5a and Npxꢀ
Lꢀ
chromatography was performed on Macherey–Nagel silica gel
230–400 mesh. Petroleum ether refers to the boiling range 40–
60 °C.
ZꢀꢁAbuꢀOH, 5b in the presence of αꢀchymotrypsin (pH
A
preliminary evaluation of the cellular toxicity of the
Transmission Electron Microscopy (TEM): TEM images were
recorded on a Morgagni 268 Transmission Electron Microscope
operating at 80 kV. The samples were prepared using the uranyl
acetate negative staining method. In a carbon coated copper
grid (400 mesh), 4 ꢂL of the hydrogel was placed and left for
30 seconds. The excess of water from the hydrogel was
removed using filter paper, and washed with water and a
solution of uranyl acetate (2 %) (3 times each).
dehydrodipeptide hydrogelators was carried out on adult human skin
fibroblasts.
Cell viability after incubation for 48 hrs with 50 ꢁM, 100 ꢁM and
500 ꢁM of hydrogelator 5d is presented in figure 10. The
dehydrodipeptide did not show toxicity, even at concentrations as
high as 500 ꢂM. Hydrogelator 5d was selected for testing due to its
higher solubility at 37 ºC in the cell culture medium, comparing to
hydrogelators 5aꢀc, thanks to its relatively high CGD (presumably
substantially higher at physiological pH). This allowed studying the
Rheometry: Rheological studies were performed on a ARESꢀ
effect of the hydrogelator on cellular viability in a wide range of G2 rheometer with a parallel plate at 25 ºC. Dynamic strain
concentrations, up to 500 ꢁM, without potential interference from
nano/microstructures that form before macroscopic gel formation
can be detected. For relevant biological applications the
biocompatibility of the hydrogels, needs also to be assessed. The
sweep and frequency sweep experiments were carried out.
During the strain sweep experiments the hydrogels were under
different oscillation strain, constant frequency (6.28 rad/s) and
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 9 of 14
Journal Name
Journal of Materials Chemistry B
View Article Online
ARTICLE
DOI: 10.1039/C5TB00501A
constant temperature (25.5 ºC). In a frequency sweep, the τT = 0.20 ps and τP = 0.10 ps. The SETTLE algorithm41 was
experiments were carried out under different frequencies (0.1 to used to constrain bond lengths and angles of water molecules,
200 rad/s), constant oscillation amplitude and temperature (25.5 while the bond lengths and angles of peptides were constrained
ºC).
with LINCS algorithm.42 For the treatment of longꢀrange
CD spectroscopy: The CD spectra were obtained in an OLIS interactions, we used the reaction field method, with cutꢀoff of
DSMꢀ20 CD spectropolarimeter operating in the UVꢀVisible 1.4 nm and dielectric constant of ε = 54 for water. The Van der
spectral region, equipped with a Peltier temperature control Waals interactions were also calculated with a cutꢀoff radius of
unit. The near UV spectra (500ꢀ260 nm) were obtained with 1 1.4 nm. The aggregation properties of each peptide system were
second accumulations every 1 nm. The far UV spectra (260ꢀ190 evaluated by identifying the occurrence of peptide clusters
nm) were obtained with 5 seconds accumulations every 1 nm. formed in the simulation box. Peptide clusters were detected by
Optical cells with path lengths ranging from 0.05 to 1.00 mm clustering peptides using the singleꢀlinkage method with a cutꢀ
were used. Baselines with the buffer used in each hydrogel off of 1.4 nm between the center of mass of each peptide. The
were obtained at 20 ºC and 80 ºC. As no relevant differences in number of clusters for each system was counted and
the spectra were observed with the variation of temperature, the characterized. The average number of intra and intermolecular
hydrogels spectra were corrected with the baselines at 20 ºC. hydrogen bonds, and also the average number of intra and
Correction in relation to the path length of the optical cell used intermolecular πꢀstacking interactions were calculated in order
was also made. The data were smoothed mathematically in to understand the interactions responsible for the formation of
Origin 8 software. The optical cells were filled with each the aggregates.
hydrogel preꢀheated at 80 ºC to form a clear solution, and then Enzymatic resistance assay: To illustrate the enzymatic
introduced in the CD spectropolarimeter with the temperature resistance of dehydrodipeptides, diluted solutions of
previously programmed to 80 ºC. The spectra were obtained compounds 5a and 5b and of Npxꢀ
10ꢀ15 minutes after each change in temperature.
mLꢀ1) were prepared in sodium phosphate buffer pH 7.47 0.1 M
Photophysical studies: Fluorescence measurements were and divided in three samples of 100 ꢂL. A solution of
LꢀPheꢀLꢀPheꢀOH (0.5 mg
α
ꢀ
performed using a Fluorolog 3 spectrofluorimeter, equipped chymotrypsin in the same buffer was also prepared (1.0 mg mLꢀ
with double monochromators in both excitation and emission, 1). All the solutions were incubated at 37 ºC and 20 rpm
GlanꢀThompson polarizers and a temperature controlled cuvette overnight. The enzyme solution (100 ꢂL) was added to each
holder. Fluorescence emission and excitation spectra were hydrogelator solution. Samples of 10 ꢂL were taken at 0 h, 2 h,
corrected for the instrumental response of the system.
Molecular Dynamic Simulations: The molecular structure of 276 nm; water/acetonitrile, 1:1 with 0.1 % TFA). The
the peptides 5aꢀe with unnatural ꢀdehydroamino acids under percentage of gelator was determined using the peptide peak
4 h, 8 h, 12 h, 24 h, 49 h and 78 h. and analyzed by HPLC (λ=
α,β
study (Scheme 1) were designed with the program Pymol.25 area in each sample and comparing it with the area of the same
These molecules were parameterized using parameters peak in the diluted solutions without the enzyme. To verify that
transferred from the natural amino acids in the GROMOS these solutions were stable at 37 ºC, samples of each peptide
54a736,37 force field. To validate the proposed parameters, the were analyzed by HPLC after 78 hours at 37 ºC and 20 rpm.
new amino acids were subjected to 12000 steps of energy MTT assay: Adult human skin fibroblasts (ASFꢀ2 cells) were
minimization calculations with the steepest descent algorithm maintained at 37 ºC in a humidified 5 % CO2 atmosphere
and 100 ps MD simulation in a cubic box solvated with Simple grown in Dulbecco´s Modified Eagle’s Medium (DMEM,
Point Charge (SPC) water model.23 The validation was done by SigmaꢀAldrich, St. Louis, MO, USA) supplemented with 10 %
analyzing the convergence of the system’s potential energy and Fetal bovine serum (FBS, Lonza, Verviers, Belgium), 10 mM
the geometry of the amino acids. The naproxen group present in Hepes and 1 % antibiotic/antimycotic solution (SigmaꢀAldrich,
all five peptides under study 5aꢀe was also parameterized St. Louis, MO, USA). Prior to culture, cells within a subꢀ
according to the GROMOS54a7 force field and the molecule confluent monolayer were trypsinized using trypsin (0.05 %)ꢀ
was subjected to the same protocol used to validate the
α
,
β
ꢀ
EDTA.4Na (0.53 mM) solution and resuspended in DMEM to
dehydroamino acids. The topologies of Npx, ꢁPhe and ꢁAbu obtain a cell concentration of around 50,000 cells per mL. The
are available upon request. The five peptides 5aꢀe were cells were plated in 96ꢀmultiwell culture plates (100 ꢂL/well)
designed and eleven copies were placed in a cubic box of size 24 hours before incubation with compound 5d. Cells were then
4.5 x 4.5 x 4.5 nm solvated with SPC water model.23 Each treated with different concentrations of 5d, prepared as follow:
system was energy minimized with the steepest descent NpxꢀLꢀAlaꢀZꢀꢁPheꢀOH (5d) was dissolved in phosphate buffer
algorithm and 60 ns of MD simulations were run, the first 40 ns 0.1 M pH 8, obtaining a solution of 5.0 mM. The 5 mM
were spent for equilibration and the last 20 ns were used for solution was used to prepare solutions of 50 ꢂM, 100 ꢂM and
analysis. In these experiments the simulation was made in 500 ꢂM in DMEM. Solutions of phosphate buffer 0.1 M pH 8
30000000 steps with integration interval of 2 fs. All simulations at 1 %, 2 % and 10 % in DMEM were prepared as controls. 100
were run with the GROMACS 4.5.4 software package.38,39 In ꢂL aliquots of buffer controls and 5d solutions were placed into
all MD simulations the system was maintained at constant the wells of the plate with the cell culture, with three replicas of
temperature and pressure of 310 K and 1 atm, respectively, each. The plate was incubated at 37 ºC for 48 hours. Cells were
using the Berendsen thermostat and barostat methods,40 with then incubated for 60 minutes with MTT [3ꢀ(4,5ꢀ
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 9

Journal of Materials Chemistry B
Page 10 of 14
VJieowuArrtnicalelONnlainme e
ARTICLE
DOI: 10.1039/C5TB00501A
dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide, Sigmaꢀ acetonitrile (1 M) followed by Boc2O (1.0 equiv) under rapid
Aldrich, St. Louis, MO, USA] to a final concentration of 0.5 stirring at room temperature. The reaction was monitored by 1H
mg mLꢀ1. Then, the medium was removed, and the formazan NMR until all the reactant had been consumed. Then TMG (2
crystals formed by the cell’s capacity to reduce MTT were % in volume) was added, stirring was continued and the
1
dissolved with a 50:50 (v/v) DMSO:ethanol solution, and reaction followed by H NMR. When all the reactant had been
absorbance measured at 570 nm (with background subtraction consumed, evaporation at reduced pressure gave a residue that
at 690 nm), in a SpectroMax Plus384 absorbance microplate was partitioned between ethyl acetate (50 mL) and KHSO4 (30
reader. The results were expressed as percentage relative to the mL, 1 M). The organic phase was thoroughly washed with
control (cells with buffer solution).
KHSO4 (1 M) and brine (2x30 mL, each), and dried with
Synthesis of βꢀhydroxydipeptides derivatives
(1a-e): The MgSO4. Removal of the solvent afforded compounds 2aꢀe.
synthesis of compound 1a 43
,
1b43 and 1e16 was described The synthesis of compounds 2a43 2b44 and 2e45 was described
,
elsewhere.
elsewhere.
Synthesis of Bocꢀ
OH (4.34 g, 20 mmol) was treated with HꢀD,LꢀPhe(
L
ꢀValꢀD,LꢀPhe(βꢀOH)ꢀOMe (1c): Bocꢀ
L
ꢀValꢀ Synthesis of Bocꢀ
ꢀOH)ꢀ g, 5 mmol) was treated according to the procedure described
OMe,HCl (4.63 g, 20 mmol) in acetonitrile using the standard above to give compound 2c (1.70, 90 %) as a white solid; mp
LꢀValꢀZꢀꢁPheꢀOMe (2c): Compound 1c (1.97
β
1
N,N’ꢀdicyclohexylcarbodiimide
hydroxybenzotriazole (HOBt) procedure, giving 1c (6.89 g, 87 6.6 Hz, 6H,
%) as an oil; 1H NMR (300 MHz, CDCl3, δ): 0.60ꢀ0.67 (dd,
1H, CH Val), 3.81 (s, 3H, OCH3), 4.01ꢀ4.16 (m, 1H,
6.3 and 7.2 Hz, 6H, CH3 Val), 0.79ꢀ0.89 (dd, = 6.9 and 9.2 Val), 5.14 (d, = 9.0 Hz, 1H, NH Val), 7.28–7.35 (m, 3H, Ar
Hz, 6H, CH3 Val), 1.40 (s, 18H, CH3 Boc), 1.83–2.00 (m, 2H, H), 7.34 (s, 1H, CH ꢁPhe), 7.48 (d, = 6.9 Hz, 2H, Ar H),
CH Val), 3.72 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.77ꢀ3ꢀ97 7.81 (s, 1H, NH ꢁPhe); 13C NMR (75.4 MHz, CDCl3, δ): 17.56
(DCC)/
1ꢀ 152.0ꢀ153.0 ºC; H NMR (300 MHz, CDCl3, δ): 1.00 (dd,
CH3 Val), 1.44 (s, 9H, CH3 Boc), 2.18–2.28 (m,
CH
J =
γ
J
=
β
α
γ
J
J
γ
β
J
β
(m, 2H,
α
CH Val), 4.86ꢀ4.92 [m, 2H,
= 9.0 and 21 Hz, 2H, NH Val), 5.30ꢀ5.38 [dd,
4.0 and 28.4 Hz, 2H, CH Phe( ꢀOH)], 7.09ꢀ7.16 [m, 2H, NH (C), 128.53 (CH), 129.43 (C), 129.70 (CH), 132.66 (CH),
Phe( CH ꢁPhe), 155.96 (C=O), 165.40 (C=O), 170.72
ꢀOH)], 7.20–7.39 (m, 10H, Ar H); 13C NMR (75.4 MHz, 133.44 (
CDCl3, δ): 17.14 ( CH3 Val), 17.74 ( CH3 Val), 18.99 ( CH3 (C=O); Anal. calcd for C20H28N2O5: C 63.81, H 7.50, N 7.44;
Val), 28.20 (CH3 Boc), 30.86 ( CH Val), 52.47 (OCH3), 52.58 found: C 63.36, H 7.36, N 7.40.
(OCH3), 58.04 [ CH Phe( ꢀOH)], 58.22 [
59.48 ( CH Val), 59.78 ( CH Val), 73.00 [
73.47 [ CH Phe( ꢀOH)], 79.91 [(CH3)3
(CH), 127.75 (CH), 127.81 (CH), 128.25 (CH), 128.30 (CH), 107.0ꢀ108.0 ºC; 1H NMR (300 MHz, CDCl3, δ): 1.38ꢀ1.44 (m,
139.73 (C), 139.78 (C), 155.89 (C=O), 155.94 (C=O), 170.73 12H, CH3 Ala and CH3 Boc), 3.79 (s, 3H, OCH3), 4.36 (brs,
(C=O), 170.99 (C=O), 171.90 (C=O), 172.01 (C=O); HRMS 1H, CH Ala), 5.27 (brs, 1H, NH Ala), 7.28–7.36 (m, 3H, Ar
(ESI) CH ꢁPhe), 7.47 (d, = 6.6 Hz, 2H, Ar H),
: [M + Na]+ calcd for C20H30N2NaO6 417.19961; H), 7.40 (s, 1H,
found, 417.19957.
8.02 (s, 1H, NH ꢁPhe); 13C NMR (75.4 MHz, CDCl3, δ): 17.87
Synthesis of Bocꢀ CH3 Ala), 28.22 (CH3 Boc), 50.22 ( CH Ala), 52.55 (OCH3),
OH (1.89 g, 10 mmol) was treated with HꢀD,LꢀPhe( ꢀOH)ꢀ 80.19 [(CH3)3 ], 123.79 ( C), 128.46 (CH), 129.44 (CH),
OMe,HCl (2.32 g, 10 mmol) in acetonitrile using the standard 129.71 (CH), 133.20 ( CH ꢁPhe), 133.46 (C), 155.58 (C=O),
DCC/HOBt procedure, giving 1d (3.50 g, 95 %) as an oil; H 165.46 (C=O), 171.54 (C=O); HRMS (ESI) m/z: [M + Na]+
NMR (300 MHz, CDCl3, δ): 1.10ꢀ1.25 (m, 6H, CH3 Ala), 1.40 calcd for C18H24N2NaO5 371.15774; found, 371.15774.
(2s, 9H, CH3 Boc), 1.42 (2s, 9H, CH3 Boc), 3.35 (brs, 2H, OH), Synthesis of dehydrodipeptides 3a-e TFA (0.3 M) was added to
3.71 (2s, 3H, OCH3), 3.73 (2s, 3H, OCH3), 4.14 (brs, 2H, CH compounds 2aꢀe. After 2 hours the mixture was taken to
Ala), 4.84ꢀ4.88 [dd, = 3.3 and 7.2 Hz, 2H, CH Phe( ꢀOH)], dryness at reduced pressure to afford the corresponding
5.08 (brs, 1H, NH Ala), 5.18 (brs, 1H, NH Ala), 5.26ꢀ5.30 [dd, dehydrodipeptide methyl ester.
= 3.0 and 6.6 Hz, 2H, CH Phe( ꢀOH)], 7.12 [brd, = 8.7 Hz, Synthesis of Hꢀ ꢀPheꢀZꢀꢁPheꢀOMe,TFA (3a): The general
2H, NH Phe(
ꢀOH)], 7.24–7.37 (m, 10H, Ar H); 13C NMR procedure described above was followed using compound 2a
(75.4 MHz, CDCl3, δ): 18.20 ( CH3 Ala), 18.53 ( CH3 Ala), (0.86 g, 1.95 mmol) giving compound 3a (0.79 g, 92 %) as a
28.21 (CH3 Boc), 49.84 ( CH Ala), 52.52 (OCH3), 52.59 white solid; mp 87.0ꢀ88.0 ºC; H NMR (400 MHz, DMSOꢀd6
(OCH3), 58.09 [ CH Phe(
73.55 [ CH Phe( ꢀOH)], 80.04 [(CH3)3
α
CH Phe(
β
ꢀOH)], 5.01ꢀ
(
γ
CH3 Val), 19.26 (
γ
CH3 Val), 28.25 (CH3 Boc), 30.49 (
βCH
5.14 (dd,
J
J
=
Val), 52.50 (OCH3), 59.99 (
α
CH Val), 80.04 [(CH3)3 ], 123.96
C
β
β
β
β
γ
γ
γ
β
α
β
α
CH Phe(
β
ꢀOH)], Synthesis of Bocꢀ
LꢀAlaꢀZꢀꢁPheꢀOMe (2d): Compound 1d (3.50
α
α
βCH Phe(
β
ꢀOH)], g, 9.5 mmol) was treated according to the procedure described
β
β
C], 125.64 (CH), 125.86 above to give compound 2d (3.05 g, 92 %,) as a white solid; mp
β
α
m
/z
β
J
LꢀAlaꢀD,LꢀPhe(βꢀOH)ꢀOMe (1d): Bocꢀ
LꢀAlaꢀ
(
β
α
β
C
α
β
1
β
:
α
J
α
β
J
β
β
J
L
β
β
β
1
α
,
α
β
ꢀOH)], 73.33 [
β
CH Phe(
β
ꢀOH)], δ): 2.94–3.00 (dd,
J
= 9.2 and 5.2 Hz, 1H,
β
CH2 Phe), 3.24–
β
β
C], 125.77 (CH), 125.90 3.29 (dd,
J
= 4.8 and 9.2 Hz, 1H,
β
CH2 Phe), 3.73 (s, 3H,
(CH), 127.90 (CH), 128.24 (CH), 128.26 (CH), 139.67 (C), OCH3), 4.25 (brs, 1H,
155.39 (C=O), 155.42 (C=O), 170.77 (C=O), 170.96 (C=O), CH ꢁPhe), 7.58ꢀ7.60 (dd,
172.93 (C=O); HRMS (ESI) m/z: [M + Na]+ calcd for (brs, 3H, NH3 ), 10.37 (s, 1H, NH ꢁPhe); 13C NMR (100.6
α
CH Phe), 7.30–7.41 (m, 9H, Ar H and
β
J
= 2.0 and 4.0 Hz, 2H, Ar H), 8.26
+
C18H26N2NaO6 389.16831; found, 389.16827.
Synthesis of dehydrodipeptides derivatives (2a-e): DMAP (0.1
equiv) was added to solutions of compounds 1aꢀe in dry (CH), 129.57 (CH), 129.72 (CH), 130.01 (CH), 132.31 (
MHz, DMSOꢀd6, δ): 36.69 (
CH Phe), 125.03 ( C), 127.31 (CH), 128.64 (CH), 128.71
CH
βCH2 Phe), 52.37 (OCH3), 53.61
(
α
α
β
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 11 of 14
Journal Name
Journal of Materials Chemistry B
View Article Online
ARTICLE
DOI: 10.1039/C5TB00501A
ꢁPhe), 132.83 (C), 134.80 (C), 164.87 (C=O), 168.30 (C=O); naproxen chloride (1 equiv) was then slowly added with
+
HRMS (micrOTOF) m/z
325.15467; found, 325.15545.
Synthesis of Hꢀ
:
[M]+ calcd for C19H21N2O3
vigorous stirring and cooling in an ice bath. After stirring at 0
°C for 30 minutes the solution was stirred at room temperature
L
ꢀPheꢀZꢀꢁAbuꢀOMe,TFA (3b): The general overnight. The reaction mixture was then concentrated and
procedure described above was followed with compound 2b partitioned between ethyl acetate (100 mL) and KHSO4 (1 M,
(1.74 g, 4.8 mmol) giving compound 3b (1.60 g, 89 %) as an 50 mL) and washed with KHSO4 (1 M), NaHCO3 (1 M) and
oil; 1H NMR (400 MHz, DMSOꢀd6, δ): 1.60 (d,
CH3 ꢁAbu), 2.99ꢀ3.05 (dd, = 8.0 and 6.0 Hz, 1H,
3.14ꢀ3.19 (dd, = 6.0 and 8.0 Hz, 1H, CH2 Phe), 3.66 (s, 3H,
OCH3), 4.18ꢀ4.20 (m, 1H, CH Phe), 6.60 (q, = 7.2 Hz, 1H, Synthesis of compound NpxꢀL
J
= 7.2 Hz, 3H, brine (3x30 mL). After drying over MgSO4 the extract was
CH2), taken to dryness at reduced pressure to afford the corresponding
ꢀprotected dehydrodipeptide methyl ester (4aꢀe).
ꢀPheꢀZꢀꢁPheꢀOMe (4a): The
γ
J
β
J
β
N
α
J
+
β
CH ꢁAbu), 7.27ꢀ7.34 (m, 5H, Ar H), 8.29 (brs, 3H, NH3 ), general procedure described above was followed with
9.89 (s, 1H, NH ꢁAbu); 13C NMR (100.6 MHz, DMSOꢀd6, δ): compound 3a (0.40 g, 0.91 mmol) giving compound 4a (0.35 g,
1
13.45 (
CH Phe), 126.71 (
(CH), 133.68 (
γ
CH3 ꢁAbu), 37.04 (
β
CH2 Phe), 52.00 (OCH3), 53.41 71 %) as a white solid; mp 176.0ꢀ177.0 ºC; H NMR (400
(
α
α
C), 127.22 (CH), 128.57 (CH), 129.57 MHz, CDCl3, δ): 1.52 (d,
J
= 7.2 Hz, 3H, CH3), 2.95ꢀ3.01 (dd,
CH2 Phe), 3.08ꢀ3.12 (dd, = 6.4 Hz, 1H,
CH2 Phe), 3.64 (q, = 7.2 Hz, 1H, CH), 3.72 (s, 3H, OCH3),
= 7.6 Hz, 1H, CH Phe), 5.96
= 8.0 Hz, 1H, NH Phe), 7.04 (d, = 7.8 Hz, 2H, Ar H),
procedure described above was followed with compound 2c 7.09 (d, = 2.4 Hz, 2H, Ar H), 7.11ꢀ7.23 (m, 7H, Ar H), 7.30ꢀ
(0.75 g, 2.0 mmol) giving compound 3c (0.70 g, 90 %) as a 7.32 (m, 3H, Ar H), 7.51 (s, 1H, ꢀCH), 7.61 (dd, = 8.4 and
white solid; mp 228.5ꢀ230.0 ºC; H NMR (300 MHz, DMSOꢀ 8.8 Hz, 2H, Ar H), 7.77 (brs, 1H, NH ꢁPhe); 13C NMR (100.6
d6, δ): 0.98 (d, = 6.9 Hz, 3H, CH3 Val), 1.04 (d, = 6.9 Hz, MHz, CDCl3, δ): 18.10 (CH3), 36.80 ( CH2 Phe), 46.85 ( CH
3H, CH3 Val), 2.19ꢀ2.30 (m, 1H, CH Val), 3.71 (s, 3H, Phe), 52.50 (OCH3), 54.41 (CH), 55.28 (OCH3), 105.59 (CH),
OCH3), 3.82 (brs, 1H, CH Val), 7.31 (s, 1H, CH ꢁPhe), 7.36ꢀ 119.15 (CH), 123.72 (CH), 125.94 (CH), 126.17 (CH), 126.90
β
CH ꢁAbu), 134.74 (C), 164.11 (C=O), 167.22
J
β
= 7.6 Hz, 1H,
β
J
(C=O); HRMS (micrOTOF) m/z: [M]+ calcd for C14H19N2O3
J
+
263.13902; found, 263.13925.
Synthesis of Hꢀ
3.93 (s, 3H, OCH3), 4.82 (q,
J
α
L
ꢀValꢀZꢀꢁPheꢀOMe,TFA (3c): The general (d,
J
J
J
J
1
J
γ
J
β
α
γ
β
α
β
7.48 (m, 3H, Ar H), 7.65ꢀ7.69 (m, 2H, Ar H), 8.27 (brs, 3H, (CH), 127.17 (CH), 127.69 (CH), 128.58 (CH), 128.93 (C),
+
NH3 Val), 10.25 (brs, 1H, NH ꢁPhe); 13C NMR (75.4 MHz, 129.21 (CH), 129.27 (C), 129.42 (CH), 129.66 (CH), 132.70
DMSOꢀd6, δ): 16.98 (
Val), 52.26 (OCH3), 57.35 (
(CH), 129.74 (CH), 130.17 (CH), 132.43 (
γ
CH3 Val), 18.31 (
CH Val), 125.30 (
CH ꢁPhe), 132.87 calcd for C33H32N2O5: C 73.86, H 6.01, N 5.22; found: C 73.45,
γ
CH3 Val), 29.91 (
βCH (CH), 133.29 (C), 133.81 (C), 135.21 (C), 136.05 (C), 157.80
α
α
C), 128.68 (CꢀO), 165.14 (C=O), 169.70 (C=O), 175.07 (C=O); Anal.
β
(C), 164.88 (C=O), 168.32 (C=O); Anal. calcd for H 6.023, N 4.924.
C17H21N2O5F3: C 52.31, H 5.42, N 7.18; found: C 51.83, H Synthesis of NpxꢀLꢀPheꢀZꢀꢁAbuꢀOMe (4b): The general
5.47, N 7.19.
procedure described above was followed with compound 3b
Synthesis of HꢀLꢀAlaꢀZꢀꢁPheꢀOMe,TFA (3d): The general (0.438 g, 1 mmol) giving compound 4b (0.433 g, 91 %) as a
procedure described above was followed with compound 2d white solid; mp164.0ꢀ165.0 ºC; 1H NMR (400 MHz, CDCl3, δ):
(1.39 g, 4.0 mmol) giving compound 3d (1.33 g, 95 %) as an 1.52ꢀ1.56 (m, 6H, CH3 and
oil; 1H NMR (300 MHz, DMSOꢀd6, δ): 1.46 (d,
= 6.9 Hz, 3H, Hz, 1H, CH2 Phe), 3.10ꢀ3.15 (dd,
CH3 Ala), 3.72 (s, 3H, OCH3), 4.08 (brt, = 5.7 Hz, 1H, CH 3.64ꢀ3.70 (m, 4H, CH and OCH3), 3.92 (s, 3H, OCH3), 4.83 (q,
Ala), 7.37–7.42 (m, 4H, Ar H and CH ꢁPhe), 7.63ꢀ7.67 (m, = 7.2 Hz, 1H, CH Phe), 6.18 (d, = 6.8 Hz, 1H, NH Phe),
2H, Ar H), 8.28 (brs, 3H, NH3 ), 10.17 (s, 1H, NH ꢁPhe); 13C 6.67 (q,
= 7.2 Hz, 1H,
NMR (75.4 MHz, CDCl3, δ): 16.77 ( CH3 Ala), 48.44 ( CH and NH ꢁAbu), 7.25 (dd,
Ala), 52.44 (OCH3), 125.16 ( C), 128.76 (CH), 129.88 (CH), H), 7.64 (dd,
CH ꢁPhe), 164.99 (C=O), MHz, CDCl3, δ): 14.15 (
CH2 Phe), 46.76 (CH), 52.11 (OCH3), 54.27 (
γ
CH3 ꢁAbu), 2.96ꢀ3.01 (dd,
J = 7.6
J
β
J
= 6.4 Hz, 1H, CH2 Phe),
β
β
J
α
β
J
α
J
+
J
β
J
CH ꢁAbu), 7.04ꢀ7.17 (m, 8H, H Ar
= 2.0 Hz, 1H, Ar H), 7.56 (s, 1H, Ar
β
α
α
J
= 8.4 and 8.8 Hz, 2H, Ar H); 13C NMR (100.6
130.12 (CH), 133.03 (C), 133.29 (
169.70 (C=O).
β
γ
CH3 ꢁAbu), 18.08 (CH3), 37.19
CH Phe),
(
β
α
Synthesis of HꢀLꢀAlaꢀZꢀꢁAbuꢀOMe,TFA (3e): The general 55.27 (OCH3), 105.53 (CH), 119.06 (CH), 125.85 (C), 126.01
procedure described above was followed with compound 2e (CH), 126.06 (CH), 126.81 (CH), 127.52 (CH), 128.47 (CH),
(0.69 g, 2.4 mmol) giving compound 3e (0.60 g, 86 %) as an 128.90 (C), 129.17 (CH), 129.24 (CH), 133.75 (C), 134.28
oil; 1H NMR (300 MHz, DMSOꢀd6, δ): 1.42 (d,
CH3 Ala), 1.69 (d, = 7.5 Hz, 3H,
OCH3), 3.99 (brs, 1H, CH Ala), 6.66 (q,
ꢁAbu), 8.19 (brs, 3H, NH3 ), 9.74 (s, 1H, NH ꢁAbu); 13C Synthesis of Npxꢀ
NMR (75.4 MHz, CDCl3, δ): 13.49 ( CH3 ꢁAbu), 17.18 ( CH3 procedure described above was followed with compound 3c
Ala), 48.25 ( CH Ala), 52.07 (OCH3), 125.83 (
CH ꢁAbu), 164.13 (C=O), 168.81 (C=O).
Synthesis of dehydrodipeptides 4a-e
J
= 6.9 Hz, 3H, (CH), 135.32 (C), 136.18 (C), 157.72 (CꢀO), 164.39 (C=O),
CH3 ꢁAbu), 3.66 (s, 3H, 169.36 (C=O), 174.91 (C=O); HRMS (micrOTOF) m/z: [M +
= 6.9 Hz, 1H,
CH Na]+ calcd for C28H30N2NaO5 497.20524; found, 497.20604.
ꢀValꢀZꢀꢁPheꢀOMe 4c): The general
β
J
γ
α
J
β
+
L
(
γ
β
α
α
C), 134.03 (0.59 g, 1.5 mmol) giving compound 4c (0.50 g, 68 %) as a
1
(
β
white solid; mp 213.0ꢀ214.0 ºC; H NMR (400 MHz, DMSOꢀ
:
Triethylamine (2.2 equiv) d6, δ): 0.91 (d,
J
= 6.8 Hz, 3H,
γCH3 Val), 0.96 (d, J = 6.4 Hz,
was added to a solution of dehydrodipeptide methyl ester 3H,
γ
CH3 Val), 1.43 (d, = 7.2 Hz, 3H, CH3), 1.97ꢀ2.07 (m,
J
hydrochloride (3aꢀe) in dichloromethane (0.1 M), and (S)ꢀ(+)ꢀ 1H,
β
CH Val), 3.60 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.96 (q,
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 11

Journal of Materials Chemistry B
Page 12 of 14
VJieowuArrtnicalelONnlainme e
ARTICLE
DOI: 10.1039/C5TB00501A
J
= 6.8 Hz, 1H, CH), 4.82 (t,
7.11 (m, 4H, Ar H), 7.18 (s, 1H,
Hz, 1H, Ar H), 7.47 (dd,
(dd, = 2.0 and 8.4 Hz, 2H, Ar H), 7.66 (d,
H), 7.69 (d, = 4.4 Hz, 1H, Ar H), 7.72 (s, 1H, Ar H), 8.14 (d, Synthesis of Npxꢀ
= 8.8 Hz, 1H, NH Val), 9.73 (brs, 1H, NH ꢁPhe); 13C NMR procedure described above was followed with compound 4a
(100.6 MHz, DMSOꢀd6, δ): 18.34 ( CH3 Val), 19.17 ( CH3 (0.268 g, 0.5 mmol) giving compound 5a (0.183 g, 70 %) as a
Val), 19.34 (CH3), 30.64 ( CH Val), 40.13 ( CH Val), 52.05 white solid; mp 195.0ꢀ196.0 ºC; H NMR (400 MHz, DMSOꢀ
(OCH3), 55.19 (OCH3), 57.70 (CH), 105.72 (CH), 118.49 (CH), d6, δ): 1.21 (d, = 7.2 Hz, 3H, CH3), 2.80ꢀ2.86 (dd, = 10.8
125.42 (CH), 125.85 (C), 126.50 (CH), 126.73 (CH), 128.41 and 13.6 Hz, 1H, CH2 Phe), 3.11ꢀ3.15 (dd, = 3.6 and 14.0
(C), 128.45 (CH), 129.10 (CH), 129.27 (CH), 129.98 (CH), Hz, 1H, CH2 Phe), 3.76 (q, = 7.2 Hz, 1H, CH), 3.83 (s, 3H,
132.37 (CH), 133.08 (C), 133.17 (C), 137.38 (C), 156.99 (Cꢀ OCH3), 4.72ꢀ4.78 (m, 1H, CH Phe), 7.09 (dd, = 2.4 and 8.8
O), 165.32 (C=O), 171.37 (C=O), 173.76 (C=O); HRMS Hz, 1H, Ar H), 7.17ꢀ7.28 (m, 8H, Ar H), 7.31ꢀ7.33 (m, 2H, Ar
(micrOTOF) m/z [M = 1.6 and 8.4 Hz, 1H, Ar H), 7.51ꢀ7.74 (m, 2H,
Na]+ calcd for C29H32N2NaO5 H), 7.37 (dd,
511.22089; found, 511.22136. Ar H), 7.65ꢀ7.68 (m, 3H, ꢀCH + Ar H), 8.31 (d, = 8.8 Hz,
Synthesis of Npxꢀ ꢀAlaꢀZꢀꢁPheꢀOMe 4d): The general 1H, NH Phe), 9.68 (s, 1H, NH ꢁPhe), 12.71 (brs, 1H, CO2H);
procedure described above was followed with compound 3d 13C NMR (100.6 MHz, DMSOꢀd6, δ): 18.86 (CH3), 37.33
(0.70 g, 2 mmol) giving compound 4d (0.62 g, 67 %) as a white CH2 Phe), 44.64 (CH), 53.80 ( CH Phe), 55.10 (OCH3),
solid; mp 169.0ꢀ170.0 ºC; H NMR (400 MHz, CDCl3, δ): 1.37 105.62 (CH), 118.37 (CH), 125.34 (CH), 126.23 (CH), 126.35
(d, = 7.2 Hz, 3H, CH3 Ala), 1.55 (d, = 7.2 Hz, 3H, CH3), (C), 126.39 (CH), 126.65 (CH), 127.99 (CH), 128.29 (C),
3.70 (s, 4H, CH and OCH3), 3.90 (s, 3H, OCH3), 4.68ꢀ4.76 (m, 128.36 (CH), 128.45 (CH), 129.04 (CH), 129.32 (CH), 129.91
1H, CH Ala), 6.29 (d, = 6.4 Hz, 1H, NH Ala), 7.05 (d, (CH), 131.83 (CH), 133.04 (C), 133.50 (C), 137.07 (C), 137.87
2.4 Hz, 1H, Ar H), 7.09ꢀ7.12 (dd, = 2.4 and 6.4 Hz, 1H, Ar (C), 156.89 (CꢀO), 166.14 (C=O), 170.93 (C=O), 173.33
H), 7.22ꢀ7.24 (m, 3H, Ar H), 7.26ꢀ7.29 (dd,
= 1.6 and 6.8 Hz, (C=O); HRMS (ESI) m/z: [M + Na]+ calcd for C32H30N2NaO5
1H, Ar H), 7.33 (s, 1H, CH ꢁPhe), 7.38ꢀ7.40 (m, 2H, Ar H), 545.20469; found, 545.20483.
7.60 (s, 2H, Ar H), 7.62 (s, 1H, Ar H), 8.02 (brs, 1H, NH Synthesis of Npxꢀ ꢀPheꢀZꢀꢁAbuꢀOH
ꢁPhe); 13C NMR (100.6 MHz, CDCl3, δ): 17.58 (
CH3 Ala), procedure described above was followed with compound 4b
18.37 (CH3), 46.73 (CH), 49.11 ( CH Ala), 52.48 (OCH3), (0.17 g, 0.36 mmol) giving compound 5b (0.160 g, 97 %) as a
55.27 (OCH3), 105.57 (CH), 119.05 (CH), 123.66 (
(CH), 126.09 (CH), 127.57 (CH), 128.46 (CH), 128.91 (C), d6, δ): 1.21 (d,
129.21 (CH), 129.47 (CH), 129.69 (CH), 133.73 ( CH ꢁPhe), CH3 ꢁAbu), 2.80ꢀ2.86 (dd,
133.33 (C), 133.73 (C), 135.64 (C), 157.68 (CꢀO), 165.21 Phe), 3.06ꢀ3.11 (dd, = 4.4 and 9.2 Hz, 1H,
(C=O), 171.00 (C=O), 174.76 (C=O); HRMS (ESI) m/z: [M + (q, = 6.8 Hz, 1H, CH), 3.84 (m, 3H, OCH3), 4.68ꢀ4.74 (m,
Na]+ calcd for C27H28N2NaO5 483.18904; found, 483.18917.
1H, CH Phe), 6.50 (q, = 7.2 Hz, 1H, CH ꢁAbu), 7.11 (dd,
Synthesis of Npxꢀ ꢀAlaꢀZꢀꢁAbuꢀOMe 4e): The general = 2.8 and 6.0 Hz, 1H, Ar H), 7.18ꢀ7.31 (m, 6H, Ar H), 7.56 (s,
procedure described above was followed with compound 3e 1H, Ar H), 7.37 (dd, = 1.6 and 6.8 Hz, 1H, Ar H) 7.64ꢀ7.73
(0.69 g, 2.4 mmol) giving compound 4e (0.89 g, 92 %) as a (m, 2H, Ar H), 8.28 (d, = 8.4 Hz, 1H, NH Phe), 9.19 (s, 1 H,
white solid; mp 150.0ꢀ151.0 ºC; H NMR (300 MHz, CDCl3, NH ꢁAbu), 12.48 (brs, 1H, CO2H); 13C NMR (100.6 MHz,
δ): 1.38 (d, = 7.2 Hz, 3H, CH3 Ala), 1.50 (d, = 7.5 Hz, 3H, DMSOꢀd6, δ): 13.52 ( CH3 ꢁAbu), 18.55 (CH3), 37.90 ( CH2
CH3 ꢁAbu), 1.58 (d, = 7.2 Hz, 3H, CH3), 3.63 (s, 3H, Phe), 44.57 (CH), 53.64 ( CH Phe), 55.10 (OCH3), 105.62
OCH3), 3.73 (q, = 7.2 Hz, 1H, CH), 3.89 (s, 3H, OCH3), 4.66ꢀ (CH), 118.42 (CH), 125.29 (CH), 126.21 (CH), 126.60 (CH),
4.76 (m, 1H, CH Ala), 6.49 (d, = 7.5 Hz, 1H, NH Ala), 6.66 127.93 (CH), 127.98 (C), 128.30 (C), 129.04 (CH), 129.16
(q, = 7.2 Hz, 1H, CH ꢁAbu), 7.06 (d, = 2.4 Hz, 1H, Ar H), (CH), 129.32 (CH), 132.11 (CH), 133.05 (C), 137.02 (C),
7.09ꢀ7.13 (dd, = 2.7 and 6.3 Hz, 1H, Ar H), 7.31ꢀ7.35 (dd, 137.79 (C), 156.91 (CꢀO), 165.37 (C=O), 169.88 (C=O),
1.5 and 6.9 Hz, 1H, Ar H), 7.62ꢀ7.66 (m, 3H, Ar H), 7.91 (brs, 173.17 (C=O); HRMS (ESI) m/z: [M + Na]+ calcd for
1H, NH ꢁAbu); 13C NMR (75.4 MHz, CDCl3, δ): 13.92 (
CH3 C27H28N2NaO5 483.18904; found, 483.18921.
ꢁAbu), 17.86 ( CH3 Ala), 18.27 (CH3), 46.61 (CH), 48.86 Synthesis of Npxꢀ ꢀValꢀZꢀꢁPheꢀOH 5c): The general
CH Ala), 52.08 (OCH3), 55.22 (OCH3), 105.48 (CH), 118.97 procedure described above was followed with compound 4c
(CH), 125.98 (CH and C), 126.44 (C), 127.40 (CH), 128.85 (0.43 g, 0.88 mmol) giving compound 5c (0.35 g, 85 %) as a
(CH), 129.17 (CH), 133.67 (C), 134.50 (
(C), 157.60 (CꢀO), 164.45 (C=O), 170.83 (C=O), 174.68 d6, δ): 0.90 (d,
(C=O); Anal. calcd for C22H26N2O5: C 66.32, H 6.58, N 7.03; 3H, CH3 Val), 1.43 (d,
CH Val), 3.84 (s, 3H, OCH3), 3.96 (q,
NaOH (1 equiv, 1 M) was CH), 4.36 (t, = 8.0 Hz, 1H, CH Val), 6.99ꢀ7.11 (m, 4H, Ar
CH ꢁPhe), 7.24 (d,
4aꢀe) in dioxane (3 mL). The solution was stirred at room 7.45ꢀ7.52 (m, 3H, Ar H), 7.67ꢀ7.72 (dd,
J
= 8.0 Hz, 1H,
α
CH Val), 7.04ꢀ temperature (the reaction was followed by TLC until no starting
β
CH ꢁPhe), 7.24 (d, J = 2.4 material was detected). The dioxane was removed under
J
= 2.0 and 8.8 Hz, 1H, Ar H), 7.51 reduced pressure and the reaction mixture was acidified to pH
= 4.8 Hz, 1H, Ar 2–3 with HCl (1 M) and the solid formed filtered.
ꢀPheꢀZꢀꢁPheꢀOH 5a): The general
J
J
J
L
(
J
γ
γ
1
β
α
J
J
β
J
β
J
α
J
:
+
J
J
L
(
(
β
α
1
J
β
J
α
J
J =
J
J
β
L
(5b): The general
β
α
1
α
C), 125.94 white solid; mp 186.0ꢀ187.0 ºC; H NMR (400 MHz, DMSOꢀ
= 7.2 Hz, 3H, CH3), 1.47 (d, = 7.2 Hz, 3H,
= 10.0 and 3.6 Hz, 1H, CH2
CH2 Phe), 3.77
J
J
β
γ
J
β
J
β
J
α
J
β
J
L
(
J
J
1
J
β
J
γ
β
γ
J
α
J
α
J
J
β
J
J
J =
γ
β
L
(
(
α
α
1
β
CH ꢁAbu), 135.66 white solid; mp 218.0ꢀ219.0 ºC; H NMR (400 MHz, DMSOꢀ
= 6.4 Hz, 3H, CH3 Val), 0.96 (d, = 6.4 Hz,
= 7.2 Hz, 3H, CH3), 2.00ꢀ2.09 (m,
= 7.2 Hz, 1H,
J
γ
J
γ
β
J
found: C 66.23, H 6.25, N 6.51.
1H,
J
Synthesis of dehydrodipeptides 5a-e
:
J
α
added to a solution of
Nꢀacyl dehydrodipeptide methyl ester H), 7.20 (s, 1H,
β
J
= 2.8 Hz, 1H, Ar H),
(
J = 2.4 and 6.4 Hz, 2H,
12 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 13 of 14
Journal Name
Journal of Materials Chemistry B
View Article Online
ARTICLE
DOI: 10.1039/C5TB00501A
observations. This allowed the rationalization of the structural
features that govern the selfꢀassembly of dehydrodipeptide
amphiphiles. Thus compounds with at least one aromatic amino
acid give hydrogels in low critical gelation concentrations.
TEM images of the new hydrogels prepared revealed that they
comprise nanofibers with different widths that entangle to give
a 3D network. All hydrogels showed a viscoelastic behaviour
with a storage modulus higher than the loss modulus and
independent from the frequency. The CD spectra of two
hydrogelators, 5a and 5b, were compared with that obtained
Ar H), 7.72 (s, 1H, Ar H), 8.11 (d,
9.54 (s, 1H, NH ꢁPhe), 12.67 (brs, 1H, CO2H); 13C NMR
(100.6 MHz, DMSOꢀd6, δ): 18.27 ( CH3 Val), 19.23 ( CH3
Val), 19.32 (CH3), 30.66 ( CH Val), 44.45 (CH), 55.11
(OCH3), 57.62 ( CH Val), 105.64 (CH), 118.39 (CH), 125.35
(CH), 126.41 (CH), 126.67 (C), 126.69 (CH), 128.24 (CH),
128.35 (C), 128.82 (CH), 129.05 (CH), 129.76 (CH), 131.81
(CH), 133.08 (C), 133.45 (C), 137.34 (C), 156.90 (CꢀO),
J = 9.2 Hz, 1H, NH Val),
γ
γ
β
α
166.13 (C=O), 170.91 (C=O), 173.59 (C=O); HRMS (ESI) m/z
:
from the dipeptide phenylalanylphenylalanine
naproxen. The CD spectra were similar and point to a structural
organization with the characteristics of a ꢀsheet arrangement.
Nꢀprotected with
[M + Na]+ calcd for C28H30N2NaO5 497.20469; found,
497.20479.
β
Synthesis of Npxꢀ
LꢀAlaꢀZꢀꢁPheꢀOH
(5d): The general
Fluorescence spectroscopy studies showed that this is a good
methodology to determine the CGC and the gelation pH.
Preliminary toxicity assays were performed using one of the
hydrogelators and it was found that this compound was not
toxic even at concentrations of 500 ꢂM. The resistance of some
of the new hydrogelators towards αꢀchymotrypsin was tested in
an 80 h assay and it was found that the presence of the
dehydroamino acid in the conjugates confers proteolytic
procedure described above was followed with compound 4d
(0.46 g, 1 mmol) giving compound 5d (0.38 g, 85 %) as a white
1
solid; mp 185.0ꢀ186.0 ºC; H NMR (400 MHz, DMSOꢀd6, δ):
1.30 (d,
CH3), 3.83ꢀ3.86 (m, 4H, OCH3 and CH), 4.44ꢀ4.52 (m, 1H,
CH Ala), 7.08ꢀ7.11 (dd, = 2.4 and 6.4 Hz, 1H, Ar H), 7.19ꢀ
7.23 (m, 5H, Ar H), 7.43ꢀ7.46 (dd,
H), 7.52ꢀ7.55 (m, 2H, CH ꢁPhe and Ar H), 7.66ꢀ7.71 (m, 3H,
Ar H), 8.23 (d, = 7.6 Hz, 1H, NH Ala), 9.43 (s, 1H, NH
ꢁPhe), 12.64 (brs, 1H, CO2H); 13C NMR (100.6 MHz, DMSOꢀ
d6, δ): 17.95 ( CH3 Ala), 18.85 (CH3), 44.41 (CH), 48.11 ( CH
J = 6.8 Hz, 3H, βCH3 Ala), 1.41 (d, J = 6.4 Hz, 3H,
α
J
J
= 1.6 and 6.8 Hz, 1H, Ar resistance to the hydrogelator. Giving the properties of this new
class of hydrogelators it is possible to conclude that they
constitute promising candidates for biomedical applications.
β
J
β
α
Ala), 55.10 (OCH3), 105.63 (CH), 118.38 (CH), 125.34 (CH),
126.28 (C), 126.43 (CH), 126.62 (CH), 128.32 (CH), 129.02
(C), 129.04 (CH), 129.05 (CH), 129.87 (CH), 131.87 (CH),
133.04 (C), 133.49 (C), 137.24 (C), 156.89 (CꢀO), 166.11
(C=O), 171.78 (C=O), 173.18 (C=O); HRMS (ESI) m/z: [M +
Na]+ calcd for C26H26N2NaO5 469.17339; found, 469.17353.
Acknowledgements
Thanks are due to Foundation for Science and Technology
(FCT) – Portugal, QREN and program FEDER/COMPETE for
financial support through Centre of Chemistry (CQꢀUM) of
University of Minho. FCT is also acknowledged for PhD grants
of G. Pereira (SFRH/BD/38766/2007), H. Vilaça
(SFRH/BD/72651/2010)
(SFRH/BD/79195/2011), coꢀfunded by the European Social
Fund. The NMR spectrometer Bruker Avance III 400 is part of
Synthesis of Npxꢀ
LꢀAlaꢀZꢀꢁAbuꢀOH
(5e): The general
procedure described above was followed with compound 4e
(0.40 g, 1 mmol) giving compound 5e (0.31 g, 80 %) as a white
and
T.
G.
Castro
1
solid; mp 170.0ꢀ171.0 ºC; H NMR (400 MHz, DMSOꢀd6, δ):
1.27 (d,
J
= 7.2 Hz, 3H,
β
CH3 Ala), 1.39 (d,
J
= 7.2 Hz, 3H, the Portuguese NMR Network (Rede/1517/RMN/2005) which
is also supported by the FCT.
CH3), 1.47 (d,
J
= 7.2 Hz, 3H,
γ
CH3 ꢁAbu), 3.84ꢀ3.86 (m, 4H,
OCH3 and CH), 4.40ꢀ4.47 (m, 1H,
Hz, 1H, CH ꢁAbu), 7.09ꢀ7.13 (dd,
H), 7.24 (d, = 2.4 Hz, 1H, Ar H), 7.42ꢀ7.45 (dd,
6.8 Hz, 1H, Ar H), 7.68ꢀ7.74 (m, 3H, H Ar), 8.21 (d,
Hz, 1H, NH Ala), 8.94 (s, 1H, NH ꢁAbu), 12.41 (brs, 1H,
CO2H); 13C NMR (100.6 MHz, DMSOꢀd6, δ): 13.46 (
CH3
ꢁAbu), 18.45 (CH3), 18.53 (CH3), 44.39 (CH), 48.05 ( CH
Ala), 55.10 (OCH3), 105.64 (CH), 118.44 (CH), 125.29 (CH),
126.45 (CH), 126.59 (CH), 127.94 ( C), 128.33 (C), 129.04
(CH), 132.05 (CH), 133.06 (C), 137.18 (C), 156.91 (CꢀO),
α
J
CH Ala), 6.47 (q,
= 2.4 and 6.4 Hz, 1H, Ar
= 2.0 and
= 7.6
J = 7.2
β
Notes and references
J
J
a Department of Chemistry
J
University of Minho
Campus de Gualtar, 4710ꢀ057 Braga, Portugal
Eꢀmail: pmf@quimica.uminho.pt .
b Department of Physics
γ
α
University of Minho
α
Campus de Gualtar, 4710ꢀ057 Braga, Portugal.
c Centre for Neuroscience and Cell Biology
University of Coimbra
165.33 (C=O), 170.88 (C=O), 173.09 (C=O); HRMS (ESI) m/z
:
[M + Na]+ calcd for C21H24N2NaO5 407.15774; found,
407.15778.
Coimbra 3004ꢀ517, Portugal
c Department of Chemistry
Brandeis University
Waltham, MA, 02454 USA.
Conclusions
Several
Nꢀaromatic dehydrodipeptide amphiphiles were
prepared and studied as new hydrogelators. Molecular dynamic
simulations were used to obtain insights into the underlying
molecular mechanism responsible for aggregation. The results
obtained were in excellent agreement with the experimental
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 13

Journal of Materials Chemistry B
Page 14 of 14
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