α,γ-Cyclic peptide ensembles with a hydroxylated cavity

Article abstract of DOI:10.1039/b911247m

Here we describe a self-assembling α,γ-cyclic tetrapeptide that contains the 4-amino-3-hydroxytetrahydrofuran-2-carboxylic acid, in which the hydroxy group is pointing towards the inner cavity of the resulting dimers.

Full text of DOI:10.1039/b911247m

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www.rsc.org/obc | Organic & Biomolecular Chemistry
a,c-Cyclic peptide ensembles with a hydroxylated cavity†
Ce´sar Reiriz, Manuel Amor´ın, Rebeca Garc´ıa-Fandin˜o, Luis Castedo and Juan R. Granja*
Received 8th June 2009, Accepted 12th August 2009
First published as an Advance Article on the web 26th August 2009
DOI: 10.1039/b911247m
Here we describe a self-assembling a,c-cyclic tetrapep-
tide that contains the 4-amino-3-hydroxytetrahydrofuran-
2-carboxylic acid, in which the hydroxy group is pointing
towards the inner cavity of the resulting dimers.
Peptides have found applications in a variety of areas in science,
ranging from drug design to nanomaterials, because of their
ease of synthesis, introduction of chemical diversity, and large
diversity and ease modulation of their 3D structures.1 One
of the simplest peptide modifications to improve biological
properties or induce conformational changes is backbone
N-methylation.2 Cyclic peptides (CPs) that contain tertiary
amides have also been used to change the pharmacological
properties of peptides, as simple models for b-sheets, turn
structures, or peptide nanotubes.3 For example, self-assembling
peptide nanotubes (SPNs)4 are formed by stacks of CPs stabilized
by b-sheet-type hydrogen-bonding interactions and dimer-
forming homochiral N-methylated CPs have been used as simple
models for the basic structure of nanotubes.5 We describe here a
new member of the dimer-forming CP family that is characterized
by a novel structure and internal cavity, in which the use of a
b-hydroxy-g-amino acid is able to stabilize the dimer but also
Scheme 1 Model for nanotube formation and synthetic strategy for
to induce the formation of one major topoisomeric dimer. In
recent years we have explored the properties of CPs composed
of a-amino acids (a-Aas) alternated with g-amino acids, more
specifically a cis-3-aminocycloalkanecarboxylic acid (g-Aca).6,7
In these a,g-CPs (e.g. 1–3, Scheme 1) the relative rigidity of
the cycloalkane ring, besides creating a hydrophobic cavity as
a result of the projection of one of the cycloalkane methylenes
into the lumen,7a ensures that the peptide backbone has the
all-trans conformation required for the CP ring to be flat and
stackable. Furthermore, a,g-CPs have the largest association
constant reported for CPs that form SPNs.7a,8 We report here
our synthetic studies towards a C2-modified g-amino acid,
namely 4-amino-3-hydroxytetrahydrofuran-2-carboxylic acid
(c-Ahf-OH), and its application in dimer-forming a,g-CPs.
C2-modified g-amino acid (c-Ahf-OH) and cyclic peptide precursor.
orientation (Scheme 1). In contrast, the all cis-isomer of 4 would
direct the OH group in a pseudoaxial orientation and thus it would
be perpendicular to the plane of the ring.
Given the above structural requirements, our synthetic objective
was the fully protected derivative Boc-c-Ahf(Bn)-OMe (Scheme 1).
We envisioned that this compound could be prepared from
D-xylose (6) by means of previously described intermediate 5,9
which could then be transformed into Boc-c-Ahf(Bn)-OMe by
benzylation, acetal/aldehyde oxidation, azide reduction and pro-
tection. The selection of appropriate orthogonal protecting groups
for the amino, hydroxy and carboxylic groups would allow the use
of this compound in either Fmoc or Boc solid and solution phase
synthesis.10 For our synthetic purposes, Boc, benzyl and methyl
groups were selected, respectively, for solution phase synthesis.
According to the proposed synthetic sequence, D-xylose was
transformed into compound 5 in five steps and 75% overall yield
(Scheme 2).9 Benzylation of the free hydroxy group by treatment of
5 with sodium hydride, benzyl bromide and tetrabutylammonium
iodide provided compound 7 in 77% yield. In order to carry out
the acetal oxidation, we initially attempted direct transformation
to the methyl ester by several methods, including treatment
with chromium salts,11 DDQ,12 oxone13 or hydrogen peroxide
and hydrogen chloride in methanol.14 However, the use of these
conditions did not give the desired ester in a reasonable yield. It
was then decided to transform the acetal in a stepwise fashion
In general, we envisaged that the 3-amino-2-hydroxy-
cycloalkanecarboxylic acid derivatives (4) should have the hydroxy
group in a trans orientation relative to the a-carboxylic and
g-amino groups, so in the flat peptide conformation the hydroxy
group would be projected into the cavity in a pseudoequatorial
Departamento de Qu´ımica Orga´nica, Laboratorios del CSIC, Facultad de
Qu´ımica, Universidad de Santiago, 15782, Santiago de Compostela, Spain.
E-mail: juanr.granja@usc.es; Fax: (+34)981 5905012; Tel: (+34)981
563100#14251
† Electronic supplementary information (ESI) available: Detailed de-
scriptions of the synthesis of the key compounds and their complete
characterization, atomic cartesian coordinates for the stationary points
calculated with basis set [B3LYP/6-31G(d)], and interaction energies for
dimers Dc-16 and Dt-16. See DOI: 10.1039/b911247m
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Scheme 2 Synthesis of Boc-c-Ahf(Bn)-OMe and dipeptide 10b.
to the aldehyde and later to the carboxylic acid derivative. The
best conditions involved heating a 1:1 mixture in methanol in
the presence of trifluoroacetic acid. The resulting aldehyde was
oxidized with N-bromosuccinimide and potassium carbonate to
give ester 8 in 91% yield.15 Hydrolysis of this ester with lithium
hydroxide gave the corresponding acid 9 in almost quantitative
yield. The 4-azidotetrahydrofurancarboxylic acid derivative can
be used directly in peptide synthesis on a solid support,16 where
the coupling of each amino acid can be carried out by means
of a Staudinger-type reaction.17 Additionally, the Staudinger
reduction with tributylphosphine in THF, followed by reaction
with Boc anhydride and diisopropylethylamine in dioxane (route
b in Scheme 2), provided the desired Boc-c-Ahf(Bn)-OMe in seven
steps and 27% overall yield.
Treatment of Boc-c-Ahf(Bn)-OMe with trifluoroacetic acid fol-
lowed by coupling with Boc-D-Leu-OH in the presence of HATU
and diisopropylethylamine in CH2Cl2 gave dipeptide 10a in 65%
yield.18 This dipeptide was also obtained in similar overall yield by
in situ Staudinger coupling of compound 8 with Boc-D-Leu-OH
in the presence of tributylphosphine, diisopropylcarbodiimide and
N-hydroxybenzotriazole.
Hydrolysis of 10a with lithium hydroxide in a methanol/water
mixture provided acid 10b in 89% yield. The resulting compound
was coupled with dipeptide 12 to provide tetrapeptide 13 in 93%
yield (Scheme 3). Dipeptide 12 was obtained by TFA treatment of
compound 11, which was prepared from Boc-MeN-c-Acp-OFm.7d,e
The use of N-methyl g-Acp was selected to block nanotube forma-
tion by introducing steric impediments and removing hydrogen-
donors on the g-face of the ring.7 Additionally, D-Tyr was selected
to simplify peptide characterization and purification because of its
spectroscopic properties. Finally, removal of N- and C-terminal
protecting groups, by subsequent treatment with piperidine and
TFA, provided the corresponding unprotected peptide 14. The
reaction of this tetrapeptide with TBTU and DIEA in CH2Cl2 in
concentrations between 5–15 mM provided cyclic tetrapeptide 15
in 66% yield.19 Finally, the benzyl-protecting group was removed
by hydrogenolysis with palladium hydroxide in methanol to give
16 in almost quantitative yield.
Scheme 3 Synthesis of CP 16.
Scheme 4 Self-assembling properties of CPs 15 and 16 and the structure
of the expected dimer registers, which differ in the relative orientation of
the g-Ahf, only CPs 16 is able to give the corresponding dimer (D16).
have denoted as cis-dimers (Dc-15 and Dc-16) those in which the
two c-Ahf residues are on the same side of the dimer, while the
trans-dimers (Dt-15 and Dt-16) are those in which the xylose-derived
g-Aa are on opposite sides. The 1H NMR spectrum of protected
CP 15 suggests that it does not undergo dimer formation (Dt-15) as
the N–H signals are not shifted downfield and are concentration
1
independent. On the other hand, the H NMR spectrum of the
unprotected peptide 16 (Fig. 1) clearly shows that the NH groups
[8.1 (NHTyr ) and 7.4 (NHLeu) ppm] are shifted downfield, suggesting
their participation in hydrogen-bonding interactions, while the
NHAhf proton that is exposed to solvent in the dimeric form gives
a signal at 6.6 ppm. The formation of dimers is also reflected
Fig. 1 1H NMR spectrum of a 6.2 mM solution of peptide 16 in CDCl3
with the 6.0–8.5 ppm region of the spectra of 6.20, 2.30, 1.32, 1.01 and
The self-assembly process of the resulting peptides should
provide two non-equivalent dimers depending on the relative
orientation between the two b-sheet-forming CPs (Scheme 4). We
0.70 mM solutions, showing the downfield shift of NHTyr and NHLeu
.
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by the downfield shift of the N–H resonances on increasing the
concentration. The association constant, determined at 298 K by
dilution experiments, was estimated to be 1.2 ¥ 104 M-1 in CDCl3,
almost two orders of magnitude larger than previously reported
tetrapeptides.20 So, the presence of the equatorially oriented
hydroxy group pointing inwards into the ensemble is clearly
participating in an intramolecular hydrogen-bonded interaction
between both peptides in the dimer that is stabilizing it. Van’t
Hoff plots for the range 273–313 K afford values of -39.31
KJ/mol for DH◦ and -53.38 J/K.mol for DS◦ and these
are consistent with dimerization being essentially an enthalpy-
driven21 hydrogen-bonding process. Dimer formation was also
supported by MS, which gave a peak arising from a singly charged
species corresponding to sodium dimer D16 (1111), suggesting self-
assembly properties and perhaps a good propensity for cation
coordination.
cis-dimer (Dc-16). These results are consistent with those observed
experimentally based on the above-mentioned lack of NOE cross-
peaks between the Ahf and Acp a- and g-protons.
Interestingly, the analogous dehydroxylated dimers preferred
the opposite trans conformation (1.59 or 3.21 kcal/mol more
stable, using the B3LYP or Truhlar’s functional, respectively) and
present intersubunit interaction energies that are 11.5 and 10.6
kcal/mol lower than the corresponding cis and trans hydroxylated
dimers, respectively. In other words, the two additional hydrogen
bonds formed in the cavity of the hydroxylated dimer are
responsible for the higher stability for the ensemble.
298
298
Conclusions
We have successfully prepared from D-xylose a new cyclic g-amino
acid
(4-amino-3-hydroxytetrahydrofuran-2-carboxylic acid,
Unfortunately, we were unable to establish the type (Dc-16 and
c-Ahf) with appropriate protecting groups that can be used in both
solution and solid phase synthesis methods. Furthermore, we used
this amino acid in the synthesis of a novel a,g-CP that form dimers
that is simple model for a,g-SPNs, in which the cavity properties
and dimer structure are modulated by the hydroxyl group of the
c-Ahf. This group, as a result of the flat conformational disposition
of the CP and its equatorial orientation, points inwards into
the ensemble and directs the self-assembly process not only to
stabilize the dimer ensemble but also the topoisomeric dimer (Dc
versus Dt) formed in solution. This cyclic peptide functionalization
should lead to nanotubes with greater selectivity as ion channels,
catalysts, receptors or molecule containers. Work is in progress to
address these possibilities.
D
t-16) or ratio of the topoisomeric dimers formed, either by
dimensional NMR or by X-ray crystallography.22 Computational
modelling provided additional insights into the structural prop-
erties of peptide nanotubes and helped in the interpretation of
experimental observations. These studies were performed using
density functional theory (DFT) calculations, with the B3LYP
functional and the standard 6-31G(d) basis set for geometry
optimizations,23 in order to evaluate the influence of the OH
groups and to explore the dimer preference. As a starting point,
we used the X-ray crystallographic data for the homodimer
c-[(D-Phe-L-MeN-g-Ach)2-].7d From this structure, one of the
N-methyl groups and the side chains of the a-amino acids were
removed, with the former change based on the low effect on
the global stability of the dimer.24 The hydroxylated structures
c-[D-Ala1-g-Ahf-D-Ala2-MeN-g-Acp-] were prepared by replacing
the corresponding aliphatic hydrogen by an OH group. The
final energies were refined by single-point calculations using two
different functionals: B3LYP and Truhlar functional M05-2x,25
both with the 6-31+G(d,p) basis set.26 The calculations were
carried out with the GAUSSIAN03 package.27 The most stable
structure found was Dc-16, in which both hydroxy groups form
a hydrogen bond and at the same time the one acting as the
acceptor also interacts with the carbonyl group present in the
monomer (Fig. 2). On the other hand in Dt-16 both OH groups
form respective hydrogen bonds with the carbonyl groups of
the opposite monomer, with N ◊ ◊ ◊ O distances slightly longer
than in Dc-16. This dimer is between 1.53 (B3LYP functional)
Acknowledgements
This work was supported by the Ministerio de Educacio´n y Cien-
cia [SAF2007-61015, and Consolider Ingenio 2010 (CSD2007-
00006)], and by the Xunta de Galicia (PGIDIT06PXIB209018PR,
PGIDIT08CSA047209PR and GRC2006/132). CR, RG-F and
MA thank the Spanish Ministerio de Ciencia e Innovacio´n for
their FPU, postdoctoral, and Ramo´n y Cajal contracts respec-
tively. We also thank the CESGA and the Centro de Computacio´n
Cient´ıfica (UAM) for computation time.
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