Folding and self-assembling with β-oligomers based on (1R,2S)-2-aminocyclobutane-1-carboxylic acid

Article abstract of DOI:10.1039/b918755c

Improved methodologies are provided to synthesize (1R,2S)-2- aminocyclobutane-1-carboxylic acid derivatives and their incorporation into β-peptides of 2-8 residues bearing different N-protecting groups. The conformational analysis of these oligomers has been carried out by using experimental techniques along with theoretical calculations. This study shows that these oligomers adopt preferentially a strand-type conformation in solution induced by the formation of intra-residue six-membered hydrogen-bonded rings, affording cis-fused [4.2.0]octane structural units that confer high rigidity on these β-peptides. Moreover, all of them are prone to self-assemble producing nano-sized fibres, as evidenced by TEM, AFM and SPFM, and, in some instances, they also form gels. These techniques and molecular modelling allowed us to suggest an aggregation model for the assembly structures in which a parallel molecular-arrangement is preferred and the conformation is similar to that observed in solution. According to this model, both hydrogen-bonding and hydrophobic interactions would account for formation of the assemblies.

Full text of DOI:10.1039/b918755c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Folding and self-assembling with b-oligomers based on
(1R,2S)-2-aminocyclobutane-1-carboxylic acid†
Elisabeth Torres,a Esther Gorrea,a Kepa K. Burusco,a Eric Da Silva,a Pau Nolis,b Federico Ru´a,a
Ste´phanie Boussert,c Ismael D´ıez-Pe´rez,d Samantha Dannenberg,a Sandra Izquierdo,a Ernest Giralt,c,e
Carlos Jaime,a Vicenc¸ Branchadella and Rosa M. Ortun˜o*a
Received 10th September 2009, Accepted 19th October 2009
First published as an Advance Article on the web 3rd December 2009
DOI: 10.1039/b918755c
Improved methodologies are provided to synthesize (1R,2S)-2-aminocyclobutane-1-carboxylic acid
derivatives and their incorporation into b-peptides of 2–8 residues bearing different N-protecting
groups. The conformational analysis of these oligomers has been carried out by using experimental
techniques along with theoretical calculations. This study shows that these oligomers adopt
preferentially a strand-type conformation in solution induced by the formation of intra-residue
six-membered hydrogen-bonded rings, affording cis-fused [4.2.0]octane structural units that confer high
rigidity on these b-peptides. Moreover, all of them are prone to self-assemble producing nano-sized
fibres, as evidenced by TEM, AFM and SPFM, and, in some instances, they also form gels. These
techniques and molecular modelling allowed us to suggest an aggregation model for the assembly
structures in which a parallel molecular-arrangement is preferred and the conformation is similar to
that observed in solution. According to this model, both hydrogen-bonding and hydrophobic
interactions would account for formation of the assemblies.
of chirality in the monomers, has allowed the synthesis of
Introduction
b-peptides with interesting structural features that include the
The use of unnatural peptides in molecular architecture presents
formation of tertiary structures such as nano-sized fibrils and
micelles.5 The obtained results have allowed a better understand-
ing of the combined influence of chirality and conformational
constraint on the molecular and supramolecular arrangement of
these compounds. The acquired knowledge has been useful in the
preparation of several materials such as nucleic acid mimics6 and
nanotubes.7
enormous possibilities for the preparation of new chiral materials
with determined properties because these products can adopt well
defined secondary, and, in some cases, tertiary and quaternary
structures.1 Among them, b-peptides are prominent. Their propen-
sity to fold forming sheets, helices and reversed turns has been
established.2 Theypresenttheadvantagewithrespectto a-peptides
that the number of residues usually needed to form foldamers is
lower than that required in a-oligomers. In addition, careful design
at the residue level can lead to enhanced secondary structural
stability among the foldamers relative to conventional peptides.
Foldamers with defined folding propensities can be endowed
with biological functions, including antibacterial, antifungal and
antiviral activities that arise from interaction of the foldamer with
a biomolecular partner.1
Although structural features of cyclohexane- and cyclopentane-
containing b-peptides are well documented,3,5 data on the con-
formational bias and supramolecular structure of b-peptides
including four-membered rings are scarce.8 Fleet et al.4 sug-
gested well-defined left-handed helical structures stabilized by ten-
membered hydrogen-bonded rings as the structural preference for
b-hexapeptides incorporating cis-substituted oxetane rings.4
Several types of cyclobutane-containing b-peptides have been
synthesized and studied in our laboratory. The ability of the
cyclobutane ring to promote defined secondary structures and
to self-assemble to form fibrils and gels has been evidenced.
Firstly, we reported on b-peptides consisting of cyclobutane
residues derived from (1R,2S)-2-aminocyclobutane-1-carboxylic
acid and b-alanine joined in alternation.9,10 A 14-helical folding
was promoted in chloroform solution in a tetrapeptide of this
series.10 Later, we have described the synthesis of a new class
of b-peptide starting from (-)-verbenone as a chiral precursor.
In these products, the cyclobutane moiety is not a part of the
The use of carbocycles3 and heterocycles4 incorporated into
2,3
b -positions of the peptide backbone, combined with the control
aDepartament de Qu´ımica, Universitat Auto`noma de Barcelona, 08193,
Bellaterra, Spain. E-mail: rosa.ortuno@uab.es; Fax: (34) 935811265
bServei de Ressona`ncia Magne`tica Nuclear, Universitat Auto`noma de
Barcelona, 08193, Bellaterra, Spain
cInstitut de Recerca Biome`dica de Barcelona, Parc Cient´ıfic de Barcelona,
Josep Samitier 1-5, 08028, Barcelona, Spain
dDepartament de Qu´ımica F´ısica, Universitat de Barcelona, Mart´ı i Franques
1, 08028, Barcelona, Spain
3
eDepartament de Qu´ımica Orga`nica, Universitat de Barcelona, 08028,
Barcelona, Spain
peptide backbone but a bulky substituent at the b -position.
These products show some conformational bias in solution and,
in the solid state, the non-cyclic b-peptides adopt a hairpin-like
molecular folding ruled by intermolecular hydrogen bonds in the
crystal packing.11
† Electronic supplementary information (ESI) available: Details on theo-
retical calculations, NMR experiments, assigned NMR spectra for 7a, 8b,
9a, 10a, 12 and 13, CD spectrum of 8b, and image of the gel formed by 8b.
See DOI: 10.1039/b918755c
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We also prepared and studied a third family of cyclobu-
tane b-peptides derived from different stereoisomers of 2-
aminocyclobutane-1-carboxylic acid, 1, shown in Chart 1. Thus,
we reported on the formation of six-membered hydrogen-bonded
rings in diastereomeric bis(cyclobutane) b-dipeptides 2 and 3, in
CDCl3 solution. These intramolecular and intra-residue hydrogen-
bonds give rise to cis-fused [4.2.0]octane structural units that
confer high rigidity on these molecules.12 We have recently
described that this preferred conformation in solution is also
manifested in b-dipeptide 4, constituted by residues derived from
amino acids (1R,2S)-1, cis, and (1S,2S)-1, trans, respectively. In
contrast, the formation of eight-membered hydrogen-bonded rings
prevails in diastereoisomeric dipeptides 5 and 6.13
In a preliminary communication14 we reported that tetramer 8a
(R = Cbz) showed the same conformational bias in solution as
dipeptides 2, 3 and 4. Moreover, this compound manifested its
tendency to the aggregation forming nano-sized fibres.
On the other hand, cyclobutane-containing b-peptides have
become very promising, since recent investigations have revealed
their biological activity as a new class of carboxypeptidase
inhibitors. Preliminary docking studies show that the conforma-
tional constraint imposed by the cyclobutane ring and its small
size contribute to suitable interaction with the enzyme.15
To gain insight on the structural features of these compounds,
in this paper, we present our results on the molecular and
supramolecular structures of all-cis cyclobutane b-peptides de-
rived from (1R,2S)-1. For this purpose, we have synthesized
and studied tetramer 8b (R = Boc) to ascertain the influence
of the protecting group on the conformational bias and self-
assembling. Hexamer 9a and octamer 10a have been synthesized
and investigated to verify if higher oligomers follow the same
trends as the smaller ones. Related to these syntheses, we provide
herein an improved methodology to prepare the meso compound
13 (Scheme 1) in a much cheaper and efficient way. Diester 13
is the starting material to prepare b-amino acids and related
peptides9–10,12–14,16 through an asymmetric and chemoenzymatic
hydrolysis to afford half-ester 14.9 The conformational analysis
of the new b-peptides has been carried out by the combined use of
NMR and CD techniques, and computational methods. Tendency
to self-assemble has also been explored for dimers 2a,b as well as
for trimer, 7a, tetramer 8b, hexamer 9a and octamer 10a. A model
based on theoretical calculations jointly with transmission electron
microscopy (TEM), atomic force microscopy (AFM) and scanning
polarization force microscopy (SPFM), is provided to understand
the way in which supramolecular aggregation takes place in this
family of b-peptides constituted by cyclobutane residues with cis
configuration.
Results and discussion
1. Synthesis of the oligomers
Half-ester 14 (Scheme 1) is the common chiral precursor to
synthesize cyclobutane b-amino acids and b-peptides according
to protocols developed in our laboratory.9,10,12–14
The simple way to prepare 14 in two steps, which involves
esterification and subsequent chemoenzymatic hydrolysis,9 is
from commercially available cyclobutane-1,2-dicarboxylic diacid.
Nevertheless, the price of this compound is very high and for this
Chart 1 Structure of some cyclobutane b-amino acids and related
b-peptides.
Scheme 1 Reagents and conditions: (a) hn (quartz), CH3CN, -35 ◦C, 4 h (quantitative); (b) MeOH, H2SO4, 50 ◦C, 5 h (88%); (c) H2 (6 atm), 5% Pd/C,
Et3N, EtOH, rt (83%).
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reason we have established a new synthetic sequence to prepare
diester 13, as shown in Scheme 1.
This synthetic route is inspired by the method reported by
Huet et al. to prepare dimethyl cyclobut-3-ene-1,2-dicarboxylate.17
Photochemical [2 + 2] cycloaddition of 1.5 equivalents of 1,2-
dichloroethylene (mixture of isomers) to maleic anhydride in a
0.08 M solution of acetonitrile, at -35 ◦C, quantitatively provided
the photoadduct 11 as a diastereoisomeric mixture. The lack of
stereoselectivity is not relevant since chlorine atoms are removed
in a later synthetic step. A similar mixture was obtained by Huet
et al. who described this cycloaddition to occur in a Pyrex reactor
in the presence of benzophenone as a photosensitizer and by
using ethyl acetate as a solvent.17 In our laboratory, better results
were achieved in the absence of a photosensitizer, working with a
quartz reactor and by using acetonitrile as a solvent. In the next
step, Fisher esterification of 11 in refluxing methanol produced
dichloro diester 12 (mixture of diastereomers, 88% yield), which
was submitted to hydrogenation in the presence of 5% Pd on
charcoal under 6 atmospheres pressure resulting in meso-diester
13 as the only defined product, in 83% yield after purification.
Another synthesis of 13 is described in the literature18 via the
photocycloaddition of ethylene to maleic anhydride by using a
Pyrex filter.19 Nevertheless, we found that 4–6 g batches of diester
13 are easily prepared in three steps and in 60% overall yield as
shown in Scheme 1. This allows us to avoid the manipulation of
a large excess of ethylene as a gas reagent and with a 20-times
cheaper cost than the product from commercial sources.
Desymmetrization of 13 was achieved by using pig liver esterase
to afford stereoselectively chiral half-ester 14.9 Conversion of this
compound into fully protected derivatives of monomer (1R,2S)-
1 was achieved by transformation of the carboxyl group into an
intermediate acyl azide and subsequent Curtius rearrangement
in the presence of benzyl or tert-butyl alcohol (series a and b,
respectively).10
Regarding the synthesis of the polycyclobutane b-peptides,
dimer 2b12 was used in the convergent synthesis of tetramer 8b
through selective deprotection of the amine and the carboxylic
acid, respectively (Scheme 2). Peptide coupling of the resultant
intermediates in the presence of pentafluorophenyl diphenyl
phosphinate (FDPP) produced 8b in 60% yield.
Additionally, hydrogenolysis of benzyl carbamate in dimer
2a gave amine 15,14 which reacted with carboxylic acid 169,12
(Scheme 3) under coupling conditions by using EDAC as a
dehydrating agent and hydroxybenzotriazole as a catalyst, in
DMF. In this way, the new b-tripeptide 7a was obtained in 40%
yield. In an alternative manner, this product was synthesized via
the Curtius rearrangement of acyl azide 18 in boiling toluene to
afford an isocyanate, which reacted in situ with carboxylic acid 17
to provide trimer 7a in 52% yield with concomitant loss of carbon
dioxide.12 Subsequent selective deprotection of the amine and the
carboxylic acid followed by coupling of the resulting compounds
resulted in hexamer 9a in 45% yield (Scheme 3).
Scheme 2 Reagents and conditions: (a) 0.25 M NaOH (94%); (b) TFA,
Et3SiH (95%); (c) FDPP, DIPEA, DMF (60%).
solvent is suitable for conformational analysis of the target
oligomers and has been successfully used in structural studies
of related cyclobutane b-peptides.10–14 In CDCl3, standard 1D
and 2D high resolution correlation NMR spectra of tetramer
8b and hexamer 9a allowed us to assign all protons and carbon
atoms (see the ESI†). Specifically, 2D 1H–1H NOESY experiments
permitted to establish intra- and inter-residue NOE connectivities.
Some of these NOE contacts and HH coupling constants led
us to secure a trans stereochemistry for all amide bonds in the
major conformer for each peptide. In the NH region of the
1H NMR spectrum, additional signals were observed, which
could be attributed to minor conformers arising from rotation
Similarly, octamer 10a was prepared by coupling of two
tetrameric units, an amine and a carboxylic acid, derived from 8a.
2. Conformational analysis in solution
=
2.1 Experimental studies by using NMR and CD techniques.
NMR experiments were carried out on CDCl3 solutions. This
around the O Cn–NH(n+1) bond in the carbamate. Particularly
significant are the strong inter-residue NOE contacts involving
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when the sample is cooled down to 288 K the doublet structure is
recovered and the strand-type conformation becomes completely
fixed (see Fig. S8 in the ESI†). Thus, the emerging conclusion is
=
that the Boc protecting group permits rotation around the O Cn–
NH(n+1) in the carbamate, while Cbz does not.
Comparison of the CD spectra in MeOH for the oligomers
of this series (R = Cbz) account for the same preferential
conformation in these b-peptides. Fig. 2 shows the superposition of
CD spectra of 0.5 mM solutions of dimer 2a, tetramer 8a, hexamer
9a and octamer 10a. The wavelength is shifted from 210 (2a) to
225 nm (10a). The intensity is enhanced from 2a to 9a, according
to the increasing number of chromophores, although the band
for the octamer 10a is similar in intensity to that of tetramer 8a.
Additionally, the normalized (per residue) CD spectra of these
oligomers show bands with very close intensities for 2a, 8a, and
9a, whereas the band for 10a is weaker (Fig. S28 in the ESI†). This
would be in accordance with a greater flexibility in 10a than in
shorter oligomers. On the other hand, CD spectrum of a 0.5 mM
solution of tetrapeptide 8b (R = Boc) compares well with that of
8a (see Fig. S26 in the ESI†).
2.2 Computational studies: simulated annealing (SA) and
molecular dynamics (MD). To begin with, conformational search
calculations for 8a, 9a and 10a were done based on SA method-
ology followed by geometry optimization. The three b-peptides
were studied by means of principal components analysis (PCA)20
to obtain their most representative conformations. The study
concluded that the flexibility in the series and, therefore, the
total number of conformations increases from 8a to 10a as a
consequence of the growing number of residues in the b-peptides.
Three structures have been selected for each b-peptide 8a, 9a and
10a on the basis of the analysis of the PCA plots. As an example,
the three selected conformations, I–III, for the hexapeptide 9a are
shown in Fig. 3.
Subsequently, the dynamic behaviour of the three selected
structures obtained by SA for each oligomer was explored by
means of MD calculations21 employing a cubic-box chloroform-
solvent model (see Fig. S3 and S4 in the ESI†). For each peptide,
the three MD calculations converge to similar results, showing
the formation of a strand-type conformation in solution. For
instance, for hexamer 9a, the average measured distances between
the hydrogens involved in NOE contacts oscillate in values around
˚
2.30 0.30 A (Table 1), which are in total accordance with the
Scheme 3 Reagents and conditions: (a) EDAC, HOBt, Et3N, DMF (40%);
(b) Et3N, toluene, reflux (52%); (c) 0.25 M NaOH (70%); (d) H2 (6 atm),
20% Pd(OH)2/C (quantitative); (e) EDAC, HOBt, Et3N, DMF (45%).
NMR results. Moreover, the calculated NH ◊ ◊ ◊ O=C distances, in
˚
the range of 2.45 0.24 A, are compatible with the formation
of six intramolecular hydrogen bonds as shown in Fig. 1. Fig. 4
shows the average conformation obtained from each trajectory for
hexamer 9a.
MD calculations done for the selected structures of tetrapeptide
8a and octapeptide 10a show the same preferential conformation
in chloroform solution (see ESI). This conclusion is in good
accordance with the most stable conformer previously predicted
for tetramer 8a when results of the conformational search were
optimized in chloroform solution at the B3LYP/6-31G(d) level of
calculation.14
Ha(i) and NH(i+1) protons since they confirmed a well-defined
strand-type conformation both for tetramer 8b (Scheme 2) and
hexamer 9a (Fig. 1).
This predominant conformation is similar to that found for
tetramer 8a and dimers 2a,b and is in excellent agreement with the
results of theoretical calculations (vide infra).
When tetramer 8b is protected with a Boc group instead of Cbz
as in 8a, it is observed that the NH4 signal becomes broad and
loses its doublet structure. This indicates a slight loss of rigidity
in the extreme of the strand-type conformation, suggesting that
the hydrogen bond is not formed in this segment. Nevertheless,
This conformational bias contrasts with the helical structures
suggested by Fleet et al. for b-hexapeptides containing cis-
substituted oxetanes.4 The presence of the oxygen ring as well as
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Fig. 1 Strand-type conformation for hexamer 9a in CDCl3 solution. Arrows show inter-residue Ha(i) and NH(i+1) NOE contacts.
3. Aggregation studies
The behaviour of all oligomers of this series was investigated
in order to verify their propensity to form aggregates. Although
tetramers 8a and 8b, hexamer 9a, and octamer 10a were obtained
as crystals, they were not suitable for structural analysis by X-ray
diffraction.
Tetramer 8a formed an organogel, which was stable for sev-
eral days, when dissolved in 3 : 2 ethyl acetate–hexane 1 mM
solution by boiling, then air cooling and left to stand. In addition
to this, a gel was obtained from 3 : 1 acetone–hexane 1 mM
solution. Unfortunately, it was less stable in this medium and
precipitated after standing for 1 d both at room temperature and
at 5 ◦C.14 Similarly, tetrapeptide 8b formed a stable gel from 1 : 3
dichloromethane–pentane 5.8 mM solution at 25 ◦C (see Fig. S27
in the ESI†).
All b-peptides in this series show strong tendency to self-
assemble from methanol solutions giving nanosized fibres whose
morphology remained unaltered after one week incubation. Fig. 5
shows selected images of fibres from different oligomers, obtained
under the optimal conditions in each case.
In the case of 10a, TEM images of 0.5 M solutions after 24 h
incubation revealed the formation of homogeneous vesicles, which
remained unaltered at room temperature for, at least, two weeks.
Their diameters measured from 70 to 100 nm on average (Fig. 6a).
Aggregates with fibrilar morphology were observed from 1 mM
solutions after a one week incubation period (Fig. 6b).
Fig. 2 CD spectra of 0.5 mM methanol solutions of 2a (blue), 8a (violet),
9a (green), and 10a (grey).
the additional substituents on the oxetanes could account for the
structural differences between cyclobutane and oxetane oligomers.
Strand-type preferential conformation had been previously
reported by Fu¨lo¨p et al.3 for cis-2-aminocyclopentanecarboxylic
acid oligomers. So, we can conclude that the restriction of the
NH–Cb–Ca–CO torsion in the gauche position facilitates extended
strand conformations in both cyclobutane and cyclopentane
derivatives.
Fig. 3 Selected conformers for hexamer 9a.
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Table 1 MD calculated average Ha(i) ◊ ◊ ◊ NH(i+1) and NH(n) ◊ ◊ ◊ OC(n) distances afor the three trajectories of hexamer 9ab
Conformer
H14 ◊ ◊ ◊ NH16
H20 ◊ ◊ ◊ NH22
H26 ◊ ◊ ◊ NH28
H32 ◊ ◊ ◊ NH34
H38 ◊ ◊ ◊ NH40
I
II
III
2.32
2.36
2.23
2.32
2.28
2.38
2.28
2.31
2.32
2.43
2.34
2.29
2.28
2.38
2.35
Conformer
NH10 ◊ ◊ ◊ OC15
NH16 ◊ ◊ ◊ OC21
NH22 ◊ ◊ ◊ OC27
NH28 ◊ ◊ ◊ OC33
NH34 ◊ ◊ ◊ OC39
NH40 ◊ ◊ ◊ OC45
I
II
III
2.58
2.44
2.37
2.67
2.60
2.58
2.70
2.69
2.57
2.77
2.64
2.42
2.15
2.46
2.44
2.05
1.92
2.14
a In angstroms. b See Fig. 1 for atom numeration.
Fig. 4 Image showing the average strand-type conformation obtained from each MD calculation for the three trajectories of hexamer 9a.
Fig. 5 TEM images of the nanosized fibres formed by (a) 2a from a 5 mM, (b) 8b from a 1 mM, (c) 9a from a 0.5 mM solution in MeOH (1 d incubation)
placed onto a carbon film-coated copper grid and stained with 2% uranyl acetate.
Accordingly, higher concentration account for the evolution
from vesicles to fibrils in MeOH solutions of 10a. A similar
behaviour has been observed for other peptides.22 Self-assembled
peptide-based vesicles have been used for biological purposes such
as DNA delivery and release in cells.22
In addition, AFM of tetrapeptide 8a onto a mica substrate
showed the formation of monolayers, which, after longer periods
of time, pile themselves up into a multilayer structure. Fig. 7a
shows a topographic image of 8a molecules. The heights measured
range from 6.4 to 8.8 ( 0.2) nm with a periodicity of 2.2 nm, which
is in agreement with the size of single molecular tetrapeptide 8a,14
as represented in Fig. 8.
characterize the local charge distribution of the self-assemblies.
It is noteworthy that the surface potential (SP) of the fibrils
increased with the size of the self-assemblies, as can be observed in
Fig. 7b.
In addition, the calculated dipole moment at the B3LYP/
6-31G(d) level of calculation14 for 8a is 11.1 D, with the positive
pole on the C-terminus. All these results suggest a molecular
arrangement as shown in Fig. 8, with the N-terminus oriented
towards the surface of the mica substrate.
MD calculations were undertaken to understand the mode of
aggregation in the monolayer. These calculations were carried
out for two cells containing nine molecules with two different
arrangements, parallel and alternate (Fig. 9).
To keep this ordering stable along the whole MD, the
eight molecules surrounding the central one are partially
The local charge distribution of the nanosized fibres was studied
by SPFM. While AFM made it possible to gain information on the
structural properties of fibrils, SPFM measurements enabled us to
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Fig. 8 Modelled vertical arrangement of the molecules of 8a in the
assemblies, as suggested by AFM and SPFM experiments and the
calculated dipole moment.
Fig. 6 Aggregates formed by 10a from (a) 0.5 mM solution in MeOH
after 1 day incubation, and (b) 1mM solution in MeOH after incubation
for a week.
retained in their positions by restraints in order to maintain
the basic cell. The restrictions were softly included into the
equilibrium distance. The molecule in the middle is absolutely
unrestrained and is therefore able to freely interact with its
environment.
˚
system by allowing distance fluctuations of 2 A from the
Fig. 7 (a) Topographic image of tetrapeptide 8a molecules deposited onto a freshly cleaved mica substrate. The heights measured range from 6.4 ( 0.2)
to 8.8 ( 0.2) nm. (b) Surface potential (SP) image of the same 8a molecules deposited onto a freshly cleaved mica substrate. The SP measurements of the
nanosized fibres increase with the size of the self-assemblies.
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Fig. 9 Parallel (a) and alternate (b) monolayer arrangements for nine molecules of tetramer 8a.
The two boxes already built, parallel and alternate, were
subjected to MD calculations. The results obtained point out
that the system exhibits a preference for the parallel arrangement
because of its lower energy (Fig. 10), which is in accordance with
SPFM results. Moreover, MD trajectories show that interactions
among the nine phenyl groups are quite favoured.
The possible inter and intramolecular hydrogen bonds involving
the central peptide molecule were analyzed during the MD
calculations (Table 2). The results show that the molecules interact
better by hydrogen bonding in the parallel aggregate (44.80%) than
in the alternate one (38.78%) (Table 2). This also points out that
the parallel monolayer is energetically favoured, which agrees with
the results in Fig. 10.
arrangements of tetramer 8a. This model could be applied to the
other oligomers in the all-cis series.
Conclusions
High-resolution NMR experiments, CD spectra and computa-
tional studies reveal that b-peptides constituted by residues de-
rived from (1R,2S)-2-aminocyclobutane-1-carboxylic acid adopt
a strand-type conformation in solution, independently of their
size and the terminal amine protecting group. The presence
of the small cyclobutane ring imposes this conformational
bias, which results from the formation of intra-residue hydrogen-
bonded six-membered rings giving rise to cis-fused [4.2.0]oc-
tane structural units that confer high rigidity on these
b-peptides. This result compares well with a similar confor-
mation described in the literature for oligomers consisting of
cis-2-aminocyclopentanecarboxylic acid residues. In both cases,
the NH–Cb–Ca–CO torsion restricted in the gauche position
These results show that both inter and intramolecular hydrogen
bonds coexist in the aggregates and that the conformation of the
peptide reproduces that observed in solution, as shown in Fig. 11.
All these results suggest that both hydrophilic (hydrogen bond-
ing) and hydrophobic interactions account for the supramolecular
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Table 2 Statistical probability for hydrogen bonding in the parallel and the antiparallel arrangements
˚
=
Box type
Parallel
Molecular interaction
Number (N)
Frequency (%)
NH ◊ ◊ ◊ O C distance/A
N–H–O angle (◦)
Inter
Intra
Total
14
5
19
20.70
24.10
44.80
3.32
3.04
3.25
40.94
51.50
43.73
Alternate
Inter
Intra
Total
8
5
13
28.83
9.95
38.78
3.30
2.04
3.20
43.30
51.97
46.63
Fig. 10 Energy histograms for the parallel and alternate monolayer
arrangements of tetramer 8a.
favours such a conformation. Otherwise, the propensity of cis-
cyclobutane b-oligomers to self-assemble and produce nanosized
fibres has been evidenced by TEM, AFM and SPFM. The
results given by these techniques and molecular modelling based
on theoretical calculations suggest a parallel alignment as the
preferential molecular arrangement in the aggregates and a
molecular conformation similar to that observed in solution. In the
assemblies, both hydrogen bonding and hydrophobic interactions
could be responsible for the aggregation. In addition, tetramers are
able to produce gels in some organic solvents. The nature of
the N-protecting group does not exert a strong influence on these
features. All these properties confer a great interest on these
compounds. Consequently, the synthesis and structural study
of new b-peptides including cyclobutane residues with diverse
stereochemistry, and also hybrid oligomers combining cyclic and
linear units is currently in progress in our laboratory. In light of
some promising results,15 the search for their possible biochemical
applications as well as their use in the design of new chiral materials
is under active investigation.
Fig. 11 Single MD parallel snapshot showing the arrangement of two
neighboring molecules with respect to the unrestrained central one in the
aggregates formed by 8a. Significant intra and intermolecular hydrogen
bonds are marked. The three molecules adopt an extended arrangement
like the predominant conformation of 8a in chloroform solution.
(geometrical optimizations) and Gaussian 9826 (determining
Merz–Kollman atomic charges). Simulated Annealing27 method-
ology (5000 steps, time-step 1.0 fs, SHAKE28 protocol and vari-
able thermal coupling29) was employed followed by geometrical
optimization (Steepest Descent30 algorithm for the first 10 steps
and then Conjugated Gradient31 algorithm until full convergence).
Detailed conditions are included in the SA.in file (for a deep
insight consult Fig. S2 and file SA in the ESI†). Molecular
dynamics calculations were also done with the AMBER 7 software
package according to a schema in 3 steps, explained in Figure S4
(see the ESI†): previous step involving geometrical optimization
and then; 300 ps. NVT heating step; 300 ps. NPT equilibration
step; and 5000 ps. NPT sampling step (sampling ratio: 1 frame
per ps. Time-step: 1.0 fs). The calculations for the parallel and
the alternate arrangements were done according to a scheme (see
the ESI, Fig. S5†): a first step involving geometrical optimization
followed by a 500 ps heating and equilibration step and, finally,
a 10000 ps. sampling step (sampling ratio: 1 frame per 10 ps.
Time-step 1.0 fs). Calculations were done in vacuum assuming
Experimental
Computational Details
The molecules 8a–10a were built taking profit of the modular
philosophy of AMBER 7,23 and the parm9924 Force Field was
employed in all cases. Several auxiliary software packages are
used to carry out side calculations such as Macromodel 9.025
572 | Org. Biomol. Chem., 2010, 8, 564–575
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The Royal Society of Chemistry 2010
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that any molecule, once the aggregate has formed, only feels in
its vicinity other molecules similar to it and not any trace of
solvent.
dried over MgSO4, filtered and evaporated under high vacuum,
obtaining the corresponding carboxylic acid (133.5 mg, 70%).
This compound was used directly in next step without further
purification.
On the other hand, tripeptide 7a (200 mg, 0.43 mmol) was
dissolved in EtOH (30 mL) and 20% Pd(OH)2/C was added
(70 mg). The mixture was stirring under hydrogen (P= 6 atm),
at room temperature for 12 h. After that, the catalyst was removed
6,7-Dichloro-3-oxabicyclo[3.2.0]heptane-2,4-dione 1117
A solution of maleic anhydride (2 g, 20.6 mmol) in acetonitrile
(250 mL) was cooled to -35 ◦C. (Z)-1,2-dichloroethylene (2.3 mL,
30.9 mmol) was added and the system was irradiated through
a quartz filter for 4 h. The solvent was removed in vacuo to
afford pure compound 11 as a white solid, as a mixture of
diastereomeric adducts (3.98 g, quantitative yield). The 1H NMR
spectrum of the mixture is in good agreement with that previously
described for these compounds.17 dH(250 MHz; CDCl3) 4.30–4.34
(complex absorption, 2H) and 5.47–5.51 (complex absorption,
2H) (one adduct). 3.85–3.91 (m, 1H), 4.40–4.49 (m, 1H) and 5.10–
5.19 (complex absorption, 2H) (one adduct). 4.07–4.09 (complex
absorption, 2H) and 5.39–5.40 (complex absorption, 2H) (one
adduct).
R
by filtering through Celiteꢀ and washed with MeOH. Then the
resulting mixture was concentrated under reduced pressure to
obtain quantitatively the free amine as a yellowish oil, which was
used in next step without further purification.
After that, the free carboxylic acid (120 mg, 0.27 mmol) and the
free amine (100 mg, 0.3 mmol) were dissolved in anhydrous DMF
(25 mL). Then, TEA (0.23 mL), EDAC (175 mg, 0.91 mmols) and
HOBt (60 mg, 0.44 mmols) were successively added. The mixture
was stirred for 20 d under a nitrogen atmosphere. After EtOAc
was added (25 mL) and organic layer was washed with aqueous
saturated solution of NaHCO3 (3 ¥ 20 mL), dried over MgSO4 and
concentrated under reduced pressure. The obtained crude product
was purified by flash column chromatography through Baker silica
gel using EtOAc as eluent, to afford the hexapeptide 9a (91 mg,
45%) as a white solid.
Dimethyl 3,4-dichlorocyclobutane-1,2-dicarboxylate 1217
A solution of cycloadduct mixture 11 (8.5 g, 43.6 mmol) and
concentrated H2SO4 (1.7 mL) in methanol (85 mL) were stirred at
50 ◦C for 5 h. Dichloromethane (150 mL) was added to the organic
phase and it was successively washed with water (2 ¥ 100 mL) and
brine (1 ¥ 100 mL). The organic layer was then dried over MgSO4,
filtered off and concentrated in order to provide the corresponding
crude as yellowish oil (9.2 g, 88% yield). dH(250 MHz; CDCl3)
3.20–3.28 (dd, J = 9.25 Hz, 1H), 3.69–3.71 (complex absorption,
2H), 3.74 (s, 6H), 3.75 (s, 3H), 3.76 (s, 6H), 3.77 (s, 3H), 3.78–3.81
(complex absorption, 2H), 3.89 (ddd, J = 9.2 Hz, J¢ = 10.1 Hz,
1H), 4.37–4.44 (dd, J = 8 Hz, J¢ = 9.1 Hz, 1H), 4.83–4.96 (complex
absorption, 5H).
◦
Hexapeptide 9a. Crystals, mp 173–175 C (EtOAc–pentane);
[a]D25 -153.0 (c 0.57, MeOH); nmax(ATR)/cm-1 3297, 2944, 1718,
1643; dH(500 MHz; CDCl3) 1.90–2.34 (complex absorption, 24H),
3.14 (complex absorption, 5H), 3.41 (m, 1H), 3.73 (s, 3H), 4.49 (m,
1H), 4.66–4.77 (complex absorption, 5H), 5.07 (dd, J = 10 Hz, J¢ =
25 Hz, 2H), 5.99 (d, J = 10 Hz, 1H), 6.52 (d, J = 10 Hz, 1H), 6.73–
6.83 (complex absorption, 4H), 7.35 (complex absorption, 5H);
dC(125 MHz; CDCl3) 19.0–19.4, 29.1–30.0, 43.9, 44.1–44.3, 44.4,
45.5, 46.0, 46.2, 51.8, 66.5, 127.9, 128.5, 136.6, 155.6, 172.2–172.6;
m/z (ESI): Found, 771.3688 [M + Na]+. Calcd. for C39H52N6O9Na:
771.3688.
Dimethyl cyclobutane-1,2-dicarboxylate 139
Octapeptide 10a. Following a similar protocol than that
described above for hexapeptide 9a, octapeptide 10a was prepared
from tetramer 8a. 70 mg, 35% yield. Crystals, mp 188–190 ◦C
(EtOAc–pentane); [a]2D5 -110.0 (c 0.74, CH2Cl2); nmax(ATR)/cm-1
3299, 2949, 1715, 1648; dH(500 MHz; CDCl3) 1.80–2.42 (complex
absorption, 32H), 3.11–3.45 (complex absorption, 8H), 3.70 (s,
3H), 4.47 (m, 1H), 4.64–4.79 (complex absorption, 7H), 5.10 (m,
2H), 5.98 (m, 1H), 6.53 (d, J = 7 Hz, 1H), 6.70–6.92 (complex
absorption, 6H), 7.34 (complex absorption, 5H); dC(125 MHz;
CDCl3) 18.9–19.6, 28.8–29.2, 43.9–46.3, 51.7, 66.5, 127.9, 128.4,
136.6, 155.5, 172.0–173.0, 174.6; m/z (MALDI-TOF): Found,
965.561 [M + Na]+. Calcd. for C49H66N8O11Na: 965.475.
TEA (8.1 mL, 9.7 mmol) was added to a solution of 12
(6.4 g, 26.5 mmol) in ethanol (15 mL). The mixture was
hydrogenated over 5% Pd/C (2.6 g) at room temperature and
at 6 atm for 58 h. The catalyst was removed by filtration
R
through Celiteꢀ and washed successively with methanol and
dichloromethane. The filtrate was evaporated in vacuo. The residue
was the residue poured into EtOAc (100 mL) and the resultant
solution was washed twice with saturated aqueous NaHCO3
(100 mL). The organic layer was dried over MgSO4, filtered
off and solvent removed to provide 13 (3.9 g, 85% yield).
dH(250 MHz; CDCl3) 2.19 (m, 2H), 2.39 (m, 2H), 3.40 (m, 2H),
3.68 (s, 3H); dC (62.5 MHz; CDCl3) 22.2 (2C), 40.7, 51.7 (2C),
173.9 (2C).
Method B: peptide coupling with FDPP
Procedures for the synthesis of peptides. Method A: peptide
coupling with EDAC
The synthesis of tetrapeptide 8b is described. On one hand,
dipeptide 2b (100 mg, 0,31 mmol) was solved in 1 : 10 THF–water
(22 mL) and 0.25 M NaOH (3 mL) was added. The mixture was
stirred at 0 ◦C for 2 h. The mixture was washed with CH2Cl2
(2 ¥ 15 mL) before being acidified to pH 2 with 2M HCl. The
aqueous layer was extracted with EtOAc (4 ¥ 40 mL) and the
organic layer was dried over MgSO4, filtered and evaporated under
high vacuum obtaining the corresponding carboxylic acid (90 mg,
As an example, the synthesis of hexapeptide 9a is described.
Tripeptide 7a (200 mg, 0.43 mmol) was dissolved in 1 : 2 THF–
water (16 mL) and 0.25 M NaOH (4.5 mL) was added. The mixture
was stirred at 0 ◦C for 4.5 h, then washed with CH2Cl2 (3 ¥ 15 mL)
before being acidified to pH 2 with 2M HCl. The aqueous layer
was extracted with EtOAc (4 ¥ 40 mL) and the organic layer was
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©

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94%). This compound was used directly in next step without
further purification.
Acknowledgements
Authors thank financial support from Spanish Ministerio de Cien-
cia e Innovacio´n (grants CTQ2007-61704/BQU, CTQ2006-08256)
and Generalitat de Catalunya (grants 2009SGR-733 and 109, and
XRQTC). They are also grateful to European Union for COST
Action CM0803. Time allocated in the Servei de Ressona`ncia
Magne`tica Nuclear and Servei de Microsco`pia Electro´nica (UAB),
and in CESCA (Centre de Supercomputacio´ de Catalunya) is
gratefully acknowledged.
On the other hand, dipeptide 2b (140 mg, 0.43 mmols) was
solved in dry CH2Cl2 (10 mL) and Et3SiH (0,17 ml, 1,10 mmol) and
TFA (0,43 ml, 5,60 mmol) were added. The mixture was stirred at
room temperature for 2 h. The reaction mixture was concentrated
under reduced pressure obtaining the free amine as a yellowish
oil (90 mg, 95%), which was used in next step without further
purification.
After that, a solution containing the free carboxylic acid (0.09 g,
0.29 mmol), the free amine (0.09 g, 0.39 mmol), DIPEA (0.1 ml,
0.48 mmol), and FDPP (0.08 g, 0.19 mmol) in anhydrous DMF (10
ml) was stirred at room temperature overnight. Then, ethyl acetate
(40 mL) was added and the combined organic layers were washed
with saturated aqueous NaHCO3 (3 ¥ 30 mL). The organic layer
was dried over MgSO4 and solvents were removed under reduced
pressure. The residue was purified by column chromatography
using dichloromethane–methanol (29 : 1) as eluent to afford 8b
(90 mg, 60%) as a white solid.
Notes and references
1 For a definition of secondary and tertiary structures in peptides, see:
S. H. Gellman, Acc. Chem. Res., 1998, 31, 173. For recent reports on
helix bundle quaternary structures of b- and a/b peptides, respectively,
see:; D. S. Daniels, E. J. Petersson, J. X. Qiu and A. Schepartz, J. Am.
Chem. Soc., 2007, 129, 1532; W. S. Horne, J. L. Price, J. L. Keck and
S. H. Gellman, J. Am. Chem. Soc., 2007, 129, 4178; S. H. Gellman,
Abstracts of Papers, 237th ACS National Meeting, Salt Lake City, UT,
United States, 2009.
2 See, for example: M. A. Gellman, and S. H. Gellman, in Enantioselective
Synthesis of b-Amino Acids, Second Edition, ed. E. Juaristi and
V. A. Soloshonok, John Wiley and Sons, New Jersey, 2005, pp. 527-
585; D. Seebach, D. F. Hook and A. Glattli, Biopolymers, 2006, 84, 23;
P. Le Grel and G. Guichard, in Foldamers: Structure, Properties and
Applications, ed. S. Hecht and I. Huc, Wiley-VCH, Weinheim, 2007,
pp. 35–74; D. Seebach and J. Gardiner, Acc. Chem. Res., 2008, 41, 1366;
W. S. Horne and S. H. Gellman, Acc. Chem. Res., 2008, 41, 1399; I. M.
Ma´ndity, E. We´ber, T. Martinek, G. Olajos, G. K. To´th, E. Vass and F.
Fu¨lo¨p, Angew. Chem., Int. Ed., 2009, 48, 2171.
3 For some representative references, see: D. H. Appella, L. A. Chris-
tianson, I. L. Karle, D. R. Powell and S. H. Gellman, J. Am. Chem.
Soc., 1996, 118, 13071; D. H. Appella, L. A. Christianson, I. L. Karle,
D. R. Powell and S. H. Gellman, J. Am. Chem. Soc., 1999, 121, 6206;
D. H. Appella, L. A. Christianson, D. A. Klein, M. R. Richards, D. R.
Powell and S. H. Gellman, J. Am. Chem. Soc., 1999, 121, 7574; K.
Tetrapeptide 8b. Crystals, mp 225–227 ◦C; [a]D -263,9 (c 0.95,
MeOH); nmax(ATR)/cm-1 3349, 3315, 2944, 1727, 1683, 1648,
1515; dH(600 MHz; CDCl3) 1.41 (s, 9H), 1.84–2.07 (complex
absorption, 8H), 2.15–2.25 (complex absorption, 4H), 2.27–2.34
(complex absorption, 4H), 3.12 (m, 1H), 3.16 (m, 2H), 3.40 (m,
1H), 3.70 (s, 3H), 4.38 (m, 1H), 4.65–4.77 (complex absorption,
3H), 5.59 (d, J = 9.4 Hz, 1H), 6.63 (d, J = 8.9 Hz, 1H), 6.71 (d, J =
8.7 Hz, 1H), 6.75 (d, J = 8.7 Hz, 1H); dC(150 MHz; CDCl3) 18.6,
19.2 (3C), 28.4 (3C), 29.1, 29.2, 29.3, 29.7, 44.0, 44.5 (3C), 45.7
(2C), 46.1, 46.3, 51.8, 79.2, 155.1, 172.3, 172.6, 172.7, 174.6; m/z
(ESI): Found, 543.2793 [M + Na]+. Calcd. for C26H40N4O7Na:
543.2789. Anal. Found: C, 59.62; H, 7.76; N, 10.36. Calcd. for
C26H40N4O7: C, 59.98; H, 7.74; N, 10.76.
M
o¨hle, R. Gu¨nther, M. Thormann, N. Sewald and H.-J. Hofmann,
Biopolymers, 1999, 50, 167; J. J. Barchi, Jr., X. Huang, D. H. Appella,
L. A. Christianson, S. L. Durrell and S. H. Gellman, J. Am. Chem.
Soc., 2000, 122, 2711; T. A. Martinek, G. To´th, E. Vass, M. Hollo´si
and F. Fu¨lo¨p, Angew. Chem., Int. Ed., 2002, 41, 1718; R. J. Doerksen,
B. Chen, J. Yuan, J. D. Winkler and M. L. Klein, Chem. Commun.,
2003, 2534; M. A. Schmitt, S. H. Choi, I. A. Guzei and S. H. Gellman,
J. Am. Chem. Soc., 2006, 128, 4538; F. Fu¨lo¨p, T. A. Martinek and G. K.
To´th, Chem. Soc. Rev., 2006, 35, 323.
Method C: reaction between a carboxylic acid and an azide12
The synthesis of tripeptide 7a is described. To a solution of acid
17 (300 mg, 0.86 mmol) in dried toluene (10 mL), were added
TEA (0.15 mL) and azide 18 (140 mg, 0.76 mmols) dissolved in
15 mL of dried toluene. The reaction mixture was stirred for 6 h
at 100 ◦C. After that, the reaction was quenched with EtOAc and
the organic layer was washed with an aqueous saturated solution
of NaHCO3 (3 ¥ 15 mL). The combined organic layers were dried
over MgSO4, filtered and evaporated. The product was purified
by flash column chromatography through Baker silica gel (1 : 1
hexane–EtOAc as eluent) to afford the tripeptide 7a (180 mg,
52%) as a white solid.
4 See, for instance: T. D. W. Claridge, J. M. Goodman, A. Moreno, D.
Angus, S. F. Barker, C. Taillefumier, M. P. Watterson and G. W. Fleet,
Tetrahedron Lett., 2001, 42, 4251.
5 A. Hete´nyi, I. M. Ma´ndity, T. A. Martinek, G. K. To´th and F. Fu¨lo¨p,
J. Am. Chem. Soc., 2005, 127, 547; T. A. Martinek, I. M. Ma´ndity, L.
Fu¨lo¨p, G. K. To´th, E. Vass, M. Hollo´si, E. Forro´ and F. Fu¨lo¨p, J. Am.
Chem. Soc., 2006, 128, 13539; T. A. Martinek, A. Hete´nyi, L. Fu¨lo¨p,
I. M. Ma´ndity, G. K. To´th, I. De´ka´ny and F. Fu¨lo¨p, Angew. Chem.,
Int. Ed., 2006, 45, 2396.
6 A. M. Bru¨ckner, P. Chakraborty, S. H. Gellman and U. Diederichsen,
Angew. Chem., Int. Ed., 2003, 42, 4395.
7 T. Hirata, F. Fujimura and S. Kimura, Chem. Commun., 2007,
1023.
8 For reviews on conformationally constrained amino acids and peptides
including cyclobutane derivatives, see: R. M. Ortun˜o, A. G. Moglioni
and G. Y. Moltrasio, Curr. Org. Chem., 2005, 9, 237; R. M. Ortun˜o,
in Enantioselective Synthesis of b-Amino Acids, Second Edition, ed.
E. Juaristi and V. A. Soloshonok, John Wiley and Sons, New Jersey,
2005, pp. 117–137.
9 M. Mart´ın-Vila`, E. Muray, G. P. Aguado, A. Alvarez-Larena, V.
Branchadell, C. Minguillo´n, E. Giralt and R. M. Ortun˜o, Tetrahedron:
Asymmetry, 2000, 11, 3569.
Tripeptide 7a. Crystals, mp 190–192 ◦C (EtOAc–pentane);
[a]2D5 -145.7 (c 1.64, CH2Cl2); nmax(ATR)/cm-1 3302, 2948, 1700,
1651, 1541; dH(500 MHz; CDCl3) 1.96 (m, 6H), 2.12–2.38 (m, 6H),
3.15 (m, 2H), 3.39 (m, 1H), 3.69 (s, 3H), 4.48 (m, 1H), 4.63–4.79
(m, 2H), 5.08 (m, 2H), 5.91 (d, J = 7.2 Hz, 1H), 6.52 (d, J =
8.7 Hz, 1H), 6.65 (d, J = 7.2 Hz, 1H), 7.34 (m, 5H); dC(125 MHz;
CDCl3) 19.8, 20.1, 20.2, 30.1, 30.2, 30.8, 44.8, 45.3, 45.4, 46.6, 47.0,
47.2, 52.7, 66.5, 128.9, 129.4 (2C), 137.5, 156.3, 173.2, 172.3 175.6.
Anal. Found: C, 63.13; H, 6.75; N, 9.19. Calcd. for C24H31N3O6:
C, 63.00; H, 6.83; N, 9.18.
´
´
10 S. Izquierdo, M. J. Kogan, T. Parella, A. G. Moglioni, V. Branchadell,
E. Giralt and R. M. Ortun˜o, J. Org. Chem., 2004, 69, 5093.
´
´
11 E. Torres, C. Acosta-Silva, F. Ru´a, A. Alvarez-larena, T. Parella, V.
Branchadell and R. M. Ortun˜o, Tetrahedron, 2009, 65, 5669.
574 | Org. Biomol. Chem., 2010, 8, 564–575
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