New cylindrical peptide assemblies defined by extended parallel β-sheets

Article abstract of DOI:10.1039/c2ob26637g

A new approach to non-covalent peptide-based nanotubular or rod-like structures is presented, whereby the monomeric units are preorganised into a β-strand geometry that templates the formation of an extended and unusual parallel β-sheet rod-like structure. The conformational constraint is introduced by Huisgen cycloaddition to give a triazole-based macrocycle, with the resulting self-assembled structures stabilized by a well-defined series of intermolecular hydrogen bonds. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2ob26637g

Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Volume 11 | Number 3 | 21 January 2013 | Pages 385–524
ISSN 1477-0520
PAPER
Andrew D. Abell et al.
New cylindrical peptide assemblies defined by extended parallel β-sheets
1477-0520(2013)11:3;1-G

Organic &
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New cylindrical peptide assemblies defined by
extended parallel β-sheets†
Cite this: Org. Biomol. Chem., 2013, 11,
425
Ashok D. Pehere, Christopher J. Sumby and Andrew D. Abell*
A new approach to non-covalent peptide-based nanotubular or rod-like structures is presented, whereby
the monomeric units are preorganised into a β-strand geometry that templates the formation of an
extended and unusual parallel β-sheet rod-like structure. The conformational constraint is introduced by
Huisgen cycloaddition to give a triazole-based macrocycle, with the resulting self-assembled structures
stabilized by a well-defined series of intermolecular hydrogen bonds.
Received 17th August 2012,
Accepted 25th September 2012
DOI: 10.1039/c2ob26637g
www.rsc.org/obc
Introduction
There is considerable interest in the design, synthesis, and
exploitation of small peptide-like motifs that can self-assemble
into higher levels of secondary structure, which mimic that
found in peptides and proteins.1–4 These extended structures
and the associated processes leading to their formation, are
central to understanding important diseases such as
Alzheimer’s5–7 and also as a basis of biological probes to study
protein structure and function. Peptide nanotubes and rods,
resulting from the self-assembly of such motifs into extended
β-sheets, are of particular fundamental interest in this context
as unique molecular architectures and more practically as anti-
microbial and chemotherapeutic agents.8,9 Tubular structures
are also found in Nature, e.g. the transmembrane channel
proteins.10,11
Synthetic peptide nanotubes are typically constructed of
cyclic peptides that stack by formation of a network of hydro-
gen bonds. The component cyclic scaffolds invariably consist
of alternating sequences of an even number of D- and
L-α-amino acids,9 β-amino acids,12,13 alternating α,γ-amino
acids,14,15 ∂-amino acids,16 N-methylated amino acids,17 non-
peptide scaffolds18–20 other such systems.21,22 With a few
exceptions,12,23 these structures associate in an antiparallel
fashion to give an extended cylindrical β-sheet that is stabi-
lized by hydrogen bonding between backbone N–H and CvO
groups that alternate up and down relative to the plane of the
ring.
Fig. 1 Previously reported37 macrocyclic peptidomimetic inhibitors (A) of
calpain and new macrocycles (1–3), examples of which self-assemble into
β-sheet stabilised nanotubular structures in solution and the solid-state.
constrained into a β-strand geometry, see Fig. 1. This species
then nucleates the formation of an extended and unusual
parallel β-sheet, with the component constraint directly
involved in the self assembly. There is a strong thermodynamic
preference for formation of an antiparallel β-sheet, over that of
the analogous parallel geometry, in peptide-based nanotubes
and also more generally in peptides and proteins.24–29 Parallel
β-sheets typically form from extended sequences that can be
covalently linked by a tether to induce a turn and a suitable
orientation to allow sheet formation.26,30–35 By contrast, anti-
parallel β-sheets form with relatively short peptides of 12–25
residues.
Here we present a new template-based approach to peptide-
based nanotubes using a ‘smart’ scaffold designed to be
The required β-strand conformational constraint was
specifically introduced by linking the P1 and P3 side chains of
a tripeptide with a 1,2,3-triazole-containing macrocycle such
that the N- and C-termini are free for subsequent functionali-
sation. There are a few reports of cyclic peptides containing a
School of Chemistry and Physics, The University of Adelaide, North Terrace, Adelaide
SA 5005, Australia. E-mail: andrew.abell@adelaide.edu.au
†Electronic supplementary information (ESI) available: Experimental details and
spectral data of new compounds. CCDC 868566. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c2ob26637g
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triazole that self-assemble, but in these the heterocycle does
not participate in intermolecular interactions, the N- and
C-termini are incorporated into the ring, and the cycle does
not define a specific conformation.36 Our new parallel β-sheet-
based structure is stabilized by a well-defined series of hydro-
gen bonds involving the backbone amide and carbonyl groups
and also the triazole ring within the macrocycle. We report the
syntheses of these constrained β-strand macrocycles and also
characterization of the resulting nanotubular structures
derived from them by IR and NMR spectroscopy, X-ray crystal-
lography, and ESI-MS. Derivatives 1a and 2a are also shown to
form extended rod-like structures in the solid-state, consistent
with bundles of nanotubes, when imaged by Scanning Elec-
tron Microscopy (SEM).
Results and discussion
We have previously reported triazole containing macrocycles as
potent inhibitors of calpain, see A in Fig. 1.37 While the com-
ponent cycle constrains the peptide backbone into a β-strand
conformation that is known to favour active site binding to the
protease,38,39 these structures do not appear to self assemble.
A second and new series reported here lacks the aryl group in
the macrocycle (see 1, 2 and 3) and is hence sterically less con-
gested and also less hydrophobic. The new structures remain
constrained into a β-strand geometry as revealed by IR, NMR
and X-ray analysis, as discussed below. The critical observation
is that these new macrocycles now self assemble into an
extended and unusual parallel β-sheet based nanotube
through a well defined network of hydrogen bonds.
A representative synthesis of the macrocycles (1a, 1b, and
1c) is shown in Scheme 1, with complete details on all com-
pounds provided in ESI.†37 In particular, the tripeptide 8 pre-
pared from L-propargylglycine40 by standard peptide coupling
sequence as shown in Scheme 1, was cyclized in the presence
of CuBr in CH2Cl2 to give the key macrocyclic ester 1a. This
was reduced with lithium borohydride to give the macrocyclic
alcohol 1b, which was purified by reverse phase HPLC. Oxi-
dation of this alcohol, with Dess–Martin periodinane (DMP),
then gave the macrocyclic aldehyde 1c (Scheme 1).
Scheme 1 Synthesis of the β-strand template.
The component triazole-H hydrogen bonds to the N2 and
N3 of an adjacent triazole ring to define the unusual parallel
β-sheet. Fig. 2b–d depict the resulting open ended tubular
structure that has an average diameter of ∼6.0 Å (0.6 nm). The
NH⋯OvC hydrogen bonds have bond lengths and angles that
are typical for a self-assembled parallel β-sheet (DNH⋯O
=
2.01–2.11 Å; dNO = 2.86–2.98 Å; angleNO = 163.1–171.1°), while
the bifurcated hydrogen bonds of the triazole that support this
nanotubular structure are slightly longer (DCH⋯N = 2.57,
2.65 Å; dCN = 3.21, 3.27 Å; angleCN = 125.3, 123.7°). The interior
of the extended peptide nanotube is hydrophilic with the
inclusion of the backbone amides and N2–N3 of the triazoles.
By comparison, the exterior of the structure presents the
exposed leucine side chains and the Cbz groups and is hence
more hydrophobic in character. The hole through the nano-
tube is small, with an average internal diameter of ∼3 Å
(taking into account the van der Waals radii), and as such is
free of solvent molecules.
X-ray crystal structure analysis
The formation of a β-sheet derived nanotube was identified by
X-ray crystallography. The 15-membered macrocycle 1a crystal-
lized from a chloroform–methanol solution in the orthorhom-
bic space group P212121 with four complete molecules in the
unit cell.41 The X-ray crystallographic structure of 1a (see
Fig. 2) revealed that the component macrocycle adopts a
β-strand conformation (Φ = −125.2° and Ψ = 122.6°) in the
solid-state, which remarkably stacks in a parallel fashion to
form an extended nanotubular structure as shown in Fig. 2b–f.
The tripeptide sequence in the macrocycle presents a planar
The crystal structure also reveals that the individual peptide
cylindrical columns are further packed into dimeric bundles
defined by edge-to-face CH⋯π contacts involving the Cbz
groups as shown in Fig. 2e and f.42 The C–H⋯πcentroid distance
is 2.99 Å, while the closest C–H⋯Caryl contact is 2.69 Å. The
crystal packing is completed by association of the bundles into
a herringbone-type arrangement in the bc plane.
NMR analysis
β-strand backbone, with the carbonyl and NH groups alternat- The 1H NMR spectral data for 1a–c, 2a–c and 3a–c in DMSO-d6
ing up and down to allow intermolecular hydrogen bonding, are consistent with all these macrocycles adopting a β-strand
3
see Fig. 2a.
conformation in solution. In particular, the JNHCαH coupling
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Fig. 2 (a) Depiction of the β-strand backbone of 1a with the triazole containing macrocyclic linkage omitted for clarity. (b) A view of the intermolecular hydrogen-
bonding observed between two macrocycles of 1a with the hydrogen atoms of the side chains omitted for clarity. Peptide backbone hydrogen bonds are shown as
yellow dashed bonds and the triazole hydrogen bonds as dashed red lines. (c) A representation of the structure of 1a showing the nanotubular structure. (d) The
structure of 1a showing extended β-sheet conformation, with the triazole containing macrocyclic linkage omitted for clarity. (e) Side view of the dimerisation of the
nanotubular structures in the crystal. (f ) Top view of the dimerised nanotubes in a space-filling representation.
constants were observed to be in the range 8.4 to 9.3 Hz. This
corresponds to literature values of 8.0 to 10.0 Hz for an Φ
torsion angle of ≈−120° and hence the all-trans backbone con-
formation of a β-strand geometry (see Table 1).43,44 ROESY
spectra of 1a, 2a, 3a revealed characteristic cross peaks
between CαHi and (i + 1NH), βHi and (i + 1NH), NHi and (i + 1c
1NH). This is also consistent with a flat β-strand conformation
Table 1 3JNHCαH coupling constantsa
Compound
P1
P2
1a
1b
9.1
ND
8.7
9.0
9.3
8.7
8.4
ND
8.4
8.4
8.4
8.4
8.9
9.1
8.9
8.7
8.6
8.8
2a
2b
2c
for the peptidic backbone that allows intermolecular hydrogen
bonding between the two parallel aligned macrocycles (see 3a
ESI†).45–47
3b
3c
Further evidence for intermolecular hydrogen bonding48,49
a Coupling constants determined in DMSO-d6. ND not determined
because of overlapping resonances.
and hence β-sheet formation arises from the observation that
1
the amide hydrogen atoms in the H NMR spectrum of 1a (in
20% DMSO/CD3OD) did not exchange over one week (see
ESI†).
Table 2 Characteristic solid-state FT-IR bands (cm−1
)
IR analysis
Solid-state FT-IR spectra of 1a–c, 2a–b and 3a–c revealed bands
at 1635–1641, 1532–1537, and 3276–3302 cm−1 that corres-
pond to the amide I, amide II and amide A stretches (see
Table 2). These signals are once again characteristic of a
Compound
Amide I
Amide II
Amide A
1a
1b
1c
1641
1637
1636
1638
1634
1635
1634
1634
1532
1535
1535
1536
1536
1534
1534
1529
3292
3281
3280
3277
3279
3302
3287
3282
β-sheet geometry for the macrocyclic nanotubes.50,51 These 2a
2b
compare favourably with literature values for a β-sheet struc-
3a
3b
ture with frequencies in the range 1612–1640 cm−1 for the
amide I stretch.51 More importantly, higher frequency bands 3c
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links the P1 and P3 side chains of natural amino acids. NMR
and FT-IR spectroscopy, mass spectrometry and X-ray diffrac-
tion data reveal that these macrocycles are intermolecularly
hydrogen bonded to form parallel β-sheets and an extended
nanotubular structure. The interior of these first-generation
nanotubes is too small for host guest chemistry, although the
synthetic approach and ideas employed provide scope for the
formation of larger component macrocycles. Importantly, both
termini of the peptidomimetic are exposed and available for
further functionalisation, which is not the case for most of the
existing peptide-based nanotubular structures. As a first
example, we prepared the C-terminal aldehyde derivatives (1c,
2c and 3c) of the core template as shown in Scheme 1. These
structures are potent inhibitors of calpain-II (IC50 = 355 nM,
582 nM, and 697 nM respectively), a result consistent with the
macrocyclic constraint of these structures defining a β-strand
geometry as is required for protease binding. Structures such
as these may be of use in drug delivery and formulation appli-
cations. The work presented here defines a new approach to
self assembled peptide-based nanotubular structures, whereby
the monomeric units are themselves constrained into a
β-strand geometry that templates the formation of an extended
β-sheet geometry.
Fig. 3 ESI spectrum of compound 2a showing the formation of oligomeric
assemblies in the gas phase [M + Na].
Fig. 4 (a) An SEM image of the rod-like assemblies of 1a. (b) A view of the
crystal of 1a used for the structure determination and the Miller indices of the
faces.
Acknowledgements
in the range 1530–1550 for the amide II stretch are consistent
with a parallel β-sheet.51 The NH stretching frequencies in the
range 3281–3302 cm−1 are good evidence of the strongly hydro-
gen bonded and ordered nanotubular structure for all com-
pounds.50,51 As representative examples, the macrocycles 1a,
2a and 3a gave the same characteristic IR bands at 3281,
1637–1638, 1539–1545 cm−1 in solution (see ESI†).
We acknowledge Mr Antonio Calabrese and Ms Jade Cottam
for assistance with the mass spectrometry, Ms Courtney Hollis
for collecting X-ray crystallographic data for 1a, and financial
support from Australian Research Council.
Remarkably, an ESI mass spectrum of 2a (1 mM solution in
9 : 1 MeOH : DMSO) gave rise to characteristic peaks consistent
with the monomeric, dimeric, trimeric, tetrameric, penta-
meric, and hexameric species, see Fig. 3. Compounds 1a and
3a also gave peaks for the corresponding oligomeric species
under the same conditions (see ESI S24†). The observation of
higher aggregates by mass spectrometry supports the for-
mation of an extended nanotubular structure that is defined
by the intermolecular hydrogen bonding of the β-sheet struc-
ture in solution and in the gas phase.
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