Solid-phase synthesis of homodimeric peptides: Preparation of covalently-linked dimers of amyloid β peptide

Article abstract of DOI:10.1039/b912784d

Covalently cross-linked homodimeric Aβ peptides have been prepared by solid-phase peptide synthesis by exploiting 'site-site interactions', and exhibit substantially increased oligomerisation and fibrillisation properties compared with the corresponding m

Full text of DOI:10.1039/b912784d

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Solid-phase synthesis of homodimeric peptides: preparation
of covalently-linked dimers of amyloid b peptidew
W. Mei Kok,abc Denis B. Scanlon,ac John A. Karas,c Luke A. Miles,cd Deborah J. Tew,bc
Michael W. Parker,cd Kevin J. Barnhambc and Craig A. Hutton*ac
Received (in Cambridge, UK) 29th June 2009, Accepted 10th August 2009
First published as an Advance Article on the web 1st September 2009
DOI: 10.1039/b912784d
Covalently cross-linked homodimeric Ab peptides have been
prepared by solid-phase peptide synthesis by exploiting ‘site–site
interactions’, and exhibit substantially increased oligomerisation
and fibrillisation properties compared with the corresponding
monomers.
dimers greater than ten residues in length, and more
importantly, attachment of a diamino diacid residue at a
position further than five residues from the C-terminus has
not been reported. Accordingly, we were keen to investigate
the preparation of dimers such as 3 (Fig. 1) in which the
position of the diamino diacid residue is distant from the
C-terminus.
Alzheimer’s disease is characterised by the formation of
amyloid deposits composed primarily of amyloid b peptide
(Ab).1 While the precise mechanism of progression of AD has
yet to be fully elucidated, it is generally accepted that Ab is
toxic and substantial evidence suggests that soluble oligomers
of Ab are the causal, neurotoxic species.2 Selkoe and
co-workers have recently shown that a Ser26Cys disulfide-
linked Ab dimer 2 shows considerably greater neurotoxicity
than the corresponding monomer (Fig. 1).3 However, this
peptide is cleavable under physiological conditions and the
cross-linking site is distant from that in the putative dityrosine
cross-linked dimer 1.4
The preparation of Ab dimers represents further challenges:
first, in that solid-phase synthesis of Ab peptides is compli-
cated by the hydrophobic nature of the C-terminal residues,
which results in folding and aggregation of the precursor
peptide on the resin and resultant low yields and purities of
the peptide monomers;10 and second, in that such peptides are
composed of B80 amino acid residues, and are therefore
approaching the limits of SPPS methodology.11
We chose diaminopimelic acid (DAP) as the cross-linking
diamino diacid to investigate the preparation of Ab peptide
dimers linked at position 10, the location of the putative
dityrosine cross-link. We initially established proof of concept
through preparation of the DAP-linked Ab(9–16) dimer
fragment 8a (Scheme 1). Preparation of the Ab(11–16) peptide
on Fmoc–L-Lys(Boc)–chlorotrityl resin 5a was followed
by coupling of Fmoc-protected DAP (Fmoc2DAP) 4 and
subsequent capping of unreacted Ab(11–16) peptide 5a with
acetic anhydride. Cleavage of the Fmoc groups with piperidine
was followed by coupling of Fmoc–Gly and cleavage from the
resin to give dimeric peptide 8a, along with the monocoupled
adduct 10a and capped Ab(11–16) peptide 9a. Conditions were
optimised with respect to the amount of Fmoc2DAP 4
employed; use of stoichiometric amounts of Fmoc2DAP 4
(0.5 equivalents with respect to loading of peptide) resulted in
capped Ab(11–16) peptide 9a as the major component,
albeit with a substantial amount of the dimer 8a. Use of
Z 2 equivalents of Fmoc2DAP 4 gave the monocoupled
adduct 10a as the major component. Use of 0.75–1.0 equiv.
4 gave the desired dimer 8a as the major product, which was
easily purified by RP HPLC (see ESIw).
Despite intense research efforts directed toward the enzyme-
and metal-catalysed oxidative dimerisation of Ab, attempts to
prepare full-length covalently-linked Ab dimers have been
unsuccessful,5,6 with oligomerisation and aggregation being
the major outcomes. Accordingly, we sought to investigate a
direct method for solid-phase peptide synthesis of Ab peptide
dimers. Solid-phase synthesis of covalently-linked peptide
dimers has been investigated previously by exploiting ‘site–site
interactions’, through the coupling of diacids to the N-terminal
amino group or lysine e-amino group of contiguous peptide
chains on the resin,7 or through cross-metathesis of olefin-
appended peptides.8 The coupling of a protected diamino
diacid to the N-terminus of two contiguous peptide chains
on a resin can enable the synthesis of covalently-linked peptide
dimers, but has received scant attention. Dimers of small
peptides (5–10 residues) have been prepared through introduction
of various diamino diacids using solid-phase synthesis.9
However, there are no reports of the preparation of peptide
N-Terminal extension of the dimeric Ab(9–16) peptide 7a
prior to cleavage gave the DAP-linked Ab(1–16) peptide dimer
a School of Chemistry, The University of Melbourne, Parkville,
VIC 3010, Australia. E-mail: chutton@unimelb.edu.au;
Fax: +61 3 9347 8124; Tel: +61 3 8344 2393
b Department of Pathology, The University of Melbourne, Parkville,
VIC 3010, Australia
c Bio21 Molecular Science and Biotechnology Institute,
The University of Melbourne, Parkville, VIC 3010, Australia
d Biota Structural Biology Laboratory, St. Vincent’s Institute of
Medical Research, 9 Princes Street, Fitzroy, VIC 3065, Australia
w Electronic supplementary information (ESI) available: Experimental
procedures for preparation of 4 and 8a–d, NMR spectra of 4 and 8b
and HPLC and MS data for 8a–d. See DOI: 10.1039/b912784d
Fig. 1 Covalently cross-linked dimers of Ab(1–40).
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Scheme 1 Solid-phase synthesis of homodimeric peptides.
Fig. 2 DLS data for Ab(1–28) peptides (repeat measurements at
0.5 mg mLÀ1 in 20 mM HEPES, pH 7.0): (a) Ab(1–28) monomer,
(b) Ab(1–28) dimer 8c, (c) Ab(1–28) dimer 8c at 12 mg mLÀ1
.
8b in good yield and purity. Preparation of longer peptide
dimers was then investigated, with Ab(11–28) resin 5c prepared
in the same manner as for the Ab(11–16) resin 5a. Coupling
of Fmoc2DAP 4, peptide extension and cleavage gave the
Ab(1–28) dimer 8c, which was purified by RP HPLC followed
by size-exclusion chromatography (see ESIw). As formation
of the peptide dimers 8a–c at standard resin loadings
(0.4–0.6 mmol gÀ1) was highly efficient, higher loadings were
not investigated.
1H NMR experiments including 2D homonuclear TOCSY
and NOESY spectra were conducted on all peptides. The
fingerprint region of the TOCSY spectrum of 8b (see ESIw)
displayed two sets of peaks for a number of amino acid
residues, indicating that the peptide exists in two distinct
conformations, presumably as a result of restricted rotation
about the DAP cross-link. The lack of nOe correlations in the
NOESY spectrum and the small chemical shift dispersion of
the amide and Ca-protons suggest that there is little secondary
structure distant from the cross-link, consistent with the CD
results. NMR spectra of 8c and 8d exhibited very broad peaks,
attributed to significant aggregation of these peptides
(vide infra).
proceed in poor yield and purity, a known problem with
longer Ab peptides.10 Accordingly, Ab(11–40) 5d was
prepared on Fmoc–Val–PEG–PS resin, which proceeded in
excellent yield and purity. Coupling of 0.5 equivalents of
Fmoc2DAP 4 was found to give optimum yields of the peptide
dimer 6d on PEG-resin; in contrast use of 1.0 equiv. of 4
generated the monocoupled adduct as the major product.
Following peptide extension and cleavage, the Ab(1–40) dimer
8d was purified by size-exclusion chromatography and RP
HPLC (see ESIw).12 The structure of the dimer 8d was
confirmed by HRMS (m/z 8487.6, calc. 8487.4, see ESIw).
CD spectroscopy was performed to determine whether the
dimerisation of Ab peptides significantly alters their
conformational properties. There were no observed changes
in the CD spectra of both the Ab(1–16) and Ab(1–28) dimers
8b and 8c when compared with their respective monomers. All
four peptides exist in a random coil conformation that remains
unchanged after aging for 6 days (data not shown). In
contrast, Ab(1–40) dimer 8d displays significantly different
properties to the Ab(1–40) monomer. Freshly-prepared
Ab(1–40) dimer 8d displays an immediate b-sheet structure,
whereas the monomer exists initially in a random coil
conformation (Fig. 3) that slowly converts to b-sheet. Aging
of the Ab(1–40) dimer 8d did not result in a change to the
b-sheet structure, but a decrease in intensity was observed,
consistent with precipitation of aggregated peptide.
Dynamic light scattering (DLS) experiments were conducted
to investigate the aggregation properties of the peptides.
Ab(1–16) and its DAP-linked dimer, 8b, displayed highly
reproducible size distribution profiles consistent with predomi-
nantly monomeric states (see ESIw). Similarly, Ab(1–28) and
the corresponding Ab(1–28) dimer, 8c, gave highly reproducible
profiles consistent with predominantly monomeric species
under the same conditions (Fig. 2). Intriguingly, a highly
concentrated sample (ca. 12 mg mLÀ1) of the Ab(1–28) dimer
8c was shown to exist in monodisperse form with a much
larger hydrodynamic radius of around 10 nM, suggesting that
the peptide dimer exists in a stable oligomeric state under these
conditions (Fig. 2c).
With Ab(1–28) dimer 8c exhibiting distinct properties
compared with Ab(1–28) monomer, extension of the SPPS
method to the full-length Ab(1–40) dimer was undertaken.
Preparation of Ab(11–40) 5d on chlorotrityl resin was found to
Fig. 3 CD of freshly-prepared Ab(1–40) monomer (blue, lmin 198 nm)
and Ab(1–40) dimer 8d (red, lmin 217 nm).
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In conclusion, we have developed synthetic methods that
enable the preparation of side-chain linked homodimeric
peptides, and have applied the method to the synthesis of an
Ab dimer containing 80 residues. This method should be
widely applicable to the synthesis of a range of side-chain
cross-linked peptide dimers. We have developed a valuable
tool for the study of discrete Ab dimers and have shown that
they exhibit increased oligomerisation and fibrillisation
compared with the corresponding monomers. Further studies
of the neurotoxicity of Ab dimers and implications for the
progression of AD will be reported in due course.
Fig. 4 DLS data for Ab(1–40) monomer (a) and Ab(1–40) dimer 8d
(b) (0.5 mg mLÀ1 in 20 mM HEPES, pH 7.0). (c) Amyloid formation
by Ab(1–40) monomer (blue) and Ab(1–40) dimer 8d (red), as
monitored by ThT fluorescence.
This work was supported by the National Health and
Medical Research Council (SRF to KJB) and the Australian
Research Council (Federation Fellowship to MWP). Access
to equipment and technical assistance through the Bio21
Collaborative Crystallisation Centre and the Bio21 Institute
Electron Microscopy and Research Transfer Facilities is
gratefully acknowledged.
Dynamic light scattering experiments conducted on the
Ab(1–40) peptides exhibited high polydispersity and yielded
different profiles for each experiment inappropriate for
cumulant data analysis. The data shown in Fig. 4 represent
size distribution profiles from single experiments, and show
both the Ab(1–40) monomer and dimer peptides exhibit highly
dynamic profiles consistent with oligomeric distribution and
high order aggregates soon after dissolution.
Notes and references
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To further ascertain the aggregation and fibrillisation properties
of the Ab dimers 8b–d, ThT fluorescence and electron
microscopy studies were performed. Both the Ab(1–16) and
Ab(1–28) dimers, 8b and 8c, showed no increase in ThT
fluorescence over time (data not shown), indicating the
absence of amyloid formation in agreement with the DLS
data. The Ab(1–40) dimer 8d, however, displayed an increase
in ThT fluorescence after 10 h, indicative of amyloid formation.
Notably, the dimer 8d exhibited a significant decrease in the
lag time to amyloid formation compared with the monomer
(10 h for dimer 8d at 7 mM compared with 42 days for
monomer at 14 mM,13 Fig. 4c). To confirm fibril formation,
the Ab(1–40) monomer and dimer were aged over 6 days and
analysed by electron microscopy. The Ab(1–40) dimer 8d
clearly shows fibril formation after just 1 day, whereas the
monomer slowly forms fibrils over the 6-day period (Fig. 5).
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Fig. 5 Negatively stained TEM images of aged solutions of Ab(1–40)
dimer 8d (top) and Ab(1–40) monomer (bottom), after 1, 2 and 6 days.
Scale bar: 50 nm.
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This journal is The Royal Society of Chemistry 2009
6230 | Chem. Commun., 2009, 6228–6230