Polymer-peptide chimeras for the multivalent display of immunogenic peptides

Article abstract of DOI:10.1039/b924112d

Chimeras of poly(n-isopropyl acrylamide) and immunogenic peptides from the cancer-associated glycoprotein MUC1 were synthesised using a combination of solid-phase peptide synthesis, RAFT polymerisation and copper-catalysed alkyne-azide cycloaddition reactions.

Full text of DOI:10.1039/b924112d

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Chemical Communications
www.rsc.org/chemcomm
Volume 46 | Number 13 | 7 April 2010 | Pages 2153–2332
ISSN 1359-7345
COMMUNICATION
Richard J. Payne and Sébastien Perrier et al.
Polymer–peptide chimeras for the multivalent display of immunogenic peptides

COMMUNICATION
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Polymer–peptide chimeras for the multivalent display
of immunogenic peptidesw
Hamilton Kakwere,a Candy K. Y. Chun,b Katrina A. Jolliffe,b Richard J. Payne*b and
a
´
Sebastien Perrier*
Received (in Cambridge, UK) 18th November 2009, Accepted 11th December 2009
First published as an Advance Article on the web 16th January 2010
DOI: 10.1039/b924112d
Chimeras of poly(n-isopropyl acrylamide) and immunogenic
peptides from the cancer-associated glycoprotein MUC1 were
synthesised using a combination of solid-phase peptide synthesis,
RAFT polymerisation and copper-catalysed alkyne–azide cyclo-
addition reactions.
In this study, we were interested in developing a synthetic
methodology for the generation of self assembling polymer–
peptide chimeras. Specifically, we proposed to link immuno-
genic peptides from the extracellular variable number tandem
repeat (VNTR) region of the cancer associated glycoprotein
MUC19 to a preformed functional polymer in a convergent
manner. A hydrophobic polymer of tailored length was
designed to complement the hydrophilic peptide segment to
afford amphiphilic chimeras. It was anticipated that these
features would afford self-assembled nanostructures in
aqueous media with the hydrophilic peptide presented at their
surface. This approach provides a novel avenue for the multi-
valent display of immunogenic peptide epitopes, a desirable
feature for a strong and sustained response in immunological
studies.10,11 The resulting nanostructures can then be utilised
to investigate the generation of immunostimulating antigens
with a view to developing vaccines for a variety of epithelial
cancers, an area which is currently under investigation with
MUC1 peptides and glycopeptides.12–18 Our synthetic strategy
was to utilise the Cu(I)-catalysed variant of the Huisgen
1,3-dipolar cycloaddition reaction,19 namely the Cu(I)-catalysed
azide–alkyne cycloaddition (CuAAC),20,21 to generate these
constructs in an efficient and concise manner. CuAAC was
chosen by virtue of its tolerance to a wide variety of solvents
and functionalities and because the reactions are chemo-
selective and high yielding. The reaction has also been
successfully employed in the preparation of a number of other
polymer–peptide conjugates.3,22–25
Chimeras are macromolecular structures that combine
biomolecules with synthetic polymers.1,2 These hybrid
molecules have recently triggered widespread enthusiasm
among physicists, chemists and biologists due to their ability
to amalgamate the properties of synthetic polymers with
the activity of biomolecules. This has seen their application
in a diverse spectrum of areas such as nanotechnology,
photonics, biotechnology and medicine.3 The major
approaches to the synthesis of these molecular structures are
either via direct polymerisation of functional biomolecule-
based monomers,4 or via the coupling of biomolecules to
preformed polymeric scaffolds. The direct polymerisation
approach has received much attention since the early 1990s,
with the development of living radical polymerisation (LRP)
techniques allowing rapid access to well-defined functional
polymers of varying molecular architecture.5 Among the LRP
methods, reversible addition–fragmentation chain transfer
(RAFT6,7) polymerisation represents an extremely versatile
means for the generation of well defined chimeras.2 However,
from
a synthetic standpoint, the direct polymerisation
approach presents some challenges. For example, the
construction of low molecular weight polymers demands large
quantities of biomolecule initiators for the polymerisation to
be carried out on a reasonable laboratory scale. Additionally,
biomolecules usually bear a range of functional groups which
may disrupt the polymerisation process e.g. the thiocarbonyl
thio chain transfer reagents used in RAFT polymerisation can
be degraded in the presence of amine groups, thus precluding
their use in peptide and protein applications.2,8 Another
potential drawback of this strategy is the potential loss
of activity of a given biomolecule due to unwanted side
reactions.
In order to generate the desired chimeras, we first
embarked on the synthesis of the requisite building blocks.
To this end, tetrapeptide N3-GSTA-OH 1 and eicosapeptide
N3-GVTSAPDTRPAPGSTAPPAH-OH 2 (representing the
complete repeat unit of the VNTR sequence) were first
synthesised via Fmoc-strategy SPPS (Scheme 1, ESIw).
Notably, azidoglycine was incorporated as the N-terminal
amino acid to facilitate conjugation to an alkyne-bearing
polymer construct via CuAAC chemistry (ESIw).26
Poly(n-isopropyl acrylamide) (PNiPAAM), a non-toxic and
biocompatible polymer, was chosen as the polymeric unit for
the generation of chimeras, and was generated via RAFT
a Key Center for Polymers and Colloids, School of Chemistry,
University of Sydney, NSW 2006, Sydney, Australia.
E-mail: s.perrier@chem.usyd.edu.au; Fax: +61 (2) 9351 3329;
Tel: +61 (2) 9351 3366
b School of Chemistry, University of Sydney, NSW 2006, Sydney,
Australia. E-mail: payne@chem.usyd.edu.au;
Fax: +61 (2) 9351 3329; Tel: +61 (2) 9351 5877
w Electronic supplementary information (ESI) available: Experimental
procedures and characterisation of 1–10. See DOI: 10.1039/b924112d
Scheme 1 SPPS of MUC1 peptides 1 and 2 bearing N-terminal
azides.
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Fig. 1 MALDI-TOF spectra of protected PNiPAAM 4 (top) and
polymer–peptide chimera 5 (bottom) after PNiPAAM deprotection
and CuAAC reaction.
showed the successful formation of conjugate 5 (Fig. 1).
SEC analysis confirmed an increase in molecular weight of
the conjugate (ESIw). In addition, the 1H NMR spectrum was
consistent with the formation of the chimera with resonances
present for polymer 4 and peptide 1 and an additional
resonance at 8 ppm corresponding to the triazole ring proton
in 5 (ESIw).
Identical CuAAC conditions were subsequently employed
for the conjugation of the azidoeicosapeptide 2 to alkyne-
functionalised PNiPAAM 4. Formation of conjugate 6 was
confirmed by 1H NMR spectroscopy and HPLC analysis
(ESIw). Unfortunately, the low solubility of 6 in SEC eluents
prevented the use of this technique for characterisation.
The amphiphilic properties of chimeras 5 and 6 were
exploited to generate self-assembled particles in water. The
presence of aggregates in water was initially ascertained for 5
via dynamic light scattering (DLS). This revealed the presence
of structures with an average diameter of 136 nm and high
polydispersity. This suggested that the structures were
Scheme 2 Synthesis of PNiPAAM–MUC1 peptide chimeras 5–10.
polymerisation.27 PNiPAAM typically exhibits a temperature-
response in water by changing its character from hydrophilic
below the lower critical solution temperature (LCST), to
hydrophobic above this temperature.28 This LCST is strongly
dependent on the molecular weight of the polymer, and short
chain PNiPAAMs have been shown to exhibit hydrophobic
properties at room temperature.29 Polymerisation of
NiPAAM, mediated by 3-(trimethylsilyl)prop-2-ynyl-2-
(butylthiocarbonothioylthio) propanoate 3, provided a low
molecular weight polymer 4 (Mn = 2000 g molÀ1) with
polydispersity below 1.2 (Scheme 2). Investigation of the
LCST by DSC28 revealed a transition temperature of 8 1C,
thus confirming the hydrophobic character of the PNiPAAM
chains at the temperatures of interest (ESIw). We chose to
incorporate a TMS-protected alkyne in 4 to avoid potential
side reactions during radical polymerisation. The silyl ether
was removed in a subsequent step by treating with TBAF,
providing the desired alkyne-terminated PNiPAAM with
non-uniform,
a result confirmed by transition electron
microscopy (TEM) imaging, which illustrated the presence
of large and poorly defined aggregates (Fig. 2). The
uncontrolled self-assembly of chimera 5 suggested that this
construct would not be useful for the multivalent presentation
of the immunogenic MUC1 tetrapeptide. In contrast chimera
6, containing the full eicosapeptide tandem repeat sequence
of MUC1, formed nearly uniform micelles with a size of
60 Æ 3 nm, according to DLS measurements, and 20 nm
according to TEM imaging. The difference observed between
DLS and TEM measurements arises from the fact that DLS
measures the hydrodynamic volume of fully hydrated micelles
whilst TEM measures the diameter of non-hydrated particles,
obtained after deposition on a slide and drying.31 The TEM
images of 6 in water clearly show well-ordered self-assembled
particles and, given the hydrophobic nature of PNiPAAM,
multiple copies of the MUC1 VNTR peptide are expected to
be presented on the surface of the micelle. Given that 5,
bearing a tetrapeptide moiety, did not form well-defined
micelles, it appears that a critical length of the hydrophilic
peptide segment must be reached in order to synergise with
1
quantitative conversion as determined by H NMR spectro-
scopy (ESIw).30
With the desired building blocks in hand, we next investi-
gated the conjugation of alkynyl-PNiPAAM to tetrapeptide 1
using the CuAAC reaction. The reaction was conducted
using CuBr and PMDETA in methanolic solvent and
proceeded to completion after 60 h as confirmed by FT-IR,
MALDI-TOF and size exclusion chromatography (SEC)
analyses (Scheme 2). Analysis by MALDI-TOF clearly
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in our laboratories for the generation of stable, crosslinked
glycopeptide–polymer chimeras for the generation of novel
vaccine candidates.
HK thanks the University of Sydney for the provision of a
Scholarship. RJP thanks the ARC for funding (DP0986632).
Notes and references
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self-assembly of 6 may require further stabilisation to avoid
loss of structure due to variations in conditions e.g. temperature,
pH, etc. One of the key benefits of the RAFT process is that it
offers polymeric chains end-capped with a thiocarbonyl thio
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maleimide (Scheme 2, ESIw).32 Pyrene was chosen as an end
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conjugated to thiols, thus offering a simple means to assess
the success of the functionalisation.33 The resulting tagged
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end groups can be functionalised, further optimisation
is required before crosslinking is employed to stabilise
such aggregates. This is currently being investigated in our
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immunogenic peptides found within the VNTR sequence of
the cancer associated protein MUC1 via CuAAC thus
generating polymer–peptide chimeras. Chimera 6, bearing
the full length eicosapeptide repeat unit, formed well defined
nanoparticles thus providing a multivalent display of the
peptide epitope at its periphery. The versatility of the RAFT
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