Multifunctional Giant Amphiphiles via simultaneous copper(i)-catalyzed azide-alkyne cycloaddition and living radical polymerization

Article abstract of DOI:10.1039/c1cc15075h

A novel class of chemically addressable, multifunctional Giant Amphiphiles was synthesized in excellent yields and polydispersity following simultaneous or sequential living radical polymerization and the click, copper(i)-catalysed azide-alkyne cycloaddition (CuAAC). This new approach allows chemical tailoring of the biomacromolecules and in situ formation of nanocontainers.

Full text of DOI:10.1039/c1cc15075h

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Cite this: Chem. Commun., 2012, 48, 1586–1588
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COMMUNICATION
Multifunctional Giant Amphiphiles via simultaneous copper(I)-catalyzed
azide–alkyne cycloaddition and living radical polymerizationwz
Eleftheria Daskalaki, Benjamin Le Droumaguet, David Gerard and Kelly Velonia*
´
a
b
b
a
Received 16th August 2011, Accepted 19th September 2011
DOI: 10.1039/c1cc15075h
11,12
(ATRP)
A novel class of chemically addressable, multifunctional Giant
Amphiphiles was synthesized in excellent yields and polydispersity
following simultaneous or sequential living radical polymerization
and the click, copper(I)-catalysed azide–alkyne cycloaddition
and Reversible Addition–Fragmentation chain
13,14
Transfer polymerization (RAFT)
of appropriately modified
biomacroinitiators were shown to bypass the multiple synthetic
and purification steps used in classical bioconjugations and
facilitate purification. We recently demonstrated that ATRP is
the method of choice also for the family of Giant Amphiphiles as
it overcomes the intrinsic synthetic limitations of the system and
displays excellent control over the polydispersity indices of the
(
CuAAC). This new approach allows chemical tailoring of the
biomacromolecules and in situ formation of nanocontainers.
The combination of the unique functional and structural
properties of biomolecules with those of synthetic molecules
and macromolecules, and the application of such chimeric
bioconjugates in medicine, bio- and nanotechnology have been
8
resulting bioconjugates. More importantly, it allows the
hierarchical in situ encapsulation of guest molecules (e.g. other
proteins) for the construction of nanoreactors.
1
One underlying goal of protein–polymer self-assembly is the
construction of multifunctional nanoarchitectures following
efficient protocols and a key approach to this direction involves
bioconjugation of multifunctional polymer moieties. Herein we
demonstrate how the facile and high-yielding ATRP mediated
grafting of the appropriate monomer from a protein–
macroinitiator I can be directly combined with the ‘‘click’’
an area of intense research during the recent decades. One of
the most straightforward approaches toward this direction
involves covalent coupling of single or multiple polymeric
chains to biomolecules with the most pronounced example
being protein PEGylation which has led to bioconjugates with
enhanced physical and pharmacological properties and wide
1
,2
applications in medicine.
1
5
copper(I)-catalysed azide–alkyne cycloaddition (CuAAC)
Protein–polymer hybrids are conventionally prepared either
through the direct conjugation of appropriately functionalized
for the in situ formation of such novel multifunctional
bioconjugates (Scheme 1, Route A). We also demonstrate
how ATRP and CuAAC can be combined in sequential steps
(Route B) and in a chemical sequence comprising other simple
chemical methodologies (Route C). The efficiency of this
3
–8
9
or cofactors, or through
macromolecules to amino acids
1
0
bioaffinity couplings. These methods have nevertheless pro-
3
–5,8–10
ven to be less efficient in the case of Giant Amphiphiles,
i.e. the subclass of protein–polymer bioconjugates in which a
hydrophobic polymer moiety conveys an overall amphiphilic
character to the biohybrids responsible for their interesting
aggregation architectures. Practical limitations posed to the
synthetic protocols primarily by this amphiphilic character or
by restrictions applied to ensure protein integrity, hamper
efficient classical synthesis and therefore limit further investigations
to unravel Giant Amphiphiles full application potential. The
recent application of living radical polymerization techniques
for the in situ preparation of polymer–protein conjugates has
revolutionized the area. Atom-Transfer Radical polymerization
a
Department of Materials Science and Technology, University of
Crete, University Campus Voutes, 71003 Heraklion, Crete, Greece.
E-mail: velonia@materials.uoc.gr; Fax: +30 2810 39 4273;
Tel: +30 2810 39 4036
b
Department of Organic Chemistry, University of Geneva, Quai
Ernest Ansermet 30, Sciences II, CH 1211 Geneva 4, Switzerland
w This article is part of the ChemComm ‘Emerging Investigators 2012’
themed issue.
z Electronic supplementary information (ESI) available: Full experi-
mental conditions and characterization data. See DOI: 10.1039/
c1cc15075h
Scheme 1 Schematic representation of one-pot or sequential ATRP/
CuAAC synthesis of Giant Amphiphiles.
1
586 Chem. Commun., 2012, 48, 1586–1588
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approach not only proves the pertinence of the grafting from
living polymerization techniques for the creation of
protein–polymer bioconjugates, it more importantly gives rise
to a new family of chemically addressable amphiphilic
bioconjugates (II, III and IV) and allows the in situ formation
of biohybrid nanocontainers.
over the biohybrid and the CuSO
4
/sodium ascorbate copper(I)
generating system. The deprotection step of Route C was
systematically pursued using TBAF, KF or K CO which
2
3
were selected on the basis of common reaction conditions
found in the literature with a significant factor directing the
choice of the reagent, the retention of protein integrity. Additional
polymerization reactions were performed under the same
conditions and in the presence of the non-polymerizable,
fluorescent dye carboxyfluorescein (CF) aiming to prove the
ability of the resulting Giant Amphiphiles to concurrently form
hierarchically assembled nanocontainers through the statistical
encapsulation of CF within the superstructures.
5
In previous studies we synthesized a hydrophilic gradient
copolymer bearing pendant alkyne-1 groups to introduce
multifuntionality to proteins and, in a later step, linked the
polymer to a protein. In the current study we reasoned that the
combination of ATRP with CuAAC should provide the means
to avoid independent polymer synthesis, improve reaction
yields, control bioconjugate polydispersity and simplify isolation
For the one-pot Route A, the SEC chromatographic beha-
viour of the dialyzed reaction mixtures revealed quantitative
formation of the biomacromolecules IIa and IIb through the
formation of new peaks possessing shorter retention times,
larger hydrodynamic volume, than BSA (Fig. 1A) and similar
molecular weight distribution as judged by their broadness. In
all SEC measurements the Refractive Index (RI) and UV
traces were in excellent agreement. The efficiency of Route A
becomes more pronounced when comparing the SEC chromato-
graphs with those obtained for Routes B and C (Fig. 1B–D).
In Route B an, at least, bimodal distribution is observed for
the case of the azido-triethylene glycol clicking, while wide
polymodal peaks point to multiple final products in the
chromatographs of Route C. The formation of the Giant
Amphiphiles IIa, IIb, III and IV was also supported by gel
electrophoresis (Fig. 1E). The polyacrylamide gels revealed for
all reaction products typical Giant Amphiphile behaviour, i.e.,
electrophoretic mobility hampered by the amphiphilicity of
the biomacromolecules. Silver staining of these gels revealed
minute quantities of the BSA–macroinitiator I and the non-
reacting BSA dimer only in the case of Route C. When
electrophoresis was performed on agarose gels, a clear differ-
ence in the migration between I and IIa,b was observed.
Infrared spectroscopy (IR) validated the structures and
provided a direct proof for the efficiency of our synthetic
8
,16
of the products.
The choice of the methacrylate monomer 1
for the polymerization reaction was dictated by the expected
simplicity and orthogonality of the CuAAC aimed at their
tailoring. BSA was utilized as a model protein for the purposes
of our experiments since it contains a unique, accessible
1
7
cysteine group (Cys 34) permitting specific couplings. The
synthesis of the biomacroinitiator I, the propargyl metha-
crylate monomer 1, the trimethylsilyl protected propargyl
methacrylate 2, the azides and the copper ligand, N-(n-propyl)-
2
-pyridylmethanimine was achieved in high yields following
,6
5
established protocols (ESIz).
The ATRP grafting from polymerization reactions were
performed in buffered aqueous solutions, under oxygen free
conditions and ambient temperature, using the copper(I)
bromide/N-(n-propyl)-2-pyridylmethanimine catalyst system
5
,6,8
and without the presence of any ‘‘sacrificial’’ initiator.
More specifically, in the novel one-pot approach (Route A)
we utilized prop-2-ynyl methacrylate 1 as a monomer in the
presence of an azide (benzyl azide or azido-triethylene glycol)
to achieve simultaneous CuAAC and living radical polymer-
1
6
ization (ESIz). In order to fully exploit this new approach,
we also performed the sequential ATRP and CuAAC de-
scribed in Route B as well as the synthetic approach described
in Route C which incorporates an intermediate deprotection
step and trimethylsilyl protected propargyl methacrylate 2 as a
monomer. The CuAAC in both latter cases was investigated
approach through the absence or presence of the characteristic
ꢀ1
5
,16
using standard conditions,
i.e. a large excess of the azide
stretching vibration of the triple bond emerging at 2130 cm
.
Fig. 1 Characterization of Giant Amphiphiles. Representative: SEC traces (A): for Route A, (B): Route B, (C) and (D): Route C, (E):
Polyacrylamide Gel Electrophoresis, lanes 1 and 14: native BSA, lane 2: III, lane 3 and 5: IIa, lane 4 and 6: IIb, lane 15: IV, lane 16: III. Agarose:
lane 7: III, lanes 8 and 9:IIb, lanes 10 and 11: IIa, lane 13: native BSA (F): FT-IR spectra, (G): MALDI-TOF spectra of the Giant Amphiphiles III
(red) and the isolated polyalkyne (blue), (H): BSA enzyme-like activity for III and IIb, (I): CFM images of III formed in the presence of CF.
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 1586–1588 1587

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[3+2] Huisgen cycloaddition. Equally important, we
presented the synthesis of a series of novel, chemically addressable,
multifunctional protein–polymer biohybrids in excellent yields
and polydispersity. This approach allows chemical tailoring of
the protein–polymer hybrids and in situ formation of nano-
containers and could be implemented in several applications.
Our current efforts are focused on the full exploitation of such
biohybrids by the introduction of secondary (catalytic)
functions for the creation of multifunctional nanoreactors.
We thank the Swiss National Science Foundation (200020-
1
13804/1) for partial financial support, D.-H. Tran and
the SVSMS/University of Geneva for MALDI-TOF,
Mrs K. Tsagaraki, IESL/FORTH for FE-SEM, Mr A. Askounis
and D. Kritsiotakis for assistance with experimental
measurements.
Notes and references
1
F. M. Veronese, Biomaterials, 2001, 22, 405; D. W. Pack,
A. S. Hoffman, S. Pun and P. S. Stayton, Nat. Rev.
Drug Discovery, 2005, 4, 581.
Fig. 2 FE-SEM micrographs (A): III, (B) and (C): IIb, (D): IIa. TEM
micrographs (E) and (F): IV (G): III upon TBAF deprotection of IV
(H) and (I): IIa.
2 F. M. Veronese and J. M. Harris, Adv. Drug Delivery Rev., 2002,
4, 453, and references cited therein.
5
This peak was present only in the BSA–polyalkyne III and
3
K. Velonia, A. E. Rowan and R. J. M. Nolte, J. Am. Chem. Soc.,
2002, 124, 4224.
was accompanied by, less clear due to the nature of the
ꢀ1
ꢀ1
4 A. J. Dirks, S. S. van Berkel, N. S. Hatzakis, J. A. Opsteen,
F. L. van Delft, J. J. L. M. Cornelissen, A. E. Rowan, J. C. M. van
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products, peaks at 3300 cm and 632 cm (Fig. 1F). All
peaks disappeared upon multiple CuAAC. Furthermore, the
ꢀ1
peak attributed to the aromatic C–H bend at 721 cm
5
B. Le Droumaguet, G. Mantovani, D. M. Haddleton and
K. Velonia, J. Mater. Chem., 2007, 17, 1916.
J. Nicolas, G. Mantovani and D. M. Haddleton, Macromol. Rapid
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emerged only in the reaction products obtained by clicking
of the benzyl azide. MALDI-TOF analysis, often problematic
in the case of Giant Amphiphiles, afforded limited data with the
most reliable showing a m/z signal of around 85 kDa for the
protein–polymer hybrid IV and a signal of approximately
6
7
7
kDa m/z for the polymer moiety itself when recovered through
HCl mediated protein degradation of II (Fig. 1G, ESIz).
8
9
B. Le Droumaguet and K. Velonia, Angew. Chem., Int. Ed., 2008,
4
Aggregation studies using Transmission Electron Microscopy
7, 6263.
(
(
TEM) and Field Emission Scanning Electron Microscopy
FE-SEM) established the amphiphilic nature of the products
M. J. Boerakker, J. M. Hannink, P. H. H. Bomans, P. M. Frederik,
R. J. M. Nolte, E. M. Meijer and N. A. J. M. Sommerdijk, Angew.
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by revealing the formation of well-defined spherical aggregates
with diameters varying from 30 to 150 nm in all products
1
1
0 J. M. Hannink, J. J. L. M. Cornelissen, J. A. Farrera, P. Foubert,
F. C. De Schryver, N. A. J. M. Sommerdijk and R. J. M. Nolte,
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(
Fig. 2). No difference was observed for the superstructures
formed in the presence of CF (Fig. 2F). In the latter, CFM
imaging demonstrated the statistical encapsulation of CF
within the superstructures (Fig. 1I), proving the ability of
the produced Giant Amphiphiles to host non-polymerizable
guests. Given that both CuAAC and ATRP could be detri-
mental for protein structure integrity due to copper poisoning,
we performed enzyme-like activity tests in products and starting
materials of all routes which established retention of activity
indicating structure integrity. For the specific step of the
alkyne deprotection in Route C, the TBAF and the KF
deprotected samples retained enzyme-like activity, showed
slightly disturbed spherical superstructures in TEM, while
CFM verified the existence of discrete fluorescent aggregates.
In conclusion, this is to the best of our knowledge, the first
synthesis of protein–polymer conjugates using simultaneous or
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1
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588 Chem. Commun., 2012, 48, 1586–1588
This journal is c The Royal Society of Chemistry 2012