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REVIEWB. Le Droumaguet and J. Nicolas Recent advances in the design of bioconjugates from controlled/living radical polymerization
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REVIEW www.rsc.org/polymers | Polymer Chemistry
Recent advances in the design of bioconjugates from controlled/livingradical polymerization
Benjamin Le Droumaguet and Julien Nicolas*
Received 29th November 2009, Accepted 5th January 2010
First published as an Advance Article on the web 29th January 2010
DOI: 10.1039/b9py00363k
Since its discovery, controlled/living radical polymerization (CLRP) has proven to be a mature
technology for building tailor-made (block) copolymers, functional polymers and polymers with a wide
range of biological recognition. Due to these considerable advantages over other synthetic approaches,
CLRP techniques have been successfully exploited to construct novel polymer-protein/peptide
bioconjugates with a high level of structural control and varied interesting, somehow unexpected,
features. A comprehensive review of the recent advances in the rapidly expanding field of
bioconjugation is presented and outlines work up to early 2010.
1 Introduction
The exciting world of polymer science has been in a state of
almost perpetual (r)evolution since the pioneering work of
Staudinger1 and has now developed into a modern and multi-
disciplinary research field. Due to the cross-fertilization of
polymer science with diverse areas, it covers not only primarily
synthetic polymers mainly devoted to structural applications
such as coatings or packaging materials, but also a broad range
of higher value-added functional applications in nanomaterials,
opto-electronic technology as well as biomedical-related areas.
Laboratoire de Physico-Chimie, Pharmacotechnie et Biopharmacie,Universit�e Paris-Sud, UMR CNRS 8612, Facult�e de Pharmacie, 5 rueJean-Baptiste Cl�ement, F-92296 Chatenay-Malabry cedex, France.E-mail: [email protected]; Fax: +33 1 46 83 59 46; Tel: +33 1 4683 58 53
Benjamin Le Droumaguet
Benjamin Le Droumaguet
obtained his Masters degree in
organic chemistry in 2004 from
the University of Rennes. Then
he moved to the research group
of Dr Kelly Velonia at the
University of Geneva to take up
a PhD position in the field of
polymer-protein bioconjugates.
After obtaining his PhD in 2008,
he came back to France for
a post-doctoral fellowship in the
research group of Prof. Patrick
Couvreur, where he is currently
working on the synthesis of
functionalized poly(alkyl cyanoacrylate) nanoparticles for active
targeting. His research interests are directed towards the conju-
gation of biological molecules with organic materials for the design
of functional ‘‘smart’’ biohybrid systems.
This journal is ª The Royal Society of Chemistry 2010
Macromolecular synthesis certainly represents one of the most
prolific sub-disciplines in polymer science, as attested by the
discovery of controlled/living radical polymerization (CLRP)
fifteen years ago,2–13 that brought about an important break-
through in the field. Indeed, this new synthetic tool provided the
polymer chemist with an efficient and easy route for achieving
various macromolecular architectures of high complexity and
functionality, unavailable by other polymerization methods.
Among the numerous arenas where CLRP has been successfully
exploited is the design of a new-class of bioconjugates.14–20
Despite the many possible definitions of the word bioconjugate,21
in this review the term refers to the coupling or the binding
between a synthetic macromolecule (here a (co)polymer synthe-
sized by CLRP) and a biomacromolecule.
Innovative drug delivery technologies are now a key compo-
nent of pharmaceutical development. Various therapeutic
peptides and proteins represent a rapidly growing section of
Julien Nicolas
Julien Nicolas graduated from
the Ecole Sup�erieure de Chimie
Organique et Min�erale
(ESCOM), France, in 2001. He
completed his PhD in 2005
under the supervision of Prof.
Bernadette Charleux at the
University Pierre and Marie
Curie, Paris, where he studied
nitroxide-mediated polymeriza-
tion. He then joined the group of
Prof. David M. Haddleton at
the University of Warwick, UK,
for a postdoctoral fellowship to
design polymer-protein bio-
conjugates by controlled/living radical polymerization. In 2007, he
was appointed as researcher at the CNRS in the group of Prof.
Patrick Couvreur, University Paris-Sud, France, where his current
research activities are focused on the synthesis of novel nano-
particles and biopolymers for drug delivery purposes.
Polym. Chem., 2010, 1, 563–598 | 563
marketed drugs and have an uncontested place alongside other
well-established therapies. However, they suffer from severe
limitations due to their inherent physicochemical properties such
as a variable solubility, a low bioavailability and a limited
stability. The attachment of polyethylene glycol (PEG) to protein
or peptide therapeutics, termed PEGylation, is an example of
a highly successful strategy that gives rise to several benefits
including increased bioavailability and plasma half-lives,
decreased immunogenicity, reduced proteolysis and enhanced
solubility and stability.22 Recent advances in PEGylation tech-
nology are enabling even more opportunities to create novel,
efficient products. Whereas the initial strategy is a random PEG
attachment on lysine residues or at the N-terminus, a recent
promising approach consists of site-specific bioconjugation23 at
areas on a pharmaceutical drug that do not interfere with its
biological activity while still making the most of the PEG shield.
Due to high robustness, flexibility and mild experimental
conditions as well as to the possibility to insert varied functional
groups within the polymer chains, CLRP is becoming a believ-
able technology regarding this public health research field.
The explosive number of publications within the past couple of
years devoted to the design of bioconjugates from CLRP
methods prompted us to provide an updated survey that includes
proteins, peptides and oligonucleotides, and which covers
achievements up to early 2010. An overview of the employed
CLRP techniques is first presented followed by a detailed
description of the various synthetic strategies to achieve well-
defined bioconjugates. A distinction is established between
whether the conjugation is performed via a covalent linkage or
using a non-covalent approach.
2 Controlled/living radical polymerization (CLRP)
CLRP techniques have emerged as simple routes for preparing
well-defined polymers of predetermined molecular weight (MW)
and narrow molecular weight distribution (MWD), as well as
various block copolymers and a wide range of complex macro-
molecular architectures.2–13 Prior to the development of CLRP,
all these features were hardly achievable. Indeed, only with living
ionic polymerization24 could such a degree of structural unifor-
mity be reached but under very drastic polymerization conditions
and with a limited choice of monomers.
In contrast to conventional free-radical polymerization where
propagating radicals exhibit a very short lifetime (�1 s) that
hampers the conception of well-defined architectures, the concept
of CLRP is to increase this lifetime up to the timescale of the
polymerization reaction, via the establishment of a reversible
equilibrium between active species, which can propagate, and
dormant/capped species, which cannot propagate. CLRP then
proceeds in such a manner that all polymer chains grow at the
same rate and the detrimental impact of irreversible termination
events over the MW and the MWD are made almost negligible.25
An ideal living polymerization system should exhibit the following
features: (i) a linear evolution of the logarithmic conversion (ln[1/
(1 � conversion)]) with time, accounting for a constant propa-
gating radicals concentration; (ii) a linear increase in the number-
average molar mass, Mn, with monomer conversion, where the
degree of polymerization, DPn, is predetermined by the consumed
monomer to initially introduced initiator molar ratio; (iii) low
564 | Polym. Chem., 2010, 1, 563–598
polydispersity indexes, Mw/Mn, close to a Poisson distribution
(Mw/Mn z 1 + 1/DPn); (iv) a quantitative a- and u-functionali-
zation and (v) the possibility for polymer chains, after monomer
consumption, to further grow when additional monomer is
introduced which allows block copolymer synthesis to be per-
formed by sequential monomer addition.25
Nowadays, a plethora of well-established CLRP methods
offers the polymer chemist an impressive toolbox for making
advanced macromolecular architectures of increasing
complexity. Among them, nitroxide-mediated polymerization
(NMP),2 atom transfer radical polymerization (ATRP)3,4,7,9,10
and reversible addition-fragmentation chain transfer
(RAFT),5,6,12,13 the latter including macromolecular design via
the interchange of xanthates (MADIX), represent the three most
well-known CLRP techniques. In addition to these famous
approaches, one can find the iniferter26–29 system and cyanoxyl-
mediated polymerization30 as well as recently emerged CLRP
techniques, such as iodine transfer polymerization (ITP),31 single
electron transfer-living radical polymerization (SET-LRP),32
organotellurium-mediated polymerization (TERP),8 organo-
stibine-mediated polymerization (SBRP)8 and cobalt-mediated
polymerization (CMRP).33,34
The following section will briefly describe the CLRP tech-
niques that have been used for the synthesis of bioconjugates so
far. The reader who would like a more exhaustive point of view
about all CLRP methods is referred to the above-mentioned
references associated to each technique.
2.1 Iniferter
In the early 80s, the use of iniferters in radical polymerization,
defined as agents (such as tetraethylthiuram disulfide, phenyl-
azotriphenylmethane or benzyl dithiocarbamate) that are able to
initiate, transfer and terminate, was actually the first successful
step to date towards CLRP.26,27 Iniferters can be either: asym-
metrical (denoted A–B), where A is a reactive radical which
participates in initiation and propagation reactions whereas B is
only involved in termination reaction, or symmetrical (denoted
C–C), where C participates in both initiation and termination
reactions. Besides this, iniferters can be activated upon heating
(thermal iniferters) or by UV irradiation (photoiniferters).28,29
Even though the use of iniferters led to broad MWDs and poor
initiation efficiencies, this technique has allowed a broad range of
monomers to be polymerized in rather mild conditions as well as
several macromolecular architectures to be prepared.28,29
2.2 Cyanoxyl-mediated polymerization
In the early 90s, it was shown that cyanoxyl radicals (cOChN)
could be successfully used as control agents during the poly-
merization of methyl (meth)acrylate, n-butyl acrylate and acrylic
acid to form homopolymers as well as block, statistical and
grafted copolymers in relatively mild experimental condi-
tions.30,35,36 Moreover, the polymerization can be carried out in
aqueous solution and is tolerant of a broad range of functional
groups. However, the use of cyanoxyl-mediated polymerization
was strongly restrained by the emergence of another type of
oxygen-centered persistent radicals as mediators for CLRP,
namely nitroxides.
This journal is ª The Royal Society of Chemistry 2010
Fig. 1 The activation–deactivation equilibrium in nitroxide-mediated
polymerization (NMP).
Fig. 2 The structures of nitroxides used as mediators in NMP: TEMPO
(N1), SG1 or DEPN (N2), TIPNO (N3) and DPAIO (N4).
Fig. 3 The activation–deactivation equilibrium in atom transfer radical
polymerization (ATRP).
2.3 Nitroxide-mediated polymerization (NMP)
NMP is based on a reversible termination reaction between
a growing radical, Pc, and a free nitroxide, Nc, to form a (mac-
ro)alkoxyamine, P–N (Fig. 1). This equilibrium between active
and dormant species presents the advantage of being a purely
thermal process: the (macro)alkoxyamine regenerates the prop-
agating radical and the nitroxide by homolytic cleavage at high
temperature (usually > 70 �C).
A typical NMP can be initiated following two different path-
ways: (i) by using a bicomponent initiating system (i.e.
a conventional radical initiator and a free nitroxide) or (ii) via
a monocomponent initiating system (i.e. a preformed alkoxy-
amine). Georges and co-workers first reported the controlled
radical polymerization of styrene with (2,2,6,6-tetramethylpi-
peridinyl-1-oxy) (TEMPO, N1, Fig. 2) as the mediator.37
However, as TEMPO was almost exclusively limited to styrenic
monomers (only a sterically hindered TEMPO derivative allowed
the control of the n-butyl acrylate polymerization),38 new acyclic
nitroxides have been designed to improved the range of poly-
merizable monomers under controlled/living conditions. More
precisely, N-tert-butyl-N-[1-diethylphosphono-(2,2-dimethyl-
propyl)] nitroxide (SG1 or DEPN, N2, Fig. 2)39–42 and N-tert-
butyl-N-[1-phenyl-2-(methylpropyl)]nitroxide (TIPNO, N3,
Fig. 2)43–47 are now able to control the polymerization of styr-
enics, alkyl acrylates, acrylic acid, acrylamides and dienes.39,43,48–50
The polymerization of methacrylic esters can be controlled either:
(i) by using a particular nitroxide such as 2,2-diphenyl-3-phenyl-
imino-2,3-dihydroindol-1-yloxyl nitroxide (DPAIO, N4, Fig. 2),51
specific to methacrylates or (ii) by a copolymerization approach
under SG1 control with a small amount of comonomer such as
styrene52–54 or acrylonitrile.55
2.4 Atom transfer radical polymerization (ATRP)
ATRP was discovered independently by Sawamoto56 and
Matyjaszewski.57–59 The ATRP process is based on a rapid
exchange of a halide atom (especially Cl or Br) between
This journal is ª The Royal Society of Chemistry 2010
a growing radical and a dormant species, via a redox process
involving a transition metal complex (Fig. 3). To ensure a good
control of the polymerization, this equilibrium is strongly shifted
towards the dormant species.3,4
Various transition metals can be employed in ATRP (Cu, Ru,
Fe, Ni, etc.). In the first ATRP process, called direct ATRP, the
transition metal complex in a lower oxidation state (Mtn/Lm,
where Mt is the metal and L is the ligand) is directly added to the
reaction as an activator and reacts reversibly with the dormant
species (P-X, with X a halogen atom) to form a deactivator
(Mtn+1X/Lm) and the active species Pc. In contrast, when the
polymerization is initiated by a conventional initiator and a metal
complex at the higher oxidation state, the process is called reverse
ATRP. The simultaneous reverse and normal initiation (SR&NI)
process takes advantage of both normal and reverse ATRP as
Cu(II) (which is tolerant to oxygen), an alkyl halide and a radical
initiator are initially present in the reaction medium.60 It provides
a way to reduce the amount of copper complex and to prepare
more complex macromolecular architectures.
Recently, new ATRP processes have been developed, namely
activators generated by electron transfer (AGET)61 and activa-
tors regenerated by electron transfer (ARGET).62 The AGET
process employs a reducing agent (e.g. ascorbic acid or tin(II) 2-
ethylhexanoate) which reacts with Mtn+1X/Lm to generate the
active catalyst (Mtn/Lm). The process then follows a direct ATRP
process. AGET ATRP allows the preparation of pure block
copolymers with no homopolymer of the second monomer. The
ARGET process uses an excess of reducing agent which allows
a significant reduction of the amount of metal in the media.
This powerful and versatile technique can be used under mild
experimental conditions and in various polymerization media
with a wide range of monomers including styrenics, alkyl
(meth)acrylates, acrylonitrile, (meth)acrylamides as well as
water-soluble monomers to give tailor-made poly-
mers.3,4,7,10,11,63,64 An additional flexibility is provided by the
possibility of using commercially available functionalized initi-
ators and to functionalize chain ends. However, if ATRP can be
used with a large range of monomers, the polymerization of
functional monomers bearing acid or amine function remains
hardly achievable.
2.5 Reversible addition fragmentation chain transfer (RAFT)
RAFT polymerization is governed by a reversible transfer reac-
tion between a growing (macro)radical (active species) and
a (macro)RAFT agent (dormant species).5,12,13,65 RAFT agents,
denoted Z–C(aS)SR, act as transfer agents by a two-step
Polym. Chem., 2010, 1, 563–598 | 565
Fig. 4 Mechanism of reversible addition fragmentation chain transfer
(RAFT).
addition-fragmentation mechanism (Fig. 4). The RAFT group is
typically a thiocarbonylthio group such as dithioester (Z ¼alkyl), trithiocarbonate (Z ¼ S-alkyl), xanthate (O-alkyl) or
dithiocarbamate (Z ¼ N(alkyl)2). The RAFT process using thi-
ocarbonylthio compounds, including dithioesters and trithio-
carbonates, was reported by the CSIRO laboratory in early
199866 whereas a similar process using xanthates as RAFT agents
(the so-called MADIX) was reported in late 1998.67 RAFT is
potentially universal and can be applied to a wide range of
functional monomers (styrenics, alkyl (meth)acrylates, acrylic
acid, vinyl acetate etc.), which allows polymers with precisely
controlled structural parameters to be prepared such as random,
block, gradient, grafted and star copolymers.5,12,13
Even though Moad, Rizzardo and co-workers recently suc-
ceeded in elaborating a switchable RAFT agent,68 one of the
major drawback of this technique was the lack of a universal
RAFT agent. In particular, dithioesters or trithiocarbonates
were suitable for controlling polymerization of more activated
monomers such as styrene (S) and derivatives, methacrylic esters
(e.g. methyl methacrylate MMA), methacrylic acid (MA),
methacrylamide (MAM), acrylic acid (AA), acrylamide (AM) or
acrylonitrile (AN). However they inhibit or retard the polymer-
ization of less activated monomers such as vinyl acetate (VAc),
N-vinylpyrrolidone (NVP) or N-vinylcarbazole (NVC), for
which xanthates or dithiocarbamates are more suitable.5,12,13 The
choice of R and Z groups is thus crucial to achieve a good control
of the polymerization.
Fig. 5 Structure of ATRP initiators empl
566 | Polym. Chem., 2010, 1, 563–598
3 Synthesis of polymer-protein/peptidebioconjugates using a covalent approach
3.1 Bioconjugation with preformed polymer: the ‘‘grafting to’’
method
The direct conjugation of preformed polymers to proteins/
peptides is certainly the most widespread approach for creating
bioconjugates, the best example being the protein PEGylation,87–90
discovered by Abuchowski et al. in the late seventies.91,92 Even
though a broad range of monomers have been polymerized by
CLRP for further coupling to proteins/peptides, comb-like
polymers based on poly(ethylene glycol) methacrylate (PEGMA)
and poly(ethylene glycol) acrylate (PEGA) were advantageously
employed as an alternative to traditional PEGylation. Such
branched PEG polymers indeed exhibit two main benefits over
traditional linear PEGs: (i) a higher flexibility of control over the
macromolecular characteristics of both the PEG chains and the
polymer backbone and (ii) a better resistance against proteolysis
and antibodies action, due to their ‘‘umbrella-like’’ shape.93,94
Moreover, one of the biggest advantages of CLRP techniques
(especially ATRP and RAFT) over traditional polymerization
methods is that they allow a fine tuning of the functionalities
present at both the a- and the u-termini of polymer chains by
using functional initiators/transfer agents (see Fig. 5 and Fig. 7)
or via post-polymerization modification steps.
3.1.1 Bioconjugation to the a-terminus. Synthesis of a-func-
tional polymers certainly remains the most exploited route for
bioconjugates as shown by the numerous examples gathered in
Table 1.
3.1.1.1 Conjugation via an amine reactive group. N-hydroxy-
succinimide terminated polymers. The Haddleton and Stenzel
groups used ATRP69 and RAFT70, respectively, to construct
well-defined N-hydroxysuccinimide (NHS) a-functional poly-
mers for protein bioconjugation purposes. For instance, NHS
oyed in the synthesis of bioconjugates.
This journal is ª The Royal Society of Chemistry 2010
Table 1 Synthesis of polymer-protein/peptide bioconjugates via the ‘‘grafting to’’ method using a-functional polymers obtained by controlled/livingradical polymerization (CLRP)
Protein a-Functional polymer
Name Target group a-Functional groupb Monomer/sc CLRP technique Ref.
lysozyme NH2 NHS PEGMA ATRP 69lysozyme NH2 NHS NVP RAFT 70lysozyme NH2 NHS MPC ATRP 71papain NH2 NHS MPC iniferter 72,73lysozyme NH2 aldehyde PEGMA ATRP 74lysozyme NH2 aldehyde MPC ATRP 71sCT NH2 (N-terminus) aldehyde PEGMA ATRP 75,76erythropoietin NH2 aldehyde MPC ATRP 71G-CSF NH2 aldehyde MPC ATRP 71lysozyme NH2 thiazolidine-2-thione HPMA, PEGMA RAFT 77BSA N3-levulinatea aminooxy NIPAAm, PEGMA, HEMA ATRP 78BSA N3-levulinatea aminooxy NIPAAm RAFT 79BSA SH maleimide PEGMA, GMA ATRP 80BSA SH maleimide GMA, PMA, HMA (terpolymer) ATRP 81T4 lysozyme SH maleimide PEGA RAFT 82BSA SH PDS HEMA ATRP 83BSA SH PDS NIPAAm ATRP 84T4 lysozyme SH PDS NIPAAm ATRP 84BSA SH PDS PEGA, NIPAAm RAFT 85IFN disulfide bridge bis-sulfone MPC ATRP 86
a via lysine residues. b NHS: N-hydroxysuccinimide; PDS: pyridyl disulfide. c PEGMA: poly(ethylene glycol) methyl ether methacrylate; NVP: N-vinylpyrrolidone; MPC: 2-methacryloyloxyethyl phosphorylcholine; HPMA: N-(2-hydroxypropyl)methacrylamide; NIPPAm: N-isopropyl acrylamide;HEMA hydroxyethyl methacrylate; GMA: glycidyl methacrylate; PEGA: poly(ethylene glycol) methyl ether acrylate.
Fig. 6 Design of sCT-poly(PEGMA) bioconjugates by a combination of
ATRP and reductive amination.75
a-functional poly[poly(ethylene glycol) methyl ether methacry-
late] (poly(PEGMA)) polymers (Mn ¼ 2.8 and 6.4 kDa, Mw/Mn
< 1.15) were synthesized from 2 different NHS-based ATRP
initiators (I1 and I2, Fig. 5) in the presence of CuBr/N-(ethyl)-2-
pyridylmethanimine and used for the bioconjugation to lysine
residues of lysozyme.69 The coupling between lysozyme and an
excess of NHS a-functional poly(PEGMA) polymers was per-
formed in anhydrous DMSO in the presence of TEA. A quan-
titative coupling was obtained after 6 h and SDS-PAGE analysis
of the bioconjugates indicated that approximately 6 to 7 polymer
chains were anchored to each protein, in good agreement with
the 7 free lysine residues available per lysozyme. More recently,
a-NHS poly(N-vinylpyrrolidone) (PVP) polymers were prepared
by RAFT polymerization from RA1 (Fig. 7) upon AIBN initi-
ation (Mn¼ 16.9–33.4 kDa, Mw/Mn¼ 1.38–1.41) and coupled to
lysozyme.70 Similarly to the ATRP route, an average of 7 PVP
chains were tethered to lysozyme.
Ishihara’s group reported the functionalization of the papain
protein with NHS a-functional water-soluble poly(2-meth-
acryloyloxyethyl phosphorylcholine) (PMPC) polymer using
a photoinduced iniferter-mediated polymerization.72,73 2-Meth-
acryloyloxyethyl phosphorylcholine (MPC) monomer and the
resulting polymers95,96 exhibit excellent biocompatibility97 as they
mimic the structure of natural phospholipids found in cell
membranes. The polymerization of MPC was triggered by the
photo-irradiation of 4-(N,N-diethyldithiocarbamoylmethyl)-
benzoic acid (BDC) at ambient temperature. It was shown that
increasing the molecular weight of the PMPC-NHS polymer
resulted in a lower degree of functionalization (from 42 to 19%).
Besides, the resulting conjugates retained 35% of the catalytic
activity of the native enzyme, whatever the molecular weight of
the PMPC-NHS moiety. Recently, ATRP has been investigated
This journal is ª The Royal Society of Chemistry 2010
by Emrick and co-workers for the preparation of NHS a-func-
tional PMPC polymers (Mn ¼ 2.3–8.2 kDa, Mw/Mn ¼ 1.2–1.5)
derived from initiators I1 and I2 (Fig. 5) for bioconjugation to
lysozyme in quantitative yields.71
Aldehyde terminated polymers. Another strategy, based on the
work of Bentley and co-workers regarding reductive amination,98
was developed by Haddleton’s group for the synthesis of alde-
hyde a-functional branched PEG polymers by ATRP and their
Polym. Chem., 2010, 1, 563–598 | 567
subsequent conjugation to proteins.74,75 In the first report,
a ketal-protected aldehyde ATRP initiator (I3, Fig. 5) was used
to initiate the polymerization of PEGMA (Mn ¼ 11 kDa, Mw/
Mn ¼ 1.14). After deprotection upon acidic catalysis, the
a-aldehyde poly(PEGMA) was reacted with lysozyme at pH 5 or
6 in the presence of sodium cyanoborohydride (NaCNBH3) to
give, after in situ reduction of the imine moiety into an amine
bond, the corresponding lysozyme-poly(PEGMA) bioconjugate.
The conjugation was quantitative and occurred faster at pH 5.
The same reductive amination pathway was also applied to the
functionalization of the N-terminal cysteine of salmon calcitonin
(sCT), a 3432 Da peptide used for the treatment of post-meno-
pausal osteoporosis and Paget’s disease, by a 6.5 kDa a-aldehyde
poly(PEGMA) in the presence of NaCNBH3 (Fig. 6).75 The
poly(PEGMA)-sCT bioconjugate (9.8 kDa) was observed to be
non-cytotoxic, even at a relatively high concentration and to
retain approximately 90% of the initial native sCT activity.
Stability studies also indicated that poly(PEGMA)-sCT dis-
played resistance toward proteolytic activity of 3 individual
intestinal enzymes (trypsin, chymotrypsin, elastase) whereas
native sCT was degraded within a few minutes. The bio-
conjugates also reduced the serum calcium levels. All these
results indicated that the poly(PEGMA) polymers have
a tremendous potential to improve the pharmacokinetics of
injected peptides in therapeutic applications. Another comple-
mentary study regarding the functionalization of sCT by poly-
(PEGMA) with Mn varying from 6.5 up to 109 kDa showed that
sCT activity was not altered by the polymer chain length.76
Fig. 7 Structure of RAFT agents emplo
568 | Polym. Chem., 2010, 1, 563–598
The reductive amination pathway was also applied to the
synthesis of bioconjugates based on granulocyte colony stimu-
lating factor (G-CSF) and erythropoietin (EPO) by Emrick and
co-workers. Briefly, ATRP of MPC from two benzaldehyde-
based ATRP initiators (I4 and I5, Fig. 5) afforded a range of well
defined aldehyde a-functional PMPC polymers (Mn¼ 4.5–7 kDa,
Mw/Mn ¼ 1.1) that were suitable for bioconjugation with the
lysine residues of the two above-mentioned proteins in the pres-
ence of NaCNBH3.71 SEC-HPLC indicated the formation of the
corresponding (G-CSF)-PMPC and EPO-PMPC bioconjugates
together with the presence of unreacted native protein.
Thiazolidine-2-thione terminated polymers. Recently, Davis
and co-workers presented the synthesis of new lysine-reactive a-
functional polymers based on the functionalization of a RAFT
agent with the thiazolidine-2-thione moiety.77 In this study, the 2-
cyano-5-oxo-5-(2-thioxothiazolidin-3-yl)pental-2-yl benzodi-
thioate RAFT agent (RA2, Fig. 7) mediated the polymerization
of N-(2-hydroxypropyl) methacrylamide (HPMA). Lysozyme
was reacted with 40 eq. of the a-thiazolidine-2-thione PHPMA
(Mn ¼ 3.5 kDa, Mw/Mn ¼ 1.09) and SEC analysis demonstrated
the efficiency of the coupling reaction. It was also shown that pH
was an important parameter as it influenced the number of
polymer chains attached to the protein (a low pH protonates the
amines that lose their nucleophilic character). Finally, the
bioactivity of the resulting lysozyme-PHPMA bioconjugates was
evaluated in the presence of a lysozyme substrate (Micrococcus
lysodeikticus cells) and gave only 4.8% of the original activity of
the native protein for the conjugate formed at pH 6.5. Besides
yed in the synthesis of bioconjugates.
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this, the same group described the successful synthesis of a well-
defined, biodegradable poly(PEGMA) using a thiazolidine-2-
thione-based RAFT agent bearing a disulfide bridge (RA3,
Fig. 7).99 After conjugation to lysozyme via amide linkages,
cleavage of the polymer chains from the conjugate was triggered
and the released protein displayed a marked increase in bioac-
tivity.
Aminooxy terminated polymers. Another option for selectively
functionalizing lysine residues of proteins with functional poly-
mers is the use of aminooxy terminated polymers. Maynard’s
group reported the design of aminooxy a-functional poly(N-
isopropylacrylamide) (PNIPAAm) polymers for the synthesis of
BSA-PNIPAAm bioconjugates via ATRP78 and RAFT79 meth-
odologies. In the first approach, Boc-protected aminooxy ATRP
initiators (I6 and I7, Fig. 5) initiated the polymerization of
NIPAAm in a controlled fashion, followed by Boc deprotection
under acidic conditions. As the use of the aminooxy coupling
requires the derivatization of the protein with ketone or aldehyde
moieties, BSA was first reacted with NHS-activated levulinate
(Fig. 8). The resulting N3-levulinyl lysine-modified BSA was then
reacted with a-aminooxy PNIPAAm to give the desired BSA-
PNIPAAm bioconjugate via oxime bonds formation, further
purified from unreacted BSA by thermal precipitation at 35 �C.
SDS PAGE analysis confirmed that the coupling was achieved
and UV-Vis turbidity experiments revealed the effective ther-
moresponsive features of the bioconjugates. This approach was
also employed with different molecular weight ATRP polymers
from PEGMA, NIPAAm and 2-hydroxyethyl methacrylate
(HEMA)78 as well as with a-aminooxy PNIPAAm (Mn ¼ 4.2
kDa, Mw/Mn < 1.15) from RAFT using RA4 (Fig. 7).79
This study also reported the possibility of modifying the tri-
thiocarbonate end-group of the PNIPAAm by aminolysis in the
presence of butylamine and tris(2-carboxyethyl)phosphine
(TCEP), to avoid disulfide coupling, and to further immobilize
the resulting thiol u-functional PNIPAAm polymer onto a gold
surface. In a final step, the authors were able to immobilize
heparin, a sulfated polysaccharide, previously decorated with
aldehyde functions by reaction with NaIO4.
Carboxylic acid terminated polymers. B€orner and co-workers
used ATRP to synthesize hybrid nanotubes composed of cyclo-
octapeptides (stacked by intermolecular hydrogen bonds) deco-
rated with two poly(n-butyl acrylate) (PnBA) polymer chains
tethered to the two opposite lysine residues of each cyclic oli-
gopeptide.100 Polymerization of nBA was initiated from benzyl
Fig. 8 Aminooxy end-functionalized polymers from
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2-bromopropionate in ACN using CuBr/CuBr2/PMDETA at
60 �C for 2 h. The benzyl ester group was then cleaved from the
PnBA polymer under reductive conditions (Mn ¼ 2.1 kDa, Mw/
Mn ¼ 1.10). Final amidation between lysine residues and car-
boxy-terminated PnBA was achieved in the presence of EDC and
DIPEA to yield a triblock-like biopolymer able to self-assemble
in nanotubular superstructures. A similar polymer was also
attached to the N-terminus of a linear dodecapeptide by classical
amidation reaction followed by an O-N-acyl switch that guided
the self assembly of the bioconjugate into densely twisted tape-
like microstructures.101 AFM and TEM were used to characterize
these helical superstructures. The stable assembly was ascribed to
the formation of antiparallel b-sheets between peptide segments
whereas PnBA tails constitute the shell of the superhelices.
3.1.1.2 Conjugation via a thiol reactive group. Amine tar-
geting via lysine residues is one of the techniques most used for
bioconjugates synthesis. However, it always results in the
formation of a broad range of bioconjugates differing in their
degree of functionalization and thus displaying different molec-
ular weights. This explains the recent increasing interest for a-
functional polymers derived from CLRP towards the targeting of
free cysteine residues that are present in protein at lower
percentages than amine counterparts.
Maleimide terminated polymers. The direct use of a maleimido
ATRP initiator is not suitable as the maleimide moiety is poly-
merizable and branching is thus likely to occur upon polymeri-
zation. Two synthetic pathways were proposed by Haddleton
and co-workers80 to efficiently circumvent this difficulty: (i) from
a Boc-protected amino ATRP initiator (I8, Fig. 5) where the
maleimide moiety was introduced during a post-polymerization
step via an amidation reaction with 3-maleimidopropionyl
chloride in the presence of DIPEA or (ii) from a furan-protected
maleimido ATRP initiator (I9, Fig. 5), the maleimide moiety
being recovered by a retro Diels–Alder reaction after polymeri-
zation. This was applied to the synthesis of well-defined and pure
maleimide a-functional poly(PEGMA) and poly(glycerol meth-
acrylate) (PGMA) polymers (Mn ¼ 4.1–35.4 kDa, Mw/Mn ¼1.06–1.27), using N-(ethyl)-2-pyridylmethanimine/CuBr as the
catalytic system. Coupling experiments were successfully per-
formed on glutathione (g-ECG) and BSA (which contains
a single free cysteine residue at position 34)102 as a model tri-
peptide and protein, respectively. SDS-PAGE and FPLC
analyses revealed the successful formation of the bioconjugates.
ATRP for selective conjugation to proteins.78
Polym. Chem., 2010, 1, 563–598 | 569
The same furan-protected maleimido ATRP initiator also
allowed BSA-polymer giant amphiphiles to be prepared in
a controlled fashion as demonstrated by Velonia and co-workers
(Fig. 9).81 Polymerization of a ketal-protected glycerol meth-
acrylate, a very small amount of hostasol methacrylate (HMA)
as a fluorescent monomer and trimethylsilyl-protected propargyl
methacrylate (incorporated in the reaction medium after �50%
conversion) yielded the corresponding terpolymer. Its subse-
quent deprotection afforded a fluorescent a-maleimide poly-
(glycerol methacrylate-co-hostasol methacrylate-co-propargyl
methacrylate) (P(GMA-co-HMA-co-PMA)) copolymer con-
taining alkyne side chains available for further click chemistry.81
After bioconjugation, hydrophilic BSA-(PGMA-co-HMA-co-
PMA) biohybrids were then hydrophobized by copper catalyzed
click chemistry reaction with small hydrophobic azides (1-
azidodecane and 1-(azidomethyl)benzene) to afford amphiphilic
protein-polymer biomacromolecules that self-assembled in
aqueous solutions into well-defined spherical superstructures.
Depending on the nature of the clicked azide, average diameters
varied from 20 to 200 nm.
Maynard’s group used RAFT polymerization for the forma-
tion of semitelechelic maleimide-containing poly(PEGA) poly-
mers for bioconjugation purposes.82 A furan-protected
maleimide a-functional RAFT agent (RA5, Fig. 7) was used to
mediate the polymerization of PEGA at 60 �C in the presence of
Fig. 9 Design of giant amphiphiles by a com
570 | Polym. Chem., 2010, 1, 563–598
AIBN to afford the corresponding poly(PEGA) polymers (Mn ¼20.0–39 kDa, Mw/Mn ¼ 1.25–1.36). In order to avoid partial
hydrolysis of the ester bond linking the maleimide to the polymer
upon retro Diels–Alder reaction, the polymer was prepared from
an amide linked RAFT agent. Maleimido poly(PEGA) was then
coupled to V131C T4 lysozyme (T4L, a mutant protein geneti-
cally engineered to present a cysteine residue at position 131
instead of a valine residue) in the presence of EDTA and TCEP
as confirmed by SDS-PAGE and SEC-HPLC.
Pyridyl disulfide terminated polymers. The use of a pyridyl
disulfide (PDS) group to target the free cysteine residues of
proteins represents a convenient alternative to the maleimide-
thiol Michael-type addition approach. Importantly, it has
a significantly higher stability in PBS compared to maleimide
group and has the advantage of being cleavable under reductive
environments, allowing for further release of the polymer/
protein.
Two complementary studies published by Maynard and co-
workers reported the synthesis of PDS a-functional polymers
from ATRP. In a first report, HEMA was polymerized from
a PDS-containing ATRP initiator (I10, Fig. 5) in the presence of
CuBr/bpy as the catalyst to give the corresponding PHEMA
(Mn ¼ 3.9, 7.9, 15.7 kDa, Mw/Mn ¼ 1.20–1.25) with 90% of PDS
functionality.83 The bioconjugation was performed with BSA
and assessed by SDS-PAGE in reducing or non-reducing
bination of ATRP and click chemistry.81
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conditions, and by Ellman’s assay in order to highlight the
specificity of the coupling on the single free cysteine residue. This
approach was then extended to BSA-PNIPAAm and T4L-PNI-
PAAm ‘‘smart’’ bioconjugates with bioconjugation yields higher
than 65% for both proteins.84 The PNIPAAm block was recov-
ered upon cleavage of the disulfide bridge and exhibited a PDI as
low as 1.34. Interestingly, in the case of the T4L-PNIPAAm
bioconjugates, no significant difference in bioactivity was
observed when compared to the native protein.
Recently, Davis and co-workers used two PDS-functionalized
RAFT agents (RA6 and R12, Fig. 7) for the synthesis of a range
of a-PDS PNIPAAm (Mn ¼ 17.2–23.3 kDa, Mw/Mn < 1.36) and
poly(PEGA) (Mn ¼ 8–18 kDa, Mw/Mn ¼ 1.14–1.45) in water or
acetonitrile. The kinetic data indicated that the PDS moiety is
largely benign in free radical polymerizations, remaining intact
for subsequent reaction with thiol groups. This has been exam-
plified by a successful conjugation to BSA, as evidenced by SEC
and PAGE analysis.85 A series of block copolymers was also
prepared from the PEG macro-RAFT agent (RA6, Fig. 7).
3.1.1.3 Disulfide bond targeting. Recently, Godwin, Lewis
and co-workers reported the formation of PMPC polymers
designed to possess a a-bis-sulfone terminal group for protein
disulfide bond targeting.86 The ATRP synthesis of PMPC poly-
mers was achieved from a a-bis-sulfide ATRP initiator (I11,
Fig. 5) for specific conjugation to interferon-2a (IFN) disulfide
bonds. A broad range of a-bis-sulfide PMPC molecular weights
was obtained (Mn¼ 29.1–56.3 kDa) in a controlled manner (Mw/
Mn < 1.23), followed by their reaction with oxone to afford
ready-for-conjugation a-bis-sulfone PMPC polymers. The bio-
conjugation of the 2 disulfide bridges of IFN with a-bis-sulfone
PMPC was undertaken at pH 8.2 in the presence of 1,4-dithio-
threitol (DTT). SDS-PAGE revealed the formation of both
mono- and di-PMPC-IFN bioconjugates which gave a marked
resistance to antibody binding while keeping similar antiviral and
antiproliferative activity compared to the native IFN. They also
exhibited an increased pharmacokinetic profile when compared
to their PEGylated counterparts.
Fig. 10 Convergent synthesis of bioconjugates by a combinat
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3.1.1.4 Conjugation using click chemistry. Another coupling
method that recently received a tremendous interest in bio-
conjugation is the copper(I)-catalyzed Huisgen 1,3-dipolar
cycloaddition reaction between an azide and an alkyne
(CuAAC).103,104 This cycloaddition belongs to the class of
chemical reactions, often referred as click chemistry, that share
several very important features: (i) a very high efficiency in terms
of both conversion and selectivity; (ii) mild experimental condi-
tions; (iii) a simple workup and (iv) little or no by products.105–108
For example, Sumerlin and co-workers reported the bio-
conjugation reaction between azide a-functional PNIPAAm and
alkyne functionalized BSA. NIPAAm was polymerized under
AIBN initiation with 2-dodecylsulfanylthiocarbonylsulfanyl-2-
methylpropionic acid 3-azidopropyl ester as a RAFT agent
(RA7, Fig. 7).109 The corresponding well-defined PNIPAAm
(Mn ¼ 16.3 kDa, Mw/Mn ¼ 1.06) was coupled to alkyne-con-
taining BSA (obtained from coupling with propargyl maleimide)
using CuSO4/sodium ascorbate as the catalyst in PBS. The
formation of BSA-PNIPAAm was demonstrated by SDS-PAGE
and SEC analysis. BSA was also reduced with TCEP prior to its
derivatization with propargyl maleimide in order to increase the
number of conjugation sites. Turbidimetry assays indicated that
the conjugates retained their thermoresponsive behaviour.
In a recent study from Lecommandoux, Taton and co-
workers, the coupling of poly(N-dimethylaminoethyl methacry-
late) (PDMAEMA) with polypeptides synthesized from the
polymerization of a-amino acid-N-carboxyanhydrides (NCAs)
(see also section 3.4.3 Combination of NCA polymerization and
CLRP) was achieved by a convergent CuAAC strategy
(Fig. 10).110 N-dimethylaminoethyl methacrylate (DMAEMA)
was polymerized from azide or propargyl a-functional ATRP
initiators (I12 and I13, Fig. 5) in the presence of CuBr/
1,1,4,7,10,10 hexamethyltriethylenetetramine (HMTETA) cata-
lytic system to afford the corresponding a-functional
PDMAEMA (Mn ¼ 8.8 or 10.4 kDa, Mw/Mn ¼ 1.12 or 1.17). In
parallel, poly(g-benzyl-L-glutamate) (PBLG) was synthesized by
NCA polymerization either from 3-azidopropylamine or from
propargylamine to afford well-controlled azide or propargyl
ion of ATRP, NCA polymerization and click chemistry.110
Polym. Chem., 2010, 1, 563–598 | 571
Fig. 11 Synthesis of BSA-poly(PEGA) bioconjugates from vinyl
sulfone-terminated polymers.117
a-functional PBLG. The final step, consisting of a CuAAC
reaction between azide a-functional PDMAEMA and propargyl
a-functional PBLG (or vice versa), was catalyzed by CuBr/
PMDETA and the PBLG-b-PDMAEMA bioconjugates were
shown to maintain low PDIs (Mw/Mn < 1.18) with molecular
weights in the 20.8–21.1 kDa range.
3.1.2 Bioconjugation to the u-terminus. The derivatization of
the u-terminal group (i.e. end-group) of polymers generated by
CLRP techniques is also an efficient way to introduce functional
moieties for further bioconjugation, providing the living fraction
of the polymer is high enough.
3.1.2.1 Conjugation via an amine reactive group. Aldehyde
terminated polymers (reductive amination). The synthesis of u-
aldehyde functionalized poly(N-vinylpyrrolidone) (PVP) by
RAFT polymerization was recently developed by Klumperman
and co-workers.111 N-vinylpyrrolidone (NVP) was polymerized
from a xanthate RAFT agent and the resulting u-thio-
carbonylthio end-group was hydrolyzed in water at 40 �C to yield
u-hydroxy PVP subsequently oxidized at high temperature to its
aldehyde form with more than 90% yield. Coupling to lysozyme
hydrochloride by the reductive amination pathway was then
undertaken and successfully assessed by SDS-PAGE analysis.
3.1.2.2 Conjugation via a thiol reactive group. Maleimide
terminated polymers. To the best of our knowledge, only one
example112 was reported in this category and relied on the design
of star polymer-protein conjugates, based on an extension of
previous studies.113,114 From a tetrafunctionalized trithiocar-
bonate RAFT agent, a four-arm PNIPAAm (Mn¼ 5.47 and 51.6
kDa, Mw/Mn < 1.06) with approximately 95% retention of chain-
end was subjected to radical cross-coupling with a furan-
protected maleimido azo-initator114 and subsequently deprotected
to display the maleimide moieties. Conjugation was achieved with
T4L at pH 7.5 in the presence of EDTA and TCEP to afford
multimeric T4L-PNIPAAm bioconjugates. A careful character-
ization indicated approximately 3 proteins per star polymer.
Pyridyl disulfide terminated polymers. RAFT polymerization
offers the advantage of allowing the trithiocarbonate or the
dithioester u-chain end to be readily converted into a thiol
moiety via aminolysis in the presence of an alkyl amine. The u-
thiol terminus can then react with a pyridyl disulfide group to
afford the desired u-functional polymer.
This pathway was illustrated by Davis and co-workers with
several different polymers such as poly(methyl methacrylate)
(PMMA), polystyrene (PS), poly(PEGA), poly(hydroxypropyl
methacrylate) (PHPMA) and PNIPAAm using 4-cyanopenta-
noic acid dithiobenzoate (CDTB) or 3-(benzylsulfanylth-
iocarbonyl sulfanyl)-propionic acid (BSPA) as RAFT agents.115
In order to maintain a high chain-end fraction, low initiator over
RAFT agent molar ratios and low monomer conversions were
selected (60–75%, Mn ¼ 1–12.5 kDa, Mw/Mn < 1.21). Depending
on the polymer structure, 65–90% of trithiocarbonate or
dithiobenzoate groups were still present after the polymerization.
Subsequent aminolysis with hexylamine followed by reaction
with 2,20-dithiopyridine (DTP) yielded the corresponding PDS-
terminated polymers. PDS u-functional PNIPAAm and
PHPMA were coupled to the NGR model peptide (GNGRGC),
572 | Polym. Chem., 2010, 1, 563–598
known to be a tumor-targeting peptide, in PBS at pH 8 with
a yield of 85–92%.116
Vinyl sulfone terminated polymers. Maynard and co-workers
recently developed a novel technique to target protein cysteine
residues using vinyl sulfone-terminated polymers (Fig. 11).117 A
poly(PEGA) prepared by RAFT (Mn ¼ 6.7 kDa and Mw/Mn ¼1.09) with a high fraction (99%) of dithiobenzoate end-groups
was subjected to a reductive amination followed by the addition
of divinylsulfone in the presence of TCEP (to avoid thiol
oxidation). After only 30 min, 99 � 6% of the poly(PEGA)
contained a vinyl sulfone terminus. Bioconjugation experiments
with BSA (previously reduced to display 3 thiol groups) were
then performed with 20 eq. of vinyl sulfone u-functional poly-
(PEGA). SDS-PAGE confirmed the formation of the desired
BSA-poly(PEGA) bioconjugate that retained 92% of the initial
activity of native BSA.
3.1.2.3 Conjugation using click chemistry. The presence of
a halide chain-end inherent to the ATRP process opened an
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avenue of opportunity for subsequent CuAAC reactions, espe-
cially from styrenic- and acrylate-based polymers.107 After
polymerization, the bromine end-group can be indeed readily
turned into the corresponding azide through a simple quantita-
tive nucleophilic substitution in the presence of azide anions
(N3�).
For example, Cornelissen and co-workers prepared BSA-PS
giant amphiphiles118 by means of CuAAC. A PS block (Mn¼ 4.2
kDa, Mw/Mn ¼ 1.15) prepared in anisole at 90 �C using CuBr/
N,N,N0,N0,N0 0-pentamethyldiethylenetriamine (PMDETA) was
reacted with azidotrimethylsilane and tetrabutylammonium
fluoride. The bioconjugation was then undertaken with a small
model peptide, Gly-Gly-Arg (GGR), tagged with 7-amino-
methylcoumarin (AMC) and functionalized at the N-terminus
with an alkyne function. The same ATRP catalyst was used for
its coupling with the u-azido PS to yield the corresponding PS-
(GGR-AMC) biohybrid. The versatility of this approach was
demonstrated with an alkyne-containing BSA for the construc-
tion of PS-BSA giant amphiphiles. Self-assembly of PS-b-(GGR-
AMC) led to 150–2000 nm vesicles with a broad particle size
distribution whereas PS-BSA bioconjugates yielded 30–70 nm
micelles.
This synthetic methodology was expanded by Lutz and co-
workers to the precise functionalization of RGD peptide
(GGRGDG) with u-azido poly(PEGA)119 and to the anchoring
of TAT peptide120 (GGYGRKKRRQRRRG), a protein trans-
duction domain (PTD) of the human immunodeficiency virus
(HIV), on u-azido PS. The RGD peptide was synthesized by
solid-phase peptide synthesis (SPPS) followed by amidation of
its N-terminus with pentynoic acid and cleavage from the
support (see also section 3.4.1 Solid-phase peptide synthesis of
peptide macroinitiators for CLRP). In parallel, a 6.85 kDa poly-
(PEGA) with low polydispersity index (Mw/Mn ¼ 1.21) was
treated with sodium azide. As a proof of concept, coupling
reactions were demonstrated with low molecular weight alkyl
azides using a CuBr/4,40-di(5-nonyl)-2,20-bipyridine (dNbpy)
catalytic system. Finally, the alkyne functionalized RGD
peptide was successfully attached onto u-azido poly(PEGA)
using CuBr/bpy. Similarly, a TAT peptide was functionalized
with pentynoic acid prior to its reaction with u-azido PS (Mn ¼2.2 kDa, Mw/Mn ¼ 1.21). The final CuAAC coupling between
the protected oligopeptide and the polymer was performed
using CuBr/bpy and afforded the PS-TAT bioconjugate in high
yield.
In a similar approach previously used by Taton and Lecom-
mandoux,110 He and co-workers reported the formation of ABC
triblock polypeptide-polymer bioconjugates using the CuAAC
methodology. In this study, azide u-functional PEG-b-PS121 or
PEG-b-PtBA122 diblock copolymers were synthesized by ATRP
from a PEG-based ATRP macroinitiator using CuBr/PMDETA
catalyst and subsequent azidation of the chain-end. In parallel,
a series of a-propargyl PBLG (Mn ¼ 2.7–26.6 kDa, Mw/Mn <
1.20) and a-propargyl PzLLys (Mn ¼ 10.5–13.1 kDa, Mw/Mn <
1.27) was synthesized by ring-opening polymerization (ROP) (see
also section 3.4.3. Combination of NCA polymerization and
CLRP) of BLG-NCA (g-benzyl-L-glutamate N-carboxyanhy-
dride) and Z-L-Lys NCA (N3-carbobenzoxy-L-lysine N-carboxy-
anhydride), respectively. Final CuAAC (CuBr/PMDETA)
between azide and alkyne derivatives led to well-defined PBLG-
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b-PS-b-PEG and PzLLys-b-PAA-b-PEG triblock copolymer
bioconjugates.
3.1.3 Bioconjugation to both a- and u-termini. Most of the
bioconjugates that consist of a single protein modified with one
or multiple polymer chains have proven to enhance the proper-
ties of protein therapeutics. Nevertheless, some biological
processes that require protein dimers or higher multimers exist.
This is why, during the past few years, a number of studies
directed toward the synthesis of a,u end-functional polymers
obtained from CLRP have flourished in the literature.
For instance, Davis and co-workers published the synthesis of
heterotelechelic polymers for the bioconjugation of proteins
using RAFT. An a-azide, u-PDS heterotelechelic RAFT agent
(RA8, Fig. 7) was synthesized and used to mediate the poly-
merization of five different monomers (MMA, HPMA,
NIPAAm, PEGA and styrene) in the presence of AIBN as
a source of radicals. The resulting difunctional polymers were
obtained with good control over the molecular weight and the
molecular weight distribution (Mn ¼ 3.2–16.2 kDa, Mw/Mn ¼1.08–1.7, conversion ¼ 46–90%).123 Those a,u-heterotelechelic
polymers were then engaged in two consecutive conjugation steps
with: (i) biotin amidopropyne clicked to the azido terminus of the
polymers through CuAAC reaction using CuSO4/sodium
ascorbate catalytic system (90% functionalization with HABA
assay) and (ii) glutathione (g-ECG), a model tripeptide con-
taining a free cysteine or BSA via the PDS group. The coupling
yield between u-PDS PNIPAAm and g-ECG was found to be
95 � 5% whereas only �10% yield was obtained with BSA,
assigned to the steric hindrance between the two macromolecules
involved in the reaction.
Another strategy is based on the synthesis of bis-functional-
ized polymers for bioconjugation to cysteine-containing
biomolecules using a post-polymerization dimerization proce-
dure.124 In this work, a a-dimethyl fulvelene-protected mal-
eimido ATRP initiator (I14, Fig. 5) initiated the polymerization
of styrene at 80 �C in the presence of CuBr/CuBr2/PMDETA
catalyst. The a-functional PS (Mn ¼ 2.31 kDa and Mw/Mn ¼1.15) was engaged in atom transfer radical dimerization reaction
triggered by CuBr/PMDETA in the presence of 4 eq. of nano-
Cu(0) at 70 �C. Bis fulvelene-protected maleimido dimeric PS
(Mn ¼ 4.2 kDa, Mw/Mn ¼ 1.32) was then deprotected and
successfully coupled at both sides with N-acetyl-L-cysteine
methyl ester as a thiol-containing model compound.
A simple route towards heterotelechelic polymers for bio-
conjugation of two different proteins was also reported using the
RAFT technique (Fig. 12). Biotin a-functional PNIPAAm
(Mn ¼ 10.9 kDa, Mw/Mn ¼ 1.08) was obtained from a bio-
tinylated trithiocarbonate RAFT agent (RA9, Fig. 7) and a pro-
tected maleimide was installed at the chain-end by radical cross
coupling at 70 �C between the trithiocarbonate moiety (93%
chain-end) and a protected maleimide azo-initiator.113 However,
to avoid a significant loss of biotin end-group upon retro Diels–
Alder deprotection at high temperature (120 �C), the RAFT
agent must contain only amide groups (RA10, Fig. 7). The
formation of BSA-PNIPAAm-SAv (see also section 4.1 Biotin/
(Strep)avidin binding) heterodimer biohybrid was achieved in two
steps: (i) first by reaction with BSA in PBS and subsequent
purification to remove unreacted polymer and (ii) by using
Polym. Chem., 2010, 1, 563–598 | 573
Fig. 12 Synthetic approach to the formation of protein-heterodimer conjugates via the RAFT technique.113
a fluorescently labeled streptavidin (SAv). The formation of the
corresponding heterodimer was demonstrated by SDS-PAGE
after visualization under UV light or after Coomassie Blue
staining. Following the same pathway but with a symmetrical
bistrithiocarbonate RAFT agent allowed homodimeric polymer-
protein bioconjugate to be obtained.114 This was applied to the
conjugation to T4L. By SDS-PAGE analysis, two new bands
were observed at �48 kDa (21%) and �26 kDa (79%) corre-
sponding respectively to dimeric and monomeric T4L-polymer
conjugates.
3.1.4 Midchain conjugation. Recently, Davis’ group reported
the coupling of a midchain-functional branched PHPMA poly-
mer to BSA.125 A PDS difunctional RAFT agent (RA11, Fig. 7)
was used for the polymerization of HPMA to yield a series of
well-defined PDS-midchain PHPMA. The subsequent coupling
to BSA in PBS at pH 7.4 using an excess of polymer ([polymer]/
[protein] ¼ 30 : 1) was successful as confirmed by SDS-PAGE in
non-reducing conditions. The use of these midchain-functional
polymers could be of great importance considering their
‘‘umbrella-like’’ shape allows a better protection against
proteolytic degradation compared to the use of linear polymers.
3.1.5 Bioconjugation to polymer side chains. As opposed to
classical bioconjugation, where at least one polymer chain is
attached to the protein/peptide (depending on the number of
reactive sites), it can be of great interest to increase the biologi-
cally active moieties/polymer molar ratio. A convenient method
for achieving this multiple attachment relies on the design of
monomers/polymers with reactive side chains towards proteins/
peptides.
The first example reported the synthesis of well-defined
poly(N-methacryloyloxysuccinimide) (PNMS) by ATRP.126 The
polymerization of N-methacryloyloxysuccinimide (NMS) from
a classical ATRP initiator was carried out using CuBr/bpy
catalyst during less than 15 min (80–96% conversion, Mn¼ 12.3–
40.7 kDa, Mw/Mn ¼ 1.13–1.20). The coupling reaction between
574 | Polym. Chem., 2010, 1, 563–598
PNMS and two model peptides (i.e. Gly-OMe and Gly-Gly-b-
naphtylamide hydrobromide) was undertaken and revealed
a good correlation with the stoichiometry of the model peptides
added (the unreacted activated esters were further coupled with
1-amino-2-propanol). With Gly-Gly-b-naphtylamide hydro-
bromide as the model peptide, the conjugation resulted in
a copolymer composed of HPMA biocompatible units89 and
Gly-Gly peptidic pendant side chains. However, it was shown
that sterically-hindered NHS side chains are prone to aminolytic
ring-opening of the succinimide moiety and intramolecular
attack by amides on neighbouring activated esters, leading to
a non-negligible fraction of glutarimide residues.127
The same methodology was also developed for the copoly-
merization of HPMA and N-methacryloyloxysuccinimide by the
RAFT process128 and its further multifunctionalization with
HTSTYWWLDGAPK peptide, which is known to inhibit the
assembly of anthrax toxin.129 Well-defined P(HPMA-co-NMS)
random copolymers (Mn ¼ 4.3–53.6 kDa, Mw/Mn < 1.22)
obtained with a constant NMS ratio of 20 mol% were subjected
to end-group removal (to favour side chains bioconjugation) and
coupled to HTSTYWWLDGAPK peptide via its lysine residue.
Subsequent addition of 1-amino-2-propanol to the reaction
mixture ensured that all NHS esters have reacted. It was shown
that the resulting bioconjugate exhibited much higher anthrax
inhibition efficiency than the monovalent counterpart.
Two other functionalizable side-chain polymers for drug
delivery purposes were successfully developed by Maynard’s
group (Fig. 13).130,131 p-Nitrophenyl methacrylate (pNPMA) and
diethoxypropyl methacrylate (DEPMA) monomers were poly-
merized by RAFT from cumyl dithiobenzoate under AIBN
initiation. The resulting poly(p-nitrophenyl methacrylate)
(PpNPMA) polymer (Mn ¼ 9.6 kDa, Mw/Mn ¼ 1.15) was sub-
jected to coupling with Gly-OMe as a model compound and
yielded a high degree of functionalization (86%). Similarly, the
acetal side chains of poly(diethoxypropyl methacrylate)
(PDEPMA) (Mn ¼ 7.7–14.4 kDa, Mw/Mn ¼ 1.29–1.26) were
subsequently turned into the corresponding aldehyde by acidic
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Fig. 13 Development of polymers with activated ester and protected
aldehyde side chains for bio-functionalization.130,131
catalytic hydrolysis. The newly formed poly(3-formyl ethyl
methacrylate) (PFEMA) polymer then allowed conjugation with
aminooxy-RGD peptide to be readily performed.
3.2 Polymerization from protein/peptide macroinitiators: the
‘‘grafting from’’ method
The opposite pathway, which consists of growing a polymer
chain by CLRP from a protein/peptide macroinitiator, the so-
called ‘‘grafting from’’ method, has recently received significant
interest due to its two main potential advantages: (i) a higher
bioconjugation efficiency is anticipated due to a lower steric
hindrance and (ii) the purification of the final materials is easier
as only small molecules have to be removed such as unreacted
monomer (and possibly ATRP catalyst or remaining radical
initiator for RAFT), in contrast to preformed polymer for the
‘‘grafting to’’ approach.
Matyjaszewski,132 Haddleton,133 Maynard84 and Velonia,134
investigated the ‘‘grafting from’’ strategy in combination with the
ATRP process. From the reaction between amino groups of
lysine residues and 2-bromoisobutyryl bromide, Matyjaszewski
and co-workers were able to control the attachment of ATRP
initiating moieties on a-chymotrypsin by varying the [ATRP
initiator]0/[a-chymotrypsin]0 molar ratio.132 A ratio of 12 : 1 gave
a single initiating site whereas increasing this ratio from 43 : 1 up
to 85 : 1 resulted in the conjugation of respectively 3–7 and 7–10
initiator moieties per protein (catalytic activity was above 90%
each time). These a-chymotrypsin ATRP macroinitiators initi-
ated the polymerization of PEGMA in PBS at pH 6.0 with
a CuBr/bpy catalyst. The almost monodisperse a-chymotrypsin-
poly(PEGMA) bioconjugates were obtained with a good control
whereas the typical ‘‘grafting to’’ method using either mono-
methoxy poly(ethylene glycol)-succinimidyl propionate
(MePEG-SPA) or NHS-terminated poly(PEGMA) afforded
a mixture of bioconjugates with higher PDI values. The authors
This journal is ª The Royal Society of Chemistry 2010
also observed that the bioconjugates retained from 50 to 86% of
the bioactivity of the native a-chymotrypsin.
Maynard and co-workers investigated the in situ formation of
protein-polymer bioconjugates using either BSA or T4L as
protein macroinitiators.84 BSA was first reduced with TCEP in
order to maximize the number of free thiols. The resulting free
cysteine residues were then coupled with a thiol-reactive PDS
a-functional ATRP initiator (I10, Fig. 5) to afford a mixture of
BSA macroinitiators with either 1 or 3 initiation sites. Then,
NIPAAm was polymerized in water with CuBr/bpy in the pres-
ence or absence of 2-bromoisobutyrate-functionalized resin as
a sacrificial initiator. The presence of the disulfide linkage
allowed for further cleavage of PNIPAAm from BSA which was
then analyzed by SEC (Mw/Mn¼ 1.34). The polymerization from
T4L was also assessed and the bioconjugate activity was main-
tained after the polymerization step.
Haddleton and co-workers turned BSA and lysozyme into
efficient protein macroinitiators with maleimido or NHS func-
tionalized ATRP initiators, respectively (I1 and unprotected I9,
Fig. 5).133 The corresponding BSA macroinitiator initiated the
polymerization of PEGMA or DMAEMA using CuBr/N-
(ethyl)-2-pyridylmethanimine as the catalytic system. In order to
better monitor the bioconjugation reaction, small amounts of
either hostasol methacrylate (HMA) or rhodamine methacrylate
(RMA) fluorescent co-monomers were added. The resulting
fluorescent bioconjugates were then successfully characterized by
SDS-PAGE, SEC-HPLC and fluorescence spectroscopy.
Conferring fluorescent properties to bioconjugates is a general
approach that facilitates their detection during biomedical
assays.
More recently, Velonia demonstrated the formation of
amphiphilic BSA-PS bioconjugates by in situ ATRP and their
further hierarchical self-assembly in aqueous solution to form
bio-nanocontainers and nanoreactors.134 Following the work
previously reported by Haddleton and co-workers,133 a BSA
ATRP macroinitiator was prepared and engaged in emulsion
polymerization (10% DMSO, PBS pH 7.4) of styrene triggered
by addition of the CuBr/N-(propyl)-2-pyridylmethanimine
catalyst. Well-defined BSA-PS bioconjugates (Mw/Mn < 1.2)
were obtained from high monomer-to-macroinitiator ratios
(1500 : 1 up to 3000 : 1). Enzymes entrapment experiments have
also been performed with fluorescently tagged papain or Horse
Radish Peroxidase (HRP). Further on, the catalytic activity of
HRP was investigated based on the oxidation of 3,30,5,50- tet-
ramethylbenzidine (TMB, substrate of HRP) in the presence of
hydrogen peroxide (H2O2). The catalytic reaction took place in
the nanoassemblies: whereas TMB was able to permeate into the
superstructures (being catalytically transformed and then
released in solution), catalytically active proteins (HRP, papain)
were retained in the superstructures. This study highlighted
a potential application in the area of biotechnology.
RAFT polymerization was also employed by Davis135,136 and
Sumerlin137 for the in situ formation of polymer-protein bio-
conjugates. Davis and co-workers functionalized BSA with
a PDS-based RAFT agent (RA12, Fig. 7) and used the resulting
BSA macro-RAFT agent to mediate the aqueous polymerization
of PEGA upon g-irradiation to initiate the polymerization.135
The formation of BSA-poly(PEGA) bioconjugate was confirmed
by SEC and by non-reducing PAGE. Aliquots from the
Polym. Chem., 2010, 1, 563–598 | 575
Fig. 15 Incorporation of amino acid initiator for precise biohybrid
synthesis.143
polymerization medium were withdrawn at regular intervals of
time and, after cleavage of the bioconjugates with TCEP aqueous
solution, the poly(PEGA) was recovered and analyzed by SEC to
show the linear evolution of poly(PEGA) molecular weight with
conversion and the low polydispersity indexes obtained with the
RAFT technique. NIPAAm and hydroxyethyl acrylate (HEA)
were also polymerized from the BSA macro-RAFT agent in PBS
at pH 6.0 (to avoid possible hydrolysis of RAFT agent and/or
denaturation of the protein) using VA044 as a free radical ini-
tator (Fig. 14).136 The linear evolution of the logarithmic
conversion was observed with time and SEC analysis revealed the
formation of biomacromolecules with hydrodynamic diameters
larger than that of native BSA.
Sumerlin and co-workers also took advantage of the ‘‘grafting
from’’ approach to achieve the synthesis of thermally responsive
BSA-PNIPAAm bioconjugates with tunable bioactivity via the
RAFT technique.137 After selective coupling of the BSA free
cysteine to a maleimido RAFT agent (RA13, Fig. 7), NIPAAm
was polymerized in PBS at pH 6 to give the corresponding BSA-
PNIPAAm bioconjugates. The logarithmic monomer conver-
sion, followed by both 1H NMR and gravimetry analysis, was
linear and suggested a constant concentration of propagating
radicals in the polymerization medium up to high monomer
conversion. Aqueous SEC and SDS-PAGE permitted the
formation of the bioconjugates to be assessed. TCEP protein
degradation allowed to characterize the PNIPAAm polymer
attached to the protein and revealed an efficient control up to
94% monomer conversion (Mn ¼ 234 kDa and Mw/Mn ¼ 1.38).
Circular dichroism (CD) spectroscopy indicated that BSA
retained its secondary structure even after coupling with the
maleimide functionalized RAFT agent or after polymerization of
Fig. 14 Synthesis of BSA-poly(HEA) and BSA-poly(NIPAAm) conju-
gates by RAFT polymerization using a BSA-macroRAFT agent.136
576 | Polym. Chem., 2010, 1, 563–598
NIPAAm. Interestingly, the responsive behaviour of the immo-
bilized polymer facilitated the isolation of the bioconjugate and
also allowed environmental modulation of its bioactivity.
Deriving from the work reported on the CLRP of nucleoside-
containing monomers and/or initiators,138–141 Venkataraman and
Wooley developed the synthesis of an amino-acid-based ATRP
initiator for bioconjugates synthesis. O-protected L-valine was
reacted with 2-bromopropionyl bromide to afford the resulting
amino-acid-based ATRP initiator that initiated the sequential
polymerization of tert-butyl acrylate (tBA) and styrene in the
presence of CuBr/PMDETA catalyst.142 All the criteria of CLRP
were obtained, leading to the formation of well-defined O-pro-
tected L-valine-PtBA-b-PS block copolymer (Mn ¼ 22.5 kDa,
Mw/Mn ¼ 1.22).
An original strategy to prepare peptide-polymer conjugates
with precise sites of attachment was reported by Maynard and
co-workers (Fig. 15).143 The concept was to design an artificial
amino acid containing an ATRP initiator moiety as a side chain,
followed by its incorporation into a peptide sequence and the
initiation of the polymerization from the resulting biomaterial.
This is exemplified by: (i) Fmoc-protected tyrosine modified with
1-chloroethyl-phenyl group suitable for ATRP of styrene and (ii)
Fmoc-Ser(OTrt)-OH modified with the 2-bromoisobutyrate
group for ATRP of methacrylates. Kinetic studies under optimal
conditions indicated a controlled polymerization with low PDI
(# 1.25).
3.3 Polymerization of peptide-based monomers: the ‘‘grafting
through’’ method
Another very interesting and promising alternative to the
anchoring of proteins/peptides on the pendant side chains of
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polymers relies on the polymerization of peptide-based mono-
mers.144,145 It offers the advantage over the ‘‘grafting to’’ method
that the functionalization is quantitative and does not require
additional post-polymerization steps.
Van Hest brought a significant breakthrough in this
domain,146 as his group synthesized several peptide-based (glu-
tamic acid,147 VPGVG,147,148 AGAG,149,150 Gramicidin S151)
methacrylates either by solution- or solid-phase peptide
synthesis. These monomers were polymerized by ATRP in the
presence of CuCl/bpy in DMSO (essential for efficient solubili-
zation) to form hybrid polymers or hybrid diblock copolymers
(Fig. 16). Glutamic acid-based methacrylate was synthesized
upon DCC coupling of O-tBu, N-Boc protected glutamic acid
with HEMA. The glutamic acid-based methacrylate (Glu-EMA)
was then polymerized either from ethyl-2-bromo isobutyrate
(EBIB) or di-a,u-bromoisobutyrate-PEG macroinitiator to
afford the corresponding homopolymer (Mn ¼ 8.7 kDa, Mw/
Mn ¼ 1.11) and diblock copolymer (Mn ¼ 10.9 kDa, Mw/Mn ¼1.22).147 Similarly, the VPGVG thermoresponsive peptide
sequence (upon LCST, a random coil to a type II b-turn tran-
sition is observed), which is predominantly present in tropoe-
lastin, was transformed into the corresponding VPGVG-based
methacrylate (VPGVG-MA) and polymerized with the same
initiators and the same catalytic system. It yielded either
a P(VPGVG-MA) homopolymer (Mn ¼ 53.0 kDa, Mw/Mn ¼1.25) or a P(VPGVG-MA)-b-PEG-b-P(VPGVG-MA) ABA tri-
block copolymer (Mn ¼ 60.0 kDa, Mw/Mn ¼ 1.24).147 More
interestingly, those materials retained the thermally-responsive
feature associated to the VPGVG peptidic sequence, as observed
by CD spectroscopy and turbidity measurements. The authors
observed that increasing the concentration of this biohybrid
triblock copolymer or increasing the peptidic block length
resulted in a decrease of the LCST whereas the increase of the pH
solution induced lower LCST.148
The polymerization of VPGVG-MA was also successfully
undertaken by Cameron and co-workers using the RAFT
Fig. 16 Polymerization of peptide-based monomers by ATRP.147–151
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polymerization with 4-cyanopentanoic acid dithiobenzoate and
V-501 initiator at 70 �C.152 Surprisingly, the authors found that
transition temperatures of those materials were much lower than
those reported for the P(VPGVG-MA)-b-PEG-b-P(VPGVG-
MA) triblock copolymer.148 This was assigned to the presence of
the PEG central block that might affect the ability of the elastin-
based polymers (EBPs) to phase separate and/or to the higher
molecular weight obtained in the work of Cameron. The influ-
ence of the pH, the EBP concentration and the EBP molecular
weight over the LCST of the polymer solutions was then inves-
tigated. A decrease of the LCST was observed by lowering the
pH. Similarly, as expected from a previous report on elastin
linear polymers (ELPs),153 a linear decrease of the LCST was
observed when the concentration of the polymer was increased.
Finally, a linear decrease of the LCST as a function of the
increasing molecular weight of the EBPs was demonstrated.154,155
N-Boc protected AGAG-based methacrylate (Fig. 16), derived
from the AGAG peptidic sequence (part of the silk protein) also
known to induce b-sheets formation, and MMA were sequen-
tially polymerized by ATRP in DMSO from 1,4-(20-bromo-20-
methylpropionato)benzene (difunctional ATRP initiator) to
afford a poly(methyl methacrylate)-b-poly(N-Boc-AGAG-MA)-
b-poly(methyl methacrylate) triblock copolymer with good
control (Mw/Mn¼ 1.19).149,150 FTIR investigations confirmed the
capability of these AGAG-based block copolymers to form b-
sheet structures. Van Hest also reported the possibility of poly-
merizing bulky cyclic peptide-based methacrylates such as
Gramicidin S (cyclic decapeptide), an analogue of an antibiotic
that had the propensity to form b-sheets (Fig. 16). Polymeriza-
tion was performed under ATRP conditions from EBIB using
CuCl/PMDETA as the catalyst, yielding the poly(Gramicidin S
methacrylate) with a narrow MWD (Mw/Mn ¼ 1.09) after 65%
monomer conversion.151 As shown by FTIR analysis, the
propensity to form intramolecular b-sheets was maintained.
3.4 In situ synthesis of polymer-peptide bioconjugates
In this section, the synthesis of both the polymer and the peptide
is undertaken, which allows great flexibility over the overall
design of the resulting bioconjugate. This can be achieved either
by solid phase peptide synthesis or by the polymerization of
NCA. Whereas, the first approach is generally limited to
medium-sized peptides, the latter allows longer peptide
sequences to be obtained in good yield and large quantities but
without controlling the primary amino acid sequence.156,157
3.4.1 Solid-phase peptide synthesis of peptide macroinitiators
for CLRP. B€orner and co-workers developed the SPPS (Fmoc
strategy) of a pentapeptide-based (DFGDG) ATRP initiator
followed by its cleavage from the resin and subsequent poly-
merization.158 Once the peptide was formed at the surface of the
resin, its N-terminus was functionalized with 2-bromopropionic
acid via a classical DCC coupling. TFA cleavage from the resin
afforded the desired ATRP macroinitiator that was used to
initiate the polymerization of nBA in DMSO using CuBr/CuBr2/
PMDETA catalyst to yield the corresponding DFGDG-PnBA
bioconjugate (Mn¼ 11.0 kDa, Mw/Mn¼ 1.19). However, as long
as polypeptides have an inherent propensity to coordinate metal
ions on their polyamide backbone, a rather-slow polymerization
Polym. Chem., 2010, 1, 563–598 | 577
rate was observed along with a non-constant concentration of
propagating radicals. Although this problem was circumvented
by increasing the catalyst-to-initiator ratio, the authors decided
to switch to RAFT polymerization via the design of dithio-
benzoate-terminated DFGDG- or GGRGDS-based RAFT
agent.159,160 The resulting DFGDG-PnBA bioconjugate (Mn ¼4.1 kDa, Mw/Mn ¼ 1.18) was well-controlled and circular
dichroism (CD) spectroscopy showed the preservation of the
chirality.
SPPS in combination with ATRP were also employed by Van
Hest and co-workers, for oligopeptide-polymer bioconjugates
synthesis (Fig. 17).161 The protected Ser-Gly-Ala-Gly-Ala-Glu-
Gly-Ala-Gly-Ala-Ser-Gly peptide was grown from Wang resin
followed by deprotection and derivatization of the two Ser resi-
dues with 2-bromoisobutyryl bromide. After cleavage from the
support, polymerization of MMA with CuCl/PMDETA
complex was undertaken. A linear first-order kinetic plot and
a molecular weight of 1.12 kDa were obtained. The two PMMA
blocks were then recovered upon basic conditions revealing
a PDI of 1.17. Self-assembly behaviour study of the PMMA-
Ser-Gly-Ala-Gly-Ala-Glu-Gly-Ala-Gly-Ala-Ser-Gly-PMMA
triblock-like copolymer bioconjugate indicated a propensity to
form hollow polymersomes and large compound micelles in
aqueous solution. Unfortunately, the initial structural confor-
mation of the oligopeptide (b-hairpin) was not maintained upon
polymerization of MMA. In contrast, it led to a random-coil
structure, assigned to the steric hindrance of the PMMA blocks
or to an aggregation process that was too fast.
Similarly, the RGD peptide (GRGDSP) was grown using
Fmoc standard chemistry, deprotected and subsequently ami-
dated with 2-bromo-2-methylpropionic acid. The released
peptide-based ATRP initiator triggered the polymerization of
dimethyl acrylamide (DMAA) in DMSO with the CuCl/tris[2-
(dimethylamino)ethyl]amine (Me6TREN) catalyst. Even though
side reactions led to non-linear first-order kinetic plots,
GRGDSP-poly(dimethyl acrylamide) (GRGDSP-PDMAA)
bioconjugates were obtained (Mn ¼ 15–30 kDa) with rather low
polydispersity indexes (1.4–1.6).162 The bioconjugates were then
Fig. 17 Synthesis of oligopeptide-polymer bioconjugates by a com
578 | Polym. Chem., 2010, 1, 563–598
tethered on a silane surface through a photochemical immobili-
zation process involving a benzophenone moiety already present
at the surface of the glass slide. The authors were able to tune the
apparent peptide film concentration by blending the GRGDSP-
PDMAA with PDMAA homopolymer. The immobilized
GRGDSP-PDMAA bioconjugate, containing a cell repelling
polymer block and a cell-promoting adhesion polypeptide (RGD
sequence), eventually showed a promoted cell adhesion feature of
human skin fibroblasts (even at 0.02 wt% of peptide in the film).
This strategy also allowed the synthesis of micropatterned
peptide-polymer films that can be devoted to live-cell biochip
applications.
Biesalski and co-workers took advantage of the ability of
a cyclic octapeptide with an even number of alternating D- and L-
amino-acids that self-assemble into well-defined nanotubes, to
carry on the polymerization by ATRP of NIPAAm from these
nanoarchitectures.163,164 The cyclooctapeptide was synthesized
by standard Fmoc protocols on a Wang-Tentagel resin, difunc-
tionalized on its 2 lysine residues by amidation with 2-bromo-N-
butyl-2-methylpropanamide and cleaved from the resin.
Following the self-assembly of these cyclic peptides by intermo-
lecular hydrogen bonding,165–169 peptide nanotubes were formed
and displayed at their periphery ATRP initiation sites suitable
for subsequent surface-initiated ATRP of NIPAAm in aqueous
dispersion.163 By tuning the polymerization time, the authors
were able to tune the length of the polymer chains grown at the
surface of the peptides nanotubes. More importantly, the length
of the peptide nanotubes remained the same until a polymeriza-
tion time of �5 h. After this time limit, small and uniform
particles appeared and the concentration of the peptide-polymer
hybrid nanotubes (PPNTs) dramatically decreased, which was
assigned to a break-up of the PPNTs into smaller and well-
defined nanoobjects.164 Complementary studies have been
recently reported using a cyclic peptide modified at 3 distinct
positions with ATRP initiation sites.170 AFM and FTIR inves-
tigations demonstrated that the PnBA-cyclic peptide adopted
core-shell rod-like nanoarchitectures with an internal b-sheet
structure surrounded by a soft PnBA coating.
bination of solid-phase peptide synthesis (SPPS) and ATRP.161
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Alkyne-terminated PNIPAAm (Mn ¼ 2 kDa, Mw/Mn < 1.2)
obtained from RAFT was successfully coupled via CuAAC to
MUC1 VNTR peptide bearing N-terminal azide group, obtained
from SPPS. By self-assembly of the conjugate in water, nearly
uniform micelles of 60 � 3 nm in diameter were obtained, pre-
senting multiple copies of MUC1 VNTR peptide on its
surface.171 Further functionalization of the thiol end-group was
also undertaken with pyrene maleimide.
3.4.2 Solid-phase peptide synthesis of bioconjugates. Alter-
natively, the whole bioconjugate can be synthesized on a resin
support as shown by Wooley and co-workers using either
ATRP172 or NMP173 techniques. In this work, shell-cross-linked
(SCK) nanoparticles (see also section 3.5.2 Bioconjugation to
nanoparticles) of poly(3-caprolactone)-b-poly(acrylic acid) (PCL-
b-PAA) block copolymers174 were fluorescently tagged with
fluorescein-5-thiosemicarbazide. In parallel, the protected TAT
peptide sequence was prepared by SPPS. The coupling between
the N-terminal residue of glycine extended peptides and the
carboxylic acid groups of the PAA shell of the SCK nano-
particles was performed in the presence of EDC as the coupling
reagent. After removal of unreacted SCK nanoparticles by
simple washings, the TAT-decorated SCK nanoparticles were
recovered from the resin by acidic cleavage that also allowed the
simultaneous TAT amino-acids deprotection and the degrada-
tion of the PCL core of the nanoparticles. TAT-derivatized
fluorescent nanocage structures175 were collected by precipitation
and purified by dialysis. Investigations regarding the interactions
of the PTD-derivatized nanocage structures with two cell types,
namely Chinese hamster ovary (CHO) or HeLa cells were also
possible thanks to the fluorescent labeling. Confocal laser scan-
ning microscopy (CLSM) indicated that the fluorescein-labeled
TAT-nanocages were located at the cell surface. Alternatively,
when the TAT peptide was functionalized with a fluorine-labeled
TIPNO-based (N3, Fig. 2) alkoxyamine for conducting sequen-
tial NMP of tBA and methyl acrylate (MA) at�130 �C, cleavage
from the resin and simultaneous deprotection of tert-butyl
groups led to well-defined TAT-poly(acrylic acid)-b-poly(methyl
acrylate) (TAT-PAA-b-PMA) hybrid block copolymers.173
To demonstrate the versatility of this methodology, Wooley
also reported the synthesis of bioconjugates from an antimicro-
bial peptide tritrpticin (VRRFPWWWPFLRR) using both
NMP or ATRP.176 Tritrpticin was synthesized by Fmoc SPPS
and coupled with fluorine-labeled TIPNO-based (N3, Fig. 2)
alkoxyamine or with 2-bromoisobutyryl bromide for conducting
NMP or ATRP, respectively. Sequential polymerization of tBA
and styrene from the immobilized tritrpticin-derivatized macro-
initiator yielded well-defined tritrpticin-PAA-b-PS block copoly-
mers. It was observed that tritrpticin-PAA-b-PS was able to self-
assemble into 51 nm micelles of narrow particle size distribution
together with a small proportion of larger-scale aggregates.
Micelles decorated with tritrpticin showed an enhancement of
the antimicrobial activity against Staphylococcus aureus (S.
aureus) and Echerichia coli (E. coli) when compared to the free
peptide.
Washburn and co-workers also developed the solid-phase
peptide synthesis of PHEMA-GRGDS bioconjugates by
ATRP.177 The GRGDS peptide functionalized with an ATRP
initiator triggered the polymerization of HEMA with CuCl/bpy
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at 50 �C. The acidic cleavage of the bioconjugate from the resin
afforded rather well-defined GRGDS-PHEMA bioconjugates
(Mw/Mn ¼ 1.47). It was shown that the bioconjugates promoted
the bioadhesion and spreading of mouse NIH-3T3 fibroblast
cells.
3.4.3 Combination of NCA polymerization and CLRP. The
polymerization of a-amino-acid-N-carboxyanhydrides, which
proceeds by a ring-opening mechanism initiated by nucleophiles
or bases such as primary amines or alkoxides,178–183 has been
successfully combined with CLRP for the conception of well-
defined polypeptide hybrid copolymers.
By using sequential ATRP and NCA polymerization, Chaikof
and co-workers prepared ABA poly(L-alanine)-b-poly(2-acryl-
oyloxyethyl-lactoside)-b-poly(L-alanine) (PLA-b-PAEL-b-PLA)
triblock bioconjugates.184,185 Dibromoxylene was used as
a difunctional ATRP initiator for the polymerization of 2-
acryloyloxyethylocta-acetyl-lactoside (AEL, G7, see Fig. 23)
with CuBr/bpy. By tuning the [monomer]0/[initiator]0 molar
ratio, a range of glycopolymers (see also section 4.3 Glycopoly-
mers and sugar-protein interaction) with Mn from 9.3 to 38.2 kDa
were prepared in a controlled fashion (Mw/Mn ¼ 1.19–1.35). The
bromine end-group was then converted into an amine to initiate
the ring-opening polymerization (ROP) of Ala-NCA, leading to
the formation of PLA-b-PAEL-b-PLA triblock hybrid copoly-
mers after deprotection of the O-protecting acetyl groups. The
same approach was used for the preparation, after removal
of the benzyl group and deacetylation of the lactose units, of
poly(L-glutamate)-b-poly(2-acryloyloxyethyl-lactoside)-b-poly-
(L-glutamate) triblock hybrid copolymers185 by NCA polymeri-
zation of b-benzyl-L-glutamate (BLG). When these amphiphilic
triblock hybrid copolymers were aggregated in aqueous solu-
tion, FT-IR spectroscopy demonstrated that the a-helix/b-
sheet ratio increased with an increase of the polypeptide block
length.
A similar approach was adopted by Brzezinska and Deming
for the synthesis by sequential ATRP and NCA polymerization
of diblock poly(g-benzyl-L-glutamate)-b-poly(methyl methacry-
late) (PBLG-b-PMMA) hybrid copolymers.186 After ATRP of
MMA, the bromine end-group of the resulting PMMA block
(Mn ¼ 3.8 kDa, Mw/Mn ¼ 1.2) was converted into an amine
moiety via a three-step reaction187 and subsequently transformed
into the required macroinitiator to initiate the ROP of g-benzyl-
L-glutamate NCA. Well-defined diblock PBLG-b-PMMA
copolymers were eventually obtained (Mw/Mn # 1.2).
Along similar lines, this strategy was used by Taton and co-
workers for the synthesis of AB2 miktoarm polystyrene-b-(poly-
(glutamic acid))2 (PS-b-(PGA)2) star copolymers.188 In this work,
styrene was polymerized by ATRP before a specific chain-end
modification step was achieved to introduce a gemini amino
group to further initiate NCA polymerization. After ring-
opening polymerization of g-benzyl-L-glutamate (BLG), a PS-b-
(PBLG)2 was obtained with low polydispersity (Mw/Mn # 1.3)
and with Mn in the 6.0–25.1 kDa range, followed by hydrolysis of
the g-benzyl to yield the corresponding PS-b-(PGA)2 miktoarm
copolymer. Upon dispersion in aqueous solution, these PS-b-
(PGA)2 copolymer self-assembled into well-defined micelles that
exhibited pH-responsive properties. A random coiled structure at
pH > 12 with RH ¼ 16 nm was observed whereas at pH < 5, the
Polym. Chem., 2010, 1, 563–598 | 579
PGA blocks took a compact a-helix conformation, leading to
a slight decrease of the average particle size (RH ¼ 11 nm). When
a trifunctional ATRP initiator was used, well-defined PS-b-
(PBLG)3 star copolymers were obtained (Mn ¼ 10.5–28.1 kDa,
Mw/Mn ¼ 1.2–1.4).189 They exhibited a higher conformational
stability than their linear counterparts, as observed by DSC and
IR spectroscopy.
The utilization of a heterobifunctional initiator for both
ATRP and ROP of NCA represents an alternative strategy to the
chain-end modification method previously described. In this
view, such a molecular initiator was developed by Menzel and co-
workers for the sequential NCA polymerization of BLG and
ATRP of MMA through a divergent chain growth. It comprised
a nickel amido-amidate terminus for NCA polymerization and
a classical ATRP initiating site (I15, Fig. 5).190 Even though poor
initiation efficiencies were observed, a good control of the BLG
polymerization was observed (Mw/Mn ¼ 1.2–1.4) and the
resulting PBLG block displayed the expected a-helical structure.
ATRP of MMA catalyzed by CuBr/HMTETA or CuBr/bpy in
DMF at 80–90 �C was then undertaken and led to Mn in the 41.0
to 110.0 kDa range with low polydispersities (Mw/Mn ¼ 1.20–
1.39).
In two more recent studies from the same group, NCA
polymerization in combination with either NMP191,192 or
ATRP191 was investigated to construct bioconjugates from
difunctional initiators (Fig. 18). ROP of BLG-NCA was initi-
ated from an amino TIPNO-based (N3, Fig. 2) alkoxyamine,
followed by the NMP polymerization of styrene via a one-pot
process to afford PBLG-b-PS bioconjugates with low PDI (Mw/
Mn � 1.1).192 In the second study, double-headed initiators were
prepared: (i) I15 (Fig. 5) was used for the sequential ROP of
BLG-NCA and ATRP of MMA while (ii) the second one, based
on the TIPNO (N3, Fig. 2) alkoxyamine, was used for the
sequential ROP of BLG-NCA and NMP polymerization of
styrene.191 Well-defined PBLG-b-PMMA were obtained with
rather-low PDI whereas the obtaining of PBLG-b-PS required
a fine tuning of the NMP reaction conditions in order to ensure
a good control.
Fig. 18 Synthesis of bioconjugates by NCA polym
580 | Polym. Chem., 2010, 1, 563–598
3.5 Bioconjugation to surfaces
CLRP methods also represent a convenient approach for the
synthesis of well-defined polymer-grafted surfaces193–196 and have
been naturally applied to design novel grafted polymer-protein/
peptide bioconjugates.
3.5.1 Bioconjugation to planar surfaces. An original immo-
bilization approach of proteins onto surfaces via CLRP was
developed by Klok and co-workers.197 A glass slide was submitted
to a pretreatment for the anchoring of ATRP initiator moieties (3-
(2-bromoisobutyramido) propyl(trimethoxy)silane). This graft-
ing allowed further surface-initiated ATRP polymerization in
aqueous conditions of different hydrophilic methacrylate mono-
mers (HEMA and PEGMA of different PEG chain lengths). The
hydroxyl terminus of the polymer brushes was functionalized by
p-nitrophenyl chloroformate (NPC) and derivatized with O6-
benzylguanine (BG) to afford immobilized BG-functionalized
polymer brushes. Taking advantage of the ability of O6-alkyl-
guanine-DNA alkyltransferase (AGT)-fusion protein to transfer
the alkyl group of O6-alkylated guanine derivatives to one of its
cysteine residues, a chemoselective immobilization of a protein of
choice, i.e. dihydrofolate reductase (DHFR, protein fusion of
AGT), onto the poly(PEGMA) and PHEMA polymer termini
was performed. By varying the amount of BG moieties at the
surface of the polymer brushes, the authors were also able to
carefully tune the surface density of the AGT fusion protein.
Kang and co-workers reported the synthesis of Si(111)-grafted
poly(glycidyl methacrylate) (PGMA) brushes for subsequent
glucose oxidase (GOD) immobilization by ATRP (Fig. 19).198
This procedure was first based on a covalent attachment of 4-
vinylbenzyl chloride (VBC) on a Si(111) surface via radical-
induced hydrosilylation. Subsequently, glycidyl methacrylate
(GMA) was polymerized in a DMF–water mixture using a CuCl/
CuCl2/bpy catalyst to obtain the Si-g-PGMA hybrid surface. In
the final step, GOD was anchored on the Si-g-PGMA polymer by
epoxy ring-opening of the PGMA with amine moieties of lysine
residues, with a concentration estimated to be 0.17–0.23 mg cm�2.
erization and subsequent ATRP or NMP.191,192
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Fig. 19 Design of Si(111)-grafted poly(glycidyl methacrylate) brushes by ATRP for glucose oxidase (GOD) immobilization.198
The authors observed that this concentration increased with the
thickness of the grafted PGMA layer until a plateau where the
accessibility of GOD to the surface was restricted by steric
hindrance due to the polymer brushes. Activity enzyme tests
revealed that the immobilized GOD displayed a good stability and
a relative activity of �60% when compared to an equivalent
amount of free native GOD, which is higher than with previously
developed immobilization methods.199–202
Not only inorganic but also biological surfaces can be of
interest for bioconjugation investigations. For example, Had-
dleton and co-workers used hair as a biological surface due to
keratin fibres that can be easily targeted with amine reactive
functional polymers.203 Well-defined a-NHS fluorescently tagged
poly(PEGMA) copolymers were prepared in toluene using CuBr/
N-(ethyl)-2-pyridylmethanimine as the catalyst (Mn ¼ 11.8–45.6
kDa, Mw/Mn¼ 1.08–1.48) from I2 (Fig. 5), followed by coupling
with hair at ambient temperature and careful rinsing. CLSM
showed intense fluorescence on the outer layer of the hair, in
agreement with a covalent polymer coating. Besides, DSC
experiments demonstrated a substantial increase of the dena-
turation temperature of the a-helical material of the keratin
fibres upon bioconjugation.
3.5.2 Bioconjugation to nanoparticles. Nanoparticles func-
tionalized with peptides/proteins in order to cross biological barriers
and/or to deliver therapeutic agents (i.e. to achieve specific target-
ing), represent a promising area of research in nanomedicine. In the
past few years, several examples reported the biofunctionalization
of nanoparticles synthesized by CLRP techniques.
Wooley and co-workers developed a method in which the TAT
peptide was coupled to SCK nanoparticles.204,205 These
This journal is ª The Royal Society of Chemistry 2010
nanoparticles were obtained by self-assembly of poly(acrylic
acid)-b-poly(methyl acrylate) (PAA-b-PMA) prepared by
sequential ATRP polymerization of tBA and MMA using CuBr/
PMDETA and successive removal of tert-butyl groups. To
ensure stability, a cross-linking reaction between carboxylic
groups of the PAA shell and 2,20-(ethylenedioxy) bis(ethylamine)
via carbodiimide activation was performed. The TAT peptide
extended by 4 additional glycine residues at the N-terminus was
synthesized by SPPS and coupled within the nanoparticle shell
with a carbodiimide-assisted coupling reaction. This study
clearly demonstrated that the presence of the TAT peptide
increased the intracellular uptake of the SCK nanoparticles.
Besides this, in vivo and in vitro evaluations of these TAT-func-
tionalized SCK nanoparticles were also undertaken and showed
a preliminary assessment of their biocompatibility.205
The versatility of the method was demonstrated by the coupling
of lysine-terminated A- or T-rich complementary peptide nucleic
acid (PNA) sequences. In this way, PNA-decorated micelles that
display different amounts of PNA moieties available at their
surface (governed by the initial stoichiometry) were prepared.
Due to the complementary bases tethered at their surface, A-rich
and T-rich PNA-decorated micelles were able, by simple base
pairing, to self-assemble into higher-ordered structures, as
observed by AFM.206 It’s worth mentioning that this strategy was
also extended to the formation of nanoparticles functionalized
with antigen for antibody-binding properties,207 saccharides for
protein recognition,208 and folic acid for cancer-cell targeting,209
thus making it a powerful and truly versatile methodology.210
ATRP in combination with another transition-metal-cata-
lyzed polymerization method, namely chain walking polymeri-
zation (CWP), has also been presented for the synthesis of
Polym. Chem., 2010, 1, 563–598 | 581
polymer-protein colloidal bioconjugates.211 CWP is a powerful
polymerization technique that offers great control over the
macromolecular structure.212–215 A dendritic macroinitiator core
was first synthesized by copolymerization of ethylene and
a comonomer bearing an ATRP initiator moiety by CWP cata-
lyzed by a chain walking palladium-a-diimine.212,216 ATRP of
PEGMA was performed on the resulting dendritic macro-
initiator at ambient temperature under CuBr/CuBr2/dNbpy
catalysis and afforded core-shell nanoparticles. By changing the
polymerization time, different copolymers were obtained with
a broad range of molecular weights (Mn ¼ 610–9180 kDa, Mw/
Mn ¼ 1.2–1.5) and with a radius of gyration, Rg, in the 19–64 nm
range. The dendritic nanoparticles were then functionalized (49%
yield) with N-acryloyloxysuccinimide by capping each methac-
rylate chain-end, for further conjugation to biomolecules
through amidation reaction. The reactivity and bioavailability of
the NHS-activated esters were assessed with fluorescein and
ovalbumin (OB). The coupling reaction yield was found to be
approximately 50% with fluorescein amine while the bio-
conjugation with OB afforded biohybrid superstructures with
approximately 40 proteins per nanoparticle.
Van Hest reported the construction of clickable polymersomes
from polystyrene-b-poly(acrylic acid) (PS-b-PAA) that were
suitable for bioconjugation.217 To this end, the PS-b-PAA block
copolymer was prepared by sequential ATRP of styrene and tBA
Fig. 20 End-functionalized poly(N-vinyl pyrrolidone) for bioconjugation
582 | Polym. Chem., 2010, 1, 563–598
(CuBr/PMDETA). The bromine terminal group was replaced by
an azide upon treatment with azido trimethylsilane and tetra-
butyl ammonium fluoride, followed by acidic hydrolysis of the
tert-butyl groups. The resulting PS-b-PAA-N3 was able to self-
assemble in aqueous solution into well-defined vesicles. The
bioavailability of the azido groups was then successfully inves-
tigated by performing click reaction with a variety of alkyne
ligands based on dansyl dye, biotin or enhanced green fluorescent
protein (EGFP), using CuSO4/sodium ascorbate/tris-(benzyl-
triazolylmethyl) amine (TBTA) catalyst. Upon clicking, the
nanoassemblies were shown to keep their initial morphology.
However, due to the dense packing of the polymer chains in the
vesicles, the optimum degree of functionalization was�25% with
the dansyl alkyne.
Earlier work on clickable polymersomes from co-self-assembly
of PS-b-PEG-N3 and PS-PIAT copolymers (where PIAT stands
for L-isocyanoalanine(2-thiophen-3-yl-ethyl)amide) via ATRP
was also reported.218,219 It was further extended to the immobi-
lization of an active enzyme (Candida Antarctica lipase B, CalB)
via its coupling with alkyne moieties displayed at the extremity of
the PEG chains in the presence of CuSO4, sodium ascorbate and
bathophenanthroline ligand. After purification of the CalB-
decorated polymersomes, the structure of the hybrid vesicles
remained unchanged and the enzymatic activity was main-
tained.220
and surface ligand immobilization onto coated silica nanoparticles.222
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Micelles of a,a0-PDS functional homotelechelic poly(PEGA)-
b-PS-b-poly(PEGA) triblock copolymers (Mn z 35.0 kDa, Mw/
Mn < 1.25) obtained from RA14 (Fig. 7) under RAFT control,
with 85% retention of the PDS end-groups, were also subjected
to bioconjugation experiments.221 The PDS group accessibility
was investigated using a thiol tethered rhodamine B or g-ECG
as a model tripeptide. In the latter case, 74% coupling was
observed.
Adsorption of functional polymers onto silica particles was
also investigated as a way of preparing coated colloidal objects
suitable for bioconjugation. As presented by Zelikin and co-
workers,222 a thiol-terminated PVP polymer from RAFT was
adsorbed onto SiO2 particles by simple mixing leading to PVP-
coated silica nanoparticles (Fig. 20). Thiol accessibility was
demonstrated upon incubation with a thiol-reactive fluorophore,
namely Alexa Fluor 488 (AF488) maleimide. These nano-
particles were stable in different aqueous solutions and organic
solvents even though desorption of the polymer was observed for
high concentration of DMF, DMSO or in the presence of poly-
ethyleneimine (PEI) that induce electrostatic PEI-silica interac-
tions. The authors finally coupled a fluorescent polypeptide
(SIINFEKL), previously reacted with succinimidyl 3-(2-pyr-
idyldithio)propionate (SPDP) to insert DTP moieties. Flow
cytometry confirmed the formation of the covalent bond between
the fluorescent peptide and the PVP-coated particles.
A recent study from Davis, Bulmus and co-workers proposed
the formation of heterotelechelic bifunctional polymers for iron
oxide nanoparticles (IONs) stabilization and biofunctionaliza-
tion.223 In this work, a small library of a-dimethylphosphonate,
u-trithiocarbonate functionalized PS, PNIPAAm and poly-
(PEGA) polymers were prepared via RAFT with good control
(Mn ¼ 3.2–62 kDa, Mw/Mn < 1.24). After transformation of the
a-dimethylphosphonate and aminolysis of the u-trithiocar-
bonate with DTP, the resulting a-phosphonic acid, u-PDS
functionalized poly(PEGA)s were grafted onto IONs surface
followed by bioconjugation experiments successfully performed
with glutathione or NGR peptide via disulfide exchange to afford
ION bioconjugates with 70 � 10% yield for both peptide. Due to
the PEG coating, IONs were also found to be resistant to protein
adsorption.
Fig. 21 LCST-driven formation of poly(N-isopropyl acryl
This journal is ª The Royal Society of Chemistry 2010
4 Synthesis of polymer/peptide-proteinbioconjugates using a non covalent approach
4.1 Biotin/(Strept)avidin binding
Biotin (5-(2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pen-
tanoic acid), also known as vitamin H or B7, is a cofactor in
the metabolism of fatty acids and leucine. It also plays a role
in gluconeogenesis and helps to transfer carbone dioxide in
several carboxylase enzymes. Only the D-(+)-biotin (among 8
isomers) is biologically active. It is also known that biotin has
a strong affinity for avidin (Av, 64 kDa glycoprotein), neu-
travidin (NAv, 60 kDa non-glycosylated form of Av) and
streptavidin (SAv, 53 kDa non-glycosylated protein). The
avidin-biotin and streptavidin-biotin complexes represent the
strongest, non-covalent, biological interaction known to date
with respectively Ka ¼ 1015 and 1013 M�1.224–227 This feature
makes these complexes extremely stable even under harsh
conditions with kinetics of dissociation exceptionally slow
compared to the time scale of most of the experimental
procedures. This explains why they have been widely used to
functionalize proteins and more recently involved in the
formation of protein-polymer bioconjugates in combination
with CLRP techniques.
4.1.2 Binding with preformed biotinylated polymers via the
‘‘grafting to’’ method. Maynard and co-workers reported the
synthesis of biotinylated PNIPAAm polymers by ATRP from
functional initiator I16 (Fig. 5).228 CuCl/CuCl2/Me6TREN was
used as a catalyst and the polymerization displayed a linear
increase of Mn with monomer conversion as well as low PDIs
(Mw/Mn < 1.2), while maintaining the biotin moiety at the a-
terminus. In a final step, the conjugation of this biotinylated
polymer with SAv was performed at room temperature for 24 h
and was evidenced by SDS-PAGE exhibiting a complete shift of
the SAv band towards higher molecular weights. More impor-
tantly, it was demonstrated using an Av/HABA assay, that an
average of 3.6 biotinylated PNIPAAm polymers were bound to
each SAv (as a comparison, 3.7 free biotin were bound). a-Biotin
PNIPAAm (Mn ¼ 12.7 kDa, Mw/Mn ¼ 1.09) was also coupled
amide)-streptavidin (PNIPAAm-SAv) nanoparticles.230
Polym. Chem., 2010, 1, 563–598 | 583
with SAv and its aqueous phase behaviour was investigated.
LCST was determined by UV-Vis turbidity experiments to be
37.4 � 0.1 �C.229
M€uller, Stayton, Hoffman and co-workers took advantage of
the RAFT process to prepare a series of PNIPAAm polymers
(Mn ¼ 2.9–25.9 kDa) with narrow MWD (Mw/Mn ¼ 1.08–
1.16).230 NIPAAm was polymerized by RAFT from 1-pyrrole-
carbodithioate using AIBN initiator. After cleavage of the
dithiocarbamate end-group in basic conditions, the thiol termi-
nated PNIPAAm was coupled to biotinamido-4-[40-(mal-
eimidomethyl)cyclohexanecarboxamido]butane (BMCC) to
yield biotin functionalized PNIPAAm polymers that were
further bound to SAv. However, steric hindrance only allowed
the presence of two PNIPAAm chains per SAv protein.231 The
PNIPAm-SAv bioconjugates displayed a LCST above which
they aggregated into uniform and stable nanoparticles with
diameters in the 250 to 900 nm range, whereas at 20 �C (below
the LCST), they were fully water-soluble (Fig. 21).
Interestingly, the colloidal characteristics of these thermores-
ponsive nanoparticles could be tuned by playing with different
factors such as the heating rate of the bioconjugate solution, the
bioconjugate concentration and the molecular weight of the
PNIPAAm block. In another complementary study, it was also
observed that a SAv-PNIPAAm-b-PAA bioconjugate exhibited
pH-dependent properties.232 Indeed, this bioconjugate was
shown to form large aggregates at pH 4.0 both below and above
the LCST (Dh ¼ 540 and 700 nm respectively), whereas much
smaller aggregates were observed below the LCST at pH 5.5
(Dh ¼ 27 nm). At neutral pH, the SAv-PNIPAAm-b-PAA
aggregation was prevented above the LCST due to the shielding
effect of the PAA block. The aggregation phenomenon can thus
be uncorrelated from the thermoresponsive behaviour of the
PNIPAAm block due to the PAA segment.
4.1.3 Binding via the ‘‘grafting from’’ method. A biotinylated
ATRP initiator (I17, Fig. 5)233 was at the origin of the only
example in the construction of SAv-polymer bioconjugates via
the ‘‘grafting from’’ method.83 After its incubation with SAv in
aqueous medium to form a tetrafunctional SAv-biotin macro-
initiator, polymerization of NIPAAm in mild aqueous condi-
tions was triggered (in the presence of sacrificial initiator
consisting in a bromoisobutyrate-modified resin) to form the
corresponding bioconjugates. The formation of the SAv-PNI-
PAAm bioconjugates was demonstrated by SDS-PAGE and
SEC analysis. To further confirm that the NIPAAm polymer was
attached to SAv through the specific biotin-SAv interaction, the
bioconjugates were treated at 90 �C in a mixture water–DMF
that triggered the disassembly of the tertrameric SAv into 4
identical monomeric subunits along with the release of the bio-
tinylated PNIPAAm chains. The resulting PNIPAAm exhibited
a molecular weight of 27 kDa and a PDI of 1.7. The polymeri-
zation of PEGMA was also tested from this SAv ATRP mac-
roinitiator to demonstrate the versatility of the method.
4.1.4 Binding to surfaces
4.1.4.1 Binding to planar surfaces. Choi and co-workers
reported the immobilization of SAv onto biotinylated polymer
brushes synthesized by a combination of ATRP and click
chemistry.234 A disulfide-containing ATRP initiator was reacted
584 | Polym. Chem., 2010, 1, 563–598
with gold substrate for 12 h at room temperature to give the
surface-immobilized ATRP initiator followed by surface-initi-
ated polymerization of PEGMA using CuBr/bpy. After 1 h, the
bromine terminus of the resulting poly(PEGMA) was substituted
with an azide upon NaN3 treatment, before the click coupling of
different alkyne-containing ligands (1-hexyne, 5-hexyn-1-ol, 4-
pentynoic acid, propargyl benzoate, alkyne-containing biotin)
using a CuSO4/sodium ascorbate catalyst. It showed that the
poly(PEGMA)-coated gold surface avoided interaction with
a number of model proteins such as BSA, fibrinogen, lysozyme,
and RNase A. The biotin-functionalized polymer brushes
showed a thickness decrease (�20 �A) upon exposure to SAv,
which was ascribed to the specific interaction between SAv and
biotin that induced a more condensed polymer layer. Finally,
upon passing a SAv solution flow on the biotin-functionalized
poly(PEGMA)-coated gold surface, a characteristic specific
ligand-receptor interaction was found, demonstrating that the
biotin was effectively attached at the poly(PEGMA) extremity.
4.1.4.2 Binding to nanoparticles. Wooley and co-workers
successfully transformed their SCK nanoparticles into bio-
tinylated colloidal scaffold from PAA-b-PMA block copolymers
synthesized by ATRP from a biotinylated initiator (I17,
Fig. 5).233 Stable and uniform biotinylated nanoparticles were
obtained by mixing different ratios of biotinylated amphiphilic
block copolymers (0–40%) and their non-biotinylated counter-
parts. By fluorescence correlation spectroscopy (FCS), they
noticed that 10 to 25% of the theoretical amount of biotin was
available at the surface of the nanoparticles.
Recently, Zhao, Liu and co-workers applied the CuAAC
methodology to generate Av-polymer bioconjugates by using
ATRP.235 In the first step, sequential ATRP of HEMA and
MMA was initiated by a functional methoxy-PEG5000 ATRP
macroinitiator, to give a PEG-b-PHEMA-b-PMMA (Mn ¼ 13
kDa, Mw/Mn ¼ 1.21). The PHEMA block was then modified by
a mesylation followed by an azidation to obtain the PEG-b-
PAzEMA-b-PMMA (PAzEMA stands for poly(azidoethyl
methacrylate)) triblock copolymer suitable for further click
reaction (Fig. 22). The functionalization of the PEG-
b-PAzEMA-b-PMMA micelles (Dh ¼ 41.4 nm) with alkynylated
biotin was performed in a mixture tert-BuOH/water (1 : 4) in the
presence of CuSO4/sodium ascorbate catalyst. Av/HABA assays
confirmed the successful click coupling on the copolymer micelles
and it was calculated that only �10% of biotin units were
accessible to Av, probably due to steric hindrance or to the
possibility that Av acted as a micelles cross-linking agent and
thus induced the formation of micelles aggregates that would
sterically hide remaining biotin units. This was demonstrated by
incubating biotin-functionalized nanoparticles with Av (biotin/
Av ratio of 2 : 1) where TEM highlighted the presence of both
micelle aggregates and isolated micelles.
Davis and co-workers used a a-biotinylated PEG macro-RAFT
agent (RA15, Fig. 7) to mediate the polymerization of pyr-
idyldisulfide ethylmethacrylate (PDSEMA) under AIBN initia-
tion (Mn ¼ 15.0–29.0 kDa, Mw/Mn ¼ 1.24–1.39), resulting in
a block copolymer able to self-assembled in methanol.236 An
additional step of cross linking by disulfide formation in the
presence of TCEP was undertaken,237 affording well-defined
spherical 54 � 4 nm micelles. SAv/HABA assays revealed that
This journal is ª The Royal Society of Chemistry 2010
Fig. 22 Bioconjugation of biotin to the interfaces of polymeric micelles by in situ click chemistry and further binding with avidin.235
75% of biotin were accessible at the surface of the core cross-linked
micelles. TEM and DLS revealed the formation of higher-order
structures with large diameters up to 1.8 � 0.4 mm. However, the
size of these particles could be lowered by using SAv with some of
its 4 binding sites already pre-occupied with biotin.
4.2 Apoenzyme/cofactor reconstitution
Even though biotin/(S)Av couples certainly exhibit nearly ideal
features in terms of binding and host guest recognition process,
the apoenzyme/cofactor reconstitution strategy represents an
interesting alternative to the construction of non-covalent poly-
mer-protein bioconjugates. This has been elegantly illustrated by
Nolte and co-workers who took advantage of the strong inter-
actions involved in the reconstitution between an apoenzyme and
its cofactor to create HRP-PEG-b-PS bioconjugates.238 The
diblock precursor was synthesized in a controlled fashion by
ATRP of styrene from a PEG based ATRP macroinitiator at
90 �C using CuBr/PMDETA as a catalyst. After nucleophilic
substitution of the bromine terminus of the PS block with an
azide, the resulting PEG-b-PS–N3 copolymer was coupled with
an alkyne derivatized heme by CuAAC using the same catalyst.
The reconstitution of apo-myoglobin (Mb) and apo-HRP was
carried out at pH 7.5 and afforded Mb-PS-b-PEG and HRP-PS-
b-PEG bioconjugates, respectively. After self-assembly, TEM
and SEM analysis of the amphiphilic biomacromolecules showed
a broad range of morphologies: micelles, micellar rods, octopi,
figure eight, toroids and micellar aggregates.
4.3 Glycopolymers and sugar-protein interaction
Carbohydrates are crucial for many biological processes, such as
inflammation, cell-to-cell communication, fertilization and
signal transmission.239–242 Indeed, sugars exhibit a great coding
capacity as they are able to store biological information in
oligosaccharide structures, glycoproteins as well as in glyco-
lipids.243 Lectins, that are carbohydrate-binding proteins, can
selectively recognise and decode the glycocode in oligosaccha-
rides. Whereas individual protein-saccharide interactions are
typically weak, the multivalent interactions employed in bio-
logical systems is characterized by high affinity (the so-called
‘‘cluster’’ glycoside effect)243,244 and high specificity. Due to their
biomimetic properties, there is an increasing interest in synthetic
glycopolymers, that are able to interact with lectins as multiva-
lent ligands in a similar manner to natural glycoproteins.
This journal is ª The Royal Society of Chemistry 2010
Synthetic glycopolymers can be obtained following two
different strategies: (i) the direct polymerization of the corre-
sponding glycomonomers (protected or not) or (ii) the synthesis
of polymer scaffolds bearing pendant reactive sites subsequently
functionalized by sugar moieties.245,246 The development of
CLRP techniques has rendered it possible to produce tailor-
made glycopolymers,247–270 well-reviewed in the recent litera-
ture.245,246 However, only examples investigating glycopolymers
biofunctionality and/or reporting further bioconjugation exper-
iments will be discussed in the following paragraphs (see Fig. 23
for the structure of the corresponding glycomonomers).
4.3.1 Synthesis of glycopolymers. Chaikof and co-workers
were the first to report the synthesis of well-defined glycopoly-
mers from cyanoxyl-mediated polymerization of a wide range
of alkene- and acrylate-based glycomonomers (G1–4, Fig. 23) in
water at 50 �C with a variable amount of acrylamide as
a comonomer (fAM0 ¼ 0–95%).271–274 Rather well-defined
glycosaminoglycan-mimetic polymers covering a broad range of
molecular weights (Mn ¼ 9.9–127 kDa, Mw/Mn ¼ 1.10–1.57)
were obtained. Bioactivity investigations showed that a low
concentration of sulfated monosaccharide-based glycopolymer
was able to enhance fibroblast growth factor-2 (FGF-2) binding
to its receptor.271 Besides this, a high anticoagulant activity has
been witnessed using sulfated disaccharide glycopolymers
contrary to monosaccharide or nonsulfated counterparts.274
In a similar synthetic way, nitroxide-mediated polymerization
of styrene carrying acetylated lactose (G5 with R ¼ Ac, Fig. 23)
was undertaken using BST-TEMPO as the alkoxyamine in DMF
at 130 �C.275 While NMP of the unprotected glycomonomer (G5
with R ¼ H, Fig. 23) led to high PDI, the polymerization of the
acetylated counterpart proceeded in a controlled fashion and
yielded well-defined glycopolymers exhibiting narrow MWDs
(Mw/Mn ¼ 1.19–1.69) in the 9.7–21.7 kDa range. The resulting
materials showed a strong CD spectra suggesting the stereospe-
cific polymerization whereas lectin recognition exhibited a clear
dependence with the number-average degree of polymerization:
the higher the DPn of these multivalent glycoclusters, the
stronger the affinity with lectin.
An original method, using chemo-enzymatic synthesis in the
presence of Candida antarctica lipase (CAL, Novozyme 435), was
used to generate 6-O-methacryloyl mannose (MaM, G6, Fig. 23)
glycomonomer, followed by its subsequent aqueous RAFT poly-
merization yielding well-defined, linear poly(6-O-methacryloyl
mannose) (PMaM) glycopolymers without the need for protecting
Polym. Chem., 2010, 1, 563–598 | 585
Fig. 23 The structures of glycomonomers polymerized by CLRP techniques for bioconjugation purposes.
group chemistry.276 RAFT polymerization kinetics using various
initial monomer to chain transfer agent concentration ratios was
investigated together with the protein binding ability of the
generated glycopolymer using Concanavalin A (Con A).
As opposed to the direct polymerization of glycomonomers for
tailor-made glycopolymers, an alternative route is to use a combi-
nation of CLRP and click chemistry, as recently reported by
Haddleton and co-workers.277 Well-defined alkyne side-chain
polymer scaffolds were obtained by polymerizing trimethylsilyl
methacrylate by ATRP using CuBr/N-(ethyl)-2-pyr-
idylmethanimine as the catalyst in toluene at 70 �C (Mn¼ 8.2–17.6
kDa, Mw/Mn ¼ 1.09–1.17), allowed for further CuAAC with azi-
dosugar derivatives (S1–3, Fig. 24). The versatility of the method
586 | Polym. Chem., 2010, 1, 563–598
was also demonstrated by using PEGMA or MMA as comono-
mers to produce well-defined random copolymers. CuAAC reac-
tions were also performed via a C-6 or an a or b anomeric azide (S4
and S5, Fig. 24) to construct a library of mannose- and galactose-
containing multidentate ligands. This was achieved following
a coclicking reaction of appropriate mixture of mannose- and
galactose-based azides leading to multivalent displays. The reac-
tivity of these glycopolymers in the presence of model lectins able
to selectively bind mannose (Con A) and galactose (Ricinus com-
munis agglutinin, RCA I) moieties was assessed. The CuAAC was
also used to attach a-mannoside, b-galactoside and b-lactoside
derivatives (S6–9, Fig. 24) via an azide functionality bound directly
to the sugar anomeric carbon to yield N-glycosyl 1,2,3-triazole
This journal is ª The Royal Society of Chemistry 2010
Fig. 24 Structure of sugar azides used for the synthesis of glycopolymers
by a combination of CLRP and click chemistry.
functional polymers.278 These studies permitted not only the
comparison of structurally identical ligands that only differ for the
nature of the sugar epitopes, but also the preparation of glyco-
polymers that only differ from each other in the length and the
nature of the linker connecting the carbohydrate units to
the macromolecular backbone. Interestingly, it has been shown by
the same group that CLRP and CuAAC could occur simulta-
neously, thus representing a new synthetic tool for the design of
functional materials.279 This novel copper-catalyzed one-pot
simultaneous CLRP-CuAAC process was then applied to the
synthesis of glycopolymers from S8 (Fig. 24), thus yielding well-
defined biomaterials in a simplified manner.
In the same spirit, the concept initially developed by M€uller
and co-workers126 was expended by Liu and co-workers for the
synthesis of glycoconjugates.280 The bulk ATRP polymerization
of N-acryloxy-succinimide (NAS) was catalyzed by CuBr/bpy.
The NHS-activated side chains of the obtained poly(N-acryloxy-
succinimide) (PNAS) were sequentially reacted with galactos-
amine and ethanolamine to obtained random coglycopolymers
composed of variable ratios of pendant galactose and N-(2-
hydroxypropyl) acrylamide (HPA) units.
4.3.2 Glycopolymer-based architectures. The ability of CLRP
to easily access complex macromolecular architectures was
naturally applied to the synthesis of novel glycopolymer-based
architectures, such as diblock/triblock269,281–287 or star285,288,289
copolymers. Due to the tunable amphiphilic properties of these
copolymers, various morphologies were obtained upon self-
assembly in aqueous solution, such as spherical core-shell
nanoparticles,283,290 spherical micelles aggregates,284,287–289,291,292
worm-like/rods aggregates,281,282,289 vesicles291 or nanocages.286
By sequential ATRP and NCA polymerization, Chaikof and
co-workers reported the synthesis of a poly(L-glutamate)-
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b-poly(2-acryloyloxyethyl lactoside)-b-poly(L-glutamate) and
poly(L-alanine)-b-poly(2-acryloyloxyethyl lactoside)-b-poly(L-
alanine) triblock copolymers from G7 (Fig. 23) and they noticed
specific interactions with RCA I lectins.184,185
Interestingly, Stenzel and co-workers synthesized amphiphilic
hybrid block copolymers based on 2-methacrylamido glucopyr-
anose (MAG, G8, Fig. 23) and 50-O-methacryloyl uridine
(MAU) under RAFT control at low polymerization temperature
(T ¼ 60–70 �C) using (4-cyanopentanoic acid)-4-dithiobenzoate
(CPADB) as a RAFT agent.282 The aim was to develop a new
colloidal carrier for short antisense oligonucleotides (ASONs),
which uses base pairing rather than electrostatic interactions to
bind the gene. Very recently, NMP was successfully employed
from 2-(20,30,40,60-tetra-O-acetyl-b-D-galactosyloxy)ethyl meth-
acrylate (AcGalEMA) glycomonomer (G9, Fig. 23) to synthesize
poly(2-(b-D-galactosyloxy)ethyl methacrylate-co-styrene)-b-
polystyrene (P(GalEMA-co-S)-b-PS) amphiphilic block copoly-
mers (Mn ¼ 18.3–79.9 kDa, Mw/Mn ¼ 1.26–1.50) under SG1
control (N2, Fig. 2), using either SG1-terminated P(AcGalEMA-
co-S) or polystyrene macroinitiators, followed by deacetylation
of AcGalEMA moieties.292 The self-assembling ability of PS-b-
P(GalEMA-co-S) amphiphilic glycopolymers was then exploited
to obtain micellar structures and honeycomb structured porous
films of intact biofunctionality. The same group also reported the
preparation of neoglycopolymers via the thiol-ene coupling
reaction between ene-functional poly[di(ethylene glycol) methyl
ether methacrylate-b-2-hydroxyethyl methacrylate)] diblock
copolymer obtained from RAFT and glucothiose under photo-
chemical conditions with 2,2-dimethoxy-2-phenylacetophenone
(DMPA) as the photoinitiator.287 The glycopolymers were shown
to undergo temperature-induced micellisation and to efficiently
bind with Con A.
Combination of ROP of either 3-caprolactone (3-CL)289 or
BLG-NCA288 with ATRP has been undertaken by Dong and
co-workers for the design of various biomimetic star-shaped
glycopolymers (Fig. 25). A small library of four-arm poly(3-
caprolactone) and poly(BLG-NCA) were synthesized and readily
transformed into ATRP macroinitiators for subsequent poly-
merization of unprotected D-gluconamidoethyl methacrylate
(GAMA, G10, Fig. 23) glycomonomer. The length of each block
was varied by playing with the initial stoichiometry of the
reagents and in all case, a nice control of the polymerization
process was reached exhibiting relatively narrow MWDs.
Recognition properties of these star-shaped glycopolymers with
Con A was also assessed.
Using a similar strategy, the same group used a difunctional
poly(3-caprolactone) ATRP macroinitiator for the preparation of
a polypseudorotaxane/glycopolymer triblock copolymer, poly-
(D-gluconamidoethyl methacrylate)-polypseudorotaxane-poly-
(D-gluconamidoethyl methacrylate) (PGAMA-b-PPR-b-PGAMA),
where inclusion complexation was performed with a-cyclodextrine
(a-CD). After ATRP with CuBr/PMDETA as the catalyst in
DMSO at 35 �C for 48 h, the resulting biomaterials exhibited
controlled molecular weights and low PDIs (Mn¼ 33–39 kDa, Mw/
Mn ¼ 1.26–1.56) together with a specific biomolecular recognition
with Con A in contrast as with BSA.291 Ring opening polymerization
and subsequent RAFT polymerization were also employed for the
synthesis of a poly(D-L-lactide)-b-poly(6-O-acryloyl-a-D-gal-
actopyranose) block copolymer (Mn z 60 kDa, Mw/Mn z 1.2)
Polym. Chem., 2010, 1, 563–598 | 587
Fig. 25 Synthesis of star-shaped poly(PBLG-b-PGAMA)4 biohybrids
via a combination of ROP of BLG-NCA and ATRP of GAMA glyco-
monomer.288
Fig. 26 Preparation of glycopolymer-stabilized gold nanoparticles.295
from glycomonomer G11 (Fig. 23).286 Subsequent cross-linking and
degradation of the core resulted in hollow sugar balls that could be
useful for drug delivery purposes.
After promising results obtained for the preparation of poly-
mers derived from serine- and tyrosine-based ATRP macro-
initiator,143 Maynard and co-workers successfully undertaken
the ATRP polymerization of glycomonomer G12 (Fig. 23) from
the serine derivatized initiator (Mn ¼ 15.4 kDa, Mw/Mn ¼ 1.19).
To demonstrate the versatility of their approach, the serine
derived ATRP initiator was deprotected and coupled using SPPS
to a model peptide, VMSVVQTK, which is well known to be O-
GlcNAc modified in the human cellular factor (HCF) protein, an
abundant chromatin-associated factor involved in cell prolifer-
ation and transcriptional regulation. Then, the C-terminus lysine
of the polypeptide was replaced with a lysine derivative modified
at the 3-position with a (7-methoxycoumarin-4-yl) acetyl (Mca)
group for bioconjugation following purposes. ATRP of G12
from the polypeptide macroinitiator afforded a glycopolymer-
peptide conjugate of 12.2 kDa after 93% conversion with a low
PDI (Mw/Mn¼ 1.14). This work opens the door to the formation
of tailor-made, well-defined polymer- or glycopolymer-peptide
conjugates in which the conjugation site is accurately chosen.
4.3.3 Hybrids glycopolymers. The attachment of glycopoly-
mers to organic/inorganic substrates to make sugar supports is of
great interest because it potentially provides an application in
a number of fields including chemical sensing, responsive
surfaces and affinity chromatography. In this area, various
supports have been used such as Wang resin beads293 as well as
gold nanoparticles294–296 and surfaces.297,298
Several authors took advantage of the fact that polymer end-
groups prepared by RAFT can be readily converted to thiol
588 | Polym. Chem., 2010, 1, 563–598
moieties and anchored to gold substrates via the formation of
Au–S covalent bonds.294–296 In their study, Cameron and co-
workers prepared a poly(2-(b-D-galactosyloxy)ethyl methacry-
late) (poly(GalEMA)) from glycomonomer G13 with CPADB as
a RAFT agent and ACPA as the radical initiator, leading to
a well defined glycopolymer (Mn ¼ 24.1 kDa, Mw/Mn ¼ 1.09).295
Formation of gold glyconanoparticles was observed upon addi-
tion of NaBH4 to a solution of glycopolymer in HAuCl2(Fig. 26), the biological activity of which was demonstrated by
agglomeration of peanut agglutinin (PNA)-coated agarose
beads.
Similar approaches were then reported for the design of either
biotinylated poly(N-acryloylmorpholine-co-6-O-acrylamido-6-
deoxy-1,2:3,4-di-O-isopropylidene-a-D-galactopyranose) (poly-
(NAM-co-GalAm)) glycopolymers from G14 (Fig. 23),296 or
polyacrylamide-based macromolecular backbones with pendant
sugar moieties, either N-acetyl-b-glucosamine (G15, Fig. 23) or
a-mannoside (G16, Fig. 23),294 subsequently anchored at the
surface of gold nanoparticles. In the latter example, molecular
recognition was studied with E. coli, which induced aggregation
of the nanoparticles at the cell periphery. Alternatively, by using
a disulfide-carrying ATRP initiator (I18, Fig. 5), Kitano and co-
workers successfully reported the coupling of galactose-con-
taining glycopolymer brushes to colloidal gold monolayer
deposited on a cover glass.297 The polymerization of lactobio-
namidoethyl methacrylate (LAMA, G17, Fig. 23) proceeded via
a divergent chain-growth with CuBr/bpy as the catalyst. It yiel-
ded a series of glycopolymers (Mn ¼ 5.5–16.0 kDa, Mw/Mn ¼1.53–2.19) followed by their accumulation via Au–S bond on the
gold substrate. The association and dissociation processes of
galactose residues on the colloidal gold with a RCA I lectin were
observed. The opposite strategy, which consists of making gly-
copolymer chains to grow from a gold surface, has also been
investigated by Vamvakaki and co-workers.298 Surface-initiated
ATRP (CuBr/bpy) of GAMA (G10, Fig. 23) and LAMA (G17,
Fig. 23) resulted in a homogeneous increase in the dry film
thickness with reaction time. It was also shown that the glyco-
polymer brushes exhibited strong binding interactions with
specific lectins via the ‘‘glycocluster’’ effect.
Covalent immobilization of a range of carbohydrates deriva-
tives onto polymer resin beads was recently described by Had-
dleton and co-workers using ATRP and CuAAC (Fig. 27).293 Two
approaches were described: (i) the direct click coupling of mannose
azides onto alkyne-derivatized Wang resin (i.e. immobilized
monosaccharide) or (ii) the synthesis of glycopolymer chains dis-
playing multiple copies of mannose epitopes grafted onto a Wang
This journal is ª The Royal Society of Chemistry 2010
Fig. 27 General approaches for the design of hybrid sugar supports: direct click coupling of mannose azides onto alkyne-derivatized Wang resin (Path
A) or synthesis of glycopolymer chains displaying multiple copies of mannose epitopes grafted onto a Wang resin by surface-initiated ATRP (Path B).293
resin by surface-initiated ATRP (i.e. immobilized glycopolymer).
These novel glyco-hybrid materials were able to efficiently recog-
nize mannose-binding model lectins such as Con A.
4.3.4 Functionalized glycopolymers. The flexibility of CLRP
allowed functional groups to be readily inserted within the gly-
copolymer structure for further coupling reactions. Many
examples related the design of well-controlled glycopolymers
bearing biotin,184,185,299–302 maleimide,303 pyridyl disulfide304 or
azide moieties.305
4.3.4.1 Functionalization with biotin. Chaikof and co-
workers used two 4-aminobenzyl-biotinamide initiators (Fig. 28)
to trigger the cyanoxyl-mediated copolymerization of 2-acryl-
aminoethyl lactoside (G4, Fig. 23) and acrylamide, giving well-
defined glycopolymers with narrow MWDs.299,300 The binding
efficiency between streptavidin and the biotinylated glycopoly-
mer was assessed by SDS-PAGE. The production of glycopoly-
mer-coated surfaces was presented from the reaction of
a glycopolymer solution with streptavidin-derivatized poly-
(ethylene terephthalate) (PET) membranes, subsequently incu-
bated with FITC-labeled galactose binding lectin.300
Similarly, ATRP initiators bearing a biotin moiety (I17 and
I19, Fig. 5) have been successfully used at room temperature to
prepare tailor-made biotin-terminated glycopolymers from
LAMA (G17, Fig. 23)301 or from a methacrylate with a pendant
N-acetylglucosamine (GlcNAc) unit (G12, Fig. 23)306 using
CuBr/bpy or CuBr/Me6TREN, respectively. Affinity with
streptavidin was then demonstrated by fluorescence displace-
ment assays301 or surface plasmon resonance.306 In the latter case,
Fig. 28 Structure of 4-aminobenzyl-biotinamide initiators.
This journal is ª The Royal Society of Chemistry 2010
an association constant, Ka, of �1015 M�1 was obtained, in good
agreement with values reported in the literature.
With the RAFT technique, different strategies were employed
to produce biotinylated glycopolymers: (i) the use of a biotin-
functionalized RAFT agent;296,307 (ii) the copolymerization
between a glycomonomer and a biotinylated monomer308 or (iii)
via the thiol-ene click chemistry involving a biotin modified
maleimide.302 By reaction of succinimido-2-[[2-phenyl-1-thio-
xo]thio]-propanoate with (+)-biotinyl-3,6-dioxaoctanediamine,
Charreyre and co-workers were able to prepare a biotinylated
RAFT309 agent (RA16, Fig. 7) further used for the copolymeri-
zation of NAM and GalAm (G14, Fig. 23) to yield biotin-
terminated gradient glycopolymers in the 2–40 kDa range.296,307
Those materials were photochemically reduced in situ for the
preparation of gold nanoparticles, at the surface of which the
biotin was still accessible for bioconjugation to streptavidin.296
Terpolymerization between 2-gluconamidoethyl methacrylamide
(GAEMA, G18, Fig. 23), biotinyl-2-aminoethyl methacrylamide
hydrochloride (BAEMA) and 2-aminoethyl methacrylamide
hydrochloride (AEMA) was undertaken in water at 70 �C in the
presence of ACVA as a water-soluble radical initiator and S,S0-
bis(R,R0-dimethyl-R0 0-acetic acid) trithiocarbonate as a RAFT
agent.308 The resulting glycopolymer (Mn ¼ 12.9 kDa, Mw/Mn ¼1.19) exhibiting pendant sugar, biotin and amine groups was
further used for the surface functionalization of quantum dots
(QDs), via standard EDC/NHS coupling chemistry, which then
demonstrated excellent water-solubility and colloidal stability as
well as lower cytotoxicity than uncoated QDs. Moreover, new
activated esters based on pentafluorophenyl acrylate (FP-A)
have been originally investigated as precursors to build well-
defined biotinylated glycopolymers under RAFT control.302
Poly(pentafluorophenyl acrylate) in the 2.8–16.0 kDa range with
PDI < 1.20 were modified in a nearly quantitative fashion by
concomitant reaction with amino functional sugar (i.e. D-
glucosamine or D-galactosamine) and in situ aminolysis of the
RAFT end-group followed by its coupling with a biotin modified
maleimide via thiol-ene click chemistry. Binding assays with
specific lectin and Con A demonstrated the biofunctionality of
these glycopolymers.
4.3.4.2 Functionalization leading to a covalent linkage.
Functionalized ATRP initiators have been judiciously employed
Polym. Chem., 2010, 1, 563–598 | 589
by several groups for the synthesis of glycopolymer bio-
conjugates. Finn and co-workers used an azide-containing
initiator (I20, Fig. 5) for the polymerization of methacryloxy-
ethyl glucoside (G19, Fig. 23) with CuBr/bpy at 25 �C to yield
azido-terminated glycopolymer (Mn¼ 13 kDa, Mw/Mn¼ 1.3). In
parallel, cowpea mosaic virus (CPMV) was derivatized with an
azide bearing N-hydroxysuccinimide for a multi-site attachment.
To form the virus-glycopolymer bioconjugate, the azido-termi-
nated glycopolymer and the modified CPMV were then
sequentially clicked onto a fluorescent dialkyne using a specific
catalytic system for each moiety (CuSO4/sodium ascorbate and
Cu-triflate/sulfonated bathophenanthroline ligand, respec-
tively).305 The number of covalently bound polymer chains per
particle was very close to the 150 CPMV available azide func-
tionalities. A larger hydrodynamic radius and molecular weight
than the native CPMV were also measured. Haddleton and
co-workers took advantage of ATRP and CuAAC to construct
well-defined neoglycopolymer-protein biohybrid materials as
glycoprotein mimics using a protected-maleimide ATRP initiator
(I9, Fig. 5). A small library of well-defined maleimide-terminated
neoglycopolymers having multiple copies of D-mannose epitopes
(Mn ¼ 8–30 kDa, Mw/Mn ¼ 1.20–1.28) or featuring different
relative amounts of D-mannose and D-galactose binding epitopes
following a co-clicking approach have been further coupled to
BSA as a single thiol-containing model protein (Fig. 29).303
Surface plasmon resonance binding studies carried out using
recombinant rat mannose-binding lectin (MBL) showed clear
and dose-dependent MBL binding to glycopolymer-conjugated
BSA. In addition, enzyme-linked immunosorbent assay (ELISA)
revealed that the neoglycopolymer-protein materials described in
this work possess significantly enhanced capacity to activate
Fig. 29 Synthesis of glycoprotein mimics by a combination of ATRP
and click chemistry.303
590 | Polym. Chem., 2010, 1, 563–598
complement via the lectin pathway when compared with native
unmodified BSA.
Very recently, Maynard and co-workers used a pyridyl disul-
fide ATRP initiator (I10, Fig. 5)310 for the polymerization of N-
acetyl-D-glucosamine (G12, Fig. 23) with CuBr/CuBr2/bpy as
a catalyst in methanol–water mixture at 30 �C to design the
corresponding end-functionalized glycopolymer (Mn ¼ 10.2
kDa, Mw/Mn ¼ 1.12), further employed to prepare a siRNA-
conjugate.304 This may offer great potential regarding siRNA
gene therapy (see also section 5 Synthesis of polymer-oligonucle-
otide bioconjugates).
The RAFT technique was employed under AIBN initiation in
THF at 60 �C with a styrene-based glycomonomer, 1,2:3,4-di-O-
isopropylidene-6-O-(20-formyl-40-vinylphenyl)-D-galactopyranose
(IVDG, G20, Fig. 23), bearing an aldehyde function for further
coupling with BSA as a model protein via the formation of Schiff
base linkage.311 The polymerization exhibited a linear evolution
of the logarithmic conversion with time as well as a linear
increase of Mn with monomer conversion and narrow molecular
weight distribution (Mn ¼ 29 kDa, Mw/Mn z 1.1). Nearly
quantitative removal of protective isopropylidene groups yielded
amphiphilic glycopolymers that self-assembled in water into
well-defined micelles. Protein-bioconjugated nanoparticles were
then successfully prepared by the immobilization of BSA onto
aldehyde-functionalized micelles.
Bertozzi and co-workers recently developed an elegant
approach for designing end-functionalized mucin-like glycopoly-
mers, suitable for integration with current microarray tech-
nology platforms, involving RAFT polymerization from RA17
(Fig. 7), oxime linkages and click chemistry (Fig. 30).312 The
strategy was to prepare a well-defined polymer intermediate
which contained: (i) pendant ketones for the attachment of a-
and b-aminooxy-GalNAc; (ii) a terminal pentafluorophenyl
(PFP) ester that offers a reactive site for further reaction with
propargyl amine to insert alkyne moieties and (iii) a trithiocar-
bonate group subsequently cleaved to release a free sulfhydryl
group for conjugation to a fluorescent probe. The structurally
uniform alkyne-terminated mucin mimetic glycopolymers were
printed on azide-functionalized chips by microcontact printing in
the presence of a copper catalyst by CuAAC. The surface-
attached glycopolymers were shown to bind lectins in a ligand-
specific manner.
5 Synthesis of polymer-oligonucleotidebioconjugates
Boasting an impressive range of well-defined polymer-protein/
peptide bioconjugates from CLRP methods, investigations have
been very recently extended to oligonucleotide bioconjugates.
Oligonucleotides (ON) are short natural RNA (ribonucleic
acid) or DNA (desoxiribonucleic acid) sequences, generally
obtained by chemical synthesis, that contain genetic information.
They have been extensively used for many years in gene therapies
for the treatment of severe diseases because they can regulate the
expression of a targeted protein or to replace a mutant allele. As
they are not able to pass through the cell membrane (especially
due to the strong negative charge of the phosphate backbone),
researchers have thus elaborated ‘‘Trojan’’ systems consisting of
viral, retroviral and adenoviral vectors to release the genetic
This journal is ª The Royal Society of Chemistry 2010
Fig. 30 Synthesis of dual-end-functionalized mucin mimics using a polymer scaffold prepared by RAFT.312
material within the cell after the membrane crossing but also non
viral vectors mainly consisting in polymers and especially poly-
cationic polymers. Regarding this, the last couple of years have
witnessed interesting studies devoted to the preparation of ON-
polymer conjugates for therapeutic purposes using CLRP
methodologies.
Fig. 31 Synthesis of reversible siRNA–polymer conjugates by the
RAFT technique.313
5.1 Direct bioconjugation to preformed polymers
5.1.1 Bioconjugation to a-functional polymers. Maynard,
Bulmus and co-workers reported on the synthesis of poly-
(PEGA) polymer by ATRP technique for gene delivery purposes.
This particular bioconjugation required the formation of a labile
binding between the polymer and the gene as the genetic material
has to be released after cell internalization. In this work the
synthesis of reversible siRNA-poly(PEGA) bioconjugates
(siRNA stands for small interfering RNA) by RAFT polymeri-
zation was described (Fig. 31).313 A pyridyl disulfide bearing
RAFT agent (RA18, Fig. 7) was involved in the polymerization
of PEGA with AIBN in DMF at 60 �C which afforded the
corresponding a-PDS poly(PEGA) (Mn ¼ 13.4 kDa, Mw/Mn ¼1.17). The bioconjugation between 50-thiol terminated siRNA
and the polymer was conducted in 100 mM sodium bicarbonate
buffer at pH 8.5. The formation of the resulting siRNA-poly-
(PEGA) bioconjugate along with the specificity of the coupling
was further confirmed by PAGE analysis and the presence of
a disulfide bridge allowed for further cleavage of the conjugates
under reductive environment.
Very recently, aptamers with a disulfide protected thiol
modification on the 30 end, have been conjugated to maleimide
activated branched PEGs of various molecular weights, obtained
using I9 (Fig. 5) as an ATRP initiator. This work demonstrated
an alternative approach to PEGylation of aptamers, and that the
effect of PEG on the affinity for the target varied according to the
structure and conformation of the synthetic polymer.314
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5.1.2 Bioconjugation to u-functional polymers. The forma-
tion of ON-polymer bioconjugates was also investigated by
RAFT polymerization combined with the aminolysis reaction.115
PNIPAAm and PHPMA functionalized with a PDS group were
then coupled to siRNA or DNA in PBS at pH 8 at 37 �C over-
night. The formation of PNIPAAm-ON bioconjugates was
confirmed by aqueous SEC and agarose gel electrophoresis.
Dendritic carbohydrate end-functional polymers from RAFT
was also investigated as a functionalizable scaffold with
siRNA.315 In this study, a-thiazolidine-2-thione dithiobenzoate
RAFT agent (RA2, Fig. 7) was used to mediate the
Polym. Chem., 2010, 1, 563–598 | 591
polymerization of HPMA. a-thiazolidine-2-thione PHPMA (Mn
¼ 4.3 and 9.9 kDa, Mw/Mn < 1.2) was reacted with an amino-
functionalized dendritic manose-derivated carbohydrate
(G2Man, designed by click chemistry). Then, the dithiobenzoate
extremity of the PHPMA-G2Man conjugate was turned into
a pyridyl disulfide moiety by aminolysis reaction in ethanolamine
in the presence of DTP. Finally, the PDS u-functional PHPMA-
G2Man conjugate was coupled to 50-sense thiol modified siRNA
through a disulfide exchange reaction in the presence of TEA and
DTT in nuclease-free water for 30 min. The achievement of the
reaction was confirmed by agarose gel electrophoresis and
HPLC.
This strategy was also exploited with a library of u-PDS
PNIPAAm polymers (Mn ranging from 7.0 to 22.2 kDa) conju-
gated to a 50-thiol-modified oligonucleotide in PBS at pH 8.0
(ratio PNIPAAm/ON: 50/1). The achievement of the bio-
conjugation was demonstrated by agarose gel electrophoresis
and the reaction yields were above 70% irrespective of the
polymer molecular weight. A release study of the ON upon
addition of DTT was undertaken and confirmed the attachment
of the ON via a reversible disulfide bond.116
The thiol-ene reaction was recently demonstrated to be
a powerful coupling method to covalently attach ON to poly-
mers.316 In this study, HPMA and NIPAAm were polymerized
by RAFT from dithioester and trithiocarbonate RAFT agents to
afford PHPMA and PNIPAAm polymers in a controlled
fashion. Then, after aminolysis reaction in the presence of 1,4-
butanediol dimethacrylate, ene u-functional PHPMA (Mn ¼ 4.4
kDa, Mw/Mn¼ 1.04) and PNIPAAm (Mn¼ 13.5 kDa, Mw/Mn¼1.10) polymers were obtained and coupled to a thiol modified
ON by thiol-ene click chemistry. The formation of the PNI-
PAAm-ON and PHPMA-ON bioconjugates was confirmed by
agarose gel electrophoresis.
5.2 Bioconjugation to surfaces
The design of DNA-polymer bioconjugates on a planar solid
support using surface-initiated RAFT polymerization has been
reported by He.317A trithiocarbonate RAFT agent (RA19, Fig. 7)
was attached to the distal point of a surface-immobilized oligo-
nucleotide and initiation of the polymerization led to controlled
growth of polymer chains. Growth kinetics of PEGMA or
HEMA atop DNA molecules was investigated by monitoring the
change of polymer film thickness as a function of reaction time.
Comparing to polymer growth atop small molecules, the exper-
imental results suggest that DNA molecules significantly accel-
erated polymer growth, which was speculated as a result of the
presence of highly charged DNA backbones and purine/pyrimi-
dine moieties surrounding the reaction sites.
The immobilization of single strand deoxyribonucleic acid
(ssDNA) on PVP coated SiO2 particles was also proposed by
Zelekin and co-workers as an interesting method to prepare
DNA biosensing systems (Fig. 20). Developing the same strategy
as already reported earlier for the immobilization of SIINFEKL
peptide at the surface of SiO2 particles, the authors coupled 50-
thiol terminated ssDNA using the disulfide exchange coupling on
activated thiol PVP previously adsorbed at the surface of SiO2
particles.222
592 | Polym. Chem., 2010, 1, 563–598
6 Conclusions
In this review, the huge potential of CLRP to prepare well-
defined and original bioconjugates has been exposed. Since their
discoveries, CLRP techniques (especially RAFT and ATRP)
have brought about a clear breakthrough in this field. They allow
the design of polymers with a high degree of control regarding
the macromolecular architecture and the accurate insertion of
functional groups in the polymer chains for further (covalent or
non covalent) bioconjugation. Moreover, these polymerization
techniques are easy to implement and can be performed with
a broad range of monomers and functional initiators as well as in
organic and aqueous solvents. All these important features make
CLRP techniques valuable tools in the actual bioconjugation
landscape. All kind of bioconjugates (polymer-protein/peptide,
polymer-ON, glycoprotein mimics etc) described herein could
have important benefits in diverse areas such as drug delivery
purposes, biomaterials, bio- and nanotechnologies, gene therapy
and much more in the near future.
The interest of the medical field for bioconjugates can be
observed by the numerous protein-polymer based therapeutics,
including blockbuster PEG-protein drugs such as Neulasta�,
Pegasys�, Pegintron�, and Mircera� that are already on the
market for the treatment of Hepatitis C, cancers and others. As
CLRP offers the possibility to accurately tune the macromolec-
ular properties of the bioconjugates, it is likely that a forth-
coming close interdisciplinary collaboration between
macromolecular synthesis and the medical/pharmaceutical
research area will result in the discovery of high value-added
bioconjugates for therapeutic purposes and for other bio-related
applications.
However, the development of this new class of biomaterials
might be hampered by the limitations and drawbacks that
CLRP techniques could potentially present. For instance with
RAFT, complete removal of the RAFT end-group has to be
efficiently performed prior to any biological application. With
ATRP, the catalyst could be an issue as many proteins exhibit
copper or iron binding pockets which may affect their bio-
logical integrities. Nevertheless, the majority of the examples
detailed in this review showed a good conservation of the
biological activity for polymer-protein conjugates or the ability
for glycopolymers to efficiently bind lectins. It thus demon-
strated the reliability and the efficiency of this approach
regarding the exciting and rapidly expanding field of polymer
science in therapeutics.
7 List of Abbreviations
aCT
This
a-chymotrypsin
AA
acrylic acidACN
acetonitrileACVA
4,40-azobis (4-cyanovaleric acid)AEL
2-acryloylethyloctaacetyllactosideAEMA
2-aminoethyl methacrylamide hydrochlorideAF488
Alexa Fluor 488AFM
atomic force microscopyAGT
O6-alkylguanine-DNA alkyltransferasejournal is ª The Royal Society of Chemistry 2010
AIBN
This journal is ª T
2,20-azobisisobutyronitrile
Ala/A
alanineAM
acrylamideAMC
7-amino-4-methylcoumarinAN
acrylonitrileArg/R
arginineASON
antisense oligonucleotideAsp/D
aspartic acidATRP
atom transfer radical polymerizationAv
avidinAzEMA
azidoethyl methacrylateBAEMA
biotinyl-2-aminoethyl methacrylamidehydrochloride
BDC
4-(N,N-diethyl)dithiocarbamoylmethylbenzoicacid
BG
O6-benzylguanineBpy
bipyridineBLG-NCA
g-benzyl-L-glutamate-N-carboxyanhydrideBMCC
1-biotinamido-4-[40-maleimidomethyl)cyclohexanecarboxamido]butane
Boc
tert-butyl carbonateBSA
bovine serum albuminBSPA
3-(benzylsulfanylthiocarbonyl sulfanyl)-propionic acid
CalB
Candida Antarctica lipase BCD
circular dichroismCDTB
4-cyanopentanoic acid dithiobenzoateCHO
Chinese hamster ovaryCL
3-caprolactoneCLRP
controlled living radical polymerizationCLSM
confocal laser scaning microscopyCon A
Concanavalin ACPADB
(4-cyanopentanoic acid)-4-dithiobenzoateCP-ini
peptide nanotube macroinitiatorCPMV
cowpea mosaic virusCTA
chain transfer agentCuAAC
copper-catalyzed azide-alkyne cycloadditionCys/C
cysteineCWP
chain walking polymerizationDCC
N-N0-dicyclohexyl carbodiimideDEPMA
diethoxypropyl methacrylateDh
hydrodynamic diameterDHFR
dihydrofolate reductaseDIPEA
N,N-diisopropylethylamineDLS
dynamic light scatteringDMAA
dimethyl acrylamideDMAEMA
dimethylaminoethyl methacrylateDMF
dimethyl formamideDMSO
dimethyl sulfoxydedNbpy
4,40-di(5-nonyl)-2,2-bipyridineDNA
desoxiribonucleic acidDPn
number-average degree of polymerizationDSC
differential scanning calorimetryDTP
2,20-dithiopyridineDTT
1,4-dithiothreitolEBIB
ethyl-2-bromo isobutyrateEBP
elastin-based polymerE. coli
Echerichia colihe Royal Society of Chemistry 2010
EDC
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
EDTA
ethylenediaminetetraacetic acidELP
elastin linear polymerEPO
erythropoietinf
initiation efficiencyfM0
initial molar fraction of monomer M ina comonomer mixture
FCS
fluorescence correlation spectroscopyFGF-2
fibroblast growth factor-2FITC
fluoresceine isothiocyanateFmoc
fluorenylmethyloxycarbonylFPLC
fast protein liquid chromatographyFT-IR
fourier-transform infrared spectroscopyGA
glutamic acidGAEMA
2-gluconamidoethyl methacrylamideGalAm
6-O-acrylamido-6-deoxy-1,2:3,4-di-O-isopropylidene-a-D-galactopyranose
GAMA
D-gluconamidoethyl methacrylateGlcNAc
N-acetyl-D-glucosamineGln/Q
glutamineGlu/E
glutamic acidGlu-EMA
glutamic acid ethyl methacrylateGly/G
glycineG-CSF
granulocyte colony stimulating factorGMA
glycidyl methacrylateGOD
glucose oxidaseGSH/g-ECG
glutathioneHEMA
2-hydroxyethyl methacrylateHis/H
histidineHABA
2-(40-hydroxyazobenzene) benzoic acidHCF
human cellular factorHEA
hydroxyethyl acrylateHIV
human immunodeficiency virusHMA
hostasol methacrylateHMTETA
1,1,4,7,10,10-hexamethyltriethylene tetramineHPA
N-(2-hydroxypropyl) acrylamideHPLC
high performance liquid chromatographyHPMA
N-(2-hydroxypropyl)methacrylamideHRP
horseradish peroxidaseIFN
interferon-2aION
iron oxide nanoparticleIVDG
1,2:3,4-di-O-isopropylidene-6-O-(20-formyl-40-vinylphenyl)-D-galactopyranose
LAMA
lactobionamidoethyl methacrylateLCST
low critical solution temperatureLeu/L
leucineCLRP
controlled/living radical polymerizationLys/K
lysineM
monomerMb
myoglobinMBL
mannose-binding lectinMca
(7-methoxycoumarin-4-yl) acetylMA
methyl acrylateMe6TREN
tris[2-(dimethylamino)ethyl]amineMeOH
methanolMMA
methyl methacrylateMn
number-average molar massPolym. Chem., 2010, 1, 563–598 | 593
MPC
594 | Polym. Chem
2-methacryloyloxyethyl phosphorylcholine
Mw
weight-average molar massMWD
molecular weight distributionNAM
N-acryloylmorpholineNAS
N-acryloyloxysuccinimideNAv
neutravidinnBA
n-butyl acrylateNCA
a-amino acid-N-carboxyanhydrideNHS
N-hydroxysuccinimideNIPAAm
N-isopropyl acrylamideNMP
nitroxide-mediated polymeriazationNMR
nuclear magnetic resonanceNMS
N-methacryloyloxysuccinimideNPC
p-nitrophenyl chloroformateNVP
N-vinyl pyrrolidoneOB
ovalbuminON
oligonucleotidePAA
poly(acrylic acid)PAEL
poly(2-acryloyloxyethyl-lactoside)PBLG
poly(g-benzyl-L-glutamate)PBS
phosphate buffer solutionPCL
poly(3-caprolactone)PDEPMA
poly(diethoxypropyl methacrylate)PDI
polydispersity index (Mw/Mn)PDMAA
poly(dimethyl acrylamide)PDMAEMA
poly(N-dimethylaminoethyl methacrylate)PDS
pyridyl disulfidePDSEMA
pyridyldisulfide ethylmethacrylatePEG
poly(ethylene) glycolPEGA
poly(ethylene glycol) methyl ether acrylatePEGMA
poly(ethylene glycol) methyl ethermethacrylate
PEI
polyethyleneiminePET
poly(ethylene terephthalate)PFEMA
poly(3-formyl ethyl methacrylate)PFP
pentafluorophenylPGMA
poly(glycerol methacrylate)Phe/P
phenylalaninePHEMA
poly(2-hydroxyethyl methacrylate)PHPMA
poly(N-(2-hydroxypropyl) methacrylamide)PIAT
poly(L-isocyanoalanine(2-thiophen-3-yl-ethyl)amide)
PLA
poly(L-alanine)PMA
propargyl methacrylatePMDETA
N,N,N0,N0,N0 0-pentamethyldiethylenetriaminePMMA
poly(methyl methacrylate)PMPC
poly(2-methacryloyloxyethylphosphorylcholine)
PNA
peptide nucleic acidPNA
peanut agglutininPNAS
poly(N-acryloxy-succinimide)PnBA
poly(n-butyl methacrylate)PNIPAAm
poly(N-isopropyl acrylamide)PNMS
poly(N-methacryloyloxysuccinimide)pNPMA
poly(p-nitrophenyl methacrylate)PpNPMA
poly(p-nitrophenyl methacrylate)PPNT
peptide-polymer hybrid nanotubePro/P
proline., 2010, 1, 563–598
PS
This
polystyrene
PtBA
poly(tert-butyl acrylate)PTD
protein transduction domainPVP
poly(N-vinyl pyrrolidone)PzLLys
poly(Z-L-lysine)QD
quantum dotRAFT
reversible addition-fragmentation transferRCA I
ricinus communis agglutininRI
refractive indexROP
ring-opening polymerizationROMP
ring-opening metathesis polymerizationS
styreneS. aureus
Staphylococcus aureusSAv
streptavidinSCK
shell-cross-linkedsCT
salmon calcitoninSDS-PAGE
sodium dodecylsulfate polyacrylamide gelelectrophoresis
SEC
size exclusion chromatographySEC-HPLC
size exclusion high performance liquidchromatography
SEM
scanning electron microscopySer/S
serineSG1
N-tert-butyl-N-[1-diethylphosphono-(2,2-dimethylpropyl)] nitroxide
SPA
succinimidyl propionateSPDP
succinimidyl 3-(2-pyridyldithio)propionateSPR
surface plasmon resonanceRg
radius of gyrationRh
hydrodynamic radiusRMA
rhodamine B methacrylateRNA
ribonucleic acidsiRNA
small interfering ribonucleic acidSPA
succinimidyl propionateSPPS
solid-phase peptide synthesisssDNA
single strand deoxyribonucleic acidt
reaction timeT4L
V131C T4 lysozymetBA
tert-butyl acrylateTBAF
tetrabutyl ammonium fluorideTCEP
tris(2-carboxyethyl)phosphineTEA
triethylamineTEM
transmission electron microscopyTEMPO
2,2,6,6-tetramethylpiperidinyl-1-oxyTHF
tetrahydrofuraneThr/T
threonineTIPNO
N-tert-butyl-N-[1-phenyl-2-(methylpropyl)]nitroxide
TMS
trimethylsilylTrp/W
tryptophanTyr/Y
tyrosineUV
ultra violetVal/V
valineVBC
4-vinylbenzyl chlorideVO
2-vinyl-4-4-dimethyl-5-oxazolonezLLys-NCA
N3-carbobenzoxy-L-lysine-N-carboxyanhydride
journal is ª The Royal Society of Chemistry 2010
Acknowledgements
We thank the European Community’s Seventh Framework
Programme (FP7/2007-2013) under grant agreement no. 212043
for funding (BLD). The French ministry of research and CNRS
are also warmly acknowledged for financial support.
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