volume 1 | number 5 | july 2010 | pages 545

ISSN 1759-9954

Polymer Chemistry

1759-9954(2010)1:5;1-O

REVIEWB. Le Droumaguet and J. Nicolas Recent advances in the design of bioconjugates from controlled/living radical polymerization

COMMUNICATIONA. Dag et al.An easy way to the preparation of multi-miktoarm star block copolymers via sequential double click reactions

www.rsc.org/polymers Volume 1 | Number 5 | July 2010 | Pages 545–756

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.


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


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.


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


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.


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


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


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


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


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-


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


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


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


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


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


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


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


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


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


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


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


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.


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


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)-


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


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.


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


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


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 acid
ACN
acetonitrile
ACVA
4,40-azobis (4-cyanovaleric acid)
AEL
2-acryloylethyloctaacetyllactoside
AEMA
2-aminoethyl methacrylamide hydrochloride
AF488
Alexa Fluor 488
AFM
atomic force microscopy
AGT
O6-alkylguanine-DNA alkyltransferase
journal is ª The Royal Society of Chemistry 2010

AIBN

This journal is ª T

2,20-azobisisobutyronitrile

Ala/A
alanine
AM
acrylamide
AMC
7-amino-4-methylcoumarin
AN
acrylonitrile
Arg/R
arginine
ASON
antisense oligonucleotide
Asp/D
aspartic acid
ATRP
atom transfer radical polymerization
Av
avidin
AzEMA
azidoethyl methacrylate
BAEMA
biotinyl-2-aminoethyl methacrylamide
hydrochloride

BDC
4-(N,N-diethyl)dithiocarbamoylmethylbenzoic
acid

BG
O6-benzylguanine
Bpy
bipyridine
BLG-NCA
g-benzyl-L-glutamate-N-carboxyanhydride
BMCC
1-biotinamido-4-[40-maleimidomethyl)
cyclohexanecarboxamido]butane

Boc
tert-butyl carbonate
BSA
bovine serum albumin
BSPA
3-(benzylsulfanylthiocarbonyl sulfanyl)-
propionic acid

CalB
Candida Antarctica lipase B
CD
circular dichroism
CDTB
4-cyanopentanoic acid dithiobenzoate
CHO
Chinese hamster ovary
CL
3-caprolactone
CLRP
controlled living radical polymerization
CLSM
confocal laser scaning microscopy
Con A
Concanavalin A
CPADB
(4-cyanopentanoic acid)-4-dithiobenzoate
CP-ini
peptide nanotube macroinitiator
CPMV
cowpea mosaic virus
CTA
chain transfer agent
CuAAC
copper-catalyzed azide-alkyne cycloaddition
Cys/C
cysteine
CWP
chain walking polymerization
DCC
N-N0-dicyclohexyl carbodiimide
DEPMA
diethoxypropyl methacrylate
Dh
hydrodynamic diameter
DHFR
dihydrofolate reductase
DIPEA
N,N-diisopropylethylamine
DLS
dynamic light scattering
DMAA
dimethyl acrylamide
DMAEMA
dimethylaminoethyl methacrylate
DMF
dimethyl formamide
DMSO
dimethyl sulfoxyde
dNbpy
4,40-di(5-nonyl)-2,2-bipyridine
DNA
desoxiribonucleic acid
DPn
number-average degree of polymerization
DSC
differential scanning calorimetry
DTP
2,20-dithiopyridine
DTT
1,4-dithiothreitol
EBIB
ethyl-2-bromo isobutyrate
EBP
elastin-based polymer
E. coli
Echerichia coli
he Royal Society of Chemistry 2010

EDC
N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide

EDTA
ethylenediaminetetraacetic acid
ELP
elastin linear polymer
EPO
erythropoietin
f
initiation efficiency
fM0
initial molar fraction of monomer M in
a comonomer mixture

FCS
fluorescence correlation spectroscopy
FGF-2
fibroblast growth factor-2
FITC
fluoresceine isothiocyanate
Fmoc
fluorenylmethyloxycarbonyl
FPLC
fast protein liquid chromatography
FT-IR
fourier-transform infrared spectroscopy
GA
glutamic acid
GAEMA
2-gluconamidoethyl methacrylamide
GalAm
6-O-acrylamido-6-deoxy-1,2:3,4-di-O-
isopropylidene-a-D-galactopyranose

GAMA
D-gluconamidoethyl methacrylate
GlcNAc
N-acetyl-D-glucosamine
Gln/Q
glutamine
Glu/E
glutamic acid
Glu-EMA
glutamic acid ethyl methacrylate
Gly/G
glycine
G-CSF
granulocyte colony stimulating factor
GMA
glycidyl methacrylate
GOD
glucose oxidase
GSH/g-ECG
glutathione
HEMA
2-hydroxyethyl methacrylate
His/H
histidine
HABA
2-(40-hydroxyazobenzene) benzoic acid
HCF
human cellular factor
HEA
hydroxyethyl acrylate
HIV
human immunodeficiency virus
HMA
hostasol methacrylate
HMTETA
1,1,4,7,10,10-hexamethyltriethylene tetramine
HPA
N-(2-hydroxypropyl) acrylamide
HPLC
high performance liquid chromatography
HPMA
N-(2-hydroxypropyl)methacrylamide
HRP
horseradish peroxidase
IFN
interferon-2a
ION
iron oxide nanoparticle
IVDG
1,2:3,4-di-O-isopropylidene-6-O-(20-formyl-40-
vinylphenyl)-D-galactopyranose

LAMA
lactobionamidoethyl methacrylate
LCST
low critical solution temperature
Leu/L
leucine
CLRP
controlled/living radical polymerization
Lys/K
lysine
M
monomer
Mb
myoglobin
MBL
mannose-binding lectin
Mca
(7-methoxycoumarin-4-yl) acetyl
MA
methyl acrylate
Me6TREN
tris[2-(dimethylamino)ethyl]amine
MeOH
methanol
MMA
methyl methacrylate
Mn
number-average molar mass
Polym. Chem., 2010, 1, 563–598 | 593

MPC

594 | Polym. Chem

2-methacryloyloxyethyl phosphorylcholine

Mw
weight-average molar mass
MWD
molecular weight distribution
NAM
N-acryloylmorpholine
NAS
N-acryloyloxysuccinimide
NAv
neutravidin
nBA
n-butyl acrylate
NCA
a-amino acid-N-carboxyanhydride
NHS
N-hydroxysuccinimide
NIPAAm
N-isopropyl acrylamide
NMP
nitroxide-mediated polymeriazation
NMR
nuclear magnetic resonance
NMS
N-methacryloyloxysuccinimide
NPC
p-nitrophenyl chloroformate
NVP
N-vinyl pyrrolidone
OB
ovalbumin
ON
oligonucleotide
PAA
poly(acrylic acid)
PAEL
poly(2-acryloyloxyethyl-lactoside)
PBLG
poly(g-benzyl-L-glutamate)
PBS
phosphate buffer solution
PCL
poly(3-caprolactone)
PDEPMA
poly(diethoxypropyl methacrylate)
PDI
polydispersity index (Mw/Mn)
PDMAA
poly(dimethyl acrylamide)
PDMAEMA
poly(N-dimethylaminoethyl methacrylate)
PDS
pyridyl disulfide
PDSEMA
pyridyldisulfide ethylmethacrylate
PEG
poly(ethylene) glycol
PEGA
poly(ethylene glycol) methyl ether acrylate
PEGMA
poly(ethylene glycol) methyl ether
methacrylate

PEI
polyethyleneimine
PET
poly(ethylene terephthalate)
PFEMA
poly(3-formyl ethyl methacrylate)
PFP
pentafluorophenyl
PGMA
poly(glycerol methacrylate)
Phe/P
phenylalanine
PHEMA
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 methacrylate
PMDETA
N,N,N0,N0,N0 0-pentamethyldiethylenetriamine
PMMA
poly(methyl methacrylate)
PMPC
poly(2-methacryloyloxyethyl
phosphorylcholine)

PNA
peptide nucleic acid
PNA
peanut agglutinin
PNAS
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 nanotube
Pro/P
proline
., 2010, 1, 563–598

PS

This

polystyrene

PtBA
poly(tert-butyl acrylate)
PTD
protein transduction domain
PVP
poly(N-vinyl pyrrolidone)
PzLLys
poly(Z-L-lysine)
QD
quantum dot
RAFT
reversible addition-fragmentation transfer
RCA I
ricinus communis agglutinin
RI
refractive index
ROP
ring-opening polymerization
ROMP
ring-opening metathesis polymerization
S
styrene
S. aureus
Staphylococcus aureus
SAv
streptavidin
SCK
shell-cross-linked
sCT
salmon calcitonin
SDS-PAGE
sodium dodecylsulfate polyacrylamide gel
electrophoresis

SEC
size exclusion chromatography
SEC-HPLC
size exclusion high performance liquid
chromatography

SEM
scanning electron microscopy
Ser/S
serine
SG1
N-tert-butyl-N-[1-diethylphosphono-(2,2-
dimethylpropyl)] nitroxide

SPA
succinimidyl propionate
SPDP
succinimidyl 3-(2-pyridyldithio)propionate
SPR
surface plasmon resonance
Rg
radius of gyration
Rh
hydrodynamic radius
RMA
rhodamine B methacrylate
RNA
ribonucleic acid
siRNA
small interfering ribonucleic acid
SPA
succinimidyl propionate
SPPS
solid-phase peptide synthesis
ssDNA
single strand deoxyribonucleic acid
t
reaction time
T4L
V131C T4 lysozyme
tBA
tert-butyl acrylate
TBAF
tetrabutyl ammonium fluoride
TCEP
tris(2-carboxyethyl)phosphine
TEA
triethylamine
TEM
transmission electron microscopy
TEMPO
2,2,6,6-tetramethylpiperidinyl-1-oxy
THF
tetrahydrofurane
Thr/T
threonine
TIPNO
N-tert-butyl-N-[1-phenyl-2-(methylpropyl)]
nitroxide

TMS
trimethylsilyl
Trp/W
tryptophan
Tyr/Y
tyrosine
UV
ultra violet
Val/V
valine
VBC
4-vinylbenzyl chloride
VO
2-vinyl-4-4-dimethyl-5-oxazolone
zLLys-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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