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Mini-review

Received: 1 August 2011 Revised: 19 October 2011 Accepted: 2 November 2011 Published online in Wiley Online Library: 13 January 2012

(wileyonlinelibrary.com) DOI 10.1002/pi.4135

Facile design of biomaterials by ‘click’


chemistry
Søren Hvilsted∗

Abstract
The advent of the so-called ‘click chemistry’ a decade ago has significantly improved the chemical toolbox for producing
novel biomaterials. This review focuses primarily on the application of Cu(I)-catalysed azide–alkyne 1,3-cycloadditon in the
preparation of numerous, diverse biomaterials and biomedical materials and concepts. In addition, the thiol–ene ‘click’ reaction
is addressed in the same manner, and the possibility of using both click reactions orthogonally is highlighted. A strategy for the
preparation of novel intriguing poly(ε-caprolactone)-based nanobiomaterials by orthogonal click chemistry is elaborated. The
present state of creating functional and biologically active surfaces by click chemistry is presented. Finally, conducting surfaces
based on an azide-functionalized polymer with prospective biological sensor potential are introduced.
c 2012 Society of Chemical Industry

Keywords: azide–alkyne cycloaddition; thiol–ene click chemistry; biomedical materials; functional surfaces; biologically active surfaces;
conducting polymer surfaces

INTRODUCTION also materials with a biological or biomedical function are


The discovery in 2001 by Meldal and co-workers1 that the almost considered.
50-year-old Huisgen 1,3-cycloaddition2 between an azide and The basic starting structure often dictates the possible final
an alkyne resulting in 1,4-linking 1,2,3-triazoles can be catalysed material use through solubility issues, controllable degradation
by Cu(I) at room temperature to afford regiospecific products rate, pH, ionic strength, temperature, and other external field
in near-quantitative yields (Fig. 1) has initiated an impressive responses like light or electrical and magnetic fields. Thus
development. Early on, the potential of this efficient Cu(I)-catalysed the following material presentations have been chosen due to
coupling reaction was elaborated by Meldal and co-workers3 that. Furthermore, we focus on what we believe are the most
and Sharpless and co-workers.4 Sharpless also termed the Cu(I)- important materials judged from availability and presence in the
catalysed 1,3-cycloadditon of an azide and an alkyne as ‘click’ literature. We stress that normally only materials with claimed
chemistry, which is now often abbreviated as CuAAC. biofunctionality are considered unless a new methodology points
to future possible biomaterial design.
The potential of cycloaddition has since then created much
As early as the material design phase a strategy aiming for
activity in the polymer community,5 – 8 in the dendrimer field9
click chemistry needs to be elaborated. This means that in
and not least in the biotechnology world.10 This contribution
the preparation for click options the appropriate functionality
will focus on many of the intriguing possibilities to create
needs to be introduced. In the case of CuAAC an alkyne or
functional biomaterials in easy ways often replacing earlier tedious
an azide should be preferably introduced; alternatively chemical
or very time-consuming methods. In fact, many applications
functionality that can be conveniently transformed into alkyne
would probably have never emerged without the click chemistry or azide should be ensured. For the latter many options
concept. It should be added that several other thermodynamically like bromo, chloro, hydroxyl or amine exist; however, harsh
favourable reactions like Diels–Alder cycloadditions and thiol–yne transformation chemistries often cannot be tolerated by the
and thiol–ene reactions have been termed click reactions.11 original ‘parent skeleton’. Furthermore, additional synthetic steps
However, in this contribution we will mainly focus on the original requiring intermittent product purification are never beneficial.
CuAAC and on the thiol–ene reactions (Fig. 2), merely because Likewise, terminal double bonds and thiols are needed when
these are by far the most extensively exploited and understood, a thiol–ene click reaction is required. The chemical functional
most probably due to the proven superior performance in many flexibility, however, also points to the options for performing
instances. It should be added, however, that a few examples orthogonal click chemistry on the parent skeleton since specific
of orthogonal ‘clicking’ of other chemical strategies that in
principle can qualify as click reactions are mentioned, especially
in combination with the thiol–ene click. This already is strong ∗
Correspondence to: Søren Hvilsted, Danish Polymer Centre, Department of
evidence that the click chemistry field is rapidly developing. Chemical and Biochemical Engineering, Technical University of Denmark,
The appearance of new click chemistries considerably widens Building 227, DK-2800 Kongens Lyngby, Denmark. E-mail: sh@kt.dtu.dk
the possibilities for preparing new biomaterials. Here the term
Danish Polymer Centre, Department of Chemical and Biochemical Engineering,
‘biomaterials’ is defined rather broadly and not only implying Technical University of Denmark, Building 227, DK-2800 Kongens Lyngby,
485

biocompatibility and biodegradability. For the sake of simplicity Denmark

Polym Int 2012; 61: 485–494 www.soci.org 


c 2012 Society of Chemical Industry
www.soci.org S Hvilsted

N R'' Cu(I)
Søren Hvilsted is a full professor of R' C CH + N
R' R''
N N
Polymer Chemistry at DTU – Technical
N N
University of Denmark. His research in-
terests entail the design, preparation 1,2,3-triazole
and testing of stimuli-responsive poly- R', R'' can be alkyls, aryls, sugars, polymers, drugs, virus etc.
mer materials. In particular attention is Cu(I): e.g from CuSO4 and ascorbic acid in water at room temperature
devoted materials for optical informa- or from Cu(I)X + amine; X = Cl, Br, I in organic solvents
tion storage, and fuel cell electrolyte
Figure 1. Cu(I)-catalysed 1,3-cycloaddition of an azide and an alkyne
membranes, as well as to fluorinated (CuAAC).
materials and polymers for biomedical,
drug release and biosensor applications.

R SH + photoinitiator
R R'
conditions for executing these two click reactions normally do R' hn S
not interfere with each other. Thus a scene is set for truly O
modular click constructions much like the potential of LEGO R SH + nucleophile R
bricks, and therefore also perfectly amenable for the construction O S R'
R'
of biomaterial libraries.
The overall theme of this review deals with facile routes for Figure 2. Thiol–ene ‘click’ reactions.11 .
the preparation of biomaterials in the broad sense by applying
various click methods. It should be added that Inglis and Barner-
Kowollik in a recent review12 attempt to provide some selection
moiety through reaction of the cysteine residue in BSA with
rules for design strategies for novel functional materials by use
propargyl maleimide and used CuAAC to couple this to an azido-
of ultra-rapid approaches to mild macromolecular conjugation in
terminated poly(N-isopropylacrylamide) (PNIPAM-N3 ) synthesized
which the various click approaches are prominent. The bulk of
by reversible addition–fragmentation chain transfer (RAFT). This
the present review is organized into four main parts, the first two
PNIPAM–BSA conjugate formed stable nanoparticles in water
sections dealing with the CuAAC and thiol–ene ‘click’ reactions,
composed of dehydrated polymer and hydrophilic protein above
respectively. This is followed by discussions of functional and
biological surfaces, and finally conducting surfaces with potential the PNIPAM lower critical solution temperature. In the light of
as sensors. multivalent dendrimers being versatile tools for the simultane-
ous presentation of biologically relevant ligands, CuAAC is very
attractive. Such materials were prepared in microwave-assisted
CU(I)-CATALYSED AZIDE–ALKYNE 1,3- CuAAC between azido di-, tetra-, octa- and hexadeca-valent pep-
tides and dendrimeric alkynes.20 These materials were claimed
CYCLOADDITON
to be useful in the preparation of synthetic vaccines or, for ex-
Shortly after the introduction of CuAAC, Kolb and Sharpless
ample, in the diagnosis and treatment of infections, where the
predicted13 the application of the method in all aspects of
biological activity can be enhanced significantly by the multi-
drug discovery, ranging from lead finding through combinatorial
valency. Assembled and scaffolded peptides could be prepared
chemistry and target-templated in situ chemistry, to proteomics
from azido and alkyne peptide and scaffold precursors through
and DNA research, using bioconjugation reactions. They claimed
CuAAC.21 The method is predicted to be particularly useful for
CuAAC to be particularly powerful due to its high degree of
dependability, its complete specificity and the biocompatibility of the generation of combinatorial libraries of assembled and scaf-
the reactants. These predictions were very soon confirmed, e.g. folded peptides through cross-combination of mutually reactive
Speers et al.14 demonstrated the ability to label enzymes in vivo azido and alkyne peptide and scaffold precursors, respectively.
and in vitro with an activity-based protein profiling probe and In addition, it is noted that the free carboxy groups of the re-
detect the labelled proteins in whole proteomes Cu(I)-catalysed sulting molecules are sites for further chemical modification, e.g.
ligation with a rhodamine–alkyne reagent. CuAAC was also through the intra- or intermolecular formation of ester and amide
employed as a potent and highly selective inhibitor of human bonds. Polymer-block-oligopeptide bioconjugates were prepared
α-1,3-fucosyltransferase.15 Link and Tirrell16 were able to label by click-ligation of azido-functional poly(ethylene glycol) (PEG)
the cell surface of Escherichia coli using CuAAC. Schultz and co- and the alkyne-functional oligopeptide GCRGDG.22,23 The route
workers17 succeeded in performing site-specific, fast, reliable and was also suggested as a ‘universal’ method towards the design
irreversible bioconjugation by the genetic incorporation of azide- of novel biomaterials such as tailor-made polymer bioconjugates
and alkyne-containing non-natural amino acids into proteins. and functional biosurfaces. A modular approach to the preparation
Versatile methods for the preparation of biohybrid am- of functional nanotapes by use of CuAAC has recently been pre-
phiphiles as models for biologically active nano-assemblies gained sented by Börner and co-workers.24 Self-assembled poly(ethylene
momentum with the advent of CuAAC. Thus terminal azide- oxide)–peptide nanotapes can be modified by a diazo transfer
functionalized polystyrene was coupled to alkyne-functionalized reaction to introduce azide groups, to which alkynated functional
GlyGlyArg tripeptides and this biohybrid amphiphile was demon- entities can be ligated through click chemistry. The functionaliza-
strated to form vesicles when injected into water from a tetrahy- tion took place while preserving the nanostructures and without
drofuran (THF) solution.18 Giant amphiphiles were furthermore interfering with the peptide organization. Many of the possibili-
constructed in a similar fashion from alkyne-functionalized bovine ties for constructing therapeutics, bioconjugates, biomaterials and
serum albumin (BSA). Sumerlin and co-workers19 prepared poly- bioactive surfaces by use of CuAAC have been recently reviewed
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mer–protein bioconjugates by functionalizing BSA with an alkyne by Lutz and Zarafshani.25

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c 2012 Society of Chemical Industry Polym Int 2012; 61: 485–494
Design of biomaterials by ‘click’ chemistry www.soci.org

The possible functionalization of micelles and shell crosslinked that circumvent these problems. Lecomte et al.35 reviewed the
nanoparticles using CuAAC was exploited by O’Reilly et al.26 In many possibilities available by the use of azide- or alkyne-
addition, the labelling of nanomaterials with active ligands was containing cyclic monomers that can be polymerized by ring-
addressed. They also advocated the introduction of bioactive opening polymerization (ROP), e.g. γ -azido-ε-caprolactone, α-
entities, which can both be tracked and detected under in vivo as propyn-1-yl-δ-valerolactone or 5-methyl-5-propargyloxycarbonyl-
well as in vitro conditions. 1,3-dioxan-2-one. It was further shown that many bioactive
Self-assembled protein capsids are interesting as a promising moieties could be ‘clicked’ onto these functionalized polyesters.
class of nanoparticles for biomedical applications due to their Maltose and glucose end-functionalization of poly(ε-caprolactone)
uniform (monodisperse) nature and versatile genetic and chemical (PCL) by CuAAC was performed by Li and co-workers.36 They
tailorability. The plasma clearance and tissue distribution in mice demonstrated the formation of stable vesicle-like aggregates
of the versatile capsid of bacteriophage Qβ were determined by of these amphiphilic polymers in water using dynamic light
means of decorating the particles with gadolinium complexes scattering and transmission electron microscopy. The saccharides
using the CuAAC reaction.27 The tobacco mosaic virus (TMV) were demonstrated to be on the surfaces of the aggregates
surface was employed as a scaffold when an alkyne was introduced by interaction with Con A, a member of the lectin family
by chemoselective modification of the tyrosine residue on the that selectively binds to α-D-mannose and α-D-glucose. It was
surface of the viral capsid.28 CuAAC was then used to couple argued that the presence of the saccharides on the aggregate
azide-functionalized compounds ranging from small molecules surface makes them useful for application in targeting drug-
to peptides to polymers onto the TMV surface with a variety of carrier systems. The azide-terminated GRGDS oligopeptide has
groups that promote or inhibit cell binding. been clicked onto an alkyne-substituted valerolactone copolymer
A C3 -symmetric triantennary N-acetyl-(1 → 6)-β-D-glucos- with caprolactone.37 The resulting amphiphilic graft copolymer
amine octadecasaccharide was convergently synthesized by was demonstrated to be biocompatible by in vitro cytotoxicity
CuAAC of the appropriate precursors.29 The target octade- evaluation paving the way for various biomaterial applications.
casaccharide showed good antitumor activity against H22 in A potent anticancer drug, camptothecin, incorporating azido
a preliminary mice test. Well-defined branched oligosaccharide moieties, and PEG-N3 have been ligated to the copolymer of ε-
mimics were prepared from dipropargylated maltoside and azido caprolactone (CL) and δ-valerolactone, which possessed pendant
maltooligosaccharides.30 These amylopectins with two isomeric
acetylenic fragments linked to the δ-valerolactone repeat units, to
hexadecasaccharide analogues have the potential for templating
afford a water-soluble and biodegradable drug delivery device.38
the formation of double helixes between two parallel mal-
In a very recent study, Müllner et al. report39 on the preparation
toheptaosyl chains attached to a core maltose unit. Various
of hybrid nanoparticles which were used as biocompatible carriers
pseudo-oligosaccharides and amino acid glycoconjugates were
for intracellular delivery as demonstrated via in vitro experiments
prepared by use of CuAAC.31 The practical ligation under neutral
with lung cancer cells. The organic polymers, which function as the
reaction conditions and the fact that several compounds con-
corona around a silica core, are produced by RAFT in combination
taining 1,2,3-triazole displayed a broad spectrum of biological
with an alkyne-carrying chain-transfer agent that mediates the
activities, such as antibacterial, herbicidal and fungicidal, anti-
polymerization and introduces a terminal alkyne, such that CuAAC
allergic and anti-HIV, were emphasized. (1 → 3)-β-D-Glucans with
can click an azido-functionalized rhodamine B dye at the periphery.
various functional attachments like lactoside, ferrocene, pyrene
CuAAC has also been demonstrated to be very effective
and porphyrin are prepared through regioselective bromination
and azidation of curdlan to 6-azido-6-deoxycurdlan followed by for couplings with DNA.11,40 Thus oligonucleotides have been
CuAAC with the functional moieties bearing a terminal alkyne.32 labelled with fluorescent dyes, sugars, peptides and other reporter
Some (1 → 3)-β-D-glucan derivatives are useful for gene carriers groups using CuAAC. Furthermore, CuAAC has been extensively
as well as redox-active and fluorescent ‘green’ materials. New en- employed to cyclize DNA, to synthesize DNA catenanes, to join
deavours in the design of neoglycopolymers were undertaken by oligonucleotides to peptide nucleic acid and to produce analogues
Haddleton and co-workers when they employed CuAAC chemistry of DNA with modified nucleobases and backbones.
to link azide-modified protected or unprotected carbohydrates The application of CuAAC click chemistry has also been much
to alkyne-backbone-functional poly(methyl methacrylate)s.33 A used for the synthesis of several different types of rotaxanes,
number of mannose- and galactose-containing multidentate lig- catenanes and molecular shuttles using passive as well as active
ands for binding studies were prepared by reacting different sugar template strategies with biological applications in mind.41 In one
azides onto the backbone. Also a fluorescent tag was added al- example the macrocycles of rotoxanes and pseudorotoxanes were
lowing for testing the reactivity of these glycopolymers in the used to create ‘nanovalves’ that by a stimuli-induced process
presence of concanavalin A and Ricinus communis agglutinin. were able to release rhodamine B dye trapped in small holes
These model lectins are able to selectively bind appropriate man- on a mesoporous silica nanoparticle (SCSN) surface.42 The pH-
nose and galactose derivatives. The potential was later exploited switchable system was based on cucurbituril (CB) pseudorotoxane
in the design of protein biohybrid materials by clicking mannopy- prepared by loading alkyne-decorated SCSNs with rhodamine
ranoside and galactopyranoside azides to polyalkyne scaffolds. It B followed by a-cucurbituril-promoted 1,3-cycloaddition. The
was further demonstrated that the materials possess enhanced pseudorotoxane-containing surface keeps the CB units on the
capacity to activate complement via the lectin pathway when axes by hydrogen bonding to two ammonium groups. At pH =
compared with native unmodified BSA and thus were able to 10 deprotonation of the ammonium groups liberates the CBs and
induce immunological function.34 releases the rhodamine B into the bulk. An enzyme-responsive
Functionalization of biodegradable aliphatic polyesters is nanovalve was prepared in a similar fashion by attaching an azide-
attractive from many points of view; however, degradation by triethyleneglycol rod to the SCSN surface before the loading.43
hydrolysis and/or transesterification are often difficult to avoid α-Cyclodextrins were threaded onto the rods and trapped by a
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during such modifications. CuAAC offers mild reaction conditions CuAAC stopping reaction. Cleavage of the ester by porcine liver

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c 2012 Society of Chemical Industry wileyonlinelibrary.com/journal/pi
www.soci.org S Hvilsted

esterase causes de-threading of the cyclodextrins and liberation oxygen and moisture leading to the corresponding thioether
of the cargo. in near-quantitative yield.54 Thiol–ene reactions comprise the
The many distinct advantages of azide–alkyne cycloadditon radical addition of thiols to electron-rich enes, and the Michael-
in the biological landscape, such as the high chemoselectivety, type addition of thiols to electron-deficient enes. The reaction
the very low background ligation rates, the facile synthetic mechanisms and the application of these reactions in the
accessibility and the stability and properties of the 1,2,3-triazole synthesis of various novel functional materials have been
products, have recently been highlighted by Mamidyala and thoroughly discussed in a number of recent reviews.53 – 56 The
Finn.44 They provide numerous examples connected with the present report, however, focuses on the radical thiol–ene click
discovery of potent inhibitors of acetylcholineesterases, carbonic reaction. Accordingly this section introduces a selection from
anhydrase, HIV-protease and chitinase. In addition, methods numerous examples of the preparation and derivatization of
for the template assembly of agents that bind DNA and macromolecules by thiol–ene coupling aiming at or pointing
proteins are presented. In another tutorial review, Holub and to new biomaterials.
Kirshenbaum thoroughly address recent developments of CuAAC Schlaad and co-workers reported modification of 1,2-
in the synthesis, modification and conformational control of polybutadiene homopolymers and block copolymers with ω-
peptidomimetic oligomers.45 A variety of issues like ligation functional mercaptans containing carboxylic acid, amine, L-amino
of peptidomimetic oligomers, synthesis of ordered ‘foldamer’ acid and fluorocarbon by free radical addition.57 The incorpo-
architectures, conjugation of ligands to peptidomimetic scaffolds ration of various desirable functionalities was possible because
and macrocyclization of peptidomimetics using triazole linkages of the availability of a vast number of thiols that allowed for
as conformational constraints are elucidated. Special attention is facile preparation of polymers with desirable functional groups
given to the widespread applications of the products, which range along the chain. However, the degree of functionalization was
from bioactive ligands to new materials. never quantitative. In following work they prepared poly[2-(3-
The CuAAC reaction employed extensively in the preparation of butenyl-2-oxazoline)] by living/controlled cationic polymerization
various biomedical materials invariably raises the question of the and clicked both hydrophobic fluoropolymers as well as water-
residual copper; the metal’s cytotoxicity is always of particular soluble glucopolymers employing 1.2–1.5 equivalents of the thiol
concern when used in living systems. Therefore much effort with respect to the unsaturation. The reactions were triggered by
has been devoted to copper-free click cycloaddition reactions exposure to UV light without additional initiator.58
in chemical biology. One feasible solution is the introduction of The specificity and reliability of the thiol–ene click reaction
a strained cyclooctyne as a source of the alkyne as originally have also been used by Hawker and co-workers to construct a
developed and recently reviewed by Bertozzi and co-workers;46 three-arm four-generation dendrimer employing 1-thioglycerol
the review also highlights the importance of bioorthogonality and 4-pentenoic anhydride that introduced the terminal alkene
to applications in vivo and in vitro. Several further modifications moieties.59 The applicability of this dendrimer was demon-
including introduction of fluorines next to the alkyne in the strated by complete functionalizing of all 48 dendrimer chain
ring system, introduction of methoxy substituents in the ring to ends with various very different monofunctional thiols. Thus
improve water solubility and increasing ring-strain by introduction thioglycolic acid was clicked in a solvent-free process, 4-(pyren-
of strained substituents on the ring to increase reaction rates 1-yl)butyl 2-mercaptoacetate could by coupled in THF and
have been carried out. All these chemical modifications of course finally 9-florenylmethoxycarbonyl cysteine was clicked in N,N-
add to the complexity of the introduction of alkyne but this dimethylformamide.
presently seems to be an unavoidable payload if copper-free In a seminal contribution, Hawker and co-workers investigated
click cycloaddition is necessary. Another type of copper-free the efficiency of photochemically and thermally induced thiol–ene
click chemistry based on nitrile oxide–alkyne cycloaddition click couplings in the modification of a series of alkene-functional
was introduced (originally copper-catalysed) in 2005 by the polymers.60 Different polymeric backbones including PEG and PCL
Sharpless group47 for coupling small molecules, and later with both single (terminal) or multiple (pendant) alkene-functional
demonstrated with ruthenium catalysis by Grecian and Fokin.48 groups along the backbone were utilized. The compatibility of the
It soon after turned out, however, that the nitrile oxide–alkyne thiol–ene reaction with various functional groups was validated
cycloaddition can be performed without a catalyst, as shown by linking a wide range of mercaptans to the unsaturations with
by Heaney and co-workers49,50 with isoxazole modification of photochemically initiated addition displaying higher efficiency
oligonucleotides. This click reaction has also been used to produce and shorter reaction times. Furthermore the click orthogonality
polyisoxazoles, which can subsequently through partial or full was verified by stepwise derivatization of a hetero-bifunctional
reduction be turned into reactive poly(β-aminoenone) or poly(β- polystyrene by thiol–ene coupling with thioglycolic acid and
aminoalcohol).51 Finally, the nitrile oxide–alkyne cycloaddition CuAAC with propargyl alcohol.60
has been used to modify polymer chain ends in an orthogonal The thiol–ene photochemical coupling has also been employed
approach together with conventional CuAAC by Lutz and in combination with the epoxy-amine reaction for building inter-
co-workers.52 nally functionalized dendrimers.61 Thus a facile access to internally
functionalized dendrimers with reactive hydroxyl groups at each
dendritic layer through efficient and orthogonal click reactions has
THIOL–ENE CLICK REACTION been devised. Such a methodology can be envisioned extended to
The thiol–ene reaction is regarded as a click reaction, now the construction of complex biomacromolecules with the present
emerging also in polymer synthesis as a simple and facile method. array of biocompatible functionality.
It is often considered orthogonal since it shows tolerance to Well-defined allyl-functionalized polyester nanoparticles were
various reaction conditions; furthermore, a wide range of easily ligated with dendritic transporter moieties bearing a thiol group
accessible starting materials is available.53 In addition, thiol–ene at the focal point and linear and cyclic peptides using a
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reactions are rapid and can be executed in the presence of thiol–ene reaction in the absence of an initiator.62 The polyester

wileyonlinelibrary.com/journal/pi 
c 2012 Society of Chemical Industry Polym Int 2012; 61: 485–494
Design of biomaterials by ‘click’ chemistry www.soci.org

nanoparticles contained various functionalities such as keto, initiated by hexyn-1-ol and the resulting PCL esterified with 4-
amine and allyl groups that could react under mild addition pentenoic acid furnishing an α-alkynyl-ω-alkenyl-PCL, which can
conditions with bioactive compounds. Thus the integrated keto undergo both CuAAC and thiol–ene click reactions. The azide-
functionality was employed in reductive amination of the N- containing L-lysine-G2 -dendron is prepared in only three steps by
terminus of peptide targeting units. Thiol–ene clicking was esterifying 3-bromo-1,2-propanediol with tert-butyloxycarbonyl
used to add a novel c-RGD and cell penetrating transporter (BOC)-protected L-lysine. The resulting dendron with a bromo
structures. Peptide–nanoparticle conjugates (NP-P-dye), dendritic moiety in the focal position is converted to the corresponding
molecular transporter nanoparticle conjugates (NP-MT-dye) as azide by the standard reaction with sodium azide. It is noted that
well as conjugates with bioactive entities (NP-P-MT-dye) were the route is much shorter than an earlier similar approach by
obtained.62 Another very important merit of the thiol–ene click Klok et al.67 using repeated protection–deprotection chemistries.
is that no metal is necessary in the reaction, which together The coupling between the alkynyl-PCL terminal and the azide-
with other metal-free reactions have expanded the possibilities containing dendron is performed using CuAAC catalysed by CuI,
for producing tailor-made macromolecules and bioconjugates, as resulting in alkenyl-PCL-block-(L-lysine-G2-dendron). The final, or-
thoroughly discussed by Schubert and co-workers.63 thogonal thiol–ene click is executed by UV irradiation of the
alkenyl-PCL-block-(L-lysine-G2-dendron) and readily available thio-
cholesteryl in the presence of the photoinitiator 2,2-dimethoxy-
2-phenylacetophenone. Deprotection of the L-lysine dendron is
NANOBIOMATERIALS BY CLICK CHEMISTRY easily achieved by trifluoroacetic acid treatment.64 The amphiphilic
Our own efforts originally focused on the design of hetero- character of the terminals is interesting; while the liquid-crystalline
bifunctional PCL, which was then employed in either orthogonal character of the cholesteryl terminal can drive self-assembly of the
CuAAC and thiol–ene click or combinations of CuAAC and rod–coil dendron, the L-lysine wedge can provide steric influ-
atom transfer radical polymerization (ATRP) to introduce various ences to direct the nanostructure formation. Furthermore, both
bioactive or medical functions.64,65 One can claim the aim has cholesterol and L-lysine can interact with cell membranes, and
been to utilize four versatile synthetic tools amongst others thus promote and facilitate cell adhesion.67 Another biomedically
to generate multifunctional amphiphilic macromolecules in a interesting amino acid, reduced L-glutathione, has been clicked to
controlled fashion for the generation of novel nanobiomaterials. α,ω-dialkenyl-PCL by the UV-triggered thiol–ene reaction.68 The
Strictly speaking the following second application belongs more to first alkene in this case is introduced by the use of allyl alcohol as
the CuAAC part. However, the initial part and the basic philosophy initiator for the ROP of CL. This example also serves the purpose
are the same and that is the claimed justification of the sequence of showing the fidelity of the thiol–ene reaction in conjunction
listed here. The concept, which is modular and thus amenable for with the appropriate substituted PCL. Thus it is foreseen that other
construction of libraries, is illustrated in Scheme 1. thiol-functional amino acids or short peptide sequences with thiol
The operational key to the approach is the well-controlled ROP functionality can be thiol–ene clicked to PCL.
of CL initiated by various functional primary alcohols and catalysed An extension of these desirable principles was exploited in
by tin octoate. This heterofunctional PCL strategy was originally the design of more robust PCL-based crosslinked nanoparticles,
developed for the preparation of an amphiphilic thiol-terminated so-called miktoarm core crosslinked star (CCS) copolymers, bear-
PCL-block-PAA block copolymer employed in the stabilization of ing multiple amine functional groups and biologically targeting
gold nanoparticles intended for bladder cancer therapy.66 After motifs on the periphery. The strategy relies on the preparation of
PCL recovery, the second functionality can be introduced at the functional methacrylic macromonomers that can be crosslinked
PCL hydroxyl terminus most often by Mitsunobu esterifications. by controlled ATRP.65 The synthetic strategy for the preparation
In the first example the target was so-called rod–coil dendrons of β-estradiol-PCL methacrylate is shown in Scheme 3. The 6-
with potential biomedical applications.51 The entire synthesis bromohexanol-initiated PCL is converted to a methacrylate after
is outlined in Scheme 2. In this case the CL polymerization is which the bromo-terminal is converted to the corresponding azide.

F1 = initiator functionality
R OH SnOct2
terminal alkyne for
CuAAC OH HOOC
O
alkene for thiol-ene
O H "click"
OH R O n
Br
terminal bromine DP ~20-40 (NMR, MALDI-TOF)
can be converted PDI ~1.1 (SEC)
HOOC
to azide for "click" OH methacrylate for macro-
monomer preparation
alkene for thiol-ene
"click"
F2 = post-functionalization
489

Scheme 1. Principle of the strategy for preparation of hetero-bifunctional PCL indicating the introduction of functional terminals.

Polym Int 2012; 61: 485–494 


c 2012 Society of Chemical Industry wileyonlinelibrary.com/journal/pi
www.soci.org S Hvilsted

BOC
NH2 O O NH
NH2 O O O O
OH O
OH O
HN OH + O
BOC
OH
SnOct2
Br OH
DCC, 4-DMAP, CH2Cl2 BOC
NH
O
BOC O
NH O
O nH
O HN O
BOC
O
NH Br
esterification with Mitsunobu protocol
BOC 4-pentenoic acid DEAD, TPP, THF
NaN3, DMF

NH
O
BOC O
BOC
NH O + O
O n
O HN
BOC O
O
NH N3
BOC L-lysine-G2-dendron CuAAC
CuI, Et3N
NH
O
BOC
BOC
NH O
O HN
BOC H
O O
NH N H rod
N O + H thiocholesteryl
N O n HS
BOC
O

DMPA (2,2-dimethoxy-
2-phenylacetophenone)
UV (@ 365 nm), Toluene
NH
O
BOC BOC
NH O
O HN
BOC H
O O
NH N H
N O H
BOC N O n S
O
rod-coil-dendron

Scheme 2. Synthetic outline of a rod–coil dendron strategy.

This is then clicked with 17β-ethynylestradiol. 17β-Estradiol is cho- with β-estradiol and (di-BOC-L-lysine)-G2 on the periphery can
sen since it is known as the most potent estrogen that enters cells be prepared as shown in Scheme 5.65 Analysis of the recov-
freely and binds to nuclear estrogen receptors (ERs).69 Since bind- ered CCS with SEC using a light scattering detector revealed
ing of β-estradiol to ERs results in conformational change initiating not only the molecular weights (Mn = 2.06 × 105 g mol−1 ,
the transcription of various genes normally upregulated in cancer- Mw = 2.65 × 105 g mol−1 ) and the resulting dispersity (1.29), but
ous cells, ERs are overexpressed in 75–80% of all breast cancers.70 also that the product did not contain unreacted macromonomers.
The multiple amine functionality is introduced by exploiting The fairly narrow dispersity furthermore suggests that the ATRP is
again the (di-BOC-L-lysine)-G2 -dendron azide that is clicked with under control, and that the contribution from possible star–star
an alkyne-terminated PCL methacrylate as illustrated in Scheme 4. coupling is negligible. By careful TGA employing also the informa-
In an optimized ATRP and by use of a conventional initia- tion obtained from analysis of the macromonomers it is possible to
tor (ethyl isobromobutyrate (EBiB)), 1,4-butanediol methacrylate estimate that the particular CCS depicted in Scheme 5 has on aver-
490

and the two functional PCL methacrylate macromomers, a CCS age 24 (di-BOC-L-lysine)-G2 -block-PCL and 27 β-estradiol-PCL arms.

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Design of biomaterials by ‘click’ chemistry www.soci.org

O O O
O Br O Br
O O
H Mitsunobu esterification m
m
with methacrylic acid
ROP of CL initiated
by 6-bromohexanol

O O
O N3 Functionalization of azide terminated
Conversion of terminal Br O macromonomer by CuAAC with
with NaN3 to azide m
17β-ethynylestradiol

N HO
O O N H OH
O N H
O
m H

Scheme 3. Synthetic strategy for preparation of β-estradiol-PCL methacrylate.

NH
O BOC
BOC
NH O
O
O HN
BOC + O
O n
O
NH N3 O
BOC
L-lysine-G2-dendron CuI/NEt3
35 °C, THF

NH
O BOC
BOC
NH O
O HN
BOC
O O
NH N
BOC N O
N O n
O

Scheme 4. Synthetic pathway for preparation of L-lysine-G2 -dendron-PCL methacrylate.

HO
N H
HO N
O O N N H OH
H N H
O N
O H OH O
m
H O
m
O O
O
BOC O
+ HN O
O
O
O BOC EBiB, CuBr/HMTETA O
O O NN O N HN BOC
50 °C, toluene
O OH H O
O N O
n N O O
HN BOC n N BOC
BOC N O N
N O H
O H
HN N
BOC BOC

Scheme 5. Preparation of CCS with β-estradiol and di-BOC-L-lysine on the periphery (only one type of each arm is shown for clarity).
491

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In addition to the examples presented where hetero- A maltose binding protein was equipped with an alkyne at
bifunctional PCL has been used in various click reactions, a the C-terminus and covalently attached to an azide-functionalized
derivatized PCL has also been employed as macroinitiator for surface.79 Biotinylated maltose used as a probe to interact with the
ATRP of tert-butyl acrylate.66 Together these examples demon- surface-linked maltose binding protein, followed by fluorescence
strate the great flexibility and fidelity of the functional, central visualization with streptavidin-Cy3 demonstrated chemoselective
PCL building block for the design of novel biomedical materials. attachment of the protein to the surface. Additionally, comparison
One conclusion is the apparent tolerance of the functional PCLs of site-specific and non-specific protein attachment showed that
to almost all other chemical functionalities. Thus these design the orientation of the protein on the surface is essential for
principles are expected to be used for the preparation of other preserving high levels of bioactivity.
valuable nanobiomaterials in the near future. The use of an alkyne-functionalized SAM allows grafting of
polymer brushes onto a passivated silicon surface using the CuAAC
strategy and azide-terminated PEG, poly(methyl methacrylate) and
FUNCTIONAL AND BIOLOGICAL SURFACES polystyrene.80 The method shows great promise for biomaterials
Surfaces with the greatest possible amount of functional groups, as demonstrated by PEG grafting. Nanopatterned multifunctional
e.g. carboxylic acids, can be prepared by use of homopolymers surfaces where one nanodomain contains an alkyne group
or copolymers cast into films.71,72 However, if only the outermost useful for CuAAC while the other microdomain carries pendant
surface is of interest this approach appears very expensive and amines, which can be functionalized with N-hydroxysuccinimide-
wasteful of resources. Exploiting surface chemistry seems a more containing compounds have been developed.81 A nanopatterned
attractive alternative. Covalently linking substances from or onto surface featuring alkyne and amine groups was realized by capillary
a surface also works in three-dimensional space; however, two force lithography of the bilayer in combination with the self-
dimensions are fixed. Many possibilities exist for adding polymer sorting of two fluorescent dyes, an azide-containing rhodamine
brushes onto various surfaces via surface-initiated controlled and an N-hydroxysuccinimide-functionalized fluorescein that was
radical polymerization.73 However, this still means that reaction performed on the patterned surface in a one-pot reaction. The
kinetics and characterization are very problematic. In particular, relatively simple fabrication and the orthogonality of the reactive
the latter is often a problem when inherent polymer properties groups are advocated as a potential platform for the synthesis of
like chain length and distribution as well as thermal behaviour are multicomponent bioactive arrays.
of importance. Click chemistry has emerged as an efficient and In a four-step BAL-based strategy involving reductive amidation,
viable coupling reaction strategy necessary for various metallic, acylation, CuAAC and trifluoroacetic acid-induced cleavage, 60 test
inorganic or polymeric surfaces that overcomes some of these compounds were screened for G-protein coupled receptor binding
problems.6,8 employing eight biogenic amine receptors. Waldmann and co-
CuAAC was exploited to attach oligosaccharides to a C14 workers have developed a new photochemical method for site-
hydrocarbon chain that non-covalently binds to a polystyrene specific immobilization and patterning of proteins with retention
microtitre well surface.74 The glycolipids non-covalently displayed of their structure and activity by use of thiol–ene coupling to
are stable under repeated aqueous washings and are functional polyamidoamine dendrimers, which were first attached to silicon
in biological screens. In addition, the saccharide arrays act as oxide surfaces.82 An aminocaproic acid spacer was attached
substrates for glycosyltransferases and are suitable for high- to the dendrimers to create distance from the surface and
throughput specificity studies of sugar–protein interactions, cystamine was coupled to the spacer, which after subsequent
enzyme inhibitor screening and diversity oriented synthesis.
reduction of the disulfide furnished the desired thiol-terminated
An early example of biological modification of a surface
surfaces. A biotin derivative was photochemically attached to
by CuAAC was the immobilization of alkyne-functionalized
the thiol-functionalized surface that after incubation with Cy5-
oligonucleotides by reaction with azide-functionalized self-
labelled streptavidin produced streptavidin-patterned surfaces,
assembled monolayers (SAMs) on gold.75 The chemistry proved
which could be imaged by fluorescence. The dimensions of the
to be extremely selective and not disturbed by typical yield-
surface patterns span the centimetre to the sub-micrometre range.
reducing reactions like hydrolysis or deactivation by elec-
The ability to control or change surfaces of polymers for
trophiles or nucleophiles. A similar approach was employed with
complex carbohydrate materials prepared in chemoenzymatic biological applications and purposes is very attractive. In general,
syntheses.76 Clicked mannose, lactose and α-Gal trisaccharide the possibility of changing chemically resistant and normally
SAMs were used in the analysis of specific carbohydrate–protein very hydrophobic polymer surfaces to biologically compatible,
interactions. non-fouling or protein-rejecting surfaces has an endless number
Carbohydrate and protein immobilization onto solid surfaces of possible applications,83 which in many cases can be done
was performed using CuAAC to an α,ω-polyethylene linker carrying by surface-initiated ATRP. However, the initial crucial step,
alkyne and cyclodiene terminal groups.77 The linker was first the introduction of the ATRP initiator, often resembles the
immobilized onto an N-(ε-maleimidocaproyl)-functionalized glass introduction of alkyne or azide functionality to a polymer
slide via an aqueous Diels–Alder synthesis. It was particularly noted surface. In principle, the ATRP initiator bromoisobutyl bromide,
that the applied diverse series of ligands, including biotin, lactose reacted onto hydroxyl groups achieved after reduction of
and a recombinant thrombomodulin protein, were stable under surface poly(ether ether ketone)s,84 or the UV-ATRP initiator
the bioconjugation conditions and did not undergo unintended benzophenonyl bromoisobutyrate, photochemically bonded to
side reactions. A backbone amide linker (BAL) was employed the surface of polypropylene,85 could easily be replaced with
for an efficient and practical parallel synthesis for both the an alkyne-containing linker or the surface bromine could be
construction of a compound library and the functionalization converted to an azide by reaction with NaN3 . This potential,
492

of formylaryloxymethyltriazole resin.78 however, still needs to be developed.

wileyonlinelibrary.com/journal/pi 
c 2012 Society of Chemical Industry Polym Int 2012; 61: 485–494
Design of biomaterials by ‘click’ chemistry www.soci.org

CONDUCTING SURFACES WITH SENSOR relatively slow click reactions experienced sometimes of the order
POTENTIAL of several tens of hours. However, a very recent study aimed at
Multiple non-interfering functionalities built into bioactive sur- circumventing these relatively slow click operations has demon-
faces for sensor applications, for example for targeting the strated that the click can take place in a few minutes by use
immobilization of large biomolecules or cellular stimulants that of a normal kitchen microwave oven.93 It should also be added
often require mild reaction conditions, are of great interest. Thus that the Bäuerle group in a recent extension demonstrated post-
the ability to functionalize a polyethylenedioxythiophene (PE- functionalization of PEDOT films with a bulky fullerene moiety.94
DOT) conducting polymer surface with azide immediately brings Moreover they also predict this strategy may be an efficient off-
multiple sensor applications to hand. This has been possible set for functionalization of polymer films with more complex
by synthesizing an azide-containing EDOT monomer (EDOT-N3 ), biomacromolecules that may pave the way for the development
which can be oxidatively (by use of Fe(III) tosylate) polymer- of novel amperometric biosensors.
ized to conducting films on a variety of surfaces including glass,
silicon wafers and polymers.86 Then various alkynes containing
a fluorophore, a short fluorinated ester or a PEG with Mn of CONCLUSIONS
5000 g mol−1 were clicked onto the surface by applying the Numerous, very different examples of both CuAAC and
conventional CuAAC protocol. All the clicked PEDOT surfaces thiol–ene click reactions employed alone or orthogonally have
showed dramatic changes of characteristics in terms of signif- demonstrated these techniques as invaluable, extremely flexible
icant changes in advancing water contact angles. Also, sensor and efficient tools for the preparation of very complex biomaterials
molecules like 2-(prop-2-ynyloxymethyl)-12-crown-4 ether, biotin and biomedical devices, which in the latter case sometimes even
prop-2-ynyl ester and ethynylferrocene could be clicked to the allows in vitro experiments. However, only brief accounts of each
PEDOT-N3 surface.87 In a similar approach Bäuerle and co-workers scenario have been provided here and the reader should consult
performed potentiodynamic electropolymerization of EDOT-N3 the original literature for full details. The total number of reports
and clicked alkynes with a neutral alkyl, an electron donor and an appears almost endless. This also reflects vigorous activities that, in
electron acceptor to the PEDOT surface.88 It should also be men- combination with the reported complexity, make it easy to predict
tioned that alkyne-containing PEDOT surfaces have recently been that many even more complex and unexpected biomaterials and
constructed by polymerizing 3,4-propylenedioxythiophene.89 biological and biomedical systems will appear in the near future.
The concept has been further developed, greatly assisted by Only fantasy seems to limit the click chemistry opportunities in
the possible spatially selective functionalization by ‘electroclick’ the biomaterials world.
chemistry.90 Here interdigitated PEDOT-azide micrometre-sized
electrodes were individually and sequentially reacted with alkyne-
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