Copper-free click chemistry for the in situ crosslinking of photodegradable star polymers

Article abstract of DOI:10.1039/b803043j

Bifunctional, fluorinated cyclooctynes were used for the in situ "click" crosslinking of azide-terminated photodegradable star polymers, yielding photodegradable polymeric model networks with well-defined structures and tunable gelation times. The Royal S

Full text of DOI:10.1039/b803043j

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Copper-free click chemistry for the in situ crosslinking of
photodegradable star polymersw
Jeremiah A. Johnson,a Jeremy M. Baskin,b Carolyn R. Bertozzi,bc Jeffrey T. Kobersteind
and Nicholas J. Turro*ad
Received (in Cambridge, UK) 21st February 2008, Accepted 25th March 2008
First published as an Advance Article on the web 24th April 2008
DOI: 10.1039/b803043j
Bifunctional, fluorinated cyclooctynes were used for the in situ
‘‘click’’ crosslinking of azide-terminated photodegradable star
polymers, yielding photodegradable polymeric model networks
with well-defined structures and tunable gelation times.
however, using this approach, chemically diverse star MACs
can be prepared from presumably any combination of the
numerous monomers polymerizable by ATRP (acrylates,
methacrylates, acrylamides, styrenics, etc. . .). For the cross-
linking step, 1 was allowed to react with a bifunctional alkyne
via CuAAC to yield MNs. As expected for the ‘‘cream of the
crop’’4 of click reactions, CuAAC crosslinking was achieved
chemoselectively in high yield.
Covalently crosslinked polymeric materials which swell, but
do not dissolve, in a given solvent (polymer gels) have found
utility in a number of applications including tissue engineer-
ing,1a drug delivery,1b chemical sensing,1c microfluidics,1d cos-
metics,1e and microelectronics.1f End-linked polymer gels
(model networks or MNs) are especially promising for drug
delivery applications due to their easily controlled, homoge-
neous pore sizes.2 As a result, much effort has been focused on
developing general synthetic strategies capable of yielding
functional MNs.
Having shown the effectiveness of ATRP and CuAAC for
preparing MACs, we chose to focus on optimizing the cross-
linking reaction because, despite the success of CuAAC for the
crosslinking of 1, it has a few major drawbacks which hinder
its general applicability to MN and polymer gel synthesis.
First, to obtain the most homogeneous MNs in organic
solvents, the crosslinking had to be performed under an inert
atmosphere, a requirement not suitable for many industrial
applications. Furthermore, the crosslinking required the use of
copper as a catalyst, as well as a ligand/base additive, so
repeated swelling of the materials in fresh solvent was neces-
sary to yield ‘‘pure’’ materials. Also, copper is known to be
cytotoxic to most bacterial and mammalian cells,7 and because
MN synthesis can be roughly divided into two stages: (1)
synthesis of a macromonomer (MAC) precursor and (2) cross-
linking. Viewing the synthetic process in this manner enables
one to design a MN bearing complex functionality by first
utilizing the well-developed tools of standard organic and
polymer synthesis to prepare functional MACs.3 Then, MN
synthesis is reduced to finding a crosslinking reaction that
proceeds in high yield and is chemoselective between the
desired crosslinking functionalities, but orthogonal to all other
functionalities present, i.e., reactions which meet the standards
of Sharpless’ click chemistry.4
As an example of this two-stage strategy, we recently
reported2c the design and synthesis of photodegradable MNs
based on a tetra-nitrobenzyloxycarbonyl (NBOC), tetra-azido
terminated, poly(tert-butyl acrylate (tBA)) star polymer (1,
Scheme 1) which was prepared by tandem copper-catalyzed
azide–alkyne cycloaddition5 (CuAAC) and atom transfer
radical polymerization (ATRP)6 followed by end group trans-
formation. The tBA monomer was chosen because it can be
easily hydrolyzed to acrylic acid thus yielding hydrogels;
a Department of Chemistry, Columbia University, 3000 Broadway,
MC3157, New York, New York 10027, United States.
E-mail: njt3@columbia.edu
b Departments of Chemistry and Molecular and Cell Biology and
Howard Hughes Medical Institute, University of California,
Berkeley, California 94720, United States
c The Molecular Foundry, Materials Sciences Division, Lawrence
Berkeley National Laboratory, Berkeley, California 94720, United
States
Scheme 1 Structures of star polymer 1, diMOFO, diDIFO, MN from
diDIFO and 1, and the corresponding linear polymer photodegrada-
tion product. To clearly depict their location in the MN structure,
photodegradable NBOC groups are shown in red and diDIFO is
shown in orange.
d Department of Chemical Engineering, Columbia University, 500
West 120th Street, New York, New York 10027, United States
w Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/b803043j
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3064 | Chem. Commun., 2008, 3064–3066

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it is very difficult to ensure, even after extensive extraction,
that all of the copper has been removed from the bulk gel, it is
highly desirable to avoid CuAAC crosslinking altogether for
biological applications. The toxicity of copper could also
complicate the use of CuAAC for biological in situ cross-
linking (i.e., crosslinking in vivo or in the presence of living
cells). Finally, CuAAC crosslinking led to rapid, uncontrolled
gelation, and for certain applications it may be desirable to
control the gelation time in order to synchronize with another
process of interest.
In search of an alternative to CuAAC for crosslinking, it
became necessary to decide whether a modification to 1 was
necessary or if an alternative reaction of azides could be
employed. As mentioned above, diversely functional polymers
possessing azide end groups can be easily prepared by ATRP
and end group modification.2c,8 Furthermore, azides are
known to be bioorthogonal.9 For these reasons, we explored
alternative reactions of azides for crosslinking rather than
change to another functionality altogether. Fortunately, all
of the drawbacks of CuAAC crosslinking described above can
indeed be overcome by using a copper-free variant that utilizes
fluorinated cyclooctyne reagents to effect Huisgen [3þ2] dipo-
lar cycloaddition with azides. This reaction, the strain-pro-
moted azide–alkyne cycloaddition9 (SPAAC), has been shown
to proceed very efficiently with high chemoselectivity even in
in vivo applications, making it perfectly suitable for in situ
crosslinking. Furthermore, since no copper or ligand/base is
required, there would be only two components to the network
and, as a result, little extractable material. Finally, as has been
shown previously, the rate of the SPAAC reaction can be
controlled by making electronic modifications to the
alkyne.9b,c
Fig. 1 Average decay curves for the loss of azide during gelation
between 1 and diMOFO or diDIFO as monitored by FTIR. Experi-
ments were performed in triplicate with error bars shown in grey. Inset
shows the azide antisymmetric stretch region of the FTIR spectrum at
different intervals during a typical gelation experiment with diDIFO.
dynamically changing environment within the gelation cavity
(i.e., increasing viscosity) during crosslinking.
We have shown previously that although no azide absor-
bance is observed after crosslinking, unreacted chains still
inevitably exist due either to incomplete reaction or error in
stoichiometry of azide
: alkyne, and thus, FTIR is not
sensitive enough to determine a yield for the crosslinking
reaction.2c,d If crosslinking occurs chemoselectively, however,
between the azide of 1 and the cyclooctyne of either cross-
linker, well-defined MNs would result which, upon photoclea-
vage of the NBOC groups, would yield soluble linear polymers
having a number average molecular weight (Mn) equal to 0.5
that of 1 (Fig. 2, inset). Furthermore, unreacted chains would
give soluble products having Mn equal to 0.25 that of 1. To
confirm chemoselective crosslinking, and to assess the yield of
Carbodiimide-mediated condensation of ethylenediamine
with two equivalents of monofluorinated cyclooctyne
(MOFO) and difluorinated cyclooctyne (DIFO) yielded cross-
linkers diMOFO and diDIFO, respectively (Scheme 1, see
ESIw). DIFO is known to react with azides more rapidly in
solution than MOFO9c and herein we report the same phe-
nomenon during the crosslinking with 1. Fourier transform
infrared spectroscopy (FTIR) can be used to monitor the
kinetics of the SPAAC reaction by monitoring the decay of
the azide antisymmetric stretch absorption and assuming that
its intensity is a linear function of the concentration of azide.
Separate millimolar stock solutions in DMF of a known
concentration of star polymer 1 and either diMOFO or
diDIFO were mixed together such that the final concentration
of azide : cyclooctyne was 1 : 1 in a total reaction volume of
10 mL. This solution was then quickly injected into the gelation
cavity (a sealed, FTIR cell with CaF2 plates and a 25 mm
Teflon spacer), and spectra were recorded continuously until
no change was observed in the azide absorbance at B2100
cmꢁ1. Fig. 1 shows the decay of the azide absorbance over a
13 h time span, confirming the faster rate of azide loss for
diDIFO versus diMOFO and thus confirming that the cross-
linking kinetics can be controlled solely by altering the elec-
tronics of the crosslinker without modifying 1. Unlike the
SPAAC reactions of MOFO or DIFO with model azides in
solution, which follow second order kinetics,9b,c these data
display more complicated kinetic behavior indicative of the
Fig. 2 SEC traces of 1 before and after photocleavage, and of the
photodegradation products of diMOFO and diDIFO derived MNs.
Inset shows schematic of crosslinking depicting the conversion of 1 to
a MN and finally to linear polymers having Mn E 0.5 that of 1.
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crosslinking, the transparent gelation cavity was submerged in
tetrahydrofuran (THF) and irradiated with 350 nm light for
2 d. The THF solution was then analyzed by size exclusion
chromatography (SEC) and the resulting chromatograms are
shown in Fig. 2. As expected, both MNs yielded a major peak
with Mn E 20 kDa which corresponds to successfully cross-
linked ends of 1. Both MNs also yielded a minor peak with Mn
E 10 kDa corresponding to unreacted or ‘‘dangling’’ chain
ends within the network. The MNs crosslinked with diDIFO
showed fewer unreacted ends, a possible result of either its
greater reactivity or, perhaps because it is smaller, its greater
mobility in the highly hindered MN environment. Finally, the
crosslinking yield for SPAAC is comparable to that found
previously for CuAAC,2c confirming the high efficiency of the
SPAAC reaction.
Notes and references
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This study represents the first example of SPAAC in
materials synthesis, specifically for the crosslinking of poly-
meric materials, and it opens a general route to complex,
functional MNs capable of biocompatible, in situ crosslinking,
controlled gelation time, and tailored degradation. Addition-
ally, this work represents the first example of monitoring the
kinetics of an in situ crosslinking process using the azide
antisymmetric FTIR stretch, an approach which can be
widely applied to studying the increasing repertoire of azide
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We thank the NSF (DMR-0214363, IGERT-0221589,
DMR-0213574, CHE04-15516, graduate fellowship), the
NIH (GM058867), the NDSEG (graduate fellowship), and
the ACS Division of Organic Chemistry (graduate fellowship)
for support of this work. We also thank J. Codelli and
B. Dickinson for technical assistance.
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This journal is The Royal Society of Chemistry 2008
3066 | Chem. Commun., 2008, 3064–3066