Synthetic approach to homodimeric protein-polymer conjugates

Article abstract of DOI:10.1039/b822799c

Synthesis of well-defined homodimeric protein-polymer conjugates using RAFT polymerization is described.

Full text of DOI:10.1039/b822799c

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Synthetic approach to homodimeric protein–polymer conjugatesw
Lei Tao,a Catherine S. Kaddis,b Rachel R. Ogorzalek Loo,b Gregory N. Grover,a
Joseph A. Loob and Heather D. Maynard*a
Received (in Cambridge, UK) 18th December 2008, Accepted 20th February 2009
First published as an Advance Article on the web 18th March 2009
DOI: 10.1039/b822799c
Synthesis of well-defined homodimeric protein–polymer conjugates
using RAFT polymerization is described.
RAFT polymerization was chosen because it enables
predictable molecular weights10 and reactive chain ends that
are susceptible to reducing or radical coupling reactions.11,12
The ability to target molecular weight is important because
biological activities of dimers can be significantly affected by
the polymer spacer length.9 The chain end reactivity provides a
convenient way to install a protein-reactive group. These post-
polymerization modification techniques have been recently
used to synthesize heterodimeric protein–polymer conjugates
by us and others.13,14 But to our knowledge, homodimeric
conjugates have not been synthesized by any controlled radical
polymerization technique.
In nature, proteins often exist as dimers or multimers, or they
self-assemble into higher order oligomers in the active state.1
In many cases, this type of multimerization is critical
for induction and regulation of biological processes.2 For
example, approximately 70% (311) of the total human
enzymes (452) are multimers, and many of the multimers are
homodimers (125).3 Cytokines, an important class of cell
signaling molecules, often dimerize in the active state, and this
association is crucial for tyrosine kinase receptor activation.
Thus, by locking proteins into conjugate designs that present
multiple copies of the biomolecules, one can study multivalent
interactions in biological processes such as signal transduction
and prepare therapeutics with higher activity than monovalent
counterparts.1,4,5 For example, epidermal growth factor
(EGF) is a well-studied cytokine that regulates cell adhesion,
proliferation, differentiation, and invasion. It is a monomeric
protein that dimerizes in the active site. This dimerization
causes two EGF receptors (EGFRs) to associate, resulting in
phosphorylation and cell signaling.6,7 This suggests that
tethering together two copies of EGF would provide a material
with superior properties. Indeed, a dendrimer modified with
an average of two EGFs exhibited significantly higher affinity
to cells that over express EGFR than did free EGF.8 In
another example, cyclic guanosine monophosphate (GMP)
polymer dimers were able to bind to cyclic-nucleotide-gated
channels with significantly higher affinity and were up to a
thousand times more potent than the ligand alone.9 Yet
despite these advantages, the majority of protein–polymer
conjugates used in therapeutics and other applications consist
of one protein bound to one or more polymers. This is likely
due to the lack of available strategies to synthesize the poly-
mers, particularly those that react with proteins to produce
conjugates of defined stoichiometry. Herein we describe the
efficient synthesis of protein-reactive telechelic polymers by
reversible addition–fragmentation chain transfer (RAFT)
polymerization and their use to produce homodimeric
protein–polymer conjugates.
The strategy we employed was polymerization from a
bistrithiocarbonate chain transfer agent (CTA), radical
exchange of the trithiocarbonates with a protein-reactive
azo-initiator, followed by conjugation of the protein
(Scheme 1). Specifically,
a difunctional polyNIPAAm
(pNIPAAm) was synthesized by RAFT polymerization
(Scheme 2a) using bisfunctionalized CTA (1). PNIPAAm
was chosen because it responds to changes in temperature,
and thus is utilized in numerous biotechnology applications.15
A small percentage (0.20% of total monomer) of a monomer
containing nitrobenz-2-oxa-1,3-diazole (NBD, 2) was
incorporated because it provides a way to verify conjugate
formation.16 Polymerization (initial ratios of [NIPAAm + 2] :
[1] : [AIBN] = 100 : 1 : 0.1) was carried out to 82% conversion
under an argon atmosphere at 80 1C, and the resultant
difunctional pNIPAAm (3) was isolated after extensive
purification by dialysis. The polymer had a low polydispersity
index (PDI) of 1.06 and a number average molecular weight
(Mn) of 15 500 Da by gel permeation chromatography (GPC)
(Fig. S2, ESIw). The Mn was higher than the expected
molecular weight of 9800 Da because the value was deter-
mined by calibration with poly(methyl methacrylate)
standards. To more accurately determine the molecular
weight, the samples were subjected to GPC with laser light
scattering (LLS) detection (Fig. S4a, ESIw), 1H NMR
spectroscopy (Fig. S5a, ESIw), and matrix-assisted laser
desorption–ionization time of flight (MALDI-TOF) mass
spectrometry (Fig. S3, ESIw) resulting in values of 11 500,
10 500, and 8990 Da, respectively. These molecular weights
a Department of Chemistry & Biochemistry and the California
NanoSystems Institute, University of California,
607 Charles E. Young Dr. East, Los Angeles, CA 90095, USA.
E-mail: maynard@chem.ucla.edu; Tel: +1 310 267 5162
b Department of Chemistry and Biochemistry, David Geffen School of
Medicine, University of California-Los Angeles, Los Angeles,
CA 90095-1570, USA. E-mail: JLoo@chem.ucla.edu
w Electronic supplementary information (ESI) available: Further
experimental procedures and data. See DOI: 10.1039/b822799c
Scheme 1 Synthesis of dimeric protein conjugates.
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polydispersity index to 1.10 (Fig. S4b, ESIw). These data
further confirmed that the chain end modification reaction
had occurred.
Deprotection of the end groups was facile. Upon heating the
polymer at 120 1C under vacuum for 1 h, the retro Diels–Alder
reaction occurred exposing the maleimides (5, Scheme 2b).
During this process, the furan byproduct was removed, and
no further purification of the polymer was necessary. The
1H NMR spectrum (Fig. S5c, ESIw) indicated that the depro-
tection had occurred, as evident by the disappearance of the
peaks at 6.51, 5.25, and 2.85 ppm and appearance of a new
peak at 6.72 ppm. The polydispersity index remained
unchanged (PDI = 1.09, Fig. S4c, ESIw). These data indicated
that side reactions were minimal and that the telechelic
maleimide polymer was formed.
Under neutral or slightly acidic pH, maleimides are known
to react with thiols approximately 1000 times faster than with
amines.17 Therefore, we investigated bioconjugation with a
model protein that contains one free cysteine in order to create
a site specific protein–polymer conjugate. A V131C mutant T4
lysozyme (T4L) was chosen as the protein. The bioconjugation
reaction was carried out at 4 1C in phosphate buffered saline
(PBS) in the presence of 10 mM of tris(2-carboxyethyl)-
phosphine hydrochloride (TCEP) and 10 mM of ethylene-
diaminetetraacetic acid (EDTA) for 16 h. The salts were
removed by centrifugal filtration (MWCO 10 000).
Scheme 2 Synthesis of telechelic maleimide pNIPAAm.
were similar to what was predicted, suggesting that the purified
polymer had the designed symmetric structure. Utilizing the
molecular weight obtained by MALDI-TOF MS, the end
group retention was 91%, indicating that the trithiocarbonate
groups were available for further elaboration.
SDS-PAGE analysis indicated that T4L–polymer conju-
gates formed (Fig. 1). The gel with Coomassie staining
had two new higher molecular weight spots at B26 kDa
and B48 kDa compared to unmodified T4L at B18 kDa
(lanes B compared to D). These new bands were attributed to
the monomer and dimer protein–polymer conjugate adducts
which amounted to 79% and 21% based on fluorescence
quantification, respectively. The spots were not visible when
the control furan-protected maleimide polymer was incubated
with the protein (lane C). Liquid chromatography tandem
mass spectrometry (LC-MS/MS) of the trypsin digests showed
that T4L was present in the bands attributed to the monomer
and dimer conjugates (see ESIw for details). The polymer
contained NDB units; thus the gel was also exposed to UV
light for visualization. The spots attributed to the monomer
and dimer conjugate were observed (Fig. 1b, lane B), confirm-
ing that polymer was attached to the proteins in these cases.
For the control conjugation, again no conjugate was observed
(lane C).
Verification of the dimeric structure was carried out by
aminolysis of the polymer with butylamine in order to cleave
the middle ester bonds, followed by MALDI-TOF MS
analysis (Fig. S3, ESIw). The molecular weight of the arm
was 4480 Da and the expected mass was 4470 Da. This result
further confirmed that the CTA was effective in producing the
desired dimeric polymer.
Radical addition of a functional azo-initiator (4) to the
polymer chain ends was chosen as a convenient method to
introduce the cysteine-reactive functionality. To avoid any
possible cross-linking reactions during radical coupling, the
maleimide was protected as the Diels–Alder adduct with
furan. Polymer 3 was heated at 70 1C in the presence of 4 in
order to form the furan-protected polymer (Scheme 2b). We
found that a 4 h reaction time was optimal for modification of
the chain ends without thermal removal of the protecting
1
group. Comparison of the H NMR spectra before and after
confirmed that the reaction had occurred (Fig. S5a and b,
ESIw). The peak at 3.34 ppm corresponding to the methylene
group of the chain end CTA disappeared while new peaks
appeared at 6.51, 5.25, and 2.85 ppm corresponding to the
furan-protected maleimide. Additional analysis by UV-Vis at
306 nm gave a zero absorbance, which indicated that all of the
trithiocarbonate chain ends had reacted. We were not able to
obtain the molecular weight by MALDI-TOF MS. However,
we were able to determine the values by NMR and GPC.
Comparison of the integral of the peak for the main chain CH
of pNIPAAm with those from the end group gave a Mn by 1H
NMR of 11 600 Da. This result was similar to what was
calculated prior to modification (10 500) suggesting a high
end group conversion. Analysis by LLS provided an Mn
of 12 700. There was only a slight broadening of the
To confirm the PAGE results, analysis by mass spectro-
metry was undertaken. The theoretical molecular weights of
Fig. 1 SDS-PAGE of the protein–polymer conjugates (a) stained
with Coomassie blue, (b) visualized with UV light (PDB #1P56).
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Fig.
2
(a) MALDI-TOF MS, (b) ESI-GEMMA of the protein–polymer conjugates. Lys: T4L, M-L: monomer linked conjugate
(T4L-pNIPAAm), and D-L: dimer linked conjugate (T4L-pNIPAAm-T4L).
the protein–polymer conjugate and protein–polymer–protein
conjugate calculated using the Mn determined by mass
spectrometry of the polymer were 27.8 kDa and 46.6 kDa,
respectively. The MALDI-TOF MS spectrum of the conjugate
(Fig. 2a) displayed peaks at 18.8 kDa, 28.8 kDa, and 48.1 kDa
corresponding to unmodified protein, monomeric and dimeric
conjugate, respectively.
dimeric protein–polymer conjugates resulting in materials with
superior biological activities compared to the monomeric
conjugates or unmodified proteins. We are currently exploring
these possibilities.
This work was supported by the NSF (CHE-0809832).
HDM thanks Amgen (New Faculty Award) and the Alfred
P. Sloan Foundation for further support. The UCLA Func-
tional Proteomics Center was established and equipped by a
grant from the W. M. Keck Foundation. JAL acknowledges
support from the NIH (RR20004). CSK thanks the
Chancellor’s Dissertation Year Fellowship and GNG thanks
the Christopher S. Foote Graduate Fellowship.
Electrospray ionization–gas-phase electrophoretic mobility
macromolecule analysis (ESI-GEMMA) was also performed
(Fig. 2b).18 ESI-GEMMA separates macromolecules based on
their electrophoretic mobility in air. ESI-GEMMA has a
higher sensitivity than MALDI-TOF for high mass proteins
and polymers. ESI-produced multiply charged particles are
subject to charge reduction such that particles detected are
mostly singly charged. Thus, the observation of complicated
ESI mass spectra of heterogeneous samples and large particles
is minimized using the ESI-GEMMA method. The electro-
phoretic mobility diameter (EMD) of the peaks identified in
the ESI-GEMMA spectra and the calculated molecular
weights were 4.6 nm and 18 kDa for T4L, 5.3 nm and
27 kDa for the polymer with one protein, and 6.5 nm and
50 kDa for the polymer with two proteins. Based on the
standard deviation of the density used to calculate the mole-
cular weights from the EMD values (see ESIw), the molecular
weights were expected to be within Æ10% of the reported
values. Combining the gel electrophoresis, MALDI-TOF MS
and ESI-GEMMA data, it was concluded that the desired
protein dimer polymer conjugate formed.
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It was not unexpected that monomeric conjugate was
observed along with the desired dimer. It is known that
macromolecular reaction dynamics can hinder reactions that
would be efficient for small molecules.19 To remove the
monomer adduct, a standard cation exchange resin was
employed and the product eluted with increasing salt concen-
trations. Analysis by SDS PAGE (Fig. S6, ESIw) indicated
that with 0.14 M NaCl, the monomeric conjugate eluted. The
dimer was retained on the column until a concentration of
0.18 M salt was introduced, providing a way to effectively
isolate the desired conjugate.
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that this straightforward strategy will provide access to
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