Visible light induced fast synthesis of protein-polymer conjugates: Controllable polymerization and protein activity

Article abstract of DOI:10.1039/c4cc02277g

Herein visible light is used to induce RAFT polymerization from protein for preparing protein-polymer conjugates at ambient temperature. Polymerization is fast and can be conveniently controlled with irradiation time. By site-specific polymerization of NI

Full text of DOI:10.1039/c4cc02277g

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Visible light induced fast synthesis
of protein–polymer conjugates: controllable
polymerization and protein activity†
Xin Li,‡a Lei Wang,‡a Gaojian Chen,*ab David M. Haddletonc and Hong Chen*a
Cite this: Chem. Commun., 2014,
50, 6506
Received 27th March 2014,
Accepted 30th April 2014
DOI: 10.1039/c4cc02277g
www.rsc.org/chemcomm
Herein visible light is used to induce RAFT polymerization from two approaches for protein conjugation – ‘‘grafting to’’ and ‘‘grafting
protein for preparing protein–polymer conjugates at ambient from’’. For example, Hoffman and coworkers successfully prepared a
temperature. Polymerization is fast and can be conveniently controlled variety of responsive protein–polymer conjugates via ‘‘grafting to’’
with irradiation time. By site-specific polymerization of NIPAm to methods which involve the reaction of a polymer that contains a
protein, the protein activity is maintained and in certain cases it presents suitable and complementary functional group for reaction at an
an efficient on–off-switchable property.
amino acid residue such as lysine, tyrosine or cysteine.2 However,
these reactions are often inefficient leading to the requirement of the
Proteins and peptides play an essential role in both highly use of an excess of functional polymers in the reaction. This
functional and structural biological applications. An ever increasing produces a mixture of conjugates and excess polymers not linked
number of therapeutic drugs in development and site specific with the protein which can lead to difficult, time consuming and
delivery agents are protein/peptide based. As bio-macromolecules often expensive separation techniques being required in this
with a very precise structure and conformation, the control of ‘‘grafting to’’ approach. The ‘‘grafting from’’ strategy is promising as
activity and stability of proteins is the key for their applications. it can simplify the purification as a low molecular weight unreacted
Often therapeutic peptides and proteins are administered by monomer is often more easily removed from the bioconjugate and
intravenous methods directly into the blood so as to avoid also the steric limitations associated with coupling the two large
complications from digestion via action of proteases. Even when macromolecules, the polymer and protein, are reduced.3 The
delivered in this direct fashion their lifetime in the body is often preparation of conjugates via ‘‘grafting from’’ has been significantly
low due to renal filtration and degradation via interactions with aided by the progress of living radical polymerization techniques.
the immune system. Conjugation of synthetic polymers with Among them, atom transfer radical polymerization (ATRP) has
proteins/peptides is a commonly employed method to improve been successfully employed for grafting polymers from proteins
protein efficacy by increasing lifetimes in vivo and decreasing with4 and without sacrificing initiators.5 However, the introduction
immunogenicity. However, this often results in reduced bioactivity of copper ions can introduce concerns regarding cytotoxicity, as
and there is an ever increasing need for conjugation to be site reported when comparing polymers prepared by ATRP and reversible
specific where interference with the active site is at a minimum. In addition-fragmentation chain transfer polymerization (RAFT).6
addition, there is increasing interest in the conjugation with RAFT, without using metal catalysts, became more popular for
stimuli responsive polymers where the activity of protein can be making protein–polymer conjugates via a ‘‘grafting from’’ approach,
turned ‘‘on’’ and ‘‘off’’ under appropriate conditions.1 There are where proteins are modified with the Z group of chain transfer
agents (CTA)7 or the R group.3b,8 Although traditional RAFT was
carried out under mild conditions for preparing protein conjugates,
polymerization from proteins often requires unsuitable long reaction
times, which is not always ideal for maintaining the bioactivity.
a The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou,
College of Chemistry, Chemical Engineering and Materials Science,
Soochow University, Suzhou, 215123, P. R. China. E-mail: chenh@suda.edu.cn;
Visible light induced RAFT came to our mind, as it can lead to well
controlled polymers at acceptably low reaction temperatures with
very acceptable fast polymerization rates.9 Thus, the ability to prepare
protein conjugates via visible light induced RAFT in aqueous media
at temperatures below 30 1C with an understanding of how this
influences protein activity is important with potential in many
therapeutic areas.
Fax: +86-512-65880827; Tel: +86-512-65880827
b Center for Soft Condensed Matter Physics and Interdisciplinary Research,
Soochow University, Suzhou, 215006, P. R. China
c Department of Chemistry, University of Warwick, Coventry, CV47AL, UK
† Electronic supplementary information (ESI) available: Detailed experimental
procedures and characterization; and additional experimental data. See DOI:
10.1039/c4cc02277g
‡ These authors contributed equally to this work.
6506 | Chem. Commun., 2014, 50, 6506--6508
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Site-specific attachment of stimuli responsive polymers
near the active site can provide a sensitive and quite precise
environmental control of the protein activity.2c Escherichia coli
inorganic pyrophosphatase (PPase) is an important protein that
provides energy and phosphate substrates for certain biosynthesis
reactions by catalyzing the hydrolysis of pyrophosphate.10 There are
only two Cys residues in the E. coli PPase, and both are buried within
the three-dimensional protein structure and show no significant
response to changes in the redox state of the environment. There-
fore, here we used site-directed mutagenesis to introduce a Cys
residue into the specific Lys148 site, which is within a conserved
sequence near the active site and is exposed to the surface of the
PPase. Site-specific modified PPase was functionalized with a RAFT
agent and subsequently used as a macro-CTA. Visible light induced
RAFT polymerization of N-isopropylacrylamide (NIPAm) was
performed and the activity of the protein–polymer conjugate
was evaluated at different temperatures (Scheme 1).
Fig. 1 Results of conjugates from visible light induced RAFT polymerization:
(a) aqueous GPC traces for PPase and PPase–PNIPAm conjugates with
different radiation time; (b) polymer molecular weight of PPase–PNIPAm
conjugates calculated from standard samples by aqueous GPC (black) and
1H NMR (red).
The maleimide functional chain transfer agent was immobilized
to PPase via its R-group to afford PPase-macroCTA, and the R-group
approach is preferred compared to the Z-group approach and the
trithiocarbonate moiety is readily accessible for RAFT chain transfer
with propagating chains in solution.8a Polymerization was carried out
in H2O at 20 1C, using (2,4,6-trimethylbenzoyl)phenyl phosphonic
acid sodium (TPO-Na) as a photoinitiator under visible light radiation
at l = 420 nm with a mild intensity of 0.2 mW cmÀ2. The reaction was
monitored using aqueous GPC (Fig. 1(a)). The GPC traces clearly
show that after 10 min, new conjugates are formed. The molecular
Fig. 2 (a) SDS-PAGE results of conjugates with different radiation time
(lane a: molecular weight marker, lane b: PPase, lane c: PPase-macroCTA,
lane d–g: 5 min/10 min/20 min/30 min radiation conjugates); (b) SDS-PAGE
results with light-on–off periodically.
To investigate the effect of visible light on the polymerization, a
weights of conjugates increased with radiation time (Fig. 1(b)) periodic light-on–off switch process was employed (Fig. 2(b)). The
according to both GPC and NMR. Sodium dodecyl sulfate- light was periodically turned off for 15 minutes between each 5 or
polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed 10 minutes radiation. PAGE results of the obtained conjugates
the results obtained via GPC and NMR as shown in Fig. 2(a). The show that the polymerization did not occur during the light-off
PPase-macroCTA mediated aqueous RAFT polymerization is rather periods as the molecular weights of conjugates remained
fast and after 30 min radiation, the molecular weight of PNIPAm on unchanged during the dark periods. After turning on the visible
the conjugates reaches about 150 kDa according to GPC. It should be light, the molecular weight increased significantly indicating that
noted that molecular weight obtained by GPC may not be precise the polymerization is turned on again with light. The periodic
due to the difference in samples and standards. By NMR, the polymerization on–off process demonstrates that the visible light
molecular weight of PNIPAm after 30 minutes is B40 kDa. exerts good control over the PPase-macroCTA mediated aqueous
Polymerization mediated by maleimide-CTA without protein RAFT polymerization. It indicates that the fast polymerization
has been carried out and it also proceeds very fast with a steady reactions occur not only through photo-initiation, but also by
increase of molecular weight (Fig. S6, ESI†), indicating that light significantly activating the fragmentation of intermediate radicals.
instead of protein is the key element for the fast polymerization. The on–off feature may also be desirable for making protein
Control polymerization reactions by replacing PPase-macroCTA conjugates with polymers of multi-blocks or more complicated
with PPase turned out to be uncontrollable and no polymers structures.
were conjugated to PPase (Fig. S10, ESI†).
PPase-macroCTA mediated RAFT under visible light radiation
can be carried out under mild conditions to afford protein–
polymer conjugates in a fast and facile fashion. And the effect
of the approach on the activity of protein is essential for its
application. In addition, the ligand–protein recognition process
and biological activity of proteins can be controlled by conjugating
polymers near the active site.11 PNIPAm is a widely used thermo-
sensitive polymer with a lower critical solution temperature
(LCST) = 32 1C, while due to the combination of PNIPAm, the
obtained conjugates have an LCST = 35 1C (Fig. S7, ESI†), which is
a little higher due to the hydrophilicity of protein. Here the effect
and controllability on the activity of PPase–PNIPAm conjugates
Scheme 1 Visible light assisted fast synthesis of protein–polymer conjugates. were investigated. PPase catalyzes the hydrolysis of inorganic
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In conclusion, visible light assisted synthesis of protein–polymer
conjugates in situ was carried out in a fast and controllable fashion
at ambient temperature for the first time. The molecular weights of
the conjugates increased with polymerization time, and in addition
temporal control was achieved with the molecular weight increase
turned on and off by controlling light radiation, which is desirable
for making conjugates of polymers bearing multi-blocks and more
complicated structures. By attaching PNIPAm site-specifically to
the position near the active site of protein PPase the activity can
be tuned by changing temperature and the molecular weight of
conjugates. In certain cases, the activity is like being turned on-and-
off between two temperatures.
This work was supported by the National Science Fund for
Distinguished Young Scholars (21125418), the National Natural
Science Foundation of China (21374069, 21334004), the Innovation
Program of Graduate Students in Jiangsu Province (CXZZ13_0810)
and the Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD). D.M.H. is a Wolfson
Royal Society Fellow. We thank Professor Xingwang Wang for
valuable discussions.
Fig. 3 Specific activity of (1) PPase-macroCTA, (2–4) PPase–PNIPAm
conjugates with polymerization time 10 min/20 min/30 min after incubation
at 25 or 45 1C for 10 min and measured at the same temperature.
pyrophosphate (PPi) into inorganic phosphate (Pi). Its catalytic
mechanism primarily relies on the lysine, arginine, tyrosine,
aspartic acid and glutamic acid residues in the active center.
The PPase catalytic activity assay for the hydrolysis of sodium
pyrophosphate were used to test the specific activity of conjugates.
We first mixed PPase with free PNIPAm and found that the free
polymer has no impact on PPase activity, no matter below or
above LCST (Fig. S9, ESI†). By chemically attaching the CTA
molecule or the polymer near the active site, the activity of
macro-CTA and conjugate decreased to an extremely low level
(approximate 0.3 kat kgÀ1) at 25 1C (Fig. 3), as compared to that of
original PPase (3.35 kat kgÀ1, Table S1, ESI†) due to the change in
the local environment (Fig. S11, ESI†). At 45 1C, a temperature
above the LCST of the PPase–PNIPAm conjugate, the activity
increased, but in different amounts. For PPase-macroCTA without
a defined LCST, activity of the proteins increased only about
twofold as the catalytic rate accelerated by temperature rise, which
was consistent with the PPase sample. However, under the same
conditions, the activity of conjugates increased about eleven fold.
This indicates that in a collapsed state of PNIPAm at 45 1C, the
polymer acts as a switch and the activity of PPase was turned on
due to the change in the local environment. Meanwhile, it is
interesting to find that above LCST the activity of conjugates with
higher molecular weight PNIPAm presents higher specific activity,
confirming the conclusion that the polymer molecular weight has
an important influence on the conjugate activity.2a,11a Although
the detailed mechanism of the activity control by attaching
PNIPAm near the active site still needs further investigation, the
effect of the visible light induced polymerization itself on the
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6508 | Chem. Commun., 2014, 50, 6506--6508
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