Reversible switching of substrate activity of poly-N-isopropylacrylamide peptide conjugates

Article abstract of DOI:10.1039/b713083j

The activity of smart polymer peptide conjugates towards chymotrypsin catalyzed hydrolysis was reversibly switched on and off using temperature as the trigger. The Royal Society of Chemistry.

Full text of DOI:10.1039/b713083j

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COMMUNICATION
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Reversible switching of substrate activity of poly-N-
isopropylacrylamide peptide conjugates{{
Kian Molawi and Armido Studer*
Received (in Cambridge, UK) 28th August 2007, Accepted 26th September 2007
First published as an Advance Article on the web 10th October 2007
DOI: 10.1039/b713083j
peptide bears more than one active side chain for covalent
The activity of smart polymer peptide conjugates towards
chymotrypsin catalyzed hydrolysis was reversibly switched on
and off using temperature as the trigger.
attachment of the initiator or the polymer, selectivity is a problem;
and (b) if more than one initiator or polymer should be added to
the peptide backbone, perfect initiator or polymer ‘‘loading’’ is
difficult to achieve. Unreacted side chains may remain. As a novel
approach, we have planned to synthesize unnatural amino acids
bearing a radical initiator moiety. Importantly, this amino acid
initiator has to be stable under standard peptide coupling
procedures. This should allow the immobilization of the amino
acid initiator at any site on the targeted peptide without
postmodification. Polymerization will eventually lead to the
conjugate. Due to the stability of the alkoxyamine functionality,
NMP has been chosen as the polymerization technique.
The development of controlled free-radical polymerization techni-
ques such as atom transfer radical polymerization (ATRP),1
RAFT (reversible addition fragmentation),2 and nitroxide-
mediated free-radical polymerization (NMP)3 has paved the way
to the synthesis of complex polymer architectures. The synthesis of
so-called smart polymers or stimuli responsive soft materials has
recently become highly important.4 Upon using an external
stimulus such as temperature, pH, light etc., the morphology
(phase behavior) of a material can be selectively altered. The
thermoresponsive poly-N-isopropylacrylamide (PNIPAM) has
often been used in that regard.5 An increase in the temperature
to above 32 uC leads to a change in the morphology and
hydrophilicity of the polymeric material in water. Below 32 uC, the
polymer is soluble in water in the chain extended highly hydrated
state. Above the so called lower critical solution temperature
(LCST), water is expelled for entropic reasons and the polymer
chains collapse and turn into their hydrophobic state. The highly
aggregated polymer is then insoluble in water. This phase switch
can be used to induce biological responses in PNIPAM
biomolecule conjugates.6,7 As recently shown by us, controlled
polymerization of N-isopropylacrylamide (NIPAM) can be
achieved via NMP using a sterically highly hindered nitroxide.8
Herein, we present the synthesis of PNIPAM peptide conjugates.
Moreover, we will show that a temperature-induced phase switch
of the smart PNIPAM tail of these novel bioconjugates can be
used to alter the substrate activity towards enzymatic hydrolysis of
the PNIPAM peptide conjugates (Fig. 1).
We decided to use the side chain of L-serine to covalently bind
the initiator moiety via ether bond formation. The bromide 1 was
readily obtained from commercially available p-ethylbromoben-
zene via radical bromination. Alkoxyamine formation using
2,2,6,6-tetramethylpiperidine-N-oxyl radical (TEMPO, 2a) or the
sterically highly hindered nitroxide 2b12 and Cu-catalysis13
afforded the alkoxyamines 3a and 3b (Scheme 1). Bromine–
lithium exchange followed by trapping of the aryllithium
compound with dimethylformamide (DMF) yielded the corre-
sponding aldehydes. Lithium aluminium hydride (LAH) reduction
and treatment of the corresponding benzylic alcohols with
Peptides have received growing attention as antibiotics and
hence temperature-induced switching of their activity is impor-
tant.9 Moreover peptides are involved in key cell surface
recognition events.10
Fig. 1 Reversible switching of the activity in PNIPAM–peptide con-
jugates—the concept.
Polymer peptide conjugates have been prepared by covalently
binding a polymerization initiator to a peptide with subsequent
polymerization (grafting from, Fig. 2). Alternatively, the con-
jugates can be synthesized by attaching a polymer to a peptide
(grafting to).11 Drawbacks of these two approaches are: (a) if the
Organic Chemistry Department, Westfa¨lische Wilhelms-Universita¨t,
Corrensstrasse 40, 48149 Mu¨nster, Germany.
E-mail: studer@uni-muenster.de; Fax: +49-(0)251-83-36523
{ Dedicated to Professor Dieter Seebach on the occasion of his 70th
birthday.
{ Electronic supplementary information (ESI) available: Experimental,
polymerization details, LCST determinations, and enzymatic hydrolysis
details. See DOI: 10.1039/b713083j
Fig. 2 Preparation of polymer peptide conjugates—different
approaches.
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Scheme 1 Synthesis of alkoxyamines 5a and 5b.
Fig. 3 NMP of styrene, butyl acrylate and N-isopropylacrylamide using
alkoxyamines 6 (n 5 0), 7 ( n 5 0) and 8 as initiators/regulators.
trimethylchlorosilane (TMSCl)–NaI in acetonitrile gave the iodides
4a and 4b in excellent overall yields. Side chain protection of Boc-
Ser-OH with the benzylic iodides 4a and 4b was achieved in DMF
using NaH to give the protected serines 5a and 5b containing
alkoxyamine moieties able to initiate the NMP.
styrene polymerization using 0.5 mol% of 8 was conducted at
105 uC for 24 h to get 9 in 80% conversion (Mn 5 16 700 g mol21
,
PDI 5 1.10). Under the same conditions, polymerization with 6
(n 5 0) or 7 (n 5 0) delivered conjugate 6 in 45% conversion
(PDI 5 1.18) or 7 in only 41% conversion (PDI 5 1.20),
respectively. As expected, the controlled polymerization of butyl
acrylate was readily achieved with 8 (1 mol%, 125 uC, 8 h) and 10
was obtained with a narrow molecular weight distribution
(PDI 5 1.13, Mn 5 14 900 g mol21, 80% conversion). However,
butyl acrylate polymerization with 6 (n 5 0) did not work at all.
Furthermore, 8 was chosen as a representative system to prove the
control/‘‘livingness’’ of the peptide initiator-mediated styrene and
butyl acrylate polymerizations. We determined the conversion as a
function of time and analyzed the molecular weight as a function
of monomer conversion (see ESI, Fig. S1 to S4{). The linear
increase in ln([M]0/[M]) versus time and molecular weight versus
monomer consumption proved the controlled character of the
polymerizations.
We successfully immobilized 5a and 5b into various peptides
using standard solution phase peptide chemistry (see ESI{). In
Fig. 3, the modified peptides 6 (n 5 0), 7 (n 5 0) and 8 used for the
initial polymerization studies are depicted. The peptides chosen are
model compounds and have no particular biological relevance.
The controlled living radical polymerization of styrene with 6
(n 5 0) or 7 (n 5 0) was performed in neat styrene at 125 uC.
The length of the polystyrene (PS) tail can be controlled by the
amount of added alkoxyamine initiator. The polymerization for
24 h using 1 mol% of 6 (n 5 0) provided the PS–peptide conjugate
6 in 72% conversion with a number average molecular weight
(Mn) of 6100 g mol21 and a polydispersity index (PDI) of
1.15 as determined from gel permeation chromatography (GPC).
Reducing the alkoxyamine concentration to 0.5 mol% provided 6
with Mn 5 11 700 g mol21 (PDI 5 1.15). Similarly, the
PS–heptapeptide 7 bearing a PS-tail was successfully synthesized
We were also able to control the polymerization of NIPAM
with 8. The polymerization was conducted in C6D6 using 100
equiv. of NIPAM at 125 uC for 8 h to give 11. The conversion
(42%) was determined using 1H NMR spectroscopy. Mn
(6500 g mol21) and PDI (1.16) were determined from mass
spectrometry.8,14 Larger PNIPAM-tails were obtained upon
increasing the reaction time and reducing the alkoxyamine
(0.5 mol% initiator, 18 h, 65% conversion, Mn 5 12 200 g mol21
,
PDI 5 1.16).
The alkoxyamines 6 (n 5 0) and 7 (n 5 0) contain the TEMPO
moiety to control the radical polymerization. However, it is well
known that controlled NMP using TEMPO as a regulator is
restricted to the polymerization of styrene and styrene derivatives.3
Therefore, we switched to 8 bearing a sterically highly hindered
nitroxide.12 As compared to the 6 (n 5 0) and 7 (n 5 0)-mediated
polymerization of styrene, reaction with 8 was more efficient. The
concentration (0.5 mol%, 20 h, 71% conversion, Mw
5
10 800 g mol21, PDI 5 1.36). For the higher molecular weight
conjugates, Mw was determined using multiple angle laser light
scattering (Mw 5 weight average molecular weight).8
5174 | Chem. Commun., 2007, 5173–5175
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Notes and references
§ We thank the Deutsche Forschungsgemeinschaft (STU 280/1–4) for
supporting our work. BACHEM AG is acknowledged for donation of
various amino acids.
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Fig. 4 Alkoxyamines 12 and 14 used in the enzymatic hydrolysis to yield
13 and 15.
For enzymatic studies, we prepared the PNIPAM peptide
conjugates 12 (Mw 5 12 700 g mol21, PDI 5 1.38) and 14
(Mw 5 10 900 g mol21, PDI 5 1.38, Fig. 4). The C-terminus of
these peptides was protected as p-nitroanilide, which was known to
undergo chymotrypsin-catalyzed hydrolysis to liberate the UV
active p-nitroaniline and the conjugates 13 and 15.
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The temperature-induced phase transitions (LCST) of the
PNIPAM peptide conjugates 11, 12 and 14 were studied using
1H NMR spectroscopy (see ESI{).15 Conjugate 11 showed a
phase switch at around 31 uC. For 12 and 14, the LCST was
found at 29 uC. It has been known that low molecular weight
PNIPAM conjugates bearing a hydrophobic moiety at the
polymer chain end have LCSTs below 32 uC, in agreement with
our results.16
The enzymatic reactions using 12 and 14 were conducted with
commercially available chymotrypsin and were followed with UV
spectroscopy (see ESI{). At 37 uC, the enzymatic hydrolysis was
completely suppressed for both substrates. At 22 uC, however, 12
and 14 are soluble in H2O and the enzymatic reaction occurred. It
is important to note that hydrolysis could be stopped by increasing
the temperature to 37 uC. Renewed cooling to 22 uC reinstalled the
active state of the system and the reaction went to completion (see
Fig. S8 in the ESI{).
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In conclusion, we showed that serines bearing an alkoxyamine
moiety able to initiate/regulate
a controlled living radical
polymerization could readily be synthesized and immobilized at
any position of a peptide. NMP of various monomers delivered
the corresponding polymer peptide conjugates. The activity of
PNIPAM peptide conjugates towards enzymatic hydrolysis was
readily switched by temperature control.§
16 For the LCST of PNIPAMs bearing different end groups, see:
P. Kujawa, F. Tanaka and F. M. Winnik, Macromolecules, 2006, 39,
3048; Q. Duan, Y. Miura, A. Narumi, X. Shen, S.-I. Sato, T. Satoh and
T. Kakuchi, J. Polym. Sci., Part A: Polym. Chem., 2006, 44, 1117;
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