Antimicrobial poly(2-methyloxazoline)s with bioswitchable activity through satellite group modification

Article abstract of DOI:10.1002/anie.201311150

Biocides are widely used for preventing the spread of microbial infections and fouling of materials. Since their use can build up microbial resistance and cause unpredictable long-term environmental problems, new biocidal agents are required. In this study, we demonstrate a concept in which an antimicrobial polymer is deactivated by the cleavage of a single group. Following the satellite group approach, a biocidal quaternary ammonium group was linked through a poly(2-methyloxazoline) to an ester satellite group. The polymer with an octyl-3-propionoate satellite group shows very good antimicrobial activity against Gram-positive bacterial strains. The biocidal polymer was also found to have low hemotoxicity, resulting in a high HC₅₀/MIC value of 120 for S. aureus. Cleaving the ester satellite group resulted in a 30-fold decrease in antimicrobial activity, proving the concept valid. The satellite group could also be cleaved by lipase showing that the antimicrobial activity of the new biocidal polymers is indeed bioswitchable. Biocides are widely used for preventing the spread of microbial infections and the fouling of materials. Since their application can build up microbial resistance and cause unpredictable long-term environmental problems, new biocidal agents are required. In a novel approach an antimicrobial polymer is deactivated by hydrolysis of an ester group through the action of a lipase. The crucial feature is the mutual interaction of the two endgroups of the polymer.

Full text of DOI:10.1002/anie.201311150

.
Angewandte
Communications
DOI: 10.1002/anie.201311150
Antimicrobial Polymers
Antimicrobial Poly(2-methyloxazoline)s with
Bioswitchable Activity through Satellite Group Modifi-
cation**
Christian Krumm, Simon Harmuth, Montasser Hijazi, Britta Neugebauer,
Anne-Larissa Kampmann, Helma Geltenpoth, Albert Sickmann, and
Joerg C. Tiller*
Angewandte
Chemie
3830
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 3830 –3834

Angewandte
Chemie
Abstract: Biocides are widely used for preventing the spread of
microbial infections and fouling of materials. Since their use
can build up microbial resistance and cause unpredictable
long-term environmental problems, new biocidal agents are
required. In this study, we demonstrate a concept in which an
antimicrobial polymer is deactivated by the cleavage of a single
group. Following the satellite group approach, a biocidal
quaternary ammonium group was linked through a poly(2-
methyloxazoline) to an ester satellite group. The polymer with
an octyl-3-propionoate satellite group shows very good anti-
microbial activity against Gram-positive bacterial strains. The
biocidal polymer was also found to have low hemotoxicity,
resulting in a high HC50/MIC value of 120 for S. aureus.
Cleaving the ester satellite group resulted in a 30-fold decrease
in antimicrobial activity, proving the concept valid. The
satellite group could also be cleaved by lipase showing that
the antimicrobial activity of the new biocidal polymers is
indeed bioswitchable.
contrast, biocidal polymers function as one molecule that is
able to destabilize and eventually destroy the cell membrane
of microorganisms resulting in cell lysis and cell death.[6,8a,b]
Thus, even antibiotic-resistant bacteria such as Methicillin-
resistent Staphylococcus aureus (MRSA) can be killed as
effectively as nonresistant S. aureus.[8c] The structure of
biocidal polymers, which by definition are composed of
nonbiocidal repeating units, is particularly interesting,
because full degradation of such polymers would inevitably
result in inactive compounds.[8d,e,13] A few examples of
potentially biodegradable biocidal polymers have been de-
scribed very recently.[9] Only one of them has actually been
explored regarding its degradation, showing deactivation only
under nonnatural conditions.[10] An alternative to the above-
mentioned polymers are biocides coupled as end groups to
inactive polymers.[8b,11] If the polymer contains a second, non-
antimicrobially active end group referred to as a satellite
group (SG), the antimicrobial activity of these polymers can
be controlled over several orders of magnitude.[8a] This might
offer the possibility of creating an antimicrobial polymer that
can be deactivated by altering a single bond in the whole
molecule. The idea is based on the fact that hydrophobic SGs
of appropriate length greatly activate the distal biocidal
group, whereas nonbasic hydrophilic groups deactivate it.
Thus, we chose to introduce an ester group as the SG, such
that the active macromolecule would be rendered inactive
just by the hydrolysis of this very function (Figure 1), possibly
biocatalyzed by anomnipresent lipase.
A
mong the most threatening health issues in modern
globalized society is the evolution of antibiotic-resistant
microorganisms.[1] Tremendous amounts of disinfectants are
used to fight pathogenic microbes but these weapons are
getting dull, because they help to build up microbial
resistance and are concentrated in the environment causing
unpredictable long-term problems[2,3a] either directly or by
their metabolites.[3] The optimal modern biocide would kill
germs in the targeted area for the required period of time and
then disappear.[4a] In the next best approach, a biocide would
biodegrade into an inactive, nontoxic form. So far, there are
only a few not fully accepted examples of biocides that are
degradable.[4]
The ester SG group was introduced at one polymer end by
a functional initiator, which was prepared by esterification of
3-bromopropanoic acid[12] and the respective alcohol, fol-
lowed by exchange of the bromide with iodide by a subsequent
Finkelstein reaction with NaI. Since the activity control of the
hydrophobic SG group is not easily predictable, a series of
ester initiators with various alkyl residues ranging from ethyl
to tetradecyl were been prepared (Table S1 in the Supporting
Information).
Antimicrobial polymers are a promising alternative to
low-molecular-weight antibiotics and disinfectants.[5] Such
macromolecules can be classified as biocide-releasing poly-
mers, polymeric biocides, and biocidal polymers.[6] A few
examples of the first two classes are designed to be
biodegradable, mostly to release biocides on demand.[7] In
The polymers were synthesized by cationic ring-opening
polymerization of 2-methyl-2-oxazoline (MOx) and termina-
tion with the known biocidal group N,N-dimethyldodecyl-
amine (DDA). All polymers were found to have more than
86% functionalization with SG as well as with the biocidal
[*] Dipl.-Ing. C. Krumm, Dipl.-Chem. S. Harmuth, M. Hijazi,
B. Neugebauer, A.-L. Kampmann, Prof. Dr. J. C. Tiller
Biomaterials and Polymer Science
1
Department of Bio- and Chemical Engineering, TU Dortmund
Emil-Figge-Strasse 66, 44227 Dortmund (Germany)
E-mail: joerg.tiller@udo.edu
DDA group according to H NMR spectroscopy.
Next, we tested whether the ester SG can be selectively
hydrolyzed. Thus, the polymer starting with octyl-3-iodopro-
panoate (OP) OP-PMOx-DDA was treated with 0.015m
aqueous NaOH at 508C overnight. The control experiment
was performed with an octyl bromide initiatedPMOx-DDA
(O-PMOx-DDA) described in previous work.[8a] According to
the 1H NMR spectra only OP-PMOx-SG was chemically
H. Geltenpoth, Prof. Dr. A. Sickmann
Leibniz-Institut fꢀr Analytische Wissenschaften –ISAS– e.V.
Otto Hahn-Strasse 6b, 44227 Dortmund (Germany)
[**] We thank Thorsten Moll for performing size-exclusion chromatog-
raphy and Dr. W. Hiller for performing 1H NMR measurements. We
also thank Andrea Breitkopf, Patrick Bolduan, and Shinthujah
Selvarasa for assistance in the laboratory and with the micro-
biological tests. All polymers were synthesized using CEM Discover
microwave reactors, which were kindly provided by CEM for
undergraduate student education. This work was supported by the
Ministerium fꢀr Innovation, Wissenschaft und Forschung des
Landes Nordrhein Westfalen and by grants from the Bildungsmi-
nisterium fꢀr Bildung und Forschung. We also thank the butcher
shop Niemann, Dortmund for providing the fresh porcine blood.
1
converted in the procedure (Figure S3). The H NMR spec-
trum of the NaOH-treated OP-PMOx-DDA (Figure S3b in
the Supporting Information) shows that the characteristic
octyl ester signals disappear after alkaline hydrolysis. All
other signals are not affected indicating that no other
modification took place.
Since 1H NMR data are not always sufficient to fully
characterize polymer chains, we performed electrospray
ionization mass spectrometry (ESI-MS) measurements of
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201311150.
Angew. Chem. Int. Ed. 2014, 53, 3830 –3834
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3831

.
Angewandte
Communications
(Figure 2c). Determination of
the MIC values for E. coli
revealed that only the OP-
PMOx-DDA
against this
was
active
bacterium
(Table S2 in the Supporting
Information). After ester
cleavage the antimicrobial
activity of the polymers
against both microorganisms
(see Figure 2c, back columns)
is completely eliminated.
These results indicate that it
is indeed possible to deacti-
vate a biocidal polymer by
cleaving a single bond and
applying the concept of the
SG effect. In the control
experiment with O-PMOx-
DDA, which has no cleavable
SG, the MIC value of this
polymer before and after
treatment
with
NaOH
remained the same.
Since the length of the
PMOx chain has little impact
on the molar activity as pre-
viously shown,[13] we prepared
an OP-PMOx-DDA with
Figure 1. Schematic representation of the membrane-active biocidal PMOx controlled by the cleavable
satellite group (SG) before and after hydrolysis.
OP-PMOx-DDA before and after treatment with NaOH. The
measurements were performed with trifluoroacetic acid
(TFA)/H2O solutions of the polymers. As seen in Figure 2a,
the mass spectrum of the polymer before hydrolysis confirms
a shorter chain length of 30 repeating units, expecting
a higher antimicrobial activity with respect to mass concen-
tration. It was found that this polymer is indeed more active
against S. aureus (MIC = 40 mgmLÀ1) but not against E. coli
(MIC = 1250 mgmLÀ1). To broaden the knowledge on the
antimicrobial spectrum, this polymer was also tested against
B. subtilis (MIC = 40 mgmLÀ1), S. epidermidis (MIC =
40 mgmLÀ1), S. mutans (MIC = 156 mgmLÀ1), and P. aerugi-
nosa (no activity). Apparently, the antibacterial activity of
OP-PMOx30-DDA is specific for Gram-positive bacteria.
Hydrolysis of the SG group of the macromolecule (weight
loss of less than 3 wt%) resulted in an MIC against S. aureus
of 1250 mgmLÀ1, indicating no full deactivation upon ester
cleavage but still an activity decrease by a factor of 30. These
results confirm previous findings, where a decyl SG afforded
high antimicrobial activity of DDA-PMOx polymers, while
these polymers with a shorter methyl SG showed 30 times
lower activity.[8a] Investigations of the interactions of these
polymers with liposomes revealed that the effect of the SG
can be attributed to controlling the adhesion of the polymers
onto and their insertion into cell membranes.[11] The addi-
tional carboxylic group might even increase the deactivating
effect of short alkyl chain SGs due to electrostatic repulsion
by the negatively net-charged outer surface of the bacterial
cytoplasmic membrane (Figure 1).
1
the results of the H NMR analysis. More than 93% of the
absolute counts represent mass peaks that correspond to
macromolecules containing the octyl ester, a varying number
of MOx repeating units, and the biocidal endgroup DDA.
After the polymer was treated with NaOH all mass peaks are
shifted to molecular weights corresponding to polymers that
contain a COOH instead of an octyl ester group, indicating
complete hydrolysis of the ester SG and no further modifi-
cation of the polymer backbone. The expected negative shift
in mass is 112 gmolÀ1, because formally the octyl group
(113 gmolÀ1) is replaced by a proton (1 gmolÀ1). All other
ester SG-PMOx-DDA polymers were treated similarly.
Next, the antimicrobial activity of the polymers was
explored with ubiquitous and clinically relevant bacterial
strains, the Gram-positive bacterium Staphylococcus aureus
and the Gram-negative bacterium Escherichia coli. The
minimal inhibitory concentration (MIC) was determined
from a factor two dilution series starting with a concentration
of 2500 mgmLÀ1.
As seen in Figure 2c (front columns) the antimicrobial
activity of the polymers strongly depends on the length of the
alkyl chain of the ester SG. In accordance to previous work,[8a]
short alkyl chain SGs, such as the ethyl ester, and very long
alkyl chain SGs, such as the tetradecyl ester, decrease the
biocidal potency of the macromolecules against S. aureus. The
highest antimicrobial activity was found for the octyl ester SG
Having established that cleaving the ester bond of the
satellite group of ester-PMOx-DDA deactivates the whole
polymer, we now explored the possibility of hydrolyzing this
group in a natural environment. Typically, nature hydrolyzes
hydrophobic esters with omnipresent lipases. In order to
3832
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 3830 –3834

Angewandte
Chemie
Figure 2. a) Electrospray ionization mass spectra of OP-PMOx-DDA a) before and b) after alkaline treatment. The peaks shown correspond to the
isotopes with the lowest molecular weight. c) Minimal inhibitory concentrations of the antimicrobial telechelic PMOx before and after hydrolysis.
monitor the lipase-induced cleavage of the ester SG, the
hydrolysis experiment with lipase was performed by treating
the most reactive polymer OP-PMOx30-DDA with lipase in
phosphate-buffered D2O at pH 7 at 378C and the whole
from porcine blood and a HC50 value of (4730 Æ 220) mgmLÀ1
was measured for OP-PMOx30-DDA. Thus, the octyl ester not
only acts as bioswitchable activating/deactivating satellite
group, but also improves the polymerꢀs hemocompatibitily by
an order of magnitude over that of previously described
biodical PMOx-DDA derivatives.[8b] The selectivity HC50/
MIC for S. aureus is 120 Æ 5, which is comparable to the best
recently described hemocompatible and antimicrobial poly-
mers.[15]
The HC50 of the coupling product of the octyl initiator and
DDA (OP-DDA), that is, the smallest molecule containing
both satellite and biocidal endgroup, was found to be
81 mgmLÀ1, which is a 60 times higher hemotoxicity than
that of OP-PMOx30-DDA. Thus, OP-DDA is not only six
times less active against S. aureus, but also more cytotoxic.
Additionally, the HC50 of the hydrolyzed OP-PMOx30-DDA
increased to a value of 21000 mgmLÀ1, proving that the switch
of the satellite group lowers the impact on living prokaryotic
as well as eukaryotic cells.
1
mixture was periodically analyzed by H NMR spectroscopy.
The degree of hydrolysis was calculated by following the
decrease of the integral of the signal at 1.56 ppm. The
degradation curve seen in Figure 3 shows that the ester is
slowly hydrolyzed by the lipase reaching 50% conversion
after 10 days.
Next, the lipase-catalyzed reaction was carried out in
aqueous phosphate buffer to directly measure the impact of
SG ester hydrolysis on the biocidal efficacy. The mixture of
buffer, lipase, and polymer was tested for its antimicrobial
activity after reaction times of 3 and 7 days. The control
mixture without polymer shows no antibacterial effect against
S. aureus. The MIC (S. aureus) values of the polymer after 3
and 7 days of contact with lipase were found to be 156 and
312 mgmLÀ1, respectively, while the polymer treated with the
buffer without lipase retains full antibacterial efficacy. This
clearly shows that lipase indeed deactivates the antimicrobial
activity of the polymer.
In closing, we could control both antibacterial activity and
the cytotoxicity of a polymer by attaching a biocleavable
satellite group. Hydrolysis of the single ester group of OP-
PMOx30-DDA achievable under lipase catalysis converts the
biocidal activity against S. aureus from very good to weak,
according to the definition given by Fortuniak et al.[16] We
believe that this concept might be applicable to other
Another important feature of biocidal polymers is their
toxicity against eukaryotic cells (cytotoxicity). This is often
tested with red blood cells by determining the HC50 value.[14]
The hemocompatibility test was performed with erythrocytes
Angew. Chem. Int. Ed. 2014, 53, 3830 –3834
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3833

.
Angewandte
Communications
d) E.-R. Kenawy, S. D. Worley, R.
Broughton, Biomacromolecules
2007, 8, 1359 – 1384.
[6] F. Siedenbiedel, J. C. Tiller, Poly-
mers (Basel Switz.) 2012, 4, 46 – 71.
[7] a) K. Kuroda, G. Caputo, Wiley
Interdiscip. Rev. Nanomed. Nano-
biotechnol. 2013, 5, 49 – 66; b) L. E.
Rosenberg, L. Carbone, U. Rçmling,
K. E. Uhrich, M. L. Chikindas, Lett.
Appl. Microbiol. 2008, 46, 593 – 599;
c) G. L. Woo, M. W. Mittelman, J. P.
Santerre, Biomaterials 2000, 21,
1235 – 1246; d) G. Baier, A. Caval-
laro, K. Vasilev, V. Mailꢂnder, A.
Musyanovych, K. Landfester, Bio-
macromolecules 2013, 14, 1103 –
1112; e) F. Nederberg, Y. Zhang,
J. P. K. Tan, K. Xu, H. Wang, Y.
Chuan, S. Gao, X. D. Gou, K.
Fukushima, L. Li, et al., Nat. Chem.
2011, 3, 409 – 414; f) L. P. OꢀMalley,
A. N. Collins, G. F. White, J. Ind.
Microbiol. Biotechnol. 2006, 33,
677 – 684; g) L. C. Paslay, B. Abel,
T. D. Brown, V. Koul, V. Choudhary,
C. L. McCormick, S. E. Morgan,
Biomacromolecules 2012, 13, 2472 –
2482; h) H. B. Kocer, I. Cerkez, S. D.
Worley, R. M. Broughton, T. S.
Huang, ACS Appl. Mater. Interfaces
2011, 3, 3189 – 3194.
Figure 3. a) A graphical summary of the performed 1H NMR investigation of the hydrolysis of n-
octanol in D2O catalyzed by lipase. b) Overview of the enzymatically catalyzed hydrolysis. c) Plot of
the postulated endgroup hydrolysis catalyzed by lipase.
[8] a) C. J. Waschinski, V. Herdes, F.
Schueler, J. C. Tiller, Macromol.
Biosci. 2005, 5, 149 – 156; b) C. P.
Fik, C. Krumm, C. Muennig, T. I. Baur, U. Salz, T. Bock, J. C.
Tiller, Biomacromolecules 2012, 13, 165 – 172; c) J. Lin, J. C.
Tiller, S. B. Lee, K. Lewis, A. M. Klibanov, Biotechnol. Lett.
2002, 24, 801 – 805; d) R. Horvath, B. Kobzi, H. Keul, M.
Moeller, E. Kiss, Int. J. Mol. Sci. 2013, 14, 9722 – 9736; e) C.
Mattheis, H. Wang, C. Meister, S. Agarwal, Macromol. Biosci.
2013, 13, 242 – 255.
biocides, such as antibiotics, and even to other bioactive
agents.
Received: December 23, 2013
Revised: January 21, 2014
Published online: March 5, 2014
[9] a) Y. Qiao, C. Yang, D. J. Coady, Z. Y. Ong, J. L. Hedrick, Y.-Y.
Yang, Biomaterials 2012, 33, 1146 – 1153; b) G. Cheng, H. Xite,
Z. Zhang, S. F. Chen, S. Y. Jiang, Angew. Chem. 2008, 120, 8963 –
8966; Angew. Chem. Int. Ed. 2008, 47, 8831 – 8834.
[10] M. Mizutani, E. F. Palermo, L. M. Thoma, K. Satoh, M.
Kamigaito, K. Kuroda, Biomacromolecules 2012, 13, 1554 – 1563.
[11] C. J. Waschinski, S. Barnert, A. Theobald, R. Schubert, F.
Kleinschmidt, A. Hoffmann, K. Saalwachter, J. C. Tiller, Bio-
macromolecules 2008, 9, 1764 – 1771.
[12] G. Bartoli, J. Boeglin, M. Bosco, M. Locatelli, M. Massaccesi, P.
Melchiorre, L. Sambri, Adv. Synth. Catal. 2005, 347, 33 – 38.
[13] C. J. Waschinski, J. C. Tiller, Biomacromolecules 2005, 6, 235 –
243.
Keywords: antimicrobial compounds · biodegradability ·
biocides · hemotoxicity · polymers
.
[1] a) J. Davies, D. Davies, Microbiol. Mol. Biol. Rev. 2010, 74, 417 –
433; b) H. F. Chambers, F. R. DeLeo, Nat. Rev. Microbiol. 2009,
7, 629 – 641; c) S. Viazis, F. Diez-Gonzalez, Adv. Agron. 2011,
111, 1 – 50.
[2] M. P. Shaffer, D. V. Belsito, Contact Dermatitis 2000, 43, 150 –
156.
[3] a) A. J. McBain, A. H. Rickard, P. Gilbert, J. Ind. Microbiol.
Biotechnol. 2002, 29, 326 – 330; b) P. Gilbert, L. E. Moore, J.
Appl. Microbiol. 2005, 99, 703 – 715; c) J. S. Chapman, Int.
Biodeterior. Biodegrad. 1998, 41, 241 – 245; d) E. Emmanuel,
G. Keck, J.-M. Blanchard, P. Vermande, Y. Perrodin, Environ.
Int. 2004, 30, 891 – 900.
[4] a) P. S. Guiamet, S. G. Gꢁmez De Saravia, Lat. Am. Appl. Res.
2005, 35, 295 – 300; b) B. N. Barman, Lubr. Eng. 1994, 50, 351 –
355; c) R. Neihof, C. Bailey, C. Patouillet, P. J. Hannan, Arch.
Environ. Contam. Toxicol. 1979, 8, 355 – 368.
[14] K. Lienkamp, G. N. Tew, Chem. Eur. J. 2009, 15, 11784 – 11800.
[15] a) K. Lienkamp, A. E. Madkour, A. Musante, C. F. Nelson, K.
Nꢃsslein, G. N. Tew, J. Am. Chem. Soc. 2008, 130, 9836 – 9843;
b) Y. Ishitsuka, L. Arnt, J. Majewski, S. Frey, M. Ratajczek, K.
Kjaer, G. N. Tew, K. Y. C. Lee, J. Am. Chem. Soc. 2006, 128,
13123 – 13129; c) E. F. Palermo, K. Kuroda, Biomacromolecules
2009, 10, 1416 – 1428; d) G. J. Gabriel, A. E. Madkour, J. M.
Dabkowski, C. F. Nelson, Biomacromolecules 2008, 9, 2980 –
2983.
[16] W. Fortuniak, U. Mizerska, J. Chojnowski, T. Basinska, S.
Slomkowski, M. M. Chehimi, A. Konopacka, K. Turecka, W.
Werel, J. Inorg. Organomet. Polym. Mater. 2011, 21, 576 – 589.
[5] a) J. C. Tiller, Adv. Polym. Sci. 2011, 240, 193 – 217; b) A. M.
Klibanov, J. Mater. Chem. 2007, 17, 2479; c) L. Timofeeva, N.
Kleshcheva, Appl. Microbiol. Biotechnol. 2011, 89, 475 – 492;
3834
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 3830 –3834