Antimicrobial polymers prepared by ROMP with unprecedented selectivity: A molecular construction kit approach

Article abstract of DOI:10.1021/ja801662y

Synthetic Mimics of Antimicrobial Peptides (SMAMPs) imitate natural host-defense peptides, a vital component of the body's immune system. This work presents a molecular construction kit that allows the easy and versatile synthesis of a broad variety of facially amphiphilic oxanorbornene-derived monomers. Their ring-opening metathesis polymerization (ROMP) and deprotection provide several series of SMAMPs. Using amphiphilicity, monomer feed ratio, and molecular weight as parameters, polymers with 533 times higher selectivitiy (selecitviy = hemolytic concentration/minimum inhibitory concentration) for bacteria over mammalian cells were discovered. Some of these polymers were 50 times more selective for Gram-positive over Gram-negative bacteria while other polymers surprisingly showed the opposite preference. This kind of "double selectivity" (bacteria over mammalian and one bacterial type over another) is unprecedented in other polymer systems and is attributed to the monomer's facial amphiphilicity.

Full text of DOI:10.1021/ja801662y

Published on Web 07/01/2008
Antimicrobial Polymers Prepared by ROMP with
Unprecedented Selectivity: A Molecular Construction Kit
Approach
Karen Lienkamp,† Ahmad E. Madkour,† Ashlan Musante,† Christopher F. Nelson,†,‡
Klaus Nu¨sslein,‡ and Gregory N. Tew*,†
Department of Polymer Science and Engineering, UniVersity of Massachusetts, Amherst,
Massachusetts 01003 and Department of Microbiology, UniVersity of Massachusetts,
Amherst, Massachusetts 01003
Received March 5, 2008; E-mail: tew@mail.pse.umass.edu
Abstract: Synthetic Mimics of Antimicrobial Peptides (SMAMPs) imitate natural host-defense peptides, a
vital component of the body’s immune system. This work presents a molecular construction kit that allows
the easy and versatile synthesis of a broad variety of facially amphiphilic oxanorbornene-derived monomers.
Their ring-opening metathesis polymerization (ROMP) and deprotection provide several series of SMAMPs.
Using amphiphilicity, monomer feed ratio, and molecular weight as parameters, polymers with 533 times
higher selectivitiy (selecitviy ) hemolytic concentration/minimum inhibitory concentration) for bacteria over
mammalian cells were discovered. Some of these polymers were 50 times more selective for Gram-positive
over Gram-negative bacteria while other polymers surprisingly showed the opposite preference. This kind
of “double selectivity” (bacteria over mammalian and one bacterial type over another) is unprecedented in
other polymer systems and is attributed to the monomer’s facial amphiphilicity.
ꢀ-amino acids,4–11 peptoids,12–14 aromatic oligomers,15–17 and
Introduction
synthetic polymers.18–23
When organisms are attacked by bacterial pathogens, natural
antimicrobial peptides (AMPs) are among the first line of
defense. These host-defense peptides have broad-spectrum
antimicrobial activity.1 Their production in the organism is much
faster than that of specific antibodies, and thus they are a vital
component of innate immunity. AMPs are found in many
species, including humans, animals, plants, and invertebrates.1
Whereas today’s common antibiotics target specific cell struc-
tures, AMPs use nonreceptor interactions, including in many
cases direct action against the bacteria’s membranes, although
other targets have been identified.1,2 The cells of the host
organism are less affected; thus AMPs can selectiVely attack
bacteria within a host organism. Bacteria could only become
immune to AMPs if they change their entire membrane
chemistry or other targets; thus resistance to AMPs is retarded
as compared to other antibiotics.3 Due to this promising feature,
there has been increasing research in the past few years aimed
at the production of synthetic mimics of antimicrobial peptides
(SMAMPs). These include the SMAMPs made of R- and
The common feature of most AMPs is their positive charge
and facial amphiphilicity. Regardless of their secondary struc-
ture, these peptides generally display one hydrophobic and one
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2002, 99, 5110.
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† Department of Polymer Science and Engineering.
‡ Department of Microbiology.
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Soc. 2004, 126, 15870.
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J. Am. Chem. Soc. 2007, 129, 12141.
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9836 J. AM. CHEM. SOC. 2008, 130, 9836–9843
10.1021/ja801662y CCC: $40.75
2008 American Chemical Society

Antimicrobial Polymers Prepared by ROMP
A R T I C L E S
hydrophilic face along their backbone.1,24,25 Due to positive
charges in the hydrophilic part, AMPs bind preferentially to
the anionic outer membranes of bacterial pathogens or other
anionic targets including proteins and DNA.3,26 In many cases,
their facial amphiphilicity allows them to insert into the bacterial
membrane and to locally change the membrane’s lipid organiza-
tion in such a way that transmembrane pores are formed,
although other mechanisms of action are also known.1 Several
mechanisms of pore formation have been proposed to describe
this, including the carpet, barrel-stave, and toroidal pore
models.1,2 This interaction may lead to a breakdown of the
membrane potential, the leaking of the cytoplasm, and eventually
the death of the pathogen cell.27
Learning how to capture the essential biological properties
of AMPs in synthetic polymers should teach us which essential
chemical features of these natural peptides are required for
antibacterial activity. In addition, access to these synthetic
polymers may open up new applications, for example, in the
materials area, where bacterial infections from medical plastics
are a current critical problem in our hospitals. Synthetic
polymers can be obtained easily and in large quantities while
still presenting facial amphiphilicity and positive charge, the
key features of AMPs. Although there have been several recent
reports of polymeric SMAMPs, their overall activities and
selectivities remain far from optimal. Examples include the
following: DeGrado and co-workers reported SMAMPs based
on poly(ammonium methylmethacrylate) salts copolymerized
with poly(butylmethacrylate) to tune the amphiphilicity;28
Klajnert et al.29 produced dendritic SMAMPs; Liu et al.30
synthesized SMAMPs from poly(maleic acid) linked to peptide
tetramers; Makovitzki et al.31 recently made SMAMPs based
on lipopeptides; and Gellman and co-workers presented a
poly(amide) based polymer with good activities (12.5 µg/mL
against E. coli and 3.1 µg/mL against S. aureus) and selectivities
up to 32 for bacterial over mammalian cells.23 Tew and co-
workers synthesized facially amphiphilic antibacterial polymers
basedonarylamides,15 urea,17 andpoly(phenyleneethynylene).20,21
SMAMPs based on poly(norbornene) derivatives were previ-
ously described by Tew and Coughlin: they reported polymers
with facially amphiphilic repeat units that had tunable antimi-
crobial activity depending on a defined ratio of hydrophobic
and hydrophilic moieties in the repeat unit. Their most selective
polymer had a hundred times higher activity toward bacteria
than against human red blood cells.19 They also very recently
reported poly(norbornenes) with quaternary pyridinium groups
(selectivities up to 20 against E. coli).32
Figure 1. “Construction kit” approach to obtain facially amphiphilic
monomers and polymers. Just as with a Lego construction kit, the synthetic
approach presented here allows the independent combination of a hydrophilic
(blue), a hydrophobic (green), and a polymerizable (yellow) part of the
monomer to yield a whole set of antimicrobial polymers with tunable activity
and selectivity.
was therefore to develop a ring-opening metathesis polymeri-
zation (ROMP) platform that (i) uses a minimum number of
building blocks and (ii) allows the easy and independent
variation of the hydrophobic and hydrophilic residues on the
monomer. Although previous work has shown that antimicrobial
activity can also be achieved with random copolymers of
hydrophilic and hydrophobic monomers,18,23 we believe that
having facially amphiphilic monomers, i.e., monomers with a
hydrophilic cationic and a hydrophobic part on the same
polymerizable unit, allows for more precise tuning of the
antibacterial activity. The key components of our molecular
construction kit are highlighted in Figure 1. The hydrophilic
(blue) and the hydrophobic component (green) are attached to
the polymerizable oxanorbornene group (yellow) and can be
varied independently. In this report we have restricted ourselves
to varying the hydrophobic component, while holding the
hydrophilic “lysine-like” primary amine constant.
Experimental Section
All experimental procedures, including monomer and polymer
synthesis, as well as the biological assays, are included in the
Supporting Information.
Results and Discussion
Monomer Synthesis. To obtain new synthetic antimicrobial
polymers via ROMP, the first task was to design an easy and
modular synthetic pathway toward facially amphiphilic mono-
mers (Figure 1). The three-step approach taken to obtain these
monomers is presented in Scheme 1. In the first step, furan and
maleic anhydride underwent a Diels-Alder reaction, yielding
exclusively the exoadduct in accordance with the literature.34
This facile step provided compound 1 containing a polymeriz-
able oxanorbornene group and a cyclic anhydride that allowed
2-fold and unsymmetrical functionalization. The anhydride 1
was ring-opened with an alcohol to introduce the desired
hydrophobic moiety R, which was varied from methyl to hexyl,
yielding a series of half-monomers 2a-f with different hydro-
The previously reported poly(norbornene) based SMAMPs
suffered from the fact that each polymer required extensive
synthetic effort to tune the amphiphilicity of the repeat units19
or did not allow copolymer synthesis.32 The aim of this work
(24) Boman, H. G. Immunol. ReV. 2000, 173, 5.
(25) Hancock, R. E. W.; Lehrer, R. Trends Biotechnol. 1998, 16, 82.
(26) Yeaman, M. R.; Yount, N. Y. Pharmacol. ReV. 2003, 55, 27.
(27) Yount, N. Y.; Bayer, A. S.; Xiong, Y. Q.; Yeaman, M. R. Biopolymers
(Peptide Sci.) 2006, 84, 435.
(28) Kuroda, K.; DeGrado, W. F. Polym. Prepr. (Am. Chem. Soc., DiV.
Polym. Chem.) 2004, 45, 610.
(29) Klajnert, B.; Janiszewska, J.; Urbanczyk-Lipkowska, Z.; Bryszewska,
M.; Shcharbin, D.; Labieniec, M. Int. J. Pharm. 2006, 309, 208.
(30) Liu, Z.; Deshazer, H.; Rice, A. J.; Chen, K.; Zhou, C.; Kallenbach,
N. R. J. Med. Chem. 2006, 49, 3436.
(31) Makovitzki, A.; Avrahami, D.; Shai, Y. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 15997.
(33) Sambhy, V.; Peterson, R. T.; Sen, A. Angew. Chem., Int. Ed. 2008,
47, 1250.
(32) Eren, T.; Som, A.; Rennie, J. R.; Nelson, C. F.; Urgina, Y.; Nu¨sslein,
K.; Coughlin, E. B.; Tew, G. N. Macromol. Chem. Phys. 2008, 209,
516.
(34) Mantovani, G.; Lecolley, F.; Tao, L.; Haddleton, D. M.; Clerx, J.;
Cornelissen, J. J. L. M.; Velonia, K. J. Am. Chem. Soc. 2005, 127,
2966.
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A R T I C L E S
Lienkamp et al.
Scheme 1. Monomer Synthesisa
It is well-known from the literature that the G3 catalyst initiates
quantitatively,35 so incomplete initiation is unlikely to be
responsible for this disparity. Analysis of the Boc-protected
Propyl_3k sample (Mn, GPC ) 9200 g/mol) by MALDI-TOF
MS (Matrix Assisted Laser Ionization and Desorption Time Of
Flight Mass Spectrometry) yields a distribution with a maximum
intensity at m/z ) 3095, which is in excellent agreement with
the targeted molecular weight. This means that GPC overesti-
mates the molecular weight for these structures, which is further
supported by the oligomer data discussed in more detail below.
Biological Activity of Homopolymers. The biological proper-
ties of the homopolymers, i.e., their antibacterial activity, MIC90,
toward growth of Escherichia coli and Staphylococcus aureus
bacteria, and their hemolytic activity, HC50, toward red blood
cells, were tested as described previously.37 HC50/MIC90 quanti-
fies the antimicrobial selectivity. The biological data obtained
for these homopolymers are included in Table 1b. The MIC90
and HC50 values are also plotted in Figure 2a and 2b. As can
be seen from this set of data, the polymer with the highest
selectivity for bacterial over mammalian cells is Ethyl_3k, with
a good selectivity of 28 for both E. coli and S. aureus. For
comparison, MSI-78, a derivative of the natural host-defense
peptide magainin, has a selectivity of 10.38 For the 3k series
shown in Figure 2a, the following trend is observed: starting
with the nontoxic (HC50 ) 2000 µg/mL) and inactive (MIC90
> 200 µg/mL) Methyl_3k, the antibacterial activity peaks for
the Propyl_3k (MIC90 ) 6.25 µg/mL for E. coli), and there
the polymers also start becoming more hemolytic (HC50 e 50
µg/mL). From Butyl_3k to Hexyl_3k the activity decreases
again until the polymers become inactive (MIC90 > 200 µg/
mL) but this time becoming strongly hemolytic. The MIC90’s
for S. aureus show the same trend; however the activities are
generally lower. The data for the 10k series are compiled in
Figure 2b. The MIC90’s for the 10k polymers are similar to the
ones for the 3k series, with a maximum activity at Propyl_10k,
but they are generally less active against E. coli than the 3k
polymers and inactive against S. aureus.
a The hydrophobic component of the facially amphiphilic monomer is
introduced in the second reaction step (R ) methyl, ethyl, propyl, butyl,
isopentyl, or hexyl), and the protected hydrophilic moiety is attached in
the last step.
phobicities. All compounds were crystallizable and thus easy
to purify. In the last step, the designated cationic group was
attached. As ROMP usually does not tolerate the presence of
unprotected amines due to their ligating properties,35 the desired
hydrophilic group (NH3+) was introduced in its protected tert-
butyl carbamate (NHBoc) form. The half-monomers 2a-f were
reacted with the Boc-protected 2-amino ethanol by DCC
coupling, yielding a series of masked amphiphilic monomers
3a-f (Scheme 1). This last step required purification by column
chromatography to yield pure products. The overall yield of all
three reaction steps was ∼40% (see Supporting Information for
all details).
Homopolymer Synthesis and Molecular Weight Character-
ization. From the monomers 3a-f, two series of homopolymers
with molecular weights of 3000 g/mol () 3k series) and 10 000
g/mol () 10 k series) were synthesized. The polymerization of
the monomers, shown in Scheme 2, was carried out using the
third generation Grubbs catalyst (G3)36 and yielded the precursor
polymers 4a-f. The facially amphiphilic SMAMPs 5a-f were
obtained by polymer analogous deprotection: the Boc protecting
group was completely removed with trifluoroacetic acid ac-
cording to NMR. Depending on the alkyl residue, the resulting
polymers were water-soluble or dispersible after workup. Details
on the workup procedure can be found in the Supporting
Information. For the reader’s convenience, instead of referring
to these polymers by compound number, for example 5a-f, a
polymer with R ) propyl and a molecular weight of 3000 g/mol
will be referred to as Propyl_3k. The organosoluble precursor
polymers were analyzed by gel permeation chromatography
(GPC) in DMF (Table 1a). The molecular weights obtained from
GPC using polystyrene standards for calibration are significantly
larger than the ones expected from the reaction stoichiometry.
These observations can be rationalized by taking the different
compositions of mammalian and bacterial membranes into
account. Human red blood cells (RBC) are predominantly
composed of cholesterol and phosphatidylcholine (outer leaflet).
In contrast, the membranes of Gram-negative bacteria like E.
coli consist mostly of phosphatidylethanolamine and anionically
charged phosphatidylglycerol (PG) while Gram-positive bacteria
like S. aureus have membranes that consist mainly of anionically
charged PG and cardiolipin. Thus bacterial membranes are more
negatively charged than RBC membranes.39 Just as observed
for natural AMPs,1 the anionic surface charge may attract the
Scheme 2. Polymer Synthesisa
a ROMP polymerization is followed by polymer analogous hydrolysis with trifluoroacetic acid to yield the facially amphiphilic polymer.
9
9838 J. AM. CHEM. SOC. VOL. 130, NO. 30, 2008

Antimicrobial Polymers Prepared by ROMP
A R T I C L E S
Table 1. Homopolymers
(a) Precursor polymer characterization by GPCa
sample
monomer
Mn. Target g mol-1 GPC Mn g mol-1
Mw/Mn
Methyl_3k
Ethyl_3k
Propyl_3k
Butyl_3k
Isopentyl_3k
Hexyl_3k
Methyl_10k
Ethyl_10k
Propyl_10k
Butyl_10k
Isopentyl_8k
methyl
ethyl
propyl
butyl
isopentyl
hexyl
methyl
ethyl
propyl
butyl
3000
3000
3000
3000
3000
11 200
9200
9200
1.08
1.10
1.10
1.08
1.11
1.08
1.05
1.10
1.07
1.07
1.06
1.16
1.04
11 500
11 400
11 000
49 000
11 300
28 200
26 700
22 600
51 500
27 600
3000
10 000
10 000
10 000
10 000
8000
isopentyl
Isopentyl_10k isopentyl
Hexyl_10k hexyl
10 000
10 000
(b) Characterization of the antimicrobial homopolymers by biological assaysb
MIC90 µg mL-1
E. coli S. aureus
selectivty
E. coli S. aureus
sample
HC50 µg mL-1
Methyl_3k
Ethyl_3k
Propyl_3k
Butyl_3k
Isopentyl_3k
Hexyl_3k
Methyl_10k
Ethyl_10k
Propyl_10k
Butyl_10k
Isopentyl_8k
Isopentyl_10k
Hexyl_10k
>200
50
6.25
15
12.5
>200
>200
>200
3.75
20
100
50
15
25
2000
1400
50
<50
<50
<50
50
1250
<50
<50
<50
<50
n.d.
<10
28
8.3
20
28
3.3
<3.3
<4.0
<0.3
<10
<6.3
13
<2.5
<0.3
<0.3
n.d.
<2.0
<1.0
<0.3
<10
<6.3
<0.25
<0.3
<0.3
<0.3
n.d.
50
>200
>200
>200
200
>200
>200
200
Figure 2. Biological data (MIC90 for E. coli and S. aureus, and HC50 for
red blood cells) for the homopolymers. (a) 3000 g/mol series, (b) 10 000
g/mol series.
50
50
100
>200
While Propyl_3k was also found to be the most toxic 3k
polymer against S. aureus, the 10k polymers were all inactive
against this pathogen (Figure 2b). This may be rationalized as
follows: Gram-positive bacteria have a 15-80 nm thick
negatively charged murein layer around the cell membrane. As
is well-known from the polyelectrolyte literature, complexation
of one polyion with an oppositely charged polyion (symplex
formation) is essentially irreversible, while complexes of a
polyion with a less charged species are reversible.40 Thus,
assuming that the negatively charged murein layer forms a
polyion-polyion complex with the positively charged SMAMPs,
the dissociation of such a complex becomes increasingly more
difficult with increasing molecular weight (more charges on the
SMAMP). Thus the higher molecular weight SMAMPs get stuck
in the murein layer of Gram-positive bacteria before reaching
the plasma membrane and as a result do not kill S. aureus cells
irrespective of their amphiphilicity. This would suggest that the
appropriately hydrophobic polymers would be active against
Gram-negative bacteria because they have a much thinner (2
nm) murein layer which is located between the outer and
cytoplasmic membranes. Figure 2b shows that the data are
consistent with this hypothesis. For example, Propyl_10k has
an MIC90 of 3.75 µg/mL against E. coli while its MIC90 against
S. aureus is 200 µg/mL, resulting in a polymer that is doubly
selective; it is >50 times more active against E.coli than against
S. aureus and 13 times more active against E.coli than against
RBCs.
a DMF, 0.01 mol L-1 LiCl, PS standards. b Inhibitory activity
towards bacterial growth of E. coli and S. aureus bacteria. (MIC90
minimal inhibitory concentration preventing 90% bacterial growth) and
hemolytic activity towards red blood cells (HC50 ) concentration lysing
50% of blood cells).
)
positively charged SMAMP, and then due to their hydrophobic
component they can subsequently penetrate the lipid bilayer if
the SMAMP’s facial amphiphilicity is rightly balanced. An
overly hydrophilic SMAMP, like Methyl_3k, is not able to
penetrate the hydrophobic core of the lipid bilayer and is
therefore inactive. Alternatively, such a hydrophilic molecule
may prefer to remain in solution as opposed to being adsorbed
to the membrane. Their high hydrophilicity also prevents
Methyl_3k and Ethyl_3k from lysing RBCs; only the more
hydrophobic homologues (Propyl_3k onward) cause significant
hemolysis (Figure 2a). An overly hydrophobic SMAMP like
Hexyl_3k has strong membrane activity and as a result is very
hemolytic. These observations lead to the maximum activity
against E. coli and S. aureus for the 3k series was from the
Propyl_3k polymer because it seems to have the optimal facial
amphiphilicity to penetrate the bacterial membrane.
(35) Slugovc, C. Macromol. Rapid Commun. 2004, 25, 1283.
(36) Dichloro-di(3-bromopyridino)-N,N′-dimesitylenoimidazolino-
RudCHPh, c.f.: Love, J. A.; Morgan, J. P.; Trnka, T. M.; Grubbs,
R. H. Angew. Chem., Int. Ed. 2002, 41, 4035.
Oligomer Synthesis and Molecular Weight Impact on
Biological Activity. The finding that biological activity is
different for the 3k and 10k series led to the following questions:
(37) MIC90 ) minimal inhibitory concentration preventing 90% bacterial
growth, HC50 ) hemolytic concentration lysing 50% of blood cells,
c.f.: Rennie, J.; Arnt, L.; Tang, H.; Nuesslein, K.; Tew, G. N.
J. Industrial Microbiol. Biotechnol. 2005, 32, 296.
(38) Gabriel, G. J.; Som, A.; Madkour, A. E.; Eren, T.; Tew Gregory, N.
Mater. Sci. Eng. ReV. 2007, 57, 28.
(40) Dautzenberg, H.; Jaeger, W.; Ko¨tz, J.; Philipp, B.; Seidel, C.;
(39) Som, A.; Tew, G. N. J. Phys. Chem. B 2008, 112, 3495.
Stscherbina, D. Polyelectrolytes; Hanser/Gardner: Munich, 1994.
9
J. AM. CHEM. SOC. VOL. 130, NO. 30, 2008 9839

A R T I C L E S
Lienkamp et al.
Table 2. Oligomers
(a) Precursor oligomer characterization by GPCa
sample
Mn Target g mol-1
GPC Mn g mol-1
nGPC
GPC Mw/Mn
MALDI Mn g mol-1
MALDI Mw/Mn
nMALDI
monomer (3c, hydrolyzed)
Oligo 1
Oligo 2
Oligo 3
Oligo 4
Oligo 5
Oligo 6
370
740
1080
3300
4300
5800
5900
6900
7400
2.9
8.9
1.04
1.08
1.09
1.07
1.09
1.10
1.08
–
–
–
1529
2151
2642
2873
3260
3935
1.11
1.09
1.07
1.11
1.08
1.09
3.7
5.3
6.7
7.0
8.2
9.7
1110
1480
1850
2220
2590
11.6
15.7
16.0
18.6
20
(b) Characterization of the antimicrobial oligomers by biological assaysb
MIC90 µg mL-1
selectivty
sample
E. coli
S. aureus
HC50 µg mL-1
E. coli
S. aureus
monomer (3c, hydrolyzed)
Oligo 1
Oligo 2
Oligo 3
Oligo 4
Oligo 5
Oligo 6
Propyl_3k
Propyl_10k
>200
200
100
200
>200
100
>200
<3.75
6.25
25
n.d.
1050
800
1250
>2000
1000
150
n.d.
5.25
8.0
6.3
>10
10
3.0
8.3
<13
n.d.
280
128
50
>40
20
6.0
3.3
<0.25
50
50
25
15
50
6.25
3.75
50
<50
200
a DMF, 0.01 mol L-1 LiCl, PS standards. b Inhibitory activity towards bacterial growth of E. coli and S. aureus bacteria (MIC90 ) minimal inhibitory
concentration preventing 90% bacterial growth) and hemolytic activity towards red blood cells (HC50 ) concentration lysing 50% of blood cells).
(i) At what molecular weight does the biological activity start,
and (ii) can tuning of the molecular weight be further exploited
to selectively target S. aureus or E. coli?
To answer the first question, all monomers were deprotected
(with trifluoroacetic acid as described for the polymers),
subjected to MIC90 testing, and found to be inactive (MIC90’s
> 200 µg/mL). This shows that a minimum chain length is
necessary to obtain any antibacterial activity, consistent with the
proposed idea that a facially amphiphilic structure and not a simple
surfactant is required for antibacterial activities in the µg/mL range.
Consequently, choosing the propyl monomer as an example, small
molecular weight oligomers were synthesized and analyzed (see
Table 2a). The GPC traces for the oligomers (selected samples
for clarity), along with the monomer, are shown in Figure 3a.
The peak maxima of all samples are in the expected order: the
higher molecular weight oligomers elute before the lower
moleculartk;2 weight ones. MALDI-TOF MS was used to
determine the actual oligomer molecular weight. The peaks of
the MALDI-TOF mass spectra are shown in Figure 3b as a
distribution function (normalized intensity vs m/z). Peaks below
m/z ) 1500 g/mol could not be used as those peaks were lost
in the background noise. From the relative peak intensities and
m/z ratio of each peak, the number average molecular weight,
Mn, and the polydispersity tk;1of the samples were calculated.
Those distributions were monomodal, unlike some the GPC
traces which appear to be multimodal due to the column
resolution. These data are included in Table 2a. The results show
once more that GPC curves calibrated with poly(styrene)
standards heavily overestimate the molecular weight, while the
average number of repeat units nMALDI is in much better
agreement with the calculated number of repeat units. However,
the smallest oligomer obtained had a degree of polymerization
of 3.7 instead of the target of 2 due to the reaction kinetics of
this particular solvent/catalyst system.
Figure 3. Characterization of propyl oligomers. (a) GPC traces and (b)
MALDI-TOF MS peaks and distributions.
activity increases by 1 order of magnitude with Oligo 6 (HC50
) 150 µg/mL) and increases further for Propyl_3k (HC50
)
50 µg/mL) and Propyl_10k (HC50 > 50 µg/mL). Figure 4 shows
that the small molecular weight oligomers are inactive against
E. coli. Starting with Oligo 5, a marked increase in activity
against E. coli (MIC90 ) 100 µg/mL) is observed, which reaches
a maximum for Propyl_10k with an MIC90 of 3.75 µg/mL. For
S. aureus, the opposite trend is observed (Figure 4). The best
activities were found for the small oligomers (MIC90’s ) 6.25
The biological data for the propyl series are summarized in
Figure 4 and Table 2b: within the error limits of the method,
all oligomers are equally nonhemolytic up to Oligo 5. Hemolytic
9
9840 J. AM. CHEM. SOC. VOL. 130, NO. 30, 2008

Antimicrobial Polymers Prepared by ROMP
A R T I C L E S
that molecular weight can be used as a parameter to selectively
target either E. coli or S. aureus, and possibly Gram-negative
or Gram-positive bacteria in general.
Previous research indicated that some SMAMPs have only
slight molecular weight dependent activity. Ilker et al.19
concluded for their system that the biological activity is
independent of molecular weight (for Mn ) 1600 to 137 000
g/mol); poly2 and poly4 (MIC90’s of 200 µg/mL against E. coli)
did not show a molecular weight dependence. Poly3 was
reported with an Mn ranging from 1650 g/mol to 57 200 g/mol
and MIC90’s of 25 µg/mL to 80 µg/mL, respectively. DeGrado18
reported poly(methacrylate) copolymers with an Mn around 1600
g/mol having an MIC90 of 16 µg/mL while polymers above 7900
g/moL exhibited an MIC90 of 80 µg/mL against E. coli. In
contrast, Oligo 1 has an Mn equal to 740 g/mol and MIC90’s <
3.75 µg/mL against S. aureus and 200 µg/mL against E. coli,
while Propyl_10k has MIC90’s of 200 µg/mL against S. aureus
and 3.75 µg/mL against E. coli. This leads to the conclusion
that antimicrobial activity is molecular weight dependent for
these SMAMPs.
Figure 4. Biological data (MIC90 for E. coli and S. aureus, and HC50 for
red blood cells) for the propyl oligomer series.
Tuning the Selectivities by Copolymer Synthesis. It was
shown previously that the incorporation of highly active but
hemolytic repeat units into an otherwise nonactive and non-
hemolytic polymer can increase the selectivity of that polymer
tremendously, making the best of both worlds.19 In our
compound library, the Methyl_3k homopolymer qualifies as
inactive and nontoxic, whereas the Propyl_3k homopolymer
is active and hemolytic. The Ethyl_3k homopolymer is neither
most active nor toxic but has the highest selectivity. Thus,
methyl, ethyl, and propyl monomers were chosen for copolym-
erization. Three series of copolymers with a target molecular
weight around 3000 g/mol and a varying monomer feed ratio
were synthesized and studied (Table 3a). The copolymer
composition was checked by NMR spectroscopy, which revealed
that the polymer composition matched with the monomer feed
ratio.
The biological data are compiled in Table 3b and in Figure 5a
to 5c. For the ethyl-propyl series, none of the copolymers
reached the high selectivity of the parent Ethyl_3k polymer:
with increasing propyl content, the ethyl-propyl copolymers
become more active but also more toxic. Both series of methyl
copolymers showed the expected trend: they become extremely
active even with little amount (10 mol%) of the more active
monomer but stay nonhemolytic. An amazing and unexpected
result however is that they are only potently active against S.
aureus (MIC90 < 3.75 µg/mL) and remain totally inactive
against E. coli (MIC90 >200 µg/mL)! These copolymers are
again doubly selective: >533 for bacterial over mammalian cells,
and >53 for S. aureus over E. coli. This double selectivity is
inverse to the one described earlier. The high selectivity of >533
for S. aureus is unprecedented in the SMAMP literature.
The reasons for this amazing and surprising double selectivity
are not yet understood and are currently under further investiga-
tion. An up to 10-fold selectivity for Gram-positive B. subtilis
over Gram-negative E. coli has been observed for peptoids12
and ꢀ-peptides;41 however this trend was not explained. One
attempt toward an explanation is that E. coli has an outer
membrane around its actual plasma membrane, while S. aureus
has only one plasma membrane below its murein layer. This
means that the SMAMPs need to rupture two membranes in
order to kill E. coli cells, which intuitively might require a higher
SMAMP concentration. On the other hand, if the SMAMPs are
able to diffuse unmolested through the murein layer of S. aureus
Figure 5. Biological data (MIC90 for E. coli and S. aureus, and HC50 for
red blood cells) for the copolymers. (a) Ethyl-propyl series, (b) methyl-
ethyl series, and (c) methyl-propyl series. Sample labeling: e.g., E1/P9 is a
copolymer with 10 mol% ethyl and 90 mol% propyl monomer.
µg/mL and < 3.75 µg/mL for Oligo 1 and Oligo 2, respec-
tively). Activity is progressively lost as the molecular weight
increases up to Propyl_10k (MIC90 ) 200 µg/mL). This is in
line with the above-discussed hypothesis that the larger SMAMPs
get stuck at the murein layer due to symplex formation. The
results of the molecular weight dependent propyl series show
9
J. AM. CHEM. SOC. VOL. 130, NO. 30, 2008 9841

A R T I C L E S
Lienkamp et al.
Table 3. Copolymers
(a) Precursor polymer characterization by GPCa
sample
monomers
Mn Target g mol-1
GPC Mn g mol-1
Mw/Mn
E1:P9
E1:P2
E1:P1
E2:P1
E9:P1
M9:E1
M1:E1
M1:E9
M9:P1
M1:P1
M1:P9
ethyl-propyl
ethyl-propyl
ethyl-propyl
ethyl-propyl
ethyl-propyl
methyl-ethyl
methyl-ethyl
methyl-ethyl
methyl-propyl
methyl-propyl
methyl-propyl
3500
3400
3000
3500
3400
3400
3500
3500
3400
3600
3700
11 600
7000
11 000
10 200
7600
13 600
14 400
14 500
15 300
15 900
14 700
1.08
1.34
1.08
1.15
1.30
1.10
1.08
1.08
1.07
1.07
1.07
(b) Characterization of the antimicrobial copolymers by biological assaysb
MIC90 µg mL-1
selectivty
sample
monomer
Mn. Target g mol-1
E. coli
S. aureus
HC50 µg mL-1
E. coli
S. aureus
Propyl_3k
E1:P9
E1:P2
E1:P1
E2:P1
propyl
3000
3500
3400
3000
3500
3400
3000
3000
3400
3500
3500
3000
3000
3400
3600
3700
3000
6.25
15
15
15
15
15
200
200
50
50
<50
8.0
<3.3
<3.3
10
10
<2.5
28
<10
<3.5
<6.0
<7
28
10
10
10
10
8.3
3.3
<3.3
<3.3
5.0
>10
10
28
20
112
96
120
28
ethyl:propyl
ethyl:propyl
ethyl:propyl
ethyl:propyl
ethyl:propyl
ethyl
<50
100
1000
>2000
500
1400
2000
700
1200
1500
1400
2000
>2000
>2000
>2000
50
>200
>200
50
>200
>200
>200
>200
50
>200
>200
>200
>200
E9:P1
Ethyl_3k
Methyl_3k
M9:E1
M1:E1
M1:E9
Ethyl_3k
Methyl_3k
M9:P1
M1:P1
M1:P9
50
methyl
100
6.25
12.5
12.5
50
100
<3.75
<3.75
<3.75
15
methyl-ethyl
methyl-ethyl
methyl-ethyl
ethyl
methyl
20
methyl-propyl
methyl-propyl
methyl-propyl
propyl
>533
>533
>533
3.3
Propyl_3k
6.25
a DMF, 0.01 mol L-1 LiCl, PS standards. b Inhibitory activity towards bacterial growth of E. coli and S. aureus bacteria (MIC90 ) minimal inhibitory
concentration preventing 90% bacterial growth) and hemolytic activity towards red blood cells (HC50 ) concentration lysing 50% of blood cells).
Sample labeling: e.g., E1:P9 is a copolymer with 10 mol% ethyl and 90 mol% propyl monomer.
Conclusion
(i.e., with no absorption or complexation), only little material
is required to disintegrate its plasma membrane.
The molecular construction kit approach described allows the
synthesis of a whole library of synthetic facially amphiphilic
antibacterial homopolymers and copolymers with tunable activ-
ity and selectivity. The hydrophilic and hydrophobic components
can be varied independently, allowing future synthesis of an
even more extensive library of antibacterial polymers. The 3k
homopolymers thus obtained show a remarkable antibacterial
activity against E. coli and S. aureus, which could be tuned
over 2 orders of magnitude by variation of the hydrophobic
residue R. Some of the 10k homopolymers showed a double
selectivity of E. coli over mammalian cells as well as over S.
aureus. It was further demonstrated that molecular weight can
be used as a parameter to tune the antibacterial activity. The
optimum activity against S. aureus was found to be at 750-1100
g/mol, and that against E. coli, at 3000 g/mol and 10000 g/mol.
We were able to push the selectivities of S. aureus over red
blood cells as high as >533 by copolymerizing an inactive/
nonhemolytic monomer with an active/hemolytic monomer.
These polymers were also doubly selective with a >533 times
higher selectivity of S. aureus over red blood cells and a >53
times more selective for S. aureus over E. coli. Such a high
selectivity against S. aureus is unprecedented. This is particularly
exciting as the Methicillin-resistant form of S. aureus, MRSA,
In summary, the selectivity of our polymers appears to be
affected by the overall hydrophobic/philic balance as previously
demonstrated by us32,19 and other groups18 and can be further
enhanced by synthesizing an optimal molecular weight. Notably
though, we have only seen this kind of high double selectivity
(bacteria over mammalian cells and one bacterial type over
another) with the specific polymers presented here, in which
the monomers themselves are facially amphiphilic. We therefore
believe that facial amphiphilicity of the monomers is an
important design principle for SMAMPs with precisely tunable
activity and selectivity. Only very recently, Shambhy et al.
reported two series of copolymers with tunable amphiphilicity.
In the first series, one repeat unit carried the cationic group and
the other the hydrophobic group (“separate centers” in the
authors’ terminology). The second series contained one facially
amphiphilic comonomer (“same centers” in the authors’ termi-
nology), and the other was chosen such that the same molecular
formula as in the previous series was obtained. The facially
amphiphilic polymers have much higher selectivities (up to 34)
than the comparable copolymers of the other series.33 This very
elegant comparison supports our hypothesis.
(41) Porter, E. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2002,
124, 7324.
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9842 J. AM. CHEM. SOC. VOL. 130, NO. 30, 2008

Antimicrobial Polymers Prepared by ROMP
A R T I C L E S
is an antibiotic-resistant “superbug” and one of today’s most
serious health threats for hospital patients.
resulting polymers. This will hopefully lead to the discovery
of polymers whose activities and selectivities will surpass even
the ones reported in this paper.
This is the clearest example thus far of how the biological
properties of cationic amphiphilic polymers can be tuned to,
first, increase the selectivity of bacteria over mammalian cells
and, second, increase the selectivity toward one bacteria family
over another. This discrimination is important for antimicrobial
agents since many bacterial types are in fact beneficial to the
body. It has yet to be shown that our doubly selective molecules
operate by the same membrane-disrupting mechanism as natural
AMPs. A detailed analysis of the interaction of these remarkably
active molecules with model membranes, for example using dye-
leakage experiments, might give insight to their mechanism of
action. These investigations are currently in progress and will
be reported in due course. In future work, we will further exploit
the versatility of this molecular construction kit approach by
varying the hydrophilic component of the monomer and
determining its influence on the antimicrobial activity of the
Acknowledgment. Dr. Abhigyan Som and Dr. Gregory J.
Gabriel are acknowledged for helpful and challenging discussions.
Mass spectral data were obtained by Dr. Steve Eyles at the
University of Massachusetts Amherst Mass Spectrometry Facility
which is supported, in part, by the National Science Foundation.
Funding by the German Research Foundation (DFG), MRSEC, and
ONR is gratefully acknowledged.
Supporting Information Available: Experimental details
(monomer and polymer synthesis, all spectroscopic data). This
material is available free of charge via the Internet at http://
pubs.acs.org.
JA801662Y
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