High potency and broad-spectrum antimicrobial peptides synthesized via ring-opening polymerization of α-Aminoacid-N-carboxyanhydrides

Article abstract of DOI:10.1021/bm900896h

Antimicrobial peptides (AMPs), particularly those effective against methicillin-resistant Staphylococcus aureus (S. aureus) and antibiotic-resistant Pseudomonas aeruginosa (P. aeruginosa), are important alternatives to antibiotics. Typical peptide synthesis methods involving solid-phase sequential synthesis are slow and costly, which are obstacles to their more widespread application. In this paper, we synthesize peptides via ring-opening polymerization of α-amino acid N-carboxyanhydrides (NCA) using a transition metal initiator. This method offers high potential for inexpensive synthesis of substantial quantities of AMPs. Lysine (K) was chosen as the hydrophilic amino acid and alanine (A), phenylalanine (F), and leucine (L) as the hydrophobic amino acids. We synthesized five series of AMPs (i.e., P(KA), P(KL), P(KF), P(KAL), and P(KFL)), varied the hydrophobic amino acid content from 0 to 100%, and determined minimal inhibitory concentrations (MICs) against clinically important Gramnegative and Gram-positive bacteria and fungi (i.e., Escherichia coli (E. coli), P. aeruginosa, Serratia marcescens (S. marcescens), and Candida albicans (C. albicans). We found that P(K₁₀F _7.5L_7.5) and P(K₁₀F₁₅) show the broadest activity against all five pathogens and have the lowest MICs against these pathogens. For P(K₁₀F_7.5L_7.5), the MICs against E. coli, P. aeruginosa, S. marcescens, S. aureus, and C. albicans are 31 μg/mL, 31 μg/mL, 250 μg/mL, 31 μg/mL, and 62.5 μg/mL, while for P(K₁₀F₁₅) the respective MICs are 31 μg/mL, 31 μg/mL, 250 μg/mL, 31 μg/mL, and 125 μg/mL. These are lower than the MICs of many naturally occurring AMPs. The membrane depolarization and SEM assays confirm that the mechanism of microbe killing by P(K₁₀F _7.5L_7.5) copeptide includes membrane disruption, which is likely to inhibit rapid induction of AMP-resistance in pathogens.

Full text of DOI:10.1021/bm900896h

60
Biomacromolecules 2010, 11, 60–67
High Potency and Broad-Spectrum Antimicrobial Peptides
Synthesized via Ring-Opening Polymerization of
r-Aminoacid-N-carboxyanhydrides
Chuncai Zhou, Xiaobao Qi, Peng Li, Wei Ning Chen, Lamrani Mouad,† Matthew W. Chang,
Susanna Su Jan Leong,* and Mary B. Chan-Park*
School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive,
Singapore 637459, Singapore, and Menicon Company, 104 rue Martres, 92583 Clichy cedex, France
Received August 6, 2009; Revised Manuscript Received November 12, 2009
Antimicrobial peptides (AMPs), particularly those effective against methicillin-resistant Staphylococcus aureus
(S. aureus) and antibiotic-resistant Pseudomonas aeruginosa (P. aeruginosa), are important alternatives to
antibiotics. Typical peptide synthesis methods involving solid-phase sequential synthesis are slow and costly,
which are obstacles to their more widespread application. In this paper, we synthesize peptides via ring-opening
polymerization of R-amino acid N-carboxyanhydrides (NCA) using a transition metal initiator. This method offers
high potential for inexpensive synthesis of substantial quantities of AMPs. Lysine (K) was chosen as the hydrophilic
amino acid and alanine (A), phenylalanine (F), and leucine (L) as the hydrophobic amino acids. We synthesized
five series of AMPs (i.e., P(KA), P(KL), P(KF), P(KAL), and P(KFL)), varied the hydrophobic amino acid content
from 0 to 100%, and determined minimal inhibitory concentrations (MICs) against clinically important Gram-
negative and Gram-positive bacteria and fungi (i.e., Escherichia coli (E. coli), P. aeruginosa, Serratia marcescens
(S. marcescens), and Candida albicans (C. albicans). We found that P(K10F7.5L7.5) and P(K10F15) show the broadest
activity against all five pathogens and have the lowest MICs against these pathogens. For P(K10F7.5L7.5), the
MICs against E. coli, P. aeruginosa, S. marcescens, S. aureus, and C. albicans are 31 µg/mL, 31 µg/mL, 250
µg/mL, 31 µg/mL, and 62.5 µg/mL, while for P(K10F15) the respective MICs are 31 µg/mL, 31 µg/mL, 250
µg/mL, 31 µg/mL, and 125 µg/mL. These are lower than the MICs of many naturally occurring AMPs. The
membrane depolarization and SEM assays confirm that the mechanism of microbe killing by P(K10F7.5L7.5) copeptide
includes membrane disruption, which is likely to inhibit rapid induction of AMP-resistance in pathogens.
coatings, handwash, detergents, filters, and so on, rather than
as therapeutic agents.26–28 On the other hand, suitable AMPs
with controlled proteolysis can be highly bioavailable but
degraded after systemic/topical treatment.29,30
1. Introduction
The increasing use in recent decades of antibiotics for
biomedical and agricultural purposes has led to the emergence
of more resistant and virulent strains of pathogens, including
methicillin-resistant S. aureus and antibiotic-resistant P. aerugi-
nosa.1 The human cost of these developments is not small; it is
estimated that annual mortality from S. aureus-associated
infections is about 90000 in the U.S. alone. There is urgent need
for highly effective antimicrobials against clinically significant
bacteria (such as S. aureus and P. aeruginosa) and fungi (such
as Candida albicans (C. albicans)). A promising approach is
the use of antimicrobial peptides (AMPs) such as defensins,
cathelicidins (LL-37), and magainins,2–6 which are components
of the natural immune system. Further, AMPs typically show
broad antimicrobial activity against bacteria, fungi, and some
types of viruses7–11 and have low propensity to induce resistance
in pathogens.12,13 In addition to poor proteolytic stability and
toxicity to mammalian cells, cost is a major obstacle to
widespread use of AMPs as antimicrobial agents.14,15
The common techniques for making AMPs, which typically
have 10-30 amino acid residues,31–33 include solid-phase
synthesis and solution-coupling.34–37 Although these serial
techniques offer versatility in the precise control of the peptide
chain composition, they are time-consuming and expensive.
Deming38,39 has developed transition metal initiators that allow
living and controlled polymerization of R-amino acid N-
carboxyanhydrides (NCA) for synthesizing peptides with nar-
rowly defined chain length and intrachain composition.40 These
NCA-based peptides have been shown to interact with cell
membranes.40 However, previous results did not address the
question of possible antimicrobial activity of these kinds of
peptides. Peptides prepared by NCA ring-opening polymeriza-
tion can easily be produced in large amounts (up to kilogram
quantities) and this method offers versatile control of peptide
composition and length. No publication to date has demonstrated
that peptides synthesized with R-amino acid NCA monomer
using transition metal initiators have antimicrobial activity and
their optimal composition.
Research has shown that polymers such as polymethacrylates,
polynorbornene, polypyridinium, and polyarylamide can be
tuned to have impressive antimicrobial activity with high
selectivity, but most of these are unsuitable for topical and
systemic treatment, as they are nonbiodegradable.15–27 Instead,
these polymers are typically used as disinfectant additives in
This paper reports the NCA ring-opening copolymerization
synthesis and characterization of five series of copeptides
containing cationic lysine and hydrophobic leucine, alanine, or
phenylalanine residues. The syntheses were performed using
Ni(COD)2 initiator (Figure 1). The five series of copeptides were
(1) poly(lysine-co-alanine) (hereafter denoted as P(KA)), (2)
* To whom correspondence should be addressed. E-mail: mbechan@
ntu.edu.sg (M.B.C.-P.); sleong@ntu.edu.sg (S.S.J.L.).
† Menicon Company.
10.1021/bm900896h
 2010 American Chemical Society
Published on Web 12/03/2009

Broad-Spectrum Antimicrobial Peptides
Biomacromolecules, Vol. 11, No. 1, 2010 61
give a homogeneous solution of the corresponding NCA in 1-3 h.
Briefly, 10 g of amino acid was suspended in 100 mL of THF, and the
mixture was heated to 50 °C, followed by the addition of an equivalent
amount of triphosgene. In the absence of a clarified solution within
1 h, 2 to 3 aliquots (0.05 equiv) of triphosgene were added to the
mixture at 30 min intervals. After 3 h, the reaction mixture was poured
into 300 mL of hexane, and the suspension was stored for 16-20 h at
-20 °C to ensure complete crystallization. The product was recrystal-
lized from THF/hexane to yield NCA-monomer crystals.
Synthesis of Poly(Nε-benzyloxycarbonyl-L-lysine-ran-L-phenylala-
nine). The initiator Ni(COD)2 (71.9 mg, 0.26 mmol) was added to a
40 mL mixture of DMF/THF (10:1) in a dried flask. NCA-L-
phenylalanine (0.578 g, 3.27 mmol) and NCA-Nε-benzyloxy carbonyl-
L-lysine (1 g, 3.27 mmol) were dissolved in DMF (10 mL) and then
added into the initiator solution. The mixture was stirred at room
temperature in argon for 4 h. The polymers were isolated by addition
of water, containing HCl (1 mmol), to the reaction mixture, causing
precipitation of the polymers. The polymers were then washed with
water and centrifuged several times to remove excess DMF. The
polymers were dried in vacuum to give poly(Nε-benzyloxycarbonyl-
L-lysine-ran-L-phenylalanine) copolymers as white solids (yield, >87%).
Synthesis of Protonated Poly(L-lysine·HBr-ran-L-phenylalanine).
The poly(Nε- benzyloxycarbonyl-L-lysine-ran-L-phenylalanine) samples
previously synthesized were each dissolved in 10 mL of TFA in a 50
mL flask. Excess HBr (30% in acetic acid) was then added and the
mixtures were stirred for 5 h at room temperature. Deprotected polymers
were isolated by addition of diethyl ether to the reaction mixtures,
causing precipitation of the peptides. The peptides were then washed
with excess diethyl ether and acetone and finally dialyzed against
deionized water and dried in vacuum at 50 °C. All other copolymers
were deprotected in the same manner.
Figure 1. Schematic of (a) R-amino NCA monomer synthesis. (b)
Synthesis of copeptides by the NCA method.
poly(lysine-co-leucine) (i.e., P(KL)), (3) poly(lysine-co-pheny-
lalanine) (i.e., P(KF)), (4) poly(lysine-co-alanine-co-leucine)
(i.e., P(KAL)), and (5) poly(lysine-co-phenylalanine-co-leucine)
(i.e., P(KFL)). The length of the copeptides was fixed at 25
residues by fixing the ratio of NCA-monomers to initiator to
25. For the P(KF) series, the effect of length variation was also
studied. The effect of composition and length of the NCA
peptides on their minimum inhibitory concentrations (MICs)
against clinically significant Gram-negative bacteria (i.e., E. coli
ATCC8739, P. aeruginosa ATCC9027, and S. marcescens
ATC13880) and Gram-positive bacteria (S. aureus ATCC6538)
and fungi (C. albicans ATCC10231) were studied. Gel perme-
ation chromatography (GPC) and proton nuclear magnetic
resonance (1H NMR) were used to characterize the peptides.
The mechanism for pathogen killing was also investigated using
the cytoplasmic membrane potential-sensitive dye DiSC3(5) and
scanning electron microscopy (SEM) to observe cell structure
change after peptide exposure. The cytotoxicity of the peptides
was characterized by hemolytic activity with red blood cells.
1
Spectroscopy Characterization. H NMR spectra were recorded
on a Bruker DMX-3000 spectrometer with either DMSO or D2O as
solvent. GPC was performed on an Agilent 1100 Series equipped with
GPC-SEC (size exclusion chromatography) data analysis software.
Peptides were dissolved in 0.1 M LiBr in DMF and the GPC eluent
was kept at 60 °C.
Measurement of Minimum Inhibitory Concentrations (MIC).
Minimal inhibitory concentrations (MICs) were determined by
dilution in the Mueller Hinton Broth (MHB) medium. Briefly, bacteria
cells were grown overnight at 37 °C in MHB medium to a midlog
phase and diluted to 104-105 colony forming units (CFU) mL-1. A
dilution series for the peptides was made by diluting 1000 mg/mL
peptide stock in MHB broth to final peptide concentrations ranging
from 1000 to 8 µg/mL. A total of 100 µL of each diluted peptide from
the serial dilutions was added to microtiter plates, followed by addition
of 100 µL of bacterial suspension, to give a final inoculum of 5 × 105
CFU/mL. The plates were incubated at 37 °C for 18 h, and microbial
growth was determined by measuring optical density absorbance at 600
nm. Two replicates were used for each pathogen, peptide, and
concentration. Positive controls containing no antimicrobial peptides
and negative controls containing no bacterial cells were also used. The
experiments were independently repeated twice. The MIC was defined
as the lowest peptide concentration that arrested bacterial growth after
18 h.41 For the fungal pathogen, C. albicans, the growth condition was
the same except the (1) growth medium was RPMI1640 supplemented
with 2% glucose, and (2) MIC end points were determined at 24 and
48 h and recorded as the minimum concentration with fungi growth.42,43
The MICs of other common AMPs, including Cecropin A, Melittin,
LL-37, Indolicidin, Magainin I, and Defensin (HNP-1), were also
measured for comparison.
2. Experimental Details
Materials. L-Alanine, L-leucine, L-phenylalanine, Nε-benzyloxycar-
bonyl-L-lysine, phosphatidylcholine (POPC), Ni(COD)2, bis(trichlo-
romethyl)carbonate, hexane, diethyl ether, tetrahydrofuran (THF),
trifluoroacetic acid (TFA), N,N-dimethylformamide (DMF), hydrogen
bromide (30% in acetic acid), and other chemical reagents were
purchased from Sigma-Aldrich and used without further purification
unless otherwise specified. Drying of THF and hexane was achieved
by heating in the presence of sodium strips and calcium hydride,
respectively, for 48 h under reflux conditions. DMF was dried using
molecular sieves. Cecropin A, Melittin, LL-37, Indolicidin, Magainin
I, and Defensin (HNP-1) were purchased from Quality Controlled
Biochemicals Ltd. (U.S.A.).
Hemolytic Activity. Peptide hemolytic activity was tested by
determining the release of hemoglobins in erythrocyte suspensions
derived from fresh human blood (5% v/v). Blood was aseptically
collected and stored for less than 2 h at 4 °C. The erythrocyte
suspensions were then centrifuged at 2000 × g for 5 min and washed
three times with Tris buffer (10 mM Tris, 150 mM NaCl, pH7.2) before
Synthesis of NCA-Monomers. One-third equivalence of triphosgene
was added to an amino acid suspension in anhydrous THF at 50 °C to

62 Biomacromolecules, Vol. 11, No. 1, 2010
Zhou et al.
being 2-fold diluted in Tris buffer. Peptides were solubilized in Tris
buffer to final concentrations of 1000, 500, 250, 125, 62.5, 32, 16, 8,
4, 2, and 1 µg/mL and incubated with the erythrocyte suspension at
equal volume ratios (i.e., 50:50 µL) under continuous shaking for 1 h
at 37 °C. The mixtures were then centrifuged at 3500 × g for 10 min.
Aliquots of 80 µL of the supernatant were transferred to a 96-well
microplate and diluted with 80 µL of distilled water.
Hemoglobin release was monitored by absorbance measurement of
the erythrocyte samples at 540 nm using a microplate reader (Biorad).
Complete hemolysis was determined in Tris buffer containing 1%
Triton-X100 (positive control). Erythrocytes in Tris buffer were used
as negative control. Percentage of hemolysis (H) was calculated as H
(%) ) 100 × [(Op - Ob)/(OT - Ob)], where Op is the peptide density
for a given peptide concentration, Ob is the Tris buffer density, and OT
is the positive control (1% Triton-X100) density. HC50 is the peptide
concentration resulting in 50% cell hemolysis.
Circular Dichroism (CD). CD spectra were recorded at room
temperature on a Chirascan CD spectrometer (Applied Photophysics
Limited, U.K.). A quartz cell with a path length of 0.5 mm was used.
Spectra were generated from 190 to 250 nm wavelengths at 0.1 nm
intervals, 50 nm/min speed, 0.5 s response time, and 1 nm bandwidth.
Peptides were dissolved to a final concentration of 1 µg/mL in 10 mM
sodium phosphate buffer (pH 7.4) containing 50 µM phosphatidylcho-
line (POPC) small unilamellar vesicles. The spectra were plotted as
mean residue ellipticity versus wavelength.
Membrane Depolarization of Intact Bacterial Cells. The peptides’
ability to depolarize membranes was determined using the membrane
potential-sensitive fluorescent dye, DiSC3-5 on intact S. aureus and C.
albicans cells, based on the methods reported by Friedrich et al.44 S.
aureus and C. albicans were grown at 37 and 28 °C, respectively, under
shaking conditions to midlog phase and harvested by centrifugation.
The cells were washed three times with wash buffer (20 mM glucose,
5 mM HEPES, pH 7.4) and resuspended to A600 ) 0.05 in the same
buffer but also containing 0.1 M KCl. A DiSC3-5 stock solution was
added to the cell suspension to a final concentration of 20 nM DiSC3-
5, and incubated until a constant fluorescence reading was achieved,
indicating the successful uptake of the dye into the bacterial membrane.
Changes in fluorescence were recorded with a luminescence spectrom-
eter (Aminco Bowman II). After the addition of peptides, membrane
depolarization was determined by monitoring the change in fluorescence
emission intensity of the DiSC3-5 dye at excitation and emission
wavelengths of 622 and 670 nm, respectively. Complete dissipation of
the membrane potential was achieved by adding gramicidin D (0.2 nM).
Morphology Change of Pathogens. Pathogen morphologies were
examined by SEM after 15 min treatment with peptide at MIC
concentration. Bacterial cells were fixed with 4% gluteraldehyde in
0.15 M sodium phosphate buffer (pH 7.4) at equal volume ratios. The
sample tube was mixed by gently inverting the tube up and down for
several minutes to prevent clumping of the cells. The fixed cell
suspension was transferred to a cover slide and allowed to air-dry. The
slides were rinsed with 0.15 M sodium phosphate buffer (pH 7.4) and
dehydrated through a graded series of ethanols (30-100%). After
ethanol dehydration, the slides were oven-dried at 60 °C for 5 min.
The slides were sputter-coated with gold-palladium for 80 s at 20 mA
and imaged with a field emission scanning electron microscope (JEOL
Field Electron Microscope, JSM-6700F, Japan).
Figure 2. NMR spectra of (A) poly(Nε-benzyloxycarbonyl-L-lysine-
ran-L-phenylalanine) (lysine mol content is 50%) in DMSO and (B)
protonated poly(L-lysine-ran-L-phenylalanine) (lysine mol content is
50%) in D2O.
Table 1. GPC Results of Poly(Nε-benzyloxycarbonyl-L-
lysine-ran-L-phenylalanine)a
ratio of NCA-monomers /initiator
Mn
Mw/Mn
15
25
35
45
100
5100
9300
13400
17600
35100
1.18
1.11
1.28
1.14
1.16
a Lysine content is 40%.
the NCA polymerization with Ni(COD)2 initiator is controllable
and the number-average molecular weight (Mn) of the peptides
can be varied from around 5K to 35K. The molecular weight
polydispersities of these peptides are narrow and between
1.1-1.3.
The hydrophobic amino acid (HAA) content of each of the
five series of copeptides was varied from 0 to 100%. The MIC
results for each series are shown in Figure 3A-E and Table 2.
The antimicrobial activity of the 100% HAA polymers was not
measured because these polymers could not dissolve in water.
Figure 3A shows that the best antibacterial activity of the P(KA)
copeptides against E. coli, P. aeruginosa, and S. aureus occurs
when the hydrophobic alanine residue content is in the 20-50%
range; outside this range, the P(KA) peptides exhibit very limited
antimicrobial activity. None of the P(KA) copeptides exhibit
significant activity against S. marcescens and C. albicans,
indicating that P(KA) shows relatively narrow antimicrobial
activity.
3. Results
Figure 2A shows the 1H NMR spectrum of a typical protected
peptide, poly(Nε-Benzyl oxycarbonyl-L-lysine-ran-L-phenyla-
lanine), and the spectrum confirms that the ring-opening reaction
(Figure 1b, step 1) proceeded as planned. After deprotection,
1
copeptides were obtained and the H NMR spectrum (Figure
2B) confirms that the deprotected peptide, specifically protonated
poly(L-lysine-ran-L-phenylalanine), has been obtained. GPC
(Table 1 and Supporting Information, Figure S1) confirms that
The antibacterial efficacy of P(KL) copeptides is shown in
Figure 3B. The P(KL) copeptides exhibit antibacterial activities
over a wider range of HAA content compared to P(KA)

Broad-Spectrum Antimicrobial Peptides
Biomacromolecules, Vol. 11, No. 1, 2010 63
Figure 3. MIC results of the peptides (A) P(KA), (B) P(KL), (C) P(KF), (D) P(KAL), (E) P(KFL), and (F) P(KF) with varying peptide length.
Table 2. MIC (µg/mL) of Optimized Copolypeptides
E. coli
(Gram-)
P. aeruginosa
(Gram-)
S. marcescens
S. aureus
(Gram+)
C. albicans
(fungi)
HC50
(µg/mL)
polypeptide
(Gram-)
P(K10F15
)
P(K10F7.5L7.5
Cecropin A
Melittin
31
31
4
31
31
64
250
250
64
31
31
128
8
125
62.5
256
16
16
)
64
64
32
32
8
LL-37
Indolicidin
Magainin I
>256
>256
128
>128
>256
128
>256
>128
>256
>256
>256
>128
>256
128
128
>128
>256
128
>100
256
>512
Defensin (HNP-1)
copeptides, and the most potent formulations of the P(KL)
copeptides are distinctly more effective with lower MICs than
the P(KA) copeptides: with 50% L content, the MICs against
E. coli, P. aeruginosa, and S. aureus are 62.5, 125, and 62.5
µg/mL compared to 125, 250, and 62.5 µg/mL, respectively,
for the most optimum composition in the P(KA) series. Like
the P(KA) copeptides, the P(KL) copeptides do not have
significant activity against S. marcescens and C. albicans.
The P(KF) copeptides are effective against all five pathogens
tested (Figure 3C) and are effective against most of them over
a broad range of phenylalanine content. It is particularly
interesting to observe that the optimal antimicrobial behavior,
or the lowest MIC, of the P(KF) copeptides series occurs at the
same F content for all five pathogens (i.e., 60%) and the MICs
are 31, 31, 250, 31, and 125 µg/mL against E. coli, P.
aeruginosa, S. marcescens, S. aureus, and C. albicans, respec-
tively. This copeptide has properties resembling that of a broad-
spectrum antimicrobial agent.
Biologically produced cationic antimicrobial peptides (CAPs)
usually carry two or more types of HAA in their amino acid

64 Biomacromolecules, Vol. 11, No. 1, 2010
Zhou et al.
Table 3. Hemolytic Activities of the Peptides (Human Red Blood
Cells)
chains will have higher than average HAA content. Peptide
chains having a higher HAA content have been shown to be
more hemolytic to human red blood cells.20,50
peptide
P(K10F15
)
P(K10F7.5L7.5
)
The influence of lipids which mimic bacterial membranes,
on peptide secondary structure was studied. CD analysis of the
P(KFL) and P(KF) peptide series in the presence of 50 µM
POPC showed that only 30 and 40% HAA P(KF) peptides
exhibit random coil structures as evidenced from the charac-
teristic peaks obtained (195 nm trough and 215 nm peak), while
the rest of the peptides did not show any distinct secondary
structures (Figure 4). No distinct R-helix, ꢀ-sheet, or random
coil structures were evident in all the KFL peptides. This result
is not unexpected considering the short peptide lengths of the
P(KFL) and P(KF) series and, hence, a lesser tendency to form
distinct secondary structures for thermodynamic stability.
Nonetheless, it is clear that the lack of a distinct secondary
structure in our P(KFL) and P(KF) peptide series did not
compromise the peptides’ antimicrobial activity.
HC50 (µg/mL)
16
16
sequences; this favors formation of energetically stable structures
and an antimicrobial peptide function.12 The simple copeptides
whose activities are shown in Figure 3A-C, however, lack the
HAA diversity inherent in natural CAPs. As a step in the
direction of exploring the effect of HAA diversity, we synthe-
sized cationic antimicrobial peptides with two different species
of hydrophobic residue. The species diversity was limited to
two for simplicity and the mole ratio of the HAAs was set to
unity. The two series tested were P(KAL) and P(KFL). As in
the syntheses of copolymers with single HAAs, the target
copolymer length was 25 residues, which was controlled through
the mole ratio of NCA-monomer to initiator.
The cytoplasmic membrane depolarization activities of the
optimum peptide, P(K10F7.5L7.5), on S. aureus (a Gram-positive
bacteria) and C. albicans (a fungi) were determined by a
fluorimetric method using the membrane potential-sensitive dye
DiSC3(5). P(K25) (0%HAA) was used as the control peptide.
The distribution of DiSC3(5) between the medium and pathogen
cells is influenced by the cytoplasmic membrane potential. The
cationic DiSC3(5) dye aggregates within the cytoplasmic
membrane, leading to self-quenching of the fluorescence.
However, upon membrane lysis, the dye is released and
dissociates into the medium, resulting in fluorescence increase.
Figure 5 shows the time course of fluorescence over 400s.
Addition of P(K10F7.5L7.5) (10 µg/mL) to the buffer resulted in
an immediate increase in fluorescence, while no significant
change was observed when P(K25) peptide was added. It is clear
that the peptide P(K10F7.5L7.5) is fast-acting on both bacterial
and fungal membranes, achieving maximum fluorescence im-
mediately upon addition of peptide to the buffer. This observa-
tion also suggests that the rates of peptide association with S.
aureus and C. albicans membranes leading to depolarization
were comparable.51
Figure 3D shows the MIC results of the P(KAL) series with
the total content of hydrophobic A and L varying from 0 to
100%. The P(KAL) copeptide has no antimicrobial activity
against C. albicans and S. marcescens, like the P(KA) and P(KL)
series (Figures 3A,B). Figure 3E shows that the P(KFL)
copeptide exhibits the best antimicrobial activity among the five
series with the lowest MICs across a broad spectrum of
pathogens. For E. coli, P. aeruginosa, and S. aureus, the
optimum range of hydrophobic F and L residues is 30-80%
but with the inclusion of S. marcescens and C. albicans, the
optimum concentration of F plus L is 60%. The average number
of peptide repeat units is 25 (Table 1) and the average reside
number is 10K, 7.5F, and 7.5L; this peptide (more precisely,
this population of peptides) is hereafter denoted P(K10F7.5L7.5).
It has MICs of 31, 31, 31, 250, and 62.5 µg/mL for E. coli, P.
aeruginosa, S. aureus, S. marcescens, and C. albicans,
respectively.
Most natural antimicrobials are less than 50 amino acids in
length (usually 10-30 residues long). Some synthetic short
peptides (e.g., those having 9 or 11 residues45,46) have also
shown excellent antibacterial activity. Of the three series of
peptides with only one kind of hydrophobic amino residue,
P(KF) appears to have a broader antimicrobial activity and we
have therefore optimized the effect of length on antimicrobial
activity using this copeptide. Figure 3F shows the MIC results
versus synthesized peptide length over the range of 15 to 100
residues for P(KF) copeptides. We find that the P(KF) copeptide
has broader antibacterial activity when it is 25 residues long
than at shorter or longer length (it is, of course, possible that
the precise minimum may lie between our sampled lengths,
particularly in the case of S. marcescens). This most effective
length for broad antimicrobial activity agrees well with the
length range of naturally occurring cationic antibacterial
peptides.12,47,48 It is possible that this length range is most ideal
for conformational stability and disruption of lipid bilayers in
pathogen cell walls.
Figure 6 shows SEM images of P(K10F7.5L7.5)-treated and
untreated E. coli, S. aureus, P. aeruginosa, C. albicans, and F.
solani. The treated pathogens exhibit obvious morphological
changes compared with the untreated controls. The surfaces of
untreated bacteria and fungi appear smooth and rounded,
whereas the peptide-treated bacteria and fungi exhibit wrinkled
and withered surfaces.
4. Discussion
We have shown for the first time that NCA ring-opening
copolymerization can be used for synthesis of copeptides that
exhibit broad spectrum antimicrobial activity against a wide
range of clinically important pathogens, including both Gram-
negative and Gram-positive bacteria and fungi. Our optimum
copeptides via NCA ring-opening polymerization are P(K10F15)
and P(K10F7.5L7.5) and the MICs of these two optimum copep-
tides were lower than those of many naturally occurring AMPs
(such as LL-37, Indolicidin, Magainin I, and defensin). Of
potentially great clinical importance, the P(K10F15) and
P(K10F7.5L7.5) copeptides are effective, even at low MIC values
(i.e., < 100 µg/mL), against antibiotic-resistant S. aureus and
P. aeruginosa.1
The hemolytic activities of two optimum copeptides (i.e.,
P(K10F15) and P(K10F7.5L7.5) were determined using human
erythrocytes (Table 3). The HC50 of these two copeptides are
both 16 µg/mL, which is slightly better than naturally derived
melittin (with HC50 of 8 µg/mL), but our peptides are more
hemolytic than other natural and synthetic peptides (e.g.,
Indolicidin with HC50 > 100 µg/mL).49 Our peptides were
synthesized from two or three kinds of amino acid monomers
using random copolymerization of NCA monomers, so the
amino acid sequences vary with different peptide chains; some
Our data suggest that the mechanism by which our AMPs
kill pathogens includes membrane disruption. Therefore, the

Broad-Spectrum Antimicrobial Peptides
Biomacromolecules, Vol. 11, No. 1, 2010 65
Figure 4. CD spectra of copolymers in the presence of 50 µM POPC lipid with varying hydrophobic monomer content: (a) P(KFL) and (b) P(KF),
where the hydrophobic monomers are (a) F and L (1:1) and (b) F.
Figure 5. Cytoplasmic membrane depolarization of (A) S. aureus and (B) C. albicans by the copeptide (I) P(K25) (0%HAA) and (II) P(K10F7.5L7.5).
likelihood of emergence of copeptide-resistant pathogenic strains
should be low as cell membranes, being a fundamental structure
of the organism, are not expected to evolve under selective
pressure over a short period of time.
the lengths or sequences of the synthesized peptides, so that a
mixture of lengths and compositions inevitably results from this
method. Even the highly potent 60% HAA samples (i.e., (K10F15)
and P(K10F7.5L7.5)) contain a significant number of low-activity
peptides. An estimate of the variance in the number of
hydrophobic residues per peptide (binomial distribution, n )
25, p ) 0.6) gives a standard deviation of the HAA% of about
10%, with about 31% of peptides outside the range of 50-70%
hydrophobic residues and only 16% of the sample having
precisely 60% (i.e., 15) hydrophobic residues. The actual
diversity in a synthesized sample would be somewhat greater
From the antimicrobial peptide database (APD),52 it has been
observed that lysine is a typical hydrophilic amino acid residue
of natural cationic AMPs; this is the hydrophilic amino acid
used in our polymer series. However, our two optimum
copeptides have higher HAA content than that of different
natural cationic AMPs, which range from 41 to 49%. The ring-
opening polymerization process does not precisely control either

66 Biomacromolecules, Vol. 11, No. 1, 2010
Zhou et al.
toxicity to mammalian cells versus pathogens. The negative
charge on bacterial cell surfaces is attributed to the presence of
hydroxylated phospholipids such as phosphatidylglycerol, car-
diolipin, and phosphatidylserine.55 On the other hand, the outer
leaflet of mammalian cell membranes is rich in zwitterionic
phospholipids such as phosphatidylethanolamine, phosphati-
dylcholine, and sphingomyelin, thus rendering mammalian
cytoplasmic membranes neutral in net charge.28 The binding
mechanisms of peptides with anionic bacterial lipid bilayer and
zwitterionic mammalian lipid bilayer are therefore expected to
be different, although other factors such as cell energetics and
cell structure orientation could also readily influence peptide
specificity. Increase of either the cationic charge or the
hydrophobicity of an AMP can help its binding to the anionic
bacterial lipid bilayers to improve the MICs.20 However,
considering that mammalian cells are electrically neutral,
increasing peptide hydrophobicity instead of cationic charge will
increase the propensity of AMP binding to mammalian cell
membranes. In this screening of NCA-polymerized copeptides,
P(K10F15) and P(K10F7.5L7.5) have the optimum compositions of
cationic and hydrophobic amino acids that kill bacteria ef-
fectively, but their high hydrophobicity (60%) or more hydro-
phobic species present may have resulted in high toxicity to
mammalian red blood cells.56 Conversely, increased cationic
charge and decreased hydrophobicity of peptides are expected
to increase selectivity. Ring-opening copolymerization of dif-
ferent NCA monomers or other types of monomers afford many
possibilities for controlling the composition, charge, and
hydrophobicity of these peptides or peptide-polymer conjugates
to generate AMPs with superior antimicrobial activities and
acceptable toxicities to mammalian cells.
We have demonstrated in this preliminary study that we can
synthesize highly effectively broad-spectrum antimicrobial
copeptides easily and inexpensively by the ring-opening of NCA
method, which will make these peptides widely applicable.
Naturally derived AMPs typically have approximately 30
residues and are expensive to synthesize by solid-phase sequen-
tial synthesis techniques which can cost 100000 dollars/gram.36,37
In contrast, medium/large scale production of our peptides would
cost less than 10 dollars/gram and kilogram quantities can be
easily produced. The production cost of these peptides is so
low that their contribution to the cost of therapeutic agents,
disinfectants, and antimicrobial surface coatings would be almost
negligible. Future research for a simple polymerization method
that could more precisely control the HAA content might further
lower the MICs and toxicities of these peptides.
Figure 6. SEM photographs of treated and untreated bacteria and
fungi with copeptide P(K10F7.5L7.5).
5. Conclusion
because there is also some variation in peptide length. The
diversity can possibly explain our higher optimal HAA content
of 60% compared to those reported for other AMPs (41-49%).
Despite the variance, the antimicrobial activities of our two
optimal peptides are still surprisingly excellent.
Although the efficacies of our peptides against clinically
important and resistant pathogens are high, they have the
disadvantage of high hemolytic activity, which is an obstacle
for their use as therapeutic agents. However, high hemolytic
activity will not impact their use in other applications such as
disinfectants or as antimicrobial surfaces. Others have shown
that the mechanism of peptide binding to cell membranes that
leads eventually to pathogen death involves a combination of
electrostatic and hydrophobic interactions.53,54 The different
compositions of bacterial and mammalian cell membranes allow
future peptide composition fine-tuning to reduce the relative
Ring-opening polymerization of NCA monomers has been
successfully applied to synthesize five series of copeptides (i.e.,
P(KA), P(KL), P(KF), P(KAL), and P(KFL)) with 0-100%
hydrophobic amino acids. We found that the P(KF) copeptides
have broader antimicrobial activity and are more effective than
P(KL) and P(KA) series. Similarly, the P(KFL) series was more
effective than the P(KAL) series. The optimum peptides against
all five pathogens appear to be P(K10F7.5L7.5) and P(K10F15) and
the MICs against E. coli, P. aeruginosa, S. marcescens, S.
aureus, and C. albicans are 31, 31, 250, 31, and 62.5 µg/mL
and 31, 31, 250, 31, and 125 µg/mL, respectively, which are
lower than values of many naturally occurring AMPs. The
membrane depolarization and SEM assays confirm the mech-
anism of microbe killing by P(K10F7.5L7.5) copeptide to include
membrane disruption, which is unlikely to lead to microbe

Broad-Spectrum Antimicrobial Peptides
Biomacromolecules, Vol. 11, No. 1, 2010 67
(23) Kawabata, N.; Nishiguchi, M. Appl. EnViron. Microbiol. 1988, 54,
2532–2535.
(24) Ikeda, T.; Tazuke, S. Makromol. Chem., Rapid Commun. 1983, 4,
resistance in clinical use. The low cost and large volume
synthesis potential of the NCA synthetic route as well as the
potential to produce highly effective antimicrobial polymers that
can kill a number of highly resistant and clinically significant
bacteria may lead to a wide future application of these peptides.
459–461.
(25) Li, G.; Shen, J.; Zhu, Y. J. Appl. Polym. Sci. 1998, 67, 1761–1768.
(26) Haldar, J.; Weight, A. K.; Klibanov, A. M. Nat. Protoc. 2007, 2 (10),
2412–2417.
Acknowledgment. This work was supported by funding from
Menicon Company (Japan) and Nanyang Technological Uni-
versity (Singapore).
(27) Mukherjee, K.; Rivera, J. J.; Klibanov, A. M. Appl. Biochem.
Biotechnol. 2008, 151 (1), 61–70.
(28) Gabriel, G. J.; Som, A.; Madkour, A. E.; Eren, T.; Tew, G. N. Mater.
Sci. Eng. R 2007, 57, 28–64.
Supporting Information Available. GPC spectra, 1H NMR
spectrum, and composition analyses of the synthesized peptides.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(29) Radzishevsky, I. S.; Rotem, S.; Bourdetsky, D.; Navon-Venezia, S.;
Carmeli, Y.; Mor, A. Nat. Biotechnol. 2007, 25 (6), 657–659.
(30) Lienkamp, K.; Madkour, A. E.; Musante, A.; Nelson, C. F.; Nusslein,
K.; Tew, G. N. J. Am. Chem. Soc. 2008, 130, 9836–9843.
(31) Brogden, K. A. Nat. ReV. Microbiol. 2005, 3 (3), 238–250.
(32) Beisswenger, C.; Bals, R. Curr. Protein Pept. Sci. 2005, 6 (3), 255–
264.
(33) Hancock, R. E.; Lehrer, R. Trends Biotechnol. 1998, 16 (2), 82–88.
(34) Poon, K. W.; Liang, N.; Datta, A. Nucleosides, Nucleotides Nucleic
Acids 2008, 27 (4), 389–407.
(35) Arnusch, C. J.; Branderhorst, H.; de Kruijff, B.; Liskamp, R. M.;
Breukink, E.; Pieters, R. J. Biochemistry 2007, 46 (46), 13437–13442.
(36) Ding, Y.; Qin, C.; Guo, Z.; Niu, W.; Zhang, R.; Li, Y. Chem.
BiodiVersity 2007, 4 (12), 2827–2834.
References and Notes
(1) Zhang, L.; Parente, J.; Harris, S. M.; Woods, D. E.; Hancock, R. E.;
Falla, T. J. Antimicrob. Agents Chemother. 2005, 49 (7), 2921–2927.
(2) Hancock, R. E.; Scott, M. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97
(16), 8856–8861.
(3) Hoffmann, J. A.; Kafatos, F. C.; Janeway, C. A.; Ezekowitz, R. A.
Science 1999, 284 (5418), 1313–1318.
(4) Baroni, A.; Donnarumma, G.; Paoletti, I.; Longanesi-Cattani, I.;
Bifulco, K.; Tufano, M. A.; Carriero, M. V. Peptides 2009, 30 (2),
267–272.
(37) Haynes, S. R.; Hagins, S. D.; Juban, M. M.; Elzer, P. H.; Hammer,
R. P. J. Pept. Res. 2005, 66 (6), 333–347.
(38) Deming, T. J. Nature 1997, 390 (6658), 386–389.
(39) Deming, T. J. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 3011–
3018.
(40) Wyrsta, M. D.; Cogen, A. L.; Deming, T. J. J. Am. Chem. Soc. 2001,
123 (51), 12919–12920.
(41) Clinical and Laboratory Standards; Clinical and Laboratory Standards
Institute: Wayne, PA, 2005; Vol 188, p 328.
(42) Lass-Florl, C.; Mayr, A.; Perkhofer, S.; Hinterberger, G.; Hausdorfer,
J.; Speth, C.; File, M. Antimicrob. Agents Chemother. 2008, 52, 3637–
3641.
(5) Schauber, J.; Gallo, R. L. J. Allergy Clin. Immunol. 2008, 122 (2),
261–266.
(6) Epand, R. F.; Umezawa, N.; Porter, E. A.; Gellman, S. H.; Epand,
R. M. Eur. J. Biochem. 2003, 270 (6), 1240–1248.
(7) Gordon, Y. J.; Romanowski, E. G.; McDermott, A. M. Curr. Eye Res.
2005, 30 (7), 505–515.
(8) Tang, Y. L.; Shi, Y. H.; Zhao, W.; Hao, G.; Le, G. W. J. Pharm.
Biomed. Anal. 2008, 48 (4), 1187–1194.
(9) Pistolesi, S.; Pogni, R.; Feix, J. B. Biophys. J. 2007, 93 (5), 1651–
1660.
(10) Lam, K. L.; Ishitsuka, Y.; Cheng, Y.; Chien, K.; Waring, A. J.; Lehrer,
R. I.; Lee, K. Y. J. Phys. Chem. B 2006, 110 (42), 21282–21286.
(11) Kristiansen, P. E.; Fimland, G.; Mantzilas, D.; Nissen-Meyer, J. J. Biol.
Chem. 2005, 280 (24), 22945–22950.
(43) Heyn, K.; Tredup, A.; Salvetmoser, S.; Muller, F. M. Antimicrob.
Agents Chemother. 2005, 49, 5157–5159.
(44) Friedrich, C. L.; Moyles, D.; Beveridge, T. J.; Hancock, R. E.
Antimicrob. Agents Chemother. 2000, 44 (8), 2086–2092.
(45) Ferre, R.; Badosa, E.; Feliu, L.; Planas, M.; Montesinos, E.; Bardaji,
E. Appl. EnViron. Microbiol. 2006, 72 (5), 3302–3308.
(46) Fasman, G. D. Prediction of protein structure and the principles of
protein conformation; Plenum: New York, 1989.
(47) Jenssen, H.; Hamill, P.; Hancock, R. E. Clin. Microbiol. ReV. 2006,
19 (3), 491–511.
(48) Brogden, K. A.; Guthmiller, J. M.; Salzet, M.; Zasloff, M. Nat.
Immunol. 2005, 6 (6), 558–564.
(12) Zasloff, M. Nature 2002, 415, 389–395.
(13) Hancock, R. E.; Sahl, H. G. Nat. Biotechnol. 2006, 24, 1551–1557.
(14) Kuroda, K.; Caputo, G. A.; DeGrado, W. F. Chem.sEur. J. 2009, 15
(5), 1123–1133.
(15) Choi, S.; Isaacs, A.; Clements, D.; Liu, D. H.; Kim, H.; Scott, R. W.;
Winkler, J. D.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A. 2009,
106 (17), 6968–6973.
(16) Kuroda, K.; DeGrado, W. F. J. Am. Chem. Soc. 2005, 127, 4128–
4129.
(49) Padmaja, J.; Satyanarayana, V.; Merrifield, R. B. J. Am. Chem. Soc.
1996, 118, 8989–8997.
(17) Ilker, M. F.; Nusslein, K.; Tew, G. N.; Coughlin, E. B. J. Am. Chem.
Soc. 2004, 126, 15870–15875.
(50) Vinardell, M. P.; Infante, M. R. Comp. Biochem. Physiol., Part C:
Toxicol. Pharmacol. 1999, 124, 117–120.
(51) Park, K. H.; Park, Y.; Park, I. S.; Hahm, K. S.; Shin, S. Y. J. Pept.
Sci. 2008, 14 (7), 876–882.
(52) Wang, Z.; Wang, G. APD: the Antimicrobial Peptide Database. Nucleic
Acids Res. 2004, 32 (Database issue), D590–D592.
(53) Shai, Y. Biochim. Biophys. Acta, Biomembr. 1999, 1462, 55–70.
(54) Brogden, K. A. Nature ReV. Microbiol. 2005, 3, 238–250.
(55) Yeaman, M. R.; Yount, N. Y. Pharmacol. ReV. 2003, 55, 27–54.
(56) Colak, S.; Nelson, C. F.; Nusslein, K.; Tew, G. N. Biomacromolecules
2009, 10, 353–359.
(18) Liu, D. H.; Choi, S.; Chen, B.; Doerksen, R. J.; Clements, D. J.;
Winkler, J. D.; Klein, M. L. Angew. Chem., Int. Ed. 2004, 43, 1158–
1162.
(19) Sambhy, V.; Peterson, B. R.; Sen, A. Angew. Chem., Int. Ed. 2008,
47 (7), 1250–1254.
(20) Al-Badri, Z. M.; Som, A.; Lyon, S.; Nelson, C. F.; Nusslein, K.; Tew,
G. N. Biomacromolecules 2008, 9, 2805–2810.
(21) Tew, G. N.; Liu, D.; Chen, B.; Doerksen, R. J.; Kaplan, J.; Carroll,
P. J.; Klein, M. L.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A.
2002, 99 (8), 5110–5114.
(22) Mowery, B. P.; Lee, S. E.; Kissounko, D. A.; Epand, R. F.; Epand,
R. M.; Weisblum, B.; Stahl, S. S.; Gellman, S. H. J. Am. Chem. Soc.
2007, 129, 15474–15476.
BM900896H