Amphiphilic cyclic peptoids that exhibit antimicrobial activity by disrupting Staphylococcus aureus membranes

Article abstract of DOI:10.1002/ejoc.201300077

There is a significant unmet need for new antimicrobial agents that can address antimicrobial resistance. One promising group of antimicrobials is the antimicrobial peptides (AMPs) and their synthetic mimics. In particular, synthetic sequence-specific oligomers of N-substituted glycine, termed peptoids , have been found to show potent antimicrobial activity against bacterial pathogens in vitro and can act against the emergence of antimicrobial resistance. In this study, we evaluate the antimicrobial activity of cyclic peptoid oligomers against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). The presence of the macrocyclic constraints can enforce a globally amphiphilic organization of the peptoid side-chains. Several of these new amphiphilic compounds show potent and selective antimicrobial activity. Electron microscopy experiments demonstrate that the peptoids target and damage the MRSA cytoplasmic membrane through the formation of pores. These results substantiate the potential of peptoids as antimicrobial therapeutic agents for the treatment of S. aureus infections.

Full text of DOI:10.1002/ejoc.201300077

FULL PAPER
DOI: 10.1002/ejoc.201300077
Amphiphilic Cyclic Peptoids That Exhibit Antimicrobial Activity by Disrupting
Staphylococcus aureus Membranes
Mia L. Huang,[a] Meredith A. Benson,[b] Sung Bin Y. Shin,[a] Victor J. Torres,*[b] and
Kent Kirshenbaum*[a]
Keywords: Medicinal chemistry / Peptidomimetics / Antibiotics / Foldamers / Macrocycles
There is a significant unmet need for new antimicrobial
Staphylococcus aureus (MRSA). The presence of the macro-
agents that can address antimicrobial resistance. One prom- cyclic constraints can enforce a globally amphiphilic organi-
ising group of antimicrobials is the antimicrobial peptides
(AMPs) and their synthetic mimics. In particular, synthetic
sequence-specific oligomers of N-substituted glycine, termed
“peptoids”, have been found to show potent antimicrobial
activity against bacterial pathogens in vitro and can act
against the emergence of antimicrobial resistance. In this
study, we evaluate the antimicrobial activity of cyclic peptoid
oligomers against clinical isolates of methicillin-resistant
zation of the peptoid side-chains. Several of these new am-
phiphilic compounds show potent and selective antimicrobial
activity. Electron microscopy experiments demonstrate that
the peptoids target and damage the MRSA cytoplasmic
membrane through the formation of pores. These results sub-
stantiate the potential of peptoids as antimicrobial thera-
peutic agents for the treatment of S. aureus infections.
antimicrobial peptidomimetics feature an amphiphilic cat-
ionic–hydrophobic structure that facilitates their interaction
with anionic bacterial cytoplasmic membranes.[7] Such anti-
microbial agents are deemed to be likely to show sustained
therapeutic efficacy due to the low frequency of resistance
observed against membrane-active antimicrobials.[8] The
challenges that confound the clinical implementation of
membrane-active antimicrobial agents include poor selec-
tivity, undesirable hemolytic activity, severe host toxicity,
and insufficient water solubility.[6a] For these reasons,
AMPs such as the decapeptide gramicidin are typically used
only as topical agents.[9]
Synthetic oligomers that can adopt defined conforma-
tions similar to peptides are a fertile area for the explora-
tion of compounds that have antimicrobial activity.[10] In-
deed, a number of such “foldamers” with antimicrobial ac-
tivity have been introduced, including bulk polymers,[11] β-
peptides,[5c] arylamides,[12] and peptoids.[13] In these exam-
ples, amphiphilicity has been implicated as an important
parameter for antimicrobial activity.
Peptoids are a particularly versatile class of foldamer
compounds, with a number of established biological activi-
ties[14] and a demonstrated potential for therapeutic devel-
opment.[15] Peptoids can potentially mimic the functional
attributes of AMPs while overcoming their limitations. For
example, the abiotic backbone in peptoid oligomers confers
protection against proteolytic degradation.[16] Peptoid
oligomers can be easily synthesized on a solid support
through iterative steps of bromoacetylation and nucleo-
philic displacement using commercially available reagents
(Figure 1).[17] The synthetic tractability of peptoid oligo-
Introduction
The spread of pathogens showing antimicrobial resis-
tance continues to challenge the clinical treatment of many
bacterial infections. The emergence of multi-drug resistant
pathogens, including methicillin resistant S. aureus
(MRSA), coupled with a diminished pace of antimicrobial
discovery, has exacerbated the need for new antimicrobial
agents.[1] From 2005–2008, approximately 21,000 cases of
invasive MRSA infections were reported in the USA, 82%
of which were healthcare associated.[2] Thus, the discovery
of antimicrobial agents that can combat bacterial infections
without the rapid induction of antimicrobial resistance
would be of significant potential benefit for clinical devel-
opment.[3]
There has been sustained interest in membrane-targeting
antimicrobial peptides (AMPs)[4] and antimicrobial pept-
ide-mimetic compounds[5] as alternative pharmacological
regimens.[6] In contrast to conventional small-molecule anti-
biotics, which have specific intracellular macromolecular
targets, AMPs and similar antimicrobial peptidomimetics
are thought to target the bacterial cytoplasmic membrane
as their primary mechanism of action. Many AMPs and
[a] Department of Chemistry, New York University,
100 Washington Square East, New York, NY 10003-6688, USA
Fax: +1-212-260-7905
E-mail: kent@nyu.edu
[b] Department of Microbiology, New York University School of
Medicine,
Fax: +1-212-263-9180
E-mail: victor.torres@nyumc.org
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300077.
3560
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 3560–3566

Amphiphilic Cyclic Peptoids
mers is advantageous for the design and optimization of macrocyclization and side-chain hydrophobic surface area
drug-like properties. Indeed, peptoids have been shown to are critical features in achieving antimicrobial potency.[19]
have potent and selective antimicrobial activities[13a,13b,18] In particular, we identified peptoid C3 as a compound with
without rapidly eliciting antimicrobial resistance in vitro.[19] potent (Table S1) and rapid (Figure S1) antimicrobial ac-
Importantly, peptoids have also been shown to have desir- tivity against MRSA (Figure 2). Using this structure as a
able pharmacokinetic characteristics in vivo that compare starting point, a library of cyclic peptoids incorporating
favorably to analogous peptide sequences.[20]
either five or six residues was synthesized from commer-
cially available primary amines and bromoacetic acid rea-
gents using an established “submonomer” protocol (Fig-
ure 1).[24] Pentamer and hexamer macrocycle sizes were
chosen to minimize molecular weights while permitting
high yields for the cyclization reactions.[23a]
Figure 1. Submonomer synthesis of cyclic peptoid oligomers. The
linear precursors were synthesized on a solid phase using 2-
chlorotrityl chloride resin. Elongation of the linear peptoid entails
iterative a) bromoacetylation (with bromoacetic acid) and b) nu-
cleophilic displacement (with a primary amine, R-NH2) steps.
Chemical diversity can easily be incorporated into the peptoid
chain by varying the R groups on the primary amine. Following
c) cleavage of the peptoid oligomer from the resin (20% HFIP/
CH2Cl2), d) head-to-tail macrocyclization can then be carried out
in solution with an activating agent (PyBOP/DIEA/DMF). Con-
version yields Ն 95% were routinely observed in the synthesis of
cyclic peptoid oligomers (see Supporting Information).
Efforts to enhance the antimicrobial activity of peptide
and peptidomimetic oligomers have been extensively report-
ed,[5c,13a,21] and these have included the use of covalent te-
thers that restrict the conformation of macrocyclic oligo-
mers.[19,22] Macrocyclization in peptoid oligomers, in par-
ticular, can result in a global amphiphilic structure that may
be advantageous in the design of membrane-active anti-
microbials.[23] X-ray crystal structures of cyclic peptoids
show that the side-chains of consecutive peptoid residues
are organized onto opposing faces of the planar macro-
cycle, such that the proper placement of cationic and hydro-
phobic residues would result in well-defined cationic and
hydrophobic surfaces.
We[19] and others,[13] have previously evaluated peptoid
macrocycles as antimicrobial agents, and have shown that
they show increased antimicrobial potency compared to
their linear analogs. The peptoids showed remarkable selec-
tivity for microbial over eukaryotic cells. This unanticipated
finding raised the question of whether the antimicrobial
compounds were acting at the bacterial cell surface, similar
to AMPs. In this study, we describe a new family of antimi-
crobial cyclic peptoids with favorable pharmacological at-
tributes. We additionally confirmed, by scanning electron
microscopy, that the bacterial cell surface is indeed an anti-
microbial site of action.
Figure 2. Chemical structures of cyclic peptoid compounds and
their antimicrobial activities. The susceptibility of MRSA USA300
to cyclic peptoid oligomers was evaluated. Three different peptoid
scaffold types were evaluated, including hexamers (scaffold A) and
pentamers (scaffolds B and C). MIC: Minimum Inhibitory Concen-
tration. Explicit chemical structures for all compounds are pro-
vided in the Supporting Information.
Amphiphilic peptoid macrocycles were synthesized with
aminopropyl groups as cationic residues, and included a
variety of hydrophobic residues (Figure 2; Table S2). The
hydrophobic residues included in the library featured aro-
matic side-chain groups including phenyl, diphenyl, naph-
thyl, fluorene, and fluoroaryl groups. The cationic and
hydrophobic residues were alternated within the oligomer
Results and Discussion
Synthesis of Cyclic Peptoid Library
The design of antimicrobial peptoids was based on con- sequence to produce cyclic peptoid hexamers (scaffold A in
sideration of our previous results, which established that Figure 2) that would allow cationic and hydrophobic sur-
Eur. J. Org. Chem. 2013, 3560–3566
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3561

V. J. Torres, K. Kirshenbaum et al.
FULL PAPER
face areas to segregate on opposite faces of the planar isomers in which the fluorene moieties are linked to the
macrocycle.[23a] Head-to-tail macrocyclization of peptoid peptoid backbone at the 2- or 9-positions of the ring,
oligomers was implemented in solution (Figure 1), follow- respectively. C106 showed a dramatic improvement in anti-
ing the stepwise assembly and cleavage of the corresponding microbial activity relative to C108 (MIC = 3.9 vs.
linear precursors on a solid support (Experimental Section). 62.5 μgmL–1, respectively), despite only small variations in
Due to the odd number of residues in cyclic pentamers, polarity (as shown by small changes in RP-HPLC retention
these compounds were synthesized to include either two time; tR = 7.3 vs. 6.8 min, respectively; Table S3). This com-
(scaffold B) or three (scaffold C) hydrophobic residues (Fig- parison suggests that small increases in hydrophobicity can
ure 2). Following RP-HPLC purification (to Ն 95% purity) significantly alter antimicrobial activity, although these
and ESI-MS characterization, a total of 25 peptoid com- variations may also influence self-assembly of the oligomers
pounds (C101–C125) were prepared, including 18 cyclic on the bacterial membrane surface, as described by Ghadiri
hexamers and 7 cyclic pentamers (Figures S2–S4; Table S3). and co-workers.[27]
Compounds incorporating phenylalkyl groups as hydro-
phobic side-chains showed moderate antimicrobial activity.
Antimicrobial Characterization of the Cyclic Peptoids
We determined the antimicrobial activities of the cyclic For example, compound C114, which features a piperonyl
peptoid oligomers against MRSA type USA300.[25] The group, showed an MIC of 250 μgmL–1 (Figure 2). Com-
USA300 clone is associated with highly invasive cases of pounds C118 and C120 are cyclic hexamers incorporating
MRSA infections, resulting in severe skin infections, septi- enantiomeric phenylethyl[28] side-chains. These compounds
cemia, and necrotizing pneumonia. In the U.S.A., this clone showed similar antimicrobial activities (MIC
=
is responsible for the majority of community-acquired 250 μgmL–1), which suggests that the chirality of the hydro-
MRSA infections.[25b] The minimum inhibitory concentra- phobic residues does not play a role in the antimicrobial
tion (MIC) was determined using standard protocols (see activity, consistent with a mechanism of action at the cell
Exp. Sect.). For these experiments, we used peptoid C3[19] membrane.[29] On the other hand, compounds incorporat-
as a positive control (Figure 2). From the 25 compounds in ing substituted phenyl rings as individual hydrophobic resi-
the library, 6 (i.e., C107, C110, C112, C115, C116, and dues showed moderate to potent antimicrobial activities, as
C119) showed insignificant antimicrobial activity (MIC Ͼ seen in compounds C122 (MIC = 15.6 μgmL–1) and C124
500 μgmL–1), 10 compounds (C101, C108, C111, C113, (MIC = 3.9 μgmL–1). Similarly to the comparison between
C114, C117, C118, and C120–C122) showed moderate anti- C106 and C108, C124 showed a fourfold enhancement in
microbial activity (15.6 μgmL–1 Յ MIC Յ 250 μgmL–1), antimicrobial activity compared to C122, despite only a
and 9 compounds (C102–C106, C109, and C123–C125) slight difference in hydrophobicity (tR = 6.6 vs. 5.9, respec-
showed potent antimicrobial activity (MIC Յ 3.9 μgmL–1). tively) imparted by an additional methyl substituent on the
The potent antimicrobial activity shown by the latter group phenyl ring (Table S3). These results indicate that a sub-
compares favorably to the antimicrobial activities reported stantial increase in hydrophobicity imparted by increasing
for natural and synthetic AMPs that target the bacterial aromatic ring size is not a strict requirement for achieving
membrane.[5b,13b,26]
potent antimicrobial activity. Moreover, appropriate substi-
A comparison of the antimicrobial activities of the dif- tution on the phenyl rings (i.e., methyl groups in C124) can
ferent peptoids revealed structure–activity relationships that provide sufficient hydrophobicity for achieving potent anti-
may aid the further development of cyclic amphiphilic microbial activity. Fluorinated versions of the peptoids gen-
oligomers with potent antimicrobial activities. The impor- erally showed increased or similar hydrophobicity, as seen
tance of a hydrophobic surface area in achieving potent an- in their RP-HPLC retention times. These compounds, ac-
timicrobial activity is clear from the differing activities of cordingly, also showed enhanced antimicrobial activities
cyclic compounds having similar chemical compositions but compared to their hydrocarbon analogs (e.g., C117 Ͼ C115,
different hydrophobic surface areas. C101 bears two hydro- C121 Ͼ C118, and C123 Ͼ C122; Figure S5).
phobic biphenyl groups and showed moderate antimicro-
bial activity (MIC = 250 μgmL–1), whereas C102 bears
three hydrophobic biphenyl groups and showed potent anti-
microbial activity (MIC = 3.9 μgmL–1). Potent antimicro-
Peptoids Show Antimicrobial Activity in the Presence of
Human Serum
The susceptibility of bacterial cells to candidate antimi-
bial activities (MIC = 3.9 μgmL–1) were observed for cyclic crobial agents was evaluated in the presence of serum to
hexamers with biphenyl (C3 and C103–C105) and naphthyl provide a measure of antimicrobial activity within a bio-
(C109) groups as hydrophobic residues. Compounds bear- medically relevant context. The binding of antimicrobial
ing indole groups also showed an increase in antimicrobial agents to serum components (e.g., proteins and fatty acids)
activity with increasing hydrophobic surface area. C112, may alter the compound’s distribution, elimination, and
bearing two indole groups, was inactive, whereas C113, biological activity, and hence the effect of serum is an im-
bearing three indole groups, showed moderate antimicro- portant pharmacological parameter to be considered.[30]
bial activity (MIC = 62.5 μgmL–1).
The antimicrobial activity in the presence of serum is par-
Cyclic peptoid oligomers bearing fluorene groups as ticularly important for amphiphilic xenobiotic agents, be-
hydrophobic residues showed unusual variations in antimi- cause their hydrophobic surface features may be susceptible
crobial activity. Compounds C106 and C108 are structural to binding to serum proteins.[31]
3562
www.eurjoc.org
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 3560–3566

Amphiphilic Cyclic Peptoids
The antimicrobial activities of compounds C3, C124, by measuring their hemolytic activity. The concentration of
C125, and gramicidin S were evaluated in the presence of antimicrobial compound required to lyse 10% of human
serum. Compound C3 was included to enable a comparison erythrocytes (HC10) following incubation for 1 h at 37 °C,
with our previous studies.[19] Compound C124 was chosen was determined (see Exp. Sect.). These experiments revealed
because it showed favorable water solubility compared to that gramicidin S showed an HC10 of 15.6 μgmL–1, whereas
other active compounds in the library. Compound C125 molecules C3 and C125 both showed HC10 values of
was chosen to evaluate the effect of fluorination on antimi- 31.3 μgmL–1 (Table 1).
crobial activity in serum. The cyclic decapeptide gramicidin
Selectivity ratio (SR) values are often calculated as the
S[9a] was included to enable a comparison between antimi- ratio of hemolytic to antimicrobial activities. SR values can
crobial peptides and peptoids. The assays were conducted be calculated using different metrics of hemolytic activity
as described above, in media supplemented with 50% nor- (e.g., HC50 or HC10, to yield SR50 and SR10 values, respec-
mal pooled human serum (NHS; Experimental Section).
tively). The determination of selectivity using SR10 values
The MIC values against MRSA USA300 in the presence is more stringent than when SR50 values are used. Large
of serum were observed to increase compared to the MICs SR10 values indicate that a compound has potent antimi-
in the absence of serum for compounds C3 (15.6 μgmL–1), crobial activity and low hemolytic activity, corresponding
C124 (15.6 μgmL–1), and C125 (62.5 μgmL–1). On the to a high selectivity for prokaryotic vs. eukaryotic cytotoxi-
other hand, the MIC remained constant (31.3 μgmL–1) for city. AMP-mimics have been reported to show values that
gramicidin S in the presence of serum. Compounds C3 and approach SR10 = 30[13b,34] and SR50 = 200.[12] In previous
C124 showed approximately fourfold decreases in antimi- work, we reported that a cyclic peptoid octamer showed a
crobial activity in the presence of serum, whereas fluori- significant 32-fold selectivity (SR10 = 32) for E. coli bacte-
nated compound C125 showed a sixteen-fold decrease in rial cells over human erythrocytes. In this work, we aimed
antimicrobial activity in the presence of serum (Table 1).
to achieve similar or greater selectivity (SR10 Ն 32) for S.
aureus vs. human erythrocytes using cyclic peptoid oligo-
mers. The results confirmed the strongly hemolytic nature
of gramicidin S, which had an SR10 of 0.5 (Table 1). Both
C3 and C125 were moderately selective, with SR10 = 8. On
the other hand, compound C124 showed substantial selec-
tivity (SR10 = 32), establishing that significant selectivity for
S. aureus over human erythrocytes can be achieved with
cyclic peptoid hexamers. The comparison between C124
and C125 reveals that fluorination reduces selectivity.
As an additional parameter of selectivity, we also evalu-
ated the compounds for toxicity towards nucleated mam-
malian cells (Figures S6–S7). The compounds were incu-
bated with HeLa cells for 1 h at 37 °C, and cell viability
was determined using a dye-based assay (see Experimental
Section). The concentration of compound that resulted in
50% lethality (LC50), was determined. Compounds C3,
Table 1. Comparison of the biological activities of selected com-
pounds. All concentration values are expressed in μgmL–1 units.
[d]
[e]
MIC[a] MIC + NHS[b] HC10[c] SR10 LC50
TI[f]
C3
C124
C125
Gramicidin S
3.9
3.9
3.9
15.6
15.6
62.5
31.3
31.3
125
31.3
15.6
8
32
8
63
180
47
16
45
12
1
31.3
0.5
31.3
[a] Minimum Inhibitory Concentrations (MIC) against MRSA
USA300. [b] MIC values against MRSA USA300 in 50% (v/v) nor-
mal human serum (NHS). [c] HC10 represents the concentration of
antimicrobial required to lyse 10% of human erythrocytes. [d] SR10
(selectivity ratio) values are defined as the ratio HC10/MIC. [e] LC50
(50% lethal concentration) represents the concentration of antimi-
crobial required to kill 50% of HeLa cells. [f] Therapeutic indices
(TI) are calculated as the ratio LC50/MIC.
Serum inactivation of antimicrobial activity has often C124, C125, and gramicidin S showed LC50 values of 63,
been observed for both small-molecule antimicrobials and 180, 47, and 31.3 μgmL–1, respectively (Table 1).
AMPs,[5b,32] and has been hypothesized to be caused by
Therapeutic indices (TI), defined as the ratio LC50/MIC,
non-specific interactions with serum proteins.[33] The sub- were determined for each compound as a measure of selec-
stantial inactivation observed for compound C125 suggests tive toxicity for bacterial over mammalian cells. All the pep-
that fluorination significantly increases the non-specific af- toids in this series showed more favorable TI values than
finity of cyclic peptoid oligomers towards serum compo- gramicidin S (TI = 1). Compound C3 had a TI of 16,
nents. The comparison between compounds C124 and C125 whereas C124 and C125 had TI values of 45 and 12, respec-
suggests that while fluorination may be an effective strategy tively. The higher TI value calculated for C124 relative to
in enhancing antimicrobial activity, the presence of serum C3 demonstrates the substantial improvement in pharma-
can significantly reduce this enhancement, and can even re- cological attributes obtained with the new library of cyclic
sult in a diminished overall antimicrobial potency.
peptoids, since C124 is smaller in size (MW = 868 Da) than
C3 (MW = 1053 Da), and is water-soluble at Ն 15 mgmL–1
concentrations.
Improved Selectivity of Peptoid C124
A detrimental property of most AMPs is their toxicity
towards host cells due to non-specific interactions with eu-
karyotic membranes. Thus, the development of antimicro-
bial oligomers is often aimed at maximizing antimicrobial
activity while minimizing eukaryotic cell toxicity. We there-
Peptoid Oligomer C124 Targets and Damages MRSA
Membranes
In general, the cationic–amphiphilic nature of AMPs en-
fore sought to determine the toxicity of the cyclic peptoids ables their interaction with negatively-charged bacterial
Eur. J. Org. Chem. 2013, 3560–3566
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3563

V. J. Torres, K. Kirshenbaum et al.
FULL PAPER
membranes.[4] Ongoing investigations continue to provide with some present near or at the division hemisphere (sep-
insight into the nature of these interactions.[35] While the tum). These features were consistently observed in micro-
exact mode of action may vary for different membrane- scopy images obtained for antimicrobial-treated bacterial
active antimicrobials, these interactions ultimately lead to cells (Figure S8).
damage of the bacterial membrane. We investigated the
These observations are consistent with the proposed
morphological changes that occur to the S. aureus cell sur- mechanism of action of membrane-active antimicrobial
face upon exposure to peptoid antimicrobials using scan- oligomers. The appearance of holes in treated S. aureus cells
ning electron microscopy (SEM).[36]
has previously been postulated for phenylene ethynylene
SEM images of untreated S. aureus cultures showed oligomers that target negatively-charged lipids in the bacte-
smooth cell surfaces with varying cell diameters from 500– rial membrane.[37] In addition, cell-surface damage has also
800 nm (Figure 3, A). The cells appeared to be roughly been observed in S. aureus and other bacterial species
spherical, and occurred in grape-like clusters. Dividing cells treated with AMPs.[36] These images indicate that antimi-
were also observed. Cells treated with sub-MIC, MIC, or crobial amphiphilic peptoids cause cell-surface damage, al-
supra-MIC concentractions of compounds C3 and C124 though other antimicrobial mechanisms may be at work.
showed cell-surface damage (Figure 3, B–L; Figure S8). The Indeed, the molecular origin of antimicrobial activity and
effects of the different compounds, incubation times (1 h vs. its correlation to the observed damage to the bacterial cell
18 h), and concentrations (sub-MIC, MIC, or supra-MIC) surfaces merits further investigation.
on cell morphologies were not readily distinguishable by
SEM. Some cell surfaces were observed to collapse upon
treatment with the antimicrobial peptoids (Figure 3G–I).
The formation of small pores (Figure 3, B–D, J, and K) and
large craters (Figure 3, E, F, and L) were also observed,
Conclusions
Many natural membrane-active antimicrobial oligomers
are composed of significantly more than 10 monomeric res-
idues, and have molecular weights much greater than
1000 Da. These compounds may therefore face intrinsic dis-
advantages regarding pharmacological development, and
are often limited to topical applications in the clinic. Biolo-
gically active synthetic oligomers, especially those that act
on the bacterial membrane, have significant potential for
clinical development.[38] Moreover, macrocyclic oligomers
have conformational stability and potential bioavailability
that may be attractive for drug discovery.[22a,39] We report
the discovery of amphiphilic cyclic peptoid oligomers that
show potent antimicrobial activity towards clinically rel-
evant isolates of S. aureus. Although these compounds are
observed to act on the bacterial surface, they show signifi-
cant antimicrobial selectivity. The potent antimicrobial ac-
tivity and non-hemolytic activity observed for these com-
pounds are comparable to those reported for other peptido-
mimetic oligomers currently in clinical development.[6a,12]
Future studies will include investigations regarding the in
vivo efficacy, the mechanism of action, and the origin of
the selectivity of the antimicrobial cyclic peptoid oligomers.
Experimental Section
Synthesis and Characterization of Cyclic Peptoid Oligomers: Pepto-
ids were synthesized and characterized as described previous-
ly.[17,19,23a] 2-Chlorotrityl chloride resin (100 mg) was swollen with
CH2Cl2 (5 min, room temp.) and treated with bromoacetic acid
(90.3 mg) and DIEA (diisopropylethylamine; 107 μL) in CH2Cl2
(2 mL) for Ͼ 40 min. Following washes with CH2Cl2 (3ϫ 1 min)
and DMF (3ϫ 1 min), the desired first amine submonomer was
added (1.0 m solution in DMF; 1.0 mL) and allowed to react for
20 min. The resins were then washed with DMF (3ϫ 1 min), and
incubated with bromoacetic acid (1.0 m solution in DMF; 1.0 mL)
and DIC (N,NЈ-diisopropylcarbodiimide; 300 μL, neat) for 20 min.
Following washings (3ϫ 1 min) with DMF, the amine displacement
and bromoacetylation steps were repeated until the final sub-
Figure 3. Membrane damage caused by cyclic peptoid oligomers.
Untreated MRSA USA300 cells (A) appear spherical with smooth
cell surfaces, whereas cells treated with antimicrobial peptoids C3
(B–F) and C124 (G–L) showed depressions (G to I), small pores
(B, C, D, J, and K), or large craters (E, F, and L). Scale bar:
300 nm. Bacterial cells were incubated with C3 at its MIC
(3.9 μgmL–1) for 1 h (C) or 18 h (B, D, E, and F), or C124 for 1 h
at sub-MIC (1.95 μgmL–1; G–J) or MIC values (3.9 μgmL–1; K
and L).
3564
www.eurjoc.org
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 3560–3566

Amphiphilic Cyclic Peptoids
monomer had been added and the desired sequence had been ob-
tained. Due to the deactivated nature of some amines featuring N-
aryl rings (e.g., aniline, p-fluoroaniline, 2-aminofluorene, 9-amino-
fluorene), the amine displacement steps were conducted overnight
at room temperature (18 h, 25 °C), and the subsequent bromoacet-
ylation steps were conducted for 1.5 h at room temperature. The
resins were then washed extensively with DMF (3ϫ 1 min) and
CH2Cl2 (3ϫ 1 min), and cleaved off the resin with HFIP (hexa-
fluoroisopropanol; 20% in CH2Cl2; 3 mL) (30 min). The cleavage
cocktail was evaporated with a N2 stream, redissolved in 50%
MeCN/H2O, frozen, and lyophilized. Cyclization reactions were
conducted with the crude linear precursors in dry deoxygenated
DMF. Typically, the linear oligomer (12 μmol) was suspended in
DMF (5.25 mL) with freshly prepared solutions of PyBOP (96 mm
solution in DMF; 375 μL) and DIEA (192 mm solution in DMF;
375 μL). The reaction vessel was sealed with parafilm to exclude
air, and the mixture was allowed to react for ca. 30 min at room
temperature. The solution was then evaporated by heating (tem-
peratures Յ 60 °C) under reduced pressure, and the resulting prod-
uct was redissolved in 50% MeCN/H2O, frozen, and lyophilized.
vine serum) in tissue-culture plates (Corning), adherent HeLa cells
were trypsinized, washed in PBS, and resuspended in medium at a
density of 1ϫ106 cells/mL. A 100 μL cell suspension was seeded
onto a 96-well plate and cultured for 25 h. Subsequently, cells were
exposed to the different compounds for 1 and 24 h, and the cell
viabilities were evaluated using the CellTiter 96® Aqueous Non-
Radioactive Cell Proliferation Assay (Promega).
Scanning Electron Microscopy (SEM) Imaging: SEM samples were
prepared following previously published procedures.[36] Mid-ex-
ponential MRSA USA300 was prepared by inoculating an over-
night culture in fresh LB medium till OD600 ≈ 0.4 (approximately
3 h at 37 °C). The culture was then treated with the antimicrobial
compound at the MIC as well as at twofold higher (supra-MIC)
and lower (sub-MIC) concentrations of the MIC (previously deter-
mined for cell density at OD600 ≈ 0.4) for 1 h or 24 h at 37 °C.
Samples were centrifuged (6000 ϫ g, 20 min at room temp.), and
the resulting cell pellets were washed and resuspended in PBS. The
resulting cell suspension was placed on a 1 cm2 piece of membrane
filter (Millipore 0.45 μm) to dry at room temp. Subsequent steps
were conducted in 24-well plates (Corning), and a fresh well was
used for each step. The samples were then fixed with glutaraldehyde
(8% v/v in PBS; Alfa Aesar) for 1 h at room temp., then they were
washed with PBS, and fixed with OsO4 (0.5% w/v in PBS; Electron
Microscopy Sciences) at 4 °C overnight. After washing with PBS,
the samples were treated with ethanol (30% aq.) for 10 min, uranyl
acetate [3% v/v in ethanol (30% aq.); Electron Microscopy Sci-
ences], ethanol (50% aq.) for 10 min, and ethanol (70% aq.) over-
night at 4 °C. The membrane pieces were then dried at room tem-
perature, attached to an aluminum pin stub with a carbon conduc-
tive adhesive (PELCO), sputter-coated (Electron Microscopy Sci-
ences coater EMS1505ES) with a 5 nm layer of gold, and imaged
with a Zeiss Merlin FESEM under SE2 detection at 2.0–4.0 kV.
The crude products were redissolved in 50% MeCN/H2O, and puri-
fied by reverse-phase (RP) HPLC (C18 semi-preparative column,
Waters, 15 μm, 100 Å, 25ϫ100 mm) using a Beckman Coulter Sys-
tem Gold Instrument operated with 32Karat software. A typical
method used for the purification was 30–100% MeCN/H2O in
40 min at a 2.5 mLmin–1 flow rate and 214–230 nm wavelength.
Relevant fractions were collected, frozen, and lyophilized. The Boc
groups were deprotected by treating the dried product with TFA/
CH2Cl2 (50%) for 30 min at r.t. The cocktail was evaporated with
N2, and the product was redissolved in 50% MeCN/H2O, frozen,
and lyophilized. Characterization of purified (to Ն 95% by RP-
HPLC) products was performed with an RP-HPLC C18 analytical
column (Peeke Scientific, 5 μm, 120 Å, 2.0ϫ50 mm) and Agilent
100 Series LC-MSD Trap XCT (Agilent Technologies).
Supporting Information (see footnote on the first page of this arti-
cle): General synthetic procedures; procedures for the synthesis of
peptoid submonomers and of gramicidin S; general biology pro-
cedures; procedures for killing assays; For cyclic peptoid oligomers:
chemical structures, MS and HPLC data, examples of HPLC and
NMR spectra; data on the antimicrobial activity, killing kinetics,
cytotoxicity and mouse toxicity of C3; data on cyctotoxic effects
of C124; comparison of the antimicrobial activities of fluorinated
and hydrocarbon cyclic peptoid oligomers.
Antimicrobial Assays: MRSA pulse-field gel electrophoresis types
USA300 (strain LAC)[40] and USA500 (BK2395)[41] were used in
this study. S. aureus were grown in tryptic soy broth (BD Biosci-
ences) as described previously.[41] Antimicrobial susceptibility as-
says were conducted in 96-well plates using the broth-macrodilu-
tion procedure outlined in document M07-A7 of the CLSI.[42] The
screenings against Staphylococcus aureus LAC were conducted in
either LB (Luria-Bertani) or RPMI media. For the assays in serum,
normal human pooled serum (NHS) was filtered through a 0.45 μm
syringe filter (Pall) prior to dilution with RPMI, such that each
well contained 50% (v/v) NHS. Experiments were conducted in
three independent replicates of three parallel trials to ensure statis-
tical significance.
Acknowledgments
This work was supported by the National Science Foundation
(NSF) (SEM imaging, MRI program, DMR-0923251; award to
K. K., CHE-1152317). Support by the National Institute of Allergy
and Infectious Diseases (United States Public Health Service Grant
K22-AI079389 to V. J. T.) and by the New York University School
of Medicine Development Funds (to V. J. T.) is acknowledged.
M. L. H. acknowledges the support by the New York University –
Graduate School of Arts and Sciences (NYU-GSAS) (Horizon Fel-
lowship). M. A. B. is supported by an American Heart Association
pre-doctoral fellowship (10PRE3420022).
Hemolysis Assays: Hemolysis assays were conducted in 96-well
plates using fresh human erythrocytes in PBS (phosphate-buffered
saline) solution. Erythrocytes treated with a detergent (1% Triton
X-100) solution were used as a positive control for erythrocyte lysis,
and vehicle buffer (PBS) was used as a negative control. Hemolytic
percentages were defined as [(A – Atestblank)/(Acontrol – Ablank)] ϫ
100, where A is the absorbance of the test well, Acontrol is the
average absorbance of wells with erythrocytes exposed to PBS but
no antimicrobial, Atestblank is the average absorbance of wells with
PBS and the antimicrobial, and Ablank is the average absorbance of
wells with PBS only. Experiments were conducted in three indepen-
dent replicates of three parallel trials to ensure statistical signifi-
cance.
[1] a) M. A. Cooper, D. Shlaes, Nature 2011, 472, 32–32; b) D. J.
Diekema, M. A. Pfaller, F. J. Schmitz, J. Smayevsky, J. Bell,
R. N. Jones, M. Beach, S. P. Grp, Clin. Infect. Dis. 2001, 32,
S114–S132; c) D. F. Sahm, M. K. Marsilio, G. Piazza, Clin.
Infect. Dis. 1999, 29, 259–263; d) F. D. Lowy, J. Clin. Invest.
2003, 111, 1265–1273; e) D. J. Payne, M. N. Gwynn, D. J.
Holmes, D. L. Pompliano, Nat. Rev. Drug Discovery 2007, 6,
29–40.
Cytotoxicity Assays: Following seeding in DMEM (Dulbecco’s mo-
died EagleЈs medium) supplemented with 10% v/v FBS (fetal bo-
Eur. J. Org. Chem. 2013, 3560–3566
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3565

V. J. Torres, K. Kirshenbaum et al.
FULL PAPER
[2]
[22] a) R. M. Kohli, C. T. Walsh, M. D. Burkart, Nature 2002, 418,
658–661; b) J. P. Tam, Y. A. Lu, J. L. Yang, K. W. Chiu, Proc.
Natl. Acad. Sci. USA 1999, 96, 8913–8918.
[23] a) S. B. Y. Shin, B. Yoo, L. J. Todaro, K. Kirshenbaum, J. Am.
Chem. Soc. 2007, 129, 3218–3225; b) M. Laurencin, M. Amor,
Y. Fleury, M. Baudy-Floc’h, J. Med. Chem. 2012, 55, 10885–
10895.
A. J. Kallen, Y. Mu, S. Bulens, A. Reingold, S. Petit, K. Gersh-
man, S. M. Ray, L. H. Harrison, R. Lynfield, G. Dumyati,
J. M. Townes, W. Schaffner, P. R. Patel, S. K. Fridkin, JAMA
J. Am. Med. Assoc. 2010, 304, 641–647.
K. Lewis, Nature 2012, 485, 439–440.
M. Zasloff, Nature 2002, 415, 389–395.
a) A. Bragonzi, Sci. Transl. Med. 2010, 2; b) S. Choi, A. Isaacs,
D. Clements, D. Liu, H. Kim, R. W. Scott, J. D. Winkler, W. F.
DeGrado, Proc. Natl. Acad. Sci. USA 2009, 106, 6968–6973;
c) T. L. Raguse, E. A. Porter, B. Weisblum, S. H. Gellman, J.
Am. Chem. Soc. 2002, 124, 12774–12785; d) S. E. Howson, A.
Bolhuis, V. Brabec, G. J. Clarkson, J. Malina, A. Rodger, P.
Scott, Nat. Chem. 2012, 4, 31–36.
a) R. E. W. Hancock, H. G. Sahl, Nat. Biotechnol. 2006, 24,
1551–1557; b) G. N. Tew, R. W. Scott, M. L. Klein, W. F. De-
grado, Acc. Chem. Res. 2010, 43, 30–39.
a) R. E. W. Hancock, R. Lehrer, Trends Biotechnol. 1998, 16,
82–88; b) P. B. Savage, Eur. J. Org. Chem. 2002, 759–768.
J. G. Hurdle, A. J. O’Neill, I. Chopra, R. E. Lee, Nat. Rev.
Microbiol. 2011, 9, 62–75.
a) G. F. Gause, M. G. Brazhnikova, Nature 1944, 154, 703–703.
D. J. Hill, M. J. Mio, R. B. Prince, T. S. Hughes, J. S. Moore,
Chem. Rev. 2001, 101, 3893–4011.
A. Song, S. G. Walker, K. A. Parker, N. S. Sampson, ACS
Chem. Biol. 2011, 6, 590–599.
[3]
[4]
[5]
[24] R. N. Zuckermann, J. M. Kerr, S. B. H. Kent, W. H. Moos, J.
Am. Chem. Soc. 1992, 114, 10646–10647.
[25] a) F. C. Tenover, L. K. McDougal, R. V. Goering, G. Killgore,
S. J. Projan, J. B. Patel, P. M. Dunman, J. Clin. Microbiol. 2006,
44, 108–118; b) R. M. Klevens, M. A. Morrison, J. Nadle, S.
Petit, K. Gershman, S. Ray, L. H. Harrison, R. Lynfield, G.
Dumyati, J. M. Townes, A. S. Craig, E. R. Zell, G. E. Fosheim,
L. K. McDougal, R. B. Carey, S. K. Fridkin, JAMA J. Am.
Med. Assoc. 2007, 298, 1763–1771.
[26] A. Cherkasov, K. Hilpert, H. Jenssen, C. D. Fjell, M. Wald-
brook, S. C. Mullaly, R. Volkmer, R. E. Hancock, ACS Chem.
Biol. 2009, 4, 65–74.
[27] L. Motiei, S. Rahimipour, D. A. Thayer, C. H. Wong, M. R.
Ghadiri, Chem. Commun. 2009, 3693–3695.
[28] P. Armand, K. Kirshenbaum, A. Falicov, R. L. Dunbrack,
K. A. Dill, R. N. Zuckermann, F. E. Cohen, Fold. Des. 1997,
2, 369–375.
[29] D. Wade, A. Boman, B. Wahlin, C. M. Drain, D. Andreu,
H. G. Boman, R. B. Merrifield, Proc. Natl. Acad. Sci. USA
1990, 87, 4761–4765.
[30] a) W. A. Craig, P. G. Welling, Clin. Pharmacokinet. 1977, 2,
252–268; b) B. Suh, W. A. Craig, A. C. England, R. L. Elliott,
J. Infect. Dis. 1981, 143.
[6]
[7]
[8]
[9]
[10]
[11]
[12] S. Choi, A. Isaacs, D. Clements, D. H. Liu, H. Kim, R. W.
Scott, J. D. Winkler, W. F. DeGrado, Proc. Natl. Acad. Sci.
USA 2009, 106, 6968–6973.
[31] G. N. Rolinson, J. Antimicrob. Chemother. 1980, 6, 311–317.
[32] J. Liu, C. Luo, P. A. Smith, J. K. Chin, M. G. Page, M. Paetzel,
F. E. Romesberg, J. Am. Chem. Soc. 2011, 133, 17869–17877.
[33] X. D. Xu, T. Henninger, D. Abbanat, K. Bush, B. Foleno, J.
Hilliard, M. Macielag, Bioorg. Med. Chem. Lett. 2005, 15, 883–
887.
[34] B. P. Mowery, S. E. Lee, D. A. Kissounko, R. F. Epand, R. M.
Epand, B. Weisblum, S. S. Stahl, S. H. Gellman, J. Am. Chem.
Soc. 2007, 129, 15474–15476.
[35] A. Ivankin, L. Livne, A. Mor, G. A. Caputo, W. F. DeGrado,
M. Meron, B. Lin, D. Gidalevitz, Angew. Chem. 2010, 122,
8640–8643; Angew. Chem. Int. Ed. 2010, 49, 8462–8465.
[36] M. Hartmann, M. Berditsch, J. Hawecker, M. F. Ardakani, D.
Gerthsen, A. S. Ulrich, Antimicrob. Agents Chemother. 2010,
54, 3132–3142.
[37] L. Yang, V. D. Gordon, D. R. Trinkle, N. W. Schmidt, M. A.
Davis, C. DeVries, A. Som, J. E. Cronan Jr., G. N. Tew, G. C.
Wong, Proc. Natl. Acad. Sci. USA 2008, 105, 20595–20600.
[38] S. K. Vooturi, S. M. Firestine, Curr. Med. Chem. 2010, 17,
2292–2300.
[13] a) J. A. Patch, A. E. Barron, J. Am. Chem. Soc. 2003, 125,
12092–12093; b) N. P. Chongsiriwatana, J. A. Patch, A. M.
Czyzewski, M. T. Dohm, A. Ivankin, D. Gidalevitz, R. N.
Zuckermann, A. E. Barron, Proc. Natl. Acad. Sci. USA 2008,
105, 2794–2799; c) D. Comegna, M. Benincasa, R. Gennaro,
I. Izzo, F. De Riccardis, Bioorg. Med. Chem. 2010, 18, 2010–
2018.
[14] a) P. M. Levine, K. Imberg, M. J. Garabedian, K. Kirshen-
baum, J. Am. Chem. Soc. 2012, 134, 6912–6915; b) Y. Utku, E.
Dehan, O. Ouerfelli, F. Piano, R. N. Zuckermann, M. Pagano,
K. Kirshenbaum, Mol. Biosyst. 2006, 2, 312–317.
[15] a) R. N. Zuckermann, T. Kodadek, Curr. Opin. Mol. Ther.
2009, 11, 299–307; b) M. T. Dohm, R. Kapoor, A. E. Barron,
Curr. Pharm. Des. 2011, 17, 2732–2747.
[16] S. M. Miller, R. J. Simon, S. Ng, R. N. Zuckermann, J. M.
Kerr, W. H. Moos, Bioorg. Med. Chem. Lett. 1994, 4, 2657–
2662.
[17] G. M. Figliozzi, R. Goldsmith, S. C. Ng, S. C. Banville, R. N.
Zuckermann, Methods Enzymol. 1996, 267, 437–447.
[18] N. P. Chongsiriwatana, M. Wetzler, A. E. Barron, Antimicrob.
Agents Chemother. 2011, 55, 5399–5402.
[19] M. L. Huang, S. B. Y. Shin, M. A. Benson, V. J. Torres, K. Kir-
shenbaum, ChemMedChem 2012, 7, 114–122.
[20] a) J. Seo, G. Ren, H. Liu, Z. Miao, M. Park, Y. Wang, T. M.
Miller, A. E. Barron, Z. Cheng, Bioconjugate Chem. 2012, 23,
1069–1079; b) C. A. Olsen, H. L. Ziegler, H. M. Nielsen, N.
Frimodt-Moller, J. W. Jaroszewski, H. Franzyk, ChemBioChem
2010, 11, 1356–1360; c) R. D. Jahnsen, N. Frimodt-Moller, H.
Franzyk, J. Med. Chem. 2012, 55, 7253–7261; d) S. A. Fowler,
H. E. Blackwell, Org. Biomol. Chem. 2009, 7, 1508–1524.
[21] a) E. F. Palermo, K. Kuroda, Appl. Microbiol. Biotechnol. 2010,
87, 1605–1615; b) K. Kuroda, G. A. Caputo, W. F. DeGrado,
Chem. Eur. J. 2009, 15, 1123–1133; c) B. P. Mowery, A. H.
Lindner, B. Weisblum, S. S. Stahl, S. H. Gellman, J. Am. Chem.
Soc. 2009, 131, 9735–9745.
[39] a) E. M. Driggers, S. P. Hale, J. Lee, N. K. Terrett, Nat. Rev.
Drug Discovery 2008, 7, 608–624; b) J. A. Raskatov, A. E. Har-
grove, A. Y. So, P. B. Dervan, J. Am. Chem. Soc. 2012, 134,
7995–7999; c) J. A. Robinson, Curr. Opin. Chem. Biol. 2011,
15, 379–386.
[40] A. D. Kennedy, M. Otto, K. R. Braughton, A. R. Whitney, L.
Chen, B. Mathema, J. R. Mediavilla, K. A. Byrne, L. D. Park-
ins, F. C. Tenover, B. N. Kreiswirth, J. M. Musser, F. R. DeLeo,
Proc. Natl. Acad. Sci. USA 2008, 105, 1327–1332.
[41] F. Alonzo III, M. A. Benson, J. Chen, R. P. Novick, B. Shop-
sin, V. J. Torres, Mol. Microbiol. 2012, 83, 423–435.
[42] Clinical and Laboratory Standards Institute. Methods for Di-
lution Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically, 7th ed., Approved Standard CLSI document M07-
A7, CLSI, Wayne, USA, 2006.
Received: January 16, 2013
Published Online: April 19, 2013
3566
www.eurjoc.org
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 3560–3566