Synthetic Mimic of Antimicrobial Peptide with Nonmembrane-Disrupting Antibacterial Properties
Polyguanidinium oxanorbornene ( PGON) was synthesized from norbornene monomers via ring-opening metathesis polymerization. This polymer was observed to be strongly antibacterial against Gram-negative and Gram-positive bacteria as well as nonhemolytic against human red blood cells. Time-kill studies indicated that this polymer is lethal and not just bacteriostatic. In sharp contrast to previously reported SMAMPs (synthetic mimics of antimicrobial peptides), PGON did not disrupt membranes in vesicle-dye leakage assays and microscopy experiments. The unique biological properties of PGON, in same ways similar to cell-penetrating peptides, strongly encourage the examination of other novel guanidino containing macromolecules as powerful and selective antimicrobial agents.
Synthetic Mimic of Antimicrobial Peptide with
Nonmembrane-Disrupting Antibacterial Properties
Gregory J. Gabriel,
Ahmad E. Madkour,
Jeffrey M. Dabkowski,
Christopher F. Nelson,
and Gregory N. Tew*
Department of Polymer Science and Engineering and Department of Microbiology, University of
Massachusetts-Amherst, Amherst, Massachusetts 01003
Received July 31, 2008; Revised Manuscript Received August 22, 2008
Polyguanidinium oxanorbornene (PGON) was synthesized from norbornene monomers via ring-opening metathesis
polymerization. This polymer was observed to be strongly antibacterial against Gram-negative and Gram-positive
bacteria as well as nonhemolytic against human red blood cells. Time-kill studies indicated that this polymer is
lethal and not just bacteriostatic. In sharp contrast to previously reported SMAMPs (synthetic mimics of
antimicrobial peptides), PGON did not disrupt membranes in vesicle-dye leakage assays and microscopy
experiments. The unique biological properties of PGON, in same ways similar to cell-penetrating peptides, strongly
encourage the examination of other novel guanidino containing macromolecules as powerful and selective
We report that poly guanidinium oxanorbornene (PGON)
possesses a remarkable combination of antimicrobial activity
against both Gram-negative and Gram-positive bacteria as well
as low hemolytic activity against human red blood cells (RBCs).
Standard antibacterial, bacteria cell-viability, and hemolysis
assays all give data that is encouraging for the development of
powerful, yet nontoxic, antibacterial materials.
the properties of PGON surpass that of other cationic polymers,
in particular Poly-1 and Poly-3 from our previous study.
Vesicle investigations show that PGON is not membrane-
disruptive, supporting a previous study indicating it also has
properties similar to poly arginine and other cell-penetrating
peptides (CPPs) like HIV-TAT.
As a result, PGON represents
an exciting and fundamentally novel entry in the expanding ﬁeld
of synthetic mimics of antimicrobial peptides (SMAMPs).
While several antimicrobial peptides (AMPs) contain the
amino acid arginine along with other basic residues, SMAMPs
have rarely used the guanidine functionality. Instead, the focus
has been almost exclusively on amines.
In this work, a new
Boc-protected guanidinium oxanorbornene monomer was po-
lymerized via ring-opening metathesis polymerization (ROMP)
using the third generation Grubbs’ catalyst to afford synthetic
poly arginine, PGON (PDI ) 1.07, MW of the deprotected
polymer-TFA salt ) 2.5 kDa,). This strategy is in contrast to a
postpolymerization functionalization technique recently pub-
lished that afforded polynorbornenes that efﬁciently promote
Lastly, polyamine norbornenes, Poly-1
and Poly-3, with similar MWs were prepared for comparison.
Materials. Solvents and reagents for the monomer synthesis were
purchased from Aldrich and used without further puriﬁcation. For the
polymer synthesis the second generation Grubbs’ catalyst, tricyclo-
thenium dichloride was purchased from Aldrich. This compound was
converted to a Grubbs’ third generation catalyst by reaction with
3-bromopyridine from Aldrich. Polymerizations were performed in dry
from Acros (sealed under nitrogen with molecular sieves).
Chloroform solutions of lipids were obtained from Avanti Polar Lipids.
Calcein dye was purchased from Aldrich. The BacLight Staining Kit
L-7012 was obtained from Invitrogen Corp.
Synthesis of PGON. The synthesis of PGON starts with oxanor-
and to this salt (1.4 g, 4.34 mmol) N,N′-di-Boc-1H-pyrazole-
1-carboxamidine (4.0 g, 13.02 mmol) and triethylamine (4.1 g, 40
mmol) were added with 60 mL of solvent (90% CH
CN, 10% H
This solution was stirred at room temperature for 12 h. The solution
was then diluted with ethyl acetate and washed twice each with H
and brine. The organic layer was evaporated and puriﬁed by silica
column chromatography using a MeOH in CH
gradient to afford
the Boc-protected guanidinium oxanorbornene monomer, 2.
This monomer was polymerized by ring-opening metathesis polym-
erization (ROMP) using a derivative of the second generation Grubbs’
The polymerization for
* To whom correspondence should be addressed. E-mail: tew@
Department of Polymer Science and Engineering.
Department of Microbiology.
Biomacromolecules 2008, 9, 2980–29832980
10.1021/bm800855t CCC: $40.75 2008 American Chemical Society
Published on Web 10/14/2008
PGON entailed adding to a test tube monomer (100 mg) plus catalyst
(27.4 mg). The test tube was capped with a septum and purged with
for 5 min, then 1 mL dry CH
was injected. The N
removed and the clear, brown solution was stirred at 28 °C for 30 min
after which 0.4 mL of ethyl vinyl ether was injected to terminate the
polymer. After stirring for 15 min, the solution was added dropwise to
300 mL of stirring pentane to precipitate the polymer. The pentane
solution was stirred an additional 30 min and left standing undisturbed
for an hour. The precipitate was then collected by a ﬁne sinter funnel.
The polymer was then redissolved in 1 mL of CH
collected, and then dried by vacuum for 8 h.
The Boc-protected polymers were deprotected by stirring 100 mg
in 8 mL of 1:1 TFA/CH
for 2 h. The solution was concentrated to
an oil by rotary evaporator set at 40 °C and residual TFA was removed
by sonicating the oil in more CH
and evaporating the solvent again
by rotary evaporator. The resulting solid was placed under vacuum for
2 h. Finally, the solid was fully dissolved in 4 mL of H
O and ﬁltered
through a polyethersulphone (PES) syringe ﬁlter (Whatman, 25 mm
diameter, 0.45 µm pore) and freeze-dried for 48 h to give an eggshell
colored soft solid. Final deprotected polymers were stored at -20 °C.
Boc-Protected Guanidinium Oxanorbornene Monomer, 2.
NMR (300 MHz, DMSO-d
): δ 11.44 (1H, s), 8.35 (1H, t, J ) 6.0
Hz), 6.56 (2H, s), 5.10 (2H, s), 3.52 (2H, m), 3.43 (2H, m), 2.85 (2H,
s), 1.47 (9H, s), 1.36 (9H, s).
C NMR (75 MHz, DMSO-d
): δ 176.3,
156.6, 153.0, 136.5, 83.3, 80.9, 47.6, 39.0, 38.4, 28.3, 28.1. HR-MS
) calcd, 451.49; found, 451.22.
H NMR (300 MHz, DMSO-d
): δ 11.51
(1H, br), 8.47 (1H, br), 5.90 (trans) and 5.70 (cis; 2H total, br), 4.89
(cis) and 4.39 (trans; 2H total, br), 1.45 (9H, s), 1.38 (9H, br), cis/
trans ratio ) 46:54.
GPC of Boc-protected PGON: M
) 3.2 kDa, PDI ) 1.07 (After
deprotection the TFA-salt PGON has an approximate weight of 2.5
H NMR (300 MHz, DMSO-d
): δ 7.88 (1H, br), 7.36 (4H,
br), 5.96 (1H, br), 5.74 (1H, br), 4.92 (1H, br), 4.43 (1H, br) 3.47 (4H,
Microbial and Hemolysis Assays. Bacteria suspensions of Ec, Sm,
Sa, and Bs,(Escherichia coli D31, Serratia marcescens ATCC 43861,
Staphylococcus aureus ATCC 25923, and Bacillus subtilis ATCC 8037)
were grown in Mueller-Hinton Broth (MHB) overnight at 37 °C,
diluted with fresh MHB to an optical density of 0.001 at 600 nm
). This OD
gave an initial bacterial concentration of ∼10
cells/mL. This suspension was mixed with different concentrations of
freshly prepared polymer solutions dissolved in DMSO (40 mg/mL
stock solution) by serial dilutions in a 96-well plate, and incubated for
6hat37°C. The OD
was measured for bacteria suspensions that
were incubated in the presence of polymer solution or only MHB.
Antibacterial activity was expressed as minimal inhibitory concentration
(MIC), the concentration at which more than 90% inhibition of growth
was observed after 6 h. All experiments were run in quadruplicate.
Hemolytic activity measurements were performed on all polymers.
Freshly drawn human red blood cells (RBC, 30 µL), were suspended
in 10 mL of Tris saline (10 mM Tris, 150 mM NaCl, pH 7.2, ﬁltered
through PES membrane with 0.22 µm pore size) and rinsed three times
by centrifugation (5 min at 1500 rpm) and resuspended in Tris saline.
Polymer solutions were prepared in DMSO at 40 mg/mL and further
diluted as necessary. Freshly prepared polymer solutions with different
concentrations were added to 100 µL of the above-prepared RBC
suspension to reach a ﬁnal volume of 200 µL on a 96-well plate. The
resulting mixture was kept at 37 °C for 30 min on a stirring plate.
Then the plate was centrifuged (IEC Centra-4B, 10 min at 1500 rpm),
and the supernatant in each well was transferred to a new plate.
Hemolysis was monitored by measuring the absorbance of the released
hemoglobin at 414 nm. Hemolysis (100%) was obtained by adding 10
µL of Triton-X (polyoxyethylene isooctylphenyl ether from Sigma-
Aldrich) solution (20 vol % in DMSO), a strong surfactant, to the above-
prepared HRBC suspension. The upper limit of polymer concentration
that was required to cause 50% hemolysis is reported as HC
, and the
absorbance from Tris saline containing no polymer was used as 0%
hemolysis. All experiments were run in quadruplicate.
Bactericidal Kinetics Studies. Bactericidal kinetics studies (also
known as time-kill assays) were performed on Sa to differentiate growth
inhibition from cell death. The protocol was adapted from literature.
Cells were grown in MHB to midexponential phase with an OD
∼0.3, and then adjusted to an OD
of 0.001. This OD
initial bacterial concentration of ∼10
cells/mL, the same as in the MIC
studies. Cells were either untreated or treated to PGON at 1× MIC or
four times the MIC
concentration and then placed under rotary
agitation at 37 °C. Aliquots were removed at different time points (0,
15, 30, 60, 120, and 240 min) and placed back at 37 °C with agitation.
Cells were diluted immediately to remove any effect of PGON. Serial
10-fold dilutions were spread on MHB agar plates and incubated at 37
°C for 18 h to determine viable colony counts. Viable cell counts were
plotted on a log-scale graph (CFU/mL vs time) to determine log-
reduction of bacterial growth compared to the untreated control.
Dye-Leakage from Large Unilamellar Vesicles. The following is
a general procedure to make large unilamellar vesicles (LUV) having
a hydrodynamic radius, R
∼ 120 nm (by dynamic light scattering).
Chloroform solutions of POPE (3.23 mg, 0.0045 mmol) and POPG
(1.15 mg, 0.0015 mmol; Avanti Polar Lipids, Inc.) were mixed in a 10
mL round-bottom ﬂask, and the chloroform was removed at room
temperature by rotary evaporator to form a uniform ﬁlm. The ﬂask
was placed under vacuum for an additional 6 h. The dried ﬁlm was
hydrated with 1 mL of a 40 mM aqueous calcein (Sigma-Aldrich)
solution in Tris buffer without added NaCl. The calcein dye solution
was adjusted to pH ) 7 prior to adding it to the ﬁlm. The solution was
subjected through ﬁve freeze/thaw cycles using liquid nitrogen and
warm water. The entire volume of 1 mL of solution was subjected to
extrusion (a total of 15 passes) through two stacked 400 nm pore PC
membranes (Avanti Polar Lipids, Inc.) at room temperature. Finally,
the solution was pressurized through a small column packed with
Sephadex-25 (Sigma-Aldrich), eluting with Tris-saline buffer to remove
nontrapped calcein dye. Fractions (∼5 drops each) were collected. The
vesicle solution can be stored in a vial at 4 °C and diluted as needed
for up to 4 days.
Diluted calcein-loaded vesicle fractions that afforded ﬂuorescence
intensities <80 au without surfactant and >800 au after addition of
surfactant, were used (Ex. ) 490 nm used and Em. ) 515 nm
monitored). A total of 50 µL of 0.2% Triton-X was used as the strong
surfactant, which causes complete vesicle disruption and leakage.
Typically, chosen vesicle fractions were diluted 6-fold and 25 µLof
this stock solution is added to 2 mL of Tris-saline buffer. A ﬂuorescence
intensity of <80 au was veriﬁed to be stable over a minute before 25
µL of a polymer stock solution was added (2.5 µg/mL ﬁnal concentra-
tion in cuvette), agitated brieﬂy with the pipettor tip, then monitored
at 515 nm. After 5 min, Triton-X was added and the corresponding
ﬂuorescence was taken as 100% leakage.
Fluorescence Microscopy of Stained Bacteria Cells. An Olympus
BX51 Reﬂected Fluorescence Microscope (Optical Analysis Corp.
Nashua, NH) with a 100 W Mercury Lamp (Chin Technical Corp.)
was used for ﬂuorescence studies. A BacLight Kit L-7012 (Invitrogen
Corp.) was used as the ﬂuorescence dye in a mixture of 1:1 propidium
iodide:SYTO9 to examine Sa in the presence of PGON. It is important
to mention that an initial bacterial concentration of ∼10
used for microscopy studies (3 orders of magnitude higher than the
MIC and bactericidal kinetics studies) for ease of visualization. The
dye mixture was incubated with the bacteria at room temperature for
15 min before adding polymer solution (75 µg/mL ﬁnal concentration).
Solution of cells, dye, and polymer were allowed to sit for 30 min
before 50 µL was placed on a slide, ﬁtted with a coverslip, and
visualized. Bacteria were viewed under a green ﬁlter (excitation/
emission, 420-480 nm/520-800 nm) or a red ﬁlter (480-550 nm/
590-800 nm). To conﬁrm that these experiments agreed with the time-
kill experiments discussed above and to conﬁrm that Poly-1 is not lethal
Communications Biomacromolecules, Vol. 9, No. 11, 2008 2981
or static and that Poly-3 is lethal, cell counts were taken directly from
the microscopy experiments. They conﬁrmed that Poly-1 is not active
with no reduction in cell count compared to the control (at 30 min and
two hours), while Poly-3 and PGON showed a reduction of 8 logs in
less than two hours. At the 30 min time point, Poly-3 and PGON
showed log reductions of 8 and 4, respectively.
Results and Discussion
Results from microbiological assays
showed that PGON had
an MIC of 6 µg/mL against E. coli (see Table 1 for abbrevia-
tions). Activity against an additional Gram-negative bacteria,
S. marcescens, which is not typically susceptible to AMPs, and
two Gram-positive bacteria, S. aureus and B. subtilis, were also
examined attesting to the broad activity of PGON. For
comparison, the Magainin derivative, MSI-78, had an MIC of
12 µg/mL against E. coli. PGON’s close structural analogue,
Poly-1, having a primary amino group instead of a guanidino
group, had MICs of 200-400 µg/mL and was thus considered
to be relatively inactive. In contrast, the MICs of the more
hydrophobic, isobutylidene-derivatized Poly-3 were found to
be between 12-25 µg/mL for all bacteria, representing good
activity. However, and in sharp contrast to Poly-3, broadly active
PGON possessed a lack of toxicity toward RBCs (HC
µg/mL). As a result of PGON’s high HC
value, its selectivity
of 250 is signiﬁcantly better than other polymeric SMAMPs.
Again, as a comparison, MSI-78 had a selectivity of 10 for E.
coli over RBCs using the HC
Bactericidal kinetics studies (also known as time-kill assays)
were performed on S. aureus to differentiate growth inhibition
from cell death and showed that PGON was lethal at 4× the
MIC with a 5-log reduction in less than 60 min (Figure 1).
Starting with ∼10
cells/mL, aliquots were removed periodically
and viable cells counted via plating techniques. The results
clearly showed PGON’s ability to kill bacteria (bactericidal
activity) not just inhibit growth (bacteriostatic activity).
Many AMPs and their structural mimics have been shown
to be membrane-active with the balance of their hydrophobic/
hydrophilic properties critical to their mode of action.
Similarly, the biological properties of Poly-1 and Poly-3 were
previously explained based on their amphiphilicity, in agreement
with the concept that the proper balance of hydrophobic and
hydrophilic groups is often essential for the activity of AMPs.
Poly-1 was the most hydrophilic and consequently not membrane-
active, presumably because it can not sufﬁciently disrupt the
lipid bilayer. In contrast, Poly-3, being signiﬁcantly more
hydrophobic, exhibited good antibacterial activity but unfortu-
nately showed high hemolysis. With its outstanding biological
properties, it appeared that PGON had attained a favorable
High performance liquid chromatography (HPLC) retention
times are routinely exploited in the evaluation of a peptide’s,
or peptidomimetic’s relative amphiphilicity.
for Poly-1, Poly-3, and PGON, were 19.9, 25.5, and 22.4 min,
respectively, indicating that the guanidinium groups of PGON
resulted in a polymer with intermediate hydrophobic character.
Therefore, from the microbiology and HPLC data, it was
expected that PGON would exhibit strong membrane-lysis in
dye-leakage assays using vesicles composed of phosphatidyl
ethanolamine (PE) and phosphatidyl glycerol (PG) phospho-
lipids, which are typical of Gram-negative bacteria.
However, these assays showed that membrane-disruption
induced by Poly-1 and Poly-3 did reﬂect their MICs, while
PGON was completely inactive (Figure 2). In other words, Poly-
1, which displayed negligible antibacterial activity, caused little
dye leakage (16%), while Poly-3, with good antibacterial
activity, had signiﬁcant dye release (87%). On the other hand,
vesicles exposed to PGON were seemingly undisturbed (4%
leakage). This lack of membrane disruption activity for PGON
was unexpected given its high antibacterial efﬁciency and the
fact that its hydrophobicity fell in between the two amine
While dye leakage assays are simple models and quite useful
in many studies,
caution should be taken when correlating
these results to biological activity. Thus, the effects of these
polymers on S. aureus were directly observed using a two-
component ﬂuorescence stain (Figure 3). This stain uses a green-
emitting SYTO9 dye that aids visualization of all cells and red-
emitting propidium iodide, which only enters cells with
From these microscopy studies, Poly-1 appeared to be inactive
(also conﬁrmed by cell viability assays under the same
and showed green intact individual
S. aureus cells (Figure 3A). In contrast, Poly-3 led to gross
aggregation and clear membrane damage inferred from the
dramatic increase in red ﬂuorescence intensity (Figure 3D),
while under the green ﬁlter the cells now appeared more yellow
compared to the Poly-1 or PGON trials (compare Figure 3C to
3A and 3E). Cell viability assays conﬁrmed Poly-3 is lethal
under these conditions.
Interestingly, PGON, which is signiﬁ
cantly bactericidal, as demonstrated by cell viability assays, did
not aggregate bacteria nor allow propidium iodide to enter the
Table 1. Biological Activities
polymer (kDa) Ec Sm Sa Bs HC
Poly-1 (3.3) 400
400 400 200
Poly-3 (2.9) 25
25 25 12
PGON (2.5) 6 50 12 12 1500 250
12 >256 12 2 120 10
MIC ) minimum inhibitory concentration, HC
) hemolytic concen
tration, Ec ) Escherichia coli, Sm ) Serratia marcescens, Sa )
Staphylococcus aureus, and Bs ) Bacillus subtilis.
Selectivity for Ec over
RBCs shown (Sel. ) HC
Previously reported MIC values
measured for Ec,
used derivatives of antimicrobial
Previously reported values.
Figure 1. Time kill plots for S. aureus. CFU ) colony forming unit;
squares ) untreated cells; white circles ) treated with PGON at 1×
MIC; and black circles ) at treated with PGON 4× MIC.
Figure 2. Vesicle dye leakage after polymer addition.
2982 Biomacromolecules, Vol. 9, No. 11, 2008 Communications
cells. These results agreed completely with the dye leakage
experiments in which Poly-1 and PGON caused little to no
membrane damage relative to Poly-3. This apparent ability to
transverse the membrane without disruption is also consistent
with CPP-like activity.
At the same time, studies with a dye-
labeled version of PGON did support interactions between
PGON and bacterial cells under the same conditions but due
to the small size of bacteria (nominally 1 µm), it was not
possible to see if PGON was inside.
Accordingly, these results suggest that PGON’s ability to
effectively kill bacteria likely occurs via a different mechanism
than gross membrane trauma. Likely targets would then be
anionic macromolecules such as essential membrane proteins
or RNA/DNA. Although no data is yet available on these other
targets, CPPs and polyarginine, are well-known to efﬁciently
cross membranes and bind DNA.
Recently, we demon
strated the ability of these PGON’s, with various chain lengths,
to traverse membranes as well as other properties reminiscent
Therefore, the proposal of intercellular targets is
There is no doubt that PGON is potently antimicrobial
without gross membrane disruption. This leads to a lack of RBC
lysis and thus improved selectivity compared to AMPs and
previous primary amine containing SMAMPs. The recent
demonstration that PGON-like polymers are effective trans-
duction domains implies intercellular targets are responsible for
the antimicrobial properties and lack of mammalian toxicity.
Although no data is yet available on these targets, it is clear
that these PGONs represent an interesting and potentially
extremely important new class of molecules. At the same time,
highly selective antimicrobial polymers, such as PGON, can
be extremely valuable in materials designed to prevent Bioﬁlm
formation. Targets of opportunity include cardiovascular, or-
thopedic, and other implants that account for 1 million implant-
associated infections and 3 billion U.S. dollars of healthcare
Acknowledgment. The National Institutes of Health and the
Ofﬁce of Naval Research are acknowledged for their generous
Supporting Information Available. Synthetic schemes,
instrumentation, and dye-labeled polymer synthesis. This mate-
rial is available free of charge via the Internet at http://
References and Notes
(1) (a) Madkour, A. E.; Tew, G. N. Polym. Int. 2008, 57, 6–10. (b) Gabriel,
G. J.; Som, A.; Madkour, A. E.; Eren, T.; Tew, G. N. Mater. Sci.
Eng., R 2007, 57, 28–64. (c) Klibanov, A. M. J. Mater. Chem. 2007,
17, 2479–2482. (d) Hetrick, E. M.; Schoenﬁsch, M. H. Chem. Soc.
ReV. 2006, 35, 780–789.
(2) Ilker, M. F.; Nu¨sslein, K.; Tew, G. N.; Coughlin, E. B. J. Am. Chem.
Soc. 2004, 126, 15870–15875.
(3) (a) Miyatake, T.; Nishihara, M.; Matile, S. J. Am. Chem. Soc. 2006,
128, 12420–12421. (b) Henriques, S. T.; Melo, M. N.; Castanho,
M. A. R. B. Biochem. J. 2006, 399, 1–7. (c) Rothbard, J. B.; Kreider,
E.; VanDeusen, C. L.; Wright, L.; Wylie, B. L.; Wender, P. A. J. Med.
Chem. 2002, 45, 3612–3618. (d) Futaki, S.; Suzuki, T.; Ohashi, W.;
Yagami, T.; Tanaka, S.; Ueda, K.; Sugiura, Y. J. Biol. Chem. 2001,
276, 5836–5840. (e) Mitchell, D. J.; Kim, D. T.; Steinman, L.;
Fathman, C. G.; Rothbard, J. B. J. Pept. Res. 2000, 56, 318–325.
(4) For polymeric examples, see: (a) Sambhy, V.; Peterson, B. R.; Sen,
A. Angew. Chem., Int. Ed. 2008, 47, 1250–1254. (b) 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. (c) Kuroda, K.; DeGrado, W. F. J. Am. Chem. Soc. 2005, 127,
4128–4129. (d) Arnt, L.; Nu¨sslein, K.; Tew, G. N. J. Polym. Sci.,
Part A: Polym. Chem. 2004, 42, 3860–3864. (e) 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, 5110–5114.
(5) For peptidomimetic examples, see (a) Gabriel, G. J.; Tew, G. N. Org.
Biomol. Chem. 2008, 6, 417–423. (b) Schmitt, M. A.; Weisblum, B.;
Gellman, S. H. J. Am. Chem. Soc. 2007, 129, 417–428. (c) Tew, G. N.;
Clements, D.; Tang, H.; Arnt, L.; Scott, R. W. Biochim. Biophys. Acta
2006, 1758, 1387–1392. (d) Patch, J. A.; Barron, A. E. J. Am. Chem.
Soc. 2003, 125, 12092–12093. (e) Porter, E. A.; Wang, X.; Lee, H.-
S.; Weisblum, B.; Gellman, S. H. Nature 2000, 404, 565–565.
(6) Radzishevsky, I. S.; Rotem, S.; Bourdetsky, D.; Navon-Venezia, S.;
Carmeli, Y.; Mor, A. Nat. Biotechnol. 2007, 25, 657–659.
(7) Kolonko, E. M.; Kiessling, L. L. J. Am. Chem. Soc. 2008, 130, 5626–
(8) See Supporting Information for details.
(9) Ge, Y.; MacDonald, D. L.; Holroyd, K. J.; Thornsberry, C.; Wexler,
H.; Zasloff, M. Antimicrob. Agents Chemother. 1999, 43, 782–788.
(10) Brogden, K. A. Nat. ReV. Microbiol. 2005, 3, 238–250.
(11) Som, A.; Tew, G. N. J. Phys. Chem. B 2008, 112, 3495–3502.
(12) Sakai, N.; Futaki, S.; Matile, S. Soft Matter 2006, 2, 636–641.
(13) (a) Pantos, A.; Tsogas, I.; Paleos, C. M. Biochim. Biophys. Acta 2008,
1778, 811–823. (b) Schroeder, T.; Niemeier, N.; Afonin, S.; Ulrich,
A. S.; Krug, H. F.; Braese, S. J. Med. Chem. 2008, 51, 376–379. (c)
Deglane, G.; Abes, S.; Michel, T.; Prevot, P.; Vives, E.; Debart, F.;
Barvik, I.; Lebleu, B.; Vasseur, J.-J. ChemBioChem 2006, 7, 684–
692. (d) Fillon, Y. A.; erson, J. P.; Chmielewski, J. J. Am. Chem.
Soc. 2005, 127, 11798–11803. (e) Funhoff, A. M.; Van Nostrum, C. F.;
Lok, M. C.; Fretz, M. M.; Crommelin, D. J. A.; Hennink, W. E.
Bioconjugate Chem. 2004, 15, 1212–1220.
(14) Hennig, A.; Gabriel, G. J.; Tew, G. N.; Matile, S. J. Am. Chem. Soc.
2008, 130, 10338-10344.
Figure 3. Fluorescence microscopy of S. aureus (cells ∼ 1 µmin
diameter) incubated with a stain to visualize membrane-disruption
and polymer for 30 min. Top row shows emission from S. aureus
using a green ﬁlter setting, while the bottom row shows the identical
ﬁeld using a red ﬁlter.
Communications Biomacromolecules, Vol. 9, No. 11, 2008 2983