Tuning the biological activity profile of antibacterial polymers via subunit substitution pattern

Article abstract of DOI:10.1021/ja500367u

Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits i

Full text of DOI:10.1021/ja500367u

Article
pubs.acs.org/JACS
Open Access on 03/07/2015
Tuning the Biological Activity Profile of Antibacterial Polymers via
Subunit Substitution Pattern
Runhui Liu,†,‡ Xinyu Chen,† Saswata Chakraborty,† Justin J. Lemke,# Zvi Hayouka,† Clara Chow,†
Rodney A. Welch,# Bernard Weisblum,§ Kristyn S. Masters,*,‡ and Samuel H. Gellman*,†
‡
#
†Department of Chemistry, Department of Biomedical Engineering, Department of Medical Microbiology and Immunology, and
§Department of Medicine, University of Wisconsin, Madison, Wisconsin 53706, United States
S
* Supporting Information
ABSTRACT: Binary nylon-3 copolymers containing cationic
and hydrophobic subunits can mimic the biological properties
of host-defense peptides, but relationships between composi-
tion and activity are not yet well understood for these materials.
Hydrophobic subunits in previously studied examples have
been limited mostly to cycloalkane-derived structures, with
cyclohexyl proving to be particularly promising. The present
study evaluates alternative hydrophobic subunits that are
isomeric or nearly isomeric with the cyclohexyl example; each
has four sp3 carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to
relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these
differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the
hydrophobic subunits. The α,α,β,β-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with
potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the
new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be
generally valuable in the development of synthetic polymers for biological applications.
do-polysaccharide,40 polycarbonate,41,42 and poly(vinyl
INTRODUCTION
■
ether).43 Most studies have focused on identifying a hydro-
Eukaryotes deploy a broad range of “host-defense peptides”
phobic-cationic balance that supports potent antibacterial
activity while limiting toxicity toward mammalian cells, which
(HDPs) to discourage infection by prokaryotes.1−3 Many of
these peptides appear to act by compromising the barrier
function of bacterial membranes, although the precise
mechanism of disruption remains uncertain, as does the relative
is typically assessed in terms of red blood cell lysis (“hemolytic
activity”).44 Hydrophobic-cationic balance is generally tuned by
varying side chain hydrophobicity (carbon atom number)
importance of membrane-directed vs alternative modes of
action.4,5 HDPs are typically rich in both hydrophobic residues
and/or altering the hydrophobic:cationic side chain proportion.
and cationic residues,6−8 and their positive charge is thought to
The research described here explores how the biological
activity profiles of cationic-hydrophobic copolymers are
underlie their preference for attacking bacterial membranes
relative to eukaryotic membranes. Numerous prokaryote-
selective synthetic oligomers, containing α-amino acid residues
and/or other types of subunits, have been designed based on
influenced by changes in the arrangement of side chain carbon
atoms within hydrophobic subunits, rather than by changes in
the number of side chain carbon atoms, which is related to
hydrophobicity. This aspect of molecular design has received
principles that are believed to underlie the function of natural
HDPs.9−26
little attention in studies of antibacterial polymers, in part
because most polymer systems explored to date would not
Designed peptides (i.e., oligomers of α-amino acids) and
easily support such changes. It is well-known that subtle
other discrete oligomers are interesting as tools to establish the
variations in the structure of α-amino acid residues exert a
features essential for a HDP-like biological activity profile, but
significant impact on the folding and function of peptides and
synthetic difficulties may hamper practical applications of this
proteins. For example, the isomers leucine and isoleucine are
type of molecule. Rigorous control of subunit sequence
comparable in terms of hydrophobicity, but they have divergent
typically requires solid-phase synthesis, which is labor-intensive
and expensive.3 This consideration has prompted recent
explorations of sequence-random copolymers as functional
conformational preferences, favoring α-helical and β-sheet
secondary structure, respectively. Glycine is more flexible
than all other residues because of the lack of a side chain.45−47
mimics of HDPs. Diverse backbones have been evaluated,
including polystyrene,27 poly(norbornene),28 polymethacry-
late,29−31 poly-β-peptide (nylon-3),32−35 polyacrylamide,36
polyolefin,37 polyvinylpyridinium-polymethyacrylate,38,39 pepti-
Received: January 16, 2014
Published: March 7, 2014
© 2014 American Chemical Society
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Many nonribosomal peptide antibiotics contain aminoisobuty-
ric acid (Aib) residues, the gem-dimethyl substitution pattern of
which causes a distinctive helix-favoring propensity.48−50
We propose that the broad range of potencies and
selectivities manifested among natural antibacterial peptides
reflects evolutionary optimization of both hydrophobic-cationic
balance and backbone conformational propensity. Based on this
hypothesis, we predict that variation of both properties could
lead to antibacterial polymers with improved properties. In
many systems, the position at which side chains are traditionally
modified is too far from the backbone to affect conformational
behavior.27−31,36,38−40 Nylon-3 polymers, on the other hand,
offer considerable latitude for modulation of conformational
propensity, because each subunit contains a pair of adjacent sp3
carbon atoms in the backbone, and the substitution pattern at
each of these positions can be varied independently. Here we
show that evaluation of a small set of related subunits, each
containing four side chain sp3 carbons, leads to new nylon-3
copolymers with diverse biological activity profiles; one of the
new polymers appears to match the profile that is characteristic
of the very best peptides and polymers previously reported.
These findings suggest that the ability to alter the subunit
substitution pattern may be an important criterion in selecting
polymer systems to be developed for specific biological
applications.
Figure 1. (a) β-Lactams used in this study, (b) representative
copolymer synthesis, (c) nylon-3 copolymers prepared from equimolar
binary β-lactam mixtures and containing 50% DM and 50% of a
hydrophobic subunit, and (d) PHMB. The DM and CH subunits are
racemic. All polymers are heterochiral.
RESULTS AND DISCUSSION
■
Experimental Design. Our previous exploration of
antimicrobial nylon-3 copolymers focused on hydrophobic
subunits with cis-cycloalkyl frameworks; hydrophobicity was
varied by changes in ring size.32,33 The cyclohexyl-based
subunit derived from β-lactam CHβ proved to be optimal in
copolymers prepared with β-lactam MMβ or DMβ, which
provide cationic subunits after side chain deprotection (Figure
1). Some copolymers derived from CHβ manifested low
hemolytic activity and moderate antibacterial activity, behavior
reminiscent of HDPs.32 The 1:1 DM:CH copolymer displayed
the strongest antibacterial activities we observed,33 with
potencies comparable to the best polymers and HDPs reported
by others. However, 1:1 DM:CH is hemolytic at low
concentrations and therefore inadequate in terms of
selectivity.33 The present study takes 1:1 DM:CH as the
starting point for examining the impact of variations in
hydrophobic subunit substitution pattern on biological activity.
New nylon-3 materials were prepared from 1:1 β-lactam
mixtures containing DMβ51 as the precursor of the cationic
subunit and one of three hydrophobic β-lactams: (1) βCPβ
(“β-cyclopentyl”), (2) βDEβ (“β-diethyl”), or (3) TMβ
(“tetramethyl”) (Figure 1). The nylon-3 subunits CH, βCP,
βDE, and TM should be comparable in terms of hydro-
phobicity because each contains four side chain carbon atoms;
however, conformational propensity is expected to vary among
these four subunits. The backbone Cα−Cβ bond of CH is
constrained by the six-membered ring. Subunits βDE and βCP
should be relatively flexible because each contains a CH2 unit in
the backbone; this expectation arises because glycine is the
most flexible α-amino acid.45−47 TM, reminiscent of Aib
because of the quaternary backbone substitution pattern at Cα
and Cβ, should have a distinctive conformational propensity.
Recently, we showed that removing the six-membered ring
constraint, by preparing copolymers from DMβ + HEβ (Figure
1) rather than DMβ + CHβ, led to a substantial (and
unfavorable) increase in hemolytic activity along with a small
diminution in antibacterial activity.52 Based on this observation,
one might have predicted that all of the new copolymers would
have less desirable activity profiles relative to 1:1 DM:CH,
because none of the new hydrophobic subunits has a cyclic
constraint on the backbone Cα−Cβ bond. This prediction
turned out to be correct for copolymers containing βDE and
βCP. We were surprised, however, to discover that polymers
containing the TM subunit display superior properties, as
explained below.
Synthesis and Evaluation of New Copolymers. All
polymers were prepared via base-catalyzed copolymerization
reactions with p-t-butylbenzoyl chloride as co-initiator.51,53,54
Thus, all polymer chains bear a p-t-butylbenzoyl group at the
N-terminus (Figure 1c). Use of 5 mol % co-initiator relative to
total β-lactam should generate 20-mer average chain lengths.
The resulting materials had polydispersity indices (PDI)
ranging from 1.05 to 1.33.
Functional comparison of 1:1 DM:CH with the three new
copolymers is summarized in Table 1. Consistent with previous
data,33 1:1 DM:CH is highly active, displaying low minimum
inhibitory concentration (MIC) values against a test panel of
four bacteria, including laboratory strains of Escherichia coli and
Bacillus subtilis and clinical strains of vancomycin-resistant
Enterococcus faecium (VREF) and methicillin-resistant Staph-
ylococcus aureus (MRSA). However, 1:1 DM:CH is quite
destructive toward red blood cells, as indicated by the relatively
low concentration at which 10% hemolysis is observed
(HC10).33 Among the three new nylon-3 copolymers, 1:1
DM:βCP and 1:1 DM:TM are similar to 1:1 DM:CH in their
antibacterial activities, while 1:1 DM:βDE is moderately less
active. Larger differences, however, are observed in the
hemolytic activities. Both copolymers with hydrophobic
subunits containing a backbone CH2 group, 1:1 DM:βCP
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MBC value that is far higher than the MIC. On the other hand,
MBC is only slightly higher than MIC for the other three
bacteria (≤4-fold), including MRSA, which indicates that the
DM:CH and DM:TM copolymers are both potent bactericidal
agents for these species.
Table 1. Antibacterial and Hemolytic Activity of Nylon-3
Copolymers
a
MIC, μg/mL
b
B.
HC10,
SI (HC /
10 c
polymer
subtilis E. coli VREF MRSA μg/mL MICMRSA)
Subunit Distribution within Polymer Chains. Variations
in polymer precursor structure can lead to differences in
reactivity. For chain-growth copolymerizations, these differ-
ences cause deviations from purely random subunit distribution
along the backbone. This factor has typically not been
considered in previous comparisons among antibacterial
copolymers with variable subunits (e.g., different subunit
hydrophobicities).29,32,33,36−39,43,56 For nylon-3 copolymeriza-
tions, differences in β-lactamate and/or chain-end reactivity can
cause preferential incorporation of one subunit in the early
stages of the reaction, which results in compositional drift along
the chains.57,58 Evaluation of the DMβ + CHβ, DMβ + βDEβ,
and DMβ + TMβ copolymerizations revealed significant
differences in terms of β-lactam incorporation preference,
implying differences in subunit distribution within the resulting
polymers (Figure 2). For 1:1 DM:CH, there is a small
1:1 DM:CH
1:1 DM:βCP
1:1 DM:βDE
1:1 DM:TM
PHMB
≤1.6
3.1
6.3
6.3
25
6.3
6.3
25
6.3
6.3
25
19
<3.1
<3.1
400
13
3
<0.5
<0.1
63
6.3
≤1.6
3.1
13
3.1
3.1
6.3
6.3
3.1
6.3
3.1
13
4
1:1 DM:CH
(“skewed”)
≤1.6
4.7
0.7
daptomycin
≤1.6
>200
6.3
6.3
>400
>63
a
MIC, which is the lowest polymer concentration that completely
inhibits bacterial growth. Polymer concentration necessary for 10%
lysis of RBC. Selectivity index (SI) was calculated based on MIC
values for MRSA. VREF is vancomycin-resistant E. faecium; MRSA is
methicillin-resistant S. aureus; and PHMB is polyhexamethylene
biguanide.
b
c
and 1:1 DM:βDE, are strongly hemolytic. In contrast, 1:1
DM:TM is only very weakly hemolytic at high concentrations.
Overall, the prokaryote-selective biological activity profile of 1:1
DM:TM is the most favorable among the four copolymers
compared here and among all nylon-3 polymers we have
examined.32,33
These nylon-3 polymers were compared with the commercial
antimicrobial polymer polyhexamethylene biguanide (PHMB)
and with the clinical antibiotic daptomycin; Figure 1d. As
expected, PHMB displays potent antibacterial activity against
the four bacteria in our panel (Table 1). However, PHMB is
highly hemolytic. Thus, the biological activity profiles of 1:1
DM:CH and PHMB are similar. Daptomycin is a lipopeptide
antibiotic that targets bacterial membranes. Therefore, this
agent is appropriate for comparison with our nylon-3 polymers,
which are believed to act on bacterial membranes. Daptomycin
displays potent activity against three Gram positive bacteria but
no activity toward E. coli. The nylon-3 copolymer 1:1 DM:TM
compares favorably to both PHMB and daptomycin in terms of
antibacterial activity and prokaryote vs eukaryote selectivity.
The antibacterial activities of the 1:1 DM:CH and 1:1
DM:TM copolymers were further compared via measurement
of minimum bactericidal concentrations (MBC) (Table 2).
Figure 2. (a) Consumption of β-lactams as a function of reaction
progress for the copolymerization of 1:1 DM:CH (×), 1:1 DM:TM
○
▲
( ), or 1:1 DM:βDE (red ). Reactions were conducted at rm temp
with an initial concentration of 50 mM for each β-lactam and 5 mM
for the co-initiator tBuBzCl (5 mol % relative to the total amount of β-
lactam), to prepare copolymers with an average 20-mer length.
Measurement of subunit incorporation is described in the Supporting
Information. (b) Cartoons of copolymers showing differences in
compositional drift along the polymer chains.
preference for incorporation of CHβ in the early stages of the
reaction, which means that the N-terminal regions of the
polymer chains are slightly enriched in CH units, and the C-
terminal regions are slightly enriched in DM units. In contrast,
for both 1:1 DM:TM and 1:1 DM:βDE there is a strong initial
preference for DM incorporation; thus, the N-terminal regions
of these polymers are highly enriched in cationic DM units,
while the C-terminal regions are highly enriched in hydro-
phobic TM or βDE units. Copolymerization of DMβ + βCPβ
was so rapid that we could not monitor β-lactam consumption
as a function of reaction progress.
The data in Figure 2 indicate that nylon-3 materials
generated via DMβ + TMβ or DMβ + βDEβ copolymerizations
have very similar subunit distribution biases along the polymer
chains. Therefore, the substantial differences in biological
activity profile between these two nylon-3 copolymers, with 1:1
DM:TM displaying higher antibacterial potency and lower
eukaryotic cell toxicity relative to 1:1 DM:βDE, can be
attributed to the different substitution patterns of the isomeric
TM and βDE subunits. This result suggests that polymers
containing a hydrophobic subunit that is expected to be more
flexible (βDE) are more strongly hemolytic than polymers
Table 2. Bactericidal Activity of Nylon-3 Copolymers
1:1 DM:CH
1:1 DM:TM
a
b
a
b
bacterium
MBC
MBC/MIC
MBC
MBC/MIC
B. subtilis
E. coli
<3.1
13
2
2
<3.1
13
2
1
VREF
>200
25
>32
4
200
64
2
MRSA
13
a
b
Minimum bactericidal concentration, μg/mL. Any polymer with
MBC/MIC ratio ≤4 is considered to be bactericidal for that species.55
MIC indicates the lowest polymer concentration at which
bacterial growth is inhibited in liquid culture, while MBC
indicates the lowest concentration at which all bacterial cells
have been killed, as demonstrated by a lack of colony formation
on solid medium after the polymer-treated liquid culture is
applied to an agar surface. The results for VREF show that
inhibition of bacterial growth need not correlate with bacterial
killing, because for this organism both copolymers display an
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Table 3. Antibacterial and Hemolytic Activities of Diblock Copolymers
a
MIC, μg/mL
b
c
block copolymer
B. subtilis
E. coli
VREF
MRSA
HC10, μg/mL
SI (HC10/MICMRSA)
(DM)10(CH)10
(DM)10(βCP)10
(DM)10(βDE)10
(DM)10(TM)10
13
13
>200
>200
>200
>200
>200
>200
>200
200
>200
>200
>200
50
300
13
<1.5
<0.07
<0.2
6
>200
6.3
38
300
a
b
c
The lowest polymer concentration that completely inhibits bacterial growth. Polymer concentration required for 10% lysis of RBC. Selectivity
index (SI) was calculated based on MIC for MRSA. VREF is vancomycin-resistant E. faecium; MRSA is methicillin-resistant S. aureus.
Figure 3. Binary hydrophobic-cationic nylon-3 copolymers containing TM, βDE, or βCP subunits and DM subunits. The DM precursor was
racemic, so all copolymers are heterochiral. Polymers within each series have variable subunit proportion; x + y = 100, with x = 40−100.
containing isomeric but more conformationally constrained
hydrophobic subunits (TM).
CH, TM, βDE, or βCP block at the C-terminal side.59 PDI
values for these block copolymers were in the range 1.07−1.27.
The antibacterial and hemolytic properties of these diblock
nylon-3 copolymers are summarized in Table 3. All four diblock
copolymers are generally ineffective at inhibiting bacterial
growth, other than for B. subtilis. There are significant
differences among the diblock nylon-3 copolymers in terms
of hemolytic activity. DM-CH and DM-TM diblock copoly-
mers are only weakly hemolytic, but DM-βDE and DM-βCP
are strongly hemolytic. Although these latter two block
copolymers are somewhat less effective at inducing hemolysis
than the corresponding copolymers generated from 1:1 β-
lactam mixtures (Table 3 vs Table 1), the trend among the four
block copolymers mirrors that among the mixed copolymers.
Specifically, the presence of hydrophobic subunits that are
expected to be relatively flexible, because they contain a
backbone CH2 unit (βDE and βCP), correlates with high
hemolytic activity.
Recent studies of hydrophobic-cationic copolymers in the
polymethacrylate and poly(vinyl ether) families indicated that
block architecture leads to diminished hemolytic activity
relative to random subunit distribution,30,43 and our results
are consistent with this trend. However, diblock and “random”
subunit distributions were comparable in terms of antibacterial
activity in these two previous studies,30,43 which contrasts
sharply with our observations for four different nylon-3
copolymer compositions, all of which display only weak
antibacterial activity in the diblock architecture. Thus, our
results suggest that there is no general relationship between
relative antibacterial potencies and diblock vs uncontrolled
subunit distribution within binary hydrophobic-cationic co-
polymers.
The data in Figure 2 indicate that one must be cautious in
drawing conclusions regarding functional differences between
the previously studied copolymer 1:1 DM:CH and the
copolymer that displays the most favorable biological activity
profile, 1:1 DM:TM. This pair differs not only in hydrophobic
subunit identity but also in subunit distribution. We therefore
undertook a modified approach to DMβ + CHβ copolymeriza-
tion in order to generate a “skewed” version of 1:1 DM:CH
with a subunit distribution comparable to that of 1:1
DM:TM.59 The mechanism of anionic β-lactam ring-opening
polymerization features a reactive chain-end (C-terminal imide)
and thus constitutes a “living” process.51,53,54 The skewed
version of 1:1 DM:CH was prepared by introduction of the β-
lactam precursors in four aliquots, each containing 25% of the
total β-lactam but with differing β-lactam proportions. The β-
lactam proportions in each aliquot were chosen to match the β-
lactam proportions incorporated into growing 1:1 DM:TM
chains at 25%, 50%, 75%, and 100% total conversion, according
to the data in Figure 2. The resulting “skewed” 1:1 DM:CH
copolymer displays antibacterial activities very similar to those
of 1:1 DM:TM, and this skewed polymer is much more highly
hemolytic than is 1:1 DM:TM (Table 1). Since the biological
activity profile of the skewed 1:1 DM:CH copolymer is very
similar to that of the original 1:1 DM:CH copolymer (prepared
by introducing all of each β-lactam to the reaction vessel at the
start of the polymerization), and since both DM:CH samples
are much more hemolytic than 1:1 DM:TM, we conclude that
the superior prokaryote vs eukaryote selectivity of the DM:TM
copolymer arises from the intrinsic properties of the hydro-
phobic TM subunit, relative to CH, rather than from variations
in subunit distribution along the polymer chains.
The similarity in antibacterial profiles among the two forms
of 1:1 DM:CH that contain different extents of compositional
bias (normal copolymer vs “skewed” in Table 1) and the
dramatic decline in antibacterial activities for the DM-CH
diblock copolymer relative to mixed copolymers indicate that
some degree of subunit intermixing is necessary to maximize
inhibition of bacterial growth. This observation may be related
to an elegant recent report on materials generated via ring-
opening polymerization of cycloalkenes, which indicated that
The subunit distribution bias within the 1:1 DM:TM
copolymer (Figure 2) led us to wonder whether complete
segregation of the cationic subunits from the hydrophobic
subunits might lead to low hemolytic activity for all pairings of
the cationic DM subunit with hydrophobic subunits. To
address this question, we prepared DM-CH, DM-βCP, DM-
βDE, and DM-TM diblock copolymers using conditions that
should provide 20-mer average chain lengths, with a 10-mer
average DM block at the N-terminal side and a 10-mer average
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Figure 4. Summary of biological activity profiles (antibacterial activities, 3T3 fibroblast toxicity, and hemolytic activities) as a function of
cationic:hydrophobic subunit proportion for the three sets of binary nylon-3 copolymers shown in Figure 3. The lines drawn for 3T3 fibroblast
toxicity and hemolysis merely connect data points. MIC is the minimum inhibitory concentration for bacterial growth; IC10 is the polymer
concentration required to induce 10% 3T3 fibroblast death; and HC10 is the polymer concentration required to cause 10% lysis of human red blood
cells. When the IC10 or HC10 value is >400 μg/mL, the plot shows a concentration at 400 μg/mL.
Figure 5. Antibacterial resistance tests for 1:1 DM:TM with E. coli and MRSA. MIC is the minimum inhibitory concentration for bacterial growth;
MBC is the minimum bactericidal concentration for 99.9% killing of the bacteria.
≥8−10 Å spacing between cationic groups along this backbone
is optimal in terms of antibacterial activity.37
from an agar plate that had been used for the measurement of
MBC in the previous passage. This colony was taken from the
plate for the polymer concentration one dilution below the
MIC measured for the previous passage, to ensure that the
bacteria could grow in the presence of a subinhibitory
concentration of 1:1 DM:TM.61 For both E. coli and MRSA,
no sign of resistance to 1:1 DM:TM was detected after 10
continuous passages (Figure 5). The variations observed in
Figure 5 correspond to a single 2-fold dilution and represent
the experimental uncertainty in these measurements. These
results suggest that it is difficult for bacteria to develop
resistance to an antibacterial nylon-3 polymer, which
strengthens the functional analogy between this polymer class
and HDPs.
Variations in the Proportion of Cationic and Hydro-
phobic Subunits. To gain a more complete understanding of
composition-activity relationships among copolymers prepared
from the hydrophobic β-lactams TMβ, βDEβ, and βCPβ, we
prepared a series of new binary copolymers via co-reaction of
each of these three β-lactams with DMβ (Figure 1). Reaction
conditions were selected to favor 20-mer average length. Within
each copolymer subset, the cationic:hydrophobic subunit
proportion was varied (Figure 3). Each polymer was analyzed
for antibacterial activity against four species (MIC), hemolysis
(HC10), and 3T3 fibroblast toxicity (IC10, the polymer
concentration that causes 10% fibroblast toxicity).
Further Antibacterial Studies with 1:1 DM:TM. In
addition to the four bacteria used in our standard antimicrobial
assessment of nylon-3 polymers (Table 1), we evaluated the
activity of the best copolymer, 1:1 DM:TM, against other
bacterial pathogens (Figure 6). This polymer displayed only
weak activity against Salmonella enterica LT2 (MIC = 200 μg/
mL), but the polymer was quite active against Bacillus cereus
ATCC14579 (MIC = 25 μg/mL), the uropathogenic E. coli
CFT073 (MIC = 50 μg/mL), and Pseudomonas aeruginosa
PA1066, a strain isolated from a cystic fibrosis patient (MIC =
12.5 μg/mL).
Figure 4 summarizes the biological activity profiles of the
new copolymer series. For both DM:βCP and DM:βDE, high
hemolytic activity and significant 3T3 fibroblast toxicity were
observed at all compositions, which indicates that nylon-3
copolymers containing the βCP or βDE subunit are generally
not selective for prokaryotic vs eukaryotic cells. In contrast,
excellent selectivity can be achieved in the DM:TM as long as
the subunit proportion is properly controlled.
Propensity of Bacteria to Develop Resistance to
Nylon-3 Copolymers. It is difficult for bacteria to develop
resistance to HDPs,60 and we wondered whether the same
would be true of nylon-3 copolymers. To evaluate this
possibility, we challenged E. coli and MRSA with the 1:1
DM:TM copolymer for 10 continuous passages. For each
passage, we determined MIC and MBC values for the polymer
using a liquid subculture derived from a single colony picked
CONCLUSIONS
■
The results reported here show that changes in backbone
substitution pattern within the hydrophobic subunit can exert a
profound impact on the biological activity profiles of binary
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solution of methylenecyclopentane (3.9 g, 47.5 mmol) in CH2Cl2 (24
mL) at rt was treated with chlorosulfonyl isocyanate (4.3 mL, 49.9
mmol), and the mixture was stirred at 60 °C for 10 h. The reaction
mixture was poured into an ice-cold buffer solution containing sodium
sulfite (12 g, 95.1 mmol) and dibasic sodium phosphate (13.5 g, 95.1
mmol). The mixture was stirred at rt overnight and then extracted with
CH2Cl2 (3 × 150 mL). The combined organic layers were washed
with brine (50 mL), dried over MgSO4, and concentrated. The crude
product was purified by silica gel chromatography (1:1 hexane:EtOAc)
1
to give β-lactam βCPβ as a light-yellow viscous oil (1.7 g, 29%). H
NMR (300 MHz, CDCl3): δ 6.23 (bs, 1H), 2.95 (dd, J = 8.1, 1.5 Hz,
1H), 1.91 (dd, J = 15, 4.5 Hz, 1H), 1.71−1.81 (m, 3H), 1.48−1.55 (m,
1H), 1.46 (bs, 3H), 1.24−1.36 (m, 1H); 13C NMR (75 MHz, CDCl3):
δ 170.24, 62.86, 60.35, 35.87, 25.85, 24.20, 23.05; EI-HRMS: m/z
calcd for C7H11NO [M]+: 125.0836; found: 125.0832.
Figure 6. Antibacterial activity of copolymer 1:1 DM:TM against four
pathogenic bacteria.
cationic-hydrophobic nylon-3 copolymers. Evolution optimizes
polypeptide properties via selection among α-amino acid
residues that vary in both hydrophobicity and conformational
propensity, and our findings suggest that exploring comparable
variations among synthetic copolymers is useful for tuning
functional properties. Implementation of this approach,
however, requires a polymer for which alternative backbone
substitution patterns can be readily accessed. Nylon-3 polymers
are very convenient in this regard, but many common polymer
families are not. In addition to their antibacterial properties,
nylon-3 polymers have displayed promising behavior in several
other areas of biological application,34,35,62−65 and the approach
described here may prove useful in the context of those
applications.
4,4-Diethylazetidine-2-one. The product was synthesized by a
modification of reported methods.67 A solution of 2-ethyl-1-butene
(10 g, 118.8 mmol) in Et2O (58 mL) at rt was treated with
chlorosulfonyl isocyanate (10.4 mL, 118.2 mmol), and the mixture was
stirred at rt for 2 h. The reaction mixture was poured into an ice-cold
buffer solution containing sodium sulfite (22.5 g, 178.2 mmol) and
dibasic sodium phosphate (25.3 g, 178.2 mmol). The mixture was
stirred at rt overnight and then extracted with CH2Cl2 (3 × 500 mL).
The combined organic layers were washed with brine (100 mL), dried
over MgSO4, and concentrated to give β-lactam βDEβ as a colorless
oil, which was used without purification (11.8 g, 78%). 1H NMR (300
MHz, CDCl3): δ 6.90 (bs, 1H), 2.51 (d, J = 1.8 Hz, 2H), 1.60 (q, J =
7.5 Hz, 4H), 0.82 (t, J = 7.5 Hz, 6H); 13C NMR (75 MHz, CDCl3): δ
168.49, 57.73, 45.94, 29.46, 8.57; EI-HRMS: m/z calcd for C7H14NO
[M + H]+: 128.1070; found: 128.1068.
EXPERIMENTAL METHODS
■
NIH 3T3 fibroblast cells were obtained from the American Type
Tissue Collection (ATCC, Manassas, VA). Dulbecco’s modified eagle
medium (DMEM) and cell culture supplies were obtained from
Invitrogen (Carlsbad, CA). CytoTox-ONE assay kits (G7892) were
obtained from Promega (Madison, WI). LB medium (244610) was
obtained from BD (Franklin Lakes, NJ). Agar (BP1423500) was
obtained from Fisher Scientific (Pittsburgh, PA). PHMB (84428-
SMP) was obtained from Lonza (Allendale, NJ) as a 20% aqueous
solution (“Vantocil IB”). The solution was lyophilized to give PHMB
as a white powder, which was used to generate solutions for biological
activity studies. All other chemicals were purchased from Sigma-
3,3,4,4-Tetramethylazetidine-2-one. The product was synthesized
by a modification of reported methods.68 A solution of 2,3-
dimethylbutene (8.5 g, 101 mmol) in CH2Cl2 (6.5 mL) was cooled
in an ice-water bath and treated with chlorosulfonyl isocyanate (8.8
mL, 101 mmol). The mixture was removed from the ice-water bath
and heated at 65 °C for 1 day. The reaction mixture was diluted with
CH2Cl2 (200 mL) and poured into an ice-cold buffer solution
containing sodium sulfite (19.1 g, 151.4 mmol) and dibasic sodium
phosphate (21.5 g, 151.4 mmol). This mixture was stirred at rt
overnight and then extracted with CH2Cl2 (3 × 250 mL). The
combined organic layers were washed with brine (100 mL), dried over
MgSO4, and concentrated. The crude product was recrystallized from
1:6 EtOAc:Hex to afford β-lactam TMβ as white needle crystals (10.8
1
Aldrich and used without purification. H and 13C NMR spectra were
collected on a Varian MercuryPlus 300 spectrometer at 300 and 75
MHz, respectively, using CDCl3 or D2O as the solvent. H NMR
1
chemical shifts were referenced to the resonance for residual
protonated solvent (δ 7.26 for CDCl3 and 4.79 for D2O). 13C NMR
chemical shifts were referenced to the solvent (δ 77.16 for CDCl3).
Mass spectra were acquired using either a Waters (Micromass) LCT
mass spectrometer or a Waters (Micromass) AutoSpec mass
spectrometer. IR spectra were acquired on a Bruker Tensor 27
instrument with an ATR attachment (Pike Technologies).
1
g, 84%), mp 101.2−101.9 °C (ref 68, mp 100−101 °C). H NMR
(400 MHz, CDCl3): δ 6.68 (bs, 1H), 1.23 (s, 6H), 1.11 (s, 6H); 13C
NMR (100 MHz, CDCl3): δ 175.30, 58.04, 54.37, 24.31, 19.01; ESI-
HRMS: m/z calcd for C7H13NNaO [M + Na]+: 150.0891; found:
150.0886.
Synthesis of β-Lactams. 1-Azaspiro[3.4]octan-2-one. The
product was synthesized by a modification of reported methods.66
A
Preparation Nylon-3 Copolymers. Regular or “random” nylon-3
copolymers were prepared in THF or DMF by adding the entire
quantity of each β-lactam to the reaction vessel before polymerization
was initiated. These copolymers were purified by precipitation from
the polymerization solution with pentane and deprotected using neat
trifluoroacetic acid (TFA) by following protocols described
previously.33 The reaction setup and polymerization operations were
conducted in a glovebox to maintain the moisture level below 5 ppm.
The reaction mixture was removed from the glovebox for purification.
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Polymers at the protected stage (with Boc protection of side chain
amine groups) were subjected to gel-permeation chromatography
(GPC) characterization using THF as the mobile phase at a flow rate
of 1 mL/min at 40 °C, using two Waters columns (Styragel HR 4E,
particle size 5 μm) linked in series. The Shimadzu GPC instrument
was equipped with a multi-angle light scattering detector (Wyatt
miniDAWN, 690 nm, 30 mW) and a refractive index detector (Wyatt
Optilab-rEX, 690 nm). Mn, Mw, and PDI were obtained with ASTRA
5.3.4.20 software using a dn/dc value of 0.1 mL/g. DP for a polymer
was calculated using the obtained Mn value and the theoretical subunit
composition based on the β-lactam proportion used for the
polymerization reaction.
The “skewed” form of 1:1 DM:CH, intended to mimic the subunit
distribution in “random” 1:1 DM:TM, as shown in Figure 2, was
prepared in THF by adding the β-lactams in four portions to the
reaction solution. The total amount of β-lactams DMβ and CHβ used
for this reaction corresponded to a 1:1 (equimolar) mixture, but the β-
lactams were divided unequally within each of the four portions. The
β-lactam distributions among the four DMβ + CHβ portions were
chosen based on the proportions of β-lactams TMβ and DMβ that
were incorporated into growing 1:1 DM:TM chains after 25%, 50%,
75%, and 100% polymerization, according to copolymerization kinetics
data (Figure 2). Thus, the first β-lactam portion used to prepare the
“skewed” 1:1 DM:CH copolymer contained 25% of the total β-lactam
precursors in an 83:17 DMβ:CHβ molar ratio. The second portion
contained 25% of the total β-lactam precursors in a 73:27 DMβ:CHβ
molar ratio. The third portion contained 25% of the total β-lactam
precursors, in a 36:64 DMβ:CHβ molar ratio, and the fourth portion
contained 25% of the total β-lactam precursors, in an 8:92 DMβ:CHβ
molar ratio. Since our studies of DMβ + CHβ copolymerization
indicated that these reactions are complete within 5 min (rt), we
allowed 20 min after the addition of the first, second, and third β-
lactam portions before adding the next β-lactam portion. The
“skewed” copolymer of 1:1 DM:CH was characterized at the protected
stage and deprotected by following the protocol used for “random”
nylon-3 copolymers.
Preparation of Nylon-3 Diblock Copolymers. Nylon-3 diblock
copolymers were prepared in DMAc in a glovebox to keep the
moisture level below 5 ppm. The β-lactam monomer for the first block
in DMAc solution was mixed with a solution of co-initiator (tert-
BuBzCl, 0.1 equiv), and a solution of base catalyst (LiHMDS, 0.25
equiv) was added. The reaction mixture was stirred for 2 h at rt, and
then a solution of the second β-lactam monomer (1 equiv relative to
the first β-lactam) was added. The reaction mixture was stirred for 36
h at rt and then removed from the glovebox. The reaction (total 4 mL)
was quenched with a few drops of MeOH, and the protected diblock
copolymer was precipitated by adding pentane (45 mL) to the
reaction mixture. The precipitated solid was collected from the bottom
of the cell culture tube after centrifugation and removal of the solvent
by decantation. The crude polymer was dissolved in THF (2 mL) and
subjected to additional precipitation/centrifugation operations. After
4−5 cycles of precipitation/centrifugation, the protected polymer was
collected and dried under N2 to give a white solid. The diblock
copolymer was deprotected by treating with neat TFA (2 mL) for 2 h
at rt and precipitated by addition of Et2O (45 mL). The precipitated
polymer was collected from the bottom of the culture tube after
centrifugation and removal of the solvent by decantation. The
collected solid was dissolved in MeOH (1 mL) and subjected to
precipitation/centrifugation operations. After a total of three cycles of
precipitation/centrifugation, the deprotected diblock copolymer was
collected and dried under N2 to give a white solid (TFA salt).
The diblock copolymer was characterized at the side-chain
protected stage as previously described.34 A Waters GPC was used
for polymer characterization using DMAc (containing 10 μM LiBr) as
the mobile phase at a flow rate of 1 mL/min at 80 °C. The GPC was
equipped with a single refractive index detector (Waters 2410) and
two Waters Styragel HR 4E columns (particle size 5 μm) linked in
series. The columns were calibrated with nine PMMA standards with
peak average molecular weight (Mp) ranging from 690 to 1 944 000.
Number-average molecular weight (Mn), weight-average molecular
weight (Mw), and polydispersity index (PDI) were calculated using
Empower software and calibration curves obtained from PMMA
standards. The degree of polymerization (DP) for a polymer was
calculated from the obtained Mn value and the theoretical subunit
composition based on the β-lactam proportion used for the
polymerization reaction.
Kinetic Studies of Copolymerization. Kinetic studies of
copolymerization for 1:1 DM:CH, 1:1 DM:TM, 1:1 DM:βCP and
1:1 DM:βDE were conducted using gas chromatography (GC) to
detect unreacted β-lactam starting materials at various time points
before each polymerization reaction was complete. The base-catalyzed
ring-opening copolymerization reactions were conducted in the same
way as preparative copolymerization reactions, but these reaction
mixtures for kinetic analysis contained triphenylmethane, which is
expected to be inert under the reaction conditions, as an internal
standard for GC analysis. At different time points during each
copolymerization reaction, an aliquot of the reaction solution was
transferred to a separate vial that contained about 10 mg of benzoic
acid, which rapidly halts the copolymerization reaction. The quenched
reaction samples were analyzed on a Shimadzu GC-17A GC
instrument equipped with an RTX-5 or a DB-wax column to obtain
the peak areas for unreacted β-lactams and the internal standard.
Subsequent calculations were based on β-lactam:internal standard peak
area ratios. For each time point, the amount of each β-lactam that
remained was determined based on a calibration curve (β-
lactam:internal standard peak area ratio plotted as a function of β-
lactam:internal standard concentration ratio). The kinetic study
revealed that the DMβ + TMβ copolymerization is much slower
than copolymerization of the other three β-lactams with DMβ. The
DMβ + TMβ copolymerization is only 81% complete after 5 h, and
92% complete after 80 h. Samples of 1:1 DM:TM used for biological
evaluation were prepared in reactions that were terminated after 16 h,
because longer reaction times can lead to byproduct formation.53
Antibacterial Assays. The MIC assay for bacteria was conducted
by following a 2-fold broth microdilution protocol previously
described.33 Eight bacteria were tested: E. coli JM 109, B. subtilis
BR151, E. faecium A634 (vancomycin-resistant), S. aureus 1206
(methicillin-resistant),33 Salmonella enterica LT2, Bacillus cereus
ATCC14579, the uropathogenic E. coli CFT073, and Pseudomonas
aeruginosa PA1066. Briefly, bacteria were cultured overnight at 37 °C
on LB agar plates and then suspended in LB medium at 2 × 106 cells/
mL. The cell suspension (50 μL) was mixed with the same volume of
polymer solutions in 2-fold serial dilutions (from 400 to 3.13 μg/mL)
in a 96-well plate, which was incubated for 6 h at 37 °C. Optical
density (OD) of each well was measured at 650 nm on a Molecular
Devices Emax precision microplate reader. Controls included on the
same plate: LB medium only (blank) and cells in LB without polymer
(uninhibited growth control). The bacterial cell growth in each well
polymer
blank
was calculated with the equation (% cell growth = (A
− A )/
650
650
control
blank
(A
650
− A ) × 100) and plotted against polymer concentration.
650
The MIC value is the minimum concentration of a given polymer
necessary to inhibit bacterial growth completely. When repeat
measurements of MIC, IC10, or HC10 oscillated between two polymer
concentrations, the average of these two is reported (e.g., 75 μg/mL is
reported when the values were 50 and 100 μg/mL). In the MIC assay
for daptomycin, 0.1 M CaCl2 was incorporated for all polymer/
bacterial cell mixture at varied polymer concentration.
The MBC for a given polymer was obtained after performing the
MIC assay described above. Aliquots of 10 μL of bacterial cell
suspension from wells containing the polymer at concentrations
ranging from one dilution below the MIC to the highest polymer
concentration were plated on LB agar. The plates were incubated
overnight at 37 °C, and bacterial colonies were then counted. The
MBC is the lowest polymer concentration to result in zero bacterial
colonies.
Antibacterial Resistance Test. A standard MIC/MBC test of 1:1
DM:TM was conducted with E. coli or MRSA using the protocol
mentioned above and beginning with the original strain of bacteria
(passage 0). The spread plate used for colony forming unit (CFU)
counting in the MBC test was used to subculture bacterial cells for this
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Article
study. Bacterial colonies were observed on the spread plate that was
inoculated with the mixture of bacterial cells and polymer at a
concentration one dilution below the MIC, i.e., the polymer
concentration is chosen as 3.1 μg/mL if MIC is identified as 6.2
μg/mL. One colony, representing a surviving cell from the previous
polymer treatment, was carefully picked from this LB-agar plate and
designated as passage 1 cells. The colony of passage 1 cells was
transferred to a centrifuge tube containing 3 mL of sterile LB medium
and dispersed under vortex mixing for 20 s. This cell suspension was
subcultured by inoculating on a LB-agar plate and incubating at 37 °C
overnight. The cultured cells at passage 1 on a LB-agar plate were
suspended in LB medium and used for the next round of standard
MIC/MBC test. This operation was repeated to evaluate the impact of
1:1 DM:TM on E. coli and MRSA for 10 successive passages.
Fibroblast Toxicity Assay. Polymer toxicity was evaluated using
NIH 3T3 fibroblasts and the CytoTox-ONE assay kit (Promega),
which measures the release of lactate dehydrogenase (LDH) from
membrane-damaged cells, as described previously.34 Briefly, 1.5 × 104
cells in DMEM were seeded in each well of a 96-well plate, which was
incubated for 24 h at 37 °C. Medium was exchanged for fresh DMEM
(phenol red- and pyruvate-free), and cells were incubated for another
2 h at 37 °C. Cells were treated with nylon-3 polymers at varied
concentrations in a 2-fold serial dilution series ranging from 400 to
3.13 μg/mL for 12 h at 37 °C. The cells in each well were then
analyzed using the CytoTox-ONE assay kit. On the same plate, wells
without polymer and wells treated with lysate solution to cause 100%
release of LDH were incorporated as the blank and positive control,
respectively. Fluorescence intensity was measured on a Tecan Infinite
M1000 microplate reader using ex/em 560/590 nm. Cell death was
calculated from (% death = (Fpolymer − Fblank)/(Fcontrol − Fblank) × 100)
and plotted against polymer concentration. The IC10 value is the
polymer concentration that causes 10% cell death.
AUTHOR INFORMATION
■
Corresponding Authors
kmasters@wisc.edu
gellman@chem.wisc.edu
Notes
The authors declare the following competing financial
interest(s): B.W. and S.H.G. are co-inventors on a patent
application that covers the polymers described here.
ACKNOWLEDGMENTS
■
This research was supported by the NIH (R21EB013259 and
R01GM093265). In addition, partial support was provided by
the UW-Madison Nanoscale Science and Engineering Center
(DMR-0832760). We thank Tammy K. McSimov of Lonza Inc.
for providing a sample of PHMB, and Alison Wendlandt and
Prof. Shannon Stahl for use of gas chromatography equipment.
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