Beyond Amphiphilic Balance: Changing Subunit Stereochemistry Alters the Pore-Forming Activity of Nylon-3 Polymers

Article abstract of DOI:10.1021/jacs.0c12731

Amphiphilic nylon-3 polymers have been reported to mimic the biological activities of natural antimicrobial peptides, with high potency against bacteria and minimal toxicity toward eukaryotic cells. Amphiphilic balance, determined by the proportions of hydrophilic and lipophilic subunits, is considered one of the most important features for achieving this activity profile for nylon-3 polymers and many other antimicrobial polymers. Insufficient hydrophobicity often correlates with weak activities against bacteria, whereas excessive hydrophobicity correlates with high toxicity toward eukaryotic cells. To ask whether factors beyond amphiphilic balance influence polymer activities, we synthesized and evaluated new nylon-3 polymers with two stereoisomeric subunits, each bearing an ethyl side chain and an aminomethyl side chain. Subunits that differ only in stereochemistry are predicted to contribute equally to amphiphilic balance, but we observed that the stereochemical difference correlates with significant changes in biological activity profile. Antibacterial activities were not strongly affected by subunit stereochemistry, but the ability to disrupt eukaryotic cell membranes varied considerably. Experiments with planar lipid bilayers and synthetic liposomes suggested that eukaryotic membrane disruption results from polymer-mediated formation of large pores. Collectively, our results suggest that factors other than amphiphilic balance influence the membrane activity profile of synthetic polymers. Subunits that differ in stereochemistry are likely to have distinct conformational propensities, which could potentially lead to differences in the average shapes of polymer chains, even when the subunits are heterochiral. These findings highlight a dimension of polymer design that should be considered more broadly in efforts to improve specificity and efficacy of antimicrobial polymers.

Full text of DOI:10.1021/jacs.0c12731

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Article
Beyond Amphiphilic Balance: Changing Subunit Stereochemistry
Alters the Pore-Forming Activity of Nylon‑3 Polymers
Lei Liu,⊥ Kevin C. Courtney,⊥ Sean W. Huth, Leslie A. Rank, Bernard Weisblum, Edwin R. Chapman,*
and Samuel H. Gellman*
Cite This: J. Am. Chem. Soc. 2021, 143, 3219−3230
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ABSTRACT: Amphiphilic nylon-3 polymers have been reported to mimic the biological activities of natural antimicrobial peptides,
with high potency against bacteria and minimal toxicity toward eukaryotic cells. Amphiphilic balance, determined by the proportions
of hydrophilic and lipophilic subunits, is considered one of the most important features for achieving this activity profile for nylon-3
polymers and many other antimicrobial polymers. Insufficient hydrophobicity often correlates with weak activities against bacteria,
whereas excessive hydrophobicity correlates with high toxicity toward eukaryotic cells. To ask whether factors beyond amphiphilic
balance influence polymer activities, we synthesized and evaluated new nylon-3 polymers with two stereoisomeric subunits, each
bearing an ethyl side chain and an aminomethyl side chain. Subunits that differ only in stereochemistry are predicted to contribute
equally to amphiphilic balance, but we observed that the stereochemical difference correlates with significant changes in biological
activity profile. Antibacterial activities were not strongly affected by subunit stereochemistry, but the ability to disrupt eukaryotic cell
membranes varied considerably. Experiments with planar lipid bilayers and synthetic liposomes suggested that eukaryotic membrane
disruption results from polymer-mediated formation of large pores. Collectively, our results suggest that factors other than
amphiphilic balance influence the membrane activity profile of synthetic polymers. Subunits that differ in stereochemistry are likely
to have distinct conformational propensities, which could potentially lead to differences in the average shapes of polymer chains,
even when the subunits are heterochiral. These findings highlight a dimension of polymer design that should be considered more
broadly in efforts to improve specificity and efficacy of antimicrobial polymers.
random polymers to mimic functions performed in biology by
sequence-specific polymers is of considerable interest.15,16
Natural AMPs have complex modes of action that are not
yet fully understood, with mechanistic variations among
specific peptides.15 Most AMPs appear to compromise
bacterial membrane integrity, a property that could lead
directly to antibacterial effects and/or enable AMPs to reach
INTRODUCTION
■
Eukaryotes deploy a wide array of antimicrobial peptides
(AMPs) to control or prevent the growth of prokaryotes.1
Collectively, these AMPs manifest diverse conformations,
including helix and sheet secondary structures, and some
adopt discrete tertiary structures (usually enforced by internal
disulfides). Many AMPs, however, do not appear to have a
strong conformational preference.2,3 The heterogeneity in
composition, sequence, and shape among AMPs has led many
research groups to consider synthetic polymers as possible
alternatives.4−14 These efforts have been motivated by the
practical consideration that polymers are generally less costly
to synthesize relative to sequence-specific peptides. From a
fundamental perspective, the prospect of using sequence-
Received: December 9, 2020
Published: February 21, 2021
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Figure 1. (a) Cationic and hydrophobic subunits of nylon-3 polymers in this study. (b) Polymerization reaction for generating a cationic-
hydrophobic nylon-3 copolymer from a β-lactam pair.
intracellular targets.17−19 AMPs are generally rich in residues
with side chains that are cationic (Lys and Arg) or
hydrophobic (such as Leu or Phe), with both functionalities
contributing to membrane interaction.19,20 The complement of
hydrophobic side chains in AMPs seems to be necessary for
disruption of the lipid bilayer. The net positive charge that is
prevalent among AMPs at neutral pH attracts them to the
surfaces of bacterial cells, which have a net negative charge.2 In
contrast, the surfaces of eukaryotic cells have little net charge
as the outer leaflet of the plasma membrane is primarily
composed of zwitterionic phospholipids.1
Common features among AMPs have inspired the design of
antibacterial polymers with a wide range of backbones and
appended functionality.6,10,21−27 Positive charges have typically
been incorporated into side chains via protonated or
quaternized nitrogen-based groups (amines or guanidines) or
quaternized phosphorus; charged groups have also been
incorporated directly into the backbone.10,28−30 Hydro-
phobicity has been incorporated via both side chains and the
backbone.31−33 By combining hydrophobic and cationic
groups in individual subunits or by mixing cationic and
hydrophobic subunits, one can achieve the amphiphilicity
necessary for an AMP-mimetic activity profile. The importance
of maintaining balance between hydrophobic and cationic
groups (“amphiphilic balance”) in antimicrobial polymer
design, to ensure specificity toward prokaryotes, has been
widely noted.31,34 Excessive hydrophobicity can lead to
disruption of eukaryotic cell membranes, a trend that is
manifested also among synthetic peptides and peptide mimics
(such as peptoids) inspired by AMPs.35−37
This study asks whether factors other than amphiphilic
balance can influence biological activity profiles of synthetic
polymers. Specifically, we evaluated a new family of nylon-3
polymers based on two stereoisomeric subunits bearing a side
chain amino group that should be protonated and therefore
cationic near-neutral pH. This stereochemical difference had
little effect on antimicrobial activity, but significant variations
were observed in terms eukaryotic cell toxicity. We interpret
these findings to suggest that the conformational propensities
of polymer subunits, which can be altered by changes in
backbone stereochemistry, influence membrane activity in a
manner that is independent of and complementary to the
influence of amphiphilic balance. Our results highlight a design
variable that has received little attention so far in efforts to
develop synthetic polymers with AMP-like activity profiles or
other biomimetic functions.
The nylon-3 system is well-suited to exploration of
stereochemistry-based strategies for tuning the polymer
function, as demonstrated by the elegant work of Grinstaff et
al. on carbohydrate-based nylon-3 polymers or “poly-amido-
saccharides” (PAS). Grinstaff et al. used enantiopure
carbohydrate-based β-lactams to generate unique synthetic
polymers that display features of both polysaccharides and
polypeptides. Homochiral polymers generated from diastereo-
meric subunits, glucose-based vs galactose-based, display
substantial aqueous solubility differences.38−40 In addition,
Grinstaff et al. found functional differences among macro-
surfactants in which the hydrophilic “head groups” are short
PAS chains. Macrosurfactants with a mixed headgroup
containing galactose- and glucose-derived units inhibited
bacterial biofilm formation, while analogous macrosurfactants
with a pure galactose-derived PAS headgroup did not. The
hydrophobic “tail” was essential for this activity; the PAS
segments themselves were not active.9 It should be noted,
however, that the PAS homopolymers are based on subunits
that are rigid and enantiopure, which allows these chains to
adopt distinct helical secondary structures depending on which
diastereomeric subunit is employed.40 Even with this
precedent, it was not obvious that membrane activities could
be altered by varying between flexible diastereomeric subunits
in a heterochiral format, as demonstrated in the work
described below.
RESULTS AND DISCUSSION
■
Polymer Design, Synthesis, and Characterization. To
examine the impact of cationic subunit stereochemistry on
polymer activities, we focused on four new nylon-3 polymers
generated by copolymerization of β-lactams that generate the
subunits shown in Figure 1a. These β-lactams, and many
others, are readily synthesized from the corresponding alkenes
via cycloaddition of N-chlorosulfonyl isocyanate (CSI)
followed by hydrolytic removal of the chlorosulfonyl group.41
The cycloaddition is stereospecific: alkene geometry (E vs Z)
specifies the relative configuration of the β-lactam that gives
rise to the ME-cis or ME-trans subunit. Base-catalyzed ring-
opening polymerization of β-lactams provides nylon-3
materials, which feature a poly-β-amino acid backbone. Use
of a strong acylating agent, such as p-tert-butylbenzoyl chloride,
in the polymerization reaction leads to formation in situ of N-
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acyl-β-lactams that initiate the polymerization process. The
identity of the acylating agent determines the identity of the
acyl group at the N-terminus of each nylon-3 polymer chain.
Copolymerization of a β-lactam pair generates chains
containing two types of subunit.42
Several groups have explored biological properties of nylon-3
materials, including antibacterial activity,9,43,44 antifungal
activity,45 lung surfactant mimicry,46 cellular growth substrate
activity,47 mucoadhesion,48 formation of low-adsorption
hydrogels,49 inhibition of ice recrystallization,50 and protein
stabilization during freeze-drying.38 Combining cationic
subunits with hydrophobic subunits in which both backbone
sp3 carbons have geminal substitution, such as the TM and
DMCH subunits (Figure 1a; TM for “tetramethyl” and
DMCH for “dimethycyclohexyl”), leads to favorable activity
profiles, with good potency toward bacteria but little
deleterious effect on eukaryotic cells.51,52
We used two new diastereomeric β-lactams to generate
polymers containing the ME-cis or ME-trans subunits (Figure
1a; ME for “monoethyl”) as the source of the positive charges
in the new nylon-3 polymers examined here. These β-lactams
were synthesized via an approach previously used for related β-
lactams bearing protected amino groups in side chains.43 The
cis and trans configurations of these compounds were
confirmed via X-ray crystal structures of synthetic intermedi-
ates (Figures S7 and S16, respectively). Comparison of these
two β-lactams via reverse-phase UPLC suggested that they
have very similar polarity (Figure S19), which supports our
hypothesis that the ME-cis and ME-trans subunits in nylon-3
polymers have similar contributions to the amphiphilic balance
of the polymer chains. These β-lactams and the one used to
generate DMCH subunits were each prepared as a racemic
mixture, and these racemates were used for all polymerization
reactions. Therefore, all of the nylon-3 polymers described
below are heterochiral because they contain subunits of both
possible absolute configurations. In contrast, the relative
configuration of the ethyl and aminomethyl side chains within
an ME-cis or ME-trans subunit is fully controlled in each
polymer based on the β-lactam precursor employed.
Table 1. Fundamental Characterizations of ME-Cis and ME-
Trans Polymers
GPC
characterization
NMR characterization
c
subunit composition -
NMR (cationic:
hydrophobic)
b
DP
DP
a
polymer
Đ
(GPC) (NMR)
ME-cis:TM
N/A
1.13
1.19
1.14
N/A
23
15
18
19
19
22
14 ME-cis:4 TM
ME-trans:TM
ME-cis:DMCH
ME-trans:DMCH
15 ME-trans:4 TM
15 ME-cis:4 DMCH
17 ME-trans:5 DMCH
14
a
Dispersity (Đ), calculated by dividing the weight-averaged molecular
b
weight by number-averaged molecular weight. Degree of polymer-
ization, the average number of repeat units in the polymer chains.
c
The average number of each subunit in the polymer chains.
deprotection and could be included in this analysis. The
results showed very similar DP values for all four polymers and
reasonable consistency with GPC-derived DP values before
deprotection in the three cases that allowed comparison. The
NMR analysis also provided insight into average subunit
proportions per chain, and the values were consistent among
the four copolymers at ∼4:1 cationic:hydrophobic subunits.
On the basis of these results, we conclude that amphiphilic
balance was very similar between these samples of ME-cis:TM
and ME-trans:TM and between these samples of ME-
cis:DMCH and ME-trans:DMCH.
Antibacterial Activities. The four copolymers were
assessed for the ability to halt bacterial growth with a small
panel of species, including one Gram-negative (Escherichia coli)
and three Gram-positive bacteria (Bacillus subtilis, Staph-
ylococcus aureus, and Enterococcus faecium) (Table 2 and Figure
Table 2. Bacteria Inhibitory Results for ME-Cis and ME-
Trans Copolymers
a
MIC (μg/mL) of copolymers
B.
E.
E. coli
b
E. coli
(EZRDM)
subtilis S. aureus
faecium
c
d
polymer
(LB)
(LB)
(LB)
(BHI)
Distinctions between polymers containing ME-cis vs ME-
trans subunits became evident during synthesis. Reactions to
produce ME-trans:DMCH or ME-trans:TM copolymers
proceeded smoothly in tetrahydrofuran (THF), but attempts
to perform comparable reactions to generate copolymers
containing ME-cis subunits were plagued by precipitation.
Therefore, reactions to produce ME-cis-containing copolymers
were performed in dimethylacetamide (DMAc). Even in this
solvent, the reaction mixture for ME-cis:TM became turbid.
Polymers were analyzed before side-chain deprotection via
gel permeation chromatography (GPC) in THF or DMAc and
after side-chain deprotection via 1H NMR in D2O. Removal of
the side-chain Boc protecting groups was accomplished by
treatment with trifluoroacetic acid (TFA) with 5% (v/v)
triisopropylsilane, and the deprotected polymers were there-
fore isolated as the trifluoroacetate salts. Table 1 summarizes
polymer properties. GPC analysis was impossible for the ME-
cis:TM copolymer because of solubility limitations, but the
other three copolymers displayed low dispersity and relatively
similar degrees of polymerization (DP). NMR analysis of the
deprotected polymers allowed an independent determination
of DP by using the unique aromatic resonances of the p-tert-
butylbenzoyl end group as an internal integration reference.
The ME-cis:TM copolymer was fully water-soluble after
ME-cis:TM
>200
>200
>200
>200
31.3
31.3
31.3
15.6
3.1
3.1
3.1
6.3
12.5
12.5
12.5
12.5
50
50
12.5
12.5
ME-trans:TM
ME-cis:DMCH
ME-trans:DMCH
a
b
MIC = minimum inhibitory concentration for bacterial growth. LB
= Luria−Bertani medium. EZRDM = EZ rich defined medium. BHI
= brain heart infusion medium.
c
d
S36). The activity profiles toward these four bacteria were
quite similar among the four copolymers, as measured via
minimum inhibitory concentration (MIC). The greatest
variation was observed with E. faecium, for which DMCH-
containing polymers were modestly more active than TM-
containing polymers. Most of these studies were conducted in
typical culture media that contain complex biologically derived
components, brain heart infusion (BHI), or lysogeny broth
(LB) medium. Moderate to good activities were observed in
these media toward the Gram-positive species, but no activity
toward E. coli was observed for any of the four copolymers in
LB medium. We evaluated E. coli also in a chemically defined
medium, EZRDM, which does not contain any biomacromo-
lecular components.53,54 In EZRDM, all four copolymers were
active against E. coli. We have previously reported similar
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Figure 2. Hemolysis activities in TBS buffer for (a) ME:TM copolymers or (b) ME:DMCH copolymers. Data obtained after 1 or 6 h incubation
time are shown. HeLa cell membrane leakage activities in PBS buffer for (c) ME:TM copolymers or (d) ME:DMCH copolymers after 2 h
incubation.
medium-dependent variations in activity toward E. coli for
other nylon-3 polymers, and this effect was traced to inhibitory
interactions between polyanionic components of the complex
medium and the polycationic polymer chains.54 On the basis of
this precedent, it is noteworthy that the four copolymers
studied here display significant activity toward Gram-positive
species in complex media, which are presumably closer to
conditions that would be encountered in vivo.55 We attempted
to determine whether polymer inhibitory activities could be
enhanced against S. aureus or E. faecium in EZRDM, but
neither bacterium would grow in the chemically defined
medium. Collectively, results from the bacterial growth assays
show that there is no discernible difference between
copolymers containing the ME-cis vs ME-trans subunits in
terms of antibacterial activities.
Lytic Activities toward Eukaryotic Cells. The most
common approach to detecting deleterious effects of AMPs or
AMP-inspired polymers on eukaryotic cells is to evaluate
hemolytic activity, i.e., damage to human red blood cell
membranes that allows hemoglobin (MW 65 kDa, 32 Å Stokes
radius) to exit.34,56−58 Our hemolysis assays were conducted in
Tris-buffered saline (TBS). We found that copolymers
containing different stereoisomeric forms of the cationic
subunit had significantly different effects on hemolytic activity.
For copolymers containing either TM or DMCH as the
hydrophobic component, hemolytic activity was high with the
ME-cis cationic subunit but low with the ME-trans cationic
subunit after a 1 h incubation (Figures 2a,b, dotted curves).
The trend was similar when the exposure was lengthened to 6
h (Figures 2a,b, solid curves). Thus, the observed differences in
hemolytic activity do not seem to reflect a difference in
membrane disruption kinetics, at least on the time scale of
several hours.
We wondered whether differences in hemolytic activity
might be correlated to differences in polymer aggregation in
aqueous solution. This concern was prompted by evidence that
emerged during synthetic studies for aggregation in organic
solvents of polymers containing ME-cis (but not ME-trans)
with side chains protected. We used a standard assay format,
based on the solubilization of a hydrophobic fluorophore,
diphenylhexatriene (DPH), to ask whether any of the polymers
formed micellar aggregates in TBS.59 No evidence for
aggregation was detected up to the highest concentration
tested, 500 μg/mL, for any of the polymers (Figure S38).
To obtain additional understanding of polymer effects on
eukaryotic cells, we used the CytoTox One assay to evaluate
interactions of the four nylon-3 copolymers with HeLa cells.60
This assay measures the leakage of lactate dehydrogenase
(LDH; tetramer MW 140 kDa, 42 Å Stokes radius) from cells,
which indicates membrane damage.61 When these assays were
conducted with cells in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum (FBS), lytic
activity was negligible. However, when the assays were
conducted in phosphate-buffered saline (PBS), significant cell
lysis was observed (Figures 2c,d). The suppression of cell lysis
in the medium containing biomacromolecular components
(DMEM-FBS) parallels observations reported above and
previously regarding polymer effects on E. coli.54 The identity
of the hydrophobic subunit had a large impact on copolymer
HeLa cell lytic activity. ME-cis:TM induced considerably more
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Figure 3. (a) Hemolysis activities for ME-cis and ME-trans homopolymers. (b) Hemolysis activities for copolymers containing ME-cis and ME-
trans subunits, with varying proportions of ME-cis and ME-trans subunits.
LDH leakage from HeLa cells in PBS than did ME-trans:TM,
which mirrors the trend observed in hemolysis assays.
However, ME-cis:DMCH and ME-trans:DMCH were similar
in their abilities to induce LDH leakage in PBS.
Collectively, the eukaryotic cell lysis experiments reveal that
amphiphilic balance cannot be the only factor influencing
membrane activity because polymers that differ only in the
stereoisomeric form of the cationic subunits (ME-cis vs ME-
trans) should be similar or identical in their amphiphilic
balance. Moreover, the observation of different trends in HeLa
cell lysis as a result of hydrophobic subunit identity, TM vs
DMCH (Figures 2c,d), suggests that the impact of amphiphilic
balance may be modulated by copolymer composition.
Because hemolytic activity was the most sensitive among all
the cell-based assays to the stereochemical variation between
ME-cis and ME-trans, we conducted additional hemolysis
studies on polymers containing only these two cationic
subunits (see the Supporting Information for GPC character-
ization). Homopolymers of ME-cis or ME-trans were highly
hemolytic and similar to one another (Figure 3a). Comparing
these data to those in Figures 2a,b indicates that introduction
of ∼20% of the hydrophobic subunit, either TM or DMCH,
causes a profound decline in hemolytic activity for polymers
containing ME-trans, while the effect on polymers containing
ME-cis is more subtle. Although it might seem surprising that
addition of hydrophobic subunits could lead to a diminution of
hemolytic activity, we note that this trend has been observed
previously with nylon-3 polymers containing the DM cationic
unit (Figure 4).51,52 The ME-cis and ME-trans homopolymers
they are similar or identical to the homopolymers in this
regard. Varying the proportion of ME-cis and ME-trans
subunits results in substantial differences in hemolytic activity.
When ME-trans is dominant, hemolytic activity is most
pronounced. In contrast, when ME-cis is dominant, little
hemolytic activity is detected. It seems puzzling that inclusion
of a small proportion of the ME-trans subunit in a polymer
largely composed of ME-cis subunits can lead to a significant
decrease of hemolytic activity at 500 μg/mL, when the ME-cis
and ME-trans homopolymers are each highly hemolytic at this
concentration. Collectively, the data in Figure 3 support the
general conclusion that factors other than amphiphilic balance
can exert a substantial effect on membrane activity, at least for
eukaryotic cells.
Experiments with Synthetic Vesicles. AMPs and their
synthetic mimics exert their effects on cells by interacting with
and destabilizing the membrane bilayer and possibly other
mechanisms.1,2,19,43 To learn more about how the nylon-3
polymers affect lipid bilayers, we undertook studies with
synthetic vesicles containing 90% 1,2-dioleoyl-sn-glycero-3-
phosphocholine (DOPC) and 10% 1,2-dioleoyl-sn-glycero-3-
phospho-L-serine (DOPS).62 DOPC has a zwitterionic head-
group; more than half of the lipids in an RBC or HeLa cell
membrane have a zwitterionic phosphocholine headgroup. The
DOPS headgroup is anionic. The proportion of zwitterionic
and anionic phospholipids in our vesicles is similar to the
proportion in RBC or HeLa cell membranes.63,64 The RBC
membrane contains ∼30% cholesterol,65 but preliminary
studies indicated that inclusion of cholesterol in our vesicles
did not affect the trends reported below (Figure S43).
Our initial in vitro studies focused on ME-cis:TM, which was
a potent inducer of leakage with both RBCs and HeLa cells. To
establish whether ME-cis:TM causes formation of pores, we
employed the black lipid membrane (BLM) method to
evaluate bilayers formed by 9:1 DOPC:DOPS (Figure 5a).
As shown in Figure 5b, addition of ME-cis:TM to the aqueous
solution on one side of the bilayer led to transient and irregular
ion conduction across the bilayer. The nature of the
conductance observed in the presence of the polymer is
consistent with transient formation of irregular/chaotic pores
with variable dimensions. These observations are not
consistent with formation of channels that have a discrete
structure, such as those formed by protegrin-166,67 or the
influenza M2 protein.68,69 The latter conclusion is consistent
with our expectations: the polymer sample contains a wide
array of chains that vary in length, composition, subunit
identity sequence, and ME-cis subunit stereochemistry
sequence, and it therefore seems exceedingly unlikely that
discrete channel structures could form.
Figure 4. Structure of the DM subunit.
displayed substantially reduced antibacterial activities (Table
S1 and Figure S37) relative to the copolymers containing these
subunits (Table 2), which demonstrates that the small
proportion of TM or DMCH in the copolymers is critical
for antibacterial efficacy.
Figure 3b shows hemolysis data obtained for three polymers
generated via copolymerization of β-lactams leading to ME-cis
or ME-trans subunits in varying proportions. These three
copolymers have similar or identical amphiphilic balance, and
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Figure 5. (a) Setup of black lipid membrane (BLM) experiments. (b) BLM results with HEPES buffer control (upper graph) and ME-cis:TM
(lower graph), with 2 μL of 250 μg/mL ME-cis:TM in PBS added to one side of the BLM chamber. (c) Giant unilamellar vesicles (GUVs) formed
from 9:1 DOPC:DOPS encapsulating 4 kDa FITC-labeled dextran, before (control panel) and after (ME-cis:TM panel) the addition of 20 μL of a
1 mg/mL ME-cis:TM solution to 100 μL of GUV suspension. Cyan color came from FITC-labeled dextran, and magenta color came from
rhodamine-labeled GUV membranes. The disappearance of cyan color from GUVs in the FITC-channel of panel c indicates leakage of FITC-
labeled dextran.
Additional studies were conducted with liposomes to ask
whether hemolytic activity arises from pores in the cell
membrane or wholesale membrane lysis. We prepared giant
unilamellar vesicles (GUVs) with 9:1 DOPC:DOPS that were
labeled with 0.1% rhodamine-tagged phosphatidyl-
ethanolamine (PE) to enable vesicle imaging via fluorescence
microscopy. Dextran with average molecular weight 4 kDa and
bearing a fluorescein tag was encapsulated within the GUVs
(Figure 5c). When these vesicles in PBS were treated with ME-
cis:TM copolymer (167 μg/mL), the cyan fluorescence inside
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Figure 6. (a). Design of large unilamellar vesicle (LUV) experiments. Before polymer treatment, fluorescence signal from entrapped FITC-labeled
dextran is quenched by entrapped TRITC-labeled dextran via FRET. TRITC-labeled dextran (155 kDa) has an average Stokes radius of 85 Å,
making it difficult to escape from the pores of liposomes. If polymer treatment causes leakage of vesicle contents, the FITC fluorescence signal will
no longer be quenched. (b) LUV dye leakage for four different FITC-labeled dextran sizes caused by TM-containing copolymers at 500 μg/mL. (c)
LUV dye leakage caused by TM-containing copolymers at 125 μg/mL. (d) LUV dye leakage caused by DMCH-containing copolymers at 500 μg/
mL. (e) LUV dye leakage caused by DMCH-containing copolymers at 125 μg/mL. In these studies, the FITC fluorescence signal is measured
immediately after addition of polymer to the LUV preparation.
the vesicles slowly dissipated, which suggests that the dextran
exited the vesicles through large, copolymer-induced pores.
The disappearance of cyan fluorescence presumably reflects
dilution of the fluorescein-labeled dextran in the large
extravesicular volume. Even after the cyan fluorescence had
dissipated, the vesicles themselves remained visible, as
indicated by fluorescence in the rhodamine channel (Figure
5c and Video S1). This observation indicates that although
pores were generated in the bilayer, the GUV structure
remained intact. In contrast, after the dextran-loaded GUVs
were treated with the detergent Triton X-100, the GUVs were
no longer visible, suggesting wholesale vesicle dissolution
(Video S2). Collectively, these initial GUV studies support the
hypothesis that ME-cis:TM forms large pores in the lipid
bilayer that enable the entrapped 4 kDaA dextran to diffuse out
and that ME-cis:TM does not completely destroy the GUVs.
We then used a quantitative dye-release assay to compare
the effects of the four nylon-3 copolymers on a lipid bilayer.
These studies employed large unilamellar vesicles (LUVs)
formed from 9:1 DOPC:DOPS. The LUVs enclosed two
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fluorophore-bearing dextrans: one of 155 kDa bearing
tetramethylrhodamine (TRITC) and the other of 4, 10, 20,
or 40 kDa bearing fluorescein (FITC) (see Table S2 for
average Stokes radii). The 20 kDa FITC-dextran has an
average Stokes radius similar to that of hemoglobin, and the 40
kDa FITC-dextran has an average Stokes radius similar to that
of LDH.58,61 When a TRITC-dextran is coentrapped with a
FITC-dextran, FITC emission is not observed because of
energy transfer to TRITC.70 If a polymer damages the lipid
bilayer, however, the smaller dextran can escape, and this
process is detected via an increase in fluorescence with
excitation at 490 nm and emission at 520 nm (Figure 6a). In
these experiments, “100%” dextran release is defined as the
fluorescence value measured after exposure of LUVs to the
lytic peptide melittin for the same incubation time as was used
for the polymers.
For experiments conducted with TM-containing copoly-
mers, at either 125 or 500 μg/mL, there was a clear distinction
between the copolymer containing ME-cis and that containing
ME-trans, with ME-cis:TM causing a greater extent of dextran
escape than ME-trans:TM (Figures 6b,c). For each polymer,
the extent of dextran escape seemed to be inversely correlated
to FITC-dextran size, although the variations were small. The
greater ability of ME-cis:TM relative to ME-trans:TM to
induce dextran leakage from the LUVs is consistent with
greater ability of ME-cis:TM relative to ME-trans:TM to
induce protein leakage from RBCs or HeLa cells (Figures 2a,c
and Figures S41a,b).
and mammalian cell-lytic activities.75−77 These observations
indicate that the mechanism underlying antibacterial and
mammalian cell-lytic activities does not involve binding to
specific biomacromolecules, such as proteins. Such targets are
chiral and occur in only one enantiomeric form; therefore, a
protein target will respond in distinct ways to the two
enantiomers of a binding partner.78,79 The precedents
involving antimicrobial peptide enantiomers are consistent
with our conclusion that activity differences we observe as a
function of subunit stereochemistry, ME-cis vs ME-trans, do
not arise from differences in engagement of specific cellular
targets by the polymers but rather from differences in the
physical properties of polymer chains containing the
diastereomeric subunits.
We propose that activity differences among polymers
containing ME-cis vs ME-trans subunits arise from variations
in the conformational propensities of these two subunits.
Figure 7 shows the conformation that is predicted to be most
The behavior of DMCH-containing polymers differed from
that of TM-containing polymers in the LUV dye leakage assays
in that the extent of dextran leakage was similar for ME-
cis:DMCH and ME-trans:DMCH (Figures 6e,f). This
similarity correlates with the similarity between these two
polymers in causing LDH leakage from HeLa cells (Figures 2d
and Figures S41c,d) and does not correlate with the significant
difference between the two polymers in hemolytic activity
(Figure 2b).
The disparity between the trends in hemolysis activities and
the trends in liposome dye leakage activities for ME-
cis:DMCH and ME-trans:DMCH could potentially be related
to differences in membrane composition. The liposome bilayer
contains no protein molecules, whereas the hRBC membrane
is approximately half protein by weight.71 RBC membrane
integrity is maintained by the cytoskeleton protein network, a
cellular feature that may prevent membrane damage from
toxins.72 Given the difference in membrane compositions
between synthetic vesicles and RBCs, we speculate that the
impact on cellular membranes exerted by polymers containing
ME-cis vs ME-trans may be differentially affected by
membrane proteins, which leads to different hemolytic
activities. The membranes of HeLa cells and hRBCs have
different protein compositions, and these compositional
differences might underlie the differences between trends in
LDH leakage compared to hemoglobin release.65,73,74 In
addition, although our GUV results suggest that the polymers
primarily act as pore-forming agents, it is possible that the large
tendency of ME-cis:DMCH to induce hemoglobin release
from hRBCs arises because a subset of the cells have their
membranes fully dissolved (as would be caused by Triton X-
100 and other detergents).
Figure 7. Newman projections showing the predicted most stable
conformations about the Cα−Cβ bond for an ME-cis subunit (a) and
an ME-trans subunit (b).
stable about the central Cα-Cβ bond of an ME-cis or ME-trans
subunit. These predictions are based on consideration of A-
values (energy difference between conformers of monosub-
stituted cyclohexane derivatives with the substituent axial vs
equatorial); A-values are well-established indicators of the
steric bulk of a substituent.80,81 Unbranched substituents with
an sp3 atom attached to the cyclohexane ring tend to have
larger A-values (i.e., are more sterically demanding) relative to
substituents with an sp2 atom attached to the ring.80 Therefore,
the conformers we anticipate to be most stable have the ethyl
and aminomethyl substituents anti to one another. This
expected preference causes the CO and NH of the ME-cis
subunit to be anti to one another, but these groups are gauche
to one another for ME-trans. Near room temperature, all three
of the staggered conformations about the central Cα−Cβ bond
of an ME-cis or ME-trans subunit should be populated to some
It has long been known that natural antimicrobial peptides,
composed of L-α-amino acid residues, and their enantiomers
(D-residues) display identical or nearly identical antibacterial
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degree, and interchange among these conformers should be
rapid. However, the expected preference for one conformer in
each case means that, on average, an ME-cis subunit favors
extended conformations of the nylon-3 backbone (CO anti
to NH), while an ME-trans subunit favors more compact
conformations (CO gauche to NH). The expected difference
in favored conformations for ME-cis and ME-trans subunits
could, in principle, lead to different global shapes of polymer
chains and thereby affect the physical and biological properties
of the polymers. These predictions may explain the substantial
differences in solubility we observed before side-chain
deprotection for polymers containing ME-cis vs ME-trans in
organic solvents like THF. Early work on discrete β-peptide
oligomers revealed that incorporation of β-amino acid residues
that favor an extended conformation tended to cause
insolubility, while use of β-amino acid residues that favor a
helical conformation (CO and NH gauche) generated
soluble β-peptides.82−84
Variations in physical properties among nylon-3 polymers
containing ME-cis vs ME-trans subunits can be viewed in the
context of research on vinyl polymers with variable tacticity.
Changes in tacticity can cause profound changes in polymer
physical properties, which presumably arise at least in part
from changes in the conformational properties of the polymer
chains. When polypropylene (PP) is atactic, for example, the
material is amorphous, but syndiotactic and isotactic PP
display crystalline domains that affect physical properties, such
as melting temperature and Young’s modulus.85,86 Because our
nylon-3 polymers are prepared from racemic β-lactams, these
polymers are necessarily atactic, whether the subunits are ME-
cis, ME-trans, or a combination of the two.
The observations reported here emphasize the importance
of varying subunit stereochemistry in antibacterial polymer
design. Stereochemistry of subunits has largely been over-
looked as a design variable in the development of antibacterial
or other bioactive polymers; some previously developed
polymers completely lack stereochemical elements.87 Many
antibacterial materials have been produced from achiral
precursors (e.g., methacrylate esters or cyclic carbonate esters)
via reactions that generate sp3 stereogenic centers within the
polymer chain but without control of subunit configuration.
The resulting polymers are therefore heterochiral; that is, they
contain subunits of both possible absolute configurations,
presumably with random distribution of the two possible
absolute configurations along each chain.88,89 Other anti-
bacterial polymers, such as most nylon-3 polymers, have been
generated in heterochiral form because racemic chiral
precursors have been used.42,90
the biological activity profiles of synthetic polymers. This
conclusion is significant, given that each copolymer sample we
evaluated contained many different chains that varied in length,
subunit composition, sequence of subunits, and sequence of
subunit configurations. The functional differences were
manifested despite this profound chain heterogeneity.
Tailoring amphiphilic balance is a critical factor in
developing synthetic polymers that are intended to display
potent inhibition of bacterial growth without harming
eukaryotic cells or to perform other biological functions
without manifesting toxic side effects.34,90 However, the data
reported here reveal that polymer properties other than
amphiphilic balance can exert substantial effects on polymer
performance, even when the polymer chains are highly
heterogeneous. This conclusion is based on functional
differences we observed between or among copolymer samples
that were comparable in all characteristics except the identity
of the ME subunits, which varied between the diastereomeric
ME-cis and ME-trans forms. These discoveries suggest that a
greater focus on harnessing subunit stereoisomerism will be
productive in future efforts directed toward design of polymers
with precisely defined bioactivity profiles.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c12731.
■
sı
Synthesis and characterizations of β-lactam monomers
and resulting nylon-3 polymers, UPLC characterization
of β-lactam monomers, assays for nylon-3 polymers
including aggregation studies, bacteria inhibitory studies,
and liposome dye leakage studies (PDF)
Video S1: 20 μL of 1 mg/mL ME-cis:TM polymer
solution in PBS was added to 100 μL of GUV
suspension in PBS buffer, with GUV composed of 90%
DOPC and 10% DOPS; FITC-dextran (4 kDa) was
encapsulated in GUVs (MOV)
Video S2: 10 μL of 10% Triton X-100 solution in PBS
was added to 100 μL of GUV suspension in PBS, where
40 kDa FITC-labeled dextran is encapsulated in the
GUVs (MOV)
Video S3: 20 μL of 1 mg/mL ME-trans:TM solution in
PBS added to 100 μL of GUV suspension in PBS buffer,
with GUV composed of 90% DOPC and 10% DOPS;
FITC-dextran (4 kDa) was encapsulated in GUVs
(MOV)
Video S4: 50 μL of 200 μg/mL melittin solution in PBS
was added to 100 μL of GUV suspension in PBS, with
40 kDa FITC-dextran encapsulated (MOV)
CONCLUSIONS
■
The data presented here show that altering the relative
configuration of the ethyl and aminomethyl side chains within
an ME-cis or ME-trans subunit exerts a substantial effect on
the biological activities of nylon-3 polymers. The antibacterial
activities we measured were not strongly affected by ME
subunit relative configuration, but activities toward mammalian
cell membranes and toward liposomes with lipid composition
similar to that of mammalian cell membranes displayed
considerable sensitivity to the presence of ME-cis vs ME-
trans. Because the diastereomeric ME subunits presumably
make similar or identical contributions to the polymer
amphiphilic balance, our findings show that factors beyond
amphiphilic balance can play a significant role in determining
Accession Codes
CCDC 2033849−2033850 contain the supplementary crys-
tallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
Corresponding Authors
■
Samuel H. Gellman − Department of Chemistry, University of
WisconsinMadison, Madison, Wisconsin 53706, United
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norbornene Derivatives. J. Am. Chem. Soc. 2004, 126 (48), 15870−
15875.
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tivity against Escherichia Coli. ACS Biomater. Sci. Eng. 2017, 3 (10),
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Biocidal Activity of Polystyrenes That Are Cationic by Virtue of
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States; orcid.org/0000-0001-5617-0058;
Email: gellman@chem.wisc.edu
Edwin R. Chapman − Department of Neuroscience and
Howard Hughes Medical Institute, University of Wisconsin
Madison, Madison, Wisconsin 53706, United States;
orcid.org/0000-0001-9787-8140; Email: chapman@
wisc.edu
Authors
Lei Liu − Department of Chemistry, University of Wisconsin
Madison, Madison, Wisconsin 53706, United States;
orcid.org/0000-0001-7782-4559
Kevin C. Courtney − Department of Neuroscience and
Howard Hughes Medical Institute, University of Wisconsin
Madison, Madison, Wisconsin 53706, United States;
orcid.org/0000-0003-1315-4917
Sean W. Huth − Department of Chemistry, University of
WisconsinMadison, Madison, Wisconsin 53706, United
States; orcid.org/0000-0002-4892-2587
Leslie A. Rank − Department of Chemistry, University of
WisconsinMadison, Madison, Wisconsin 53706, United
States; orcid.org/0000-0001-9551-5004
Bernard Weisblum − Department of Pharmacology,
University of WisconsinMadison, Madison, Wisconsin
53706, United States
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Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c12731
Author Contributions
⊥L.L. and K.C.C. contributed equally to this work as co-first
authors.
Notes
The authors declare the following competing financial
interest(s): S.H.G. is an inventor on patents and patent
applications covering antimicrobial nylon-3 polymers. L.A.R. is
an inventor on a patent application covering antimicrobial
nylon-3 polymers.
ACKNOWLEDGMENTS
■
We thank I. Guzei for X-ray crystal structure determination.
This research was supported in part by the U.S. National
Institutes of Health (R01 GM093265 and R33 AI121684 for
S.H.G.; R35 NS097362 and R01 MH061876 for E.R.C.).
E.R.C. is an Investigator of the Howard Hughes Medical
Institute.
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