Structure-activity relationships among antifungal nylon-3 polymers: Identification of materials active against drug-resistant strains of Candida albicans

Article abstract of DOI:10.1021/ja500036r

Fungal infections are a major challenge to human health that is heightened by pathogen resistance to current therapeutic agents. Previously, we were inspired by host-defense peptides to develop nylon-3 polymers (poly-β-peptides) that are toxic toward the fungal pathogen Candida albicans but exert little effect on mammalian cells. Based on subsequent analysis of structure-activity relationships among antifungal nylon-3 polymers, we have now identified readily prepared cationic homopolymers active against strains of C. albicans that are resistant to the antifungal drugs fluconazole and amphotericin B. These nylon-3 polymers are nonhemolytic. In addition, we have identified cationic-hydrophobic copolymers that are highly active against a second fungal pathogen, Cryptococcus neoformans, and moderately active against a third pathogen, Aspergillus fumigatus.

Full text of DOI:10.1021/ja500036r

Article
pubs.acs.org/JACS
Structure−Activity Relationships among Antifungal Nylon‑3
Polymers: Identification of Materials Active against Drug-Resistant
Strains of Candida albicans
Runhui Liu,†,‡ Xinyu Chen,† Shaun P. Falk,§ Brendan P. Mowery,† Amy J. Karlsson,∥,⊥
Bernard Weisblum,§ Sean P. Palecek,∥ Kristyn S. Masters,*,‡ and Samuel H. Gellman*,†
‡
§
∥
†Department of Chemistry, Department of Biomedical Engineering, Department of Medicine, and Department of Chemical and
Biological Engineering, University of Wisconsin, Madison, Wisconsin 53706, United States
S
* Supporting Information
ABSTRACT: Fungal infections are a major challenge to human
health that is heightened by pathogen resistance to current
therapeutic agents. Previously, we were inspired by host-defense
peptides to develop nylon-3 polymers (poly-β-peptides) that are
toxic toward the fungal pathogen Candida albicans but exert
little effect on mammalian cells. Based on subsequent analysis of
structure−activity relationships among antifungal nylon-3
polymers, we have now identified readily prepared cationic
homopolymers active against strains of C. albicans that are
resistant to the antifungal drugs fluconazole and amphotericin B. These nylon-3 polymers are nonhemolytic. In addition, we have
identified cationic−hydrophobic copolymers that are highly active against a second fungal pathogen, Cryptococcus neoformans, and
moderately active against a third pathogen, Aspergillus fumigatus.
Over the past decade, a variety of synthetic polymers have been
reported to display prokaryote-specific toxicity.21,23−35 Selectiv-
ity has typically been evaluated by assessing a polymer’s ability
to lyse human red blood cells (“hemolysis”); antibacterial activity
is often thought to depend on disruption of bacterial
membranes.36,37 The ideal profile is low minimum inhibitory
concentration (MIC) for bacteria but high minimum hemolytic
concentration (MHC) or concentration for 50% hemolysis
(HC50). In contrast to the numerous examples of antibacterial
polymers, there have been few reports of synthetic polymers
with antifungal activity. Chan-Park et al. recently described
cationic peptidopolysaccharides with good activity against a
drug-susceptible strain of Candida albicans (MIC = 20 μg/mL)
and little or no hemolytic activity.33 Earlier examples feature
nonphysiological assay conditions and/or low activities, and they
lack information regarding hemolysis or other measures of
toxicity toward mammalian cells.38,39
Nylon-3 materials were among the first synthetic polymers
reported to display an HDP-mimetic activity profile (substantial
growth inhibition for diverse bacterial species and low hemolytic
activity).27 Although many earlier studies had identified synthetic
polymers with antibacterial activity,40−48 such materials were
typically not evaluated in terms of toxic effects on mammalian
cells (e.g., hemolysis).21 We were attracted to the nylon-3
polymer class for biological applications because the protein-like
backbone, comprised of β-amino acid residues, seemed likely to
INTRODUCTION
■
Fungal infections are major threats to human health and difficult
to treat.1 Systemic fungal infections are especially dangerous
because they are associated with a high mortality rate, which
reflects the low efficacy and multiple side effects of current
antifungal drugs as well as the emergence of strains resistant to
current therapeutic agents.1 Pathogen resistance to fluconazole is
widespread,2−4 and resistance to amphotericin B (AmpB) is on
the rise.2,3,5 Identifying agents that specifically inhibit fungal
growth without deleterious effects on patients is challenging
because both fungi and humans are eukaryotes.1 The discovery of
host-defense peptides (HDPs) in the 1980s sparked extensive
exploration of these natural antimicrobial agents and synthetic
analogues for potential therapeutic applications.6−9 Many HDP
analogues that display antibacterial activity have been docu-
mented.7,10 In contrast, relatively few studies have characterized
the effects of HDPs and their analogues on fungi.11 The antifungal
activities reported are very sensitive to conditions, and some
assays may not be biologically relevant.12,13
The use of conventional peptides and other sequence-specific
oligomers (e.g., D-peptides,14,15 peptoids,16,17 β-peptides,18,19
α/β-peptides20) as antimicrobial agents is problematic because
production of these types of compounds is extremely costly. This
limitation has led many groups to examine synthetic polymers
as antimicrobial agents in recent years.21 Such efforts have
been motivated by the observation that diverse natural HDP
sequences display similar growth-inhibitory activities toward
bacteria, which suggests that the crucial feature may be simply an
appropriate balance of hydrophobic and cationic side chains.22
Received: January 2, 2014
Published: March 7, 2014
© 2014 American Chemical Society
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Figure 1. Racemic β-lactams and corresponding heterochiral cationic homopolymers.
Figure 2. Synthesis of the new β-lactam αNMβ (racemic).
confer biocompatibility.29,49−57 The identity of hydrophobic and
cationic subunits can be readily varied among nylon-3
copolymers, which facilitates the optimization of a particular
activity profile.27,28 However, our initial efforts to identify nylon-
3 polymers with selective toxicity toward the fungal pathogen
C. albicans were unsuccessful; antifungal polymers were always
found to be quite hemolytic (unpublished).
Recently, we evaluated a new family of nylon-3 polymers
that contain the βNM subunit (Figure 1).29 To our surprise,
poly-βNM with ∼20-mer average length displayed strong and
selective activity against C. albicans strain K1.58 This homo-
polymer was fungicidal at the MIC (3.1 μg/mL), but very little
hemolysis or toxicity toward 3T3 fibroblasts was detected even at
400 μg/mL. In contrast, the cationic homopolymer poly-MM
was only weakly active against C. albicans K1 (MIC = 200 μg/mL).
Cationic poly-DM was highly active against C. albicans (MIC =
6.3 μg/mL) but also highly hemolytic and moderately toxic to 3T3
fibroblasts.29
Favorable preliminary observations regarding the antifungal
properties of poly-βNM prompted us to undertake the additional
studies reported here. We have made comparisons among diverse
nylon-3 materials, and the results have elucidated the polymer
parameters that are crucial for achieving selective antifungal
activity; this information will support future efforts to optimize
materials for specific applications. We have identified nylon-3
polymers with substantial activity against pathogenic strains of
C. albicans that are resistant to AmpB and fluconazole. In
addition, we have found nylon-3 polymers that are active against
two other pathogenic fungi that commonly cause invasive human
infections, Cryptococcus neoformans and Aspergillus fumigatus.1
on Cβ for βNM. In order to determine whether the position
of the aminomethyl side chain is important in terms of bio-
logical activity profile, we have now prepared poly-αNM.
Nylon-3 materials are synthesized via anionic ring-opening
polymerization (AROP) of β-lactams;59,60 synthesis of a
precursor for poly-αNM, the β-lactam αNMβ, is outlined in
Figure 2. Racemic β-lactam 1 was prepared according to a reported
protocol61 and then converted to 2 via tosylate displacement.
Reduction of the azide to an amino group and Boc protection
provided 3, which was converted to αNMβ via reductive
debenzylation. AROP of this new β-lactam proceeded smoothly
under conditions previously described (Figure 3).29,60 An acid
chloride serves as a co-initiator by forming an N-acyl imide in situ;
the acyl group remains at the N-terminus of the resulting
polymer chains. Two acyl groups were examined in this work,
p-t-butylbenzoyl (tBuBz) and acetyl (Ac). The side-chain-
protected polymer displayed low polydispersity (Table 1).
Deprotection under acidic conditions provided poly-αNM.
Poly-αNM is indistinguishable from previously described
poly-βNM in terms of biological activity profile, which indicates
that the location of the aminomethyl side chain is not
functionally significant (Table 1; p-t-butylbenzoyl group at the
N-termini in both cases). Both nylon-3 polymers are fungicidal at
the MIC. Both are substantially more active than α-poly-L-lysine,
magainin 2 (an HDP from amphibians), or fluconazole (an
antifungal drug). Both nylon-3 polymers are slightly less active
than AmpB against C. albicans K1; however, both polymers are
superior to AmpB in terms of selectivity, based on comparisons
of the lowest concentrations required to cause 10% lysis in a
sample of human red blood cells (HC10). We define the
selectivity index (SI) as the ratio HC10/MIC, with higher values
corresponding to greater selectivity for fungal cell destruction.
SI > 130 for poly-αNM and poly-βNM, while SI = 1 for AmpB.
Because these two nylon-3 polymers have indistinguishable
activities, and because the synthesis of αNMβ is considerably
more time consuming than the synthesis of βNMβ, all
subsequent studies focused on poly-βNM.
RESULTS AND DISCUSSION
■
Location of the Aminomethyl Side Chain Is Unimportant:
Poly-αNM vs Poly-βNM. The nylon-3 subunit designated βNM
in Figure 1 (previously designated “NM”29) differs in two
ways from the MM and DM subunits. First, MM and DM both
have methyl substituents, while βNM does not. Second, the
aminomethyl side chain is located on Cα for MM and DM but
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Figure 3. Synthesis of heterochiral poly-αNM.
a
Table 1. Biological Activity Comparison of Poly-αNM, Poly-βNM, and Related Materials
b
c
d
e
f
g
polymer
DP
16
20
PDI
MIC (μg/mL)
MFC (μg/mL)
HC10 (μg/mL)
SI
poly-αNM
1.4
1.1
3.1
3.1
>400
>400
ND
ND
ND
0.8
>130
>130
ND
ND
ND
1
h
poly-βNM
3.1
3.1
α-poly-L-lysine
magainin 2
fluconazole
AmpB
27−103
NA
ND
NA
NA
NA
>200
>100
>200
ND
ND
ND
NA
h
h
NA
0.8
0.8
a
b
NA means not applicable. ND means not determined. Average degree of polymerization; both nylon-3 homopolymers have a p-t-butylbenzoyl at
c
d
e
the N-terminus. Polydispersity index. Minimum inhibitory concentration for C. albicans (K1 strain) growth in planktonic form. Minimum
f
fungicidal concentration, defined as >99.9% killing of C. albicans (K1 strain) cells. Concentration necessary for 10% lysis of human red blood cells.
g
h
Selectivity index (SI) calculated from HC10/MIC. All data for poly-βNM and antifungal data for AmpB were reported previously.29
a
Table 2. N-Terminal Group and Polymer Length Effects on the Antifungal and Hemolytic Activities of Poly-βNM
b
MIC (μg/mL)
c
c
c
c
d
d
d
N-terminal group
4-mer
6-mer
8-mer
20-mer
45-mer
70-mer
104-mer
HC10 (μg/mL)
t-BuBz
12.5
50
6.3
6.3
6.3
3.1
3.1
3.1
3.1
3.1
3.1
3.1
>400 for all polymers
Ac
12.5
ND
≥400 for all polymers
a
b
The K1 strain of C. albicans was tested in planktonic form. Minimum inhibitory concentration for C. albicans (K1 strain) growth in planktonic
form. The MIC value was also the MFC value. The polymer length was determined by both GPC and proton NMR. The polymer length was
determined by proton NMR only because of low solubility in the GPC mobile phase. ND indicates the MIC and HC10 values were not determined.
c
d
Drug-Susceptible vs Drug-Resistant Strains of C.
albicans: Importance of Poly-βNM Chain Length. The
impact of average chain length on the activity of poly-βNM
against C. albicans K1 was assessed for two polymer series, one
bearing p-t-butylbenzoyl groups and the other bearing acetyl
groups at the N-termini (Table 2). For the p-t-butylbenzoyl
series, very short chains, with averages in the 4-mer to 8-mer
range, are slightly less active than chains with average lengths of
20-mer or higher, up to a 104-mer average. A similar trend is seen
in the acetyl series. In all cases, the polymer samples displayed
little or no hemolytic activity. The identity of the N-terminal
group, p-t-butylbenzoyl vs acetyl, influenced antifungal activity
only among the very shortest oligomers, for which p-t-
butylbenzoyl seemed to be slightly more favorable.
The K1 strain of C. albicans is highly susceptible to antifungal
drug AmpB but less susceptible to fluconazole (Table 1). K1
appears to be resistant to fluconazole when evaluated in terms
of the MIC, the lowest concentration that allows no detectable
C. albicans growth; however, this drug is seen to display modest
activity if MIC50 (the concentration that inhibits growth by 50%)
is considered instead. MIC50 is widely used as a measure of
antifungal activity; we find that MIC50 = 3.1 μg/mL for
fluconazole against the K1 strain (Table 3).
We broadened our evaluation of polymer activity to C. albicans
strains other than K1, focusing on strains that are resistant to
standard drugs used clinically for human infections. The Gu5
strain is resistant to fluconazole,62 and the C4 and E4 strains are
resistant to both fluconazole and AmpB.63 Table 3 compares
activities against all four C. albicans strains for the poly-βNM
series with varying average chain length and p-t-butylbenzoyl at
the N-terminus. The pattern manifested for the Gu5 strain is
nearly identical to that for the K1 strain. Poly-βNM appears to be
less active against the C4 and E4 strains; however, significant
activity is observed when the polymer chains are sufficiently long
(maximal effect against C4 at 45-mer average length and against
E4 at 20-mer average length). Poly-βNM is fungicidal at the
MIC in all cases. The activity against C. albicans C4 and E4 is
noteworthy because neither of the clinical drugs, fluconazole or
AmpB, displays activity against these strains. The Gu5 strain is
resistant to fluconazole because of overexpression of the CDR1
and CDR2 genes, which encode the transmembrane drug
transporter proteins Cdr1p and Cdr2p.62 The C4 and E4 strains
are resistant to both fluconazole and AmpB because their
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Table 3. Antifungal Activities of Nylon-3 Polymers toward Drug-Resistant Strains of C. albicans
a
MIC (μg/mL)
compd
K1
Gu5
>200
C4
E4
fluconazole
>200
>200
>200
b
b
b
b
fluconazole
MIC50 = 3.1
MIC50 > 200
MIC50 > 200
MIC50 > 200
>200
>200
100
AmpB
0.8
12.5
6.3
6.3
3.1
3.1
3.1
3.1
0.8
12.5
6.3
6.3
6.3
3.1
3.1
3.1
≥200
>200
200
200
50
tBuBz-(βNM)4
tBuBz-(βNM)6
tBuBz-(βNM)8
tBuBz-(βNM)20
tBuBz-(βNM)45
tBuBz-(βNM)70
tBuBz-(βNM)105
50
12.5
25
12.5
25
12.5
25
12.5
a
b
Minimum inhibitory concentration for C. albicans growth in planktonic form. The MIC values were also the MFC values. The concentration to
inhibit 50% C. albicans growth in planktonic form.
Figure 4. Racemic β-lactams used to prepare nylon-3 polymers in this study.
Figure 5. Representative synthesis of βNM:CO nylon-3 copolymers. R could be the side chain of either subunit. All other nylon-3 copolymers in this
study were prepared in a similar manner using either THF or DMAc as the solvent.
membranes have reduced ergosterol content relative to other
C. albicans strains.63 The high activity of poly-βNM against the
Gu5 strain suggests that transmembrane drug transporter proteins
Cdr1p and Cdr2p cannot effectively counter the antifungal effects
of the nylon-3 polymers. The somewhat reduced activity of longer
poly-βNM toward the C4 and E4 strains of C. albicans, relative
to the K1 strain, suggests that ergosterol may play a role in the
nylon-3 mode of antifungal activity. However, since the longer
forms of poly-βNM manifest very significant activity against the
C4 and E4 strains, we conclude that ergosterol is either not the
direct target or not the only direct target of these polymers.
Cationic−Hydrophobic Nylon-3 Copolymers. Previ-
ously, we conducted an extensive survey of binary nylon-3
copolymers containing both cationic and hydrophobic subunits
in an effort to identify the hydrophilic−lipophilic balance that
is optimal for inhibition of bacterial growth without harm to
eukaryotic cell membranes.27,28 Two cationic subunits were
considered, those derived from MMβ and DMβ (Figure 4). This
study revealed that the identities of the cationic and hydrophobic
subunits and their proportion were critical determinants of
antibacterial and hemolytic activities. We have now conducted a
comparable study of the antifungal effects of binary nylon-3
copolymers containing βNM and varying hydrophobic subunits,
including cyclopentyl (CP), cyclohexyl (CH), cycloheptyl
(CHp), cyclooctyl (CO), and cyclododecyl (CD) (Figures 5
and 6). The impact of copolymer composition and proportion on
MIC for C. albicans K1 and on hemolysis, as indicated by HC10, are
summarized in Figure 7A. For comparison, data for two binary
copolymer series involving the MM cationic subunit are shown in
Figure 7B and data for two binary copolymer series involving the
DM cationic subunit are shown in Figure 7C.
Data for each of the five copolymer series containing the βNM
cationic subunit are depicted in separate plots in Figure 7A. The
leftmost data points, for the βNM homopolymer, are identical in
all five plots. Tracking the hemolytic data across the five plots
reveals a trend consistent with previous work: increasing
hydrophobic content leads to polymers with greater hemolytic
activity (lower HC10). For the least hydrophobic subunit, CP,
none of the binary copolymers display significant hemolytic
activity. For the more hydrophobic CH subunit, very modest
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Figure 6. Nylon-3 polymers used for structure−activity relationship studies. All polymers were heterochiral and prepared at an average length of 20-mer.
The MM:CHp and MM:CO copolymers had similar antifungal activity profiles; we provide data for only MM:CO copolymers. The MM:CP and
MM:CH copolymers displayed low antifungal activity; we provide data for only MM:CH copolymers. Most of the MM:CD, DM:CO, and DM:CD
copolymers had limited solubility in water and displayed high hemolytic activity; these polymers were not studied carefully.
hemolytic activity is detected only when the CH content reaches
50%. Increasing subunit hydrophobicity, with CHp or CO, leads
to substantial hemolytic activity for ≥20% hydrophobic subunit
content. For the most hydrophobic subunit, CD, considerable
hemolytic activity is evident even at 10% content.
two series shown, introduction of a hydrophobic subunit leads
to a modest increase in antifungal activity (lower MIC), with the
more hydrophobic CO subunit exerting a stronger antifungal
effect than the less hydrophobic CH subunit. In contrast, the data
for the binary nylon-3 copolymers containing DM (Figure 7C)
suggest that this cationic subunit behaves more like βNM than
MM in that DM units seem to exert an intrinsic antifungal effect.
In this set, antifungal activity declines when DM is partially replaced
with hydrophobic subunits. However, DM differs from βNM in
that the DM subunit seems to have an intrinsic hemolytic effect
(HC10 increases as DM subunits are replaced by hydrophobic
subunits), while the βNM subunit does not. The nature of the
apparent intrinsic antifungal effects of the βNM and DM cationic
subunits is unclear at present, as is the origin of the seemingly
discontinuous trend among the three cationic subunits, with
addition of one methyl group to the backbone (replacement of
αNM or βNM with MM) strongly diminishing the antifungal effect
but addition of a second methyl group (replacement of MM with
DM) restoring the intrinsic antifungal effect.
The trend in antifungal activities among the βNM-containing
copolymers requires a more complex interpretation than the
hydrophobicity-based explanation provided for hemolytic
activity trends in the preceding paragraph. Specifically, the data
in Figure 7A suggest that the βNM subunit manifests intrinsic
antifungal activity in nylon-3 chains, and that a sufficiently
hydrophobic nylon-3 subunit can compensate, at least partially,
for the removal of βNM subunits. The data for the βNM-CP
copolymer series supports the hypothesis that the βNM subunit
is intrinsically antifungal because introduction of only 10% CP
causes a substantial decline in antifungal activity (increase in
MIC). Further decline is observed as the CP proportion grows.
A comparable but smaller effect is observed in the βNM-CH
series, where inclusion of ≥30% CH leads to a decline in
antifungal activity. Declines in antifungal activity are observed
also with the more hydrophobic subunits; however, these
decreases in antifungal effect are muted relative to that seen for
CP, which suggests that the more hydrophobic subunits manifest
an antifungal effect that partially compensates for the loss of
βNM subunits.
The data for the MM and DM series in Figure 7B and C show
that antifungal activity cannot be achieved without promoting
hemolytic activity in these binary nylon-3 polymer families. In
other words, we were unable to identify a selective antifungal
agent within these polymer sets. In this context, the polymers
containing αNM or βNM cationic units stand out for their
selective antifungal activity profiles.
The data for the binary nylon-3 copolymers containing MM
(Figure 7B) suggest that this subunit does not manifest an
intrinsic antifungal effect, in contrast to the βNM subunit. For the
Activity against Other Pathogenic Fungi. We evaluated
nylon-3 polymers for the ability to inhibit growth of two other
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Figure 7. Antifungal activities (MIC) toward C. albicans (K1 strain) and hemolytic activities (HC10) of nylon-3 copolymers as a function of subunit
composition. Black crosses indicate MIC values, and red diamonds indicate HC10 values. All polymers had a p-t-butylbenzoyl group at the N-terminus. The lines
are drawn simply to connect the data points. Structures of the polymers are shown in Figure 6. Results for the βNM:CH polymers were reported previously.29
fungal species, C. neoformans and A. fumigatus, both of which
cause invasive infections in humans and are associated with
high mortality rates.1 The data in Table 4 show that poly-βNM
and βNM:CH copolymers display potent activity against
C. neoformans and excellent selectivity relative to mammalian
cells, nearly matching the performance of fluconazole. AmpB is
slightly more active than the polymers, but as noted above, this
drug has poor selectivity. Cationic polycarbonates active against
C. neoformans have very recently been described.64 A. fumigatus
proved to be a more challenging target. Poly-βNM is only very
weakly active, but moderate activity is observed for βNM:CH
copolymers with 40−50% CH. A. fumigatus is resistant to
fluconazole, but AmpB is active against this species.
βNMβ, MMβ, and DMβ for antifungal activity. However,
none of theses β-lactams displayed any activity against either the
K1 or Gu5 strain of C. albicans, even at 500 μg/mL (Table S11,
Supporting Information).
Conclusions. Data presented here show that nylon-3
polymers containing the cationic βNM subunit display favorable
antifungal behavior with little or no propensity to induce lysis of
human red blood cells. The βNM subunit appears to manifest
an intrinsic antifungal effect, enabling homopolymers to kill
strains of C. albicans that are resistant to the widely used drugs
AmpB and fluconazole. The isomeric αNM and βNM subunits
are comparable in terms of biological activity profile, but the
β-lactam precursor for αNM is time consuming to prepare,
so βNM-containing nylon-3 polymers are preferable from a
practical perspective. Poly-βNM displays potent activity against a
second human pathogen, C. neoformans, but this homopolymer
is not active against A. fumigatus. Supplementing βNM subunits
Table 2 shows that even very short versions of poly-βNM
display significant activity against the K1 strain of C. albicans.
All nylon-3 chains bear a C-terminal imide, which contains a
β-lactam unit. We therefore evaluated β-lactam starting materials
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the solvent. 1H NMR chemical shifts were referenced to residual
protonated solvent (δ 7.26 for CDCl3 and δ 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.
Synthesis of β-Lactams. 1-Benzyl-3-((p-toluenesulfonyloxy)-
methyl)-azetidin-2-one (1). Racemic β-lactam 1 was prepared as a
racemic mixture by following the reported method,61 which pro-
Table 4. Antifungal Activities against C. neoformans and
A. fumigatus
HC10
(μg/mL)
a
b
MIC (MIC50 ) (μg/mL)
C.
d
compd
neoformans
A. fumigatus
red blood cell
SI
fluconazole
1.6
0.8
6.3
3.1
3.1
3.1
3.1
3.1
3.1
>200 (>200)
6.3 (4.7)
>400
0.8
>250
1
1
vided the compound as a white solid. mp 100.8−101.7 °C. H NMR
AmpB
(400 MHz, CDCl3): δ 7.75 (d, J = 6.5 Hz, 2H), 7.28−7.35 (m, 5H), 7.21
(dd, J = 7.6, 1.8 Hz, 2H), 4.23 (d, J = 5.1 Hz, 1H), 4.20−4.31 (m, 3H),
3.45 (m, 1H), 3.23 (t, J = 5.6 Hz, 1H), 3.08 (dd, J = 5.9, 2.5 Hz, 1H). 13C
NMR (100 MHz, CDCl3): δ 165.33, 145.22, 135.02, 132.32, 130.01,
128.90, 128.04, 127.99, 127.85, 66.59, 49.02, 46.07, 42.45, 21.68.
HRESI-MS: m/z calcd for C18H23N2O4S [M+NH4]+, 363.1367; found,
363.1374.
50:50 βNM:CH
60:40 βNM:CH
70:30 βNM:CH
80:20 βNM:CH
90:10 βNM:CH
tBuBz-(βNM)20
tBuBz-(βNM)80
150 (50)
200
32
c
150 (50)
>400
>130
>130
>130
>130
>130
>130
c
200 (75)
>400
c
200 (100)
>200 (100)
>200 (200)
>200 (200)
>400
c
>400
c
>400
1-Benzyl-3-azidomethyl-azetidin-2-one (2). A mixture of β-lactam
1 (3.7 g) and sodium azide (2.1 g) in 6:1 EtOH:water (42 mL) was
heated at 60 °C for 10 h. After cooling, the mixture was concentrated
in vacuo to give a yellow oil. DI water (80 mL) was added to the reaction
mixture with shaking, and the crude product was extracted with CH2Cl2
(3 × 200 mL). The combined organic layers were washed with brine
(80 mL), dried over MgSO4, and concentrated in vacuo. The crude
product was purified by silica gel chromatography using 1:1
hexane:EtOAc as the eluent to give racemic β-lactam 2 as a colorless
oil (2.1 g, 91% yield). 1H NMR (400 MHz, CDCl3): δ 7.28−7.35 (m,
3H), 7.23 (d, J = 7.3 Hz, 2H), 4.30−4.44 (m, 2H), 3.62−3.65 (m, 2H),
3.38 (m, 1H), 3.24 (t, J = 5.5 Hz, 1H), 3.04 (dd, J = 5.8, 2.4 Hz, 1H).
13C NMR (100 MHz, CDCl3): δ 166.94, 135.29, 128.93, 128.19, 127.91,
49.26, 49.08, 46.14, 42.81. HRESI-MS: m/z calcd for C11H13N4O
[M+H]+, 217.1084; found, 217.1083.
1-Benzyl-3-(tert-butyloxycarbonyl aminomethyl)-azetidin-2-one
(3). Pd(OH)2 on activated charcoal (1.0 g) was added to a solution of
β-lactam 2 (2.0 g) in MeOH (50 mL), and the suspension was stirred
under a hydrogen atmosphere (∼1 atm) overnight. The reaction
mixture was then filtered, and the filtrate was concentrated in vacuo
to give a colorless oil. The oil was dissolved in MeOH (44 mL); Boc2O
(5.3 mL) and Et3N (258 μL) were added to this solution. The mixture
was heated to reflux for 6 h. After cooling, the mixture was concentrated
in vacuo to give an oil. DI water (80 mL) was added to the residue
with shaking, and the crude product was extracted with CH2Cl2 (3 × 200
mL). The combined organic layers were washed with brine (80 mL),
dried over MgSO4, and concentrated in vacuo. The crude product was
purified by silica gel chromatography using 1:1 hexane:EtOAc as the
eluent to give racemic β-lactam 3 as a colorless viscous oil (1.68 g, 63%
overall yield). 1H NMR (400 MHz, CDCl3): δ 7.28−7.36 (m, 3H), 7.23
(dd, J = 6.6, 1.8 Hz, 2H), 4.91 (br, 1H), 4.45 (d, J = 15.1 Hz, 1H), 4.30
(d, J = 15.1 Hz, 1H), 3.47 (m, 2H), 3.32 (m, 1H), 3.21 (t, J = 5.6 Hz,
1H), 3.00 (dd, J = 5.9, 2.4 Hz, 1H), 1.40 (s, 9H). 13C NMR (100 MHz,
CDCl3): δ 168.62, 156.19, 135.54, 128.95, 128.18, 127.86, 79.63, 50.18,
46.10, 42.77, 38.71, 28.42. HRESI-MS: m/z calcd for C16H23N2O3
[M+H]+, 291.1704; found, 291.1706.
3-(tert-Butyloxycarbonyl aminomethyl)-azetidin-2-one (αNMβ).
To a solution of β-lactam 3 (1.4 g) in EtOH (3 mL) at −78 °C, freshly
dried liquid ammonia (100 mL) was added. Sodium metal was added
slowly at −78 °C until the reaction solution became dark blue. The
reaction mixture was maintained at −78 °C for 15 min and then warmed
slowly to rt. After all ammonia had been removed from the reaction
flask by a flow of N2, the residue was mixed with aqueous NH4Cl. The
crude product was extracted with EtOAc (3 × 200 mL). The combined
organic layers were washed with brine (80 mL), dried over MgSO4, and
concentrated in vacuo. The crude product was purified by silica gel
chromatography using 80:1 CH2Cl2:MeOH to give racemic αNMβ as a
a white solid (0.68 g, 71% yield). mp 132−133 °C. 1H NMR (400 MHz,
CDCl3): δ 5.75 (br, 1H), 4.91 (br, 1H), 3.45−3.58 (m, 2H), 3.40 (m,
2H), 3.17 (m, 1H), 1.44 (s, 9H). 13C NMR (125 MHz, CDCl3): δ
170.03, 156.24, 79.68, 51.74, 39.63, 38.78, 28.42. HRESI-MS: m/z calcd
for C9H17N2O3 [M+H]+, 201.1234; found, 201.1241.
>400
a
Minimum inhibitory concentration for fungal cell growth in
b
planktonic form. The concentration to inhibit 50% of A. fumigatus
growth in planktonic form. These data were reported previously.29
c
d
Selectivity index for C. neoformans (HC10/MIC). ND means HC10
was not determined. The MIC50 results for these polymers against
A. fumigatus are included because MIC50 is a recommended activity
measurement method by the Clinical and Laboratory Standards
Institute (CLSI, previously known as NCCLS).65
with hydrophobic CH subunits is required to generate nylon-3
copolymers that are moderately inhibitory toward A. fumigatus.
The antifungal potency and low mammalian cell toxicity of
nylon-3 materials based on βNM are significant because fungal
infections remain major threats to human health, and new agents
that can treat or prevent such infections are therefore valuable.
Our interest in antimicrobial properties of nylon-3 polymers was
originally inspired by HDPs, but it is noteworthy that the ability
of many HDPs to inhibit fungal growth may be limited. Synthetic
polymers have a substantial synthetic advantage relative to
sequence-specific peptides, because peptides require a laborious
step-by-step approach while polymers can be rapidly generated
on a large scale. The resulting polymeric materials contain many
different chain lengths, but our data show that variations in poly-
βNM chain length have little effect on either antifungal activity or
hemolytic activity. The ease with which βNM-based nylon-3
polymers can be prepared raises the possibility that they may find
use for killing fungi on surfaces or perhaps even as clinical
antifungal agents.
EXPERIMENTAL METHODS
■
Materials and Methods. The K1 strain of C. albicans was a
generous gift from Professor David Andes at University of Wisconsin at
Madison; the Gu5 strain (MYA-574), the C4 strain (38245), and the E4
strain of C. albicans (38248), C. neoformans (ATCC MYA-737), and
A. fumigatus (ATCC 96918) were obtained from American Type
Culture Collection; amphotericin B (46006), fluconazole (PHR1160),
α-poly-L-lysine 4−15 KDa (P6516), 3-(N-morpholino) propanesulfonic
acid (MOPS, M3183), yeast extract peptone dextrose broth (YPD,
Y1375), and Sabouraud dextrose broth (S3306) were obtained from
Sigma-Aldrich (St. Louis, MO); magainin 2 (20640) was obtained
from AnaSpec; RPMI 1640 (31800-089) was obtained from Life
Technologies (Grand Island, NY); LB medium (244610) was obtained
from BD (Franklin Lakes, NJ); agar (BP1423500) was obtained from
Fisher Scientific (Pittsburgh, PA); and Easivial polymethyl methacrylate
(PMMA) standards for GPC column calibration (PL2020-0200) were
obtained from Polymer Varian (Palo Alto, CA). All other chemicals were
1
purchased from Sigma-Aldrich and used without purification. H and
13C NMR spectra were collected either on a Varian MercuryPlus 300
spectrometer at 300 and 75 MHz, respectively, or on a Bruker Avance III
spectrometer at 400 and 100 MHz, respectively, using CDCl3 or D2O as
Synthesis and Characterization of Nylon-3 Polymers. In a
moisture-controlled glovebox under dry N2, β-lactam monomers were
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Journal of the American Chemical Society
Article
weighed out into a predried glass vial and dissolved in 3 mL of
polymerization solvent, dimethylacetamide (DMAc) or THF, depend-
ing on the solubility of the resulting polymers. A 0.5 mL solution of
co-initiator (t-BuBzCl or AcCl) in DMAc or THF was added to the
reaction vessel in a predetermined ratio relative to β-lactams to control
the polymer length; e.g., a 1:20 co-initiator:β-lactam molar ratio was
used to prepare polymers with 20-mer average length. The monomers
and co-initiator were mixed with strong magnetic stirring, and the poly-
merization reaction was started by adding a 0.1−1 mL solution of
lithium bis(trimethylsilyl)amide (LiHMDS) in DMAc or THF to the
reaction mixture. The reaction mixture was adjusted to total 4 mL in
volume by adding more polymerization solvent and stirred at rt for 12 h.
Then, the reaction mixture was removed from the glovebox, and the
reaction was quenched by adding a few drops of MeOH. The resulting
polymer was isolated by precipitation after a “poor” solvent had been
added (see following paragraph).
Polymers containing αNM or βNM were prepared in DMAc. In these
cases, the quenched reaction mixture was combined with pentane
(40 mL), which caused a light yellow oil to separate from the solution.
The oil was collected after centrifugation and dissolved in THF (2 mL)
with the assistance of vortex mixing and ultrasonication. Pentane
(40 mL) was then added to induce polymer precipitation. If the oil
collected from the first phase separation would not dissolve in THF,
MeOH (0.5−1 mL) was used instead, and then Et2O (43 mL) was
added to induce polymer precipitation. After four dissolution/
precipitation cycles, the side chain amine-protected polymer was
collected as a white solid. This material was dried under vacuum
overnight and then subjected to gel permeation chromatography (GPC)
characterization using DMAc as the mobile phase.
Polymers containing MM or DM were prepared in THF. In these
cases, the quenched reaction mixture was combined with pentane
(40 mL), which caused precipitation. The precipitate was collected after
centrifugation and dissolved in THF (2 mL), and then, pentane (40 mL)
was added to induce polymer precipitation. After three dissolution/
precipitation cycles, the side chain amine-protected polymer was collected
as a white solid. This material was dried under vacuum overnight and then
subjected to GPC characterization using THF as the mobile phase.
To remove Boc protecting groups from side chain amino groups,
the protected polymer was treated with neat trifluoroacetic acid (TFA,
2 mL) at rt for 2 h with shaking. The deprotected polymer was
precipitated as a white fluffy solid by addition of Et2O (40 mL) into the
TFA solution. The solid was collected after centrifugation and dissolved
in MeOH (2 mL), and then precipitated again by addition of Et2O
(40 mL). After three dissolution/precipitation cycles, the deprotected
polymer was collected, dissolved in Milli-Q water, and lyophilized to
provide a white powder (TFA salt of the polymer). For the homo-
polymers poly-αNM and poly-βNM, the collected solid was sometimes
yellowish.
Polymer Characterization by GPC Using DMAc as the Mobile
Phase. The side chain amine-protected polymer was dissolved in
DMAc (supplemented with 10 μM LiBr) at 2 mg/mL. The polymer
was swollen in DMAc for at least 20 min and then passed through
a 0.2 μm polytetrafluoroethylene (PTFE) filter before GPC analysis.
A Waters GPC instrument was used. The instrument was equipped with
a refractive index detector (Waters 2410) and two Waters Styragel HR
4E columns (particle size 5 μm) linked in series. The mobile phase ran
at 1 mL/min at a column temperature of 80 °C. The number-average
molecular weight (Mn), weight-average molecular weight (Mw), and
polydispersity index (PDI) were calculated using the Empower software
provided by Waters and calibration curves obtained from at least nine
PMMA standards (peak average molecular weight ranging from 690 to
1 944 000). The degree of polymerization (DP) for a particular polymer
was calculated on the basis of the deduced Mn value, the initial ratio of
β-lactam monomers in the reaction mixture, and the molecular weight of
the β-lactam monomers, as described previously.28
with a multiangle light scattering detector (Wyatt miniDAWN, 690 nm,
30 mW), a refractive index detector (Wyatt Optilab-rEX, 690 nm), and
two Waters columns (Styragel HR 4E, particle size 5 μm) linked in
series. The mobile phase ran at 1 mL/min at a column temperature of
40 °C. Mn, Mw, and PDI were calculated using ASTRA 5.3.4.20 software
based on a dn/dc value of 0.1 mL/g. DP for a particular polymer was
calculated on the basis of the deduced Mn value, the initial ratio of
β-lactam monomers in the reaction mixture, and the molecular weight of
the β-lactam monomers, as described previously.28
Antifungal Activity Assay (MIC, MIC50, and MFC). The MIC and
MIC50 assays for C. albicans and C. neoformans were conducted
according to a previously described protocol.66 The MIC and MIC50
assays for A. fumigatus were conducted using the M38-A2 protocol
suggested by the Clinical and Laboratory Standards Institute (CLSI,
previously known as NCCLS).65 C. albicans and C. neoformans cells
were inoculated in YPD broth (for C4 and E4 strains of C. albicans)
or RPMI medium (for K1 and Gu5 strains of C. albicans and
C. neoformans), and the sample was incubated at 30 °C for 2−3 days.
The YPD medium for C4 strain culture was supplemented with
150 units/mL of nystatin.
The cultured C. albicans or C. neoformans cells were collected by
centrifugation and suspended in 0.145 M NaCl at 2.5 × 106 cells/mL to
generate the stock suspension. A. fumigatus cells were inoculated on a
Sabouraud dextrose agar plate and incubated at 37 °C for 4−5 days. The
plate surface was flooded with 15 mL of RPMI and gently rubbed with a
sterile cotton swab. The RPMI suspension was transferred to a 50 mL
centrifuge tube and vortexed vigorously for 15 s. After the mixture stood
for 15 min, the supernatant was transferred to a new centrifuge tube, and
cells were collected after centrifugation.67 The collected pellet was
dispersed in RPMI and subjected to two precipitation/dispersion cycles.
The cells were then suspended in RPMI with the density adjusted to
(0.5−1) × 107 cells/mL to generate the stock suspension. The working
suspension for each type of fungal cell was prepared as a 1:1000 dilution
of the stock suspension using adjusted RPMI 1640 medium (containing
L-glutamine but not sodium bicarbonate) buffered with 0.145 M MOPS.
Two-fold serial dilution of nylon-3 polymers, fluconazole, and AmpB
was conducted in a 96-well plate using adjusted RPMI to obtain con-
centrations from 400 to 0.4 μg/mL. Each sample well had a 100 μL
compound solution after the 2-fold serial dilution. Then, 100 μL of the
cell working suspension was added to each well (except the cell-free
blank control), followed by gentle shaking of the plate for 10 s. The plate
was then incubated at 37 °C for 2−3 days (E4 strains of C. albicans cells
were incubated for 4 days), and fungal cell growth was evaluated visually.
On the same plate, wells containing RPMI medium only were used as
the negative control (cell-free blank); wells containing cells in RPMI
without any polymer or antifungal drug were used as the positive
control. The antifungal MIC was the lowest concentration of a polymer
or antifungal drug that completely inhibited fungal cell growth; that is,
no cell colony was visible in the well. The antifungal MIC50 was the
concentration of a polymer or antifungal drug that inhibited 50% of
fungal cell growth. Each experiment was performed in duplicate on a
given day, and experiments were repeated on two different days.
The minimum fungicidal concentration (MFC) assay for C. albicans
was conducted after the MIC test. Aliquots of 10 μL of well-mixed
suspension were transferred to YPD agar plates from cell−polymer
mixtures containing polymer concentrations from one dilution below
the MIC to the highest polymer concentration. The YPD agar plates
were incubated at 37 °C for 48 h and then inspected visually for
C. albicans colony formation. The MFC was defined as the lowest
polymer concentration to kill all (>99.9%) the cells, which means no
C. albicans colony was observed.
Hemolysis Assay. Hemolysis assays were conducted as previously
described using human red blood cells (RBCs).66,68 Human whole
blood (5 mL) was washed three times with Tris-buffered saline (TBS,
pH 7.2) that was composed of 10 mM Tris and 150 mM NaCl. The
collected RBCs were suspended in TBS (250 mL) to obtain a working
suspension of 2% RBC relative to total RBCs in the whole blood. Two-
fold serial dilution of nylon-3 polymers was conducted in a 96-well plate
in TBS to obtain concentrations ranging from 800 to 6.25 μg/mL. Each
sample well had 100 μL of compound solution after the 2-fold serial
Polymer Characterization by GPC Using THF as the Mobile
Phase. The side chain amine-protected polymer was dissolved in
THF at 10 mg/mL. The polymer was swollen in THF for at least 20 min
and then passed through a 0.2 μm PTFE filter before GPC analysis.
A Shimadzu GPC instrument was used. The instrument was equipped
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Journal of the American Chemical Society
Article
dilution. Then, 100 μL of the RBC working suspension was added to
each well, followed by gentle shaking of the plate for 10 s. On the same
plate, wells containing TBS without polymer were used as the blank;
wells containing Triton X-100 (3.2 mg/mL in TBS) were used as the
positive control. The plate was incubated at 37 °C for 1 h, and then
centrifuged at 3700 rpm for 5 min to precipitate the RBCs. An aliquot
of 80 μL of the supernatant from each well was transferred to the
corresponding well in a new 96-well plate, and the optical density (OD)
at 405 nm was measured using a Molecular Devices Emax precision
microplate reader. Measurements were performed in duplicate, and
each measurement was repeated on two different days. The percentage
of hemolysis at each polymer concentration was calculated from
“% hemolysis = 100 × (Apolymer − Ablank)/(Acontrol − Ablank)” and plotted
against polymer concentration to give the dose−response curves for
hemolysis for each polymer. The HC10 value for each polymer was
defined as the polymer concentration to cause 10% lysis of RBCs.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Bioassay results and compound characterization spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(27) 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,
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AUTHOR INFORMATION
Corresponding Authors
kmasters@wisc.edu
■
(28) Mowery, B. P.; Lindner, A. H.; Weisblum, B.; Stahl, S. S.; Gellman,
gellman@chem.wisc.edu
S. H. J. Am. Chem. Soc. 2009, 131, 9735.
(29) Liu, R. H.; Chen, X. Y.; Hayouka, Z.; Chakraborty, S.; Falk, S. P.;
Weisblum, B.; Masters, K. S.; Gellman, S. H. J. Am. Chem. Soc. 2013, 135,
5270.
(30) Song, A. R.; Walker, S. G.; Parker, K. A.; Sampson, N. S. ACS
Chem. Biol. 2011, 6, 590.
Present Address
⊥Department of Chemical and Biomolecular Engineering,
University of Maryland, College Park, MD. 20742
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.
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ACKNOWLEDGMENTS
■
This research was supported by the NIH (R21EB013259,
R01AI092225, and R01GM093265). In addition, we are grateful
for support from the UW-Madison Nanoscale Science and
Engineering Center (DMR-0832760) during early phases of the
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