Amphipathic β-strand mimics as potential membrane disruptive antibiotics

Article abstract of DOI:10.1021/jo900933r

(Chemical Equation Presented) In recent years, there have been increasing numbers of bacterial strains emerging that are resistant to the currently available antibiotics. In the search for new antibiotics, attention has been focused on natural antimicrobi

Full text of DOI:10.1021/jo900933r

pubs.acs.org/joc
Amphipathic β-Strand Mimics as Potential Membrane
Disruptive Antibiotics
Jessica L. Watson† and Elizabeth R. Gillies*,†,‡
†Department of Chemistry and ‡Department of Chemical and Biochemical Engineering,
The University of Western Ontario, 1151 Richmond Street, London, Ontario, Canada N6A 5B7
egillie@uwo.ca
Received May 4, 2009
In recent years, there have been increasing numbers of bacterial strains emerging that are resistant to
the currently available antibiotics. In the search for new antibiotics, attention has been focused on
natural antimicrobial peptides that act by selectively disrupting the membranes of bacterial cells, a
mechanism that is thought to be nonconducive to the development of resistance. It is desirable to
mimic the structures and activities of these peptides while introducing properties such as resistance to
proteolytic degradation, which make molecules more ideal for development as drugs. Described here
is the design and synthesis of β-strand mimetic oligomers based on alternating R-amino acids and
azacyclohexenone units that segregate cationic lysine and hydrophobic valine side chains on opposite
1
faces of the β-strand. H NMR dilution studies demonstrated that despite the incorporation of
alternating D- and L-amino acids in order to obtain facial amphiphilicity, these oligomers are capable
of dimerizing to β-sheet mimics in a manner similar to the oligomers containing all L-amino acids. The
ability of the molecules to disrupt phospholipid vesicles mimicking the membranes of both bacterial
and mammalian cells was investigated using a fluorescent dye leakage assay. Several of the oligomers
were found to exhibit activity and selectivity for the bacterial over mammalian membranes. Overall,
these studies demonstrate the promise of this class of molecules for the development of new potential
antibiotics and provide information on the structural features that are important for activity.
Introduction
and defensins,6 exhibit a diverse range of structures and
activities, but a common feature underlying these classes is
the ability of the molecules to adopt conformations in which
hydrophobic and cationic amino acids are spatially clustered
in discrete regions or faces of the molecules.1 This structural
feature is referred to as amphipathicity. It is proposed that
the cationic groups interact selectively with anionic phos-
pholipids,7 lipopolysaccharides,8 or teichoic acids9 on the
surfaces of bacterial cells while the hydrophobic groups
The increasing emergence of bacterial strains that are resis-
tant to available antibiotics is currently motivating significant
interest in new approaches to the treatment of bacterial infec-
tions. In natural systems, antimicrobial peptides are often the
first line of defense against bacteria.1,2 These molecules, includ-
ing families such as the cecropins,3 magainins,4 protegrins,5
(1) Zasloff, M. Nature 2002, 415, 389–395.
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DOI: 10.1021/jo900933r
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Watson and Gillies
JOCArticle
facilitate membrane penetration and disruption via toroidal
pore,10 carpet,11 or barrel stave mechanisms,12 leading to the
leakage of the cell contents and ultimately resulting in
bacterial cell death. This mode of action is of particular
interest for the development of new therapeutics as it is
thought to be relatively nonspecific and thus not very con-
ducive to the development of resistance.13
Despite the significant progress in the development of
synthetic membrane-disruptive oligomers, the mechanism
of action and structure-property relationships are still not
fully understood, and new antibiotics are still badly needed.
In addition, there has been very little work thus far on
peptide-based oligomers that assume amphipathic linear or
β-strand-like conformations. Prior successes using the linear
aromatic oligomers25-27 as well as an example of a highly
active R-peptide capable of adopting an amphipathic β-sheet
structure33 suggest that such a design would be successful.
Although synthetic analogues of protegrins have been devel-
oped to mimic their rigid antiparallel two-stranded β-sheet
structure which is stabilized by disulfide bonds, these struc-
tures have focused on the incorporation of the new turn
inducing elements, and like the protegrins, they contain
segregated domains of cationic groups at each end of the
β-sheet.34,35 Reported here is the first example of an amino
acid based β-strand peptidomimetic that is designed to have
an amphipathic structure in which cationic amino acid
side chains are directed to one face of the strand and
hydrophobic groups are directed toward the opposite face,
with the aim of developing a new scaffold for membrane-
disruptive antibiotics.
Several synthetic oligomers designed to mimic an elon-
gated β-strand conformation have been developed, although
a limited number of these allow for the incorporation of the
functional side chains required to access the target amphi-
pathic structures.36-39 For the current work, oligomers
based on alternating R-amino acids and azacyclohexenone
(Ach) units (Figure 1a), developed and termed @-tides by
Bartlett and co-workers, were selected as the backbone.39
The replacement of alternating amino acids with the Ach
units provides conformational restriction which favors elon-
gated conformations and the tertiary amide limits hydrogen
bonding to one edge of the strand, preventing uncontrolled
aggregation. They also possess attractive features such as
resistance to proteolytic degradation40 and the easy incor-
poration of the Ach unit by a flexible, modified peptide
synthesis.39,41 Similar analogues have been demonstrated to
bind to the PDZ domains of proteins more strongly than
their natural β-sheet ligands.40 In the work described here,
new amphipathic @-tides were designed and synthesized,
and their abilities to potentially serve as membrane active
antibiotics were investigated by studying the release of
encapsulated fluorescent dye molecules from phospho-
lipid vesicle models of both bacterial and mammalian cell
Inspired by their structures and activities, there has been
significant interest in the development of new synthetic
mimics of the naturally occurring antimicrobial peptides.
These synthetic molecules are providing important insights
into the mechanism of action of membrane-disruptive pep-
tides14-16 while at the same time introducing simplified
sequences and resistance to proteolytic degradation,17
a
problem that plagues natural R-peptides as potential drug
candidates. Thus far, synthetic oligomers based on R- and
β-amino acids18-23 as well as peptoids24 that adopt helical
amphipathic structures in the presence of membranes have
been developed, providing good antibiotic activity and se-
lectivity. Aromatic oligomers based on amides25 and ureas,26
as well as oligo(phenylene ethynylene)s27 that exhibit ex-
tended conformations, have also been investigated, provid-
ing promising results. Recent work has also shown that
through careful balancing of their cationic charge and
hydrophobicity, amphiphilic polymers can provide desirable
activities.28-32
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peptidomimetics have generally consisted of larger fractions
of hydrophobic than hydrophilic residues.20,21,23 However,
it was anticipated that the azacyclohexenone units in the
@-tides would introduce significant hydrophobicity to the
structures, so in order to obtain at least the minimal
water solubility required for the subsequent evaluation of
the molecules, structures with equal or greater numbers of
lysine relative to valine groups were targeted.
Synthesis. The target molecules were synthesized by a
convergent solution-phase approach based on modifications
to the method reported by Phillips et al.41 First, as shown in
Scheme 1, the previously reported benzyl carbamate (Cbz)
protected @-tide unit 141 was reacted with either D-valine or
ε-Boc-L-lysine to provide the corresponding condensation
products 2 and 3, respectively, which will be referred to as
dimers throughout this discussion. Without further purifica-
tion, the L-lysine derivative 3 was converted to the methyl
ester 4. After removal of the Cbz group by catalytic hydro-
genolysis in methanol, the resulting dimer amine 5 was
coupled with dimer acid 2 using O-(7-azabenzotriazol-
1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate
(HATU) as the coupling agent in N,N-diisopropylethyl-
amine (DIPEA) and DMF to provide the tetramer 6.
The Cbz protecting group on the tetramer 6 was then
removed by hydrogenolysis, providing 7, which was then
coupled to R-acetyl-ε-Boc-L-lysine44 to provide the pentamer
8 as shown in Scheme 2. Alternatively, the tetramer was
coupled with the dimer 3 to provide the hexamer 9. Depro-
tected versions of the pentamer and hexamer were prepared
for evaluation of the membrane disruptive capabilities. The
Boc protecting group on the pentamer 8 was removed by
treatment with a 1/1 TFA/CH2Cl2 solution to provide the
dicationic pentamer 10. The Boc groups on the hexamer 9
were removed under the same conditions to provide the
dicationic oligomer 11.
FIGURE 1. Structure of an @-tide comprising (a) all L-amino acids
alternating with azacyclohexenone units and (b) hydrophobic
D-amino acids and hydrophilic L-amino acids alternating with
azacyclohexenone units.
membranes. In addition, the propensity of these modified
oligomers to assemble into β-sheet structures in solution was
investigated and compared to the results obtained for the
original @-tides described by Bartlett and co-workers.39
Several of the oligomers were found to exhibit membrane
disruptive activity as well as selectivity for the bacterial over
mammalian cell membrane models. These studies therefore
introduce a promising new template for the development of
potential antibiotics and provide important insights into the
structural features that are critical for activity in this class of
molecules.
Results and Discussion
In addition, it was of interest to investigate the effect of
incorporating the alternating D- and L-amino acids to poten-
tially provide facially amphiphilic structures in the linear
β-strand conformations, in comparison with oligomers con-
taining all L-amino acids. Thus, molecules corresponding to
2, 6, 8, 9, 10, and 11, but containing only L-amino acids were
prepared by the same methods described above. These
molecules will be referred to as 20, 60, 80, 90, 100, and 110
respectively. It is also noteworthy that while none of the
oligomers described above are very long and do not possess
highly charged states, high antibiotic activity and selectivity
have been previously observed for aromatic amide- and urea-
based oligomers of similar lengths.15,25,26 Therefore, this
initial series of molecules was expected to give valuable
insights into the activity of this class of molecules.
Design. The common structural feature of most membrane
disruptive antimicrobial peptides is the segregation of hydro-
phobic and cationic groups into different regions of the
molecule, such as on opposite faces of a helix.1 In a β-strand
based entirely on L-R-amino acids, the side chain groups will
naturally diverge to opposite faces of the strand. However, in
the @-tides, as every second amino acid is replaced by an
azacyclohexenone unit, all of the side chains are expected to
be directed to the same face of the strand in its linear
conformation as shown in Figure 1a.39 Therefore, the main
design modification made to the previously reported @-tide
template was to alternate L- and D-amino acids. By subse-
quently alternating hydrophobic and cationic amino acids as
shown in Figure 1b, it was anticipated that an amphipathic
structure should be presented in the @-tide’s linear confor-
mation. As a cationic residue, L-lysine was selected because
aliphatic amines have been one of the most commonly and
successfully used cations in the development of antimicrobial
peptidomimetics.19,25,29 D-Valine was selected as the hydro-
phobic residue because of its propensity to form β-strands
and β-sheets.42,43 The previously reported antimicrobial
Oligomers up to the tetramer length (6, 60) were characte-
rized by the standard methods used for small molecules,
1
using techniques including H NMR, 13C NMR, and IR
spectroscopy as well as HRMS. All characterization data
were consistent with the proposed structures. Due to the
increasing broadness and complexity of the NMR spectra
with increasing length and solubility constraints, the penta-
mers and hexamers were characterized mainly by HPLC and
HRMS, techniques that are standard for the characteriza-
tion of oligopeptides, along with 1H NMR spectroscopy.
(42) Rossmeisl, J.; Kristensen, I.; Gregersen, M.; Jacobsen, K. W.;
Norskov, J. K. J. Am. Chem. Soc. 2003, 125, 16383–16386.
(43) Fasman, G. D. In Prediction of Protein Structure and the Principles of
Protein Conformation; Fasman, G. D., Ed.; Plenum Publishing: New York,
1989.
(44) Zheng, Y.; Duanmu, C.; Gao, Y. Org. Lett. 2006, 8, 3215–3217.
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SCHEME 1
SCHEME 2
N-H proton were measured in pure CDCl3, 99/1 CDCl3/
CD3OH, and 97.5/2.5 CDCl3/CD3OH as a function of con-
centration and are shown in Figure 2. Unfortunately, despite
the use of 2D NMR experiments including NOESY, COSY,
and HMBC it was not possible to unambiguously assign this
peak to a specific N-H of 8 due to the high symmetry of the
molecule relative to those previously reported,39 as well as
the broadness and overlap of many of the peaks in the
spectra. However, to determine the dissociation constant
Kd for the dimerization, the NMR chemical shift data was fit
to eq 1 using a nonlinear curve fitting procedure,46 where δs is
the chemical shift of the nondimerized oligomer, Δδ is the
difference in chemical shifts between the dimerized and
nondimerized oligomer, and co is the concentration of the
oligomer. This provided Kd’s of 0.2 ( 0.4, 0.7 ( 0.4, and
1.6 ( 0.6 mM in pure CDCl3, 99/1 CDCl3/CD3OH, and 97.5/
2.5 CDCl3/CD3OH, respectively. The increasing Kd’s with
increasing CD3OH content are consistent with the disruptive
effect of CD3OH on the expected hydrogen-bonded dimers.
To investigate the effect of oligomer length, the Kd’s for both
the tetramer 6 and hexamer 9 were also measured using the
same method. In 97.5/2.5 CDCl3/CD3OH their respective
Kd’s were 34 ( 3 and 5.7 ( 1.3 mM. The higher Kd for the
tetramer 6 was expected because its shorter length allows for
the formation of a maximum of four hydrogen bonds in the
dimerized state. In contrast, both the pentamer 8 and the
hexamer 9 are capable of forming a maximum of six hydro-
gen bonds in the dimerized state and were therefore expected
to exhibit similar Kd’s, which was indeed observed. These
values are of approximately the same order of magnitude
as those obtained by Phillips et al. for the dimerization of
@-tides of similar lengths in these solvents.39
Self Association of @-tides in CDCl3/CD3OH Mixtures.
Phillips et al. have reported extensive studies to demonstrate
that @-tides of various lengths and compositions assemble in
organic and aqueous solutions to form β-sheets.39,45 While
the formation of such assemblies is probably not critical to
achieving membrane disruptive activity, it is nevertheless of
interest to investigate the effect of alternating the amino acid
stereochemistry on this potential dimerization, as it can
provide some insight into the conformational preferences
of the molecules and their potential to assume amphipathic
conformations in the presence of membranes. Therefore, the
approximate self-association constants for several oligomers
in CDCl3 and CDCl3/CD3OH mixtures were determined.
These studies were carried out using the NMR dilution
method.46 First, a solution of 8 was prepared and was
gradually diluted. The chemical shifts of the most downfield
Overall, the magnitudes of the Kd’s, their dependence on
the CD3OH concentration, and their dependence on the
oligomer length suggest that the oligomers containing alter-
nating D- and L-amino acids are capable of dimerizing to
form β-sheet mimics in the same manner as the previously
well-characterized @-tides containing all L-amino acids.
This indicates that under some conditions these oligo-
mers should be capable of forming elongated amphipathic
(45) Phillips, S. T.; Blasdel, L. K.; Bartlett, P. A. J. Am. Chem. Soc. 2005,
127, 4193–4198.
(46) Wilcox, C. S. In Frontiers in Supramolecular Organic Chemistry and
Photochemistry; Schneider, H. J., Durr, H., Eds.; VCH: New York, 1991; pp
123-143.
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contain negatively charged phospholipids, lipopolysacchar-
ides, and teichoic acids on their surfaces.47 The phospho-
lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was
chosen for the preparation of SUVs mimicking mammalian
cell membranes as eukaryotic cell membranes are rich in PC
lipids.15
Thus far, several techniques have been used to investi-
gate the disruption of phospholipid membranes by antimi-
crobial peptides and peptidomimetics,14,15 but by far the
most widely used methods involve fluorescence.15,16,22,25
Generally, a water-soluble dye is entrapped in the vesicle
core during vesicle formation at a concentration that is
sufficiently high to provide fluorescence quenching. Upon
the addition of the membrane-active molecules, and rapid
disruption of the membranes, the dye molecules are released
from the vesicles, resulting in a significant dilution and the
recovery of their fluorescence. In the current work, the
8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS)
and p-xylene-bis(N-pyridinium bromide) (DPX) fluorescent
probe system was selected.48 HPTS undergoes efficient self-
quenching at moderately high concentrations in the presence
of DPX, a collisional quencher.49 Thus, the new oligomers
were added to the HPTS-DPX-loaded vesicles at varying
concentrations from DMSO solutions. Small volumes of
DMSO were used to dissolve the oligomers due to difficulties
in dissolving some of the more hydrophobic molecules
directly in the aqueous buffer. However, none of the oligo-
mers were found to precipitate upon addition of the DMSO
solutions to the aqueous buffer at the concentrations eval-
uated. The initial fluorescence intensity was taken as 0%
HPTS-DPX release, and it was verified that the small
quantities of DMSO that were used to dissolve the @-tides
for their addition to the vesicles did not lead to any changes
in fluorescence. At the end of each experiment, Triton X-100,
a well-known membrane disruptive surfactant, was added to
completely lyse the vesicles and the observed fluorescence
intensity was used to indicate 100% HPTS-DPX release.
In preliminary work, it was found that @-tide oligomers
with a carboxylic acid terminus did not lyse either the DOPE/
DOPG or the DOPC vesicles to any measurable degree. This
may be due to an insufficient cationic charge as the terminal
carboxylic acid would cancel the charge of one of the two
lysines, leaving an overall positive charge of only þ1 on the
molecule. Alternatively, the presence of the charged carboxy-
late ion may make the molecule insufficiently hydrophobic
for membrane disruptive activity. Therefore, the current
efforts focused on only the evaluation of the methyl ester
derivatives. As shown in Figure 3a, the pentamer 10, with an
overall positive charge of þ2, exhibited a significant degree
of DOPE/DOPG membrane lysis at 100 μg/mL, with greater
than 50% of the dye molecules released in 3 min. Hexamer
11, also having an overall charge of þ2, exhibited a similar
degree of membrane lysis after 3 min, but the kinetics of dye
release were somewhat slower than for the pentamer 10.
To investigate the role of the cationic ε-amines of lysine in
the membrane disruptive activity, the protected tetramer 6,
FIGURE 2. Chemical shifts of the farthest downfield N-H proton
of pentamer 8 in 1H NMR spectroscopy as a function of solvent and
concentration. The concentration dependence is indicative of
dimerization of 8 to β-sheet mimics.
conformations. However, the relatively high association
constants and the strong effect of hydrogen-bonding sol-
vents observed in our studies and previously reported by
Phillips et al.39,45 for oligomers of these relatively short
lengths suggests that they would not spontaneously assemble
into dimers in aqueous solution at the concentrations rele-
vant for antimicrobial activity, prior to their interactions
with membranes. In addition, it should be noted that while
circular dichroism (CD) spectroscopy has been previously
used to elucidate the extent of @-tide dimerization in a
variety of solvents, this technique was not suitable for the
analysis of oligomers containing D- and L-amino acids due to
the requirement of having two L-amino acids surrounding
the @-unit in order to observe the characteristic signal near
280 nm.39,45
2
3
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢀ
ꢁ
ꢀ
ꢁ
Kd
Kd
Kd
c0
4
5
δobs ¼ δs þΔδ 1þ
-
2þ
ð1Þ
2c0
2c0
Assessment of Membrane Disruptive Potential Using a
Vesicle Leakage Assay. Numerous studies have demon-
strated that the ability of membrane-disruptive antimicro-
bial peptides to kill bacterial cells via membrane lysis
generally coincides with their ability to disrupt and lyse
the phospholipid membranes of small unilamellar vesicles
(SUVs).15,16,22,25 Such studies have provided insight into the
mechanism of action of these molecules. In addition, in order to
predict the selectivity of the molecules for bacterial over mam-
malian cell membranes, it is possible to choose SUVs that mimic
either bacterial or mammalian cell membranes.15,16 In this
study, as previously reported by Yang et al.,15 an 80/20 ratio
of the lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE)/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]-
(sodium salt) (DOPG) were selected to mimic bacterial cell
membranes. Most Gram-negative bacterial membranes are
rich in PE lipids, which have a relatively small headgroup and
therefore a tendency to promote negative curvature.16 DOPG
is anionic, which is characteristic of bacterial membranes that
(47) Brock, T. D. Biology of Microorganisms, 2nd ed.; Prentice Hall:
Englewood Cliffs, NJ, 1974.
(48) Bai, Y.; Louis, K. L.; Murphy, R. S. J. Photochem. Photobiol. A 2007,
192, 130–141.
(49) Daleke, D. L.; Hong, K.; Papahahjopoulos, D. Biochim. Biophys.
Acta 1990, 1024, 352–366.
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FIGURE 3. Detection of membrane disruption based on the leakage of encapsulated HPTS and its quencher DPX from vesicles: (a) oligomers
based on alternating D- and L-amino acids assessed in vesicles of 80/20 DOPE/DOPG (bacterial mimic); (b) oligomers based on alternating
D- and L-amino acids assessed in vesicles of DOPC (mammalian mimic); (c) oligomers based on all L-amino acids assessed in vesicles of
80/20 DOPE/DOPG; (d) oligomers based on all L-amino acids assessed in vesicles of DOPC. The oligomer concentration was 100 μg/mL
in each case.
pentamer 8, and hexamer 9 were also investigated. Quite
unexpectedly, all of these molecules were found to be active
in DOPE/DOPG vesicles. All three molecules provided
approximately 80% release of HPTS-DPX after 3 min, with
the hexamer again exhibiting slower release kinetics than the
tetramer or pentamer. Overall, these results indicate that a
cationic charge is not essential for activity in this class of
molecules and that perhaps their activity may be enhanced
by increasing their hydrophobicity. While 6, 8, and 9 are not
cationic, they may still be amphipathic with the hydrogen-
bonding edge of the molecule being relatively hydrophilic
and the opposite edge with the valine side chain and the
Boc- protected amine groups being hydrophobic in the
β-strand conformation. The results thus far also suggest that
there would not be significant benefits to the preparation of
longer @-tide oligomers in terms of the degree of membrane
disruption or the rate.
their potential applications as antibiotics, it is also important
to evaluate their abilities to lyse the DOPC-based mimics
of mammalian membranes, as this may provide early
indications of their potential toxicity to mammalian cells.
Therefore, all of the molecules described above, which
exhibited activity in the DOPE/DOPG vesicles, were eval-
uated in DOPC vesicles with encapsulated HPTS-DPX. As
shown in Figure 3b, it was found that at a concentration of
100 μg/mL, all of the molecules exhibited greatly reduced
membrane disruptive activity in the DOPC vesicles, with less
than 10% of the HPTS-DPX released during the course of
the experiment. The reduced activities of the molecules in the
DOPC vesicles may be due partly to the lack of anionic
charge on these vesicle membranes. However, for the non-
cationic @-tides 6, 8, and 9, the reduced activity cannot be
attributed only to charge effects but may be due at least
partly to the intrinsic negative curvature of the DOPE lipids
relative to the DOPC lipids and thus their increased suscepti-
bility to lysis.
While the abilities of the molecules to lyse the DOPE/
DOPG-based mimics of bacterial membranes is critical to
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JOCArticle
In order to evaluate the role of the three-dimensional
structure on the membrane disruptive activity, the analogues
60, 80, 90, 100, and 110, containing only L-amino acids, were
also tested in the vesicle lysis assay. As illustrated in Figure 1
and described above, it was not expected that these molecules
would display facially amphiphilic conformations, as all of
the amino acid side chains would be directed to the same side
of the β-strand in the linear conformation. As shown in
Figure 3c, all of these all L-amino acid analogues exhibited
greatly reduced lysis activity in DOPE/DOPG vesicles rela-
tive to their alternating D,L counterparts, with the exception
of the hexamer 110 which had similar activity to 11. This
suggests that the designed amphipathicity of the molecules
does have an important role in their activity and that while
the molecules would not be expected to preorganize into
β-sheet mimics in aqueous solution, they may be able to
assume conformations resembling β-strands in the presence
of membranes. In the case of natural antimicrobial peptides
such as magainin, it has been found that while the molecules
are capable of displaying amphipathic conformations, they
are often unstructured in aqueous solution in the absence
of membranes or membrane mimics.1,50 Nevertheless, this
result is surprising in light of the high activities of the
protected oligomers 6, 8, and 9, which suggested that
the presence of hydrophobic and cationic residues on oppo-
site faces of the molecule was not essential for activity and
that instead an amphipathicity based on the hydrogen
bonding edge and the edge presenting the amino acid side
chains might be sufficient. Compounds 60, 80, 90, 100, and 110
were all also evaluated in the DOPC vesicles to probe their
selectivity for bacterial over mammalian cell membranes,
and as observed for the alternating D,L analogues, less than
10% of the HTPS-DPX was released.
Overall, these vesicle lysis assays have shown that many of
the @-tides described here have moderate membrane dis-
ruptive activity and also promising selectivity for bacterial
over mammalian membranes. It is also apparent that for
membrane disruptive activity there is an advantage to the
preparation of @-tides from alternating D- and L-amino
acids such that alternating side chains diverge to opposite
sides of the molecule in the linear conformation. The surpris-
ing activities of the protected oligomers suggest that the
azacyclohexenone units of the @-tides may not contribute as
much hydrophobicity as expected and that higher levels of
activity might be achieved by the use of higher ratios of
hydrophobic/hydrophilic amino acid side chains as well as
the incorporation of amino acids or terminal groups with
higher hydrophobicity.
demonstrated that these new oligomers containing both
D- and L-amino acids could dimerize to β-sheet mimics in
CDCl3/CD3OH solutions with similar affinities to the oligo-
mers containing all L-amino acids, which were previously
reported. This indicates that the incorporation of D-amino
acids likely does not dramatically alter the conformational
preferences of the molecules and that they are capable of
exhibiting amphipathic conformations. In vesicle leakage
assays using membranes designed to mimic those of bacteria,
it was found that several oligomers exhibited moderate
membrane disruptive activity. No significant effects based
on oligomer length were observed in this series of molecules
ranging from tetramers to hexamers, although the hexamer
exhibited slower leakage kinetics. Surprisingly, the oligomers
with the ε-Boc protecting groups on the lysine units were as
active or more active than the corresponding deprotected
oligomers with pendant cationic amines, demonstrating that
a cationic charge was not essential for activity in this class of
molecules and that there may be some advantage to the
increased hydrophobicity of the protected oligomers. In
addition, it was found that oligomers based on alternating
D- and L-amino acids generally exhibited significantly higher
activity than the corresponding oligomers containing all L-
amino acids. This indicates that there is some advantage to
having the amino acid side chains diverging to opposite sides
of the strand. Furthermore, much lower activity was ob-
served for all of the oligomers with membranes mimicking
those of eukaryotic cells in comparison with those of bacter-
ial cells, suggesting that this class of molecules may be
capable of selectively killing bacteria in the presence of
mammalian cells. Overall, this work represents the first
example of membrane disruptive oligomers developed from
a β-strand mimic based on R-amino acids. Although the
membrane-disruptiveactivitiesobtained thusfar are relatively
modest, several important insights were gained into the
structural features that are important for activity, providing
the groundwork for further exploration of @-tides as poten-
tial antimicrobials. These structures are highly tunable as
a diverse range of R-amino acids and oligomer terminal
functionalities can be readily incorporated using the same
synthetic routes described here. Thus, the careful design,
syntheses, and evaluation of additional series of @-tides based
on the discoveries described here can likely lead to new
molecules with high antimicrobial activity and selectivity.
Experimental Section
Synthesis of Dimer 3 and Representative Dimer Synthesis. Com-
pound 141 (4.5 g, 18 mmol, 1.0 equiv) and ε-Boc-L-lysine51,52 (4.8 g,
20 mmol, 1.1 equiv) were dissolved in dry MeOH (270 mL) under a
N2 atmosphere. The reaction was heated to 60 ꢀC and maintained
at this temperature overnight. The solution was then cooled to
room temperature and concentrated. The crude product was
redissolved in EtOAc/MeOH (220 mL/9 mL) and extracted with
1 M KHSO4 followed by brine. The organic layer was isolated,
dried with MgSO4, filtered, and concentrated. The product was
purified by silica gel chromatography using a gradient of EtOAc/
hexane (95/5) to remove impurities followed by EtOAc/MeOH
(90/10) to elute the product (5.97 g, 68%) as a viscous oil.
Conclusions
β-Strand mimetic oligomers based on alternating R-amino
acids and azacyclohexenone units were designed as potential
membrane disruptive antibiotics. D-Valine and L-lysine were
incorporated in an alternating manner with the aim of
obtaining amphipathic structures having a cationic and a
hydrophobic face in the linear β-strand conformation. The
molecules were successfully synthesized by a solution phase
convergent approach. Using NMR dilution studies, it was
(51) Scott, J. W.; Parker, D.; Parrish, D. R. Synth. Commun. 1981, 11,
303–314.
(50) Bechinger, B.; Zasloff, M.; Opella, S. J. Protein Sci. 1993, 2, 2077–
(52) Nowshuddin, S.; Reddy, A. R. Tetrahedron Lett. 2006, 47, 5159–
2084.
5161.
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Watson and Gillies
JOCArticle
The product was taken to the next step without further purifica-
peaks at higher concentrations in CDCl3. The same procedure
was carried out for pentamer 8 in 99/1 CDCl3/CD3OH and 97.5/
2.5 CDCl3/CD3OH but at concentrations ranging from 125 to
1 mM. The tetramer 6 and hexamer 9 were evaluated in 97.5/2.5
CDCl3/CD3OH at concentrations ranging from 125 to 1 mM.
Vesicle Leakage Assay. Vesicles formed from DOPC were
used as models for mammalian cell membranes, and vesicles
formed from an 80/20 mixture of DOPE/DOPG were used as
models for bacterial membranes. HPTS and its quencher DPX
were encapsulated in these vesicles. The following solutions were
used in the vesicle experiments: 4-(2-hydroxyethyl)piperazine-
1-ethanesulfonic acid (HEPES) buffer (10 mM HEPES, 145 mM
NaCl, 0.1 mM EDTA, NaHCO3 pH 7.25), HPTS (10 mM) in
HEPES buffer, DPX (100 mM) in HEPES buffer, and HPTS/
DPX/HEPES buffer (3.0, 1.8, and 5.2 mL, respectively, of the
HPTS, DPX, and HEPES buffers). A 1.1 mL portion of a
DOPC stock solution (25 mg/mL) was dried under N2 and then
dried under high vacuum for 2 h. A 0.88 mL portion of a DOPE
stock solution (25 mg/mL) in chloroform was mixed with
0.22 mL of a DOPG stock solution (25 mg/mL) in chloroform
to a total volume of 1.1 mL, and then the resulting solution was
dried under N2 followed by under high vacuum for 2 h. The
resulting films were hydrated with 0.6 mL of HPTS/DPX/
HEPES buffer for 2 h. The suspensions were then subjected to
five freeze-thaw/sonication cycles and were subsequently
extruded through a 1 μm Whatman polycarbonate membrane
>10 times yielding about 0.5 mL, which was then diluted to
2.5 mL with HEPES buffer. The excess dye was removed by gel
filtration chromatography (Nap-10 columns, GE Healthcare)
using HEPES buffer, resulting in 4.5 mL of vesicle solution.
Twenty microliters of this solution was added to 1.98 mL of
HEPES buffer in a quartz fluorescence cuvette. Solutions of the
oligomers in DMSO (5-40 μL of a 5 mg/mL solution) were
added to provide final oligomer concentrations ranging from
12.5 to 100 μg/mL in the 2 mL of vesicle solution. The solutions
1
tion. H NMR (400 MHz, CDCl3/CD3OD (2/1)): δ 1.31-1.57
(m, 13H), 1.68-1.95 (m, 2H), 3.02 (t, 2H, J = 6.3 Hz), 3.83-3.94
(m, 1H), 3.96-4.12 (m, 2H), 4.25-4.37 (m, 2H), 5.04-5.11 (m,
1H), 5.12-5.18 (m, 2H), 7.29-7.39 (m, 5H).
Synthesis of Dimer 4. The acid 3 (6.0 g, 13 mmol, 1.0 equiv)
was dissolved in THF/H2O (386 mL/45 mL) with stirring.
A 20% solution of Cs2CO3 was added to the reaction mixture
slowly until a pH of 7 was obtained. The mixture was then
concentrated, redissolved in THF, and concentrated again.
Methyl iodide (0.94 mL, 15 mmol, 1.2 equiv) in DMF (162 mL)
was added to the resulting oil, and the reaction was stirred at
room temperature for 30 min. The solvent was evaporated, and
the viscous oil was triterated with distilled water to remove salts.
The product was filtered and taken back up in MeOH and then
concentrated. The product was further purified by silica gel
chromatography using a gradient of CH2Cl2 to CH2Cl2/MeOH
(98/2) to remove the impurities, followed by CH2Cl2/MeOH
(90/10) to elute the product (2.9 g, 48%) as a glassy solid.
1H NMR (600 MHz, CD3OD): δ 1.26-1.51 (m, 13H), 1.69-
1.90 (m, 2H), 2.97-3.07 (m, 2H), 3.71 (s, 3H), 3.96 (t, 1H,
J=6.9), 4.00-4.07 (m, 2H), 4.24-4.33 (m, 2H), 5.04 (s, 1H),
5.12 (s, 2H), 7.26-7.37 (m, 5H). 13C NMR (150 MHz, CDCl3/
CD3OD (2/1)): δ 23.9, 29.0, 30.5, 32.2, 40.9, 48.9, 51.4, 53.1,
56.7, 68.7, 79.7, 95.2, 128.9, 129.2, 129.6, 137.5, 156.1, 158.2,
173.1, 193.3. IR (cm-1, film from CH2Cl2): 3269, 3056, 2925,
2859, 1739, 1658. HRMS: calcd for [M þ H]þ (C25H35N3O7)
489.2475, found (ESþ) 489.2480.
Synthesis of Tetramer 6 and General Coupling Procedure. The
dimer 2 (1.0 g, 2.9 mmol, 1.1 equiv), dimer 5 (0.93 g, 2.6 mmol,
1.0 equiv), HATU (3.6 g, 9.5 mmol, 3.3 equiv), and DIPEA
(1.1 mL, 6.4 mmol, 2.2 equiv) were dissolved in dry DMF
(84 mL) under N2, and the reaction mixture was stirred for
24 h. The reaction progress was monitored by thin-layer chro-
matography (100% EtOAc), and upon completion the solution
was concentrated, redissolved in EtOAc (124 mL), and washed
with 1 M KHSO4 followed by satd NaHCO3. The organic layer
was isolated, dried over MgSO4, filtered, and evaporated to
yield a glassy solid. The product was purified by silica gel
chromatography using EtOAc to elute the impurities, followed
by EtOAc/MeOH (95/5) to elute the product (1.1 g, 63%) as a
were stirred, and the fluorescence emission intensities It (λem
=
520 nm, λex = 460 nm) were monitored as a function of time (t).
Twenty microliters of 20% Triton X-100 in DMSO was then
added to provide complete vesicle lysis. The curves were normal-
ized to percent leakage [(It - I0)/(I¥ - I0)] ꢀ 100. I0 is the emission
intensity before the addition of any of the polymers, and I¥ is the
emission intensity after the addition of Triton X-100.
1
glassy solid. H NMR (600 MHz, CD3OD): δ 0.81-1.09 (m,
6H), 1.25-1.55 (m, 15H), 1.71-1.95 (m, 2H), 2.02-2.16 (m,
1H), 2.96-3.09, (m, 2H), 3.66-3.78 (m, 3H), 3.94-4.14 (m, 3H),
4.14-4.25 (m, 1H), 4.28-4.62 (m, 5H), 4.73 (t, 1H, J=17.0),
5.06-5.28 (m, 4H), 6.52 (br s, 1H), 7.24-7.38 (m, 5H).
13C NMR (150 MHz, CD3OD): δ 18.7, 19.6, 24.0, 28.9, 30.5,
32.5, 41.0, 43.7, 45.1, 51.5, 53.1, 56.9, 59.0, 68.8, 79.8, 95.0, 95.5,
129.0, 129.3, 129.7, 137.6, 156.4, 158.4, 163.4, 164.9, 170.8,
171.2, 173.0, 173.2, 192.4, 193.7. IR (cm-1, film from CH2Cl2):
3283, 3057, 2960, 2927, 2862, 1736, 1689. HRMS: calcd for
[M þ Na]þ (C35H49N5O9Na)þ 706.3422, found (ESþ) 706.3428.
HPLC: tR 9.7 min (MeCN/H2O (20/80)).
Acknowledgment. This work was supported by the
Natural Sciences and Engineering Research Council of
Canada, an Early Researcher Award from the Government
of Ontario, the Canada Foundation for Innovation, and the
Ontario Research Fund. We thank Doug Hairsine for mass
spectrometry data and Darryl Knight and Aneta Borecki for
help with HPLC.
Supporting Information Available: General procedures and
materials section, syntheses of oligomers 2, 5, 7, 8, 9, 10, 11, 20,
60, 80, 90, 100, and 110, NMR spectra of compounds 2/20, 4, 6, 60, 8,
80, 9, 90, 10, 100, 11, and 110, and HPLC chromatograms of
compounds 6, 60, 8, 80, 9, 90, 10, 100, 11, and 110. This material is
available free of charge via the Internet at http://pubs.acs.org.
1H NMR Dilution Experiment. The pentamer 8 was dissolved
in CDCl3 at a concentration of 50 mM, and serial 2-fold
dilutions were carried out to a concentration of 0.78 mM.
1H NMR spectra were obtained at 600 MHz for each concen-
tration. The upper concentration was limited by broadness of
5960 J. Org. Chem. Vol. 74, No. 16, 2009