Length-Dependent Formation of Transmembrane Pores by 310-Helical α-Aminoisobutyric Acid Foldamers

Article abstract of DOI:10.1021/jacs.5b12057

The synthetic biology toolbox lacks extendable and conformationally controllable yet easy-to-synthesize building blocks that are long enough to span membranes. To meet this need, an iterative synthesis of α-aminoisobutyric acid (Aib) oligomers was used to create a library of homologous rigid-rod 3₁₀-helical foldamers, which have incrementally increasing lengths and functionalizable N- and C-termini. This library was used to probe the inter-relationship of foldamer length, self-association strength, and ionophoric ability, which is poorly understood. Although foldamer self-association in nonpolar chloroform increased with length, with a ～14-fold increase in dimerization constant from Aib₆ to Aib₁₁, ionophoric activity in bilayers showed a stronger length dependence, with the observed rate constant for Aib₁₁ ～70-fold greater than that of Aib₆. The strongest ionophoric activity was observed for foldamers with >10 Aib residues, which have end-to-end distances greater than the hydrophobic width of the bilayers used (～2.8 nm); X-ray crystallography showed that Aib₁₁ is 2.93 nm long. These studies suggest that being long enough to span the membrane is more important for good ionophoric activity than strong self-association in the bilayer. Planar bilayer conductance measurements showed that Aib₁₁ and Aib₁₃, but not Aib₇, could form pores. This pore-forming behavior is strong evidence that Aib_m (m ≥ 10) building blocks can span bilayers.

Full text of DOI:10.1021/jacs.5b12057

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Article
Length-dependent formation of transmembrane
10
pores by 3 helical Aib foldamers
Jennifer E. Jones, Vincent Diemer, Catherine Adam, James Raftery, Rebecca E. Ruscoe, Jason T.
Sengel, Mark I. Wallace, Antoine Bader, Scott L. Cockroft, Jonathan Clayden, and Simon J. Webb
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b12057 • Publication Date (Web): 23 Dec 2015
Downloaded from http://pubs.acs.org on December 29, 2015
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Length-dependent formation of transmembrane pores by 310
helical Aib foldamers
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Jennifer E. Jones,‡,1,2 Vincent Diemer,‡,1,2 Catherine Adam,1,5 James Raftery,1 Rebecca E. Ruscoe,1
Jason T. Sengel,3 Mark I. Wallace,3 Antoine Bader,4 Scott L. Cockroft,4 Jonathan Clayden1,5,* and
Simon J. Webb1,2,*
1. School of Chemistry, University of Manchester, Oxford Road, Manchester M13 9PL, U.K.
2. Manchester Institute of Biotechnology, University of Manchester, 131 Princess St, Manchester M1 7DN, U.K. E-
mail: S.Webb@manchester.ac.uk
3. Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, U.K.
4. EaStCHEM School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edin-
burgh EH9 3FJ, U.K.
5. School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, U.K. E-mail: j.clayden@bristol.ac.uk.
ABSTRACT: The synthetic biology toolbox lacks extendable, conformationally controllable yet easy-to-synthesize build-
ing blocks that are long enough to span membranes. To meet this need, an iterative synthesis of α-aminoisobutyric acid
(Aib) oligomers was used to create a library of homologous rigid-rod 310-helical foldamers, which have incrementally in-
creasing lengths and functionalizable N- and C-termini. This library was used to probe the interrelationship of foldamer
length, self-association strength and ionophoric ability, which is poorly understood. Although foldamer self-association in
non-polar chloroform increased with length, with a ~14-fold increase in dimerization constant from Aib6 to Aib11, iono-
phoric activity in bilayers showed a stronger length dependence, with the observed rate constant for Aib11 ~70-fold greater
than that of Aib6. The strongest ionophoric activity was observed for foldamers with >10 Aib residues, which have end-to-
end distances greater than the hydrophobic width of the bilayers used (~2.8 nm); X-ray crystallography showed that Aib11
is 2.93 nm long. These studies suggest that being long enough to span to the membrane is more important for good iono-
phoric activity than strong self-association in the bilayer. Planar bilayer conductance measurements showed that Aib11 and
Aib13, but not Aib7, could form pores. This pore-forming behavior is strong evidence that Aibm (m ≥ 10) building blocks
can span bilayers.
gested to be behind AMP antibacterial activity.6 However,
INTRODUCTION
for many AMPs and foldamers7 the mechanisms behind
cell toxicity are poorly understood.
Foldamers are synthetic oligomers that can mimic some
of the structural complexity of proteins and peptides
without the constraints imposed by natural biopolymers.
Like many peptides, in solution these oligomers can fold
into conformationally stable structures which have large
and structurally well-defined surfaces that are able to in-
teract with individual biopolymers or biomolecular as-
semblies. For example, foldamers have been used to repli-
cate protein-protein interactions1 and to form foldamer-
DNA complexes for gene delivery,2 suggesting a role in a
number of biomedical applications.
Peptide foldamers containing high proportions of α-
aminoisobutyric acid (Aib) have a number of attractive
features as membrane-spanning building blocks, includ-
ing high hydrophobicity and a propensity to adopt rigid-
rod 310-helical secondary structures in a variety of sol-
vents. Moreover, extended sequences of Aib are found in
peptaibols, a class of naturally occurring AMPs produced
by Trichoderma fungi. These AMPs are mostly between 11
and 21 residues long, often with a high Aib content, and
are typically terminated by an N-terminal acyl group and
a C-terminal 1,2-aminoalcohol residue.8 Since Aib residues
stabilize 310 helices,9 shown schematically in Figure 1a,
such peptaibols can have 310-helical and/or α-helical sec-
ondary structures.10
In recent years there have been a number of reports of
foldamers displaying membrane activity, for example
mimicking cell-penetrating peptides3 and antimicrobial
agents,4 with the latter implying that foldamers could
address the growing problem of antibiotic resistance.5
Like the antimicrobial peptides (AMPs), these foldamers
may produce pores after spanning the membrane, alt-
hough membrane disruption is another mechanism sug-
One well-studied peptaibol is alamethicin, which can
adopt membrane-spanning orientations in membranes
and produce ion channels.11,12 The mixed 310- and α-helical
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conformation of this 19-residue peptide13 results in suffi-
Page 2 of 18
1
cient length to span the 2.0 to 2.8 Å hydrophobic section
of the phospholipid bilayer at the center of the cell mem-
brane.13a,14 Alamethicin acts through a ‘barrel-stave’ mech-
anism in which multimeric channels are formed by the
self-association of 3-12 helical monomers, yielding parallel
bundles with a central hydrophilic pore.11,12 The number of
alamethicin peptides in a bundle is dynamic, with differ-
ent conductance levels observed for bundles of different
molecularity.15 Nonetheless, shorter peptaibols that can-
not span a bilayer, such as the trichogins,16 are also mem-
brane active, including 6-residue trichodecinin I17 and 4-
residue peptaibolin,18 showing that the relationship be-
tween peptaibol length and ionophoric activity is un-
clear.19 A family of foldamers with incrementally increas-
ing lengths may throw light on this relationship, with
such a study also identifying the minimum foldamer
lengths needed to span different phospholipid bilayers.
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Figure 1: (a) 310 Helical Aib foldamers 1 to 9. (b) Schematic
There have been a handful of investigations into the re-
lationship between ionophore length and membrane ac-
tivity, with most using conformationally flexible systems.
Although ionophoric activity was not assessed, a small
family of homologous lipopeptaibols structurally related
to the trichogins was assayed for membrane disruption.
Release of dye from vesicles occurred upon addition of 11-,
15- and 19-residue peptides, with the 15-mer most active,
but not the 7- residue homologue.20 Sakai and Matile re-
ported that a rigid rod tetriphenyl exhibited no mem-
brane activity, but homologous rigid rod sexiphenyls (2.6
nm long) and octiphenyls (3.4 nm long) acted as iono-
phores; the latter was three times more active than the
former.21 Gokel et al. investigated flexible channel-
forming ionophores, which comprised three 4,13-diaza-18-
crown-6 macrocycles connected by incrementally longer
alkyl chains (–(CH2CH2)n–, n = 4, 5, 6, 7, 8). The rate con-
stant for sodium ion transport across vesicle membranes
increased 200-fold from n = 4 to n = 6, then halved for n =
8.22 Interestingly these compounds had antibiotic activity,
with the n = 6 compound 13 times more effective at killing
ampicillin-resistant E. coli than the n = 4 homologue.23
representation of Aib foldamer helices interacting with a
bilayer to form a pore with activity determined by foldamer
length (a, black arrows) and foldamer-foldamer interactions
(b, white arrows).
We now describe the synthesis and analysis of a series
of 310-helical foldamers built exclusively from Aib resi-
dues, ranging from five to thirteen residues in length
(Figure 1a). A flexible strategy was developed for the syn-
thesis of the “Aibm” oligomers that gave incrementally
extendable mimics of the pore-forming peptaibols, struc-
tures with chemical functionality at both termini and
without complicating side chain functionality. The iono-
phoric activity of each of these compounds was assessed
and used to probe how the activity of helical peptides in
bilayer membranes depended on both self-association
strength and chain length (Figure 1b), with the adoption
of membrane-spanning conformations confirmed using
planar bilayer conductance assays.
RESULTS AND DISCUSSION
Design and synthesis of Aib foldamers 1 – 9: A ho-
mologous series of foldamers was synthesized that varied
only in their length (Scheme 1). As Aib is an achiral resi-
due, these oligomers exist as an equal ratio of M and P 310-
helical conformers that interconvert on a sub-millisecond
timescale at room temperature.24 The shortest Aib folda-
mer 1 was designed to have sufficient length to give one
turn of a 310 helix (> 4 residues) in solution,25 and this core
unit was extended by an iterative synthetic strategy
(Scheme 1). By this method, a family of foldamers was
created with lengths predicted to be up to and beyond the
thickness of a typical bilayer. Groups were incorporated at
the termini (azido at the N-terminus and 2-
(trimethylsilyl)ethyl, CH2CH2SiMe3, at the C-terminus)
which not only facilitated this iterative synthetic proce-
dure but also minimized end-to-end intermolecular inter-
actions.25 These terminal groups will permit the synthesis
of functionalized derivatives, either after deprotection or
through “click” chemistry. In a 310 helical conformation
with a typical rise-per-residue of 1.94 Å, the lengths of
compounds 1 to 9 were anticipated to lie between 1.8 nm
Although these studies imply that longer compounds
should be more membrane-active than shorter ones and
hint that an optimal length may exist, several questions
remain unanswered. For example the relationship be-
tween length and self-association strength is poorly un-
derstood, which is especially important for the multimeric
barrel-stave channels formed by oligophenyls or peptai-
bols. Ionophore flexibility also complicates the analysis of
length dependent behavior, as long, flexible compounds
might still adopt conformations with high membrane
activity. The ideal system for a systematic study would be
based on conformationally-defined structures that can be
extended in small increments. Such a study would pro-
duce a nanoscale scaffold optimized for spanning a given
bilayer and useable as a potential building block for truly
synthetic biology in a membrane or new generation anti-
biotics. Furthermore, any building block will need chemi-
cal functionality at both ends that allows it to be integrat-
ed into more complex constructs.
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at the C-terminus (NH…O distance 3.038 Å, Figure 2 e)
to 3.4 nm.26 These foldamers are all relatively hydropho-
1
2
3
4
5
6
7
8
bic, facilitating partitioning into phospholipid bilayers
from the buffer.27
and the NH of the second N-terminal Aib and the final
amide link at the C-terminus (NH…O distance 2.941 Å,
Figure 2 f). Octamer 4 only showed intermolecular hydro-
gen bonding to acetonitrile of crystallization (Figure 2b,
NH…N distance 3.067 Å), and unlike 3 and 7 both the az-
ido group and CH2CH2TMS tail folded back onto the he-
lix. Undecamer 7 showed intermolecular hydrogen bonds
analogous to those observed for 3, involving the first and
second N-terminal Aib residues and the C-terminus of a
neighboring foldamer (i.e. NH…O distances 2.999 Å and
3.130 Å respectively).
i
ii
i
N3Aib2OtBu
N3Aib3OtBu
HAibOtBu
HAib2OtBu
10
13
14
11
iii
ii
i
iii
iv
N3Aib4OH
N3Aib4OtBu
HAib3OtBu
N3Aib3OH
18
15
12
17
iii
16
N3Aib5OtBu
v, vi
N3Aib5OH 19
11 + 17
9
ii, vii
10
11
12
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N3AibO(CH2)2TMS
HCl.HAibO(CH2)2TMS
BrC(CH3)2CO2H
i
22
20
21
ii
N3Aib2O(CH2)2TMS
HAib2O(CH2)2TMS
23
24
ii
iv with
N3Aib5O(CH2)2TMS
HAib5O(CH2)2TMS
24
1
("Aib5")
25
ii
iv with
25
N3Aib3OH
N3Aib8O(CH2)2TMS
HAib8O(CH2)2TMS
17
4 ("Aib8")
26
iv with
26
N3Aib11O(CH2)2TMS
7 ("Aib11")
iv with
N3Aib9O(CH2)2TMS
25
N3Aib4OH
5
("Aib9")
iv with
18
N3Aib12O(CH2)2TMS
26
8 ("Aib12")
ii, i
iv with
N3Aib7O(CH2)2TMS
N3Aib6O(CH2)2TMS
22
2 ("Aib6")
3
("Aib7")
iv with
N3Aib5OH
N3Aib10O(CH2)2TMS
25
19
6
("Aib10")
iv with
N3Aib13O(CH2)2TMS
26
9
("Aib13")
Scheme 1: Synthesis of foldamers 1-9. i) N3AibCl, Et3N,
CH2Cl2, RT. ii) Pd/C, MeOH or EtOH, H2, RT. iii) TFA,
CH2Cl2, RT. iv) Ac2O, 120 °C or EDC.HCl, CH2Cl2, RT then
HAibmOR (R = t-Bu or (CH2)2TMS), CH3CN, 80 °C. v) NaN3,
DMF, RT. vi) 2-(Trimethylsilyl)ethanol, EDC.HCl, DMAP,
CH2Cl2, RT. vii) HCl in Et2O.
Solid state structures: Crystal structures were ob-
tained for N3Aib7OCH2CH2TMS 3, N3Aib8OCH2CH2TMS 4
and N3Aib11OCH2CH2TMS 7, which confirmed the adop-
tion of 310 helical conformations stabilized by intramolec-
ular hydrogen bonds (Figure 2 a-c).
Figure 2: X-ray crystal structures of (a) N3Aib7OCH2CH2TMS
3, (b) N3Aib8OCH2CH2TMS
4 with CH3CN and (c)
N3Aib11OCH2CH2TMS 7. Foldamer-foldamer interactions of
N3Aib7OCH2CH2TMS 3 in the solid state, involving hydrogen
bonds from (d) the first and (e) the second N-terminal Aib
residue to the C-terminus of a neighboring foldamer. Select-
ed hydrogen bonds are shown in green, black arrows indicate
the continuation of the foldamer chains.
The extended conformations adopted by 3 and 7 gave
head-to-tail distances (CH3 to N3) of 2.25 nm and 2.93 nm.
These values compare well to anticipated lengths for the-
se compounds, calculated as 2.24 nm and 3.01 nm respec-
tively using 0.194 nm per Aib in a 310 helix, with 0.24 nm
added for the azido group and 0.64 nm added for the
OCH2CH2SiMe3 group. All compounds were found to pack
in offset head-to-tail arrangements (see Supporting In-
formation).25,28 The unit cell also revealed side-to-side
packing of the helices, a geometry important for the for-
mation of membrane-spanning pores/channels. The hep-
tamer 3 contained head-to-tail intermolecular hydrogen
bonds between foldamers involving the NH of the first N-
terminal Aib and carbonyl of the penultimate amide link
Foldamer self-association: The self-association of Aib
oligomers in CDCl3 may be quantified by fitting the con-
centration dependence of NH chemical shifts to either
dimerization or isodesmic self-association (with equal K
values) models.29 Such self-association in non-polar envi-
ronments is a key aspect of most proposed mechanisms of
action of peptaibols. However, we and others have found
that aggregates larger than dimers do not form to a signif-
icant extent for similar Aib oligomers in the millimolar
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Page 4 of 18
concentration range in chloroform.30 After fitting data to
a dimerization model, we have found that longer Aib
foldamers self-associate more strongly in non-polar sol-
vents, as well as displaying lower solubility.25
8-Hydroxypyrenetrisulfonate (HPTS) assays of ion-
1
2
3
4
5
6
7
8
ophoric activity: Foldamers 1 to 9 were assessed for ei-
ther Na+/H+ antiport or Na+/OH– symport by means of
standard HPTS assays,34 using 1:4 cholesterol:egg yolk
phosphatidylcholine (EYPC) vesicles with an interior pH
of 7.4 and an exterior pH of 8.4.35 Aliquots of foldamers
Aib5 1 to Aib13 9 in methanol were equilibrated with sus-
pensions of large unilamellar vesicles (LUVs) for 180 s,
before the addition of the base pulse. The change in HPTS
fluorescence was measured for 27 minutes, followed by
addition of Triton X-100 to lyse the vesicles and obtain
the maximum change in HPTS fluorescence for data nor-
malization (Figure 3 a).
As found during this previous work, compounds 1 to 9
showed concentration-dependent NH chemical shifts in
CDCl3 but not in CD3OD or other hydrogen bond do-
nor/acceptor solvents.25 These shifts in CDCl3, a solvent
that replicates the low polarity at the center of the bi-
layer,31 allowed the dimerization constant K of 1, 2, 4, 7-9
9
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1
to be quantified by H NMR spectroscopy (Table 1, corre-
sponding dissociation constants, Kd, and curve fitting er-
rors shown). The NH resonances of these foldamers were
monitored as the solutions were diluted, with marked
changes in chemical shift observed for some NHs in the
longer foldamers even at relatively low concentrations.
For example, a 0.4 ppm shift for the N-terminal NH of
Aib11 7 was measured upon dilution from 40 to 1 mM,
compared to a 0.05 ppm shift for the N-terminal NH pro-
ton of Aib5 1 over the same concentration range. From
these data the dimerization constant for each compound
was calculated by standard iterative curve fitting using
different minimization algorithms (see the Supporting
Information).25
Table 1: Experimental NMR dimerization constants K
(dissociation constants, Kd) for foldamers 1, 2, 4, 7-9
in CDCl3 at 298 K
Oligomer
Aib5 1
K / M−1 (Kd / mM)
< 1 (> 1000)
Aib6 2
1.2 ± 0.2 (830 ± 140)
3.0 ± 1.3 (330 ± 140)
17.3 ± 3.1 (58 ± 10)
13.6 ± 0.6 (74 ± 3)
16.9 ± 0.4 (59 ± 1)
Aib8 4
Aib11 7
Aib12 8
Aib13 9
The dimerization constants were < 3 M–1 for the shorter
foldamers 1, 2 and 4, but 14 to 17 M–1 for the longer folda-
mers 7-9. These values, equivalent to ΔG = 7 kJ mol-1 for
dimerization of Aib13 9,32 are consistent with previous
findings that Aib foldamers dimerize more strongly in
chloroform as oligomer length increases.25,30 Although
these dimerization constants are low, partitioning into
the membrane can lead to very large effective concentra-
tions, as the volume of the membrane is much less that
the total volume of the sample.33 For instance, 1 mol %
foldamer in the membrane is equivalent to ~8 mM folda-
mer within the volume occupied by the bilayer. At these
“in membrane” concentrations, an increase in dimeriza-
tion constant from 3 to 17 M–1 (foldamers Aib8 4 and Aib13
9 respectively) would give an approximately proportion-
ate increase in dimerization, with a simple model giving
4.4 % of Aib8 4 present as dimer at 1 mol % foldamer in-
corporation but 18.2 % of Aib13 9 is present as the dimer at
the same membrane loading (see the Supporting Infor-
mation).
Figure 3: (a) HPTS fluorescence changes for foldamers 7
(Aib11, blue) 5 (Aib9, green), 4 (Aib8, grey) 3 (Aib7, red) and
methanol blank (black). Additions of foldamer solution in
methanol (20 ꢀL, 60 μM) to LUVs (20% cholesterol/EYPC,
0.76 mM lipid, 2 mL). Base pulse of NaOH (13 ꢀL, 1 M) at 180
s (dashed line). Data normalized to maximum fluorescence
after addition of Triton X-100 (10% v/v in MOPS buffer) at
1800 s. (b) Plot of [foldamer] (log scale) vs. pseudo-first order
rate constants of ion leakage from LUVs for foldamers 7
(Aib11, blue), 4 (Aib8, grey) and 1 (Aib5, brown). Curve fits to
guide the eye.
These HPTS fluorescence assays showed an initial
‘burst’ of activity (the “burst phase”), due to rapid ion
transport through pores/channels that formed in the vesi-
cles before the addition of the base pulse,36 followed by
slower pH discharge over the next 1620 s. Foldamers 1 to 9
could all discharge the transmembrane pH gradient, albe-
it with strong concentration dependence (data for 1, 4 and
7 shown in Figure 3 b). For the shorter Aibm foldamers (m
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that there is no sudden appearance of ionophoric activity
= 5, 6, 7, 8), a high concentration of 10 μM was required
1
2
3
4
5
6
7
8
for activity, whereas for the longer Aib m-mers (m = 9, 10,
11, 12, 13) a 0.6 μM concentration was sufficient to show
high activity. As these data suggest, the ionophoric activi-
ty of these oligomers was markedly length-dependent,
with longer molecules significantly more active. For ex-
ample, 0.08 mol % of undecamer 7 (0.6 μM of Aib11) was
able to discharge 70 % of the pH gradient within 10
minutes, but 0.08 mol % heptamer 3 (0.6 μM of Aib7) only
discharged 7 % over the same time period (Figure 3 a).
Shorter oligomers such as pentamer Aib5 1 only dis-
charged most of the pH gradient at concentrations >200
μM (>26 mol %, see the Supporting Information).
when these incremental changes in foldamer length pro-
duce compounds long enough to span the bilayer. The
activity of the shorter foldamers and the absence of a
step-change in ionophoric activity at a certain length of
foldamer suggest the mechanism for foldamer activity
may be more nuanced than simply the formation of the
“barrel-stave” ion channels suggested to explain the activ-
ity of alamethicin.11,12
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The slower change in fluorescence after the “burst
phase” encompasses several molecular level events, in-
cluding the rate of intervesicle transfer of foldamers.36
Nonetheless to gauge the relative effectiveness of the
compounds as ionophores, all the data was fitted to first
order reaction kinetics as an approximation; this provided
consistent observed rate constant values within an error
of 5%. Control experiments using methanol only were
subtracted from the experimental data to correct for leak-
age caused by the solvent.
These observed rate constants (kobs, Figure 4) confirmed
that the longer oligomers, such as Aib11 7 (kobs = (3.3 ± 0.4)
× 10−3 s−1), show very powerful activity. Under the same
conditions (0.6 μM peptide in EYPC/cholesterol), an
HPTS assay showed the natural Aib-containing peptaibol
alamethicin had kobs = (2.7 ± 0.2) × 10−3 s−1, similar to that
of foldamer Aib9, 5 (kobs = (2.4 ± 0.3) × 10−3 s−1) and smaller
than that of dodecamer Aib12 8 (kobs = (5.0 ± 0.2) × 10−3 s−1).
With a reported37 hydrophobic width of EYPC/cholesterol
bilayers in the region of 2.8 nm, the length mismatch be-
tween the foldamer and bilayer decreases from 1 to 9 (1.8
nm to 3.4 nm), with the 310 helical decamer Aib10 6 closest
in length to the hydrophobic length estimated for α-
helical 19-residue alamethicin (ca. 2.8 nm).13,14
Figure 4: Plot of apparent first-order rate constants for Na+
transport vs. number of Aib residues for foldamers (a) Aib5 1
to Aib8 4 at 10 μM (b) Aib8 4 to Aib13 9 at 0.6 μM. Additions of
foldamer stock solution in methanol (20 ꢀL) to 0.76 mM
LUVs (20% cholesterol/EYPC, 2 mL). (c) Estimation of rela-
tive rate constants with foldamer length, at 10 μM (dashed
line) and 0.6 μM (solid line), normalized to relative rate of
Aib8 4 at both concentrations (krel = 1).
The activities of the shorter foldamers 1 to 3 could not
be distinguished from the background level at the lower
0.6 μM concentration, but their activities could be differ-
entiated at 10 μM. The octamer Aib8 4 was then analyzed
at both 10 μM and 0.6 μM to bridge between the two data
sets. Setting the activity of the octamer as 1.0 allowed the
activity of all the other Aibm foldamers to be approximate-
ly ranked (Figure 4).38 The relative activities were calcu-
lated to be 0.04 (m = 5, 10 μM), 0.16 (m = 6, 10 μM), 0.28
(m = 7, 10 μM), 1.0 (m = 8), 9 (m = 9), 14 (m = 10), 11 (m =
11), 17 (m = 12), 8 (m = 13), revealing an approximately 400-
fold difference in relative activity between the most and
least active foldamers. The sharpest increase in activity
(nine-fold) occurs for the change from octamer to
nonamer. This suggests that the length of Aib9 5 (estimat-
ed as 2.62 nm, calculated as described earlier) represents
the minimum length able to span the hydrophobic core of
the bilayer (ca. 2.8 nm for EYPC, see the Supporting In-
formation for a representation of 310 helical Aib11 7 in an
idealized bilayer).39 Beyond foldamer Aib10 6, there ap-
pears to be a levelling off or even a diminution of activity
with further increases in length. Interestingly it is clear
The HPTS assay cannot easily discriminate between ion
channel/pore,11,12 membrane disruption40 and ion carrier
mechanisms,41 all of which have been suggested to occur
for the structurally related peptaibols. An ion carrier
mechanism for Na+ transport by this family of foldamers
was discounted as no activity was observed over 20 h for 1,
7 and 8 in simple U-tube assays (see the Supporting In-
formation). To test for membrane disruption, the dye 5/6-
carboxyfluorescein (5/6-CF) was encapsulated within ves-
icles at self-quenching concentrations. If an added com-
pound disrupts the bilayer and/or forms pores greater
than the size of 5/6-CF (ca. 10 Å for 5/6-CF42 compared to
ca. 2 Å for sodium43), the dye will escape from the vesicle
lumen and give a recovery in emission at 517 nm (λex. 492
nm). As with the HPTS assay, the addition of Triton X-100
releases all entrapped 5/6-CF and allows normalization of
the data. When Aib foldamers Aib7 3, Aib11 7 and Aib13 9
were tested for 5/6-CF release from EYPC/cholesterol
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vesicles it was clear that very little dye release occurred; broke (see Supporting Information). In contrast, current-
1
2
3
4
5
6
7
8
foldamers 3, 7 and 9 caused only 3%, 3% and 5% leakage
of 5/6-CF over 1200 s at concentrations of 10 μM, 0.6 μM
and 0.6 μM respectively. The same compounds gave ion-
ophoric activities of 60-75% at the same concentrations
under analogous conditions (Figure 5). This observation
shows that membrane disruption by these compounds is
not significant under these conditions and suggests the
majority of ion channels or pores formed by 3, 7 and 9 at
these membrane loadings are not large enough to allow
5/6-CF release.44
voltage sweeps from -100 to +100 mV for compounds Aib11
7 and Aib13 9 at 0.6 μM displayed a range of ensemble
conductances. The flux passed was seen to gradually in-
crease until a maximum conductance was reached after
ca. 2.5 h, after which time the conductance reached an
equilibrium value between 50-80 pA/0.5-0.8 nS at an ap-
plied voltage of 100 mV (see Supporting Information).
9
10
11
12
13
14
15
16
17
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19
20
21
22
23
24
25
26
27
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5: Co-plot of normalized HPTS ion transport data
(filled circles) and 5/6-CF release data (lines) for 3 (Aib7, 1
mM, red), 7 (Aib11, 60 μM, blue) and 9 (Aib13, 60 μM purple).
Additions of foldamer solution in methanol to LUVs (20%
cholesterol/EYPC, 0.76 mM lipid, 2 mL) at 0 s (HPTS data,
base pulse at 180 s) or 180 s (5/6-CF data).
Planar bilayer conductance (PBC) assays of iono-
phoric activity: PBC measurements allow ionophoric
activity due to pores and/or channels to be identified and
the stability of these conducting structures to be assessed,
although the mechanism of ion conductance in HPTS
vesicle experiments is not necessarily the same under the
applied potential used in PBC measurements.45 After ap-
plication of a potential across the bilayer, if channels or
pores form then sudden and intermittent increases in
current occur, the length of which correlate to the kinetic
stability of an “open” channel.46 Such voltage-clamp tech-
niques can be used to observe single channel activities,
multiple conductance states for self-assembled channels
and ensemble conductance from multiple channels.47
Figure 6: Typical planar bilayer conductance behavior for 7
and 9. a) 7 (Aib11, 60 μM), applied voltage -100 mV, current
passed -150 pA, 1.5 nS. b) 9 (Aib13, 60 μM), applied voltage
+100 mV, current passed +145 pA, with smaller conducting
state also observed at +130 pA. 1.45 nS or 1.3 nS respectively c)
9 (Aib13), 60 μM concentration, applied voltage +100 mV,
current passed +145 pA. 1.45 pS. d) Example I-V traces: 9
(Aib13, purple) and 7 (Aib11, blue) traces approximately 1 h
after addition of compound.
At a set voltage, both 7 and 9 gave intermittent increas-
es in current over time (Figure 6a-c). Both compounds
exhibited multiple conductance states and maintained
this reproducible behavior for at least 16 h, after which
time the bilayer was still intact. Only a few seconds were
required for this channel/pore behavior to start appear-
ing, indicating fast diffusion into and across the bilayer.
Classical “square-top” traces were not observed, with the
traces instead showing flickering conductance events,
with poorly-defined current levels and short transition
lifetimes. Integer steps in the conductance levels were not
observed, suggesting multiple openings of the same type
of pore do not occur. Instead the different conductance
states, which range from 1.3 to 1.5 nS (Figure 6 a-c), may
result from pores with differing molecularities and corre-
Selected foldamers 3, 4, 7, 9 (N3AibmOCH2CH2TMS, m =
7, 8, 11, 13) were assessed for their ability to conduct ions
through planar phospholipid bilayer membranes com-
posed of 1:4 cholesterol:EYPC. The shorter foldamers Aib7
3 and Aib8 4 at concentrations of 250 or 500 μM generally
produced irregular and spiky conductances in the cur-
rent-voltage sweeps (I-V sweeps from +100 mV to −100
mV), which indicates transient membrane disruption by
the peptides. At higher concentrations the bilayers be-
came unstable, e.g. 1 mM Aib8 4 gave multilevel activity
after ~0.5 h at high positive or high negative applied po-
tential but the shape of the I-V curves suggest progressive
membrane weakening until eventually the membranes
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Journal of the American Chemical Society
Many natural and artificial channel/pore formers are
spondingly different conductances, similar in behavior to
1
2
3
4
5
6
7
8
the amphiphilic heptapeptides reported by Ferdani and
Gokel.48 This voltage-dependent formation of short-lived,
multi-level pores/ion channels is also characteristic for
some neutral and many cationic toroidal or α-barrel-
forming peptides like melittin and oligo(Ala-Aib-Ala-Aib-
Ala).49 Synthetic ion channels typically give conductances
between 1 and 100 pS, whereas pores give conductances
between 0.1 and 5 nS,50 suggesting that foldamers 7 and 9
form pore-like structures. Very low foldamer concentra-
tions (5 to 50 nM) were assessed for channel formation
using droplet-interface bilayers (DIBs, see the Supporting
Information).51 However DIB studies on 2 (6-mer) and 7
(11-mer) did not indicate that defects of a pre-defined ra-
dius were formed, but at these very low concentrations
the foldamers induced points of weakness that nucleated
electropores.52
believed to achieve activity by self-associating in the
membrane, with “barrel-stave”, “carpet” and “toroidal-
pore”6 mechanisms invoking the formation of defined or
ill-defined aggregates. We found that increasing the
length of Aib foldamers increased the strength of self-
association in non-polar environments, a principle that
may extend to other peptides or foldamers in bilayers.
However, the increase in ionophoric activity with length
was stronger than the increase in self-association with
length; for example, Aib11 7 was 91 times more membrane
active than Aib6 2 and 12 times more active than Aib8 4,
whereas K for Aib11 7 was only 14 times greater than K for
Aib6 2 and 5.8 times greater than K for Aib8 4. These data
suggest a key factor for good ionophoric activity is the
ability to achieve a membrane-spanning conformation.
Ionophoric activity was similar for foldamers with m = 10
and 13 (lengths from ~2.8 to ~3.4 nm), implying that 2.8
nm is the ideal length for these foldamers to span
EYPC/cholesterol bilayers (~2.8 nm hydrophobic width)
and further increases in foldamer length have little influ-
ence on activity.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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39
40
41
42
43
44
45
46
47
48
49
50
51
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53
54
55
56
57
58
59
60
The current-voltage sweeps for Aib11 7 and Aib13 9 at 60
μM showed a non-linear relationship between current
and voltage, with higher conductance at high voltages
(Figure 6 d), non-ohmic behavior similar to that found for
other antimicrobial peptides including alamethicin.12b,15
The increase in conductance at high voltages could be
due to a reduction in the energy barrier to pore for-
mation,52 or the interaction of the stronger applied field
with the foldamer dipole causing more transmembrane
geometries.53 The I-V curves also show asymmetric con-
ductance behavior (see the Supporting Information), be-
havior that is different to that observed for symmetric
pores based on β-sheets (where the opposing backbone
orientations produce no net macrodipole) and dipole-free
octiphenyl rigid-rod channels.49,54 This behavior suggests
a lack of symmetry when interacting with the membrane.
Peptide foldamers, such as Aib11 7 and Aib13 9, have differ-
ent termini and intrinsic end-to-end asymmetry that pro-
duces a significant dipole moment.55 Since these com-
pounds are added solely to the cis side of the PBC bilayer,
easier insertion of the small azido group into the bilayer
over the bulky TMS group would produce an asymmetric
foldamer alignment in the bilayer, which may either
match or oppose the changing applied voltage.56
The potent ionophoric activity of the longer foldamers
suggests they may have rich potential in new generation
antibiotics. Much like the Aib-rich peptaibols themselves,
they should be resistant to proteases, yet the azido and
CH2CH2TMS protecting groups allow simple synthetic
modification of the N- and/or C- termini. Simple modifi-
cations such as adding chiral28,58 or hydrogen-bonding
groups to the termini could increase activity by strength-
ening aggregation in the bilayer.25 Alternatively, specific
cell-targeting groups, such as saccharides or biotin, could
be added.35,59
The membrane activity reported here for conforma-
tionally defined Aib foldamers gives a hint of the wider
potential of these nanoscale building blocks. These stud-
ies have shown that an Aibm (m = 10-12) core is sufficient
achieve a membrane spanning geometry. Since both the
N- and C- termini can be easily modified, these helical
transmembrane units could therefore be used as scaffolds
for the construction of functional GPCR mimics. For ex-
ample, foldamers 6-9 exist as racemic mixtures of inter-
converting right and left-handed helices, but controlling
the helical screw-sense distribution can provide a means
of relaying information along the helix.60 Given that, non-
covalent reversible switching of chiral control should lead
to end-to-end transmission of conformational change,
and ultimately information communication across a bi-
layer.
CONCLUSIONS
Simple and versatile synthetic methodology has given
access to a homologous family of functionalizable 310 heli-
cal Aib foldamers containing from 5 to 13 Aib residues. All
members were active as ionophores, but only foldamers
that are able to form 310 helices longer than the thickness
of the hydrophobic core of the bilayer gave high conduct-
ance pores. Aib foldamers that were too short to span the
membrane were much less active. They may act through
an “amyloid-like” mechanism that has been suggested for
other short Aib foldamers,57 where at high concentrations
the foldamers assemble in or around the membrane sur-
face, porating and weakening the bilayer. The longer
foldamers were remarkably effective ionophores. In the
HPTS assay the observed rate constant for transport by
dodecamer Aib12 8 was twice that of the naturally occur-
ring 19-residue antibiotic alamethicin.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.10221/jacs.XXXXXXX.
1
Full experimental details including H and 13C NMR spectra
of new compounds, fitting of HPTS data, U-tube data, PBC
data/calculations, dimerization constant data/calculations
and DIB procedures/data (PDF). Crystallographic data for
compounds 3, 4 and 7 (CIF). Additional research data sup-
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Page 8 of 18
H.; Arnt, L.; Scott, R.W. Biochim. Biophys. Acta 2006, 1758, 1387-
porting this publication are available from the eScholar re-
pository at http://dx.doi.org/XXXXX.
1
2
3
4
5
6
1392 (g) Chen, X.; Tang, H.; Even, M. A.; Wang, J.; Tew, G. N.;
Chen, Z. J. Am. Chem. Soc. 2006, 128, 2711-2714 (h) Arnt, L.; Ren-
nie, J. R.; Linser, S.; Willumeit, R.; Tew, G. N. J. Phys. Chem. B
2006, 110, 3527-3532 (i) Epand, R. F.; Raguse, T. L.; Gellman, S.
H.; Epand, R. M. Biochemistry 2004, 43, 9527-9535 (j) Epand, R.
F.; Schmitt, M. A.; Gellman, S. H.; Epand, R. M. Biochim. Bio-
phys. Acta 2006, 1758, 1343-1350
AUTHOR INFORMATION
Corresponding Author
* j.clayden@bristol.ac.uk or S.Webb@manchester.ac.uk.
7
8
9
Author Contributions
(8) Peptaibols (Eds.: C. Toniolo, H. Brückner), Wiley-VCH,
Weinheim, 2009.
(9) Marshall, G. R.; Hodgkin, E. E.; Langs, D. A.; Smith, G. D.;
Zabrocki, J.; Leplawy, M. T. Proc. Natl. Acad. Sci. USA 1990, 87,
487-491
‡ These authors contributed equally.
Notes
The authors declare no competing financial interest.
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11
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23
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37
38
39
40
41
42
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60
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phys. Biomol. Struct. 1994, 23, 141-165
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ACKNOWLEDGMENT
We gratefully acknowledge Dr. J. Sanderson for the “NMR
dilution fit” Excel spreadsheet used for dimerization constant
determination. This work was supported by the BBSRC
(grant ref. I007962), ESPRC (grant refs. EP/N009134/1 and
EP/K039547) and the ERC (Advanced Grant ROCOCO).
ABBREVIATIONS
AMP, antimicrobial peptide; Aib, α-aminoisobutyric acid;
5/6-CF,
(dimethylamino)pyridine;
5/6-carboxyfluorescein;
EDC,
DMAP,
4-
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; EYPC, egg yolk phos-
phatidylcholine; HPTS, 8-hydroxypyrenetrisulfonate; LUV,
large
unilamellar
vesicle;
MOPS,
3-(N-
morpholino)propanesulfonic acid; PBC, Planar bilayer con-
ductance; RT, room temperature; TFA, trifluoroacetic acid;
TMS, trimethylsilyl.
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Org. Biomol. Chem. 2015, 13, 9580-9584.
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(58) (a) Brown, R. A.; Marcelli, T.; De Poli, M.; Solà, J.; Clay-
den, J. Angew. Chem. Int. Ed. 2012, 51, 1395-1399 (b) Orcel, U.; De
Poli, M.; De Zotti, M.; Clayden, J. Chem. Eur. J. 2013, 19, 16357-
16365 (c) Pike, S. J.; De Poli, M.; Zawodny, W.; Raftery, J.; Webb,
S. J.; Clayden, J. Org. Biomol. Chem. 2013, 11, 3168-3176 (d) De
Poli, M.; Byrne, L.; Brown, R. A.; Solà, J.; Castellanos, A.;
Boddaert, T.; Wechsel, R.; Beadle, J. D.; Clayden, J. J. Org. Chem.
2014, 69, 4659-4675 (e) Byrne, L.; Solà, J.; Boddaert, T.; Marcelli,
T.; Adams R. W.; Morris, G. A.; Clayden, J. Angew. Chem. Int. Ed.
2014, 53, 151-155 (f) De Poli, M.; Clayden, J. Org. Biomol. Chem.,
2014, 12, 836-843 (g) Le Bailly, B. A. F.; Clayden, J. Chem. Com-
mun. 2014, 50, 7949-7952
(59) (a) Coxon, T. P.; Fallows, T. W.; Gough J. E.; Webb, S.J.
Org. Biomol. Chem. 2015, 13, 10751–10761. (b) de Cogan, F.;
Gough, J. E.; Webb, S. J. J. Mater. Sci.-Mater. M. 2011, 22, 1405.
(60) Brown, R.; Diemer, V.; Webb, S. J.; Clayden, J. Nature
Chem. 2013, 5, 853-860
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(38) A low background value of 0.004 s-1, which was not due to
methanol, was apparent when fitting the rate data for foldamers
1, 4 and 7 at different concentrations. Above this value, the ob-
served rate constants are approximately proportional to concen-
tration (see the Supporting Information).
(39) Altering the width of the bilayer using DLPC (~2.1 nm
hydrocarbon core thickness) did not provide useful information
as these bilayers were too leaky.
(40) (a) Monaco, V.; Formaggio, F.; Crisma, M.; Toniolo, C.;
Hanson, P.; Millhauser, G. L. Biopolymers 1999, 50, 239-253 (b)
Eid, M.; Rippa, S.; Castano, S.; Desbat, B.; Chopineau, J.; Rossi,
C.; Béven, L. J. Biophys 2010, 2010, 179641
(41) Duclohier, H.; Snook, C. F.; Wallace, B. A. Biochim. Bio-
phys. Acta 1998, 1415, 255-260
(42) Pajewski, R.; Ferdani, R.; Pajewska, J.; Djedovič, N.; Schle-
singer, P. A.; Gokel, G. W. Org. Biomol. Chem. 2005, 3, 619-625
(43) Zhou, J.; Lu, X.; Wang, Y.; Shi, J. Fluid Phase Equilibria
2002, 194-197, 257–27
(44) 9.3 μM alamethicin can release 50% of 5/6-CF encapsu-
lated in 1:1 EYPC: cholesterol vesicles after 20 minutes, although
at a much higher membrane loading of 47 mol %. See: Stella, L.;
Burattini, M.; Mazzuca, C.; Palleschi, A.; Venanzi, M.; Coin, I.;
Peggion, C.; Toniolo, C.; Pispisa, B. Chem. Biodiversity 2007, 4,
1299-1312
(45) Fyles, T. M. Chem. Soc. Rev. 2007, 36, 335-347
(46) Andersen, O. S.; Koeppe, R. E.; Roux, B., Gramicidin
Channels: Versatile Tools; In Biological Membrane Ion Channels,
Chung, S. H., Andersen, O. S., Krishnamurty, V., Eds.; Springer:
New York, 2007, pp 36
(47) Chui, J. K. W.; Fyles, T. M. Chem. Soc. Rev. 2012, 41, 148-
175
(48) Ferdani, R.; Gokel, G. W. Org. Biomol. Chem. 2006, 4,
3746-3750
(49) Sakai, N.; Houdebert, D.; Matile, S. Chem. Eur. J. 2003, 9,
223-232
(50) Sakai, N.; Mareda, J.; Matile, S. Acc. Chem. Res. 2005, 38,
79-87
(51) (a) Bayley, H.; Cronin, B.; Heron, A.; Holden, M. A.;
Hwang, W. L.; Syeda, R.; Thompson, J.; Wallace, M. Mol. Biosyst.
2008, 4, 1191-1208 (b) 0.5 μM alamethicin has been successfully
used in DIBs. See: Harriss, L. M.; Cronin, B.; Thompson, J. R.;
Wallace, M. I. J. Am. Chem. Soc. 2011, 133, 14507-14509
(52) Heimburg, T. Biophys. Chem. 2012, 150, 2-22
(53) Duclohier, H.; Wróblewski, H. J. Membrane Biol. 2001,
184, 1–12.
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(a) 310 Helical Aib foldamers 1 to 9. (b) Schematic representation of Aib foldamer helices interacting with a
bilayer to form a pore with activity determined by foldamer length (a, black arrows) and foldamer-foldamer
interactions (b, white arrows).
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Synthesis of foldamers 1-9 . i) N3AibCl, Et3N, CH2Cl2, RT. ii) Pd/C, MeOH or EtOH, H2, RT. iii) TFA, CH2Cl2,
RT. iv) Ac2O, 120 °C or EDC.HCl, CH2Cl2, RT then HAibmOR (R = t-Bu or (CH2)2TMS), CH3CN, 80 °C. v) NaN3,
DMF, RT. vi) 2-(Trimethylsilyl)ethanol, EDC.HCl, DMAP, CH2Cl2, RT. vii) HCl in Et2O.
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X-ray crystal structures of (a) N3Aib7OCH2CH2TMS 3, (b) N3Aib8OCH2CH2TMS 4 with CH3CN and (c)
N3Aib11OCH2CH2TMS 7. Foldamer-foldamer interactions of N3Aib7OCH2CH3TMS 3 in the solid state, involving
hydrogen bonds from (d) the first and (e) the second N-terminal Aib residue to the C-terminus of a
neighboring foldamer. Selected hydrogen bonds are shown in green, black arrows indicate the continuation
of the foldamer chains.
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(a) HPTS fluorescence changes for foldamers 7 (Aib11, blue) 5 (Aib9, green), 4 (Aib8, grey) 3 (Aib7, red) and
methanol blank (black). Additions of foldamer solution in methanol (20 µL, 60 µM) to LUVs (20%
cholesterol/EYPC, 0.76 mM lipid, 2 mL). Base pulse of NaOH (13 µL, 1 M) at 180 s (dashed line). Data
normalized to maximum fluorescence after addition of Triton X-100 (10% v/v in MOPS buffer) at 1800 s. (b)
Plot of [foldamer] (log scale) vs. pseudo-first order rate constants of ion leakage from LUVs for foldamers 7
(Aib11, blue), 4 (Aib8, grey) and 1 (Aib5, brown). Curve fits to guide the eye.
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Plot of apparent first-order rate constants for Na+ transport vs. number of Aib residues for foldamers (a)
Aib5 1 to Aib8 4 at 10 µM (b) Aib8 4 to Aib13 9 at 0.6 µM. Additions of foldamer stock solution in methanol
(20 µL) to 0.76 mM LUVs (20% cholesterol/EYPC, 2 mL). (c) Estimation of relative rate constants with
foldamer length, at 10 µM (dashed line) and 0.6 µM (solid line), normalized to relative rate of Aib8 4 at both
concentrations (krel = 1).
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Co-plot of normalized HPTS ion transport data (filled circles) and 5/6-CF release data (lines) for 3 (Aib7, 1
mM, red), 7 (Aib11, 60 µM, blue) and 9 (Aib13, 60 µM purple). Additions of foldamer solution in methanol to
LUVs (20% cholesterol/EYPC, 0.76 mM lipid, 2 mL) at 0 s (HPTS data, base pulse at 180 s) or 180 s (5/6-CF
data).
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Typical planar bilayer conductance behavior for 7 and 9. a) 7 (Aib11, 60 µM), applied voltage -100 mV,
current passed -150 pA, 1.5 nS. b) 9 (Aib13, 60 µM), applied voltage +100 mV, current passed +145 pA,
with smaller conducting state also observed at +130 pA. 1.45 nS or 1.3 nS respectively c) 9 (Aib13), 60 µM
concentration, applied voltage +100 mV, current passed +145 pA. 1.45 pS. d) Example I-V traces: 9 (Aib13
purple) and 7 (Aib11, blue) traces approximately 1 h after addition of compound.
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