Carboxylate anion diminishes chloride transport through a synthetic, self-assembled transmembrane pore

Article abstract of DOI:10.1002/chem.200701071

Six amphiphilic heptapeptides with the structure (C₁₈H ₃₇)₂NCO-CH₂OCH₂CO-(Gly) ₃-Pro-(Gly)_n-(Glx)-(Gly)_m-O(CH ₂)₆CH₃, in which Glx represents glutamic acid or its benzyl ester and n + m = 2, have been studied. In addition, the glutamate residue in the GGGPGGE sequence was esterified by fluorescent 1-pyrenemethanol. These compounds insert into phospholipid bilayers and form anion-conducting pores. Hill plots based on carboxyfluorescein release indicate that the pores are at least dimeric. Studies that involved ion-selective electrode techniques showed that transport of chloride varied with the position of glutamate within the peptide chain and whether glutamic acid was present as the free acid or its benzyl ester. Chloride transport activity was significantly higher for the glutamate esters than for free carboxylates irrespective of the glutamate position. Activity was highest when the glutamate residue in - (Gly) ₃-Pro-(Xxx)₃- was closest to the C terminus of the peptide. A fluorescent pyrene residue was introduced to probe the aggregation state of the amphiphile. The selectivity of the pore for Cl^- over K⁺ was maintained even when the carboxylate anion was present within it. Complexation of Cl^- by the ionophoric peptides was confirmed by negative ion mass spectrometry. Planar bilayer voltage clamp experiments confirmed that pores with more than one conductance state may form in these dynamic, self-assembled pores.

Full text of DOI:10.1002/chem.200701071

DOI: 10.1002/chem.200701071
Carboxylate Anion Diminishes Chloride Transport through a Synthetic, Self-
Assembled Transmembrane Pore
[a]
Lei You,[a] Riccardo Ferdani,[b] RuiqiongLi, Joseph P. Kramer,[b]
Rudolph Ernst K. Winter,[b] and George W. Gokel*[a, b, c]
Abstract: Six amphiphilic heptapepti-
des with the structure (C18H37)2NCO-
CH2OCH2CO-(Gly)3-Pro-(Gly)n-(Glx)-
(Gly)m-O(CH2)6CH3, in which Glx rep-
resents glutamic acid or its benzyl ester
and n+m=2, have been studied. In
addition, the glutamate residue in the
GGGPGGE sequence was esterified
niques showed that transport of chlo-
ride varied with the position of gluta-
mate within the peptide chain and
whether glutamic acid was present as
the free acid or its benzyl ester. Chlo-
ride transport activity was significantly
higher for the glutamate esters than for
free carboxylates irrespective of the
glutamate position. Activity was high-
est when the glutamate residue in
~(Gly)3-Pro-(Xxx)3 ~ was closest to the
C terminus of the peptide. A fluores-
cent pyrene residue was introduced to
probe the aggregation state of the am-
phiphile. The selectivity of the pore for
Clꢀ over K+ was maintained even
when the carboxylate anion was pres-
ent within it. Complexation of Clꢀ by
the ionophoric peptides was confirmed
by negative ion mass spectrometry.
Planar bilayer voltage clamp experi-
ments confirmed that pores with more
than one conductance state may form
in these dynamic, self-assembled pores.
AHCTREUNG
G
G
ACHTREUNG
ACHTREUNG
by
fluorescent
1-pyrenemethanol.
These compounds insert into phospho-
lipid bilayers and form anion-conduct-
ing pores. Hill plots based on carboxy-
fluorescein release indicate that the
pores are at least dimeric. Studies that
involved ion-selective electrode tech-
Keywords: anion transport · mem-
branes · peptides · self-assembly ·
supramolecular chemistry
Introduction
tion of a transmembrane, anion-conducting pore would
mimic some functions of the ClC family of chloride-trans-
porting proteins.[9,10,11,12]
Over the last ten years, numerous anion-complexing
agents[1,2] have been designed, prepared, and studied.[3,4,5] In
many cases, the ability of these receptor molecules to com-
plex anions has been characterized by X-ray crystallography.
Solution complexation of various anions has also been stud-
ied for cyclic[6] and acyclic receptors.[7] Our particular inter-
est has been in designing molecules that can form pores in
phospholipid bilayers and not only complex anions, but
transport them across a membrane.[8] The successful forma-
The first solid-state structure of a ClC protein was report-
ed in 2002.[13] The amino acid sequence G
(K/R)EGP at posi-
A
tions 146 to 150, which is conserved in ClC Clꢀ transporting
proteins, was thought to be part of the conductance pore.
Subsequent study led to the proposal that chloride transport
was gated, at least in part, by a glutamate residue at position
148 (E-148).[14] The solid-state structure of a Glu148!Ala148
mutant revealed a fully open channel. The ion path suggest-
ed by structural studies for chloride transport is complex
and involves interactions with at least a main-chain amide
NH, tyrosine and serine side chains, and a phenylalanine.[14]
Glutamic acid has a side chain carboxyl pKa of ꢁ4.3. We
presume that there is a significant concentration of water
within the self-assembled pores. At physiological pH, the
glutamate carboxyl group should be completely deprotonat-
ed. A carboxylate anion should certainly repel a chloride
anion, but the effect of a carboxylate anion present within a
pore on transport is less clear. Within a pore, a carboxyl
group could repel Clꢀ and prevent passage of this ion. Alter-
natively, the combination of negative charges could cause
[a] L. You, R. Li, Prof. Dr. G. W. Gokel
Department of Chemistry, Washington University
1 Brookings Drive, St. Louis, MO 63130 (USA)
Fax : (+1)314-516-5321
E-mail: gokelg@umsl.edu
[b] Dr. R. Ferdani, Dr. J. P. Kramer, Dr. R. E. K. Winter,
Prof. Dr. G. W. Gokel
Departments of Chemistry & Biochemistry and Biology
University of Missouri—St. Louis, St. Louis, MO 63121 (USA)
[c] Prof. Dr. G. W. Gokel
Center for Nanoscience, University of Missouri—St. Louis
St. Louis, MO 63121 (USA)
382
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Chem. Eur. J. 2008, 14, 382 – 396

FULL PAPER
the pore to collapse. A third possibility is that if the Clꢀ con-
centration gradient was large, the carboxyl could be repelled
by Clꢀ, the pore opened to a wide diameter, and ions (possi-
bly of both charge types) could traverse the membrane. This
would be a simple charge-based
ther, hinge bend sequences involving proline were typically
GxxP. The simplest emulation of GxxP is GGGP, which was
employed as ~(Gly)3-Pro-(Gly)3 ~, as the first example in
this system.
gating mechanism. A challenge
is to develop a dynamic trans-
port system that functions
within the bilayer and incorpo-
rates features of complex
modern proteins.
In extensive previous stud-
ies,[15] we have found that com-
pounds of the general form
(CnH2n+1)2NCOCH2OCH2CO-
(Gly)3-Pro-(Gly)3-OR insert in
phospholipid liposomes to form
a chloride-selective, ion-perme-
able pore. Variations in both
the N- (CnH2n+1) and C-termi-
nal (OR or NHR) alkyl groups
altered the properties of the
pores that formed.[15c,e,g] Studies
of carboxyfluorescein anion
(CFꢀ) release from liposomes
mediated by these compounds
suggested that pore formation
requires at least a dimer aggre-
gate.[15j] These synthetic pep-
tides have also been shown to
bind Clꢀ ions in CDCl3 in a
cation-dependent fashion.[15g,k]
In the work presented herein,
we have varied the triad of
amino acids on the C-terminal
side of proline in heptapeptides
GGGPXXX. Glycines in posi-
tions five to seven were systematically replaced by either
glutamic acid or by its benzyl ester. This modification per-
mitted us to assess the effect of a negative charge in each of
the three “X” positions and to compare it with the presence
of a sterically similar, but uncharged, side chain. Glutamic
acid, rather than aspartic acid, was chosen for this study be-
cause it is glutamate that is present in the putative ion path-
way of the ClC proteins.
At the C-terminal side of proline, triglycine (~GlyGlyG-
ly~) was systematically replaced by ~GlxGlyGly~,
~GlyGlxGly~, or ~GlyGlyGlx~, in which Glx represents
both glutamic acid and its benzyl ester. The suite of com-
pounds thus comprises three pairs of related structures:
1 and 2, 3 and 4, and 5 and 6. The first pair (1 and 2)
can
(C18H37)2NCOCH2OCH2CO-(Gly)3-Pro-Glx-(Gly)2-O-
(CH2)6CH3. I n1, Glx is glutamic acid and its side chain car-
be
described
by
the
general
formula
AHCTREUNG
boxyl group is benzylated. In 2, Glx is glutamic acid (Glu),
which has a free carboxyl group. Similarly, peptide sequen-
ces for 3 and 4 are (Gly)3-Pro-Gly-Glx-Gly in which Glx is
benzylated Glu in 3 and Glu in 4. The final two peptides, 5
and 6, have the sequence (Gly)3-Pro-Gly-Gly-Glx in which
Glx is benzylated Glu in 5, Glu in 6, and in 7 Glx is Glu
that has been esterified by 1-pyrenemethanol. All eight hep-
tapeptides (1–8), have the (C18H37)2NCOCH2OCH2CO~
group at the N terminus of the peptide.
Results and Discussion
Compounds used in the study: Seven amphiphilic heptapep-
tides of the type known to form chloride-conducting
pores[11] were prepared for use in the present study. Six of
these compounds (1–6) all have the basic structure ~(Gly)3-
Pro-(Xxx)3 ~. A seventh (7), discussed below, is a fluores-
cent analogue of 5. The sequence was inspired by two obser-
vations. First, the GKxGP sequence, noted above as G
R)EGP, was conserved in the ClC transporter ion path. Fur-
A
The twin octadecyl chains in 1–8 are intended to commin-
gle with the fatty acid chains in the bilayer, thus anchoring
Chem. Eur. J. 2008, 14, 382 – 396
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383

G. W. Gokel et al.
the amphiphilic peptide within the membrane[16] and the C-
terminal heptyl group serves as a secondary anchor.[15c]
From an evolutionary perspective, we surmise that the earli-
est transmembrane pores were formed by molecules that re-
sembled the membrane monomers themselves. In modern
bilayers, the monomers are phospholipids. We thus incorpo-
rated the diglycoyl residue as a spacer and connector that
links the twin hydrocarbon chains to the peptide. Diglycolic
(H37C18)2NCOCH2OCH2CON-
H-GGGPGGG-OCH2Ph.[15h]
Hill plots based on the concen-
tration dependence of ion re-
lease showed that a minimum
of two amphiphilic peptides
were required for ions to pass
through a liposomal bilayer.[15h]
Thus, pore formation can occur
by insertion of the amphiphiles
into the outer leaflet of the bi-
layer. This will place glutamate
residues in the chloride ion
pathway; this is illustrated sche-
matically in Figure 1.
acid (O
converted into its anhydride, which can then be opened by
treatment with secondary amine (R2NH) to give
(CH2COOH)2) was chosen because it can be easily
a
R2NCOCH2OCH2COOH. Equally important, however, is
the fact that the polar atoms in the diglycoyl residue are
positioned to emulate the polar elements of the glyceryl
subunit of a phospholipid.
Figure 1. Schematic represen-
tation of a pore formed by the
association of two amphiphilic
heptapeptides.
The alkyl chain span (C-18!N) is ꢁ22.4 Š and the digly-
colic acid chain adds ꢁ6.4 Š (total length ꢁ29 Š). Together
with the diglycolic acid subunit, the twin chains approximate
the length and polarity of the diacylglycerol unit of phos-
pholipids. The span of the insulator or hydrocarbon regime
of a bilayer is typically estimated to be 30 to 35 Š. The hy-
drocarbon chains of 1 to 8 will not span this distance,
but a pore can form by rearrangement of the phospholipid
monomer head groups in the lower leaflet of the
bilayer.[17] Both Clꢀ and CFꢀ release are mediated by
Synthetic access to compounds
1–7: The series under study
comprises three pairs of compounds, 1 and 2, 3 and 4, and 5
and 6. Compound 7 is a pyrenyl ester analogue of 5. The
production of each of the free acids and esters within the
series confronts similar synthetic issues. Thus, we show the
preparation of 5 and 6, the pair of compounds that have the
~(Gly)3-Pro-(Gly)2Glx~
Scheme 1.
heptapeptide
sequence,
in
Scheme 1. Synthesis of compounds
5 and 6. i) 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine
(DMAP), 1-heptanol, CH2Cl2, 88%; ii) 1. HCl, dioxane, 2. Boc-Gly-OH, EDCI, 1-hydroxybenzotriazole (HOBt), Et3N, CH2Cl2, 76%, iii) 1. HCl, dioxane,
2. Boc-Gly-OH, EDCI, HOBt, Et3N, CH2Cl2, 84%; iv) 1. HCl, dioxane, 2. Boc-Pro-OH, EDCI, HOBt, Et3N, CH2Cl2, 74%; v) 1. HCl, dioxane,
2. 182DGA-GGG-OH, EDCI, HOBt, Et3N, CH2Cl2, 47%; vi) 10% Pd/C, EtOH, H2, 93%.
384
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Self-Assembled Transmembrane Pores
FULL PAPER
2-t-Butoxycarbonylaminopentanedioic acid 5-benzyl ester
(N-Boc l-glutamic acid g-benzyl ester) was esterified with n-
heptanol, the t-Boc group was removed (HCl/dioxane), and
the heptyl ester was coupled to t-Boc-Gly-OH. The resulting
dipeptide was coupled to t-Boc-Gly-OH to give Boc-Gly-
The chloride release data in Figure 2 show that initial
chloride transport is similar (0–250 s) for 3 and 4. We infer
that the insertion of either compound into the bilayer occurs
with similar dynamics. In contrast with the initial behavior,
Clꢀ release after 1800 s is significantly higher for 3 than for
4. Structurally, these compounds differ only by the presence
or absence of the benzyl ester on the glutamic acid residue
at position 6 in the peptide. Although the modes of insertion
and pore formation remain speculative, it is clear that 4
must present a negative charge in or near the pore and 3
does not. If the pore is dimeric or oligomeric, more than
one negative charge will be present proximate to the pore
(see Figure 1). The side chain carboxyl of glutamic acid is
reported to have a pKa value of 4.32, so it will be completely
ionized beyond about pH 6.8; the experiment is conducted
at pH 7.
Gly-Glu(OCH2Ph)-OC7H15 after Boc deprotection. Sequen-
A
tial reaction with HCl/dioxane and then t-Boc-Pro-OH
gave the t-Boc-protected tetrapeptide Pro-(Gly)2-Glu. We
have
previously
reported
the
preparation
of
(C18H37)2NCOCH2OCH2CO-(Gly)3-OH, which was coupled
to the deprotected tetrapeptide to form diester 5.[11d] The
side chain benzyl ester in 5 was hydrogenolytically removed
to give 6. Details of the preparation of compounds 1 to 7
are given in the Experimental Section.
Chloride release from liposomes mediated by 1–6: Chloride
transport can be assessed in various ways. Planar bilayer
clamp studies give the most detailed picture of a conduc-
tance pore, but the method is complex and time consuming.
For the compounds described herein, we used an assay in
which Clꢀ release from liposomes was detected by an Accu-
met chloride-selective electrode. Thus, liposomes (200 nm
average diameter) were prepared from a 7:3 (w/w) mixture
of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and
1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) as described in
the Experimental Section. The vesicles were loaded with
KCl (600 mm) in HEPES buffer (pH 7). The external buffer
was chloride free K2SO4 (400 mm) in HEPES (pH 7). With
the electrode inserted into the aqueous liposome suspension,
the ionophore under study was introduced as a solution in
2-propanol and the response of the electrode was recorded
over 1800 s.
Typically, the chloride electrode began to respond to ion
release within five seconds after ionophore addition. Data
collection is automated and a value was typically recorded
every second. At the conclusion of an experiment, Triton X-
100 detergent was added to lyse the vesicles and a final
value was determined for chloride concentration. This value
was arbitrarily set to one and the release data presented are
the fraction of total chloride measured after vesicular lysis.
Each data set is the average of at least three independent
experiments. Typical results are shown in Figure 2 for 3, 4, 5
and 6.
Compounds
6
and
5
are identical (sequence:
GGGPGGE) except that the former is the free acid and the
latter is its benzyl ester. Thus, the glutamate residue is at
the C-terminal (position 7) end of the heptapeptide. Com-
pound
4 is a free acid with the peptide sequence
GGGPGEG; chloride release for free acids 4 and 6 is very
similar, both in terms of curve shape and ultimate ion re-
lease. Chloride release mediated by benzyl ester 5, however,
is about twice (0.65 vs. 0.32) that observed for the benzyl
ester of 4, that is, 3. The curve shapes exhibited by ion re-
lease can be difficult to interpret, but either insertion or the
initial organization into a pore clearly occurs differently for
esters 5 and 3.
The chloride transport activity of glutamic acid derivative
2 (G3PEGG) is slightly less than, but similar to, that shown
above for 4 (G3PGEG) and 6 (G3PGGE). In all three cases,
however, the ability of free acid compounds 2, 4, and 6 to
transport Clꢀ is less than that observed for esterified ana-
logues 1, 3, and 5. We infer from this that the presence of
one or more negative charges in the ion pathway impeded
Clꢀ entry into the pore and/or transport through it. In terms
of electrostatic interactions, it seems reasonable that this
should be so, although the curve shape does not reveal
which, if either, of these variables is dominant. In the ClC
channel, the Glu148!Ala148 mutation appeared to foster an
open ion conduction pathway.[18] The lower activity of 2, 4,
and 6 (charged) than 1, 3, and 5 (neutral) suggests that the
self-assembled pores described herein are similarly affected
by a charge in the ion path.
Comparison of transport rates: Compounds 1–6 are similar
in overall structure, but the variations in transport rates are
significant. In addition, the curve shapes differ. In all cases,
any divergence in the curve shapes occurs primarily in the
first 0 to 300 seconds of observation. The various curve
shapes must reflect differences in the rates of amphiphile in-
sertions and the dynamics of pore formation. The details of
these processes remain to be characterized, but the curves
exhibit reasonably regular shapes, at least after this initial
period.
Figure 2. Chloride release from phospholipid liposomes mediated by 3
and 4, 5 and 6 (lipids (0.31 mm), compounds (65 mm), pH 7.0).
Chem. Eur. J. 2008, 14, 382 – 396
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385

G. W. Gokel et al.
Chloride ion release data are summarized in Table 1.
Compound 5 showed the greatest ion release at the arbitra-
rily selected time point of 1500 s. Its release value at 1500 s
Table 1. Comparison of fractional chloride release from liposomes medi-
ated by 1–6 at 1500 s
[c]
Compound Peptide sequence
Release at
1500 s[a]
Relative
rate[b]
%
ꢀ
ꢀ
ꢀ
1
2
3
4
5
6
GGGPE
GGGPEGG
GGGPGE
GGGPGEG
GGGPGGE
GGGPGGE
G
65
22
49
30
100
38
33%
0.14
0.20
0.25
62%
38%
Figure 3. Difference in Clꢀ release from liposomes for the amphiphile
pairs 1–2 (middle, R2 =0.92), 3–4 (bottom, R2 =0.91), and 5–6 (top, R2 =
0.96). The solid line in each case is the calculated fit.
[a] Fraction of Clꢀ detected after liposomal lysis. [b] Comparison of 1–6;
release for 5 arbitrarily set to 100. [c] Percentage of activity for the less
effective compound in each pair.
Comparison of ester compounds: In the ester series (1, 3,
and 5), 5 is the most active and 3 is the least active Clꢀ
transporter. The presence of a bulky benzyl ester side chain
is expected to alter the conformation of the peptide and to
have an especially large effect when it is near either of the
was therefore set to 100 and the values shown in column 4
of Table 1 were calculated accordingly. Thus, 22 to 100 rep-
resents the overall range of chloride release values.
Thus far, the synthetic anion transporter (SAT) deriva-
tives have not yielded crystals suitable for solid-state struc-
7
amide protons (5Gly or Gly), which were identified above
as the key donor residues. We surmise that the benzyl group
will have the greatest effect where it is most flexible (C ter-
minus) and the least effect near the rigid proline. Computa-
tional studies were conducted by using Gaussian 03[19] (gas
phase, see the Experimental Section) to gain insight into
this structural question.
The calculations were performed on analogues of
(C18H37)2N-COCH2OCH2CO-(Gly)3-Pro-(Gly)3-OCH2Ph in
which the N-terminal octadecyl alkyl chains and the C-ter-
minal benzyl ester were replaced by methyl groups. This
simplified the calculation, which was intended to focus on
the heptapeptide chain. The structures that correspond to 1,
3, and 5 are arranged in Figure 4 with the proline residue at
or near the apex of each structure. The N-terminal side of
the chain descends from it to the left and the C-terminal
chain, which includes the glutamate benzyl ester, appears at
the right.
tural analysis.
A detailed solution NMR analysis on
(C18H37)2N-COCH2OCH2CO-(Gly)3-Pro-(Gly)3-OCH2Ph in
the presence of Bu4NCl showed a folded structure in which
7
the amide NH hydrogen atoms on 5Gly and Gly strongly in-
teract with chloride anions in homogeneous solution.[15k,l]
Both 1D and 2D NMR indicated that the amide protons of
the fifth and seventh residues (i.e., the fourth and sixth gly-
cines) are hydrogen bonded to Clꢀ in solution in CDCl3, but
the NH of glycine-5 (the sixth residue) is not. Replacement
of either glycine residue by glutamate will obviously have
conformational consequences on this NH donor interaction.
The data presented in Table 1 confirm that a Gly!Glu mu-
tation at positions 5 (5Gly) or 7 (7Gly), alters the Clꢀ trans-
port rate more than a corresponding alteration at position 6.
To confirm the validity of comparisons between the am-
phiphile pairs 1–2, 3–4, and 5–6, we computed the difference
in transport rate for each pair
at time values between 300 and
1500 s. This gave three 1200-
point lines that are shown in
Figure 3. Each of the three data
sets is overlaid by a calculated
second order fit. The scatter in
each line is clearly modest and
the calculated correlation factor
(R2) is at least 0.91 in each
case. We infer that although the
dynamics of pore formation
may vary, which is reflected in
the 0 to 300 s time frame, ion
release is reasonably consistent
thereafter.
Figure 4. Calculated structures for 1 (left), 3 (center), and 5 (right).
386
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Self-Assembled Transmembrane Pores
FULL PAPER
A key finding of the calculated structures (Figure 4) is
that the benzyl group of 5 (right structure) may serve as an
additional anchor to the bilayer. Assuming that a better
anchored monomer contributes to a more stable conduction
pore, then 5 should be superior to either 1 or 3 as an ion
transporter. This accounts, at least in part, for the higher
transport activity observed for 5 in this group of three close-
ly related structures. It is interesting to note that the ben-
zene ring in the peptide of 3 is very close to the amide NH
of the sixth glycine (position-7, C terminus). The NMR re-
sults described above[15k] clearly show that this NH is critical
for binding Clꢀ. An NH···p hydrogen-bond contact between
position adjacent to proline (2) towards the C terminus of
the peptide. Thus, at 1800 s, the fractional Clꢀ release values
are 0.13 for 2, 0.20 for 4, and 0.25 for 6. The release profiles
(line shapes) are similar in all three cases (see Figures 2 and
3). Based simply on charge–charge repulsion, it seems rea-
sonable that Clꢀ transport would be hindered by encounter-
ing a negative charge within the pore. It was also plausible
that charge–charge repulsions between Clꢀ and carboxylate
could force the self-assembled monomers apart, which re-
sults in making the pore larger, increasing ion flux, and re-
ducing selectivity.
7
the benzyl ester and GNH could further diminish the trans-
Anion versus cation transport: Early studies of (C18H37)2N-
COCH2OCH2CO-(Gly)3-Pro-(Gly)3-OCH2Ph showed a min-
imum ten-fold selectivity for Clꢀ over K+.[15b] This informa-
tion was obtained by the cumbersome planar bilayer con-
ductance method. An alternative approach is to measure po-
tassium efflux and compare it with chloride release data. Po-
tassium cation transport experiments were conducted as de-
scribed for the corresponding Clꢀ experiments, except that
the external buffer was a mixture of choline chloride
(600 mm) and HEPES (10 mm) held at pH 7.0. Compounds
1 to 6 all mediated K+ release from DOPC/DOPA lipo-
somes to a small extent. The most active chloride transport-
er (5) showed 12% K+ release at 1500 s compared with
65% for Clꢀ release. The least active Clꢀ transporter (2)
showed approximately 1% K+ release compared with 14%
Clꢀ release at 1500 s. These data correspond to selectivity
ratios of ꢁ5:1 to 14:1 for Clꢀ over K+. The key finding of
this part of the study is that the self-assembled transmem-
brane pores remain Clꢀ selective even when the free carbox-
ylate anion is present within or proximate to the pore. A
negatively charged side chain within the pore could electro-
statically attract K+. Effective transport depends on both
transient complexation and release. If K+ enters the pore,
but is neither released nor blocks the pore, Clꢀ transport
will dominate and selectivity will be maintained. This ex-
planation is consistent with our recent finding (unpublished)
that a positive charge within the pore does not enhance
anion transport.
port activity of 3 relative to 1 and 5 by hindering access of
the transient chloride ion to the molecule.
Secondary anchor effect: The pores formed by molecules 1
to 6 are self-assembled. As such, the effect of residues that
can help to anchor and/or organize the monomers in the bi-
layer is potentially very significant. Diglycolic acid is used in
our SAT design as the glycerol-mimetic transition from the
peptide to the N-terminal alkyl chain anchors. The similarity
of diglycolic and glutamic acids to each other and to the
glyceryl residue is apparent (Figure 5). It is plausible that
glutamic acid could serve as a transitional component for a
benzyl group anchor.
Figure 5. The structures of the diacylglyceryl and diglycolic acid subunits,
which are similar to that of the glutamic acid subunit.
Negative ion mass spectral study: Electrospray mass spec-
trometry (ES-MS) is a powerful structural tool that has
been used relatively little to probe anion complexation.[22,23]
We have used it to characterize the SAT molecules and the
Clꢀ complexes that are the subject of this paper.
It is interesting to note that Tirrell and co-workers have
used a similar structural motif as an N-terminal midpolar
regime mimetic.[20] They have also observed antibiotic activi-
ty for their compounds.[21] The peptides reported herein
have not yet been screened for antibacterial activity, but our
hydraphile cation-selective channels have shown significant
cytotoxicity.[15]
When
(C18H37)2N-COCH2OCH2CO-(Gly)3-Pro-(Gly)2-
Glu-O(CH2)6CH3 (6) in CH3CN/2-PrOH (1:1 v/v) was
AHCTREUNG
sprayed, only two peaks with ion abundances greater than
20% were observed in the mass range 600 to 2400. The base
peak was observed at m/z 1245.6 and corresponds to
[6ꢀH]ꢀ. The other peak in the spectrum occurred at m/z
1217.6 and corresponds to loss of a proton and CO from 6.
Loss of 28 mass units could correspond to CO or to ethyl-
ene. We were unable to find any precedent for this decom-
position mode of glutamate in negative ion MS. There is,
however, a report of CO loss from glutaric acid esters that
Acid position on the heptapeptide chain: The chloride trans-
port activity of free acids 2, 4, and 6 are all lower than their
esterified analogues. Their transport efficacy increases
almost linearly as the glutamate residue is moved from its
Chem. Eur. J. 2008, 14, 382 – 396
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387

G. W. Gokel et al.
suggests it is CO rather than CH2=CH2 that is lost in the
present case.[24]
therefore, poorer ion-recognition capability of 6 compared
with 5 may contribute to the lower transport efficacy of 6.
When a 1:3 molar ratio mixture of 6 and Clꢀ was sprayed
in CH3CN, four major peaks were observed in the 1192 to
1314 mass range, m/z 1217.6 (46%), 1245.6 (100%), 1253.5
(18%), and 1281.5 (27%). These ions correspond to
[6ꢀHꢀ28]ꢀ, [6ꢀH]ꢀ, [6+Clꢀ28]ꢀ, and [6+Cl]ꢀ, respective-
ly. As in the absence of Clꢀ, the base peak is deprotonated
6. Its complex with Clꢀ has an abundance of 27%. This is a
Stoichiometry of pore formation: Chloride ion transport, as
shown above, may be measured by using ion-selective elec-
trodes. This method is reproducible and reliable, but sensi-
tivity and the speed of the response are both limited. Car-
boxyfluorescein (CFꢀ) is an anion whose efflux from vesicles
can be quantitatively monitored by fluorescence technique-
s.[15a] The increased sensitivity also permits a broader con-
centration range to be surveyed than that for chloride re-
lease. Chloride and carboxyfluorescein may seem to be too
different for comparison, but they have behaved similarly in
a number of previous studies.[15h]
ratio of [6ꢀH]ꢀ/[6+Cl]ꢀ of ꢁ3.7:1. This ratio was un-
N
changed when the 6/Clꢀ molar ratio was increased from 1:3
to 1:12. Similarly, the decarbonylated derivative of 6 has a
ratio of parent to complex of ꢁ2.4.
Similar experiments were conducted for 5. When a 1:3
molar ratio mixture of 5 and Bu4NCl was sprayed, only two
major peaks were detected above 1000, m/z 1343.6 (50%)
and m/z 1371.6 (100%). These ions are [5+Clꢀ28]ꢀ and
[5+Cl]ꢀ, respectively. Of course, free neutral 5 cannot be
detected by negative ion ES-MS.
Release of CFꢀ from DOPC/DOPA liposomes (see the
Experimental Section) was monitored over a concentration
range from 0.99 to 7.86 mm. The ion-release data are shown
in Figure 7a. Carboxyfluorescein release is consistent and
It would be interesting to use ES-MS to observe the
extent of complexation by 6 compared to its ester analogue
5. Therefore, competition experiments were performed. A
mixture of 5, 6, and Bu4NCl (molar ratio 1:1:1) was sprayed.
Six species were observed above 1000, 1217.6 (35%)
[6ꢀHꢀ28]ꢀ, 1245.6 (100%) [6ꢀH]ꢀ, 1253.5 (12%)
[6+Clꢀ28]ꢀ, 1281.5 (20%) [6+Cl]ꢀ, 1343.6 (30%)
[5+Clꢀ28]ꢀ, and 1371.6 (72%) [5+Cl]ꢀ. The results are
shown in Figure 6. When a 1:1:12 molar ratio of 5, 6, and
Figure 6. Negative ion mass spectrum of a 1:1 CH3CN/2-PrOH solution
Figure 7. a) Carboxyfluorescein release from DOPC:DOPA vesicles
mediated by 5 at the following concentrations (bottom to top): 0.99, 1.98,
2.47, 3.45, 5.66, and 7.86 mm. b) Hill plot of CF release by 5.
of 5/6/Clꢀ in a 1:1:1 molar ratio.
Clꢀ was used, the abundances for [5+Clꢀ28]ꢀ and [5+Cl]ꢀ
increased to 47 and 94%, respectively. The peak intensity
ratio for [5+Cl]ꢀ over [6+Cl]ꢀ was 3.6 for the 1:1:1 case
and 4.7 when the molar ratios were 1:1:12.
It is apparent from these experiments that neutral 5 binds
chloride anions. This result is expected from solution NMR
results reported previously.[15k] The results for 5 are in con-
trasts with the behavior of 6, which shows poor complexa-
tion behavior. Compound 6 is deprotonated to the extent
that the negative charge will repel the chloride anion and
result in poor binding. The poorer chloride binding, and
concentration dependent. The Hill equation[25] was applied
to the release data to determine the minimum number of
monomers required for pore formation. The plot is shown in
the Figure 7b; the slope of 2.4 suggests that at least two
monomers are required, although more amphiphiles may be
present in some pores.
Previous
compounds
of
the
general
type
R1 NCOCH2OCH2CO-(Gly)3-Pro-(Gly)3-OR2, which have
2
been studied, to date, formed pores that were at least dimer-
ic.[15a] Additional evidence for dimeric pore formation was
acquired by covalently linking two amphiphilic monomers
388
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Chem. Eur. J. 2008, 14, 382 – 396

Self-Assembled Transmembrane Pores
FULL PAPER
either at the two C- or N-terminal ends.[15j] The latter study
is strongly suggestive that dimeric pore formation occurs,
but is limited by the C-terminal to C-terminal and N-termi-
nal to N-terminal linkages. In fact, the organization of the
dimer pore may occur in a C-terminal to N-terminal fashion,
as intuition suggests. To date, there is no direct experimental
evidence to confirm a C!N organization in the bilayer. It
ꢀ
ꢀ
should be noted, however, that both the C C and N N
pseudo-dimers mediated Clꢀ release from liposomes more
effectively than twice the concentration of monomers.[15j]
We note that the Clꢀ and CFꢀ release experiments were
run under different conditions because the anions differ in
structure and presumably in hydration state. These differen-
ces notwithstanding, the line shapes observed for release of
these two ions are similar. Further, separate studies in which
the two anions were compared, revealed comparable release
profiles from vesicles when mediated by this family of iono-
phores.[15h]
Figure 9. Fluorescence titration curves determined in HEPES buffer for
7. The concentrations of 7 used were, from bottom to top, 0.25, 0.62, 1.25,
1.87, 2.49, 3.10, and 3.72 mm. The excitation wavelength was 345 nm.
aqueous solution. A plot of fluorescence intensity versus
concentration was linear (R2 =0.99, data not shown).
Formation of the well-known pyrene excimer by amphi-
philic 7 in a polar solvent was not surprising. However, we
wished to determine if aggregation was controlled by the
ionophoric peptide rather than by pyrene at these concen-
trations. Fluorescence spectra were therefore determined for
1-pyrenylmethanol (Figure 10a) and for 7 (Figure 10b) in
CH2Cl2, EtOH, and HEPES buffer. The concentrations of 1-
pyrenylmethanol (0.62 mm) and 7 (1.25 mm) were adjusted to
afford similar fluorescent intensities and thereby to facilitate
comparison. In these solvents at these concentrations, only 7
showed an excimer band. No excimer band was detected for
Fluorescent heptapeptide monomers: Pyrenyl ester 7 is
identical to 5 except that the glutamic acid residue is esteri-
fied by hydroxymethylpyrene rather than hydroxymethyl-
benzene. It was prepared specifically to be used as a probe
of membrane insertion and dynamics. Pyrene fluorescence
can afford information on the polarity of the environment
experienced by the fluorophore and excimer formation,
when it is observed, indicates association. The experimental
studies are described below. First, however, it was essential
to demonstrate that the presence of a pyrenyl ester did not
alter the Clꢀ transport behavior of the SAT. Figure 8 shows
Figure 8. Fractional chloride release from DOPC/DOPA liposomes medi-
ated by 5 and 7.
the fractional chloride release from liposomes mediated by
benzyl ester 5 and by pyrenylmethyl ester 7. The curves are
not identical, but they are nearly so. If anything, the pyre-
nylmethyl ester appears to give a cleaner sigmoidal release
than 5.
The fluorescence spectrum of 7 was determined in aque-
ous HEPES buffer in the concentration range 0.25 to
3.72 mm by using an excitation wavelength of 345 nm
(Figure 9). The major band was centered at ꢁ470 nm in all
spectra. This broad band is typical of excimer formation for
pyrene, which suggests aggregation of the amphiphiles in
Figure 10. a) Solvent dependence of 1-pyrenylmethanol in HEPES
buffer, CH2Cl2, and EtOH. b) Solvent dependence of 7 in HEPES buffer,
CH2Cl2, and EtOH. The excimer band centered at l=470 nm is observed
only for 7 in aqueous buffer.
Chem. Eur. J. 2008, 14, 382 – 396
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389

G. W. Gokel et al.
1-pyrenylmethanol in buffer, CH2Cl2, or ethanol. The inten-
sities of the spectra diminished in the order HEPES>
CH2Cl2 >EtOH. The approximate dielectric constants of
these solvents are 80 in aqueous buffer, 24 in EtOH, and 5
in CH2Cl2. Thus, the spectral intensity order does not follow
the polarity order, which suggests that other forces are at
work.
The striking difference in solvent dependence between 1-
pyrenylmethanol and 7 shows that aggregation results from
peptide interactions rather than an aggregation of pyrene
itself. Of course, the excimer emission shows that pyrene–
pyrene contact occurs in 72, even though many other resi-
dues are present. The aggregation suggests that 7 dimerizes
or oligomerizes in aqueous suspension before inserting into
a vesicular membrane.
mation in homogeneous solution only in aqueous buffer and
not in CH2Cl2 or EtOH (Figure 10b). When the excimer of 7
was observed in HEPES buffer, no monomer peak was ap-
parent. In either liposomal preparation, however, monomer
and excimer peaks were observed. Our interpretation of
these observations is that 7 aggregates to form at least
dimers in aqueous solution. These dimers or oligomers then
contact the liposome surface and partition into the mem-
brane where they form pores. The overall process must be
rapid because both Clꢀ and CFꢀ release are detected rapidly.
To the extent that monomer and dimer both exist in the bi-
layer, the equilibrium could account for the observed open–
close behavior (see below) of the synthetic channels.
To further determine if excimer formation influenced ag-
gregation and/or pore formation, a solution of 7 (1.87 mm)
was titrated with (C18H37)2NCOCH2OCH2CO-(Gly)3-Pro-
Two comparisons are of interest concerning Figure 11.
First, we note that there is relatively little difference in the
fluorescence spectra obtained for 7 in DOPC vesicles (Fig-
(Gly)3-O(CH2)CH3 (8). The latter is nonfluorescent, but
G
structurally similar to 7. The amounts of 8 added ranged
from about two to twenty equivalents. As the amount of 8
increased, The fluorescence arising from the monomer in-
creased. The monomer/excimer intensity ratio was mea-
sured. The fluorescence intensity ratio was plotted as a func-
tion of concentration (0–40 mm). A straight line (slope 0.05)
was obtained with an R2 value of 0.97. We thus infer that 7
behaves in a similar manner to other amphiphilic iono-
phores in this class and pyrene serves effectively as a report-
er.
Planar bilayer conductance: The formation of ion-conduct-
ing pores and channels can be distinguished from the action
of carrier molecules by using planar bilayer conductance
methods. In this experiment, a bilayer membrane is created
in a plate that separates two aqueous salt reservoirs. The bi-
layer membrane is an insulator in the absence of added ion-
ophore. Thereafter, ion transport may be observed and
monitored. Figure 12 shows a representative group of re-
cordings obtained when 6 was added to a DOPC/DOPA
(2:1 mol/mol, 7:3 w/w) membrane. The two reservoirs (cuv-
ette and chamber) each contained a KCl (450 mm) buffer so-
lution (10 mm HEPES, pH 7).
Figure 11. a) Fluorescence spectra of 7 in DOPC liposomes (0.31 mm)
suspended in HEPES buffer. b) Fluorescence spectra of 7 in DOPC-
DOPA liposomes (0.31 mm) suspended in HEPES buffer.
ure 11a) compared to liposomes formed from 7:3 DOPC/
DOPA (Figure 11b). DOPA has an anionic head group and
DOPC has a cationic head group. Differences in amphiphile
insertion into the membrane and/or aggregation of the iono-
phores might have been apparent, but were not. Similar re-
sults were obtained for Clꢀ release from the two different
types of liposomes (data not shown).
Divergence in behavior is more apparent when the fluo-
rescence spectra of 7, obtained in solutions or vesicular sus-
pension, are compared. Compound 7 showed excimer for-
Figure 12. Planar bilayer conductance results for 6 in DOPC/DOPA bi-
layer membranes.
390
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Chem. Eur. J. 2008, 14, 382 – 396

Self-Assembled Transmembrane Pores
FULL PAPER
The recordings shown clearly illustrate that the channels
or pores formed have regular, open–close states. The record-
ings shown here illustrate the predominant conductance
state. During analyses of this system, more than one conduc-
tance state was observed, which is not surprising because
multiple monomers may interact to give different pores si-
multaneously. The Hill analysis suggests that at least two
monomers are involved in the formation of a pore. Thus,
both 2- and 3-monomer pores are reasonable and expected.
Of course, the ion release plots shown in this paper illustrate
the average release over time from all pores that occur
within the liposomes. A plot of current versus voltage (I–V
curve) gave the expected straight line, which indicated that
the pore was nonrectifying. The conductance calculated for
the state illustrated in Figure 12 is 29.8 pS. Two other states
were observed in different experiments that had conduc-
tance values of ꢁ6 and ꢁ100 pS.
charge effect in these heptapeptides by preparing liposomes
at pH 4. At this pH, glutamate should be largely protonated
and differences between acid and ester should be mini-
mized. Unfortunately, at pH 4, the vesicles proved to be un-
stable.
Negative ion ES-MS has clearly shown that chloride ion
binding occurs with the neutral ionophoric peptides, but less
well when a free carboxyl is present. Selectivity studies
showed that the self-assembled pores favor Clꢀ over K+
transport even when the free and ionizable carboxyl group
is present on the peptide. It is proposed that the ClC pro-
teins have a similar electrostatic influence in their ion-con-
duction pathway. It is postulated that in the closed state the
pore is blocked by E-148 by a hydrogen-bond interaction
with the main-chain amide. The possibility that glutamate
could serve as a charged gate in a more rigid protein system
is certainly plausible in light of the present results.
Conclusion
Experimental Section
General: 1H NMR spectra were recorded in CDCl3 unless otherwise
specified at 300 MHz on a Varian Gemini 300 MHz NMR spectrometer
and are reported in the following manner: chemical shifts reported in
ppm (d) downfield from internal (CH3)4Si (multiplicity (br=broad, s=
singlet, d=doublet, t=triplet, q=quartet, brs=broad singlet, m=mul-
tiplet, etc.), coupling constants in Hz, integrated intensity, assignment).
13C NMR spectra (in CDCl3 unless otherwise noted) were obtained at
75 MHz and are referenced to CDCl3 (77.23 ppm). Infrared spectra were
recorded by using a Perkin–Elmer 1710 Fourier transform infrared spec-
trometer. Melting points were determined on a Thomas Hoover appara-
tus in open capillaries and are uncorrected. Thin layer chromatography
analyses were performed on silica gel 60-F-254 with a thickness of
0.2 mm. Preparative chromatography columns were packed with silica gel
(Kieselgel 60, 70–230 mesh or Merck grade 9385, 230–400 mesh, 60 Š).
The family of amphiphilic heptapeptides presented herein
insert into phospholipid bilayers and form anion-conducting
pores. These pores are at least dimeric, and planar bilayer
experiments show that more than one conductance state
may form. Ion-selective electrode and fluorescence tech-
niques showed that chloride and carboxyfluorescein anions
pass through the pores and the behavior of the average pore
varies with the position of glutamate. Activity is highest
when the glutamate residue is farthest from the central pro-
line bend (i.e., closest to the C terminus). This suggests that
charge is most influential when it is closest to the center of
the ion pathway. Activity is also higher for glutamate esters
than for the free carboxylate residue irrespective of the glu-
tamate position. The influence of carboxyl compared with
Reagents were the best (non-LC) grade commercially available and were
distilled, recrystallized, or used without further purification as appropri-
ate. CH2Cl2 was distilled from calcium hydride. DGA represents diglyco-
yl, ~COCH2OCH2CO~. Combustion analyses were performed by M-H-
W Laboratories, Phoenix, AZ, and are reported as percentages.
5
7
ester is largest for Glx and Glx, the two residues known
from previous NMR studies to involve direct NH contact to
anions.
Synthesis182DGA-GGG-OH: Compound 182DGA-GGG-OH was pre-
pared as previously reported.[11d]
As noted above, 7 is identical to 5 except that the gluta-
mate residue is esterified by 1-pyrenemethanol rather than
benzyl alcohol. Despite the significantly larger size of the ar-
omatic residue, Clꢀ transport by these two compounds is
similar. This is an important observation because concern is
sometimes expressed that the presence of a fluorescent resi-
due necessarily alters the experiment. In the present case,
esterification by hydroxymethylpyrene rather than hydroxy-
methylbenzene shows no deleterious effect. On the contrary,
the fluorescence of pyrene permits an assessment of the en-
vironment it experiences within the liposome or in aqueous
suspension. The assembly of peptide amphiphiles and inser-
tion into liposomes can be monitored by the intensity
changes observed in the monomer and excimer peaks.
Although these compounds are dynamic pore formers, the
influence of the glutamate residue on ion transport is clearly
in evidence. The presence of the negatively charged side
chain within the heptapeptide sequence clearly diminished
Clꢀ transport. An attempt was made to reverse the negative
TsOH·GG-OC7H15: Glyclglycine (GG) (1.00 g, 7.57 mmol) and TsOH
monohydrate (1.59 g, 8.36 mmol) were added to a mixture of 1-heptanol
(4.5 mL, 31.8 mmol) and toluene (50 mL). The mixture was heated to
reflux and water was removed by using a Dean-Stark adapter for 7 h.
The mixture was cooled to room temperature, diluted with diethyl ether
(50 mL) and cooled at 08C overnight. The solid was collected and recrys-
tallized from MeOH/Et2O to give a white solid (2.46 g, 81%). M.p. 131–
1338C; 1H NMR (300 MHz, CDCl3): d=0.88 (t, 3J
CH2CH3), 1.26 (pseudo-s, 8H; OCH2CH
2(CH2)4CH3), 1.52 (t, 3J
6.6 Hz, 2H; OCH2CH2(CH2)4CH3), 2.29 (s, 3H; CH3C6H4SO3), 3.77 (d,
3J
(H,H)=5.1 Hz, 2H; Gly CH2), 3.95–4.00 (m, 4H; Gly CH2 and
OCH2CH2 (H,H)=8.1 Hz, 2H; Tosyl HAr), 7.64–
(CH2)4CH3), 7.01 (d, 3J
ACHTREUNG
A
ACHTREUNG
AHCTREUNG
AHCTREUNG
A
ACHTREUNG
3
7.53 (m, 5H; Tosyl HAr and Gly NH3), 8.10 ppm (t, J(H,H)=5.4 Hz, 1H;
A
Gly NH); 13C NMR (75 MHz, CDCl3): d=14.3, 21.5, 22.8, 26.0, 28.6,
29.2, 31.9, 41.3, 65.8, 126.2, 129.2, 140.5, 141.3, 167.1, 170.0 ppm; IR
(CHCl3): 3299, 3164, 2956, 2931, 2858, 1758, 1697, 1575, 1513, 1467, 1442,
1397, 1375, 1323, 1279, 1216, 1170, 1124, 1037, 1011 cmꢀ1
.
Boc-E(g-benzyl
ester)GG-OC7H15
:
TsOH·GG-OC7H15
(0.60 g,
1.49 mmol), Boc-E-g-benzyl ester (0.50 g, 1.49 mmol), EDCI(0.31 g,
1.64 mmol), and HOBt (0.22 g, 1.64 mol) were dissolved in CH2Cl2
(40 mL) and Et3N (0.62 mL) was added. The mixture was stirred at 08C
for 0.5 h, at RT for 12 h, and the solvent was evaporated in vacuo. The
Chem. Eur. J. 2008, 14, 382 – 396
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391

G. W. Gokel et al.
residue was purified by column chromatography (silica gel, CHCl3/
CH3OH 98:2) to give an oil (0.73 g, 89%). 1H NMR (300 MHz, CDCl3):
shaken under H2 (60 psi) for 3 h in a Parr apparatus. The reaction mix-
ture was heated to reflux for 5 min and the hot mixture was filtered
through a Celite pad. The solvent was evaporated in vacuo to afford a
slight yellow solid (100 mg, 89%). 1H NMR (300 MHz, CDCl3): d=0.89
d=0.89 (t, 3J
OCH2CH
6.8 Hz, 2H; OCH2CH2
ACHTREUNG
A
N
ACHTREUNG
N
(t, 3J
(CH2)15CH2CH2N, OCH2CH
OCH2CH2(CH2)4CH3), 1.98–2.38 (m, 6H; Glu CH2CH2CO and Pro
NCH2CH2CH2), 2.47 (brt, 2H; Glu CH2CH2CO), 3.12 (brt, 2H; CH3-
(CH2)15CH2CH2N), 3.30 (brt, 2H; CH3(CH2)15CH2CH2N), 3.49–4.51 (m,
20H; Pro NCH2CH2CH2, Gly CH2, OCH2CH2(CH2)4CH3, COCH2O, Pro
ACHTREUNG
2.12–2.23 (m, 1H; Glu CH2CH2CO), 2.41–2.61 (m, 2H; Glu
A
N
ACHTREUNG
CH2CH2CO), 3.98–4.04 (m, 4H; 2” Gly CH2), 4.09–4.21 (m, 3H; Glu
ACHTREUNG
3
CH and OCH2CH2
(CH2)4CH3), 5.13 (s, 2H; OCH2Ph), 5.50 (d, J
G
7.2 Hz, 1H; Glu NH), 6.99–7.13 (m, 2H; two Gly NH), 7.32–7.40 ppm
(m, 5H; Ph HAr); 13C NMR (75 MHz, CDCl3): d=14.2, 22.8, 25.9, 27.5,
28.5, 28.7, 29.0, 30.6, 31.9, 41.4, 43.1, 54.5, 65.9, 66.9, 80.6, 128.5, 128.6,
128.8, 135.8, 169.2, 169.9, 172.4, 173.4 ppm; IR (CHCl3): 3321, 2957, 2931,
2858, 1739, 1662, 1529, 1455, 1392, 1367, 1250, 1171, 1049, 1028, 864, 749,
A
ACHTREUNG
AHCTREUNG
CH and Glu CH), 7.63 (brs, 1H; NH), 7.79 (brs, 2H; NH), 8.10 (brs,
1H; NH), 8.39 (brs, 1H; NH), 8.64 ppm (brs, 1H; NH); 13C NMR
(75 MHz, CDCl3): d=14.2, 14.3, 22.8, 22.9, 25.2, 26.0, 27.1, 27.3, 27.8,
28.7, 29.0, 29.1, 29.5, 29.6, 29.8, 29.9, 30.4, 31.9, 32.1, 41.4, 42.6, 43.1, 46.8,
47.0, 47.3, 53.8, 61.9, 65.9, 69.6, 71.5, 169.0, 170.0, 170.4, 170.6, 170.8,
171.3, 172.1, 173.0, 177.0 ppm; IR (CHCl3): 3307, 3080, 2920, 2851, 1738,
1651, 1539, 1467, 1411, 1378, 1339, 1247, 1206, 1129, 1031 cmꢀ1; elemental
analysis calcd (%) for C67H122N8O13: C 64.49, H 9.86, N 8.98; found: C
64.44, H 9.73, N 9.07.
698, 666 cmꢀ1
Boc-PE(g-benzyl ester)GG-OC7H15
.
:
Boc-E(g-benzyl ester)GG-OC7H15
was deprotected with 4n HCl in dioxane for 1 h. HCl·E(g-benzyl es-
ter)GG-OC7H15 (0.60 g, 1.23 mmol), Boc-Pro-OH (0.27 g, 1.23 mmol),
EDCI(0.26 g, 1.36 mmol), and HOBt (0.18 g, 1.36 mmol) were dissolved
in CH2Cl2 (35 mL) and Et3N (0.52 mL) was added. The mixture was
stirred at 08C for 0.5 h and then at RT for 12 h. The solvent was evapo-
rated in vacuo. The residue was purified by column chromatography
(silica gel, CHCl3/CH3OH=98:2!97:3) to give a white solid (0.72 g,
90%). M.p. 99–1018C; 1H NMR (300 MHz, CDCl3): d=0.88 (t, 3J-
Glycine n-heptyl ester tosylate: A solution of glycine (2.00 g, 26.7 mmol),
TsOH monohydrate (5.70 g, 30.0 mmol), and 1-heptanol (15 mL,
106 mmol) in toluene (18 mL) was heated at reflux for 12 h. Water was
removed from the reaction mixture by using a Dean-Stark adapter. The
solution was cooled to room temperature, Et2O (50 mL) was added, and
the solution was then cooled at 08C overnight. The solid was collected
and recrystallized from MeOH/Et2O to afford white crystals (7.07 g,
77%). M.p. 105–1068C; 1H NMR (300 MHz, CDCl3): d=0.87 (t, 3J-
ACHTREUNG
A
N
ACHTREUNG
ACHTREUNG
NCH2CH2CH2), 2.48–2.70 (m, 2H; Glu CH2CH2CO), 3.39–3.60 (m, 2H;
Pro NCH2CH2CH2), 3.75–3.85 (m, 1H; Gly CH2), 3.91–4.07 (m, 2H; Gly
AHCTREUNG
CH2), 4.10 (t, 3J
N
G
A
N
ACHTREUNG
A
N
ACHTREUNG
CH), 5.14 (s, 2H; OCH2Ph), 7.07 (brt, 1H; Gly NH), 7.30–7.41 (m, 5H;
Ph HAr), 7.85–7.95 ppm (m, 2H; Gly NH and Glu NH); 13C NMR
(75 MHz, CDCl3): d=14.2, 22.8, 24.9, 25.6, 25.9, 28.6, 28.7, 29.1, 29.8,
31.0, 31.9, 41.4, 43.2, 47.4, 54.5, 61.4, 65.6, 67.1, 81.2, 128.3, 128.7, 128.9,
135.5, 169.8, 171.8, 174.0, 175.0 ppm; IR (CHCl3): 3307, 2956, 2931, 1739,
C
G
ACHTREUNG
AHCTREUNG
1662, 1543, 1455, 1399, 1367, 1167, 1125, 1090, 1031 cmꢀ1
.
.
182DGA-GGGPE(g-benzyl ester)GG-OC7H15 (1): Boc-PE(g-benzyl es-
ter)GG-OC7H15 was deprotected with 4n HCl in dioxane for 1 h.
HCl·PE(g-benzyl ester)GG-OC7H15 (0.31 g, 0.51 mmol), 182DGA-GGG-
OH (0.41 g, 0.51 mmol), EDCI(0.11 g, 0.56 mmol), and HOBt (0.08 g,
0.56 mmol) were suspended in CH2Cl2 (35 mL). Et3N (0.21 mL) was then
added. The mixture was stirred at 08C for 0.5 h and then at RT for 48 h.
The solvent was removed in vacuo. The residue was dissolved in CH2Cl2
(35 mL), washed with 5% citric acid (25 mL), H2O (25 mL), 5%
NaHCO3 (25 mL), and brine (25 mL), dried over MgSO4 and evaporated.
Column chromatography (silica gel, CHCl3/CH3OH 97:3!95:5) to afford
ACHTREUNG
G
G
ACHTREUNG
a
waxy solid (0.18 g, 26%). M.p. 120–1228C; 1H NMR (300 MHz,
ACHTREUNG
3
CDCl3): d=0.88 (t, J
ACHTREUNG
3
CH
CH
C
N
(H,H)=6.6 Hz, 2H;
N
G
ACHTREUNG
CH2CH2CO, Pro NCH2CH2CH2), 2.43–2.60 (m, 2H; Glu CH2CH2CO),
3.07 (t, 3J (CH2)15CH2CH2N), 3.27 (t, 3J
(H,H)=7.6 Hz, 2H; CH3 (H,H)=
7.6 Hz, 2H; CH3(CH2)15CH2CH2N), 3.50–3.60 (m, 1H; Pro
NCH2CH2CH2), 3.77–4.15 (m, 15H; Pro NCH2CH2CH2, five Gly CH2,
OCH2CH2(CH2)4CH3 and COCH2O), 4.30 (s, 2H; COCH2O), 4.32–4.42
ACHTREUNG
A
G
A
ACHTREUNG
ACHTREUNG
(m, 2H; Pro CH and Glu CH), 5.10 (s, 2H; OCH2Ph), 7.30–7.38 (m, 5H;
Ph HAr), 7.44 (brt, 1H; NH), 7.57–7.65 (m, 2H; NH), 7.80–7.88 (m, 2H;
NH), 8.47 ppm (brt, 1H; NH); 13C NMR (75 MHz, CDCl3): d=14.2,
14.3, 22.8, 22.9, 25.2, 25.7, 26.0, 27.1, 27.3, 27.8, 28.7, 29.1, 29.5, 29.6, 29.8,
29.9, 31.1, 31.9, 32.1, 41.5, 42.6, 43.1, 43.4, 46.6, 47.2, 47.4, 53.6, 62.0, 65.8,
66.9, 69.9, 71.9, 128.3, 128.5, 128.8, 136.0, 168.8, 169.8, 170.1, 170.3, 170.6,
170.9, 171.7, 172.1, 172.6, 174.2 ppm; IR (CHCl3): 3307, 3068, 2924, 2854,
1739, 1660, 1545, 1456, 1378, 1337, 1248, 1205, 1130, 1030 cmꢀ1; elemental
analysis calcd (%) for C74H128N8O13: C 66.43, H 9.64, N 8.38; found: C
66.48, H 9.47, N 8.48.
ACHTREUNG
182DGA-GGGPEGG-OC7H15 (2): 182DGA-GGGPE(g-benzyl es-
ter)GG-OC7H15 (120 mg, 0.090 mmol) was dissolved in hot absolute
EtOH (15 mL), 10% Pd/C (0.06 g) was added and this mixture was
E
ACHTREUNG
AHCTREUNG
392
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 382 – 396

Self-Assembled Transmembrane Pores
FULL PAPER
3
CH2CH2CO), 3.76 (d, J
G
(t, 3J
NCH2CH2CH2), 3.82–4.54 (m, 18H; Gly CH2, Pro CH, Glu CH,
OCH2CH2
(CH2)4CH3 and COCH2O), 7.54 (brs, 1H; Gly NH), 7.62 (d, 3J-
(H,H)=6.9 Hz, 1H; Glu NH), 7.76 (brs, 1H; Gly NH), 7.92 (brs, 1H;
Gly NH), 8.03 (brs, 1H; Gly NH), 8.15 ppm (brs, 1H; Gly NH);
13C NMR (75 MHz, CDCl3): d=14.2, 14.3, 22.8, 22.9, 25.2, 26.0, 27.1,
27.3, 27.8, 28.7, 29.1, 29.6, 29.8, 29.9, 30.9, 31.9, 32.1, 41.4, 42.4, 43.0, 43.2,
43.7, 46.7, 47.2, 47.4, 53.0, 61.6, 65.7, 69.2, 71.2, 168.8, 169.1, 170.2, 170.8,
171.4, 172.3, 173.4, 176.2 ppm; IR (CHCl3): 3302, 3068, 2924, 2854, 1738,
1658, 1536, 1466, 1456, 1378, 1338, 1242, 1193, 1166, 1130, 1029 cmꢀ1; ele-
mental analysis calcd (%) for C67H122N8O13: C 64.49, H 9.86, N 8.98;
found: C 64.55, H 9.69, N 9.08.
U
N
3
Gly CH2), 4.12 (t, J
(H,H)=6.8 Hz, 2H; OCH2CH2
ACHTREUNG
(m, 1H; Glu CH), 5.13 (q, 3J
E
1H; Gly NH), 7.08–7.21 (m, 2H; Gly NH and Glu NH), 7.30–7.40 ppm
(m, 5H; Ph HAr); 13C NMR (75 MHz, CDCl3): d=14.2, 22.7, 26.0, 27.6,
28.5, 28.7, 29.0, 30.6, 31.9, 41.5, 44.7, 52.6, 65.9, 66.9, 80.7, 128.4, 128.5,
128.8, 136.0, 169.7, 170.0, 171.4, 173.7 ppm; IR (CHCl3): 3307, 2957, 2931,
2858, 1736, 1658, 1528, 1456, 1392, 1367, 1250, 1171, 1051, 1030,
ACHTREUNG
1003 cmꢀ1
Boc-PGE(g-benzyl ester)G-OC7H15
was deprotected with 4n HCl in dioxane for 1 h. HCl·GE(g-benzyl
ester)-OC7H15 (0.33 g, 0.68 mmol), Boc-Pro-OH (0.15 g, 0.68 mmol),
EDCI(0.14 g, 0.75 mmol), and HOBt (0.10 g, 0.75 mmol) were dissolved
in CH2Cl2 (20 mL) and Et3N (0.28 mL) was added. The mixture was
stirred at 08C for 0.5 h and at RT for 24 h. The solvent was evaporated
and the residue was purified by column chromatography (silica gel,
.
:
Boc-GE(g-benzyl ester)G-OC7H15
Boc-E(g-benzyl ester)-OC7H15
: Boc-Glu(g-benzyl ester)-OH (0.60 g,
1.78 mmol), 1-heptanol (0.28 mL) and DMAP (0.035 g, 0.28 mmol) were
dissolved in CH2Cl2 (20 mL). The mixture was cooled in an ice bath and
protected from moisture (CaCl2 drying tube). EDCI(0.37 g, 1.96 mmol)
was added and the reaction was stirred for 6 h at 08C. The solution was
concentrated to dryness; redissolved in EtOAc (50 mL); and washed suc-
cessively with H2O (3”25 mL), 5% citric acid (25 mL), H2O (25 mL),
5% NaHCO3 (25 mL), and brine (25 mL). Purification by column chro-
CHCl3/MeOH 97:3) to give a solid (0.40 g, 89%). M.p. 119–1218C;
1H NMR (300 MHz, CDCl3): d=0.88 (t, J
(H,H)=6.6 Hz, 3H; CH2CH3),
3
1.20–1.40 (m, 8H; OCH2CH
U
(CH3)3), 1.63
3
(quintet, J
A
ACHTREUNG
Pro NCH2CH2CH2 and Glu CH2CH2CO), 2.42–2.66 (m, 2H; Glu
CH2CH2CO), 3.35–3.52 (m, 2H; Pro NCH2CH2CH2), 3.84–4.06 (m, 4H;
matography (silica gel, hexane/EtOAc 6:1) afforded an oil (0.68 g, 88%).
3
1H NMR (300 MHz, CDCl3): d=0.89 (t, J
(H,H)=6.9 Hz, 3H; CH2CH3),
Gly CH2), 4.11 (t, 3J
A
N
1.20–1.40 (m, 8H; OCH2CH
1.68 (m, 2H; OCH2CH2
CH2CH2CO), 2.15–2.26 (m, 1H; Glu CH2CH2CO), 2.38–2.55 (m, 2H;
Glu CH2CH2CO), 4.12 (t, 3J
(H,H)=6.8 Hz, 2H; OCH2CH2(CH2)4CH3),
4.33 (q, 3J
A
N
N
ACHTREUNG
U
A
ACHTREUNG
13C NMR (75 MHz, CDCl3): d=14.2, 22.7, 24.7, 25.9, 27.1, 28.6, 28.7,
29.0, 29.8, 30.7, 31.8, 41.4, 43.5, 47.5, 52.8, 60.8, 65.7, 66.7, 80.9, 128.4,
128.7, 136.0, 169.9, 171.5, 173.3, 173.7 ppm; IR (CHCl3): 3301, 2956, 2930,
2872, 2858, 1738, 1665, 1534, 1477, 1455, 1395, 1367, 1257, 1191, 1165,
A
OCH2Ph), 7.30–7.40 ppm (m, 5H; Ph HAr); 13C NMR (75 MHz, CDCl3):
d=14.2, 22.8, 26.0, 28.2, 28.5, 28.7, 29.0, 30.6, 31.9, 53.2, 65.9, 66.7, 128.4,
128.5, 128.8, 136.1, 172.4, 172.7 ppm; IR (CHCl3): 3373, 2957, 2931, 2858,
1128, 1090, 1032, 1003 cmꢀ1
.
1739, 1716, 1500, 1455, 1391, 1367, 1254, 1167, 1051, 1028, 1004 cmꢀ1
.
182DGA-GGGPGE(g-benzyl ester)G-OC7H15 (3): Boc-PGE(g-benzyl
ester)G-OC7H15 was deprotected with 4n HCl in dioxane for 1 h.
HCl·PGE(g-benzyl ester)G-OC7H15 (0.29 g, 0.46 mmol), 182DGA-GGG-
OH (0.40 g, 0.46 mmol), EDCI(0.098 g, 0.51 mmol), and HOBt (0.069 g,
0.51 mmol) were suspended in CH2Cl2 (20 mL) and Et3N (0.19 mL) was
added. The mixture was stirred at 08C for 0.5 h and at RT for 48 h. The
solvent was evaporated and the residue was washed successively with 5%
citric acid (20 mL), H2O (20 mL), 5% NaHCO3 (20 mL), and brine
(20 mL). The product was purified by column chromatography (silica gel,
CHCl3/MeOH 98:2!97:3) and the resulting solid was recrystallized from
MeOH to afford a white solid (162 mg, 26%). M.p. 96–988C; 1H NMR
Boc-GE(g-benzyl ester)-OC7H15: Boc-E(g-benzyl ester)-OC7H15 was de-
protected with 4n HCl in dioxane for 1 h. HCl·E(g-benzyl ester)-OC7H15
(0.91 g, 2.46 mmol), Boc-Gly-OH (0.43 g, 2.46 mmol), EDCI(0.52 g,
2.70 mmol), and HOBt (0.37 g, 2.70 mmol) were dissolved in CH2Cl2
(50 mL). Et3N (1.03 mL) was then added. The reaction mixture was
stirred at 08C for 0.5 h and then at RT for 48 h. The solvent was evapo-
rated and the residue was washed successively with 5% citric acid (2”
20 mL), H2O (2”20 mL), 5% NaHCO3 (2”20 mL), and brine (2”
20 mL). Purification by column chromatography (silica gel, hexane/
EtOAc 3: 1) afforded an oil (0.92 g, 76%). 1H NMR (300 MHz, CDCl3):
d=0.88 (t, 3J
OCH2CH2(CH2)4CH3), 1.45 (s, 9H; C
7.1 Hz, 2H; OCH2CH2(CH2)4CH3), 1.97–2.08 (m, 1H; Glu CH2CH2CO),
ACHTREUNG
(300 MHz, CDCl3): d=0.88 (t, 3J
(pseudo-s, 68H; OCH2CH2(CH2)4CH3 and CH
1.68 (m, 6H; OCH2CH2(CH2)4CH3 and CH3(CH2)15CH2CH2N), 1.90–2.30
(m, 6H; Pro NCH2CH2CH2 and Glu CH2CH2CO), 2.40–2.58 (m, 2H;
Glu CH2CH2CO), 3.07 (brt, 2H; CH3(CH2)15CH2CH2N), 3.28 (brt, 2H;
CH3(CH2)15CH2CH2N), 3.46–4.54 (m, 20H; Pro NCH2CH2CH2, Gly CH2,
Pro CH, Glu CH, OCH2CH2(CH2)4CH3 and COCH2O), 5.10 (s, 2H;
A
A
N
ACHTREUNG
A
N
ACHTREUNG
A
N
2.20–2.31 (m, 1H; Glu CH2CH2CO), 2.36–2.57 (m, 2H; Glu
CH2CH2CO), 3.73–3.88 (m, 2H; Gly CH2), 4.08–4.20 (m, 2H; OCH2CH2-
E
A
H
OCH2Ph), 6.78 (d, 3J
(H,H)=7.8 Hz, 1H; Glu NH), 7.30–7.40 ppm (m,
G
5H; Ph HAr); 13C NMR (75 MHz, CDCl3): d=14.2, 22.7, 25.9, 27.6, 28.5,
28.6, 28.7, 29.0, 30.4, 31.8, 44.6, 51.9, 66.1, 66.8, 128.4, 128.5, 128.8, 136.0,
169.6, 171.8, 172.7 ppm; IR (CHCl3): 3327, 2957, 2931, 1739, 1680, 1522,
OCH2Ph), 7.28–7.38 (m, 5H; Ph HAr), 7.42 (brs, 1H; NH), 7.48–7.62 (m,
2H; NH), 7.84 (brs, 2H; NH), 8.41 ppm (brs, 1H; NH); 13C NMR
(75 MHz, CDCl3): d=14.3, 22.8, 22.9, 25.3, 26.0, 27.1, 27.3, 27.8, 28.7,
29.1, 29.3, 29.6, 29.9, 30.8, 31.9, 32.1, 41.4, 43.0, 46.6, 47.1, 52.7, 61.4, 65.8,
66.8, 69.7, 128.3, 128.4, 128.7, 135.9, 168.8, 170.1, 170.5, 172.0, 173.0,
174.1 ppm; IR (CHCl3): 3304, 3078, 2924, 2854, 1735, 1651, 1544, 1466,
1455, 1420, 1392, 1367, 1327, 1251, 1169, 1082, 1052, 1029, 1003 cmꢀ1
Boc-GGE(g-benzyl ester)-OC7H15: Boc-GE(g-benzyl ester)-OC7H15 was
deprotected with 4n HCl in dioxane for 1 h. HCl·GE(g-benzyl ester)-
OC7H15 (0.53 g, 1.23 mmol), Boc-Gly-OH (0.22 g, 1.23 mmol), EDCI
(0.26 g, 1.36 mmol), and HOBt (0.18 g, 1.36 mmol) were dissolved in
CH2Cl2 (30 mL). Et3N (0.52 mL) was added and the reaction mixture was
stirred at 08C for 0.5 h and then at RT for 48 h. The solvent was evapo-
rated and the residue was dissolved in CH2Cl2 (50 mL) and washed suc-
cessively with 5% citric acid (2” 20 mL), H2O (2” 20 mL), 5%
NaHCO3 (2”20 mL), and brine (2”20 mL). Purification by column chro-
matography (silica gel, CHCl3/MeOH 98:2) afforded an oil (0.58 g,
1377, 1199, 1130, 1030 cmꢀ1
C74H128N8O13: C 66.43, H 9.64, N 8.38; found: C 66.40, H 9.80, N 8.33.
182DGA-GGGPGEG-OC7H15 (4): 182DGA-GGGPGE(g-benzyl
; elemental analysis calcd (%) for
ester)G-OC7H15 (80 mg, 0.060 mmol) was dissolved in hot absolute EtOH
(10 mL), 10% Pd/C (0.045 g) was added and this mixture was shaken
under H2 (60 psi) for 3 h in a Parr apparatus. The reaction mixture was
heated to reflux for 5 min and the hot mixture was filtered through a
Celite pad. The solvent was evaporated in vacuo to afford a white solid
84%). 1H NMR (300 MHz, CDCl3): d=0.88 (t, 3J
CH2CH3), 1.20–1.40 (m, 8H; OCH2CH2(CH2)4CH3), 1.44 (s, 9H; C-
(CH3)3), 1.63 (quintet, 3J
(H,H)=7.0 Hz, 2H; OCH2CH2(CH2)4CH3),
A
(70 mg, 94%). 1H NMR (300 MHz, CDCl3): d=0.88 (t, 3J
9H; CH2CH3), 1.25 (pseudo-s, 68H; OCH2CH2(CH2)4CH3 and CH3-
(CH2)15CH2CH2N), 1.40–1.68 (m, 6H; OCH2CH2(CH2)4CH3 and CH3-
(CH2)15CH2CH2N), 1.94–2.50 (m, 8H; Pro NCH2CH2CH2 and Glu
CH2CH2CO), 3.09 (t, 3J
(H,H)=6.3 Hz, 2H; CH3(CH2)15CH2CH2N), 3.28
A
ACHTREUNG
N
A
N
ACHTREUNG
A
N
1.97–2.10 (m, 1H; Glu CH2CH2CO), 2.17–2.30 (m, 1H; Glu
A
CH2CH2CO), 2.38–2.58 (m, 2H; Glu CH2CH2CO), 3.84 (d, 3J
4.5 Hz, 2H; Gly CH2), 3.88–4.07 (m, 2H; Gly CH2), 4.11 (t, 3J
ACHTREUNG
A
N
ACHTREUNG
Chem. Eur. J. 2008, 14, 382 – 396
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
393

G. W. Gokel et al.
6.8 Hz, 2H; OCH2CH2
2H; OCH2Ph), 5.30 (brt, 1H; Gly NH), 6.86 (brt, 1H; Gly NH), 7.03 (d,
A
2H; CH3
(CH2)15CH2CH2N), 3.50–3.79 (m, 2H; Pro NCH2CH2CH2), 3.80–4.20 (m,
14H; Gly CH2, OCH2CH2(CH2)4CH3 and COCH2O), 4.30 (s, 2H;
COCH2O), 4.42 (brt, 1H; Pro CH), 4.49–4.62 (m, 1H; Glu CH), 7.44 (d,
3J
(H,H)=7.2 Hz, 1H; Glu NH), 7.70–7.83 (m, 2H; Gly NH), 7.98–8.16
G
U
AHCTREUNG
3J
C
ACHTREUNG
AHCTREUNG
(m, 2H; Gly NH), 8.38 ppm (brt, 1H; Gly NH); 13C NMR (75 MHz,
CDCl3): d=14.2, 14.3, 22.8, 22.9, 25.3, 26.0, 27.1, 27.3, 27.8, 28.7, 29.1,
29.6, 29.9, 30.7, 31.9, 32.1, 42.2, 43.4, 46.6, 47.2, 52.3, 61.6, 66.1, 69.5, 71.4,
168.7, 169.6, 170.4, 170.6, 170.7, 171.0, 171.6, 171.8, 173.2, 175.5 ppm; IR
(CHCl3): 3303, 3076, 2924, 2854, 1737, 1658, 1543, 1466, 1253, 1208, 1130,
1029 cmꢀ1; elemental analysis calcd (%) for C67H122N8O13: C 64.49, H
9.86, N 8.98; found: C 64.37, H 9.80, N 9.12.
1529, 1455, 1391, 1367, 1334, 1252, 1169, 1083, 1051, 1029, 1003 cmꢀ1
Boc-PGGE(g-benzyl ester)-OC7H15
(0.56 g, 1.02 mmol) was deprotected with 4n HCl in dioxane for 1 h.
HCl·GGE(g-benzyl ester)-OC7H15, Boc-Pro-OH (0.22 g, 1.02 mmol),
EDCI(0.21 g, 1.12 mmol), and HOBt (0.15 g, 1.12 mmol) were dissolved
in CH2Cl2 (30 mL). Et3N (0.43 mL) was added and the reaction mixture
was stirred at 08C for 0.5 h and then at RT for 48 h. The solvent was
evaporated and the residue was washed successively with 5% citric acid
(20 mL), H2O (20 mL), 5% NaHCO3 (20 mL), and brine (20 mL). Purifi-
cation by column chromatography (silica gel, CHCl3/MeOH 98:2) gave
182DGA-GGGPGGE(O-1-pyrenemethylene)-OC7H15
(7):
182DGA-
GGGPGGE-OC7H15 (0.20 g, 0.16 mmol) was suspended in CH2Cl2
(15 mL) at 08C. EDCI(0.034 g, 0.18 mmol), DMAP (0.010 g, 0.08 mmol),
and 1-pyrenemethanol (0.038 g, 0.16 mmol) were then added successively.
The mixture was stirred at ambient temperatures for 4 d. The solvent was
evaporated and the residue was purified by column chromatography
(silica gel, CHCl3/MeOH 95:5) and recrystallized from CHCl3/MeOH to
an oil (0.49 g, 74%). 1H NMR (300 MHz, CDCl3): d=0.88 (t, 3J
6.4 Hz, 3H; CH2CH3), 1.20–1.40 (m, 8H; OCH2CH2(CH2)4CH3), 1.42 (s,
9H; (H,H)=6.9 Hz, 2H; OCH2CH2-
(CH3)3), 1.62 (quintet, 3J
(CH2)4CH3), 1.78–2.29 (m, 6H; Pro NCH2CH2CH2 and Glu CH2CH2CO),
2.47 (t, 3J
(H,H)=7.5 Hz, 2H; Glu CH2CH2CO), 3.35–3.55 (m, 2H; Pro
NCH2CH2CH2), 3.79–4.17 (m, 7H; Gly CH2, Pro CH and OCH2CH2-
(CH2)4CH3), 4.52–4.60 (m, 1H; Glu CH), 5.10 (s, 2H; OCH2Ph), 6.99–
C
A
give
(300 MHz, CDCl3): d=0.88 (t, 3J
(pseudo-s, 68H, NCH2CH2(CH2)15CH3 and OCH2CH
1.67 (m, 6H; NCH2CH2(CH2)15CH3 and OCH2CH2(CH2)4CH3), 1.71–2.62
(m, 8H; Pro NCH2CH2CH2 and Glu CH2CH2CO), 2.93–3.51 (m, 5H;
NCH2CH2(CH2)15CH3 and Pro NCH2CH2CH2), 3.65–4.45 (m, 19H; Pro
NCH2CH2CH2, Gly CH2, Pro CH, Glu CH, OCH2CH2(CH2)4CH3 and
a
yellowish solid (100 mg, 43%). M.p. 144–1468C; 1H NMR
(H,H)=7.5 Hz, 9H; CH2CH3), 1.25
2(CH2)4CH3), 1.40–
ACHTREUNG
AHCTREUNG
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
ACHTREUNG
7.10 (m, 2H; Gly NH and Glu NH), 7.30–7.40 (m, 5H; Ph HAr), 7.74 ppm
(brt, 1H; Gly NH); 13C NMR (75 MHz, CDCl3): d=14.2, 22.8, 24.9, 25.9,
27.2, 28.5, 28.6, 29.0, 29.7, 30.4, 31.8, 43.2, 43.6, 47.4, 51.8, 61.0, 65.9, 66.6,
81.2, 128.3, 128.4, 128.7, 135.9, 169.3, 169.9, 171.6, 173.0, 173.8 ppm; IR
(CHCl3): 3310, 3067, 2956, 2931, 2872, 2858, 1740, 1668, 1543, 1478, 1455,
AHCTREUNG
AHCTREUNG
OCH2CO), 5.83 (s, 2H; OCH2Ar), 7.23 (overlap with CDCl3 signal, 1H;
Glu NH), 7.42 (brs, 1H; Gly NH), 7.71–8.46 ppm (m, 13H; Gly NH and
HAr); 13C NMR (75 MHz, CDCl3): d=14.2, 14.3, 22.8, 22.9, 25.2, 26.0,
26.5, 27.1, 27.3, 27.8, 28.6, 29.1, 29.5, 29.6, 29.9, 30.4, 31.8, 32.1, 42.0, 42.9,
43.2, 43.9, 46.7, 47.0, 47.3, 52.3, 61.4, 65.1, 66.1, 69.7, 71.7, 123.1, 124.8,
125.0, 125.7, 125.8, 126.4, 127.5, 128.0, 128.1, 128.5, 129.0, 129.7, 130.8,
131.4, 131.9, 168.9, 170.0, 170.4, 171.0, 172.4, 172.9, 174.0, 174.9 ppm; IR
(CHCl3): 3307, 3052, 2922, 2852, 1738, 1660, 1651, 1544, 1467, 1415, 1377,
1411, 1367, 1331, 1257, 1208, 1165, 1134, 1091, 1029 cmꢀ1
182DGA-GGGPGGE(g-benzyl ester)-OC7H15 (5): Boc-PGGE(g-benzyl
ester)-OC7H15 was deprotected with 4n HCl in dioxane for 1 h.
HCl·PGGE(g-benzyl ester)-OC7H15 (0.44 g, 0.76 mmol), 182DGA-GGG-
OH (0.61 g, 0.76 mmol), EDCI(0.16 g, 0.83 mmol), and HOBt (0.11 g,
0.83 mmol) were suspended in CH2Cl2 (30 mL). Et3N (0.32 mL) was
added and the mixture was stirred at 08C for 0.5 h and at RT for 48 h.
The solvent was evaporated and the residue was crystallized from
MeOH. The crude product was purified by column chromatography
(silica gel, CHCl3/MeOH 95: 5) and crystallized from MeOH to afford a
white solid (0.48 g, 47%). M.p. 137–1398C; 1H NMR (300 MHz, CDCl3):
1335, 1246, 1130, 1029 cmꢀ1
; elemental analysis calcd (%) for
C84H132N8O13.H2O: C 68.17, H 9.13, N 7.57; found: C 67.95, H 8.92, N
7.43.
A
was prepared as previously reported.[11d]
d=0.88 (t, 3J
ACHTREUNG
Vesicle preparation and chloride release measurement: Chloride release
was assayed directly on ꢁ200 nm phospholipid vesicles prepared from a
7:3 mixture of DOPC and DOPA (both from Avanti Polar Lipids) by
OCH2CH
A
OCH2CH2
A
NCH2CH2CH2 and Glu CH2CH2CO), 2.52 (t, 3J
(H,H)=7.4 Hz, 2H; Glu
(CH2)15CH2CH2N), 3.18–
(CH2)15CH2CH2N), 3.45–3.64 (m, 2H; Pro
NCH2CH2CH2), 3.70–4.40 (m, 18H; Gly CH2, Pro CH, Glu CH,
OCH2CH2 (H,H)=9.0 Hz, 2H;
(CH2)4CH3 and COCH2O), 5.10 (q, 3J
OCH2Ph), 7.24 (d, J(H,H)=6.6 Hz, 1H; Glu NH), 7.30–7.40 (m, 5H; Ph
using
a chloride-selective electrode (Accumet Chloride Combination
3
Electrode). Vesicles were prepared in the presence of an internal, chlo-
ride-containing buffer (KCl (600 mm), HEPES (10 mm), adjusted to
pH 7.0). After extrusion and exchange of the internal solution with a
chloride-free buffer (K2SO4 (400 mm), HEPES (10 mm), adjusted to
pH 7.0), vesicles were suspended in the same external buffer (final phos-
pholipid concentrationꢁ0.31 mm). The electrode was introduced in the
solution and it was allowed to equilibrate. The voltage output was record-
ed, and after the baseline became flat, aliquots of the solution of the
compound being studied (9 mm in isopropanol) were added. No more
than 20 mL of 2-propanol were added in any experiment to avoid any
affect of solvent on the liposomes. Complete lysis of the vesicles was in-
duced by the addition of a 2% aqueous solution of Triton X100 (100 mL)
and the data collected were normalized to this value. The data were col-
lected by using Axoscope 9.0 with a DigiData 1322 A series interface.
CH2CH2CO), 3.08 (t, J
3.32 (m, 2H; CH3
G
3
ACHTREUNG
HAr), 7.45 (brt, 1H; Gly NH), 7.81 (brt, 1H; Gly NH), 7.95–8.05 (m,
2H; Gly NH), 8.33 ppm (brt, 1H; Gly NH); 13C NMR (75 MHz, CDCl3):
d=14.2, 14.3, 22.8, 22.9, 25.4, 26.0, 26.4, 27.1, 27.3, 27.8, 28.7, 29.1, 29.5,
29.6, 29.8, 29.9, 30.4, 31.9, 32.1, 42.0, 42.8, 43.0, 44.0, 46.5, 47.2, 52.4, 61.4,
66.0, 66.6, 69.8, 71.6, 128.3, 128.4, 128.7, 136.0, 168.6, 168.8, 169.9, 170.3,
170.6, 170.9, 171.3, 172.5, 172.8, 174.1 ppm; IR (CHCl3): 3305, 3067, 2924,
2853, 1740, 1659, 1537, 1455, 1378, 1336, 1259, 1209, 1164, 1130,
1029 cmꢀ1; elemental analysis calcd (%) for C74H128N8O13: C 66.43, H
9.64, N 8.38; found: C 66.32, H 9.67, N 8.16.
Carboxyfluorescein dequenchingfrom vesicles : A 7:3 mixture (15 mg) of
DOPC and DOPA was dissolved in Et2O (0.75 mL) and internal buffer
(0.75 mL, CFꢀ (20 mm), HEPES (10 mm), pH 7.0) was added. The mix-
ture was sonicated and diethyl ether was removed under mild vacuum.
The remaining suspension was filtered (200 nm filter membrane) and
then passed through a Sephadex G25 column eluted by external buffer
(KCl (100 mm), HEPES (10 mm), pH 7.0). The size of the liposomes was
analyzed by laser light scattering to be around 200 nm.
182DGA-GGGPGGE-OC7H15
(6):
182DGA-GGGPGGE(g-benzyl
ester)-OC7H15 (0.22 g, 0.16 mmol) was dissolved in hot absolute EtOH
(20 mL), 10% Pd/C (0.10 g) was added and this mixture was shaken
under H2 (60 psi) for 3 h in a Parr apparatus. The reaction mixture was
heated to reflux for 5 min and the hot mixture was filtered through a
Celite pad. The solvent was evaporated in vacuo to afford a white solid
(0.20 g, 98%). 1H NMR (300 MHz, CDCl3): d=0.88 (t, 3J
9H; CH2CH3), 1.26 (pseudo-s, 68H; OCH2CH2(CH2)4CH3 and CH3-
(CH2)15CH2CH2N), 1.42–1.70 (m, 6H; OCH2CH2(CH2)4CH3 and CH3-
(CH2)15CH2CH2N), 1.90–2.34 (m, 6H; Pro NCH2CH2CH2 and Glu
CH2CH2CO), 2.40 (brt, 2H; Glu CH2CH2CO), 3.08 (t, 3J
(H,H)=7.4 Hz,
A
AHCTREUNG
A
G
Carboxyfluorescein-containing vesicles were diluted to 0.9 mm in external
buffer (2 mL). The fluorescence (excitation 497 nm and emission 520 nm
with 2 nm bandpass) was monitored at 258C. Compounds were added as
ACHTREUNG
AHCTREUNG
394
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 382 – 396

Self-Assembled Transmembrane Pores
FULL PAPER
a solution (1 mm) in 2-propanol with mixing to the desired concentration.
Dequenching, F520, was computed as the fraction of total release upon ad-
dition of 2% aqueous solution of Triton X100 (100 mL) and is shown in
Equation (1):
Acknowledgement
We thank the NIH for grants (GM 36262, GM 63190) to support this
work.
FꢀF0
ð1Þ
F520
¼
FTritonꢀF0
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Received: July 12, 2007
Published online: October 10, 2007
396
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Chem. Eur. J. 2008, 14, 382 – 396