Molecular design and synthesis of artificial ion channels based on cyclic peptides containing unnatural amino acids

Article abstract of DOI:10.1021/jo001079t

A series of novel cyclic peptides composed of 3 to 5 dipeptide units with alternating natural-unnatural amino acid units, have been designed and synthesized, employing 5-(N-alkanoylamino)-3-aminobenzoic acid with a long alkanoyl chain as the unnatural amino acid. All cyclic peptides with systematically varying pore size, shape, and lipophilicity are found to form ion channels with a conductance of ca. 9 pS in aqueous KCl (500 mM) upon examination by the voltage clamp method. These peptide channels are cation selective with the permeability ratio P_Cl-/P_K+ of around 0.17. The ion channels formed by the neutral, cationic, and anionic cyclic peptides containing L-alanine, L-lysine, and L-aspartate, respectively, show the monovalent cation selectivity with the permeability ratio P_Na+/P_K+ of ca. 0.39. On the basis of structural information provided by voltage-dependent blockade of the single channel current of all the tested peptides by Ca²⁺, we inferred that each channel is formed from a dimer of the peptide with its peptide ring constructing the channel entrance and its alkanoyl chains lining across the membrane to build up the channel pore. The experimental results are consistent with an idea that the rate of ion conduction is determined by the nature of the hydrophobic alkanoyl chain region, which is common to all the channels.

Full text of DOI:10.1021/jo001079t

2978
J . Org. Chem. 2001, 66, 2978-2989
Molecu la r Design a n d Syn th esis of Ar tificia l Ion Ch a n n els Ba sed
on Cyclic P ep tid es Con ta in in g Un n a tu r a l Am in o Acid s
Hitoshi Ishida,*,† Zhi Qi,‡ Masahiro Sokabe,*,‡,§ Kiyoshi Donowaki,| and Yoshihisa Inoue†,|
Inoue Photochirogenesis Project, ERATO, J apan Science and Technology, 4-6-3 Kamishinden,
Toyonaka, Osaka 560-0085, J apan, Department of Physiology, Nagoya University School of Medicine,
65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, J apan, Cell Mechanosensing Project, ICORP,
J apan Science and Technology, 65 Tsurumai, Nagoya 466-8550, J apan, and Department of Molecular
Chemistry, Osaka University, Yamada-oka, Suita, Osaka 565-0871, J apan
ishida@inoue.jst.go.jp
Received October 24, 2000
A series of novel cyclic peptides composed of 3 to 5 dipeptide units with alternating natural-
unnatural amino acid units, have been designed and synthesized, employing 5-(N-alkanoylamino)-
3-aminobenzoic acid with a long alkanoyl chain as the unnatural amino acid. All cyclic peptides
with systematically varying pore size, shape, and lipophilicity are found to form ion channels with
a conductance of ca. 9 pS in aqueous KCl (500 mM) upon examination by the voltage clamp method.
-
+
These peptide channels are cation selective with the permeability ratio PCl /PK of around 0.17.
The ion channels formed by the neutral, cationic, and anionic cyclic peptides containing L-alanine,
L-lysine, and L-aspartate, respectively, show the monovalent cation selectivity with the permeability
+
+
ratio PNa /PK of ca. 0.39. On the basis of structural information provided by voltage-dependent
blockade of the single channel current of all the tested peptides by Ca2+, we inferred that each
channel is formed from a dimer of the peptide with its peptide ring constructing the channel entrance
and its alkanoyl chains lining across the membrane to build up the channel pore. The experimental
results are consistent with an idea that the rate of ion conduction is determined by the nature of
the hydrophobic alkanoyl chain region, which is common to all the channels.
In tr od u ction
category also includes model peptides employed as the
segments in the active site of natural ion channels17-19
and amphipathic polypeptides.20 Some of the peptides,
e.g., gramicidin, form a single molecular helix, the central
pore of which can permeate ions.2,5,6,9-12 However, most
of the peptides, e.g., alamethicin, aggregate to form a
barrellike structure, forming a hydrophilic pore in its
center in bilayer membranes.3,4,13-15 In the latter cases,
the domain peptides have been gathered by using the
template-assembled synthetic protein (TASP) method,21,22
Ion channels are huge membrane proteins which
generate electrical and calcium signals in most of the cells
including neurons.1 Synthetic low molecular weight
compounds which exhibit ion channel activity have been
investigated as models for the native ion channels. These
model studies may have two general advantages: (1) We
can elucidate the biochemical or biophysical properties
of the natural ion channels through the knowledge
obtained from the investigation of the ion channel
behavior of model compounds, because the comprehensive
understanding of the structure and dynamics of the
native ion channels seems difficult to achieve at present.
(2) We can obtain knowledge to design and construct
molecular devices that can convert chemical signals to
electrical ones, mimicking the biological signal transduc-
tion system.
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* Corresponding authors: E-mail for M. Sokabe: msokabe@
med.nagoya-u.ac.jp.
† Inoue Photochirogenesis Project, ERATO.
‡ Nagoya University School of Medicine.
§ Cell Mechanosensing Project, ICORP.
| Osaka University.
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10.1021/jo001079t CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/03/2001

Artificial Ion Channels
J . Org. Chem., Vol. 66, No. 9, 2001 2979
and furthermore their function-structure relationships
have been investigated by controlling the assembling
numbers.15,22-25 As an interesting example, Ghadiri et al.
have reported that cyclic peptides composed of alternat-
ing D- and L-amino acids form a peptide nano tube
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the hydrophilic sites. For example, there have been
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hydrophobic sterol47 or phospholipids,48 have also been
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ether chains to form a hydrophilic pore, the polyethers
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phobic long alkyl compounds with cationic headgroups
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ion channels.44-46 Combinations of crown ether and
polyether also behave as artificial ion channels.49-57
Furthermore, polyol with rigid, hydrophobic oligo(p-
phenylene) skeleton58 and polyesters such as poly(2-
ethylacrylic acid)59 or poly-(3-hydroxybutyrate)60 have
been reported to function as ion channels which do not
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long alkyl chains have been reported to exhibit ion
channel activity. Tabushi et al. reported that â-cyclodex-
trin modified by four long alkyl chains transports cobalt-
(II) ions across a bilayer membrane, although this
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2980 J . Org. Chem., Vol. 66, No. 9, 2001
Ishida et al.
Sch em e 1
F igu r e 1. Cyclic peptides (1-7). Abbreviations: A, D, K, and
S for L-alanine, L-aspartate, L-lysine, and L-serine, and C10
,
C
16, and C18 for decanoyl, hexadecanoyl, and octadecanoyl side
chains, respectively.
peptides by fixing the position and/or orientation of the
side chain of the amino acid residues, both of which are
known to be important factors determining the function.
By using this strategy we have designed cyclic peptides,
which are composed of an alternating natural amino acid/
3-aminobenzoic acid sequence, and have shown indeed
that these cyclic peptides exhibit the expected functions,
such as strong binding of phosphates65 and ester hydro-
lytic activity.66 We have developed the molecular design
of the cyclic peptide for an artificial ion channel and have
actually synthesized cyclic peptides containing an alter-
nating natural and unnatural amino acid sequence, the
latter of which is 5-(N-alkanoylamino)-3-aminobenzoic
acid with a long alkyl chain for feasible incorporation of
the peptides into bilayer membranes (Figure 1).67,68 In
this design, a variety of natural amino acids can be
introduced into the cyclic peptides, and the number of
the component amino acids and the length of alkanoyl
chains are readily altered. These cyclic peptides could be
compared with the cyclic resorcinol tetramer bearing long
alkyl chains,62 which is more difficult to change the ring
size due to some synthetic reasons. By investigating the
ion channel properties of such cyclic peptides, we first
examine whether the hydrophobic long alkanoyl (alkyl)
chains can form a pore which acts as an ion channel.
Moreover, we expect that such a simple ion channel
should provide valuable information to elucidate the
structure-function relationship of natural ion channels
and to develop molecular devices which can convert
chemical signals into electrical ones.
Resu lts a n d Discu ssion
Syn th esis a n d Str u ctu r e of Cyclic P ep tid es. We
synthesized cyclic peptides (1-7) containing an un-
natural amino acid, 5-(N-alkanoylamino)-3-aminobenzoic
acid, by coupling the precursor dipeptides prepared
separately, because the unnatural amino acid is readily
oxidized when the amino group is deprotected. For the
amino acids which do not require protection of the side
chain (in this work, L-alanine), ethyl 5-(N-alkanoyl-
amino)-3-aminobenzoate was used as the protecting
precursor (Scheme 1). In this synthesis, after 3,5-diami-
nobenzoic acid (8) was esterified to produce the ethyl
ester (9), one of the two amino groups was protected with
tert-butyloxycarbonyl (Boc) group to yield 10. A C10 or
C16 alkanoyl group was attached to the remaining amino
group to give the corresponding compound 11a or 11b,
respectively. Then, the Boc group was removed from
11a ,b, and the resulting 5-(N-alkanoylamino)-3-ami-
nobenzoic acid ethyl ester was combined with a Boc-L-
alanine. Finally, the ethyl ester protective group was
removed with NaOH in aqueous ethanol to give the
dipeptides (14), which are used in the solid-phase syn-
thesis.
In this study, we have synthesized a series of sym-
metrical cyclic peptides with different ring size and
alkanoyl chain, where each peptide contains a single kind
of natural and unnatural amino acid. Here, the abbrevia-
tion for the cyclic peptides is given as a combination of
the natural amino acid used, the length of alkanoyl chain,
and the number (n) of the dipeptide units in peptides:
e.g., (AC10)3 corresponds to the cyclic peptide having
L-alanine, N-decanoyl group (C10), and three dipeptide
units (n ) 3). We report herein the synthesis of the cyclic
peptides with a structural variety and their ion channel
properties evaluated from the single channel study.
On the other hand, the peptides containing such amino
acids that require the side chain protection (e.g., L-
aspartate, L-lysine, and L-serine) were synthesized as

Artificial Ion Channels
J . Org. Chem., Vol. 66, No. 9, 2001 2981
Sch em e 2
Sch em e 3
therefore allowed to react with trimethylsilyl trifluo-
romethanesulfonate (TMSOTf) in trifluoroacetic acid
(TFA) at 0 °C for 1 h.72 All peptides synthesized in this
work were identified by means of NMR and MS spec-
troscopy.
1H NMR spectra of all cyclic peptides showed one
equivalent signal for each proton in the component
natural amino acid and 5-(N-alkanoyl)-3-aminobenzoic
acid moieties, indicating that all cyclic peptides have
symmetrical structures. Moreover, the 1H NMR of (AC10)3
and (AC10)4 were almost independent of temperature from
25 through 80 °C in DMSO-d6, although the amide
protons showed downfield shifts of 0.3-0.4 ppm, probably
as a result of aggregation through the hydrogen bonding
interaction with water molecules present in the NMR
solvent (see the Supporting Information). These results
obtained from the NMR spectral study evidently indicate
that all of the cyclic peptides are symmetrical and the
conformations are thermodynamically stable. The 1H
NMR of (AC16)5 showed broad peaks at 25 °C and sharp
signals at higher temperatures, suggesting that the
conformation is slightly flexible, although the peptide
keeps the symmetrical structure at room temperature.
These structural features are consistent with the molec-
ular modeling structures of (AC10)3, (AC10)4, and (AC16)5
which are obtained by the Insight/Discover program
(from MSI, San Diego, CA). Figure 2 shows the top views
of (AC10)3, (AC10)4, and (AC16)5 exhibiting a triangular,
square, and pentagonal shape with C3, C4, and C5
symmetry, respectively. Each cyclic peptide has a cavity
at the center due to the rigidity of the unnatural amino
acid component. Since these peptides have a common
shown in Scheme 2. In this synthesis, the phenacyl (Pac)
group, which can be removed by reduction with zinc
powder, was used for the protection of the benzoic acid
to avoid the possible side reaction of the peptides upon
removal of the protecting group. One of the amino groups
of the unnatural amino acid was protected with Boc, the
carboxylate with Pac by reacting with phenacyl bromide,
and then the remaining free amino group was acylated
by the DCC (N,N-dicyclohexylcarbodiimide)/HOBt (1-
hydroxybenzotriazole) method. The Boc group of the
compounds (15) was detached by treating with HCl in
dioxane to produce the compounds (16) which were
hardly crystallized, whereas the corresponding ethyl
esters (12), which readily crystallize, were more easily
purified and better handled. The compounds (16) were
reacted with the amino acids with the protected side
chain to yield dipeptides (17-19), followed by detaching
the phenacyl group so as to be applicable to the solid-
phase synthesis.
These dipeptides (14, 20-22) were coupled and cyclized
on an oxime resin to produce the cyclic peptides as shown
in Scheme 3.69-71 The cyclic peptides (23-25) containing
L-aspartate, L-lysine, and L-serine, which further require
removal of the protecting groups of the side chains, were
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1987, 274.

2982 J . Org. Chem., Vol. 66, No. 9, 2001
Ishida et al.
Ta ble 1. Con d u cta n ce Va lu es of th e Ch a n n els in a Sym m etr ica l Solu tion of 500 m M KCl
(AC10 (AC10 (AC16 (AC16 (DC16 (KC18
channel
)
)
4
)
4
)
)
4
)
4
(SC18)
4
3
5
γ (pS) ( SDa
9.0 ( 1.4
14
8.0 ( 0.8
8
8.8 ( 1.4
22
9.1 ( 1.7
9
10.3 ( 1.8
14
8.6 ( 0.9
6
8.7 (0.9
4
nb
a
b
SD means standard deviation. Numbers of the experiments.
F igu r e 2. Top view of CPK models of the peptides composed
of (a) 3, (b) 4, and (c) 5 dipeptide units. The alkanoyl chains
are not shown for simplicity.
distance of 9.6 Å between the R-carbons of adjacent
L-alanines located at the corners, the diameters of their
cavities are ca. 4, 6, 11 Å for (AC10)3, (AC10)4, and (AC16)5.
The alkanoyl chains are aligned parallel in the same
direction so that they can penetrate into bilayer mem-
branes.
F igu r e 3. Representative single channel current traces
observed for the membranes containing the cyclic peptides at
+100 mV in a symmetric solution of 500 mM KCl.
Sin gle Ion Ch a n n el P r op er ties of Cyclic P ep tid es.
We have already reported that the cyclic octa-peptide
(AC16)4 forms an ion channel.68 The detailed studies on
the ion channel properties have revealed that the (AC16)4
channel has the single-channel conductance of ca. 9 pS
in a symmetrical 500 mM KCl solution and favors
permeation of cations over anions with a permeability
+
-
+
ratio (PCl /PK ) of 0.15. The channel is also K selective
over Na+ cation with a permeability ratio (PK /PNa ) of
ca. l2.5, and furthermore the single-channel current is
blocked by Ca2+. From all available structural informa-
tion, we have inferred that the (AC16)4 channel is formed
upon the tail-to-tail association of two (AC16)4 molecules
incorporated in bilayer membranes. We describe here the
ion channel properties of the newly synthesized cyclic
peptides as well as (AC16)4.
+
+
F igu r e 4. Current-voltage relationship of the (DC16)4 channel
in symmetrical (cis/trans, 500/500 mM KCl) and asymmetrical
solutions (cis/trans, 500/100 mM KCl). Curves fitted to the data
are drawn according to the GHK current equation.1
Sin gle Ch a n n el Con d u cta n ce. The single channel
conductance of individual cyclic peptides was examined
in a symmetrical aqueous solution of KCl (500 mM) under
the voltage clamp condition. All the cyclic peptides
exhibited typical single channel characteristics, i.e., rapid
transitions between the open and closed states, and
stable current levels, when incorporated in lipid bilayer
membranes (Figure 3). The latter behavior indicates that
the structure of the open channel in the bilayer, once
established, is very stable. The current-voltage (I-V)
relationships of all the channels were linear under a
symmetrical ionic condition (a typical result for (DC16)4
in Figure 4). The single channel conductance was deter-
mined from the slope of I-V curve obtained with a
symmetrical 500 mM KCl solution. The results are
summarized in Table 1 with the number (n) of reported
runs. No significant difference in the single channel
conductance is seen despite the diversities in the alkanoyl
chain length (10, 16, and 18), the number of constituted
amino acids (6, 8, 10), and even the polarity of the
peptides (containing neutral L-alanine ((AC16)4) and L-
serine ((SC18)4), anionic L-aspartate ((DC16)4), and cationic
L-lysine ((KC18)4)). It should be noted that these values
are consistent with each other at 8.9 ( 0.7 pS, which is
analogous to that (6.1 ( 0.8 pS) reported for the ion
channel formed by the resorcinol tetramer, which also
bears long C17 alkyl chains.62

Artificial Ion Channels
J . Org. Chem., Vol. 66, No. 9, 2001 2983
Ta ble 2. Ch a r ge Selectivity (Cl-/K+) of th e Ch a n n els in Cis/Tr a n s 500/100 m M KCl
a
channel
(AC10
)
(AC10
)
4
(AC16
)
4
(AC16
)
5
(DC16
)
4
(KC18
)
(SC18)
4
3
4
Vr [mV] ( SD
-23.1 ( 1.9
-24.4 ( 3.2
-25.4 ( 1.1
-23.7 ( 1.7
-25.3 ( 2.2
-25.8 ( 3.1
-23.4 ( 2.6
n
4
5
3
4
3
3
3
-
+
PCl /PK
0.19
0.17
0.15
0.18
0.15
0.14
0.19
a
Reference 68.
Ch a r ge Selectivity. To determine whether the chan-
nels favor the permeation of cation or anion, similar
experiment was performed under asymmetric ionic con-
ditions (e.g. cis/trans 500/100 mM KCl), from which a
curved I-V relationship was obtained (see Figure 4, the
broken line). This curvature is due to the charge selectiv-
ity of the channels and the concentration gradient of
permeant ion. If a channel favors the permeation of K+
over Cl-, the cation, rather than the anion, moves from
the cis to the trans side under the asymmetric condition.
Such movement gives an excess positive charge in the
trans side. As the trans side is usually defined as 0 mV,
such ion movement builds up a negative electrical
potential difference across the membrane. Under the
voltage-clamp condition, the flow of current will reverse
its direction at this potential (thus reversal potential).
At the same time, currents (K+) flow from cis to trans
would be larger than that from trans to the cis, giving a
super linear curve crossing at a negative voltage. Simi-
larly, if a channel favors the permeation of Cl- over K+,
we should observe an I-V curve with a positive reversal
potential. Therefore, by measuring the reversal potential,
we can determine the charge selectivity of the channel
F igu r e 5. Current-voltage relationships of the (DC16)4 chan-
nel in biionic solutions. Curves fitted to the data are drawn
according to the GHK current equation.1
+
+
Ta ble 3. Ca tion Selectivity (P Na /P K ) of th e Ch a n n els
u n d er Biion ic Con d ition of 500/500 (K+/Na + cis/tr a n s m M)
a
channel
(AC10
)
(AC16
)
(AC16
)
5
(DC16)
4
-
+
4
4
and furthermore the permeability ratio, PCl /PK , with the
Goldman-Hodgkin-Katz (GHK) equation.1 The results
are listed in Table 2.
Vr [mV] ( SD -21.2 ( 2.8 -22.1 ( 0.8 -23.6 ( 1.9 -21.7 ( 3.3
n
4
0.41
3
0.39
3
0.37
3
0.40
+
+
PNa /PK
The (AC16)4 channel has been already reported to favor
permeation of K+ over Cl- with the permeation ratio
a
Reference 68.
PCl /PK of 0.15.68 The other cyclic peptides consisting of
L-alanine, (AC10)3, (AC10)4, and (AC16)5 showed similar
permeation ratios, 0.17-0.19. This is probably due to the
partially negatively charged carbonyl oxygens on the
ring, which are situated at the entrance of the channel
to facilitate permeation of K+ rather than Cl-.68 This
hypothesis would lead to an idea that the charge selectiv-
ity can be altered by changing the natural amino acids
from L-alanine to charged ones such as L-aspartate
(negative) and L-lysine (positive). However, the Cl-/K+
selectivity obtained for the (DC16)4 and (KC18)4 channels
were 0.15 and 0.14, which are comparable to that
obtained for the channels containing L-alanine. Moreover,
(SC18)4, which is neutral and probably more hydrophilic
than (AC16)4, has a similar Cl-/K+ selectivity as that of
(AC16)4. No significant difference was observed among the
different channels despite their varied polarity of the side
chain (alanine (or serine), aspartate, and lysine), ring size
(3, 4, and 5 dipeptides), and chain lengths (10, 16, and
18 carbon atoms). It is noteworthy that the permeability
ratios (0.14-0.19) we observed are slightly larger (less
selective) than that (0.05) of the channel formed by the
resorcinol tetramer with four C17 alkyl chains.62 The
results imply that the charge selectivity is determined
by the structure of the cyclic headgroup. A possible reason
of why the variation within the cyclic peptides scarcely
affects the permeation ratio is discussed in the section
of “Blockade by Ca2+”.
-
+
which were limited to the L-alanine and L-aspartate
peptides. The monovalent cation selectivity was exam-
ined under biionic conditions, which were established by
adding the same amounts of Na+ and K+ gluconate
solutions to the trans and cis side of the membrane,
respectively, where gluconate anion is assumed not to
permeate through the membrane. If the channel favors
permeation of K+ over Na+, the negative reversal poten-
tial will be observed. As can be seen from Figure 5 (solid
line), the (DC16)4 channel shows a reversal potential of
-21.7 mV under a biionic condition of cis/trans 500/500
mM [K+]/[Na+]. The permeability ratio PNa /PK , calcu-
lated from the reversal potential, is 0.40, indicating that
the channel is more permeable for K+ than for Na+. In
contrast, Li+ could not permeate through the channel (for
(DC16)4, see the broken line in Figure 5) even though its
ionic diameter (1.56 Å (Goldschmidt)) is much smaller
than that of K+ (2.66 Å).73 This is probably due to
difficulty of stripping some of water molecules surround-
ing Li+, which is a necessary step for ion permeation
through channels.1 A similar tendency has been observed
for the peptide channels constituted of L-alanine.68 As
+
+
+
+
,
summarized in Table 3, the permeability ratios, PNa /PK
among the channels are almost similar regardless of
varying chain length (compare (AC10)4 with (AC16)4), ring
size (compare (AC16)4 with (AC16)5), or charges (compare
(AC16)4 with (DC16)4).
Ca tion Selectivity. We examined the monovalent
cation selectivity between K+ and Na+ of the channels,
(73) Sinha, A. P. Struct. Bonding 1976, 25, 69.

2984 J . Org. Chem., Vol. 66, No. 9, 2001
Ishida et al.
Ta ble 4. Block in g P a r a m eter s of Ca 2+ on th e Ch a n n els
a
peptide
(AC16
)
4
(AC16
)
5
(DC16)
4
zδ ( SD
Kd(0) [mM] ( SD
0.07 ( 0.03
0.81 ( 0.11
0.12 ( 0.04
0.78 ( 0.14
0.11 ( 0.03
0.87 ( 0.16
a
Reference 68.
the single channel currents in a one to one fashion. From
these titration curves, the dissociation constant of Ca2+
at +100 mV was calculated to be ca. 0.6 mM throughout
the channel tested ((AC16)4, (AC10)3, and (AC16)5) despite
their different pore sizes.
The blocking effect of Ca2+ was voltage dependent. The
single channel current for the peptide channels was
reduced more effectively at positive voltages than at
negative ones in the presence of 0.5 mM Ca2+ in the cis
side. As a typical example, the I-V relationships for the
(DC16)4 channel in the presence and absence of 0.5 mM
Ca2+ in the cis side are shown in Figure 6D. For the
voltage-dependent blockade by Ca2+, the relative con-
ductance value (γ/γ0) is defined as:
γ/γ0 ) {1 + [Ca2+]/Kd(0)exp(zδFV/RT)}-1 (2)
according to the Woodhull’s blocking theory,74 where
Kd(0) is an apparent zero voltage dissociation constant
for Ca2+ and zδ is a fractional electrical distance between
the binding site and the cis entrance of the channel. Thus,
analysis of the I-V data in the presence and absence of
Ca2+ gives the dissociation constant at 0 mV (Kd(0)) and
the electric distance (zδ). These values for the (DC16)4
channel are summarized in Table 4 along with those for
(AC16)4 and (AC16)5. The Kd(0) values are kept constant
at ca. 0.8-0.9 mM regardless of their different structures.
The electric distances (zδ) are also similar (0.07-0.12)
for these three cyclic peptides. The zδ values are quite
small, indicating that the Ca2+ binding site is located very
close to the channel entrance. It should be noted that
Ca2+ can block the channels from either side of the
membrane in the same manner, suggesting that these
channels have two equivalent binding sites at the both
sides of the membrane and therefore the symmetrical
structure throughout the bilayer membrane. Taking into
account the highly hydrophilic nature of Ca2+, the most
hydrophilic cyclic peptide moiety is thought to be the
binding site for Ca2+. Therefore, the small zδ values
suggest that the peptide ring should be situated at the
entrance of the channel. As electrophysiological proper-
ties of all the channels are similar, we may speculate that
all the channels take similar structure in the bilayer
membrane as that of the (AC16)4 channel.68 That is, each
channel is formed from a dimer of the cyclic peptide, the
peptide ring of which forms the channel entrance while
its alkanoyl chains line across the membrane to form the
channel pore.
F igu r e 6. Dose and voltage dependent blockade of single
channel currents by Ca2+. (A) Single channel current traces
for the (AC16)4 channel at +100 mV in the presence of 0 and
0.2 mM Ca2+. (B) Relative single channel currents (I/I0) in the
presence of 0.2 mM Ca2+. I and I0 are the currents at +100
mV in the presence and absence of Ca2+. Curves fitted to the
data are drown according to eq 1. (C) Dose-dependent blockade
of single channel currents by Ca2+ at +100 mV. (D) Voltage-
dependent blockade of single channel current: current-voltage
relationships of the (DC16)4 channel before and after adding
0.5 mM Ca2+ into the cis side of the solution.
Block a d e by Ca 2+. Addition of 0.2 mM Ca2+ to one
side of the chamber containing a KCl (500 mM) solution
decreased the single channel currents of the (AC16)4
channel (Figure 6A). This result indicates that Ca2+
blocks the ion channel. The relative currents (I/I0) in the
presence and absence of 0.2 mM Ca2+ are almost similar
for all the channels tested despite their different struc-
tures (Figure 6B). This suggests that Ca2+ blocks these
cyclic peptides in a similar manner. Furthermore, relative
single-channel currents (I/I0) could be fitted with a single
site titration curve according to
Despite the different structural features between each
of the channels, all the single channel properties were
similar. The reason for this discrepancy is not exactly
known, but might be explained by the following consid-
erations. The fact that the charge selectivity and the
Ca2+-blocking behavior are scarcely affected by the dif-
ference in side chain charge of the cyclic peptides may
be accounted for if the charged residues are located far
from the channel entrance. Actually, the molecular
I/I0 ) {(1 + [Ca2+]/Kd(V))}-1
(1)
where Kd (V) is the dissociation constant at a given
voltage V, [Ca2+] is the concentration of Ca2+ in the
solution, I and I0 are the current at given voltage with
or without Ca2+. This result suggests that Ca2+ blocks
(74) Woodhull, A. M. J . Gen. Physiol. 1973, 61, 687.

Artificial Ion Channels
J . Org. Chem., Vol. 66, No. 9, 2001 2985
modelings by the InsightII/Discover program indicate
that the distance between the carboxyl oxygen in Asp and
the peptide ring is over 6 Å and that between the nitrogen
atom in Lys and the ring is over 9 Å. According to the
Gouy-Chapman’s surface potential theory, the Debye
length, which is a distance to which the electrostatic
effect of a surface charge can reach in solution, is only
4.6 Å in seawater.1 Therefore, charged atoms of the side
chains are too far to significantly affect the ion perme-
ation.
chain length, the ring size of cyclic peptide, and the type
of natural amino acid, have been synthesized. All the
peptides were demonstrated to form ion channels by the
single-channel recording technique. The single channel
properties of the cyclic peptides were investigated in
terms of single channel conductance, charge selectivity,
and cation selectivity. These properties were almost
similar among the cyclic peptide channels despite the
divergent structural variations. The channels were blocked
by Ca2+ in a voltage-dependent manner. The blocking
experiments revealed that Ca2+ block the ion channels
from both sides, clearly indicating that the cyclic peptide
aggregates to give a symmetrical channel. The parameter
of zδ is quite small, suggesting that the binding site is
very close to the entrance of the channel. These results
are consistent with an idea that each channel is formed
from a dimer of the peptide, the ring of which forms the
channel entrance while its alkanoyl chains line the
channel pore. On the basis of this structure, we concluded
that the rate-limiting step of ion conduction occurs at the
hydrophobic alkanoyl chain region.76 Since the hydro-
phobic region exists widely in natural channel proteins,
this work provided a good model to study the mechanism
of ion permeation through a hydrophobic region of
natural channels.
It is expected that the larger the size of the peptide
ring, the easier the ions to enter into the pore of the
channel, and consequently the larger the conductance of
the channel. But no significant difference of the conduc-
tance between different peptides with ring size ranging
from 3 to 5 was observed. This might be explained by a
consideration that the enlargement will weaken the
interaction between the ion and the peptide ring at the
same time. Based on the proposed structures of the
channels, the interaction between the ion and channel
during the ion permeation can be decomposed into the
interaction between the ion and the peptide ring, and the
interaction between the ion and the alkanoyl chains.
Obviously, the only common part of the interaction
between the ion and all of the peptide is the alkanoyl
chains. Therefore, the small influence of the structural
variation on the single channel conductance suggests that
the process of ion passing through the hydrophobic
alkanoyl chain region, is the rate-limiting step for ions
permeation through all of these channels. This inference
is consistent with energy profiles for ions permeation
through the (AC16)4 channel, which was calculated based
on a three-energy-barrier two-binding-site (3B2S) model
of Eyring rate theory.68 It is well-known that the current
of an ion permeating across a channel is an integration
through the whole energy profile the ion felt during its
permeation.75 Therefore, the alkanoyl chain region, which
is much longer than the peptide ring region, contributes
significantly to the current, therefore the conductance,
of the peptide channels. The length of the alkanoyl chain
in the channels did not influence the conductance,
although one would expect a lower conductance level for
a longer pore. Such apparently unexpected behavior
would be rationalized by assuming that the degree of
interdigitation of the alkanoyl chains increases with
increasing chain length to afford a more ordered pore,
which facilitates the ion permeation eventually giving
analogous conductances for a series of peptides with
different alkanoyl chains.
Exp er im en ta l Section
Gen er a l. The Boc-L-amino acids were purchased from
Watanabe Chemical Industries, Ltd., except for Boc-L-Ala-OH
which was prepared in our laboratory. The other chemicals of
reagent grade were purchased from Tokyo Kasei Kogyo Co.,
1
Ltd. and Nacalai Tesque, Inc. H and 13C NMR spectra were
recorded in CDCl3 or DMSO-d6 at 400 MHz with TMS as an
internal standard. Structural models of the channels were
constructed on a Silicon Graphics Indigo2 workstation by using
Insight II 95 and Discover 95 (MSI, San Diego, CA) programs
as previously described.77
Syn th eses of Cyclic P ep tid es
3,5-Dia m in oben zoic Acid Eth yl Ester (9). Thionyl chlo-
ride (10 cm3) was added dropwise to dry ethanol (30 cm3) in
an ice bath. 3,5-Diaminobenzoic acid (8, 5 g) was added to the
solution, which was further refluxed for 1 day. After evapora-
tion, the resulting solid was dissolved in ethyl acetate, and
the solution was washed with 4% aqueous solution of sodium
hydrogen carbonate (NaHCO3) (10 cm3 × 3) and then with
water to remove the remaining starting material 8. The ethyl
acetate solution was dried over anhydrous MgSO4 and then
evaporated to yield the product 9 as a solid: 5.5 g, 93% yield.
1H NMR (CDCl3) δ 6.79 (2H, s), 6.18 (1H, s), 4.44 (2H, q), 3.13
(4H, br), 1.36 (3H, t). Anal. Calcd for C9H12O2N2: C, 59.99; H,
6.71; N, 15.55. Found: C, 60.02; H, 6.60; N, 15.47.
3-(N-ter t-Bu toxyca r bon yla m in o)-5-a m in oben zoic Acid
Eth yl Ester (10). To 60 cm3 of dioxane-water (2:1 v/v)
solution were added 9 (2 g), triethylamine (Et3N, 3.3 cm3), and
di-tert-butyl dicarbonate ((Boc)2O, 2.1 g). The solution was
gently stirred at 0 °C for 1 h and at room temperature for 10
h, and then the solvent was evaporated. The oil residue was
dissolved in ethyl acetate (50 cm3), and the solution was
Con clu sion
We have designed and synthesized artificial ion chan-
nels based on cyclic peptides which consist of alternating
natural and unnatural amino acids. The unnatural amino
acid, 5-(N-alkanoylamino)-3-aminobenzoic acid, renders
the cyclic peptides structurally rigid, endowing an inher-
ent pore through which ions can permeate. As has been
demonstrated for the (AC16)4 channel,68 the cyclic peptide
builds up a single ion channel by forming a tail-to-tail
associated dimer so as to align the hydrophobic alkanoyl
chains in bilayer membranes. In this work, the cyclic
peptides with structural varieties, including the alkanoyl
(76) An alternative channel structure model would involve a pore
formed by aggregation of the cyclic peptides. Although the present
results cannot rigorously exclude such a possibility, this model is not
compatible with the fact that the ion channel properties, such as gating
kinetics, are not appreciably affected by the charge of the cyclic
peptides; charged peptides are more difficult to aggregate with each
other than neutral ones. This model can reasonably account for the
observed channel properties independent of the charge, as the rate-
limiting step is the ion permeation through the hydrophobic alkanoyl
chain region.
(75) Levitt, D. G. Annu. Rev. Biophys. Chem. 1986, 15, 29.
(77) Qi, Z.; Sokabe, M. Biophys. Chem. 1998, 71, 35.

2986 J . Org. Chem., Vol. 66, No. 9, 2001
Ishida et al.
washed with 10% aqueous citric acid solution (20 cm3 × 3) and
with water. The ethyl acetate solution was dried over MgSO4
and evaporated to give the product 10: 1.8 g, 83% yield. 1H
NMR (CDCl3) δ 7.26 (1H, s), 7.13 (1H, s), 7.02 (1H, s), 6.55
(1H, br), 4.45 (2H, q), 1.51 (9H, s), 1.36 (3H, t). Anal. Calcd
for C14H20O4N2: C, 59.99; H, 7.19; N, 9.99. Found: C, 59.99;
H, 7.29; N, 9.83.
(30 cm3) and 10% aqueous citric acid solution (20 cm3). The
solution was stirred for 30 min and was filtrated in order to
remove DCUrea. The ethyl acetate phase was washed succes-
sively with 10% aqueous citric acid solution (20 cm3 × 3), water
(20 cm3), 4% NaHCO3 aqueous solution (20 cm3 × 3), and water
(20 cm3). The ethyl acetate solution was dried over MgSO4 and
evaporated. The resulting oil was purified by silica gel chro-
matography (Wakogel C-200) with CHCl3-CH3OH (30:1 v/v).
A mixture of ether-petroleum ether was added to the oily
compound to produce a solid: 4.5 g, 88% yield. 1H NMR
(DMSO-d6) δ 10.13 (1H, s), 10.09 (1H, s), 8.24 (1H, s), 7.93
(1H, s), 7.91 (1H, s), 7.07 (1H, d, J ) 6.84 Hz, amide(Ala)),
4.32 (2H, q, CH2(Et)), 4.10 (1H, m), 2.49 (2H, t), 1.57 (2H, br),
1.36 (9H, s), 1.32 (3H, t), 1.26 (3H, d, J ) 6.84 Hz, â-CH3-
(Ala)), 1.21 (12H, br), 0.84 (3H, t). Anal. Calcd for C27H43O6-
N3: C, 64.13; H, 8.57; N, 8.31. Found: C, 64.08; H, 8.57; N,
8.29.
3-(N-ter t-Bu toxycar bon ylam in o)-5-(N′-decan oylam in o)-
ben zoic Acid Eth yl Ester (11a ). Triethylamine (3 cm3) and
decanoic anhydride (4 g) were added to 10 (5 g) in chloroform
(50 cm3). The mixture was stirred at room temperature for 1
day, and then the solvent was evaporated. The oily residue
was dissolved in ether-petroleum ether, and the solution was
allowed to stand at room temperature for 3 h to give the
1
product 11a as crystals: 5.8 g, 75% yield. H NMR (CDCl3) δ
8.05 (1H, s), 7.83 (1H, s), 7.77 (1H, s), 7.66 (1H, s), 6.95 (1H,
s), 4.38 (2H, q), 2.36 (2H, t), 1.72 (2H, m), 1.51 (9H, s), 1.38
(3H, t), 1.26 (12H, br), 0.89 (3H, t); 13C NMR (CDCl3) δ 169.3,
166.2, 152.7, 139.3, 138.8, 131.7, 115.0 (2C), 114.0, 81.0, 61.2,
37.7, 33.8, 29.4 (3C), 28.3 (3C), 25.5, 22.6 (2C), 14.3 (2C). Anal.
Calcd for C24H38O5N2: C, 66.33; H, 8.81; N, 6.45. Found: C,
66.35; H, 8.77; N, 6.47.
3-[N-(N-ter t-Bu toxyca r bon yl-L-a la n yl)a m in o]-5-(N′-p a l-
m itoyla m in o)ben zoic Acid Eth yl Ester (13b). The titled
compound was synthesized from Boc-Ala-OH (4.5 g) and 12b
(5 g) according to the same procedures employed for 13a : 4 g,
1
60% yield. H NMR (DMSO-d6) δ 10.13 (1H, s), 10.08 (1H, s),
8.24 (1H, s), 7.93 (2H, s), 7.08 (1H, d, J ) 6.83 Hz, amide-
(Ala)), 4.33 (2H, q), 4.12 (1H, m), 2.32 (2H, t), 1.60 (2H, br),
1.38 (9H, s), 1.33 (3H, t), 1.30 (3H, d), 1.22 (24H, br), 0.84 (3H,
t); 13C NMR (DMSO-d6) δ 172.5, 171.6, 165.5, 139.9 (2C), 130.5,
114.4 (2C), 112.5, 78.0, 60.8, 50.2, 36.4, 31.3-28.2 (12C), 25.0,
22.1, 17.8, 14.2 (2C). Anal. Calcd for C33H55O6N3: C, 67.20; H,
9.40; N, 7.13. Found: C, 66.97; H, 9.39; N, 7.31.
3-(N-ter t-Bu toxyca r bon yla m in o)-5-(N′-h exa d eca n oyl-
a m in o)ben zoic Acid Eth yl Ester (11b). Triethylamine (5.6
cm3) and dicyclohexylcarbodiimide (DCC, 10 g) were added to
chloroform (60 cm3) containing 10 (9.3 g), hexadecanoic acid
(10 g), and 1-hydroxybenzotriazole (HOBt, 6.2 g) at 0 °C. The
mixture was stirred continuously at 0 °C for 1 h and then at
room temperature for 20 h. The solution was evaporated to
yield an oily residue, which was dissolved in ethyl acetate (60
cm3) and 10% aqueous citric acid solution (30 cm3). The solu-
tion was stirred for 30 min to generate N,N′-dicyclohexylurea
(DCUrea), which was filtered off. The ethyl acetate phase was
washed successively with 10% aqueous citric acid solution (30
cm3 × 3), water, 4% aqueous NaHCO3 solution (30 cm3 × 3),
and then water (30 cm3 × 1). The ethyl acetate solution was
dried over MgSO4 and was evaporated. The resultant was
dissolved in ether-petroleum ether, and the solution was
allowed to stand at room temperature for 5 h to produce the
3-[N -(N -t er t -Bu t oxyca r b on yl-L-a la n yl)a m in o]-5-(N ′-
deca n oyla m in o)ben zoic Acid (14a ). To an ethanol solution
(10 cm3) of 13a (6 g) was added 1 M NaOH aqueous solution
(15 cm3), and the resultant mixture was vigorously stirred at
room temperature for 1 day. After evaporation, the residue
was dissolved in ethyl acetate (30 cm3), which was washed with
10% aqueous citric acid solution (30 cm3 × 3) and with water
(30 cm3 × 1). The ethyl acetate phase was further dried over
MgSO4 and evaporated. A mixture of ether-petroleum ether
was added to the resulting oil to produce a solid: 4.2 g, 73%
1
yield. H NMR (DMSO-d6) δ 10.09 (1H, s), 10.05 (1H, s), 8.18
1
product 11b as crystals: 16 g, 93% yield. H NMR (CDCl3) δ
(1H, s), 7.91 (1H, s), 7.89 (1H, s), 7.09 (1H, d, J ) 6.96 Hz,
amide(Ala)), 4.11 (1H, m), 2.50 (2H, t), 1.60 (2H, br), 1.38 (9H,
s), 1.25 (15H, br), 0.87 (3H, t). Anal. Calcd for C25H39O6N3: C,
62.86; H, 8.23; N, 8.80. Found: C, 62.51; H, 8.25; N, 8.70.
3-[N-(N-ter t-Bu toxycar bon yl-L-alan yl)am in o]-5-(N′-h exa-
d eca n oyla m in o)ben zoic Acid (14b). The synthesis of 14b
was performed by using 13b (4 g) according to the same
procedures employed for 14a : 3 g, 76% yield. 1H NMR (DMSO-
d6) δ 10.08 (1H, s), 10.03 (1H, s), 8.18 (1H, s), 7.92 (2H, s),
7.06 (1H, d, J ) 6.83 Hz, amide(Ala)), 4.12 (1H, m), 2.31 (2H,
t), 1.58 (2H, br), 1.26 (9H, s), 1.23 (27H, d), 0.86 (3H, t). Anal.
Calcd for C31H51O6N3‚0.5H2O: C, 65.23; H, 9.18; N, 7.36.
Found: C, 65.45; H, 8.29; N, 7.32.
3-(N-ter t-Bu toxyca r bon yla m in o)-5-(N′-h exa d eca n oyl-
a m in o)ben zoic Acid P h en a cyl Ester (15a ). In the synthesis
of 15a , one amino group of 8 with tert-Boc was first protected
by using (Boc)2O (21.5 g) and 8 (15 g) according to the similar
procedures employed for 10 to produce 3-(tert-butoxycarbonyl-
amino)-5-aminobenzoic acid: 20 g, 82% yield. Esterification
of the carboxylic group of this compound with phenacyl group
was performed by reacting the intermediate product (6.7 g)
mentioned above with triethylamine (3.7 cm3) and 2-bromo-
acetophenone (4.8 g) in ethyl acetate to produce 3-(N-tert-
butoxycarbonylamino)-5-aminobenzoic acid phenacyl ester: 9.4
g, 95% yield. The title compound 15a was synthesized by
coupling of the phenacyl ester (6.9 g) with hexadecanoic acid
(4.8 g) according to the same procedure employed for 11b. The
crude product 15a was purified by using a column chroma-
tography (Wakogel C-200) with CHCl3-CH3OH (30:1 v/v)
eluent and was recrystallized from ether-petroleum ether: 8.3
g, 75% yield. 1H NMR (CDCl3) δ 8.00 (1H, br), 7.96 (3H, d),
7.81 (1H, s), 7.64 (1H, t), 7.51 (3H, d), 6.85 (1H, br), 5.55 (2H,
s), 2.35 (2H, t), 1.69 (2H, br), 1.50 (9H, s), 1.25 (24H, br), 0.89
(3H, t). Anal. Calcd for C36H52O6N2: C, 71.02; H, 8.61; N, 4.60.
Found: C, 68.21; H, 7.64; N, 5.15.
8.11 (1H, s), 7.83 (1H, s), 7.77 (1H, s), 7.42 (1H, s), 6.78 (1H,
s), 4.38 (2H, q), 2.38 (2H, t), 1.74 (2H, m), 1.50 (9H, s), 1.36
(3H, t), 1.26 (24H, br), 0.91 (3H, t); 13C NMR (CDCl3) δ 171.6,
166.1, 152.6, 139.3, 138.8, 131.8, 115.0 (2C), 113.8, 81.0, 61.2,
37.8, 31.9, 29.7 (9C), 28.3 (3C), 25.5, 22.7 (2C), 14.3 (2C). Anal.
Calcd for C30H50O5N2: C, 69.46; H, 9.72; N, 5.40. Found: C,
69.44; H, 9.78; N, 5.28.
3-Am in o-5-(N-d eca n oyla m in o)ben zoic Acid Eth yl Es-
ter (12a ). To dry dioxane (35 cm3) containing 11a (5 g), 4 M
HCl-dioxane (35 cm3) was added, and the solution was
allowed to stand at room temperature for 3 h to yield 12a as
crystals. By adding ether (100 cm3) to the solution, the further
precipitated crystals were collected by filtration: 3.4 g, 73%
yield. 1H NMR (DMSO-d6) δ 7.29 (1H, s), 7.28 (1H, s), 7.21
(1H, s), 4.28 (2H, q), 2.27 (2H, t), 1.56 (2H, br), 1.31 (3H, t),
1.25 (12H, br), 0.87 (3H, t). Anal. Calcd for C19H30O3N2: C,
68.23; H, 9.04; N, 8.38. Found: C, 68.12; H, 9.13; N, 8.32.
3-Am in o-5-(N-h exa d eca n oyla m in o)ben zoic Acid Eth yl
Ester (12b). Synthesis of 12b was performed from the
compound 11b (16.0 g) according to the same procedures
1
employed in the synthesis of 12a : 13 g, 99% yield. H NMR
(CDCl3) δ 7.57 (1H, s), 7.26 (1H, s), 7.18 (1H, s), 7.17 (1H, s),
4.36 (2H, q), 2.35 (2H, t), 1.72 (2H, br), 1.37 (3H, t), 1.25 (24H,
br), 0.90 (3H, t); 13C NMR (CDCl3) δ 171.6, 166.4, 147.4 (2C),
139.1, 111.5, 110.5, 110.4, 61.0, 37.9, 31.9, 29.7 (6C), 25.6, 22.7
(2C), 14.3 (2C). Anal. Calcd for C25H42O3N2: C, 71.73; H, 10.11;
N, 6.69. Found: C, 71.74; H, 10.24; N, 6.69.
3-[N -(N -t er t -Bu t oxyca r b on yl-L-a la n yl)a m in o]-5-(N ′-
d eca n oyla m in o)ben zoic Acid Eth yl Ester (13a ). Boc-Ala-
OH (2.3 g) and 12a (3.4 g) were dissolved in DMF (20 cm3), to
which HOBt (0.3 g), triethylamine (14.0 cm3), and DCC (2.5
g) were added at 0 °C. The solution was stirred at 0 °C for 1
h and then at room temperature for additional 20 h. After
evaporation, the oily residue was dissolved in ethyl acetate

Artificial Ion Channels
J . Org. Chem., Vol. 66, No. 9, 2001 2987
3-(N-ter t-Bu t oxyca r b on yla m in o)-5-(N′-oct a d eca n oyl-
a m in o)ben zoic Acid P h en a cyl Ester (15b). The compound
15b was similarly synthesized according to the procedures
employed for 15a by using octadecanoic acid (6.8 g) and 3-(tert-
butoxycarbonylamino)-5-aminobenzoic acid phenacyl ester (7.4
recrystallized with ether-petroleum ether: 2.1 g, 77% yield.
1H NMR (DMSO-d6) δ 10.23 (1H, s), 10.05 (1H, s), 8.19 (1H,
s), 7.92 (2H, d), 7.35 (5H, m), 7.27 (1H), 5.11 (2H, s), 4.51 (1H,
br), 2.87-2.69 (2H, m), 2.31 (2H, t), 1.58 (2H, br), 1.39 (9H,
s), 1.23 (24H, br), 0.86 (3H, t). Anal. Calcd for C39H57O8N3: C,
67.31; H, 8.26; N, 6.04. Found: C, 66.41; H, 8.44; N, 5.98.
3-[N-(N-ter t-Bu toxycar bon yl-N′-E-2-ch lor ocar boben zoxy-
L-lysyl)am in o]-5-(N′-octadecan oylam in o)ben zoic Acid (21).
The compound 21 was synthesized from 18 (3.4 g) according
to the similar procedures as described above: 2.2 g, 74% yield.
1H NMR (DMSO-d6) δ 10.10 (1H, s), 10.04 (1H, s), 8.20 (1H,
s), 7.92 (2H, d), 7.47 (2H, d), 7.36 (2H, d), 6.99 (1H, d, J )
7.33 Hz, amide(Lys)), 5.07 (2H, s), 4.03 (1H, br), 3.00 (2H, br),
2.31 (2H, t), 1.58 (4H, br), 1.38 (11H, s), 1.22 (30H, br), 0.86
(3H, t). Anal. Calcd for C44H67O8N4Cl: C, 64.80; H, 8.28; N,
6.87. Found: C, 64.01; H, 8.98; N, 6.67.
1
g) in the final step: 10 g, 80% yield. H NMR (CDCl3) δ 8.03
(1H, s), 7.96 (1H, s), 7.94 (2H, d), 7.83 (1H, s), 7.80 (2H, s),
7.59 (1H, t), 6.79 (1H, s), 5.54 (2H, s), 2.35 (2H, t), 1.61 (2H,
br), 1.51 (9H, s), 1.25 (28H, br), 0.89 (3H, t). Anal. Calcd for
C
38H56O6N2: C, 71.67; H, 8.86; N, 4.40. Found: C, 72.67; H,
10.41; N, 3.94.
3-Am in o-5-(N-h exa d eca n oyla m in o)ben zoic Acid P h en -
a cyl Ester (16a ). Deprotection of the Boc group of 15a was
performed by adding 4 M HCl-dioxane (47 cm3) to the dry
dioxane solution (47 cm3) of 15a (9.7 g). The solution was
allowed to stand at room temperature for 3 h and evaporated.
The resulting solid was dissolved in ethyl acetate (60 cm3),
and the solution was washed with 10% aqueous citric acid
solution (50 cm3 × 4) and then water (50 cm3 × 1). The ethyl
acetate solution was dried with MgSO4 and evaporated to yield
16a : 7.4 g, 75% yield. 1H NMR (DMSO-d6) δ 10.42 (1H, s),
8.03 (3H, d), 7.95 (1H, s), 7.75 (1H, t), 7.61 (3H, d), 5.79 (2H,
s), 2.37 (2H, t), 1.60 (2H, br), 1.25 (24H, br), 0.87 (3H, t). Anal.
Calcd for C31H44O4N2‚HCl: C, 68.30; H, 8.32; N, 5.14. Found:
C, 64.96; H, 7.17; N, 6.07.
3-[N-(N-ter t-Bu toxyca r bon yl-O-ben zyl-L-ser yl)a m in o]-
5-(N′-octa d eca n oyla m in o)ben zoic Acid (22). The com-
pound 22 was similarly synthesized from 19 (2.5 g): 1.7 g, 80%
1
yield. H NMR (DMSO-d6) δ 10.23 (1H, s), 10.05 (1H, s), 8.21
(1H, s), 7.93 (1H, d), 7.90 (1H, d), 7.29 (5H, m), 7.04 (1H, br),
4.50 (2H, s), 4.38 (1H, br), 3.64 (2H, br), 2.30 (2H, t), 1.58 (2H,
br), 1.39 (9H, s), 1.22 (28H, br), 0.84 (3H, t). Anal. Calcd for
C
40H61O7N3‚2H2O: C, 65.64; H, 8.95; N, 5.74. Found: C, 65.46;
H, 8.63; N, 5.94.
3-Am in o-5-(N-stea r oyla m in o)ben zoic Acid P h en a cyl
Ester (16b). The synthesis of 16b was performed from 15b
(6 g) according to the same procedures employed for 16a (3.2
Cou p lin g a n d Cycliza tion of P ep tid es on Oxim e Resin .
The cyclic peptides (1-4, 23, 24, 25) were synthesized by
stepwise coupling of the corresponding dipeptides and further
cyclization on oxime resin. The dry CH2Cl2 solution (18 cm3)
of the dipeptide (2 mmol) and DCC (2 mmol) was mixed with
the oxime resin (2.0 g). The reaction mixture was allowed to
shake at room temperature for 1 day. The solvent was filtered
off, and the resin was washed with CH2Cl2 (× 2), CH2Cl2-
C2H5OH (1:1 v/v) (× 2), and CH2Cl2 (× 2) and then dried in
vacuo. The substitution level of the dipeptide to the oxime resin
was estimated from the picrate assay; normally 0.23-0.28
mmol/g-resin.
Further coupling of dipeptides was performed as follows:
the Boc-dipeptide-oxime resin, which was prepared as men-
tioned above, was washed with 25% TFA in CH2Cl2, and the
resin was shaken in 25% TFA-CH2Cl2 at room temperature
for 30 min. The solvent was filtered off, and the resin was
washed with CH2Cl2 (× 2), 2-propanol (× 1), CH2Cl2 (× 2), and
DMF (× 1). To the resin was added a DMF solution (18 mL)
containing the dipeptide (3 equiv), [(benzotriazole-1-yl)oxy]tris-
(dimethylamino)phosphonium hexafluorophosphate (BOP) (3
equiv), HOBt (3 equiv), and triethylamine (5 equiv), and the
resulted mixture was shaken at room temperature for 1 h.
Then, the solvent was filtered off, and the resin was washed
with DMF (× 2), and CH2Cl2 (× 2). The termination of the
coupling reaction was checked by the Kaiser test. The same
procedures were repeated to further couple the dipeptides.
To cyclize the peptides on the oxime resin, the Boc group
was removed with 25% TFA-CH2Cl2. To the resulted peptide-
bound resin, a DMF solution (18 mL) containing acetic acid
(2 equiv of the peptides) and triethylamine (2 equiv) was
added. The resin in the solution was shaken at room temper-
ature for 1 day, the resultant mixture was filtered, and the
resin was washed with DMF. The combined filtrate was
evaporated in vacuo to give an oily residue, to which was added
water to crystallize the peptides. Purification of the cyclic
peptides was performed by chromatography over silica gel
(Wakogel C-200) and Sephadex (LH-20), with CHCl3:CH3OH
(30:1 v/v) and DMF as eluents, respectively. The yields and
spectral data of the cyclic peptides are shown below.
1
g, 59% yield). H NMR (DMSO-d6) δ 10.22 (1H, s), 8.02 (1H,
d), 8.00 (1H, s), 7.91 (1H, s), 7.78 (1H, s), 7.73 (1H, t), 7.60
(2H, d), 5.76 (2H, s), 2.33 (2H, t), 1.58 (2H, br), 1.22 (28H, br),
0.86 (3H, t). Anal. Calcd for C33H48O4N2‚HCl: C, 69.14; H, 8.62;
N, 4.89. Found: C, 68.51; H, 8.42; N, 4.85.
3-[N-(N-ter t-Bu t oxyca r b on yl-O-â-b en zyl-L-a sp a r t yl)-
a m in o]-5-(N′-p a lm itoyla m in o)ben zoic Acid P h en a cyl Es-
ter (17). The dipeptide 17 was synthesized from Boc-Asp-
(OBzl)-OH (2.1 g) and 16a (3 g) by the same procedure as
described for 13a : 3.3 g, 67% yield. 1H NMR (DMSO-d6) δ
10.34 (1H, s), 10.15 (1H, s), 8.27 (2H, d), 8.03 (2H, d), 7.74
(1H, s), 7.61 (2H, t), 7.36 (5H, m), 7.30 (1H), 5.76 (2H, s), 5.11
(2H, s), 4.53 (1H, br), 2.89-2.68 (2H, m), 2.33 (2H, t), 1.59
(2H, br), 1.39 (9H, s), 1.23 (24H, br), 0.86 (3H, t). Anal. Calcd
for C47H63O9N3: C, 69.34; H, 7.80; N, 5.16. Found: C, 68.95;
H, 8.29; N, 5.52.
3-[N-(N-ter t-Bu toxycar bon yl-N′-E-2-ch lor ocar boben zoxy-
L-lysyl)a m in o]-5-(N′-oct a d eca n oyla m in o)b en zoic Acid
P h en a cyl Ester (18). The synthesis of 18 was similarly
performed from Boc-Lys(Cl-Z)-OH (1.2 g) and 16b (1.3 g): 1.8
1
g, 80% yield. H NMR (DMSO-d6) δ 10.20 (1H, s), 10.13 (1H,
s), 8.27 (1H, s), 8.03 (2H, d), 8.01 (2H, d), 7.73 (1H, s), 7.61
(2H, t), 7.36 (2H, d), 7.34 (3H, d), 7.01 (1H, d, J ) 7.32 Hz,
amide(Lys)), 5.76 (2H, s), 5.07 (2H, s), 4.05 (1H, br), 3.01 (2H,
br), 2.33 (2H, t), 1.60 (4H, br), 1.38 (11H, s), 1.22 (30H, br),
0.85 (3H, t). Anal. Calcd for C52H73O9N4Cl: C, 66.90; H, 7.88;
N, 6.00. Found: C, 64.40; H, 7.52; N, 6.24.
3-[N-(N-ter t-Bu toxyca r bon yl-O-ben zyl-L-ser yl)a m in o]-
5-(N′-octa d eca n oyla m in o)ben zoic Acid P h en a cyl Ester
(19). The dipeptide 19 was similarly synthesized from Boc-
Ser(Bzl)-OH (1.7 g) and 16b (2.7 g): 2.5 g, 65% yield. 1H NMR
(DMSO-d6) δ 10.32 (1H, s), 10.12 (1H, s), 8.28 (2H, d), 8.05
(2H, d), 7.72 (1H, s), 7.60 (2H, t), 7.32 (5H, m), 7.05 (1H, br),
5.74 (2H, s), 4.53 (2H, s), 4.43 (1H, br), 3.65 (2H, m), 2.33 (2H,
t), 1.59 (2H, br), 1.39 (9H, s), 1.23 (28H, br), 0.86 (3H, t). Anal.
Calcd for C48H67O8N3: C, 70.82; H, 8.30; N, 5.16. Found: C,
70.82; H, 8.38; N, 5.18.
3-[N-(N-ter t-Bu t oxyca r b on yl-O-â-b en zyl-L-a sp a r t yl)-
a m in o]-5-(N′-h exa d eca n oyla m in o)ben zoic Acid (20). To
the 90% acetic acid solution (15 cm3) of 17 (3.3 g) was added
zinc powder (8.0 g), and the mixture was vigorously stirred at
room temperature for 1 day. To remove the unreacted zinc
powder, the solution was filtrated and then evaporated. The
resulting oil was dissolved in ethyl acetate (50 cm3), which was
washed with 10% aqueous citric acid solution (30 cm3 × 4) and
with water (30 cm3 × 1). The ethyl acetate solution was dried
over MgSO4 and was evaporated. The crude compound was
Cyclo{-tr i[3-(N-L-a la n yl-a m in o)-5-(N′-d eca n oyl-a m in o)-
p h en ylca r bon yl]-} (1): 17 mg, 2.6% yield, H NMR (DMSO-
1
d6) δ 10.34 (3H, s), 10.05 (3H, s), 8.31 (3H, d, J ) 7.32 Hz,
amide(Ala)), 8.26 (3H, s), 7.98(3H, s), 7.61 (3H, s), 4.70 (3H,
m), 2.33 (6H, t), 1.59 (6H, br), 1.44 (9H, d, J ) 7.33 Hz, â-CH3-
(Ala)), 1.28 (36H, br), 0.87 (9H, t); 13C NMR (DMSO-d6) δ 171.6
(3C), 171.3 (3C), 166.1 (3C), 139.7 (3C), 139.2 (3C), 135.6 (3C),
113.5 (3C), 112.7 (3C), 112.1 (3C), 59.2 (3C), 36.4 (3C), 31.3
(3C), 28.6 (12C), 25.1 (3C), 22.1 (3C), 18.2 (3C), 14.0 (3C), MS
m/z calcd for (M + Na) C60H87O9N9 1101.6, found 1100.6. Anal.

2988 J . Org. Chem., Vol. 66, No. 9, 2001
Ishida et al.
Calcd for C60H87O9N9‚2H2O: C, 64.66; H, 8.23; N, 11.31.
Found: C, 64.62; H, 7.91; N, 11.08.
washed with ether. The solid was purified by chromatography
on a Sephadex column (LH-20) with DMF.
Cyclo{-tetr a[3-(N-l-aspar tyl-am in o)-5-(N′-h exadecan oyl-
a m in o)p h en ylca r bon yl]-} (5). The compound was synthe-
sized from 23 (30 mg) and was recrystallized from DMF-4%
aqueous NaHCO3 solution: 17 mg, 67% yield. 1H NMR
(DMSO-d6) δ 10.25-10.01 (8H, m), 9.10-8.68 (4H, m), 8.07-
7.35 (12H, m), 4.89 (4H, br), 2.95-2.67 (8H, br), 2.30 (8H, br),
1.58 (8H, br), 1.25 (96H, br), 0.85 (12H, t). MS m/z calcd for
Cyclo{-tetr a [3-(N-L-a la n yl-a m in o)-5-(N′-d eca n oyl-a m i-
n o)ph en ylcar bon yl]-} (2): 70 mg, 9% yield, 1H NMR (DMSO-
d6) δ 10.10(4H, s), 10.01(4H, s), 8.47(4H, d, J ) 6.83 Hz,
amide(Ala)), 7.99(4H, s), 7.91(4H, s), 7.64(4H, s), 4.53(4H, q),
2.29(8H, t, J ) 6.83 Hz, 2-alkanoyl), 1.59(8H, br), 1.40(12H,
d, J ) 7.33 Hz, â-CH3), 1.25(48H, br), 0.83(12H, t, J ) 6.35
Hz, 10-alkanoyl); 13C NMR (DMSO-d6) d 171.5 (8C), 166.6 (4C),
139.4 (4C), 139.1 (4C), 135.4 (4C), 113.6 (4C), 113.2 (4C), 112.8
(4C), 50.1 (4C), 37.7 (4C), 31.3 (4C), 28.9 (16C), 25.1 (4C), 22.1
C
O
108H164O20N12 1950.6, found 1951.1. Anal. Calcd for C108H160-
20N12Na4‚H2O: C, 63.07; H, 7.94; N, 8.18. Found: C, 63.36;
H, 8.11; N, 7.93.
(4C), 17.6 (4C), 13.9 (4C); MS m/z calcd for (M + Na) C80H116
-
‚
Cyclo{-t et r a [3-(N-l-lysyl-a m in o)-5-(N′-oct a d eca n oyl-
a m in o)p h en ylca r bon yl]-} (6). The compound was synthe-
sized from 24 (50 mg). After purification by chromatography,
trifluoroacetic acid and then ether was added to the eluted
solution in order to obtain the solid as a TFA salt. The solid
was recrystallized from DMF-water: 31 mg, 83% yield. 1H
NMR (DMSO-d6) δ 10.38-10.03 (8H, br), 8.46-8.18 (4H, br),
8.04 (4H, s), 7.97 (4H, s), 7.84 (4H, s), 4.52 (4H, br), 2.89 (8H,
br), 2.30 (8H, br), 1.58 (16H, br), 1.39 (8H, br), 1.23 (120H,
br), 0.85 (13H, t); MS m/z calcd for C124H208O12N16 2115.1, found
2114.3. Anal. Calcd for C124H208O12N16‚4(CF3COOH)‚10H2O: C,
57.62; H, 8.50; N, 8.15. Found: C, 57.30; H, 8.08; N, 8.17.
Cyclo{-tetr a[3-(N-l-ser yl-am in o)-5-(N′-octadecan oylam i-
n o)p h en ylca r bon yl]-} (7). The compound was synthesized
from 25 (60 mg) and was recrystallized from DMF-water: 44
mg, 87% yield. 1H NMR (DMSO-d6) δ 10.44-10.04 (8H, br),
8.89 (4H, br), 8.17-7.67 (12H, br), 4.66 (4H, br), 3.80 (8H, br),
2.30 (8H, br), 1.58 (8H, br), 1.23 (112H, br), 0.85 (12H, t); MS
m/z calcd for (M + Na) C112H180O16N12 1973.7, found 1973.2.
Anal. Calcd for C112H180O16N12‚10H2O: C, 63.13; H, 9.46; N,
7.89. Found: C, 63.42; H, 8.78; N, 7.60.
O12N12 1461.1, found 1457.2. Anal. Calcd for C80H116O12N12
4H2O: C, 63.63; H, 8.28; N, 11.13. Found: C, 63.95; H, 8.08;
N, 11.22.
Cyclo{-tetr a [3-(N-L-a la n yl-a m in o)-5-(N′-p a lm itoyl-a m i-
n o)p h en ylca r bon yl]-} (3): 177 mg, 17% yield, 1H NMR
(DMSO-d6) δ 10.33-10.01(6H, m), 8.48-8.27(4H, m), 8.19-
7.58(12H, m), 4.70-4.53(4H, m), 2.33-2.29(8H, m), 1.59(8H,
br), 1.43(9H, br), 1.25(36H, br), 0.83(9H, t, J ) 6.35 Hz, 10-
alkanoyl); 13C NMR (DMSO-d6) δ 171.6 (8C), 166.6 (4C), 139.7
(4C), 139.2 (4C), 135.6 (4C), 113.6 (4C), 113.2 (4C), 112.7 (4C),
50.0 (4C), 38.9 (4C), 31.3 (4C), 29.0 (40C), 25.1 (4C), 22.1 (4C),
17.6 (4C), 13.9 (4C); MS m/z calcd for (M + Na) C104H164O12N12
1797.5, found 1797.1. Anal. Calcd for C104H164O12N12‚5H2O: C,
66.99; H, 9.41; N, 9.02. Found: C, 66.97; H, 9.12; N, 9.34.
Cyclo{-pen ta[3-(N-L-alan yl-am in o)-5-(N′-palm itoyl-am i-
n o)p h en ylca r bon yl]-} (4): 40 mg, 3.6% yield, 1H NMR
(DMSO-d6) δ 10.16 (5H, s), 10.03 (5H, s), 8.53 (5H, br), 8.10-
7.66(15H, br), 4.55 (5H, m), 2.29 (10H, br), 1.57 (10H, br), 1.40
(15H, d, J ) 5.86 Hz, âCH3), 1.22 (120H, br), 0.85 (15H, t).
MS m/z calcd for (M + Na) C130H205O15N15 2241.1, found
2240.7. Anal. Calcd for C130H205O15N15‚4H2O: C, 68.17; H, 9.38;
N, 9.18. Found: C, 67.93; H, 9.30; N, 9.30.
Mea su r em en ts of Sin gle Ch a n n el Cu r r en ts. Planar
bilayers were formed by applying phosphatidylcholine (PC,
asolectin type IV, Sigma Chemical Co., St. Louis, MO) solu-
tions (40 mg/mL in n-decane) to a pore (300-500 µm in
diameter) in a polypropylene partition separating two solution-
filled chambers as described earlier.78 A dimethyl sulfoxide
(DMSO, Wako Co., J apan) solution of the peptide (1 mg/mL,
ca. 0.5 mM) was premixed with the phospholipid solution (1:
9, v/v) in a tube and was bath sonicated for 1 min to force the
amount of peptide into the lipid solution as much as possible.
Several minutes after sonication, the mixed solution automati-
cally separated into two phases, where an n-decane solution
formed an upper phase and a DMSO solution a lower phase.
The bilayer membrane was formed from the n-decane solution,
into which a certain amount of the peptide was expected to
move. The same procedure for control experiment with DMSO
only could not generate any channel activity in bilayers. To
eliminate the possibility that the channel was formed from
some contaminations in a high concentration of the peptide
solution, we gradually lowered the concentration of the peptide
in DMSO solution down to 1 µM, but no noticeable difference
was observed for the single channel behavior. Curiously, the
success rate of channel formation was only slightly improved
by the high concentration of the peptide. Due to their poor
solubility in lipid phase, it is suspected that most of the peptide
molecules form an aggregation in the lipid solution such that
they cannot participate in the formation of an ion channel.
However, we used higher concentrations in most experiments
to improve the success rate as much as possible.
Cyclo{-tetr a [3-(N-(O-â-ben zyl-L-a sp a r tyl)a m in o)-5-(N′-
p a lm itoyl-a m in o)p h en yl ca r bon yl]-} (23): 35 mg, 3.2%
yield, H NMR (DMSO-d6) δ 10.18 (4H, s), 10.01 (4H, s), 8.75
(4H, d), 8.09 (4H, s), 7.79 (4H, s), 7.70 (4H, s), 5.10 (8H, s),
4.99 (4H, m), 2.97 (8H, m), 2.29 (8H, br), 1.57 (8H, br), 1.22
1
(96H, br), 0.84 (12H, t) MS m/z calcd for (M + Na) C136H188
-
‚
O
20N12 2334.1, found 2333.1. Anal. Calcd for C136H188O20N12
10H2O: C, 65.57; H, 8.42; N, 6.75. Found: C, 65.74; H, 8.03;
N, 6.63.
Cyclo{-tetr a [3-(N-(N′-E-2-ch lor oca r boben zoxy-l-lysyl)-
a m in o)-5-(N′-stea r oyl-a m in o)p h en yl ca r bon yl]-} (24): 106
1
mg, 5.6% yield, H NMR (DMSO-d6) δ 10.38-10.00 (8H, br),
8.41-8.20 (4H, br), 8.20 (4H, s), 8.03 (4H, s), 7.96 (4H, s), 7.42
(8H, s), 7.32 (8H, d), 5.05 (8H, s), 4.50 (4H, br), 2.98 (8H, br),
2.29 (8H, br), 1.58 (16H, br), 1.44 (8H, br), 1.22 (120H, br),
0.85 (13H, t); MS m/z calcd for (M + Na) C156H228O20N16Cl4
2812.8, found 2808.5. Anal. Calcd for C156H228O20N16Cl4‚
3H2O: C, 65.89; H, 8.30; N, 7.88. Found: C, 65.99; H, 8.26; N,
7.86.
Cyclo{-tetr a [3-(N-(O-ben zyl-l-ser yl)a m in o)-5-(N′-stea -
r oyl-a m in o)p h en yl ca r bon yl]-} (25): 172 mg, 11% yield, 1H
NMR (DMSO-d6) δ 10.52 (4H, s), 10.09 (4H, s), 8.40 (4H, s),
8.23 (4H, br), 8.00 (4H, s), 7.65 (4H, s), 7.29 (20H, m), 4.93
(4H, br), 4.55 (8H, s), 3.84 (8H, br), 2.32 (8H, t), 1.60 (8H, br),
1.22 (112H, br), 0.85 (12H, t); MS m/z calcd for (M + Na) C140
-
-
H
204O16N12 2334.2, found 2333.5. Anal. Calcd for C140H204O16
N12‚4H2O: C, 70.55; H, 8.97; N, 7.05. Found: C, 70.26; H, 8.96;
N, 6.76.
Transmembrane currents across voltage-clamped bilayers
were low-pass filtered at 10 kHz through a bilayer-clamp
amplifier (model BC-525C, Warner Instrument Co. USA) and
recorded on a magnetic tape by means of a PCM recorder (VR-
10B, Instrutech Co.). The side to which lipid solution was
smeared was defined as cis. The opposite side, which was
connected to the signal ground of the amplifier, was defined
as trans. The voltage was referred to the cis side with respect
to the trans side. All the solutions used were buffered by 5
mM HEPES and adjusted with Tris base to pH 7.2. All the
experiments were carried out at room temperature (20-25 °C).
Dep r otection of th e Cyclic P ep tid es 23-25. Removal
of the protecting group, i.e., the benzyl group for aspartic acid
and serine and the 2-chlorocarbobenzoxy group for lysine, in
the cyclic peptides (23-25) was performed as follows. The
protected cyclic peptides (30-60 mg) were dissolved in tri-
fluoroacetic acid at -10 °C under N2. To the solution, 3-cresol
(0.06 cm3), thioanisole (0.18 cm3), and trimethylsilyl trifluo-
romethanesulfonate (TMSOTf; 0.28 cm3) were added under N2.
The solution was sealed in a flask under N2 and stirred at 0
°C for 1 h. The reaction solution was added to cold ether, and
the mixture was allowed to stand in a refrigerator. The
resultant powder was collected by centrifugation and was
(78) Nomura, K.; Sokabe, M. J . Membrane Biol. 1991, 124, 53.

Artificial Ion Channels
J . Org. Chem., Vol. 66, No. 9, 2001 2989
[aK+]c - [aK+]texp(-VrF/RT)
Da ta An a lyses. Single channel currents recorded on the
tape were replayed, postfiltered at 0.10 kHz, and digitized at
0.50 ms/point by means of an analogue to digital converter
(model 2801A, Data Translation Co.). Amplitude of the single
channel currents was measured as a peak-to-peak distance
(each peak represents open and closed levels) on the amplitude
histogram constructed by a single channel analysis program,
PAT (ver. 6.2).79 Single channel conductance values at a
symmetrical salt solution were determined by the slope of the
current-voltage (I-V) curve, where the I-V data could be well
fitted to a straight line. Single-channel conductance values
under asymmetrical ionic conditions were obtained by the slope
of I-V curves at higher voltages (g60 mV), where nonlinear
I-V curves became asymptotic to straight lines. The perme-
PCl
PK
-
)
(4)
[aCl-]cexp(-VrF/RT) - [aCl
]
-
t
+
under asymmetrical conditions,
where the reversal potential Vr was obtained by fitting the
I-V curve to the Goldman-Hodgkin-Katz (GHK) voltage
equation,1 aK and aNa are the activity of K+ and Na+,
subscripts c and t represent cis and trans sides of the chamber,
F is the Faraday constant, R is the gas constant, and T means
absolute temperature.
+
+
Ack n ow led gm en t. This work was funded in part
by the J apan Science and Technology, by the Naito
Memorial Science Foundation (H.I.), and by a grant for
a future program from J SPS (M.S.).
+
+
ability ratios of the channel (PNa /PK ) were defiened as
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
PNa+/PK+ ) (aK+/aNa+)exp(VrF/RT)
(3)
spectra for (AC10
)
3
and (AC10)4; temperature dependent 1H
NMR for (AC10)3, (AC10)4, and (AC16)5; NMR peak assignments
for all compounds except for 8. This material is available free
of charge via the Internet at http://pubs.acs.org.
under biionic conditions,
(79) Dempster, J . Computer Analysis of Electrophysiological Signals;
Academic Press: New York, 1993.
J O001079T