The influence of varied amide bond positions on hydraphile ion channel activity

Article abstract of DOI:10.1039/b510863m

Hydraphile compounds have been prepared in which certain amine nitrogens have been replaced by amide residues. The amide bonds are present either in the side chain, the spacer chain, or the central relay. Sodium cation transport through phospholipid vesicles mediated by each hydraphile was assessed. All of the amide-containing hydraphiles showed increased levels of Na⁺ transport compared to the parent compound, but the most dramatic rate increase was observed for sidearm amine to amide replacement. We attribute this enhancement to stabilization of the sidearm in the bilayer to achieve a better conformation for ion conduction. Biological studies of the amide hydraphiles with Escherichia coli and Bacillus subtilis showed significant toxicity only with the latter. Further, the consistency between the efficacies of ion transport and toxicity previously observed for non-amidic hydraphiles was not in evidence. the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006.

Full text of DOI:10.1039/b510863m

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/njc | New Journal of Chemistry
The influence of varied amide bond positions on hydraphile ion channel
activity
Michelle E. Weber,a Wei Wang,b Sarah E. Steinhardt,b Michael R. Gokel,a
W. Matthew Leevya and George W. Gokel*ab
Received (in Durham, UK) 29th July 2005, Accepted 8th December 2005
First published as an Advance Article on the web 19th January 2006
DOI: 10.1039/b510863m
Hydraphile compounds have been prepared in which certain amine nitrogens have been replaced
by amide residues. The amide bonds are present either in the side chain, the spacer chain, or the
central relay. Sodium cation transport through phospholipid vesicles mediated by each hydraphile
was assessed. All of the amide-containing hydraphiles showed increased levels of Na1 transport
compared to the parent compound, but the most dramatic rate increase was observed for sidearm
amine to amide replacement. We attribute this enhancement to stabilization of the sidearm in the
bilayer to achieve a better conformation for ion conduction. Biological studies of the amide
hydraphiles with Escherichia coli and Bacillus subtilis showed significant toxicity only with the
latter. Further, the consistency between the efficacies of ion transport and toxicity previously
observed for non-amidic hydraphiles was not in evidence.
lowered by the presence of the formal positive charge. In either
Introduction
event, however, the oxygen donors are directed away from the
Crown ethers are known to complex metal and organic
internal, complexing cavity. This is illustrated in the CPK
cations.1 They can also complex or include in their crystalline
molecular model shown with the resonance forms in Fig. 1.
lattices neutral compounds such as nitromethane, acetonitrile,
The cation complexing ability of relatively few macrocyclic
and urea.2 The donor elements that best complement alkali
lactams has been studied. Even when such studies have been
metal or alkaline earth cations are oxygen and nitrogen.
undertaken, direct comparisons to complexation strengths and
Complexation to transition metal cations is favored by the
selectivities of the corresponding crowns or azacrowns have
not been reported.4–6 When tertiary amide donors were pre-
presence of sulfur, but nitrogen is often an effective donor
element for these cations as well. Oxygen and sulfur are
sent in flexible lariat ether sidearms, and therefore accessible to
ring-bound cations, complexation of Na1 in methanol in-
typically neutral and divalent in their complexing role, but
nitrogen within a macrocycle may be secondary or tertiary and
creased by an order of magnitude compared to the macrocycle
lacking a sidearm.7,8 In other studies, it was found that when
neutral or protonated. Further, nitrogen may be present either
as an amine or as an amide residue.
two amide residues are present in lariat ether sidearms,
calcium over sodium cation selectivity is enhanced.9
An amine’s nitrogen is electron rich and, unless it is
sterically hindered, it is readily accessible to a Lewis acid.
When crown ethers are incorporated into a synthetic chan-
nel system,10 such as the hydraphiles,11 transport dynamics
The electron donating ability of nitrogen is diminished by
proximate electron withdrawing substituents and it is rendered
and complexation strengths are at odds with each other.
partially positive by resonance when it is in the amide (R2N–
Channels must obviously recognize a cation, but selectivity
CO–R0) form. An amide is thus a poorer donor at nitrogen but
in the channel context means that a particular cation is passed
a strong donor at oxygen by virtue of its formal negative
most readily, not held most strongly. The hydraphiles are less
charge in the enol canonical form.
Numerous macrocyclic amides (lactams) are known.3–6 In
principle, incorporation of an amide into a typical 15- or
18-membered ring macrocycle will rigidify the ring. The utility
of an amide as a donor in a macrocycle depends upon whether
or not the carbonyl residue is available for complexation. Fig.
1 shows 3-oxo-4-aza-15-crown-5 in its two resonance forms (A
and B). The amide oxygen is a more powerful donor in form B,
but cation complexation by the macroring is expected to be
a Department of Molecular Biology & Pharmacology, Washington
University School of Medicine, Campus Box 8103, 660 S. Euclid
Ave., St. Louis, MO 63110, USA. E-mail: ggokel@wustl.edu;
Fax: þ1 314-362-9298; Tel: þ1 314-362-9297
b Department of Chemistry, Washington University, 1 Brookings
Fig. 1 Resonance forms (A and B) and CPK molecular model of 3-
Drive, St. Louis, MO 63130, USA
oxo-4-aza-15-crown-5.
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 177–184 | 177

View Article Online
structurally complex than protein channels but many of their
functions are similar. For example, they transport ions in
planar bilayers,12 in liposomes,13 and in the membranes of
live organisms.14
The hydraphiles are constructed in four modules. Two distal
diaza-18-crown-6 macrocycles serve as head groups for ion
entry and egress. Covalent spacer chains link the two distal
macrocycles to the central relay15 and define the channel’s
overall length. In the compounds studied here, the spacers are
1,12-dodecyl. Finally, sidearms are appended to the distal
macrocycles. These sidearms were designed to serve as intra-
membrane anchors to stabilize the channel in the bilayer.16
The compounds reported here incorporate the features de-
scribed above but integrate amide residues at various positions
within the channel. The influence of these strategically placed
amide residues on sodium cation transport has been assayed
analytically and in vivo and the results are reported below.
Results and discussion
Compounds studied
Three new hydraphile structures were prepared for this study.
They are illustrated as 2–4 along with the parent structure (1),17
which contains all of the same structural features but lacks any
amide function. In compound 2 (C12hN18NiC11COhN18Ni
COC11hN18NiC12), the amide residues are adjacent to the
central relay. In 3 (C12hN18NiCOC11hN18NiC11COhN18Ni
C12), the amides comprise the proximal nitrogen atoms in the
two distal macrocycles. The amides in 4 (C11COhN18Ni
C12hN18NiC12hN18NiCOC11) comprise the distal nitrogens in
the same macrocycles. Thus, the three structurally distinct
positions adjacent to the macrocycles have been cataloged by
this small library.
each hydraphile (Na1 selective electrode, see experimental
section for details). We note that the Na1 release profile for
1 has previously been reported in another context.19
Synthetic access to 1–4
The release profile for 4 is shown in Fig. 2. Each line is the
average of at least three separate determinations. The graph
clearly shows the release of sodium cations from these phos-
pholipid liposomes mediated by 4 in a concentration-depen-
dent fashion. When the concentration of 4 is ([4] =) 12 mM,
B60% of the total Na1 is released under these conditions.
Total release is achieved in B500 s when [4] = 36 mM. The
concentration range in this experiment is from 2 mM to 36 mM
or a nearly 20-fold increase. The quality of the data and the
broad concentration range give us considerable confidence in
this method for comparing compound activity. It is interesting
to note that channel 4 shows 10-fold higher Na1 transport at
12 mM than does the same concentration of the parent channel
(119), which lacks amide bonds.19
Different synthetic strategies were required for the preparation
of 2, 3, and 4. Channel 1, although previously described,17 was
synthesized for this study by the reduction of 4. Channel 4 was
made from the reaction of HhN18NiC12hN18NiC12hN18NiH
with commercially available dodecanoic acid to afford a
yellow solid in 79% yield. This compound was then reduced
with BH3 to afford 1 in 67% yield. Channel 2 was prepared by
acylation of diazacrown18 with 12-bromododecanoyl chloride.
The resulting dibromide was then alkylated with
CH3(CH2)11hN18NiH to afford 2 as a white powder. The
route to hydraphile 3 reversed the first steps used for com-
pound 2: diazacrown was dialkylated with 12-bromododeca-
noic acid to afford the diacid. This product was then converted
to the acid chloride and reacted with C12hN18NiH to afford 3
as an orange oil. All compounds were characterized by 1H and
13C-NMR and had satisfactory elemental analyses. Synthetic
details are recorded in the experimental section.
Since amide-containing channel 4 proved to be more active
than 1,19 comparisons with channels 2 and 3 were undertaken.
These are structural variations in which the amide residues are
adjacent to the central macrocycle (2) or on the proximal sides
of the distal macrocycles (3). Sodium release from phospholi-
pid vesicles mediated by 1–4 is shown in the graph of Fig. 3.
At 12 mM, the percentage release values determined for 1, 2,
3, and 4 were 6%, 8%, 11%, and 60%, respectively. Based on
these values, we can assign the following activity trend: 4 4 3
4 2 4 1. The concentration of 12 mM was chosen arbitrarily
Ion transport studies for 1–4
We have recently developed a method that uses ion selective
electrodes (ISEs) to monitor sodium cation release from
phospholipid vesicles (liposomes).13 Using this technique, a
concentration-dependent set of release curves was obtained for
ꢀc
This journal is the Royal Society of Chemistry the Centre National de la Recherche Scientifique 2006
178 | New J. Chem., 2006, 30, 177–184

View Article Online
Fig. 2 Fractional Na1 release from phospholipid vesicles mediated
by 4. Transporter concentrations are, from bottom to top, 2, 4, 8, 12,
20, and 36 mM. The concentration of l,2-dioleoyl-sn-glycero-3-phos-
phocholine (DOPC) vesicles was 0.4 mM.
in order to simplify comparisons. As noted above, total release
of Na1 is observed in B500 s when [4] = 36 mM. In contrast,
119 and 3 give full release in 500 s when their concentrations
are 72 mM and 48 mM, respectively. While 4 is by far the most
active compound in this series, the efficacies of compounds 1–3
can be differentiated at higher concentrations. Thus, while 2
and 3 are both more active than parent compound 1, 3 exhibits
higher Na1 transport than does 2.
Comparison with previously studied hydraphiles
We have previously obtained transport data for a few amide-
containing hydraphile compounds.20 In our two earlier stu-
dies, the goals were to assess sub-structural units other than
the amide residue(s). As a consequence, comparisons of only
two related structures are available. Moreover, the methods
used to assay transport differed from the ISE technique used
here. A final and problematic variable is that comparisons
made by different methods were made against different stan-
dards. Notwithstanding these difficulties, the data are valuable
because the syntheses of these compounds are complicated
enough that a fully consistent series will be difficult to obtain.
The compounds shown as 5–10 have previously been reported
and may be compared pairwise.
Sodium transport was determined for 5–10 by using the
method of Riddell and Hayer.21 In this approach, phospholi-
pid vesicles are created in a sodium-containing buffer. Sodium-
23 is an NMR active nucleus that may be observed directly.
The vesicle suspension shows a single 23Na line spectrum even
though some cations are within the liposomes and most are in
the bulk aqueous phase. When a dysprosium shift reagent is
added to the suspension, external and internal Na1 are
apparent as different NMR resonances. When the hydraphile
is added, it inserts in the liposomal bilayer, and allows passive,
equilibrium transport of both internal and external Na1
through the membrane. From changes in the two linewidths,
an exchange constant may be calculated. This constant is
compared with the exchange rate observed for the same system
with a standard. In most of our previous work, the standard
was the bacterial ion channel gramicidin. In other studies, it
was the hydraphile we call ‘‘dansyl channel.’’ The latter is
identical to 5 except that the terminal benzyl groups are
replaced by dansyl (dimethylaminonaphthylsulfonyl) residues.
Data were obtained in two previous studies (shown in Table
1).20 In the three cases where direct comparisons can be made,
the presence of amide functions reduced sodium transport
activity. Amide residues are adjacent to the medial macrocycle
in all three of these cases (6, 8, 10). Compound 10 has carbonyl
groups in positions that correspond to those in 4. If the medial
amides deactivate and the distal amides activate, then 9 and 10
should show similar activities. Indeed, 10 retains 74% of the
activity of 9. The amide position is not immediately adjacent
Fig. 3 Fractional Na1 release, determined by the ISE method, for
1–4 (12 mM each). The concentration of DOPC vesicles was 0.4
mM. Total Na1 release of o11% was observed for 1–3.
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 177–184 | 179

View Article Online
Table 1 Sodium transport activity loss between hydraphile pairs in
which amides replace amine residues
a macrocyclic lactam also affects the macrocycle’s rigidity. The
lack of flexibility and structural adaptation is often detrimen-
tal to cation complexation. Of course, when the amide is
present in a sidearm that can serve as a lariat ether donor,
the effect is favorable.24
Compound
Function
Amide placement
Relative activity
5
6
7
8
9
10
X = H2
X = O
X = H2
X = O
X = H2
X = O
None
Medial
None
Medial, lariat
None
Medial, distal
100%a
33%a
100%a
65%a
Amide and amine geometry
100%b
74%b
In order to better understand the influence of amide and amine
residues, we energy minimized N-butylmorpholine and N-
morpholinobutyramide. Their structures are shown in Fig. 4.
The results are unsurprising but the key points deserve com-
ment. First, in the case of N-butylmorpholine, the morpholine
ring is in the chair conformation and the butyl chain is
equatorial and fully staggered. The nitrogen is approximately
tetrahedral and its electron pair is unhindered. The structure
of N-morpholinobutyramide is quite different. The 6-mem-
bered ring is nearly planar, as is the amide residue. The amide
oxygen is roughly in a plane with the morpholine ring but it is
pointed away from it. Inversion of the morpholine ring would
give a bidentate O and N donor array focused in the same
direction. No conformational variation of the amide will
permit the two oxygen donors to comprise a bidentate co-
ordination array.
a
b
See ref. 20a. See ref. 20b.
to the distal macroring in 8, which is only 65% as active as 7.
The most striking contrast in activity is between 5 and 6. The
latter loses two thirds of its activity when the medial macro-
cycle’s nitrogens are amidated.
Cation binding data for macrocyclic lactams and lactones
Numerous macrocyclic amides have been reported and a lesser
number of macrocyclic lactones have been prepared. Cation
binding data have been reported for several of the latter, but
the cation complexing ability of relatively few macrocyclic
lactams is represented in the literature. One reason for this is
that many lactams have been prepared as intermediates in the
syntheses either of azacrowns or cryptands (D - C).3,22 As
such, they were not studied directly for cation complexation.
Moreover, an unsubstituted lactam reduces to a secondary
azacrown and these typically show low binding affinities for
cations in polar solvents.
Amide residues and the hydraphile central relay
We have prepared several hydraphile derivatives in which the
central macrocycle (‘‘central relay’’) is replaced by other
residues.15 From these studies, we concluded that the central
relay served a water-binding function rather than directly
coordinating a transient cation. This analysis comports with
the studies conducted by MacKinnon and coworkers on the
KcsA K1 channel of Streptomyces lividans.25 Crown ether
derivatives are known to bind water26 as well as various
cations.1 Binding occurs by hydrogen bond formation between
water and alternate oxygen or nitrogen donors within the
macrocycle.
An 18-membered ring macrocycle is shown in which the
donors are numbered 1–6. Water may bridge positions 1–3,
1–5, 2–4, 2–6, 3–5, and 4–6. If donors 1 and 4 are removed as
by amidation of nitrogens at those positions, the only alternate
H-bond sites remaining for water would be 2–6 and 3–5. Thus,
binding of water would be less effective when the amides are
part of the central macrocycle. In turn, the absence of an
effective central relay for cations should diminish transport, as
observed in all of these cases.
The presence of an ester residue within the macrocycle does
not significantly alter cation complexation strength or selec-
tivity (cf. A, B).23 This is expected because the carbonyl group
is turned outward and the ester’s ether oxygen is not strongly
affected by the adjacent carbonyl. In contrast, an embedded
amide confers upon the linkage both a partial positive charge
and partial double bond character (cf. E, F). Thus, the
principal donor is altered from nitrogen within the ring to
carbonyl, which is turned outward. The formal double bond in
Sidearm amide residues
Conversion of a sidearm amine to its corresponding amide will
have two obvious consequences. We know from previous
ꢀc
This journal is the Royal Society of Chemistry the Centre National de la Recherche Scientifique 2006
180 | New J. Chem., 2006, 30, 177–184

View Article Online
sidearms and also possess integral amides. The results ob-
tained in the present study confirm the expectation that small
structural changes in hydraphile structure can mediate large
changes in biological activity. Even so, it is surprising that
compounds 2–4 were active in the 0.5 to 2 mM range to B.
subtilis but inactive to E. coli. The differences in activity are
shown in the graph of Fig. 5. Note that a higher value
indicates better transport but lower toxicity.
The differences in toxicity to these two microorganisms may
reflect the disparity in membrane structure. Gram-positive B.
subtilis have a cytoplasmic membrane surrounded by a thick
peptidoglycan cell wall. Gram-negative E. coli have a cyto-
plasmic membrane, thin peptidoglycan layer, and an addi-
tional lipopolysaccharide membrane.28 These membranes are
very different from each other and from the synthetic lipo-
somes tested previously. Notwithstanding these differences in
organisms, the correspondence between ion transport activity
and biological activity for amide-containing crowns stands in
contrast to the results obtained previously for non-amidic,
benzyl sidearmed hydraphiles.
Fig. 4 Framework molecular models of N-butylmorpholine (top) and
N-morpholinobutyramide (bottom).
studies that the macroring nitrogens of dibutyldiaza-18-
crown-6 have pK1 and pK2 values in the range 7–9.17 Proto-
nation of a macroring nitrogen is expected to diminish cation
transport. Transport reduction most likely would result from
charge–charge repulsion between the protonated nitrogen and
the transient cation. Protonation will also make the nitrogen
to which it is attached quaternary, diminishing flexibility. This
could have either positive or negative consequences depending
upon the conformational requirements for transport. In these
dynamic systems, as for protein channels, this issue remains
largely unresolved.
Conclusion
The studies reported here present evidence for enhanced Na1
ion transport activity of amide-containing hydraphiles com-
pared to analogous structures lacking the amide residues. The
data obtained presented two surprises. First, we anticipated
that when amide donors replaced amines, the diminished
molecular flexibility would impair ion transport. Instead, more
flexible 1 showed significantly lower transport than that ob-
served for 4. Second, the previously observed correspondence
between cation transport rates and biological activity is not
apparent in this series of structures. The compounds pre-
viously studied possessed only saturated oxygen and/or nitro-
gen and the present channels contain amide functional groups.
This accounts for, but does not explain, the differences in
activity, which are under further investigation.
The dramatic difference in transport efficacy between 3 and
4 suggests that stabilization and rigidification of the sidearm is
more important than a corresponding alteration to the inner
distal nitrogen atoms. The presence of an amide in either
position should make the amide nitrogen atom both a poorer
donor and a more rigid functional group. Indeed, both effects
may be operating but the influence is clearly greater in the
outer distal nitrogen position. The hydraphiles were conceived
as flexible molecules that could adapt to the existing bilayer
structure as necessary. This study suggests that the flexibility
of the dodecyl side chain is not necessarily a positive attribute
in these systems. Better Na1 transport is observed when the
sidearm is conformationally restricted, even marginally.
Experimental
General
Biological studies
1H-NMR were recorded at 300 MHz in CDC13 solvents and
are reported in ppm (d) downfield from internal TMS unless
otherwise noted. 13C-NMR were recorded at 75 MHz in
CDC13 unless otherwise stated. Infrared spectra were recorded
on a Perkin-Elmer 1710 Fourier Transform Infrared Spectro-
photometer and were calibrated against the 1601 cmꢁ1 band of
polystyrene. Melting points were determined on a Thomas
Hoover apparatus in open capillaries and are uncorrected.
Studies of sodium cation transport mediated by 1–4 in syn-
thetic liposomes show that amide bonds can dramatically
affect hydraphile activity. In previous studies of non-amide
hydraphiles, bacterial toxicity data showed a remarkable
correspondence to hydraphile activity.27 We thus undertook
similar biological studies using compounds 1–4. The minimum
inhibitory concentrations for 1–4 against Escherichia coli and
Bacillus subtilis are recorded in Table 2.
Table 2 MICa (mM) of 1–4 to E. coli and B. subtilis
The results shown in Table 2 do not correspond well with
Na1 transport rate data, as was the case for their saturated
analogs.28 While compound 1 is active against E. coli (2 mM),
its amide-containing counterparts (2–4) are at least 16-fold less
cytotoxic (34–67 mM) to this bacterium. We note that a
previous comparative study28 involved hydraphiles in which
the side chains were benzyl groups and only the overall lengths
were varied. The compounds examined here have alkyl chain
Compound
E. coli
B. subtilis
1
2
3
4
a
2.1
467
34
0.5
0.5
2
467
1
Minimum Inhibitory Concentration.
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 177–184 | 181

View Article Online
reported30 and vesicle size was measured by a Coulter N4MD
submicron particle analyzer. The vesicles used in the transport
studies had diameters of B200 nm.
Na1 transport measurements
All data were collected by Axoscope 7.0 using a Digidata
1322A series interface. Sodium transport was measured using
a Micro-Combination pH/sodium electrode (Thermo-Orion)
in aqueous sodium-free buffer (750 mM cholineCl–15 mM
HEPES, pH 7.0). After equilibration in external buffer, the
electrode was placed in a 5 mL disposable beaker containing
external buffer and vesicle solution to achieve a lipid concen-
tration of 0.4 mM and a total volume of 2 mL. In order to set
the baseline, the voltage output was recorded for five minutes
prior to addition of the channel. The channel solution in 2-
propanol was added and channel conductivity was measured
over an appropriate time range, typically 25 minutes. Addition
of 200 mL 10% aqueous n-octylglucoside induced complete
lysis of the vesicles to achieve total sodium release. All experi-
ments were performed at room temperature.
Fig. 5 Graphical illustration of the relationship between Na1 trans-
port (open squares) and toxicity of compounds 1–4 (x axis) to E. coli
(filled circles) and B. subtilis (open circles). The units for biological
activity are mM and for ion transport are percentage release from
liposomes at 1500 s.
Thin layer chromatographic (TLC) analyses were performed
on aluminium oxide 60 (F-254 neutral, Type E) with a 0.2 mm
layer thickness or on silica gel 60 F-254 with a 0.2 mm layer
thickness. Preparative chromatography columns were packed
with activated aluminium oxide (MCB 80–325 mesh, chroma-
tographic grade, AX 611) or with Kieselgel 60 (70–230 mesh).
All reactions were conducted under dry N2 unless otherwise
stated. All reagents were the best (non-LC) grade commer-
cially available and were distilled, recrystallized, or used with-
out further purification, as appropriate. Molecular distillation
temperatures refer to the oven temperature of a Kugelrohr
apparatus. Combustion analyses were performed by MWH
Laboratories, Phoenix, AZ, and are reported as percentages.
Where sodium is factored into the analytical data, high
resolution mass spectroscopy (Washington University) con-
firmed its presence.
Data analysis
The data were collected in units of mV and converted to units
of concentration, using the appropriate electrode calibration
curve. The data were normalized (OriginPro 7) to the total
sodium concentration, as determined by lysing the vesicles
with n-octylglucoside.
Biological studies
A series of test tubes was prepared in which each contained
2 mL of Luria Bertani (LB) Miller media (10 g Lꢁ1 peptone,
5 g Lꢁ1 yeast extract, 10 g Lꢁ1 NaCl). Each series contained a
set of serial 2-fold dilutions of an individual hydraphile
compound. 1 ꢂ 106 colony forming units (C.F.U.) of bacteria
were added to the 2 mL aliquots to give an innoculum of 5 ꢂ
105 C.F.U. mLꢁ1, as outlined by the NCCLS.31 The MIC was
taken as the lowest hydraphile concentration that inhibited
growth after 24 h, as judged by visual turbidity. Each MIC
value was confirmed by three independent trials for each
concentration and bacterium tested.
Vesicle experiments
Hydroxyethylpiperazine-N0-2-ethanesulfonic acid (HEPES)
and the inorganic salts NaCl and cholineCl were all purchased
from Sigma-Aldrich. The water used was Milli-Q Plus quality,
which is essential to avoid salt contamination in the buffer
systems. n-Octylglucoside was purchased from CalBioChem.
The vesicles used were prepared using the reverse evapora-
tion method of Szoka and Papahadjopoulos.29 l,2-Dioleoyl-sn-
glycero-3-phosphocholine (DOPC) was purchased from Avan-
ti Polar Lipids as a chloroform solution, which was dried to a
lipid film and stored at ambient temperature under high
vacuum. The vesicles were prepared by dissolving a dry lipid
film in 0.3 mL diethyl ether and 0.3 mL buffer (750 mM NaCl–
15 mM N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid
(HEPES), pH 7.0). The mixture was sonicated for B20 s to
give an opaque solution. Solvent was removed (in vacuo) and
the solution was passed through a mini extruder containing a
0.2 mm polycarbonate membrane filter. The residual, external
buffer solution was exchanged for a sodium-free buffer (750
mM cholineCl–15 mM HEPES, pH 7.0) via passage over a
Sephadex G25 column. Vesicle concentration was measured as
N,N0-bis{12-[N-(N0-dodecyl)-diaza-18-crown-6]-dodecyl}-
diaza-18-crown-6, C12H25hN18Ni(CH2)12hN18Ni
(CH2)12hN18NiC12H25, 1
1 was prepared from the reduction of 4 (0.35 g, 0.23 mmol, see
below) in BH3–THF (6 mL). The reaction was stirred (rt, N2)
for 3 d. Dropwise addition of H2O quenched the remaining
hydride and the reaction was concentrated under reduced
pressure. The resulting white solid was dissolved in 24%
HBr (10 mL) and heated to reflux for 3 h. The pH was
adjusted to B9 by addition of NaOH pellets at 0 1C. The
aqueous solution was extracted 3ꢂ with CH2C12, washed with
brine (2 ꢂ 50 mL) and NaHCO3 (50 mL), dried over MgSO4,
and the solvent removed in vacuo to afford 1 (240 mg, 67%) as
white crystals, mp 60–61 1C (lit.,17 61–63 1C). H-NMR: 0.86
(6H, t, J = 6.9 Hz), 1.24 (68H, s), 1.42 (12H, m), 2.46–2.51
(12H, m), 2.76–2.80 (24H, m), 3.58–3.62 (48H, m). 13C-NMR:
1
ꢀc
This journal is the Royal Society of Chemistry the Centre National de la Recherche Scientifique 2006
182 | New J. Chem., 2006, 30, 177–184

View Article Online
14.01, 22.60, 27.03, 27.44, 29.26, 29.57, 31.84, 53.93, 55.90,
69.81, 70.67.
days. The mixture was filtered and concentrated. The residue
was dissolved in Et2O and the resulting solid removed by
filtration. The product was purified by column chromatogra-
phy (SiO2, acetone - 2% Et3N–acetone) to afford a white
H25C12hN18Ni(CH2)11COhN18NiCO(CH2)11hN18Ni
1
C12H25, 2
solid (0.63 g, 59%, mp 45–46 1C). H-NMR: 1.22 (28H, s),
1.42 (4H, t), 1.58 (4H, t), 2.27 (4H, t), 2.49 (4H, t), 2.78–2.80
(8H, m), 3.58–3.63 (16H, m). 13C-NMR: 24.81, 27.05, 27.30,
29.08, 29.11, 29.32, 29.40, 33.91, 51.34, 53.89, 55.90, 69.99,
70.60, 174.41.
N-Dodecyl-4,13-diaza-18-crown-6, C12hN18NiH. A solution
of 4,13-diaza-18-crown-6 (1.0 g, 3.81 mmol), 1-bromodode-
cane (0.85 g, 3.43 mmol), Na2CO3 (4.08 g, 0.038 mol), and KI
(cat.) in n-PrCN was heated to reflux for 1.5 h. The reaction
was cooled, filtered and concentrated. The product was pur-
ified (SiO2, 2% Et3N–acetone - 10% Et3N–acetone) to
afford 500 mg (32%) of a yellow solid, mp 34 1C (lit.,17
HOOC(CH2)11hN18Ni(CH2)11COOH. The diazacrown di-
ester (0.63 g, 0.91 mmol) was dissolved in 2 M NaOH (20 mL)
and heated to reflux for 24 h. The pH was adjusted to B4 with
2 M HC1. The organic layer was extracted with MeCN (3 ꢂ 50
mL), dried over MgSO4 and concentrated to afford 0.56 g
(93%) of a white solid, mp 242–244 1C. 1H-NMR (D2O): 1.05–
1.10 (28H, bs), 1.34 (4H, m), 1.49 (4H, m), 2.12 (4H, t), 3.00
(4H, t), 3.24 (8H, t), 3.50 (8H, s), 3.62 (8H, s). 13C-NMR
(CD3OD): 24.4, 26.1, 27.6, 30.2, 30.4, 35.1, 54.2, 55.4, 65.8,
71.4, 174.4.
1
33.5 1C). H-NMR: 0.84 (3H, t), 1.22 (18H, s), 1.50 (2H, bt),
2.49 (2H, t), 2.72–2.81 (16H, m). 13C-NMR: 14.10, 22.78,
27.41, 28.43, 29.70, 31.92, 50.01, 55.87, 69.07, 70.28, 71.25.
Br(CH2)11COhN18NiCO(CH2)11Br. Oxaloyl chloride (2.72
g, 21.5 mmol) was added (0 1C, N2) to a solution of 12-
bromododecanoic acid (1.00 g, 3.58 mmol) in anhydrous
CH2C12 (20 mL). The reaction was stirred at ambient tem-
perature for 2 h. The reaction was concentrated and dried
under high vacuum for 2 h. The residue was dissolved in
CH2C12 and added (0 1C, N2) to a solution of 4,13-diaza-18-
crown-618 (0.57 g, 1.63 mmol), Et3N (0.45 g, 6.52 mmol), and
4-dimethylaminopyridine (cat.) in anhydrous CH2C12 (30
mL). The reaction was stirred (rt) for 20 h. The reaction
mixture was concentrated, dissolved in CHC13, washed with
aq. citric acid (2 ꢂ 25 mL), sat. Na2CO3 (2 ꢂ 25 mL), and
brine solution (2 ꢂ 25 mL). The filtrate was dried over MgSO4
and concentrated to afford a slightly yellow solid (1.10 g, 77%,
C12H25hN18NiCO(CH2)11hN18i(CH2)11COhN18NiC12H25,
3. DMF (2 drops) and oxaloyl chloride (213 mg, 1.68 mmol)
were added (N2, 0 1C, dropwise) into a solution of the
diazacrown diacid (180 mg, 0.27 mmol) in CH2C12 (20 mL).
The reaction was stirred at rt for 2 h. The yellow solution was
concentrated under vacuum and dried under high vacuum for
1 h. The residue was added (N2, 0 1C, dropwise) to a solution
of N-dodecyl-4,13-diaza-18-crown-6 (260 mg, 0.6 mmol), Et3N
(165.8 mg, 2.4 mmol), and 4-dimethylaminopyridine (20 mg,
cat.) in anhydrous CH2C12 (20 mL). The reaction was stirred
at rt for 2 d. The solvent was removed and the residue washed
with 5% Na2CO3 (2 ꢂ 25 mL), followed by brine solution (2 ꢂ
25 mL). The organic phase was dried over MgSO4, filtered and
concentrated. The product was purified over SiO2 (2% Et3N–
acetone) to afford the product as a yellow oil (40 mg). 1H-
NMR: 0.87 (6H, t), 1.25 (64H, bs), 1.43 (8H, m), 1.61 (4H, m),
2.31 (4H, t), 2.48 (8H, t), 2.78 (16H, m), 3.59–3.64 (56H, m).
13C-NMR: 14.10, 22.66, 25.32, 27.20, 27.49, 29.32, 29.51,
29.61, 31.89, 33.18, 46.84, 46.98, 48.81, 53.87, 56.02, 69.56,
69.99, 70.43, 70.53, 70.72,173.34 ppm. Anal. calc. for
C84H166N6O14 ꢃ 2Na: C, 65.93; H, 10.93; N, 5.49%. Found:
C, 66.00; H, 11.04; N, 5.49%. Complexation of 2Na1 ions was
confirmed by high-resolution mass spectroscopy.
1
mp 77–78 1C) that was used without further purification. H-
NMR: 1.27–1.43 (32H, pseudo-s), 1.61 (4H, m), 1.84 (4H, m),
2.31 (4H, m), 3.40 (4H, t), 3.57–3.70 (16H, m).
H25C12hN18Ni(CH2)11COhN18NiCO(CH2)11hN18NiC12-
H25, 2. A solution of Br(CH2)11COhN18NiCO(CH2)11Br
(0.76 g, 0.87 mmol), C12H25hN18NiH (0.75 g,1.74 mmol),
Na2CO3 (3.69 g, 34.8 mmol), and KI (20 mg) in n-PrCN (40
mL) was heated at reflux (N2) for 4 days. The reaction mixture
was cooled, filtered, and concentrated. The resulting residue
was dissolved in CHC13 (50 mL), washed with aqueous 5%
NaHCO3(2 ꢂ 25 mL), brine (2 ꢂ 25 mL), and dried over
MgSO4. The filtrate was concentrated to obtain a brown oil.
The product was purified by column chromatography (SiO2,
acetone - 2% Et3N–acetone) and crystallized from acetone
at 4 1C to afford 100 mg of white crystals (mp 51–52 1C). 1H-
NMR: 0.88 (6H, t), 1.25 (68H, s), 1.46 (8H, t), 1.66 (4H, m),
2.31 (4H, t), 2.50 (8H, t), 2.80 (16H, m), 3.58–3.64 (56H, m).
13C-NMR: 14.10, 22.67, 22.95, 23.71, 25.33, 27.27, 28.88,
29.48, 29.52, 29.60, 30.33, 31.89, 33.15, 38.69, 46.81, 48.71,
53.44, 68.12, 69.73, 70.15, 70.50, 70.82, 173.31. Anal. calc. for
C84H166N6O14: C, 67.97; H, 11.27; N, 5.66%. Found: C, 67.77;
H, 11.36; N, 5.70%.
C11H23COhN18Ni(CH2)12hN18Ni(CH2)12hN18NiCOC11H23, 4
Dodecanoic acid (0.2 g, 1.03 mmol) was dissolved by the slow
addition of SOC12 (4 mL, 6.5 mmol) at 0 1C. The reaction
was heated to reflux (N2) for 1 h. The SOC12 was removed
in vacuo and the residue washed with toluene (2 ꢂ 5 mL).
The resulting acid chloride was dissolved in toluene (5 mL)
and added dropwise (0 1C, N2) to a toluene solution of
HhN18Ni(CH2)12hN18Ni(CH2)12hN18NiH17 (0.5 g, 0.45
mmol), dry Et3N (100 mL) and 4-dimethylaminopyridine
(cat.). The reaction was stirred at rt for 2 d. It was filtered,
concentrated in vacuo, and washed with sat. NaHCO3 (2 ꢂ 25
mL) and brine (2 ꢂ 25 mL). The product was purified by
column chromatography (SiO2, 2% Et3N in acetone) to afford
C12H25hN18NiCO(CH2)11hN18i(CH2)11COhN18NiC12H25, 3
CH3OCO(CH2)11hN18SNi(CH2)11COOCH3. A solution of
4,13-diaza-18-crown-618 (0.406 g, 1.55 mmol), Br(CH2)11
COOCH3 (1.00 g, 3.41 mmol), Na2CO3 (3.62 g, 34.1 mmol),
and KI (20 mg) in 40 mL n-PrCN was heated to reflux for 2
1
4 (520 mg, 79%) as a yellow solid, mp 48–49 1C. H-NMR:
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 177–184 | 183

View Article Online
0.87 (6H, t, J = 6.9 Hz), 1.24 (64H, pseudo-s,), 1.42 (8H, m),
1.61 (4H, m), 2.28–2.33 (4H, m), 2.44–2.49 (8H, m), 2.75–2.80
(16H, m), 3.58–3.66 (56H, m). 13C-NMR: 14.07, 22.63, 25.33,
27.18, 27.47, 29.29, 29.46, 29.58, 31.86, 33.12, 46.89, 48.77,
53.87, 56.01, 69.62, 69.96, 70.08, 70.25, 70.32, 70.47, 70.58,
70.70, 70.96, 173.34. Anal. calculated for C84H166N6O14: C,
67.97; H, 11.27; N, 5.66%. Found: C, 68.02; H, 11.36; N,
5.60%.
Pajewska, R. Pajewski and M. E. Weber, Bioorg. Med. Chem.,
2004, 12(6), 1291–304.
11 (a) G. W. Gokel, Chem. Commun., 2000, 1–9; (b) G. W. Gokel, Cell
Biochem. Biophys., 2001, 35(3), 211–31.
12 O. Murillo, I. Suzuki, E. Abel, C. L. Murray, E. S. Meadows, T.
Jin and G. W. Gokel, J. Am. Chem. Soc., 1997, 119, 5540–5549.
13 M. E. Weber, P. H. Schlesinger and G. W. Gokel, J. Am. Chem.
Soc., 2005, 127, 636–642.
14 W. M. Leevy, J. E. Huettner, R. Pajewski, P. H. Schlesinger and
G. W. Gokel, J. Am. Chem. Soc., 2004, 126, 15747–15753.
15 C. L. Murray, H. Shabany and G. W. Gokel, Chem. Commun.,
2000, 2371–2372.
16 A. Nakano, Q. Xie, J. V. Mallen, L. Echegoyen and G. W. Gokel,
J. Am. Chem. Soc., 1990, 112, 1287–9.
Acknowledgements
17 O. Murillo, S. Watanabe, A. Nakano and G. W. Gokel, J. Am.
Chem. Soc., 1995, 117, 7665–7679.
18 V. J. Gatto, S. R. Miller and G. W. Gokel, Org. Synth., 1989, 68,
227–233.
19 A. E. Meyer, W. M. Leevy, R. Pajewski, I. Suzuki, M. E. Weber
and G. W. Gokel, Bioorg. Med. Chem., 2005, 13, 3321–3327.
20 (a) H. Shabany and G. W. Gokel, Chem. Commun., 2000, 2373–
2374; (b) H. Shabany, R. Pajewski, E. Abel, A. Mukhopadhyay
and G. W. Gokel, Heterocycl. Chem., 2001, 38, 1393–1400.
21 F. G. Riddell and M. K. Hayer, Biochem. Biophys. Acta, 1985, 817,
313–317.
We thank the NIH for a grant (GM-36262) that supported this
work and for Training Grant Fellowships under grants T32-
GM08785 and T32-GM04892 to MEW and WML, respec-
tively.
References
1 Y. Inoue and G. W. Gokel, in Cation Binding by Macrocycles, ed.
Marcel Dekker, New York, 1990, 761 pp.
2 G. W. Gokel, Crown Ethers and Cryptands, The Royal Society of
Chemistry, London, England, 1991, vol. 3, p. 190.
3 G. W. Gokel and S. H. Korzeniowski, Macrocyclic Polyether
Syntheses, Springer-Verlag, Berlin, 1982, 410 pp.
22 J. S. Bradshaw, K. E. Krakowiak and R. M. Izatt, Aza-Crown
Compounds, Wiley, New York, 1993, vol. 51, p. 885.
23 R. M. Izatt, K. Pawlak and J. S. Bradshaw, Chem. Rev., 1995, 95,
2529–2586.
4 H.-J. Choi, M.-O. Kwak and J.-M. Kim, Tetrahedron Lett., 1997,
38, 6217–6220.
5 S. Kumar, M. S. Hundal, N. Kaur, R. Singh and H. Singh, Org.
Chem., 1996, 61, 7819–7825.
6 S. Kumar, R. Singh and H. Singh, J. Chem. Soc., Perkin Trans. 1,
1992, 3049–3053.
24 K. A. Arnold, J. C. Hernandez, C. Li, J. V. Mallen, A. Nakano,
O. F. Schall, J. E. Trafton, M. Tsesarskaja, B. D. White and
G. W. Gokel, Supramol. Chem., 1995, 5, 45–60.
25 D. A. Doyle, J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis,
S. L. Cohen, B. T. Chait and R. MacKinnon, Science, 1998, 280,
69–77.
7 R. Kataky, D. Parker, A. Teasdale, J. P. Hutchinson and H.-J.
Buschmann, J. Chem. Soc., Perkin Trans. 1, 1992, 1347–1351.
8 J. C. Hernandez, J. E. Trafton and G. W. Gokel, Tetrahedron
Lett., 1991, 6269–6272.
9 (a) B. D. White, K. A. Arnold and G. W. Gokel, Tetrahedron Lett.,
1987, 1749–1752; (b) B. D. White, F. R. Fronczek, R. D. Gandour
and G. W. Gokel, Tetrahedron Lett., 1987, 1753–1756.
10 (a) G. W. Gokel and O. Murillo, Acc. Chem. Res., 1996, 29, 425–
432; (b) G. W. Gokel and A. Mukhopadhyay, Chem. Soc. Rev.,
2001, 30, 274–286; (c) G. W. Gokel, P. H. Schlesinger, N. K.
Djedovic, R. Ferdani, E. C. Harder, J. Hu, W. M. Leevy, J.
26 G. W. Gokel and B. J. Garcia, Tetrahedron Lett., 1977, 317–20.
27 W. M. Leevy, M. E. Weber, P. H. Schlesinger and G. W. Gokel,
Chem. Commun., 2005, 89–91.
28 D. A. Reaveley and R. E. Burge, Adv. Microb. Phys., 1972, 1–81.
29 F. J. Szoka and D. Papahadjopoulos, Proc. Natl. Acad. Sci. USA,
1978, 75, 4194–4198.
30 J. C. M. Stewart, Anal. Biochem., 1980, 104, 10–14.
31 National Committee for Clinical Laboratory Standards, Methods
for Dilution Antimicrobial Susceptibility Tests for Bacteria that
grow Aerobically, NCCLS, Wayne, Pennsylvania, 2000, 5th edn,
M7-A5.
ꢀc
This journal is the Royal Society of Chemistry the Centre National de la Recherche Scientifique 2006
184 | New J. Chem., 2006, 30, 177–184