Structure-activity relationships, kinetics, selectivity, and mechanistic studies of synthetic hydraphile channels in bacterial and mammalian cells

Article abstract of DOI:10.1039/b508157b

Hydraphile compounds are shown to be cytotoxic to Gram-negative and Gram-positive bacteria, yeast, and mammalian cells. Their cellular toxicity compares favorably with other synthetic ionophores and rivals that potency of natural antibiotics. The effects

Full text of DOI:10.1039/b508157b

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A R T I C L E
Structure–activity relationships, kinetics, selectivity, and mechanistic
studies of synthetic hydraphile channels in bacterial and
mammalian cells
W. Matthew Leevy,a Seth T. Gammon,b Tatiana Levchenko,e David D. Daranciang,a
Oscar Murillo,a Vladimir Torchilin,e David Piwnica-Worms,a,b James E. Huettnerc and
George W. Gokel*a,d
a Department of Molecular Biology & Pharmacology, Washington University School of
Medicine, Campus Box 8103, 660 S. Euclid Avenue, St. Louis, MO, 63110, USA.
E-mail: ggokel@wustl.edu; Tel: +1 314 362 9297
b Departments of Radiology, Washington University School of Medicine, Campus Box 8103,
660 S. Euclid Avenue, St. Louis, MO, 63110, USA
c Departments of Cell Biology and Physiology, Washington University School of Medicine,
Campus Box 8103, 660 S. Euclid Avenue, St. Louis, MO, 63110, USA
d Department of Chemistry, Washington University, 1 Brookings Drive, St. Louis, MO, 63130,
USA
e Department of Pharmaceutical Sciences, Bouve College of Health Sciences, Northeastern
University, Boston, MA, 02115, USA
Received 9th June 2005, Accepted 11th August 2005
First published as an Advance Article on the web 26th August 2005
Hydraphile compounds are shown to be cytotoxic to Gram-negative and Gram-positive bacteria, yeast, and
mammalian cells. Their cellular toxicity compares favorably with other synthetic ionophores and rivals that potency
of natural antibiotics. The effects of structural variations on toxicity are described. The effects of these variations
correlate well with previous studies of ion transport in liposomes. Whole cell patch clamping with mammalian cells
confirms a channel mechanism in living cells suggesting that this family may comprise novel and flexible
pharmacological agents.
ural systems.3 Early contributions to the field came from the
Introduction
groups of Tabushi,4 Lehn,5 Fyles,6 and our own laboratory.7
Biological membranes are complex but ubiquitous structures
More recent contributions include the peptide nanotubes
that comprise the boundary layer of cells and organelles.
of Ghadiri,8 the peptide-linked crown ethers of Voyer,9 our
Natural bilayers are formed by contact between the non-polar
chloride-selective heptapeptide channels,10 and others.11 Many
hydrocarbon chains of two leaflets. The polar headgroups of
of the synthetic ion channel compounds have demonstrated
phospholipids contact the aqueous cytosol or periplasm. In
classic open/closed channel behavior in planar bilayer voltage
vital systems, the bilayer is usually asymmetric and contains nu-
clamp experiments3,12,13 and have shown activity in synthetic
merous other species, including proteins, sterols, sphingolipids,
liposomes.14–16 The peptide back-boned crown system9 is thought
and other lipids. Bilayer membranes provide the fundamental
protective barrier between the cell and the outside environment,
to form an a-helix in the membrane, which then aligns the
macrocycles to form tubes through the bilayer. The cyclic D,L-
but they are also involved in cell respiration, nerve conduction,
amino acid nanotubes8 stack to form an extended, hydrogen-
and many other physiological activities. Despite decades of in-
bonded tunnel that permits the passage of ions or sugars
tense research, uncertainty remains over many of their features,
across the membrane. The hydraphiles mimic the structure of
including the exact head group orientation and the extent of
known protein channels by having an entry and exit portal
connected to a centrally hydrated relay. Fig. 1 shows a schematic
hydrocarbon interdigitation between leaflets.1
Membrane-spanning proteins have garnered attention for
representation of these three ion conducting compounds or
their roles in regulating numerous cellular processes, including
assemblies as they are thought to function in the bilayer.
ion balance, cell signaling, and the uptake of organic substrates.
Of particular interest have been protein channels that mediate
the passage of ions through membranes. Channel proteins play
the remarkable role of moving charged species through the
˚
approximate 30 A thickness of membrane hydrocarbons. The
“hydrocarbon slab” or “insulator regime” is thought to have a
dielectric constant of 2–3. Nature’s rigorous maintenance
of cellular salt concentrations demonstrates the importance
of ion transport, which must occur through the insulating
bilayer. The protein ion channels that mediate ion conductance
have been studied for decades, but structural details have
only recently become available.2 Moreover, mechanistic details
remain obscure in most cases.
Because channel proteins are so complex, chemists as early
as the 1980s responded to the challenge of developing synthetic
ionophores that are working models of these remarkable nat-
Fig. 1 Presumed channel conformations for (left) peptide nanotubes,
(center) hydraphiles, and (right) multi-crown peptide.
3 5 4 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 4 4 – 3 5 5 0
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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Natural ionophores are known to exhibit toxic effects in cel-
lular systems. Examples include valinomycin,17 the cecropins,18
gramicidin,19 and melittin.20 The synthetic ionophores described
above are also biologically active. Specifically, the peptide-linked
crown ethers cause red blood cell haemolysis but are inactive
against E. coli,21 while the cyclic peptide nanotubes and the
synthetic hydraphiles act as potent antimicrobial agents.22,23 The
cyclic peptide nanotubes are quite active, killing bacteria at
concentrations as low as 2.5 lM.24 The hydraphiles, studies
of which are reported here, are also cytotoxic to bacteria at
concentrations as low as 0.6 lM. Other synthetic channels25
presumably hold this potential, although, to our knowledge, it
is currently unrealized.
The toxicity of synthetic channels is presumably the result
of rapid, unregulated ion flux through the plasma membrane
that disrupts cellular ion gradients causing osmotic stress and,
ultimately, cell death. Two mechanisms have been proposed to
describe this process.26 Previous studies using membrane poten-
tial sensitive dyes showed that hydraphiles act as ionophores in
the E. coli membranes.23 The hydraphiles were designed to be
modular and are available in a range of lengths and polarities. We
have assayed the biological activity of 13 synthetic ionophores
and report here the observed structure–activity relationships. In
addition, evidence for the mechanism of action is presented for
the hydraphile family of compounds.
Compounds 1–11 and 13 contain three macrocycles linked
covalently. The distal macrocycles of 2–11 are 4,13-diaza-18-
crown-6, but in 1 the corresponding macrocycle is aza-18-crown-
6. The sidearms in structures 4–10 are benzyl and n-dodecyl in
11. Compounds 4–10 differ in spacer chain length. The shortest
chain is octylene (4) and the longest is eicosylene (10). The spacer
chains in tris(macrocycle)s 1, 2, 6, 11 and 13 are all dodecylene.
Compound 12 differs from the other structures in this study
because a fourth macrocycle is present and linked covalently to
the distal macrocycles. This symmetrical structure is thought to
form a “tunnel” within the bilayer and is the most active sodium
ion transporter reported in this study. Finally, compound 13 is
identical to 6 except that para-fluoro substituents are present on
the benzyl groups.
Structural and fluorescence studies indicate that the hy-
draphiles arrange in the bilayer with the distal macrocycles
at opposite ends of the membrane’s insulator regime while
the hydrocarbon chains (e.g. the dodecyl groups of 11) align
with the fatty acid chains.29 Compound 1 does not possess a
side chain; instead the distal macrocycles are aza-18-crown-
Ą
6 residues. In this compound, the hydraphile’s typical ĚN–R
side chain is replaced by a single oxygen atom. Compound 1
showed no detectable sodium transport activity in 23Na-NMR
experiments conducted in liposomes.14 In these same studies,
compounds 2, 6, and 11 had relative rates of 28, 39, and 28
(compared to the transport activity of gramicidin (arbitrarily set
to 100) determined simultaneously). Compound 12, the dodecyl
chain “tunnel” structure, shows a relative rate for sodium ion
transport of approximately 81 compared to the gramicidin
Results and discussion
Ą
standard.30 These sodium transport results indicate that an ĚN–
Compounds studied
H (not an ether link) is the minimum sidearm commensurate
with transport activity. Other side chains and spacer chain
lengths alter transport activity. These results are mirrored in
the biological results described below.
Hydraphiles are typically comprised of three diaza-18-crown-
6 units separated by two 12-carbon spacers. The two distal
crowns act as head groups and as portals for ion entry and
egress. The central macrocycle is an ion relay27 that serves the
same purpose as the “water and ion-filled capsule” recently
identified in the solid state structure of the KcsA channel of
Streptomyces lividans.28 The hydraphile compounds used in this
study are shown as 1–13.
Biological activity
Hydraphiles are cytotoxic to the Gram-negative bacterium E.
coli.23 Toxicity studies (IC50 curves) showed benzyl channel (6)
to be active in the 10 lM range, while the shorter C8 benzyl
channel (4) was 13-fold less active. Control compounds N,Nꢀ-
didodecyl-4,13-diaza-18-crown-6 and N-benzyl-4,13-diaza-18-
crown-6 were both inactive to E. coli when assessed using the
disk diffusion method. In addition, a compound in which the
macrocyclic central relay was replaced by a biphenyl group and
known27 not to transport ions in vitro, was also inactive. These
controls suggest that toxicity is caused by channel formation
in the membrane rather than a non-specific detergent interac-
tion. The fluorescent dansyl channel was used in microscopy
experiments to show that hydraphile compounds localized to
the membrane of E. coli.
Side arm effects
Compounds 1–12 were prepared and assayed for toxicity to
bacteria. The toxicity results for 1–12 and their activity in
synthetic liposomes were comparable. The minimum bactericidal
concentration (MBC) and minimum f ungicidal concentration
(MFC) for each hydraphile are reported in Tables 1 and 2 as
the lowest serial 2-fold dilution that killed 99.9% of a cultured
bacterium or yeast, as described by the National Committee
for Clinical Laboratory Standards (NCCLS).31,32 Compound 1
has a MBC of 87 lM to B. subtilis, and 175 lM to E. coli
and S. cerevisiae. We consider this to be largely inactive, a
result in accord with the previously noted sodium ion transport
data.
Replacing the macroring oxygen of 1 by NH results in 2,
which shows a significant increase in toxicity to both E. coli and
B. subtilis (MBC = 22 lM and 11 lM), while the yeast remains
resistant. “Benzyl channel”, 6, is formed when a benzyl group
replaces the hydrogen of the N–H group of 2. The activity of
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 4 4 – 3 5 5 0
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Table 1 Comparison of experimentally determined toxicity values
(in lM) in organisms with Na+ transport rate in liposomes
phospholipids used to prepare different vesicles were identical,
including a single cis-unsaturation. The data obtained for 4–10
confirmed the trend previously observed by NMR for 4–8. In
addition, hydraphile length was found to generally correlate with
phospholipid fatty acid chain length, i.e., membrane thickness.15
This critical correspondence between hydraphile length and
membrane thickness was explored in the biological context using
E. coli, B. subtilis, and S. cerevisiae.
Compound
1
2
6
11
4.2
0.53
8.4
12
E. colia
175
87
22
11
9.4
1.2
2.0
0.5
64
81
B. subtilisa
S. cerevisiaeb
175 170 38
<2 28 39
The benzyl channel compounds (4–10) that were studied
had spacer chains of 8, 10, 12, 14, 16, 18, and 20 methylenes.
Table 2 records the response of microorganisms to the presence
of these compounds. The C8 benzyl channel (4) is the least active
compound to the bacteria. This result mirrors those of previous
studies conducted in phospholipid vesicles. A modest increase
in toxicity to both E. coli and B. subtilis is observed for 5 (C10
Na+ transport in liposomesc
28
a Minimum bactericidal concentration. b Minimum fungicidal concen-
tration. c Relative to gramicidin (=100)
6 increases compared to 2 by about 2-fold for E. coli (MBC =
9.4 lM) and 10-fold for B. subtilis (MBC = 1.2 lM). A moderate
increase in activity is also observed for S. cerevisiae (MFC = 39).
The toxicity of these compounds rates well as antibiotics are
generally considered effective at MBCs of 10 lM and below.33
When the distal macrocycles of the hydraphile framework are
terminated by 12-carbon side chains (11), activity increases
again by about two-fold to 2.1 lM for E. coli and 0.53 lM
for B. subtilis. The yeast sustain an approximate 5-fold increase
in activity to 8.4 lM.
Linking the dodecyl side chains through an additional macro-
cycle (12) leads to another two-fold enhancement in activity to
∼2 lM for E. coli, while B. subtilis remains at the 500 nM
level of inhibition. We note that 12 was the most active sodium
transporter studied in liposomes.30 Yeast are resistant to the
action of 12 but they are susceptible to 11 (8.4 lM). Some
of the difference in activity may result from differences in the
environments in which the organisms were grown. S. cerevisiae
are grown in YPD media that has pH = 6.5, compared to LB
media (bacteria) that has pH = 7. Previous studies showed that
higher acidity reduced the toxicity of 2, 6, and 12 to bacteria.
Surprisingly, 11 was found to be more toxic at lower pH. The
yeast are grown at a lower pH and, as noted for bacteria, 11 is
the most active compound, while 2, 6, and 12, are less cytotoxic.
˚
benzyl channel), which is approximately 5 A longer overall than
is 4. Addition of two more methylenes in each spacer chain
gives 6 (C12 benzyl channel) and engenders a sizable increase in
˚
activity. This further 5 A of channel length affords a ∼9-fold
increase in toxicity to E. coli and to B. subtilis. Maximal toxicity
is reached for E. coli in the C14–C16 range and activity declines as
length increases further (9, C18 and 10, C20 hydraphiles). These
results correlate well with Na+ transport activity monitored in
synthetic liposomes.34 Maximal toxicity to B. subtilis, 0.56 lM,
is observed for the C14 and C16 channels (7, 8). This value of
∼0.6 lM compares with a toxic concentration for penicillin of
∼8 lM and is among the most potent examples of ionophore-
mediated toxicity reported, natural or synthetic.
Kinetics of toxicity to E. coli determined by using bioluminescent
bacteria
The potent toxicity of the hydraphiles to both E. coli and B.
subtilis is apparent from the final MBC values. How rapidly
the bacteria are killed is a question that could be addressed
directly by use of bioluminescent bacteria. E. coli bacteria
expressing the lux operon genes of Photorhabdus luminescens
on a pT7-3 plasmid were used for the kinetic study. These
bioluminescent bacteria emit light during log phase growth
and the disappearance of light is a quantitative measure of
cell death.35 The experiments reported here monitored the
loss of photon emission from the luminescent E. coli after
treatment with hydraphile at the concentration determined for
the MBC. The studies were performed in 96-well plates and
photon output was read using a CCD camera (see Experimental
section). Fig. 2 shows the decay of light from the bacteria as a
percentage of the light emitted from untreated control wells.
These data highlight the potency of hydraphiles. When tested
at their MBCs, 6, 7, and 3 killed half of the E. coli population in
8.5, 9.1, and 12.5 minutes. This compares with a halftime of the
known antibiotic kanamycin (MBC = 1.3 lM) of 44.8 minutes
at 10–20 times the concentration of hydraphile. Although less
active than the hydraphiles, kanamycin shows high selectivity
for bacteria over mammalian cells, while the hydraphiles do not
(see below).
Hydraphile channel length effects on transport and toxicity
Membrane thickness depends on numerous factors. These
include, but are not limited to, the extent of hydrocarbon
chain interdigitation, the chain length of the lipid hydrocarbon
chains, fatty acid unsaturation and double bond geometry,
differences in head groups, and the presence or absence of
such membrane thickening agents as cholesterol. In a previous
study,27 we examined Na+ transport mediated by analogs of
6 that are identical except that their spacer chains vary in
length. Initially, we studied sodium equilibration mediated by
4–8 in phospholipid liposomes by using the 23Na NMR method.
Compounds 6 and 7 were found to be about equally active and
more active than shorter or longer analogs.
A more recent study expanded the range from 4–10; these
compounds differ in spacer chain length from 8 to 20 methyl-
ene units. In this work, an ion selective electrode (ISE) method
was used to monitor Na+ efflux in synthetic liposomes.15 The
Table 2 MBCa values of seven benzyl channel compounds having varying hydrocarbon chain lengths
Compound
4
5
6
7
8
9
10
Spacer chain length
E. coli
(CH2)8
170
42
170
∼2
(CH2)10
80
10
160
23
(CH2)12
9.4
1.2
38
84
(CH2)14
2.3
0.56
4.6
(CH2)16
4.6
0.6
2.3
95
(CH2)18
(CH2)20
160
75
1
—
B. subtilis
2
—
b
b
S. cerevisiae
Na+ releasec
100
39
14
a Minimum bactericidal concentration (in lM). b Not determined. c Percent release of Na+ from liposomes determined by using an ISE technique.15
3 5 4 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 4 4 – 3 5 5 0

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Fig. 2 Kinetics of killing bioluminescent E. coli with 3, 6, and 7 at the
indicated concentrations. Also shown are data for kanamycin.
Fig. 4 Cytotoxicity of hydraphiles to CaCo2 cells. [Hydraphile] is
micromolar.
Hydraphile aggregation
more resistant to 6 than are HEK cells. A possible explanation
is that the apoptosis machinery normally present in non-cancer
cells such as HEK 293 is suppressed.
Some amphiphilic molecules are known to exhibit toxicity
through membrane disruption effects caused by the formation
of micelles. A study of hydraphile aggregation in buffer was
conducted to determine their critical micelle concentrations
(CMCs). Compound 6 was tested at concentrations as high
as 38 lM in buffer (0.5 mM HEPES, 67 mM KCl), and no
aggregation was detected by laser light scattering analysis (see
Experimental section). The benzyl channel derivatives typically
exhibit cytotoxicity to bacteria in the 0.5–10 lM range. Since this
range is significantly below 38 lM, we conclude that aggregation
of hydraphiles in solution does not influence their toxicity.
The use of electrophysiology to probe mechanism
The whole cell patch clamping technique is widely used and
accepted by biologists to study ion channel behavior. We have
used this method to show that indeed hydraphiles are acting as
ionophores in the bilayers of living organisms, and generate
levels of current on a par with native protein ion channels
present in the bilayer. Whole cell patch clamping experiments
were undertaken to evaluate the membrane channel activity of
hydraphiles in a biological setting and to better understand their
behavior in the bilayer of a living cell.37 HEK 293 cells were held
in whole cell mode at −20 mV (with respect to the outside of
the cell) giving the baseline current represented as the solid line
shown in the left panel of Fig. 5. The points during each phase
of the experiment represent 10 ms voltage steps from −100 to
+60 mM, and appear as a continuous line during saline rinse.
The left panel of Fig. 5 shows the membrane current of HEK
293 cells during rinse with saline solution, followed by a brief
application of p-fluorobenzyl hydraphile 13. This compound is
identical to 6 (illustrated) except that a fluorine atom is present
in the para- or 4-position of the benzyl group. After application
of 13, the cell is rinsed with saline solution. Compound 13 was
tested because it had previously shown long open times during
planar bilayer conductance experiments.38
Mammalian cell cytotoxicity
Toxicity studies were performed on Human Embryonic Kidney
(HEK 293) and colonic carcinoma (CaCo2) cells to ascertain
the inherent selectivity of hydraphiles between mammalian
and bacterial cells, and also to assess anti-tumor efficacy.
HEK 293 cells were incubated with 6 and 7 (C12 and C14
benzyl channels) for 24 h and assayed for their ability to re-
duce 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium salt (MTS assay).36 The re-
sults using HEK 293 cells are shown graphically in Fig. 3.
Hydraphiles 3, 5, and 6 have LD50 values in the range of
2 lM. The effectiveness of hydraphile channel compounds across
all types of cells is apparent in these data, as both bacteria and
mammalian cell types are killed in the same concentration range.
The right panel of Fig. 5 shows the current–voltage (I–V) plot
of the cell during its first rinse with saline solution (open circles),
during treatment with 13 (black circles), and during extended
rinsing (grey circles). Membrane conductance increases from
about 1 nS to 7 nS upon treatment with 4-fluorobenzyl
compound 13. The cell exhibited a quick return to homeostasis
after hydraphile application was stopped. Even after prolonged
rinsing, a residual conductance of 1.8 nS persisted, possibly
indicating stable inclusion of the compound in the cellular
bilayer. On average, application of the compound caused a 3.7
1.2 fold (n = 4) increase in membrane conductance. Based on
these results, the use of hydraphiles at subtoxic concentrations
may hold utility in the recently posited pharmaceutical approach
known as channel replacement therapy.39
The HEK 293 cells are bathed in Tyrode’s solution, which
contains 150 mM NaCl as the highest concentration salt. The
cells are clamped at an inside potential of −20 mV with respect
to the outside. Thus, positively charged Na+ ions pass into the
cell through a hydraphile, while negatively charged particles
depart. The concentrations of Na+ and Cl− are low within
the cell but the K+ concentration is high. Glucuronate, a large
organic anion, is also present. The latter presumably cannot
pass through the hydraphile channel. We therefore reason that
Na+ is the primary ion driving the sizable currents noted at
Fig. 3 Toxicity of hydraphiles 3, 6, and 7 to HEK 293 cells.
This MTS assay was further used to assess the antitumor
activity of compounds 4, 6, and 11 against CaCo2 cells.
Compounds 6 and 11 showed LD50 values of ∼12 lM while the
C8 benzyl hydraphile, 4, was inactive at concentrations as high
as 20 lM (Fig. 4). These CaCo2 cancer cells are about 6-fold
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 4 4 – 3 5 5 0
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Fig. 5 Cellular electrical response during a brief application of 13 to an HEK 293 cell (black bar) under whole cell patch clamp conditions.
negative holding potentials. When the cell is clamped at positive
potentials, we observe sizable currents equal to those just noted.
These currents may be carried by K+ leaving the cell, and/or by
Cl− entering the cell.
7,16-Bis-[12-(16-benzyl-1,4,10,13-tetraoxa-7,16-diazacycloocta-
dec-7-yl)-dodecyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane,
3, HꢀN18Nꢁ(CH2)16ꢀN18Nꢁ(CH2)16ꢀN18NꢁH. A solution of
PhꢁN18Nꢂ(CH2)16ꢁN18Nꢂ(CH2)16ꢁN18NꢂPh (0.3 g, 0.212 mmol)
and Pd/C catalyst (5 wt% on activated carbon, 100 mg) in
30 mL EtOH was shaken in a Parr series 3900 hydrogenation
apparatus at 60 psi H2 for 24 h. The reaction was filtered over
a pad of Celite, and concentrated in vacuo. Crystallization from
15 mL EtOAc gave HꢁN18Nꢂ(CH2)16ꢁN18Nꢂ(CH2)16ꢁN18NꢂH
(0.15 g, 57%) as a light yellow solid (mp 47–49 ◦C). 1H-
NMR: 1.1–1.35 (48H, s, NCH2CH2(CH2)12CH2CH2N),
1.35–1.5 (8H, s, NCH2CH2(CH2)12CH2CH2N), 2.3–2.5 (8H, m,
NCH2(CH2)14CH2N), 2.65–2.85 (24H, m, OCH2CH2N), 3.48–
3.7 (48H, overlapping signals due to CH2CH2OCH2CH2N).
13C-NMR: 27.38, 27.75, 29.89, 29.91, 49.42, 53.54, 54.16, 56.27,
69.81, 70.20, 70.48, 70.95. Elem. Anal. Calcd for C68H138N6O12:
C, 66.30;H, 11.29;N, 6.82%. Found:C, 66.45;H 11.64;N, 6.49%.
Conclusion
The present study demonstrates the potent cytotoxicity of
hydraphile compounds to Gram-negative and Gram-positive
bacteria, yeast, and mammalian cells. These compounds com-
pare favorably with other synthetic ionophores in toxicity,
and rival those developed by nature over millions of years.
Flexible synthetic access to these compounds allows structural
components such as length to be optimized to maximize toxicity.
Further, sidearms may be changed completely or adjusted to
vary steric bulk, hydrophobicity, electron richness, etc. The
structural dependence of toxicity corresponds well with our pre-
vious ion transport experiments utilizing liposomes. In addition,
whole cell patch clamping with mammalian cells confirms a
channel mechanism in these living systems. The hydraphile class
of compounds provides a functional model to study channel
mediated ion transport in a biological setting, and furthermore
offers a strong architecture for the pursuit of novel and flexible
pharmacological agents.
7,16-Bis-[12-(16-benzyl-1,4,10,13-tetraoxa-7,16-diazacycloocta-
dec-7-yl)-octyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane,
4. Compound 4 was prepared as previously reported.27
7,16-Bis-[12-(16-benzyl-1,4,10,13-tetraoxa-7,16-diazacycloocta-
dec-7-yl)-decyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane,
5. Compound 5 was prepared as previously reported.27
7,16-Bis-[12-(16-benzyl-1,4,10,13-tetraoxa-7,16-diazacyclo-
octadec-7-yl)-dodecyl]-1,4,10,13-tetraoxa-7,16-diazacycloocta-
decane, 6. Compound 6 was prepared as previously reported.14
Experimental
General
1H-NMR were recorded at 300 MHz in CDCl3 solvents and
are reported in ppm (d) downfield from internal (CH3)4Si. 13C-
NMR were recorded at 75 MHz in CDCl3 unless otherwise
stated. Infrared spectra were recorded on a Perkin-Elmer
1710 Fourier transform infrared spectrophotometer and were
calibrated against the 1601 cm−1 band of polystyrene. Melting
points were determined on a Thomas Hoover apparatus in open
capillaries and are uncorrected. 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. All reactions were
conducted under dry N2 unless otherwise stated. All reagents
were the best (non-LC) grade commercially available and were
distilled, recrystallized, or used without further purification, as
appropriate. Combustion analyses were performed by Atlantic
Microlab, Inc., Atlanta, GA, and are reported as percents.
7,16-Bis-[12-(16-benzyl-1,4,10,13-tetraoxa-7,16-diazacyclo-
octadec-7-yl)-tetradecyl]-1,4,10,13-tetraoxa-7,16-diazacycloocta-
decane, 7. Compound 7 was prepared as previously reported.37
7,16-Bis-[12-(16-benzyl-1,4,10,13-tetraoxa-7,16-diazacyclo-
octadec-7-yl)-hexadecyl]-1,4,10,13-tetraoxa-7,16-diazacycloocta-
decane, 8. Compound 8 was prepared as previously reported.27
7,16-Bis-[12-(16-benzyl-1,4,10,13-tetraoxa-7,16-diazacyclo-
octadec-7-yl)-octadecyl]-1,4,10,13-tetraoxa-7,16-diazacycloocta-
decane, 9. Compound 9 was prepared as previously reported.15
7,16-Bis-[12-(16-benzyl-1,4,10,13-tetraoxa-7,16-diazacyclo-
octadec-7-yl)-eicosyl]-1,4,10,13-tetraoxa-7,16-diazacycloocta-
decane, 10. Compound 10 was prepared as previously
reported.15
7,16-Bis-[12-(16-benzyl-1,4,10,13-tetraoxa-7,16-diazacyclo-
octadec-7-yl)-dodecyl]-1,4,10,13-tetraoxa-7,16-diazacycloocta-
decane, 11. Compound 11 was prepared as previously
reported.27
7,16-Bis-[12-(1,4,7,10,13-pentaoxa-16-aza-cyclooctadec-16-
yl)-dodecyl]-1,4,10,13-tetraoxa-7,16-diaza-cyclooctadecane, 1.
Compound 1 was prepared as previously reported.14
7,16-Bis-[12-(1,4,10,13-tetraoxa-7,16-diazacyclooctadec-7-
yl)-dodecyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane, 2.
Compound 2 was prepared as previously reported.14
Tetrakis{16-[12-(1,4,10,13-tetraoxa-7,16-diaza-cyclooctadec-
7-yl)]-dodecane}, 12. Compound 12 was prepared as previously
reported.37
3 5 4 8
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incubated for 4 h. This assay is based on the bioreduction of
MTS tetrazolium compound (Owen’s reagent) into a colored
soluble formazan product. The viability of cells was measured
using a plate-reader (Multiscan MCC/340, Fisher Scientific) at
490 nm. Relative viability was calculated from reference cells
treated with medium alone as a control.
7,16-Bis-{12-[16-(4-fluorobenzyl)-1,4,10,13-tetraoxa-7,16-
diazacyclooctadec-7-yl]-dodecyl}-1,4,10,13-tetraoxa-7,16-diaza-
cyclooctadecane, 13. Compound 13 was prepared from N-4-
fluorobenzyl-4,13-diaza-18-crown-6 and previously reported
N,Nꢀ-di-(n-12-bromododecyl)-4,13-diaza-18-crown-6.14
N-4-Fluorobenzyl-4,13-diaza-18-crown-6. Diaza-18-crown-6
(3.55 g, 13.5 mmol), 4-fluorobenzyl bromide (2.20 g, 11.6 mmol),
Na2CO3 (28.8 g, 272 mmol), KI (0.32 g, 1.9 mmol), and
CH3CH2CH2CN (135 mL) were heated under reflux (72 h) in
a 250 mL round bottomed flask. The reaction mixture was
evaporated and the residue dissolved in CH2Cl2 (100 mL),
washed with H2O (3 × 50 mL), dried (MgSO4), and evaporated.
Chromatography over Al2O3 (2% 2-PrOH in CH2Cl2) gave the
HEK 293 cytotoxicity assay
HEK 293 cells were regularly cultured in
a 5% CO2
100% humidity tissue culture incubator. Cells were split
regularly into DMEM (Invitrogen) supplemented with 10%
heat inactivated fetal bovine serum (DFBS), 1% Gln, 0.1%
penicillin/streptomycin–0.1% fungizone.
◦
title compound (1.14 g, 41%) as a white solid (mp 83–84 C).
After being freshly split, cells were counted on a hemacytome-
ter and plated at a density of 20 000 cells per well in a 96-well
plate and grown for 24 hours. Ethanol stocks of each compound
were diluted 1 : 100 into DMEM supplemented with 10% DFBS
NMR: 2.8–2.9 (m, 8H); 3.6–3.7 (m, 19H); 6.95–7.05 (pseudo-T,
2H); 7.3–7.4 (pseudo-T, 2H).
7,16-Bis-{12-[16-(4-Fluorobenzyl)-1,4,10,13-tetraoxa-7,16-
diazacyclooctadec-7-yl]-dodecyl}-1,4,10,13-tetraoxa-7,16-diaza-
cyclooctadecane, 13. N-4-Fluorobenzyldiaza-18-crown-6 (0.8 g,
2.2 mmol), N,Nꢀ-di-(n-12-bromododecyl)-4,13-diaza-18-crown-
6 (0.82 g, 1.08 mmol), Na2CO3 (2.3 g, 21.7 mmol), KI (14 mg),
and CH3CH2CH2CN (14 mL) were heated at reflux for 25 h. The
reaction mixture was evaporated to a thick oil (1.98 g). Column
chromatography (Al2O3, 10–15% 2-propanol in hexanes) gave
13 (0.41 g, 28%) as a waxy solid. NMR: 1.2–1.6 (m, 40H),
2.5–2.6 (broad S, 8H), 2.7–3.0 (m, 24H), 3.5–3.7 (broad s, 52H),
7.0, 7.3–7.4 (m, m, 8 H). Calculated for C74H132F2N6O12, High
resolution mass spectrometry, exact mass: 1334.99. Found,
(M + 1)+ m/z 1335.996.
1
and 1% Gln in triplicate. They were then serially diluted
2
into DMEM supplemented with 10% DFBS, 1% Gln, and 1%
ethanol. The original media was then removed from the cells
and replaced with media containing the desired concentration
of compound. As a positive control for growth, three wells
containing cells were treated with DMEM supplemented with
10% DFBS, 1% Gln, and 1% ethanol. As a negative control, three
wells without cells were treated with DMEM supplemented with
10% DFBS, 1% Gln, and 1% ethanol. The remaining empty wells
on the plate were filled with 100 lL of phosphate buffered saline
to minimize evaporation. After another 24 hours of culture,
20 lL of Cell Titer 96 Aqueous One (Promega) was added to
each well and then developed in the tissue culture hood between 1
and 2 hours as per the manufacturer’s protocol. The absorbance
was measured at 490 nm and at 630 nm to correct for nonspecific
absorbance. The data were plotted as OD490–OD630 and fit to a
sigmoidal dose response curve using GraphPad 4 software.
Electrophysiology
The methods used were those reported previously.37
Microbial cytotoxicity
The minimum inhibitory concentration (MIC) for each hy-
draphile is reported as the lowest serial 2-fold dilution that
prevented bacterial growth as outlined by the National Com-
mittee for Clinical Laboratory Standards (NCCLS).31,32 E. coli
DH5a cells with a pBluescript plasmid having AMP resistance
were tested using the standard inoculum size of 5 × 105 CFU
mL−1. Cells were grown at 37 ◦C in 2 mL of Luria Bertani
(LB) Miller media (10 g L−1 peptone, 5 g L−1 yeast extract,
10 g L−1 NaCl, 100 lg mL−1 ampicillin) that were 2-fold serially
diluted with hydraphile test compound. Bacillus subtilis (JH642
WT) cells were tested in similar fashion using Luria broth (no
AMP) at the same inoculum size, and grown at 30 ◦C. The MIC
was taken as the lowest hydraphile concentration that inhibited
growth after 24 h as judged by visual turbidity. Each compound
was assayed three times at every reported concentration to
each bacterium, using an independent dilution of compound
for each experiment. The minimum bactericidal concentration
(MBC) was determined as the lowest macrodilution that killed
>99.9% of bacteria in the culture. This was determined by
plating aliquots of the test suspensions onto Petri dishes and
counting the number of colony forming units after overnight
growth.
Kinetics of hydraphile cytotoxicity
TOP10 E. coli were transformed with the pT7-3 plasmid
containing the lux operon from Photorhabdus luminescens. A
single colony of the transformed E. coli was picked from a freshly
streaked LB-Amp (100 lg mL−1) plate. The culture was grown
in LB-Amp (100 lg mL−1) at 37 ◦C until the optical density was
0.5. The culture was then diluted to an OD = 0.1 and 50 lL was
added to wells in a black-walled 96-well plate.
To study the dose response of hydraphile toxicity, each
hydraphile was dissolved in neat ethanol to a concentration of
10 mg mL−1 for 3 and 2.5 mg mL−1 for 6 and 7. The hydraphile
ethanol stocks were then diluted 1:50 into LB-Amp (100 lg
mL−1) in triplicate. As a vehicle control, ethanol was also diluted
into LB-Amp (100 lg mL−1) in triplicate. The initial 1 : 50
dilution of hydraphile into LB was then further serially diluted
1 : 2 into LB-Amp (100 lg mL−1) and 2% ethanol (to maintain
a constant concentration of vehicle). Finally, the procedure was
repeated for kanamycin (20 mg mL−1 dissolved milli-Q water)
except that the vehicle control was a 1 : 50 dilution of milli-Q
water into LB-Amp (100 lg mL−1), and the media for the serial
dilution was LB-Amp (100 lg mL−1) 2% milli-Q water.
Saccharomyces cerevisiae MIC/MFC experiments were con-
ducted in similar fashion. Cells were grown in YPD media
(10 g◦L−1 yeast extract, 20 g L−1 peptone, 20 g L−1 dextrose)
at 30 C. An inoculum of 5 × 103 cells was used for MIC studies,
and allowed to grow for 48 hours to achieve turbidity.
The diluted compound (50 lL) was then added using a
multichannel pipette to the appropriate wells containing the
transformed E. coli. The result was that each well contained:
100 lL of lux operon transformed E. coli at OD = 0.05 in LB-
Amp (100 lg mL−1), 1% ethanol, and the specified concentration
of either hydraphile or Kanamycin.
CaCo2 Cytotoxicity
The plates were then imaged for photon emission from the
transformed E. coli by using a commercial IVIS 100 (Xenogen)
CCD camera and computer system (settings: no filter, binning
8, f-stop = 1, exposure time = 30 s at the indicated time points).
CaCo2 cells were seeded in 96-well tissue culture microtiter
plates at a density of 20 000 cells per well. After 24 h, the culture
medium was removed and the cells were treated with 4, 6, and
11 in serum-free medium. After 24 h, CellTiter 96 Aqueous One
solution (Promega) was added to each well, and the plates re-
◦
For the first 30 min, the plates remained in the IVIS at 37 C,
and then were transferred to an incubator in which the bacteria
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 4 4 – 3 5 5 0
3 5 4 9

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◦
were grown at 37 C with gentle shaking (∼210 rpm) between
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time points.
Data were analyzed by normalizing to the pre-drug photon
flux from each well and then expressed as a percent of the vehicle
treated wells at any given time point [eqn. (1)]. The standard error
of the mean was propagated through eqn (1).
19 H. W. Huang, Novartis Found. Symposium: Gramicidin and Related
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20 L. Be´ven and H. Wro`blewski, Res. Microbiol., 1997, 148, 163–175.
21 Y. R. Vandenburg, B. D. Smith, E. Biron and N. Voyer, Chem.
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fluxtestwells
%Vehicle =
(1)
fluxvehiclewells
22 S. A. Fernandez-Lopez, H.-S. Kim, E. C. Choi, M. Delgado, J. R.
Granja, A. Khasanov, K. Kraehenbuehl, G. Long, D. A. Weinberger,
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24 This value is based on a molecular weight of 1214 for KQRWLWLW
at 3 lg mL−1 for B. subtilis.
Acknowledgements
We thank the NIH for grants GM-36262 to GWG, NS-30888 to
JEH, and P50 CA94056 to DPW that supported this work and
for a Training Grant Fellowship T32-GM04892 to WML.
25 G. W. Gokel and O. Murillo, Acc. Chem. Res., 1996, 29, 425–32.
26 Y. Shai, Curr. Pharm. Des., 2002, 8, 715–725.
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3 5 5 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 5 4 4 – 3 5 5 0