Anion transport properties of amine and amide-sidechained peptides are affected by charge and phospholipid composition

Article abstract of DOI:10.1039/b800530c

Four synthetic anion transporters (SATs) having the general formula (n-C₁₈H₃₇)₂N-COCH₂OCH ₂CO-(Gly)₃Pro-Lys(ε-N-R)-(Gly)₂-O-n-C ₇H₁₅ were prepared and studied. The group R was Cbz, H (TFA salt), t-Boc, and dansyl in peptides 1, 2, 3, and 4 respectively. The glutamine analog (GGGPQAG sequence) was also included. A dansyl-substituted fluorescent SAT was used to probe peptide insertion; the dansyl sidechain resides in an environment near the bilayer's midpolar regime. When the lysine sidechain was free or protected amine, little effect was noted on final Cl ^- transport rate in DOPC: DOPA (7: 3) liposomes. This stands in contrast to the significant retardation of transport previously observed when a negative glutamate residue was present in the peptide sequence. It was also found that Cl^- release from liposomes depended on the phospholipid composition of the vesicles. Chloride transport diminished significantly for the free lysine containing SAT, 2, when the lipid was altered from DOPC: DOPA to pure DOPC. Amide-sidechained SATs 1 and 5 showed a relatively small decrease in Cl^- transport. The effect of lipid composition on Cl^- transport was explained by differences in electrostatic interaction between amino acid sidechain and lipid headgroup, which was modeled by computation. The Royal Society of Chemistry.

Full text of DOI:10.1039/b800530c

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Anion transport properties of amine and amide-sidechained peptides are
affected by charge and phospholipid composition†
Lei You,a Ruiqiong Lia and George W. Gokel*a,b
Received 11th January 2008, Accepted 16th April 2008
First published as an Advance Article on the web 16th June 2008
DOI: 10.1039/b800530c
Four synthetic anion transporters (SATs) having the general formula
(n-C18H37)2N-COCH2OCH2CO-(Gly)3Pro-Lys(e-N-R)-(Gly)2-O-n-C7H15 were prepared and studied.
The group R was Cbz, H (TFA salt), t-Boc, and dansyl in peptides 1, 2, 3, and 4 respectively. The
glutamine analog (GGGPQAG sequence) was also included. A dansyl-substituted fluorescent SAT was
used to probe peptide insertion; the dansyl sidechain resides in an environment near the bilayer’s
midpolar regime. When the lysine sidechain was free or protected amine, little effect was noted on final
Cl− transport rate in DOPC : DOPA (7 : 3) liposomes. This stands in contrast to the significant
retardation of transport previously observed when a negative glutamate residue was present in the
peptide sequence. It was also found that Cl− release from liposomes depended on the phospholipid
composition of the vesicles. Chloride transport diminished significantly for the free lysine containing
SAT, 2, when the lipid was altered from DOPC : DOPA to pure DOPC. Amide-sidechained SATs 1 and
5 showed a relatively small decrease in Cl− transport. The effect of lipid composition on Cl− transport
was explained by differences in electrostatic interaction between amino acid sidechain and lipid
headgroup, which was modeled by computation.
A broad range of synthetic, membrane-active peptides has been
developed in recent years. Examples include antibacterial peptides
Introduction
developed in the labs of Ghadiri10 and Tirrell.11 Pore-forming
peptides have been reported by Voyer12 and by Matile,13 and their
coworkers. At least the compounds reported by Ghadiri, Voyer,
and Matile form pores that appear to disrupt cellular osmotic
balance. Our non-peptidic hydraphile molecules do likewise.14
Various synthetic non-peptide receptors, which can mediate anion
transport through bilayer membrane, have also been designed,
prepared and characterized.15–19 We have prepared a family
of pore-forming synthetic anion transporters (SATs) typically
of the formula (C18H37)2N-COCH2OCH2CO-(Gly)3-Pro-(Gly)3-
OR.20 Although not active as antibiotics against E. coli, they show
chloride transport activity in vital mammalian cells. In the latter
sense, they have potential as pharmaceutical agents.21 The Cl−
transport efficacy of many members of this peptide family has
been documented by studies involving liposomes22 and at least 10-
fold selectivity for chloride over potassium was observed by planar
bilayer conductance measurements for the ∼(Gly)3-Pro-(Gly)3-
OC18H37 derivative.20,23 Both planar bilayer conductance studies
and Hill plots comport with pore formation.22,23
In recent work, we systematically replaced each of the glycine
residues in the sequence ∼(Gly)3-Pro-(Gly)3∼ on the C-terminal
side of proline with glutamic acid.24,25 The goal was to determine if
the presence of a negatively charged residue within the pore formed
by these compounds altered the Cl− transport rate. A negative
charge is, of course, expected to repel other negative charges, but
this study sought to characterize the behavior of a pore formed
within a phospholipid bilayer rather than in any bulk phase. The
question was inspired by the proposal that a glutamic acid residue
in the conductance pore of the ClC protein chloride transporter
serves as a gate.26,27 Indeed, the SAT containing a glutamate
Phospholipid membranes occur in astonishing variety even within
organelles of the same cell.1 Variations are found in all ele-
ments of the phospholipid monomer, including the hydrophobic
tail(s), headgroup, etc. Common headgroups include choline,
ethanolamine, serine, and inositol, among many others. These
headgroups are typically linked through a phosphoryl residue
to glycerol. Two fatty acids, esterified to primary and secondary
glyceryl hydroxyl groups, generally provide the membrane’s hy-
drophobic element.2
The great variability in phospholipid headgroups means that
they may interact differently with their environment and/or with
structures embedded within them.3 Variations in the headgroup’s
charge state may alter the rate at which ions or molecules are
transported through the bilayer. In recent studies conducted with
hydraphile synthetic ion channels, we found that cation–p interac-
tions involving phosphocholine headgroups4 played a significant
role in transport efficacy.5 Further, organizational elements such as
hydrogen bond donors or acceptors, residues that can participate
in salt bridge formation, etc., may exhibit unique interactions
with certain membrane compositions.6 It is known, for example,
that many antibiotic peptides incorporate basic amino acids in
their sequences, which enhance their affinity for Gram-negative
bacteria, the outer membrane of which is negatively charged.7–9
aDepartment of Chemistry, Washington University, St. Louis, MO 63130,
USA
bDepartments of Chemistry & Biochemistry and Biology, Center for
Nanoscience, University of Missouri-Saint Louis, One University Boulevard,
Saint Louis, MO 63121, USA. E-mail: gokelg@umsl.edu
† Electronic supplementary information (ESI) available: Computational
results. See DOI: 10.1039/b800530c
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residue, was a measurably poorer Cl− transporter than was the
glutamate benzyl ester analog. In the present study, we pose two
questions. First, does lysine, which should be protonated and posi-
tive within the conductance pore, affect Cl− transport? Second, do
variations in the membrane monomers and their charge balance
affect Cl− transport? In order to address these issues, we have
developed a fluorescent probe, described below, that was used to
gain insight into the function of SATs within the bilayer.
transport efficacy of these SATs. The results are described in the
sections below.
Compound preparation
Standard solution coupling methods were used to prepare 1–
5. The e-amino group of lysine was protected during peptide
coupling. During the preparation of peptide 1, Fmoc protected
the main chain amino group and the sidechain amino group was
masked by Cbz. The “orthogonal” protecting groups Cbz and
Fmoc can be removed by hydrogenolysis or by treatment with
base, respectively. The C-terminal, three amino acid sequence was
prepared by coupling the lysine derivative with glycylglycine n-
heptyl ester tosylate (EDCI, HOBt, NMM, CH2Cl2) to afford
tripeptide Fmoc-(e-N-Cbz)KGG-OC7H15. The Fmoc group was
removed in the presence of Et2NH. The free amine was then treated
with Boc-proline to give Boc-P(e-N-Cbz)KGG-OC7H15. After
cleavage of the Boc group (HOAc : TFA, 3 : 7), the amine salt was
coupled with previously reported22 (C18H37)2N-COCH2OCH2CO-
(Gly)3-OH to afford 1. Compounds 2–5 were prepared by using a
similar strategy. Synthetic access to 1 is summarized in Scheme 1
and details are recorded in the Experimental section.
Results and discussion
Compounds studied
We have studied anion transport for numerous compounds of the
type (R1)2N-COCH2OCH2CO-(Gly)3-Pro-(Gly)3-OR2, in which
R1 ranged from methyl to octadecyl. The most common identity
for R1 was n-octadecyl. A number of derivatives were studied
in which R2 was varied, but in most examples R2 was either
benzyl or n-heptyl. The peptide sequences studied have been
varied as well but were predominantly (Gly)3-Pro-(Gly)3.28 Since
most of the data were acquired with bis(octadecyl) derivatives,
such as (C18H37)2N-COCH2OCH2CO-(Gly)3-Pro-(Gly)3-OR2, and
since transport activity was typically highest when R2 was n-
heptyl,22 the compounds studied for the present report can
be summarized as (C18H37)2N-COCH2OCH2CO-(Gly)3-Pro-Lyx-
(Gly)2-O(CH2)6CH3. The abbreviation Lyx is used to connote
a lysine derivative having various substituents on the e-amino
group. The lysine derivatives 1–4 are illustrated, along with 5,
which has the structure (C18H37)2N-COCH2OCH2CO-(Gly)3-Pro-
Gln-Ala-Gly-O(CH2)6CH3.
Use of dansyl-SAT 4 as a fluorescent membrane probe
The dimethylaminonaphthylsulfonyl or dansyl group is highly
fluorescent. It can readily be conjugated to the sidechain amino
group of lysine to serve as a probe of amphiphile self-assembly
and to probe insertion into the bilayer. Data are shown in
Fig. 1 for chloride release mediated by 1–4 from vesicles prepared
from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-
dioleoyl-sn-glycero-3-phosphate monosodium salt (DOPA, struc-
tures shown). Dansyl-SAT 4 shows essentially the same chloride
release behavior as does 3. Fig. 1 shows that 2 appears to form a
pore faster than 1, 3, or 4 but 2–4 show similar Cl− release profiles
over the observation period. Compound 1 shows an unexpected
L-Lysine was incorporated in the fifth position of the sequence
noted above to give ∼(Gly)3-Pro-Lys-(Gly)2∼ (∼GGGPKGG∼).
The e-amino group of lysine was functionalized as follows:
1, benzyloxycarbonyl (Cbz); 2, free amine, trifluoroacetic acid
salt; 3, tert-butoxycarbonyl (t-Boc); 4, dansyl. This range of
compounds permitted us to assess three issues. First, dansyl
derivative 4 was used to determine if it, and by inference the
lysine derivatives, inserts into the bilayer. Second, compound 2
places a positive charge (protonated amino group) within the
conductance pore. Evidence obtained in previous studies suggests
that the conductance pore forms from at least two monomers,22
so two (or more) positive charges should be present. A third issue
concerns the influence of changes in bilayer composition on the
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Scheme 1 Preparation of compound 1.
The fluorescence maximum (kmax, kexc = 340 nm) was determined
for 4 in solvents of varying polarity in order to better understand
the dansyl group’s position in the bilayer. This would, by inference,
indicate the position of the lysine. Thus, the emission maximum
for dansyl-SAT 4 was measured in hexane, dioxane, ethyl acetate,
dichloromethane, 2-propanol, butanol, ethanol, and methanol
(kmax ∼475–510 nm). The maxima were plotted on the ordinate
vs. Reichardt’s ET polarity parameter (low to high values) on
the abscissa.29 The plot gave a straight line relationship (slope =
1.19, y intercept = 439.9 nm, r2 = 0.97) over the range of
solvents. The fluorescence maximum for 4 in DOPC : DOPA (7 :
3) liposomes was 492 nm. This corresponds to an ET value of
43. Common solvents having similar ET values are acetone, 42;
dimethylformamide (DMF), 43; and butyrolactone, 44. It is clear
that the dansyl group is neither exposed to the aqueous interface
(high polarity) or embedded in the insulator/hydrocarbon regime
(very low polarity) of the bilayer. This places dansyl at or near the
midpolar (glyceryl) regime of the bilayer leaflet.30 The fluorescence
method does not permit greater precision in the present case
because SAT insertion and pore formation are dynamic processes.
Fig. 1 Chloride release from DOPC : DOPA (7 : 3) liposomes mediated
by 1, 2, 3, and 4 (0.31 mM lipids, 65 lM compound, pH= 7.0).
increase in activity after about 1200 s. Although there are certainly
differences in Cl− release activity among these compounds, these
data show that the fluorescent reporter in 4 does not fundamentally
alter the peptide’s behavior as is sometimes the case for fluorescent
derivatives.
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The fluorescence maximum (kmax) observed for 4 suspended
in HEPES buffer was 481 nm. Interpolation results in an ET
value of 35, which lies between hexane and dioxane on this scale.
The aqueous buffer is very polar; the low polarity environment
experienced by 4 shows that the amphiphilic peptide aggregates
in aqueous solution, although the extent of this association is
currently unknown.
Typically, the chloride electrode showed ion release within
seconds after ionophore addition. Automated data collection
recorded an electrode potential every second. At the conclusion
of an experiment, Triton X-100 detergent was added to lyse the
vesicles and a final reading established [Cl−] = 100%. The release
data were normalized to the final chloride concentration and are
presented as fractional chloride release. Each data set is the average
of at least three independent experiments.
Effect of membrane composition on the fluorescence spectrum of 4
Effect of lysine on chloride transport
In studies with hydraphile cation channels, we noted a significant
effect of membrane composition on membrane transport. We
have attributed this to cation–p interactions between the channel-
former and the membrane monomer’s headgroups.5 We were
thus interested to determine if the SAT pore-formers exhibited a
membrane composition-dependent variation in anion transport.
This was of particular significance in the present case as the
positive charge of 2 could interact with the negatively charged
phosphoryl residues in the membrane monomers.
The emission maximum of dansyl-SAT 4 was found to be
492 nm when it was recorded in vesicles prepared from a 7 : 3
(w/w) DOPC : DOPA mixture. Based on the polarity dependence
study described above, the dansyl group of 4 should be near
the midpolar/insulator (hydrocarbon) interface of the membrane
in this monomer mixture. When liposomes were prepared from
DOPC alone, an emission maximum was reproducibly observed
at 490 nm for 4, suggesting that dansyl resides in a slightly less
polar environment than when present in DOPC : DOPA vesicles.
Fig. 2 shows the variation in emission maximum (kmax) in buffer,
pure DOPC and the 7 : 3 DOPC : DOPA mixture. Based on these
data and the polarity survey described above, the dansyl residue of
4 experiences diminishing polarity in the order DOPC : DOPA >
DOPC > HEPES buffer.
One of the key questions we wished to address was whether
or not protonated lysine affects Cl− release from liposomes.
Structurally, compounds 1–4 differ only by the presence or absence
of the functional group on the lysine residue in the peptide’s fifth
(GGGPXGG) position. The sidechain amino group of lysine is
reported to have a pKA of 10.54, so it will be completely protonated
at any pH below 8. Fig. 1 (above) shows that chloride release
from DOPC : DOPA vesicles by 1–4 is generally similar, although
ultimate release by 1 is greater and pore formation by 2 is faster
than the others. Release of Cl− by 1 before 1200 s is about the same
as for 3–4 and only about 50% higher at 0.5 h. The reason for these
two variations is obviously of interest but beyond the scope of the
present work. The fluorescence results show that 4 aggregates in
buffer so the dynamics of both insertion and pore formation could
both differ somewhat for each of the structures.
The key finding here is that the positively charged lysine
sidechain in 2 appears not to have a significant overall effect on
the Cl− transport rate, although the initial rate is somewhat faster.
In previous work, we found that a similarly placed glutamate
significantly diminished chloride transport under comparable
conditions.24,25 The effect of positive charge in or near the
conductance pore is clearly less significant than is the presence
of negative charge.
There are at least two explanations for the differences in
charge effect. In one sense it is obvious that a negative charge
(ionized glutamic acid) in a confined space should repel another
negative charge and a positive charge (protonated lysine) should
attract it. In the latter case, attraction of the anion may favor
transport although too strong an interaction could diminish it.
The lack of an observed effect may be due to offsetting effects.31
Alternately, it may be that the amide residues of the GGGPXGG
sequence bind32,33 the carboxylate anion, partly blocking the pore
or otherwise reorganizing it. The amides are less likely to interact
with the ammonium ion although some H-bonding may occur
there as well.
Fig. 2 The fluorescence emission spectra of 4 in aqueous buffer, DOPC,
and DOPC : DOPA liposome suspension.
Effect of lipid composition on chloride transport
Chloride release mediated by compounds 1–4
The second major question of the present study concerned
the effect, if any, of lipid composition on the efficacy of Cl−
release mediated by 1–4. Vesicles were prepared either from pure
DOPC or 7 : 3 DOPC : DOPA as described above and Cl−
release was monitored. The headgroup of DOPA is negatively
charged, whereas DOPC is overall neutral but is terminated by a
trimethylammonium residue (see structures above). The results of
Cl− release from DOPC vesicles, mediated by 1–4, are shown in
Fig. 3. Protectedsidechainaminederivatives 3and4showbehavior
similar to that exhibited in DOPC : DOPA vesicles while 1 is
The Cl− release profiles of 1–4 in DOPC : DOPA liposomes are
shown above in Fig. 1. The experiments were conducted as follows.
The vesicles were loaded with 600 mM KCl in HEPES buffer
(pH 7). The external buffer was chloride free, 400 mM K2SO4 in
HEPES (pH 7). An Accumet chloride combination electrode was
calibrated and the vesicle system was checked for leakage. With
the electrode inserted into the aqueous liposome suspension, the
ionophore under study was introduced (typically as a 2-propanol
solution) and the electrode response was recorded during 0.5–1 h.
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of the former is diminished and the latter is not. Our main focus
here was to determine if there was lipid dependence on anion
transport mediated by 2. Such an effect was observed. The lipid
effect on 1 remains unclear but it is only half that observed for 2.
Compound 2, which has a positively charged sidechain, is
less active as a chloride transporter than either 1 or 5 at the
arbitrarily chosen 1500 s time point. The Cl− release mediated
by 2 at 1500 s decreases by 60% when the lipid composition is
changed. Since there was little change in the transport rate between
protonated and neutral sidechained 2–4 in DOPC : DOPA (7 : 3)
liposomes, we infer that there is a significant interaction between
transporter 2 and lipid monomer. This interaction must be
weaker for 3 and 4. Possible interactions between a phospholipid
headgroup and a SAT sidechain can involve H-bonding, salt
bridge formation, charge–charge, or dipole–dipole interactions.
Four plausible interactions are shown in Fig. 5.
Fig. 3 Chloride release from DOPC liposomes mediated by 1, 2, 3, and
4 (0.31 mM lipids, 65 lM compound, pH= 7.0).
slightly less active in DOPC vesicles. Positively charged 2, however,
was significantly less active in pure DOPC liposomes. The chloride
release from DOPC mediated by glutamate containing SATs has
similar result as that from DOPC : DOPA (7 : 3) liposomes.
Comparison of transport rates
It is clear from the data that transport by these synthetic anion
transporters differs according to membrane composition. Since
the release curves vary from DOPC : DOPA (7 : 3) to DOPC,
especially from 0–200 s, we chose an arbitrary time point for
comparison.5 The fractional chloride release at 1500 s for each
compound is summarized in Fig. 4. The left hand (open) bar is
the transport rate for the compound in the 7 : 3 DOPC : DOPA
mixture. The right hand (filled) bar is for an identical experiment
conducted in DOPC only lipids. Each data set is the average
of at least three independent measurements (error bars shown).
Previously reported 5, (C18H37)2N-COCH2OCH2CO-(Gly)3-Pro-
Gln-Ala-Gly-OC7H15, was used as a control compound since it
contains a sidechain amide moiety, as do compounds 1 and 3.
Fig. 5 Plausible interactions between phospholipid headgroup and
ammonium (left) or amide residues (right).
Molecular modeling study
We applied computational methods to simplified models of both
the lipids and the SAT in order to gain insight into these
possible interactions and to assess geometrical constraints. Such
calculations could be done on an ensemble of lipid monomers34
and SAT molecules. Such calculations require a huge amount
of CPU time even with the most capable processors. We thus
simplified the system as follows. The phospholipids were modeled
as methyl phosphate. DOPA was modeled as dimethylphosphate
monosodium salt while DOPC was modeled as dimethylphos-
phocholine. The isolated N-terminus of lysine or glutamine was
capped with an acetyl group to mimic an amide residue. The
peptide’s C-terminus was simplified as a methyl ketone to minimize
the number of additional H-bond donor and acceptor interactions.
We note that when more laborious calculations were done on a
compound in which the C-terminus was a methyl ester rather than
a methyl ketone, similar results were obtained.
The DFT method of Gaussian 03 was used to optimize
geometries and to minimize energies (see Experimental section).
Selected results are shown in Fig. 6. The calculations reported
here were conducted for the gas phase. The limitations of a gas
phase calculation are obvious but the dynamics of a membrane
and the uncertain water content make simple alternatives unten-
able. Moreover, the structural information thus obtained will be
internally comparable.
Fig. 4 Comparison of fractional chloride release at 1500 s mediated by
1–5 in DOPC or DOPC : DOPA liposomes.
The transport results for compounds 1–5 can be summarized in
three groups. The Cl− transport rates for 3 and 4 are unchanged
within experimental error, irrespective of lipid composition. Com-
pounds 1 and 5, which have neutral but polar carbamate and
amide sidechains, respectively, show about 70% of the transport
activity in DOPC membranes compared to the DOPC : DOPA
mixture. Lysine-containing 2 shows a drop in Cl− transport ability
to only 40% of its previous value. We note that both 1 and 3
incorporate carbamate residues in their sidechains, but the activity
Bearing in mind the caveats noted above concerning both the gas
phase and the choice of models and the tendency to over-interpret
models, certain differences are apparent. The model for the
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when the lipid composition is changed from DOPC : DOPA to
pure DOPC. This activity decrease is explained by differences in
hydrogen bond interactions between the lipid phosphoryl group
and the sidechain ammonium ion. Computational results using
model systems showed that a phosphate–lysine interaction is
stronger than a phosphocholine–lysine interaction. This is not
surprising and it is known that basic amino acids show enhanced
affinity for Gram-negative bacteria, the outer membrane of which
is negatively charged.7–9
The Cl− transport activity of sidechain amide containing
compounds 1 and 5 also decreased when the DOPC : DOPA
lipid composition was changed to DOPC. The effect is not as
pronounced as that observed for amine derivative 2, but this
sidechain amide effect is notable because it is less well documented.
The formation of a doubly bridged hydrogen bond interaction
between amide and phosphate may play an important role that is
supported by the computational models reported here.
Fig. 6 Calculation results for four model complexes: phosphate–lysine
(upper left), phosphocholine–lysine (lower left), phosphate–glutamine
(upper right) and phosphocholine–glutamine (lower right).
Experimental section
lipid–ammonium sidechain interaction of 2 shows two or three H-
bond associations (shown as dotted lines, left panels). The amide–
lipid association that models the amide sidechain of 5 shows one
or two such contacts (right panels). The calculated bond distances
are recorded in the Supplementary Material†. The key issue here is
that greater changes are predicted when 2 or 5 interacts with phos-
phate (DOPA model) than with phosphocholine (DOPC model).
The calculations show that the interaction between phosphate and
either the lysine or glutamine sidechain is more favorable than
the corresponding phosphocholine interaction. These interactions
may stabilize the active conformation of pore-formers more in
DOPC : DOPA liposomes than in pure DOPC. Simplified as these
models are, they comport with the transport data and with the
suggestion offered above of differences in H-bond interactions
within the pore.
General
1H-NMR spectra were recorded on a Gemini 300 spectrometer
and are reported in the following manner: chemical shifts reported
in ppm (d) downfield from internal (CH3)4Si (integrated intensity,
multiplicity (b = broad, s = singlet, d = doublet, t = triplet,
q = quartet, bs = broad singlet, m = multiplet, etc.), coupling
constants in Hz, assignment). 13C-NMR spectra were obtained at
75 MHz and referenced to CDCl3 (77.23 ppm). NMR spectra
were obtained in CDCl3 unless otherwise specified. Infrared
spectra were recorded on a Perkin-Elmer 1710 Fourier transform
infrared spectrometer. Melting points were determined on a
Thomas Hoover apparatus in open capillaries and are uncorrected
for crystalline compounds. Thin layer chromatography analyses
were performed on silica gel 60-F-254 with a 0.2 mm thickness.
Preparative chromatography columns were packed with silica gel
(Kieselgel 60, 70–230 mesh or Merck grade 9385, 230–400 mesh,
Conclusion
˚
60 A).
Reagents were of the best grade commercially available and
Three important findings emerge from the present study. First, a
positively charged residue (ammonium cation) within the chloride
transporting pore does not favor final chloride transport rate
through the bilayer mediated by the SAT molecules described here.
Indeed, in the DOPC : DOPA (7 : 3) lipid system, the ammonium-
sidechained SAT and those having a protected amine function
exhibit similar activity. This result is strikingly different from the
previously reported inhibitory influence of a negatively charged
sidechain.24,25
A fluorescent dansyl residue was incorporated into the peptide
sequence to probe the SAT’s position in the bilayer. Dansyl
fluorescence is solvent dependent and a correlation line between
solvent polarity and fluorescence emission maxima was used
to estimate the environment experienced by the fluorophore. In
aqueous buffer, the amphiphilic peptide SATs aggregate. After
insertion into the membrane, however, the emission is red shifted
and the dansyl sidechain resides in an environment near the
bilayer’s midpolar regime.
were distilled, recrystallized, or used without further purification,
as appropriate. CH2Cl2 was distilled from calcium hydride. EDCI
represents 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hy-
drochloride. HOBt represents 1-hydroxybenzotriazole. DMAP
represents 4-dimethylaminopyridine. DGA represents diglycolyl,
∼COCH2OCH2CO∼. Combustion analyses were performed by
M-H-W Laboratories, Phoenix, AZ, and are reported as percents.
High resolution mass spectra were obtained from the University
of Missouri-Saint Louis mass spectrometry facility.
Preparation of (C18H37)2NCOCH2OCH2CO-(Gly)3-Pro-Lys(e-N-
Cbz)-(Gly)2-OC7H15, 1
Fmoc-K(e-N-Cbz)GG-OC7H15. TsOH·GG-OC7H15 (0.60 g,
1.49 mmol), Fmoc-K(e- N-Cbz)-OH (0.75 g, 1.49 mmol), EDCI
(0.31 g, 1.64 mmol) and HOBt (0.22 g, 1.64 mmol) were dissolved
in CH2Cl2 (35 mL). NMM (0.18 mL) was added. The mixture
was stirred at 0 ◦C for 0.5 h and then at room temperature for
24 h. The solvent was removed in vacuo. The residue was dissolved
in CH2Cl2 (50 mL), washed with 5% citric acid (2 × 25 mL),
The chloride transporting activity of amine- and amide-
sidechained SATs is dependent on the liposome’s phospholipid
composition. The Cl− transport ability of 2 decreases significantly
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H2O (2 × 25 mL), 5% NaHCO3 (25 mL) and brine (25 mL),
dried over MgSO4 and evaporated. Column chromatography
(silica gel, CHCl3 : CH3OH = 98 : 2) afforded a white solid
(0.94 g, 88%). 1H-NMR: 0.87 (3H, t, J = 6.8 Hz, CH2CH3),
1.26 (8H, pseudo-s, OCH2CH2(CH2)4CH3), 1.34–1.62 (6H, m,
Lys CH2CH2CH2CH2NH and OCH2CH2(CH2)4CH3), 1.62–1.93
(2H, m, Lys CH2CH2CH2CH2NH), 3.17 (2H, d, J = 4.8 Hz, Lys
CH2CH2CH2CH2NH), 3.96 (4H, d, J = 5.1 Hz, two Gly CH2),
4.04 (2H, t, J = 6.9 Hz, OCH2CH2(CH2)4CH3), 4.08–4.20 (2H, m,
Lys CH and Fmoc CH), 4.39 (2H, d, J = 6.6 Hz, Fmoc CH2), 5.06
(2H, s, OCH2Ph), 5.10 (1H, bt, CONH), 5.86 (1H, bd, CONH),
6.96–7.07 (2H, m, two CONH), 7.21–7.40 (9H, m, Ph HAr and
Fmoc HAr), 7.51–7.60 (2H, m, Fmoc HAr), 7.74 (2H, d, J = 7.5 Hz,
Fmoc HAr). 13C-NMR: 14.2, 22.5, 22.8, 25.9, 28.6, 29.0, 29.6, 31.9,
40.3, 41.4, 43.1, 47.3, 55.4, 65.9, 66.8, 67.3, 120.2, 125.2, 127.3,
128.0, 128.3, 128.7, 136.8, 141.5, 143.9, 157.0, 169.2, 170.0, 172.7.
IR (CHCl3, cm−1): 3301, 3066, 2931, 2858, 1739, 1689, 1645, 1537,
1451, 1406, 1263, 1134, 1104, 1090, 1032.
CF3COOH·P(e-N-Cbz)KGG-OC7H15. Boc-P(e-N-Cbz)KGG-
OC7H15 (0.42 g, 0.61 mmol) was dissolved in HOAc (1.5 mL).
The solution was cooled in ice and TFA (3.5 mL) was added.
The mixture was stirred at rt for 50 min. The acids were
rapidly removed in vacuo and the residue was triturated
with ether. Vacuum filtration afforded a white solid (0.31 g,
72%). 1H-NMR: 0.88 (3H, t, J = 6.9 Hz, CH2CH3), 1.20–
1.45 (8H, m, OCH2CH2(CH2)4CH3), 1.50–2.21 (12H, m,
OCH2CH2(CH2)4CH3, Lys CH2CH2CH2CH2NH and Pro
NCH2CH2CH2), 3.14 (2H, bs, Lys CH2CH2CH2CH2NH), 3.43
(2H, bs, Pro NCH2CH2CH2), 3.60–4.30 (8H, m, two Gly CH2,
OCH2CH2(CH2)4CH3, Pro CH and Lys CH), 4.93 (2H, s,
OCH2Ph), 5.15 (1H, bs, NH), 6.20 (1H, bs, NH), 7.16 (5H,
pseudo-s, Ph HAr), 7.35 (1H, s, NH), 8.20 (1H, bs, NH), 9.17 (1H,
bs, NH), 11.53 (1H, bs, NH). 13C-NMR: 14.2, 14.6, 22.8, 22.9,
25.9, 26.1, 28.7, 29.1, 29.3, 31.9, 36.2, 41.4, 43.3, 65.8, 74.6, 89.6,
128.2, 128.6, 160.2, 170.0, 206.5. IR (CHCl3, cm−1): 3320, 3069,
2932, 2860, 1743, 1671, 1546, 1455, 1410, 1369, 1256, 1204, 1136,
1029.
H2N-K(e-N-Cbz)GG-OC7H15. Fmoc-K(e-N-Cbz)GG-OC7H15
(0.83 g, 1.16 mmol) was dissolved in DMF (10 mL). Et2NH
(1.2 mL) was added and the reaction was stirred at rt for 2 h. The
reaction was quenched by water (3 × 25 mL) and extracted with
CH2Cl2 (3 × 25 mL). Organic layers were combined and washed
with 5% NaHCO3 (2 × 25 mL) and brine (2 × 25 mL). Dry over
MgSO4. The solvent was evaporated in vacuo and the residue was
triturated with ether. Vacuum filtration afforded a white solid
(0.32 g, 60%). 1H-NMR: 0.89 (3H, t, J = 6.8 Hz, CH2CH3),
1.28 (8H, pseudo-s, OCH2CH2(CH2)4CH3), 1.40–1.93 (8H,
OCH2CH2(CH2)4CH3 and Lys CH2CH2CH2CH2NH), 3.10–3.23
(2H, m, CH2CH2CH2CH2NH), 3.67 (1H, bs, NH), 3.85–4.17
(7H, m, two Gly CH2, Lys CH and OCH2CH2(CH2)4CH3), 5.08
(2H, s, OCH2Ph), 5.21 (1H, bt, NH), 7.07 (1H, bs, NH), 7.27–7.40
(6H, m, Ph HAr and NH), 8.11 (1H, bs, NH). 13C-NMR: 14.2,
22.5, 22.8, 26.0, 28.7, 29.1, 29.7, 31.9, 40.8, 41.4, 43.9, 66.0, 128.3,
128.7, 137.0, 169.6. IR (CHCl3, cm−1): 3318, 3067, 2929, 2858,
1742, 1690, 1657, 1542, 1455, 1411, 1370, 1260, 1212, 1137, 1028,
913.
182DGA-GGGPK(e-N-Cbz)GG-OC7H15, 1. 182DGA-GGG-
OH (0.36 g, 0.44 mmol), CF3COOH.PK(e-N-Cbz)GG-OC7H15
(0.31 g, 0.44 mmol), EDCI (0.093 g, 0.48 mmol) and HOBt
(0.065 g, 0.48 mmol) were suspended in CH2Cl2 (20 mL). Et3N
(0.18 mL) was then added. The mixture was stirred at 0 ◦C for
0.5 h and then at room temperature for 48 h. The solvent was
evaporated and the residue was crystallized from MeOH. The
crude product was chromatographed (silica gel, CHCl3 : MeOH
95 : 5). Recrystallization from MeOH afforded a white solid
(204 mg, 33%). 1H-NMR (CDCl3, almost all of the peaks
are quite broad): 0.88 (3H, t, J = 6.6 Hz, CH2CH3), 1.20–
1.70 (74H, m, OCH2CH2(CH2)4CH3, CH3(CH2)15CH2CH2N,
OCH2CH2(CH2)4CH3 and CH3(CH2)15CH2CH2N), 1.70–2.30
(10H, m, Pro NCH2CH2CH2 and Lys (CH2)3CH2NH),
3.00–3.46 (8H, m, CH3(CH2)15CH2CH2N, Lys (CH2)3CH2NH
and Pro NCH2CH2CH2), 3.60–4.44 (18H, m, Gly CH2,
OCH2CH2(CH2)4CH3, COCH2O, Pro CH, and Lys CH), 5.00–
5.13 (2H, m, OCH2Ph), 5.53 (1H, bs, NH), 7.30–7.40 (5H, m, Ph
HAr), 7.53 (2H, bs, NH), 7.86 (2H, bs, NH), 8.64 (1H, bs, NH).
13C-NMR: 14.2, 14.3, 22.8, 22.9, 23.1, 25.1, 26.0, 27.1, 27.3, 27.8,
28.7, 29.0, 29.1, 29.5, 29.6, 29.7, 29.8, 29.9, 31.9, 32.1, 40.4, 41.5,
42.7, 43.0, 43.4, 46.6, 47.2, 47.6, 54.0, 61.8, 65.8, 66.7, 69.8, 71.9,
128.2, 128.3, 128.7, 137.0, 156.8, 168.7, 169.8, 170.1, 170.3, 170.6,
171.6, 171.8, 172.5, 173.1. IR (CHCl3, cm−1): 3306, 3069, 2920,
2851, 1743, 1647, 1540, 1467, 1411, 1377, 1338, 1250, 1208, 1130,
1029. Anal. calcd for C76H133N9O13: C, 66.10; H, 9.71; N, 9.13%.
Found: C, 66.04; H, 9.87; N, 9.15%.
Boc-PK(e-N-Cbz)GG-OC7H15. H2N-(e-N-Cbz)KGG-OC7H15
(0.32 g, 0.65 mmol), Boc-Pro-OH (0.14 g, 0.65 mmol), EDCI
(0.14 g, 0.71 mmol) and HOBt (0.10 g, 0.71 mmol) were dissolved
in CH2Cl2 (25 m◦L). Et3N (0.27 mL) was then added. The mixture
was stirred at 0 C for 0.5 h and then at rt for 48 h. The solvent
was evaporated and the residue was chromatographed (silica gel,
CHCl3 : CH3OH 98 : 2–97 : 3) to afford a yellow oil (0.42 g, 94%).
1H-NMR: 0.88 (3H, t, J = 6.8 Hz, CH2CH3), 1.19–1.37 (8H,
m, OCH2CH2(CH2)4CH3), 1.44 (9H, s, C(CH3)3), 1.48–2.23
(12H, m, OCH2CH2(CH2)4CH3, Lys CH2CH2CH2CH2NH and
Pro NCH2CH2CH2), 3.18 (2H, bt, Lys CH2CH2CH2CH2NH),
3.34–3.51 (2H, m, ProNCH2CH2CH2), 3.88–4.06 (4H, m, two Gly
CH2), 4.11 (2H, t, J = 6.9 Hz, OCH2CH2(CH2)4CH3), 4.16–4.38
(2H, m, Pro CH and Lys CH), 5.00–5.18 (3H, m, OCH2Ph and
NH), 7.00 (1H, bs, NH), 7.09 (1H, bs, NH), 7.28–7.38 (5H, m, Ph
HAr), 7.46 (1H, bs, NH). 13C-NMR: 14.2, 22.8, 22.9, 26.0, 28.6,
28.7, 29.1, 29.6, 31.9, 37.5, 41.4, 43.3, 47.5, 58.8, 60.9, 65.8, 66.8,
77.4, 83.9, 128.2, 128.3, 128.7, 136.9, 161.2, 170.0, 170.9, 173.4.
IR (CHCl3, cm−1): 3309, 3068, 2931, 2860, 1748, 1674, 1535, 1455,
1404, 1367, 1250, 1204, 1164, 1128, 1090, 1029.
Preparation of (C18H37)2NCOCH2OCH2CO-(Gly)3-Pro-Lys-
(Gly)2-OC7H15·TFA, 2
182DGA-GGGPKGG-OC7H15·TFA, 2. 182DGA-GGGPK(e-
N-Boc)GG-OC7H15, 3 (64 mg, 0.048 mmol) was dissolved
in CH2Cl2 (2 mL). TFA (2 mL) was then added. The
mixture was stirred at room temperature for 2 h. Solvents
were removed in vacuo. Toluene was added and the mixture
evaporated to remove residual acid. High vacuum drying
overnight afforded a white waxy solid (60 mg, 93%). 1H-
NMR (CDCl3, all peaks are broad): 0.88 (9H, t,
6.5 Hz, CH2CH3), 1.20–2.30 (84H, m, NCH2CH2(CH2)15CH3,
J
=
2920 | Org. Biomol. Chem., 2008, 6, 2914–2923
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OCH2CH2(CH2)4CH3, NCH2CH2(CH2)15CH3, OCH2CH2-
(CH2)4CH3, Lys CH2CH2CH2CH2NH and Pro NCH2CH2-
CH2), 2.80–3.20 (4H, m, NCH2CH2(CH2)15CH3 and Lys
CH2CH2CH2CH2NH), 3.26 (2H, bt, NCH2CH2(CH2)15CH3),
3.44–3.78 (2H, m, Pro NCH2CH2CH2), 3.80–4.20 (15H, m, Gly
CH2, OCH2CH2(CH2)4CH3, Pro CH and OCH2CO), 4.26–4.47
(3H, m, OCH2CO and Lys CH), 7.71 (1H, bs, NH), 7.86 (4H,
bs, NH), 8.14 (2H, bs, NH), 8.29 (1H, bs, NH), 8.44 (1H, bs,
NH). 13C-NMR: 14.2, 14.3, 22.8, 22.9, 25.2, 26.0, 26.6, 27.1,
27.3, 27.8, 28.7, 29.0, 29.1, 29.5, 29.6, 29.8, 29.9, 31.9, 32.1, 39.9,
41.4, 42.4, 42.9, 43.1, 46.0, 46.6, 47.2, 53.7, 61.5, 65.9, 69.4, 71.3,
168.8, 169.3, 170.4, 170.5, 170.9, 171.1, 171.4, 173.0, 173.3. IR
(CHCl3, cm−1): 3296, 3079, 2920, 2851, 1743, 1658, 1652, 1547,
1467, 1411, 1378, 1341, 1203, 1132, 1031. HRMS (FAB): Calcd
for C68H128N9O11 [M − CF3COO]+, 1246.9733; found, 1246.9696.
Boc-QAG-OC7H15. Boc-AG-OC7H15 was deprotected in the
usual way (4 N HCl in dioxane for 1 h). HCl·AG-OC7H15 (0.48 g,
1.71 mmol), Boc-Gln-OH (0.42 g, 1.71 mmol), EDCI (0.36 g,
1.88 mmol) and HOBt (0.25 g, 1.88 mmol) were dissolved in
◦
CH2Cl2 (35 mL). The mixture was stirred at 0 C for 0.5 h and
then at room temperature for 12 h. The residue was dissolved in
CH2Cl2 (35 mL), washed with 5% citric acid (2 × 25 mL), H2O
(2 × 25 mL), 5% NaHCO3 (2 × 25 mL) and brine (25 mL), dried
over MgSO4 and evaporated. Column chromatography (silica
gel, CHCl3 : CH3OH = 97 : 3) afforded a solid (0.54 g, 67%).
1H-NMR: 0.88 (3H, t, J = 6.6 Hz, CH2CH3), 1.20–1.38 (8H,
m, OCH2CH2(CH2)4CH3), 1.40–1.49 (12H, m, Ala CH3 and
C(CH3)3), 1.63 (2H, quintet, J = 6.3 Hz, OCH2CH2(CH2)4CH3),
1.98–2.15 (2H, m, Gln CH2CH2CO), 2.40 (2H, t, J = 6.2 Hz, Gln
CH2CH2CO), 3.91–4.03 (2H, m, Gly CH2), 4.11 (2H, t, J = 6.8 Hz,
OCH2CH2(CH2)4CH3), 4.24 (1H, bs, Gln CH), 4.55 (1H, quintet,
J = 7.2 Hz, Ala CH), 5.79 (1H, bs, Gln CONH2), 6.40 (1H, bs,
Gln CONH2), 7.02 (1H, bs, Gln NH), 7.30 (1H, bt, Gly NH),
7.68 (1H, bd, Ala NH). 13C-NMR: 14.2, 17.8, 22.7, 26.0, 28.5,
28.7, 29.0, 29.1, 31.9, 41.5, 49.3, 53.9, 65.9, 80.5, 170.3, 172.1,
173.0, 176.1. IR (CHCl3, cm−1): 3433, 3318, 3076, 2959, 2930,
2857, 1735, 1688, 1644, 1527, 1451, 1392, 1366, 1346, 1287, 1271,
1246, 1172, 1052, 1028, 872, 780, 665.
Preparation of (C18H37)2NCOCH2OCH2CO-(Gly)3-Pro-
Gln-Ala-Gly-OC7H15, 5
L-Glycine heptyl ester tosylate. A solution of glycine (2.00 g,
26.7 mmol), TsOH monohydrate (5.70 g, 30.0 mmol) and 1-
heptanol (15 mL, 106 mmol) in toluene (18 mL) was refluxed
for 12 h. Water was removed from the reaction mixture by using
a Dean–Stark adapter. The reaction mixture was cooled to room
temperature, diethyl ether added (50 mL) and the mixture cooled
at 0 ◦C for 2 h. The solid was collected and recrystallized from
methanol–ether to afford white crystals (4.91 g, 53%), mp 105–
106 ◦C. 1H-NMR: 0.87 (3H, t, J = 6.9 Hz, CH2CH3), 1.23
(8H, pseudo-s, OCH2CH2(CH2)4CH3), 1.49 (2H, quintet, J =
6.8 Hz, OCH2CH2(CH2)4CH3), 2.33 (3H, s, CH3C6H4SO3), 3.65
(2H, q, J = 5.8 Hz, NH3CH2CO), 3.99 (2H, t, J = 6.9 Hz,
OCH2CH2(CH2)4CH3), 7.10 (2H, d, J = 7.8 Hz, Tosyl HAr), 7.72
(2H, d, J = 7.8 Hz, Tosyl HAr), 8.03 (3H, bt, NH3CH2CO). 13C-
NMR: 14.0, 21.3, 22.6, 25.6, 28.2, 28.9, 31.7, 40.3, 66.3, 126.0,
128.9, 140.4, 141.2, 167.4. IR (CHCl3, cm−1): 3473, 3030, 2961,
2929, 2856, 2730, 2643, 2220, 1996, 1911, 1747, 1616, 1600, 1519,
1471, 1428, 1380, 1194, 1127, 1105, 1056, 1036, 1012.
Boc-PQAG-OC7H15. Boc-QAG-OC7H15 was deprotected in 4
N HCl in dioxane for 1 h. HCl·QAG-OC7H15 (0.37 g, 0.91 mmol),
Boc-Pro-OH (0.20 g, 0.91 mmol), EDCI (0.19 g, 1.00 mmol) and
HOBt (0.14 g, 1.00 mmol) were dissolved in CH2Cl2 (35 mL).
Et3N (0.38 mL) was added and the mixture was stirred at 0 ◦C
for 0.5 h and then at rt for 12 h. The residue was dissolved in
CH2Cl2 (35 mL), washed with 5% citric acid (2 × 25 mL), H2O
(2 × 25 mL), 5% NaHCO3 (2 × 25 mL) and brine (25 mL),
dried over MgSO4, and evaporated. The residue was purified by
column chromatography (silica gel, CHCl3 : CH3OH 95 : 5) to
give a white solid (0.41 g, 79%). 1H-NMR: 0.88 (3H, t, J = 6.6 Hz,
CH2CH3), 1.20–1.37 (8H, m, OCH2CH2(CH2)4CH3), 1.40–1.50
(12H, m, Ala CH3 and C(CH3)3), 1.62 (2H, quintet, J = 6.9 Hz,
OCH2CH2(CH2)4CH3), 1.83–2.48 (8H, m, Pro NCH2CH2CH2
and Gln CH2CH2CO), 3.39–3.49 (1H, m, Pro NCH2CH2CH2),
3.64–3.74 (1H, m, Pro NCH2CH2CH2), 3.88–4.04 (2H, m, Gly
CH2), 4.10 (2H, t, J = 6.8 Hz, OCH2CH2(CH2)4CH3), 4.18 (1H,
dd, J = 8.6, 4.6 Hz, Pro CH), 4.27 (1H, q, J = 5.7 Hz, Gln
CH), 4.46 (1H, quintet, J = 7.5 Hz, Ala CH), 5.68 (1H, bs, Gln
CONH2), 6.55 (1H, bs, Gln CONH2), 7.40 (1H, t, J = 5.4 Hz, Gly
NH), 8.02 (1H, d, J = 7.2 Hz, Ala NH), 8.78 (1H, bd, Gln NH).
13C-NMR: 14.2, 17.5, 22.8, 24.9, 26.0, 28.6, 28.7, 29.1, 30.3, 31.6,
31.9, 41.5, 47.4, 49.9, 55.0, 57.0, 61.7, 65.5, 81.0, 165.6, 170.0,
171.6, 174.8, 176.6, 179.3. IR (CHCl3, cm−1): 3409, 3296, 3079,
2959, 2930, 2858, 1744, 1698, 1662, 1634, 1547, 1454, 1406, 1366,
1286, 1211, 1163, 1123, 1091, 978, 771, 666.
Boc-AG-OC7H15. TsOH·G-OC7H15 (0.51 g, 1.49 mmol), Boc-
L-Ala (0.28 g, 1.48 mmol), EDCI (0.31 g, 1.62 mmol) and HOBt
(0.22 g, 1.63 mmol) were dissolved in CH2Cl2 (40 mL). Et3N
(0.61 mL) was then added. The mixture was stirred at 0 ◦C for 0.5 h
and then at room temperature for 48 h. The solvent was evaporated
and the residue was dissolved in CH2Cl2 (40 mL) and washed
with 5% citric acid (2 × 20 ml), H2O (2 × 20 mL), 5% NaHCO3
(2 × 20 mL) and brine (2 × 20 mL), dried over MgSO4 and
evaporated. The residue was purified by column chromatography
(silica gel, EtOAc : hexane = 40 : 60) to give an oil (0.42 g,
1
83%). H-NMR: 0.87 (3H, t, J = 6.3 Hz, CH2CH3), 1.20–1.35
(8H, m, OCH2CH2(CH2)4CH3), 1.37 (3H, d, J = 7.2 Hz, Ala
CH3), 1.44 (9H, s, C(CH3)3), 1.63 (2H, quintet, J = 6.6 Hz,
OCH2CH2(CH2)4CH3), 4.02 (2H, d, J = 5.1 Hz, Gly CH2),
4.13 (2H, t, J = 6.8 Hz, OCH2CH2(CH2)4CH3), 4.23 (1H, broad
quintet, Ala CH), 5.11 (1H, d, J = 6.6 Hz, Ala NH), 6.79 (1H, bs,
Gly NH). 13C-NMR: 14.2, 18.5, 22.8, 26.0, 28.5, 28.7, 29.0, 31.9,
41.5, 65.9, 80.5, 100.2, 170.0, 173.0. IR (CHCl3, cm−1): 3320, 3088,
2958, 2932, 2859, 1753, 1715, 1668, 1531, 1455, 1392, 1367, 1291,
1250, 1172, 1068, 1048, 1028.
182DGA-GGGPQAG-OC7H15, 5. Boc-PQAG-OC7H15 was
deprotected by using 4 N HCl in dioxane for 1 h. 182DGA-GGG-
OH (0.40 g, 0.46 mmol), HCl·PQAG-OC7H15 (0.24 g, 0.46 mmol),
EDCI (0.10 g, 0.50 mmol) and HOBt (0.07 g, 0.50 mmol) were
suspended in CH2Cl2 (30 mL). To this mixture Et3N (0.19 mL)
was added. The mixture was stirred at 0 ◦C for 0.5 h and then
at rt for 48 h. The solvent was evaporated in vacuo and the
residue was crystallized from MeOH. The crude product was
chromatographed (silica gel, CHCl3 : CH3OH : HOAc 90 : 10 :
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1
0.1) to afford a solid (0.18 g, 31%). H-NMR: 0.88 (3H, t, J =
was 0.31 mM (same as chloride release experiment). The excitation
wavelength was 340 nm and the emission spectrum was recorded
between 300–600 nm (2.5 nm slit width, 400 nm min−1 scan speed,
average three scans).
6.4 Hz, CH2CH3), 1.26 (68H, pseudo-s, CH3(CH2)15CH2CH2N
and OCH2CH2(CH2)4CH3), 1.45–1.66 (9H, m, Ala CH3,
CH3(CH2)15CH2CH2N and OCH2CH2(CH2)4CH3), 1.98–2.53
(8H, m, Pro NCH2CH2CH2 and Gln CH2CH2CO), 3.09 (2H,
t, J = 7.2 Hz, CH3(CH2)15CH2CH2N), 3.28 (2H, t, J = 7.4 Hz,
CH3(CH2)15CH2CH2N), 3.45–3.55 (2H, m, Pro NCH2CH2CH2),
3.78–4.18 (13H, m, four Gly CH2, OCH2CH2(CH2)4CH3,
COCH2O and Pro CH), 4.32 (2H, s, COCH2O), 4.40–4.54 (2H,
m, Gln CH and Ala CH), 6.48 (1H, bs, Gln CONH2), 7.12 (1H,
bs, Gln CONH2), 7.50–7.60 (2H, m, two Gly NH), 7.68 (1H, d,
J = 7.5 Hz, Ala NH), 7.99 (1H, bt, Gly NH), 8.42 (1H, bt, Gly
NH), 8.84 (1H, bd, Gln NH). 13C-NMR: 14.3, 17.6, 22.8, 22.9,
25.2, 26.0, 27.1, 27.3, 27.8, 28.7, 29.0, 29.1, 29.5, 29.6, 29.8, 29.9,
31.9, 32.1, 41.4, 42.4, 43.3, 46.7, 47.2, 49.7, 54.1, 61.8, 65.6, 69.5,
71.6, 168.8, 169.3, 170.1, 170.3, 170.4, 171.3, 171.4, 173.0, 173.6,
177.4. IR (CHCl3, cm−1): 3302, 3077, 2923, 2854, 1747, 1660, 1544,
1466, 1378, 1260, 1201, 1130, 1028. Anal. calcd for C68H125N9O12:
C, 64.78; H, 9.99; N, 10.00%. Found: C, 64.80; H, 9.86; N, 10.11%.
Computational method
All calculations were performed using the Gaussian 03 suite
of computer programs. Geometry optimization and frequency
analysis were done by using density functional theory method
(B3LYP/6–31G*). All geometries were completely optimized in
the gas phase; the structures reported here are true minima on
the potential energy surfaces. This was confirmed by vibration
calculations without imaginary vibrational frequencies. For those
complexes which have multiple-conformations, several conforma-
tions have been calculated and the ones with lowest energy were
selected for the final structure.
Acknowledgements
Preparation of (C18H37)2NCOCH2OCH2CO-(Gly)3-Pro-Lys(e-N-
Boc)-(Gly)2-OC7H15, 3 and (C18H37)2NCOCH2OCH2CO-
(Gly)3-Pro-Lys(e-N-dansyl)-(Gly)2-OC7H15, 4
We thank the NIH for grants (GM 36262, GM 63190) for support
of this work.
Preparation of 3 and 4 have been reported previously.35
References
1 R. B. Gennis, Biomembranes: Molecular Structure and Function,
Springer Verlag, New York, 1989, p. 533.
Vesicle preparation and chloride release measurement
2 P. Yeagle, The structure of biological membranes, CRC Press, Boca
Phospholipid vesicles from 7 : 3 1,2-dioleyl-sn-glycero-3-phospho-
choline (DOPC) and 1,2-dioleyl-sn-glycero-3-phosphate mono-
sodium salt (DOPA, both from Avanti Polar Lipids) or DOPC
were prepared in the presence of an internal, chloride containing
buffer (600 mM KCl, 10 mM HEPES, adjusted to pH = 7).
After extrusion through a 200 nm filter and exchange of external
solution with a chloride-free buffer (400 mM K2SO4, 10 mM
HEPES, adjusted to pH = 7), vesicles were suspended in the same
external buffer (final phospholipid concentration about 0.31 mM).
An approximate vesicle size of 200 nm was confirmed by using a
particle analyzer. The electrode was introduced into the solution
and allowed to equilibrate. The voltage output was recorded
and after the baseline became flat, aliquots of the solution of
compound at study (9 mM in isopropanol) were added. Complete
lysis of the vesicles was induced by addition of a 2% aqueous
solution of Triton X100 (100 lL) and the data collected were
normalized to this value. The data were collected by Axoscope 9.0
using a DigiData 1322A series interface.
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