Recognition of polarized lipid bilayers by p-oligophenyl ion channels: From push-pull rods to push-pull barrels

Article abstract of DOI:10.1021/ja017497c

Design, synthesis, and evaluation of 1⁴-methoxy-8⁴-methylsulfonyl-2²,3³,4²,5³,6²,7³-hexa(Gla-Leu-Lys-Leu-NH₂)-p-octiphenyl (1) and 1⁴,8⁴

Full text of DOI:10.1021/ja017497c

Published on Web 01/26/2002
Recognition of Polarized Lipid Bilayers by p-Oligophenyl Ion Channels: From
Push-Pull Rods to Push-Pull Barrels
Naomi Sakai* and Stefan Matile*
Department of Organic Chemistry, UniVersity of GeneVa, CH-1211 GeneVa, Switzerland
Received November 9, 2001
The rational design of synthetic ion channels has seen remarkable
progress during the last two decades.1 The construction of synthetic
ion channels that act selectively, however, remains a central
challenge in bioorganic chemistry. Recognition of bilayer mem-
branes by specific characteristics such as thickness,2 charge,3
specific components,4 and potential5 is an aspect of ion-channel
selectivity with medicinal relevance. To elucidate the importance
of dipole-potential interactions in the recognition of highly polarized
membranes (e.g., bacterial plasma membranes) by electronically
asymmetric “rods” (e.g., R-helical peptides), we have recently
developed a strategy to turn the axial dipole of rigid p-octiphenyl
scaffolds “on” and “off” without major changes in global structure.6
Although the expected increase in activity was observed with push-
pull ionophores 1 and 2 in polarized bilayer membranes, their
selectivity and, in particular, their activity were quite modest (Figure
1). In a different line of research, we found that p-octiphenyls
equipped with eight lateral peptide strands of the sequence GLKL-
NH2 can self-assemble into hexameric rigid-rod â-barrels 3 with
high ion-channel activity.7 In this report, we unify the activity of
synthetic rigid-rod â-barrels and the selectivity of push-pull rods
in push-pull â-barrel 4vvvv
.
Push-pull hexa(GLKL-NH2)-p-octiphenyl 4 was synthesized
from hexa(GOtBu)-p-sexiphenyl 5 (Scheme 1).6a Suzuki-coupling
with methyl ether 6 and methyl sulfide 7 gave p-octiphenyls 8, 9
and 10. The axial dipole in rod 9 was “turned on” by oxidation of
the sulfide π donor into the sulfone π acceptor in 11. After
deprotection, lateral peptide strands were introduced by the coupling
with tripeptide 12,7a and removal of the Boc groups in p-octiphenyl
13 provided the desired push-pull rod 4. The symmetric push-
push rod 14 was prepared from p-octiphenyl 10 along the same
route.6b
Figure 1. Push-pull rods1 and 2, rigid-rod â-barrel 3, and parallel/
antiparallel push-pull â-barrels 4vvvv/4vVvV. â-Sheets are given as arrows
pointing to the C terminus, amino acid residues (one-letter abbreviation, G
) -OCH2CO-) pointing toward the barrel exterior are black on white,
internal residues white on black.
Activity and selectivity of push-pull rod 4 were assessed with
small unilamellar vesicles (SUVs) composed of fresh egg yolk
phosphatidylcholine (EYPC). In the employed assay, changes in
transmembrane pH gradient and potential were simultaneously
monitored by entrapped pH-sensitive fluorophore HPTS, and
potential-sensitive dye safranin O, respectively (Figure 2a).6a An
inside negative Nernst potential was applied by addition of
potassium carrier valinomycin to vesicles with a transmembrane
potassium concentration gradient and quantified by comparing the
emission intensity of safranin O with calibration curves (see
Supporting Information). Application of a pH gradient by extra-
vesicular addition of base completed preparation of the system for
the evaluation of push-pull rod 4.
Information). The activity of push-pull rod 4 in the presence of
increasingly polarized membranes exhibited an exponential increase
(Figure 2c, b). Application of eq 18
Gvd ) g0 exp(zgeE/kT)
(1)
where Gvd is the observed activity, e the elementary charge, k the
Boltzmann constant, and T the absolute temperature, gave a gating
charge zg ) 0.85/channel for push-pull rod 4. A gating charge zg
) 1.2/channel reported for melittin8b,c suggested that cell membrane
recognition by push-pull rods may compare well with that by
R-helix bundles which comprise invariable axial dipoles due to
uniform orientation of the backbone amides.5
This substantial potential dependence was consistent with
quantitative amplification of the axial dipole of push-pull rod 4
by self-assembly into parallel push-pull â-barrel 4vvvv in the presence
of polarized bilayer membranes (Figure 1). Self-assembly into
Figure 2b shows the differential changes in HPTS emission after
addition of push-pull rod 4 to polarized spherical bilayers. The
molecular origin of these changes (i.e., HPTS efflux, ion selectivity,
etc.) was not determined; absence of lysis was, however, demon-
strated by the unchanged membrane potential (see Supporting
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1184 VOL. 124, NO. 7, 2002 J. AM. CHEM. SOC.
10.1021/ja017497c CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1 a
kobs ) k0 + kint [monomer]n/KD
(2)
where KD is the dissociation constant, kobs the observed transport
rate, k0 the rate without pore, and kint an intrinsic rate constant,
suggested that n ) 3.9 ( 1.6 monomers 4 are required for
thermodynamically unfavorable self-assembly of an active pore 4n.
Since push-pull rod 2 exhibited linear concentration dependence6a
in agreement with a monomeric active structure, it was not possible
to compare the activity of tetramer 4vvvv and monomer 2 quantita-
tively. The different concentrations needed to detect substantial
activity, i.e., 100 nM tetramer 4vvvv (Figure 2d) versus 10 µM
monomer 2, however, may illustrate an increase that extends clearly
beyond 2 orders of magnitude.
In summary, we show that push-pull â-barrels recognize
polarized bilayer membranes at nanomolar concentrations with a
gating charge of zg ) 0.85 per channel. Together with fourth-order
dependence of activity on monomer concentration, these findings
identify parallel self-assembly in polarized membranes as a powerful
strategy to amplify the selectivity and activity of ion channels
formed by rigid push-pull rods. Comprehensive, ongoing studies
on structure and function of push-pull â-barrels in planar and
spherical bilayer membranes and in solution support the conclusions
made in this communication and will be reported in due course.
a (a) Pd(PPh)3, Na2CO3, 24% (8), 51% (9), 20% (10). (b) 1. mCPBA, 2.
KMnO4, MgSO4, 65%. (c) 1. TFA, 2. 12, HBTU, TEA, 47%. (d) 1. TFA,
2. 12, HBTU, TEA, 58%. (e) TFA, 94%. (f) TFA, 78%.
Acknowledgment. We thank A. Pinto, J.-P. Saulnier, and the
group of F. Gu¨lac¸ar for NMR and MS, respectively, and the Swiss
NSF (21-57059.99 and 2000-064818.01) for financial support.
Supporting Information Available: Preparation of new compounds
and depolarization assays (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
References
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2524. (b) Push-push (14) rather than pull-pull rods were selected because
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controls with pull-pull 14,84-dicyano-p-octiphenyls have been reported.5a,6a
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Figure 2. Activity of p-octiphenyl 4 in polarized EYPC-SUVs. (a)
Schematic presentation of the assay (see text). (b) Representative differential
changes in fluorescence intensity I of HPTS (λem ) 510 nm, λex ) 450/
405 nm) within EYPC-SUVs (pH 6.4, inside: 10 mM KmHnPO4, 100 mM
KCl, 90 µM HPTS; outside: n/10 mM KmHnPO4, 10 - n/10 mM
NamHnPO4, n mM KCl, 100 - n mM NaCl, n ) 2.02 (-100 mV), 0.425
(-140 mV), 0.195 (-160 mV), 0.09 (-180 mV)) as a function of time
during addition of safranin O (6 nM), NaOH (5 mM), valinomycin (0.6
µM), 4 (A; 1 µM) and excess melittin (B; I∞). Curves are obtained at (from
top to bottom) -180, -160, -140, and -100 mV. (c) Dependence of
activity (e.g., % emission change 100 sec after rod addition) of 4 (1 µM,
b) and 14 (1 µM, O) on membrane potential with exponential (solid) and
linear (dotted) curve fit. (d) Dependence of activity on the concentration of
4 at E ) -180 mV. Experimental details: see Supporting Information.
antiparallel tetramers 4vVvV without axial dipole, expected to be
favored in isotropic media, can thus be excluded in polarized
membranes. The near independence of the activity of push-push
rod 14 on membrane polarization confirmed that the dipole moments
of peptide backbone amides are canceled out in rigid-rod â-barrels
as expected for â-sheet conformation (Figure 2c, O).
Formation of a tetrameric supramolecule 4vvvv was indicated at
-180 mV by the nonlinear dependence of activity on the
concentration of monomeric push-pull rod 4 (Figure 2d). The best
curve fit to eq 22a,3a,4a
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