Rigid push-pull oligo(p-phenylene) rods: Depolarization of bilayer membranes with negative membrane potential [16]

Full text of DOI:10.1021/ja991133r

J. Am. Chem. Soc. 1999, 121, 7961-7962
7961
Rigid Push-Pull Oligo(p-Phenylene) Rods:
Depolarization of Bilayer Membranes with Negative
Membrane Potential
Jean-Yves Winum and Stefan Matile*,†
Department of Chemistry, Georgetown UniVersity
Washington, D.C. 20057
ReceiVed April 12, 1999
In light of steadily emerging multi-drug-resistant bacteria,1 the
molecular mechanism of natural antibiotics is of high interest for
the development of new routes toward antimicrobials that can be
hoped to cause minimal microbial resistance.2 Many natural
antibiotics are cationic, R-helical peptides that presumably act
by recognizing and depolarizing anionic, highly polarized bacterial
cell membranes.2 The alignment of their dipole moment with
negative membrane potentials in particular has been proposed to
be critical for their specificity.2,3 To study this central aspect of
the molecular recognition mechanism of natural antibiotics, one
would need the development of novel functional biomimetics3,4
with permanently oriented dipole moments that can be systemati-
cally Varied in magnitude without significant modification of the
global structure of the synthetic model. As an example of the
usefulness of rigid-rod molecules to address bioorganic topics of
current concern,5 we here report the design, synthesis, and
evaluation of push-pull rigid-rod ionophore 1 that is, in clear
contrast to octi(p-phenylene) 2 with nearly identical structure but
Figure 1. Structure of push-pull (1) and pull-pull (2) rigid-rod
ionophores; f, permanently fixed dipole moments; O, 18-azacrown-6.
The rigid-rod molecules 1 and 2 are composed of three subunits.
Octi(p-phenylene) scaffolds were selected because they adapt
transmembrane orientation in hydrophobically matching bilayer
membranes (Figure 1).5b,c,f,g The terminal cyano and methoxy
groups were chosen as π acceptors (µ ≈ 3.9 D) and donors (µ ≈
1.3 D),6 respectively, to control magnitude and orientation of the
permanently fixed dipole moments in 1 and 2. 18-Azacrown-6
was placed as a lateral side chain because its capacity as ion-
transporting “relays” is well established.4
Scheme 1a
without permanently fixed dipole moment, capable of depolarizing
bilayer membranes with negative membrane potential (Figure 1).
† Present address: Department of Organic Chemistry, University of Geneva,
CH-1211 Geneva 4, Switzerland.
(1) (a) Niccolai, D.; Tarsi, L.; Thomas, R. Chem. Commun. 1997, 2333.
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(4) Reviews on nonpeptide ion channels and channel models: (a) Gokel,
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Straaten-Nijenhuis, W. F. In ComprehensiVe Supramolecular Chemistry;
Reinhouldt, N. D., Ed.; Elsevier: Oxford, U.K., 1996; Vol. 10, p 53. (c) Voyer,
N. Top. Curr. Chem. 1996, 184, 1. Some recent references: (d) Li, C.; Budge,
L. P.; Driscoll, C. D.; Willardson, B. M.; Allman, G. W.; Savage, P. B. J.
Am. Chem. Soc. 1999, 121, 931. (e) Fritz, M. G.; Walde, P.; Seebach, D.
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Prados, P.; Meadows, E. S.; Gokel, G. W. Angew. Chem., Int. Ed. Engl. 1998,
37, 1534. (h) Fyles, T. M.; Loock, D.; Zhou, X. J. Am. Chem. Soc. 1998,
120, 2997. (i) Meillon, J.-C.; Voyer, N. Angew. Chem., Int. Ed. Engl. 1997,
36, 967. (j) Pechulis, A. D.; Thompson, R. J.; Fojtik, J. P.; Schwartz, H. M.;
Lisek, C. A.; Frye, L. L. Bioorg. Med. Chem. 1997, 5, 1893. (k) Wagner, H.;
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a (a) Pd(PPh3)4, Na2CO3, 10%; (b) BBr3; (c) tert-butyl bromoacetate,
Cs2CO3, 46% from 5; (d) p-methoxyphenylboronic acid, Pd(PPh3)4,
Na2CO3, 40% (conversion yield, 54%; dimethoxy octamer, 26%); (e)
p-cyanophenylboronic acid, Pd(PPh3)4, Na2CO3, 34%; (f) TFA/CH2Cl2;
(g) 18-azacrown-6, PyBOP, DIPEA, 57% from 9; (h) see (e) 35%
(conversion yield: 50%); (i) BBr3; (j) see (c), 42% from 6; (k) TFA/
CH2Cl2; (l) see (g), 42% from 13.7
The synthesis of 1 and 2 was more difficult than expected
(Scheme 1). In contrast to that of shorter oligo(p-phenylene)s,5h
(5) (a) Sakai, N.; Majumdar, N.; Matile, S. J. Am. Chem. Soc. 1999, 121,
4294. (b) Ghebremariam, B.; Sidorov, V.; Matile, S. Tetrahedron Lett. 1999,
40, 1445. (c) Tedesco, M. M.; Ghebremariam, B.; Sakai, N.; Matile, S. Angew.
Chem., Int Ed. Engl. 1999, 38, 540. (d) Sakai, N.; Ni, C.; Bezrukov, S. M.;
Matile, S. Bioorg. Med. Chem. Lett. 1998, 8, 2743. (e) Ghebremariam, B.;
Matile, S. Tetrahedron Lett. 1998, 39, 5335. (f) Ni, C.; Matile, S. Chem.
Commun. 1998, 755. (g) Weiss, L. A.; Sakai, N.; Ghebremariam, B.; Ni, C.;
Matile, S. J. Am. Chem. Soc. 1997, 119, 12142. (h) Sakai, N.; Brennan, K.
C.; Weiss, L. A.; Matile, S. J. Am. Chem. Soc. 1997, 119, 8726. (i)
Ghebremariam, B.; Matile, S. Enantiomer 1999, 4, 127 and 139.
10.1021/ja991133r CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/13/1999

7962 J. Am. Chem. Soc., Vol. 121, No. 34, 1999
Communications to the Editor
the diiodo tetraester 7 with p-methoxyphenylboronic acid gave
heptamer 8 in excellent 54% conversion yield and subsequent
coupling of 8 with p-cyanophenylboronic acid yielded octamer
9. Cleavage of ester 9 with TFA and coupling of acid 10 with
18-azacrown-6 gave ionophore 1.7
The capacity of rigid-rod ionophores 1 and 2 to depolarize
polarized small unilamellar vesicles (SUVs) composed of egg yolk
phosphatidylcholine (EYPC) was assessed by double-channel
fluorescence kinetics using the membrane potential sensitive
safranin O (excitation: 522 nm, emission: 581 nm)8 as an
extravesicular probe and the pH sensitive 8-hydroxypyrene-1,3,6-
trisulfonic acid (HPTS, excitation: 450 nm, emission: 510
nm)5a,c,f-h as an intravesicular fluorescence probe. Uniformly sized
EYPC-SUVs in saline phosphate buffer, pH 6.4, with internal
HPTS and KCl (100 mM) and external safranin O (60 nM), KCl
(36 µM), and NaCl (100 mM) were prepared by the dialytic
detergent removal method described before.5c,g,7 External addition
of the selective K+ carrier valinomycin established an inside
negative membrane potential that is, according to Nernst′ equa-
tion,7 comparable to that of bacteria (-200 mV).2c The assay
system was completed by the application of a pH gradient, and
both membrane potential (Figure 2A, curves a-c) and intra-
vesicular pH (Figure 2A, curves d, e) were subsequently moni-
tored by changes of the emission intensity of safranin O and
HPTS, respectively. The presence of 10 µM ionophore 2 affected
neither membrane potential nor proton gradient significantly
(Figure 2A, curves b and e). In clear contrast, push-pull rod 1
rapidly depolarized the EYPC-SUVs (Figure 2C and A, curve
c) and, after depolarization, caused pH gradient collapse (Figure
2D and A, curve d).
Control experiments showed that both ionophores have identical
activity to mediate intravesicular pH changes in unpolarized
EYPC- and anionic EYPG-SUVs (EYPG ) egg yolk phosphati-
dylglycerol).7 The increased activity of push-pull rod 1 in
polarized SUVs can thus be unambiguously attributed to its
permanently fixed dipole moment. Although other explanations
are possible,3b the presence of a push-pull scaffold is likely to
increase transmembrane binding to polarized bilayers due to
favorable alignment of the fixed dipole with the membrane
potential3c that results in the recognition of polarized membranes.
The delayed pH gradient collapse (Figure 2D and A, curve d vs
e) further demonstrated that push-pull rod 1 acts with M+ > H+
selectivity and not by lysis.
Figure 2. Activity of push-pull (1) and pull-pull (2) rods in polarized
EYPC-SUVs. (A) RepresentatiVe curves for simultaneously detected
changes of membrane potential (emission of external safranin O at 581
nm; λex 522 nm, curves a-c) and pH gradient (emission of internal HPTS
at 510 nm; λex 450 nm, curves d and e) during the addition of valinomycin
(6 nM), base (NaOH, ∆pH 1), sample (curve b, e, 10 µM 2; curve c, d,
10 µM 1; curve a, negative control) and melittin as a function of time.
All curves were normalized to 100% at 0 s and 0% at 320 s; in curves
d and e, the negative control curves were subtracted after normalization.7
(B-D) Schematic representation for ion flux mediated by push-pull rod
1. Addition of 1 to doubly labeled, polarized SUVs with low internal pH
(B) resulted in membrane depolarization by K+/Na+ antiport (C) followed
by H+ gradient collapse presumably due to H+/Na+ antiport (D); less
likely transport mechanisms are not shown for clarity.
In summary, we have shown that a permanent dipole moment
along a hydrophobically matching rigid-rod scaffold as in 1 is
sufficient for recognition and depolarization of polarized bilayer
membranes. These findings imply the promise of push-pull rods
as potential antimicrobials.
Acknowledgment. We are grateful to L. A. Weiss for experimental
assistance and to Dr. K. Lu and C. Ladd (University of Maryland, College
Park) for measuring the mass spectra. This research was supported by
NIH (GM56147), an award from Research Corporation, the donors of
the Petroleum Research Fund, administered by the American Chemical
Society, Suntory Institute for Bioorganic Research (SUNBOR Grant),
and Georgetown University.
iodination of the termini of substituted sexi(p-phenylene)s is
hampered by poor solubility and regioselectivity. However, Suzuki
coupling of an excess of diiodo dimer 35e,h with diboronic acid 4
provided direct access to the key intermediate 5 in 10% yield.
Coupling of diiodo hexamer 5 with p-cyanophenylboronic acid
gave octamer 6, which was readily converted into a pull-pull
ionophore 2 following our previously established protocols,5a,h
but the intermediate heptamer needed to prepare octi(p-phen-
ylene)s with different termini was not observed. This problem
was solved by attaching the “solubilizing” tert-butyl glycolate
side chain5a on the hexamer stage. Indeed, Suzuki coupling of
Supporting Information Available: Experimental procedures, vesicle
preparation, and flux assays. This material is available free of charge via
the Internet at http://pubs.acs.org.
JA991133R
(7) See Supporting Information.
(8) (a) Woolley, G. A.; Deber, C. M. Biopolymers 1989, 28, 267. (b)
Woolley, G. A.; Kapral, M. K.; Deber, C. M. FEBS Lett. 1987, 224, 337.
(6) McClellan, A. L. Tables of experimental dipole moments; W. H.
Freeman and Co.: San Francisco, CA, 1963; pp 233 and 254.