Toward biomimetic ion channels formed by rigid-rod molecules: Length-dependent ion-transport activity of substituted oligo(p-phenylene)s

Full text of DOI:10.1021/ja971513h

8726
J. Am. Chem. Soc. 1997, 119, 8726-8727
Toward Biomimetic Ion Channels Formed by
Rigid-Rod Molecules: Length-Dependent
Ion-Transport Activity of Substituted
Oligo(p-Phenylene)s
Naomi Sakai, Kevin C. Brennan, Linnea A. Weiss, and
Stefan Matile*
Department of Chemistry
Georgetown UniVersity
Washington, D.C. 20057-1227
Figure 1. Amphotericin B (AmB 1), composed of mycosamine
(“anchoring”) (A), polyene (“rigid-rod”) (B), and polyol (“relay”)
subunits (C).
ReceiVed May 12, 1997
Polyene macrolide antibiotics, e.g. amphotericin B (AmB 1,
Figure 1), are unique examples of nonpeptide natural products
which form ion channels in biomembranes.1,2 Three structural
subunits are essential for the formation of “barrel stave”-type
AmB aggregates: the mycosamine group (Figure 1A) “anchors”
AmB 1 at the bilayer/water interface, the hydrophobic polyene
rods (Figure 1B) allow cooperative van der Waals forces with
components of cell membranes, and the polyols (Figure 1C)
function as ionophoric “relays”. While numerous alternative
“anchors” and “relays” have been explored by means of
synthetic, nonpeptide ion channel models,3 specific hydrophobic
interactions have not received equal attention, even though they
apparently give rise to the cell membrane specificity of AmB
1. Here, as a first step toward artificial ion channels which
specifically recognize (bio)membranes by their thickness, we
report syntheses and activities of substituted oligo(p-phenylene)s
2-4 (Figure 2).
Figure 2. In-scale planar structures of membrane-bound oligo(p-
phenylene)s 2-4.
ecules because of the immense synthetic possibilities to modify
the hydrophobic skeleton, as well as their luminescence,
conductivity, and conformational flexibility along the long
molecular axis.
On the basis of the findings by Ourisson and co-workers,4
we hypothesized that the ion transport activity of substituted
rigid-rod molecules5,6 would be maximized if their length
matches the thickness of lipid bilayers. Shape and length of
the rigid-rod skeletons should further control their organization
in lipid bilayers, and the attachment of “relays” along a rigid-
rod skeleton may result in versatile, artificial ion channels.
Oligo(p-phenylene)s7 were selected as model rigid-rod mol-
Syntheses of oligomers 2-4 are shown in the Scheme 1.
Chelation-controlled lithiation of 3,3′-bianisole 5 with the
sterically demanding t-BuLi followed by iodination gave 6 and
7 as major products in a mixture of regioisomers. Oxidative
coupling of 6 with n-BuLi/CuCl2 furnished tetramer 8.7d,e For
the preparation of oligomers 9 and 10, dimer 6 was converted
into boronic acid 11. Suzuki coupling8 of 11 and bisiodo
derivative 7 afforded hexamer 9. Regioselectively iodinated
tetramer 12 was elongated identically to give 10. The methyl
groups of oligomers 8-10 were removed, and the resulting
oligophenols 13-15 were converted into acetonides 16-18 with
racemic tosylate 19. Acid-catalyzed deprotection yielded poly-
ols 2-4.
The initial evaluation of ion transport activities was made
with uniformly sized, small unilamellar vesicles (SUVs) com-
posed of fresh egg yolk phosphatidylcholine (EYPC). The
thickness of the hydrophobic part of EYPC bilayers, which
consist of ∼70% 16:0 and 18:1 PC, is thought to be nearly equal
to that of pure di-18:1 PC bilayers (36 Å).9 In contrast to
tetramer 2 (17 Å) and hexamer 3 (26 Å), octamer 4 (34 Å)
should thus be able to span this bilayer subunit (Figure 2).
EYPC-SUVs containing entrapped HPTS (8-hydroxypyrene-
1,3,6-trisulfonic acid) were prepared by the dialytic detergent
removal technique (100 mM HEPES, 100 mM NaCl, 0.05 mM
KCl, pH 7.0).10 Increase of extravesicular pH from 7.0 to 7.6
led to negligible changes in the emission of the intravesicular
HPTS.11 The presence of 20 µM tetramer 2 had a minor effect
on the intravesicular pH compared to the negative control
(1) (a) Bolard, J. Biochim. Biophys. Acta 1985, 864, 257. Recent leading
references include: (b) Yamashita, K.; Janout, V.; Bernard, E. M.;
Armstrong, D.; Regen, S. L. J. Am. Chem. Soc. 1995, 117, 6249. (c) Stadler,
E.; Dedek, P.; Yamashita, K.; Regen, S. L. J. Am. Chem. Soc. 1994, 116,
6677. (d) Bolard, J.; Legrand, P.; Heitz, F.; Cybulska, B. Biochemistry 1991,
30, 5707.
(2) For other nonpeptide natural products and their analogues of interest
see: (a) Sakai, N.; Matile, S. Tetrahedron Lett. 1997, 38, 2613. (b) Matile,
S.; Nakanishi, K. Angew. Chem., Int. Ed. Engl. 1996, 35, 757. (c) Deng,
G.; Dewa, T.; Regen, S. L. J. Am. Chem. Soc. 1996, 118, 8975.
(3) Gokel, G. W.; Murillo, O. Acc. Chem. Res. 1996, 29, 425.
(4) Milon, A.; Wolff, G.; Ourisson, G.; Nakatani, Y. HelV. Chim. Acta
1986, 69, 12.
(5) In contrast to their poorly explored role as membrane-related
biomaterials,6 rigid-rod molecules have attracted a great deal of attention
for the preparation of novel materials and as spacers to probe intramolecular
interactions. Selected recent references: (a) Maddux, T.; Li, W.; Yu, L. J.
Am. Chem. Soc. 1997, 119, 844. (b) Nuding, G.; Vo¨gtle, F.; Danielmeier,
K.; Steckhan, E. Synthesis 1996, 71. (c) Li, W.; Fox, M. A. J. Am. Chem.
Soc. 1996, 118, 11752. (d) Ayres, F. D.; Khan, S.; Chapman, O. L.
Tetrahedron Lett. 1994, 35, 8561. (e) Eaton, P. E.; Galoppini, E.; Gilardi,
R. J. Am. Chem. Soc. 1994, 116, 7588. (f) Kaszynski, P.; Friedli, A. C.;
Michl, J. J. Am. Chem. Soc. 1992, 114, 601. (g) Yang, X.; Jiang, W.;
Knobler, C. B.; Hawthorne, M. F. J. Am. Chem. Soc. 1992, 114, 9719. (h)
Feldman, K. S.; Bobo, J. S.; Ensel, S. M.; Lee, Y. B.; Weinreb, P. H. J.
Org. Chem. 1990, 55, 474.
(6) Lehn, J.-M. In Frontiers in Supramolecular Organic Chemistry and
Photochemistry; Schneider, H.-J., Du¨rr, H., Eds.; VCH: Weinheim, 1991;
p 1.
(8) Miyaura, N.; Yanagi, T.; Suzuki, A. Synth. Commun. 1981, 11, 513.
(9) (a) Caffrey, M.; Feigenson, G. W. Biochemistry 1981, 20, 1949. (b)
Kleinfeld, A. M. Curr. Top. Membr. Transp. 1987, 29, 1.
(10) (a) Zumbu¨hl, O.; Weder, H. G. Biochim. Biophys. Acta 1981, 640,
252. (b) Mimms, L. T.; Zampighi, G.; Nozaki, Y.; Tanford, C.; Reynolds,
J. A. Biochemistry 1981, 20, 833.
(11) (a) Venema, K.; Gibrat, R.; Grouzis, J.-P.; Grignon, C. Biochim.
Biophys. Acta 1993, 1146, 87. (b) Kano, K.; Fendler, J. H. Biochim. Biophys.
Acta 1978, 509, 289.
(7) Some pertinent references on oligo(p-phenylene)s: (a) Rajca, A.;
Safronov, A.; Rajca, S.; Shoemaker, R. Angew. Chem., Int. Ed. Engl. 1997,
36, 448. (b) Keegstra, M. A.; De Feyter, S.; De Schryver, F.; Mu¨llen, K.
Angew. Chem., Int. Ed. Engl. 1996, 35, 775. (c) Baker, K. N.; Fratini, A.
V.; Resch, T.; Knachel, H. C.; Adams, W. W.; Socci, E. P.; Farmer, B. L.
Polymer 1993, 34, 1571. (d) Rinke, M.; Gu¨sten, H.; Ache, H. J. J. Phys.
Chem. 1985, 90, 2661. (e) Heitz, W.; Ullrich, R. Makromol. Chem. 1966,
98, 29.
S0002-7863(97)01513-8 CCC: $14.00 © 1997 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 119, No. 37, 1997 8727
Scheme 1a
Figure 4. Same experiments (A) including schematic representation
(B) as in Figure 3 for 20 µM octamer 4 and AmB 1 in the absence
(curves a, b) and presence (curves c, d) of 10 µM FCCP (curve e: 40
µL of MeOH).
M+ or OH-/Cl- exchange (Figure 3B) or by the formation rate
of the active suprastructures.14 For instance, the 57-fold increase
in ion flux in the presence of the proton carrier FCCP (carbonyl
cyanide 4-(trifluoromethoxy)phenylhydrazone) indicates that
proton efflux limits the rate of AmB mediated ion exchange
(Figure 4, curves b and d).1d In contrast, FCCP did not
significantly affect the activity of octamer 4; proton transport
is therefore not limiting the rate of intravesicular pH change
(Figure 4, curves a and c).13
Although partition coefficients obtained by reverse-phase
HPLC methods should be interpreted with caution,15 the nearly
identical retention times of 2 (6.86 min), 3 (6.73 min), and 4
(6.78 min) on a C18 column with methanol as the mobile phase
should originate from roughly equal hydrophobic parameters.
The observed 2 < 3 < 4 transport activity of the three oligomers
thus arises with all likelihood from differences in length and
not from unequal partition coefficients.
a (a) (1) t-BuLi; (2) I2, Et2O. Yields: 7, 4%; 6, 34%. (b) n-BuLi,
CuCl2. Yield: 64%. (c) (1) n-BuLi; (2) B(O-i-Pr)3; (3) HCl. Yield:
75%. (d) Pd(PPh3)4, Na2CO3. Yield: 77%. (e) (1) t-BuLi; (2) I2.
Yield: 29%. (f) See d. Yield: 66%. (g) BBr3. (h) 19, Cs2CO3. Yield:
82% from 8. (i) TFA. Yield: 85%. (j) See h. Yield: 21% from 9. (k)
TFA. Yield: 88%. (l) See h. Yield: 25% from 10. (m) TFA. Yield:
92%.
In conclusion, we have demonstrated that rigid-rod molecules
carrying hydrophilic substituents facilitate ion transport across
lipid bilayers. The ion flux rate mediated by octa(p-phenylene)
4, having a length of 34 Å which nearly matches the hydro-
phobic part of EYPC bilayers, exceeds that of hexamer 3 (26
Å) by a factor of 3.0, while the tetramer 2 (17 Å) is almost
inactive. The mechanism of transport remains to be elucidated,
although the significant length dependence disfavors a mobile
carrier mechanism, and the induction of membrane defects in a
detergent-like manner is unlikely.13 Vigorous studies on
transport mechanism and active suprastructure of substituted
rigid-rod oligomers are ongoing.
Acknowledgment. We thank the donors of the Petroleum Research
Fund, administered by the American Chemical Society, and Georgetown
University for support of this work. We also thank Dr. Karel Ubik
from the Academy of Science of the Czech Republic for measuring
the mass spectra.
Figure 3. (A) Change in fluorescent intensity ((It - I0)/(I∞
-
I0)×100%) of vesicle-entrapped HPTS as a function of time after the
addition of 40 nmoles of octamer 4 (curve a), hexamer 3 (curve b),
tetramer 2 (curve c), and 40 µL of MeOH (curve d, negative control)
followed by 40 µL of 1.2% triton X-100 (pHin ) 7.0, pHout ) 7.6);
excitation at 460 nm, emission at 510 nm. (B) Schematic representation
of the transport experiments.
Supporting Information Available: Experimental procedures and
physical data of product(s), vesicle preparation, and HPTS-assay (7
pages). See any current masthead page for ordering and Internet access
instructions.
(Figure 3, curves c and d). Increased ion flux rates were
observed with hexamer 3 (k ) 4.6 × 10-4 s-1) and octamer 4
(k ) 13.8 × 10-4 s-1, Figure 3, curves a and b). Under these
conditions, octamer 4 transports ions across an EYPC bilayer
3.8 times faster than AmB 1 (k ) 3.6 × 10-4 s-1, Figure 4,
curve b).12
The above method measures the change in intravesicular pH
assuming HPTS leakage caused by the rigid-rod molecules 2-4
does not occur.13 The observed rate of internal pH change is
limited either by the slowest ion transport rate of facilitated H+/
JA971513H
(13) Preliminary results show that the ion flux rate mediated by octamer
4 strongly increases in the presence of small amounts of the K+ carrier
valinomycin (e.g., 16-fold with 12 nM valinomycin). This effect implies
that octamer 4 acts as a proton/cation exchanger with H+ > K+ selectivity
and not as a detergent-like defect inducer; dye-release experiments fully
corroborate the latter implication.
(14) Herve´, M.; Cybulska, B.; Gary-Bobo, C. M. Eur. Biophys. J. 1985,
12, 121.
(15) Leo, A. J. Methods Enzymol. 1991, 202, 544.
(12) The corresponding octamethoxy derivative 10 and octaphenol 15
were completely inactive; octaacetonide 18 had minor activity.