Self-assembled rigid-rod ionophores

Full text of DOI:10.1021/ja983893s

4294
J. Am. Chem. Soc. 1999, 121, 4294-4295
Self-Assembled Rigid-Rod Ionophores
Naomi Sakai, Nirmalya Majumdar, and Stefan Matile*
Department of Chemistry, Georgetown UniVersity
Washington, D.C. 20057
ReceiVed NoVember 10, 1998
ReVised Manuscript ReceiVed March 15, 1999
During the course of recent efforts to explore the scope and
limitations of rigid-rod molecules as versatile biomimetics,1 we
have observed giant ion channels formed at high voltages in planar
lipid bilayers with a lifetime of less than 0.1 ms (Figure 1A).1c
The proposed structure of this voltage-dependent ion channel, i.e.,
self-assembly 1n, could not be further studied because of its
transient nature, and design and synthesis of more stable analogues
was essential to determine structure and activity of toroidal rigid-
rod supramolecules. The use of interdigitating peptidic side chains
to establish â-sheets between rigid-rod scaffolds (e.g., 2n, Figure
1A) was particularly inviting because many biological toroidal
supramolecules have been shown to consist of â-barrels,2 and
cylindrical â-barrels formed by self-assembly of synthetic circular
peptides have been studied extensively.3 Most importantly, the
feasibility to create supramolecular, antiparallel â-sheets by
interdigitation has been demonstrated in an elegant model study,4
and the formation of â-sheets in, e.g., 2n is further likely to occur
with high cooperativity5 because of the preorganizing rigid-rod
scaffold. It is further interesting to note that despite their functional
versatility in biological systems, â-barrels have attracted little
attention for biomimetic modeling of complex biomolecules such
as ion channel proteins.1,3,6 Here we wish to report a model study
to define the minimal requirements for â-sheet formation inbe-
tween rigid-rod scaffolds, focusing on self-assembled, presumably
dimeric rigid-rod ionophore 22 (Figure 1B).7
Figure 1. (A) Hypothetical toroidal rigid-rod supramolecules with lateral
side chains containing vicinal diols (1n)1c or peptides (2n). (B) Self-
assembled dimer 22.
Scheme 1a
The rigid-rod octamers 2 and 3 were prepared from octa- and
tetraanisoles 41a and 5,1b respectively (Scheme 1). While tetra-
Leu 3 was readily prepared from tetraanisole 5 by aryl ether
cleavage, conversion of tetraphenol 6 to tetraglycolate 7, ester
hydrolysis, and amide formation of the resulting tetraacid 8, severe
solubility problems with octamethylglycolate 9 required modifica-
tions of this sequence to prepare octa-Leu 2. In sharp contrast to
methylglycolate 9, the corresponding tert-butyl ester 10 (obtained
from octaphenol 11 and tert-butyl bromoacetate) exhibited
a Key: (a) BBr3; (b) methyl bromoacetate, Cs2CO3, 76% from 5; (c)
1 M aqueous NaOH; (d) H-Leu-NH2, EDC, HOBt, NEt3, DMF, 68%
from 7; (e) BBr3; (f) see footnote b, 55% from 4; (g) tert-butyl
bromoacetate, Cs2CO3, 62% from 4; (h) TFA/CH2Cl2; (i) see footnote d,
14% from 10.
(1) (a) Weiss, L. A.; Sakai, N.; Ghebremariam, B.; Ni, C.; Matile, S. J.
Am. Chem. Soc. 1997, 119, 12142. (b) Ghebremariam, B.; Matile, S.
Tetrahedron Lett. 1998, 39, 5335. (c) Sakai, N.; Ni, C.; Bezrukov, S. M.;
Matile, S. Bioorg. Med. Chem. Lett. 1998, 8, 2743 and references therein.
(2) (a) Henning, M.; D’Arcy, A.; Hampele, I. C.; Page, M. P. G. Nature
Struct. Biol. 1998, 5, 357. (b) Kim, K. K.; Kim, R.; Kim, S.-H. Nature 1998,
394, 595. (c) Song, L.; Hobaugh, M. R.; Shustack, C.; Cheley, S.; Bayley,
H.; Gouaux, J. E. Science 1996, 274, 1859 and references therein.
(3) Clark, T. D.; Buriak, J. M.; Kobayashi, K.; Isler, M. P.; McRee, D. E.;
Ghadiri, M. R. J. Am. Chem. Soc. 1998, 120, 8949 and references therein.
(4) LaBrenz, S. R.; Kelly, J. W. J. Am. Chem. Soc. 1995, 117, 1655.
(5) (a) Sharman, G. J.; Searle, M. S. J. Am. Chem. Soc. 1998, 120, 5291.
(b) Schenck, H. L.; Gellman, S. H. J. Am. Chem. Soc. 1998, 120, 4869. (c)
Kortemme, T.; Ramirez-Alvarado, M.; Serrano, L. Science 1998, 281, 253.
(6) For selected examples of artificial ion channels/channel models, see:
(a) Merritt, M.; Lanier, M.; Deng, G.; Regen, S. L. J. Am. Chem. Soc. 1998,
120, 8494. (b) de Mendoza, J.; Cuevas, F.; Prados, P.; Meadows, E. S.; Gokel,
G. W. Angew. Chem., Int. Ed. Engl. 1998, 37, 1534. (c) Fyles, T. M.; Loock,
D.; Zhou, X. J. Am. Chem. Soc. 1998, 120, 2997. (d) Meillon, J.-C.; Voyer,
N. Angew. Chem., Int. Ed. Engl. 1997, 36, 967. (e) Lear, J. D.; Schneider, J.
P.; Kienker, P. K.; DeGrado, W. F. J. Am. Chem. Soc. 1997, 119, 3212. (f)
Wagner, H.; Harms, K.; Koert, U.; Meder, S.; Boheim, G. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 2643. (g) Tanaka, Y.; Kobuke, Y.; Sokabe, M. Angew.
Chem., Int. Ed. Engl. 1995, 34, 693.
excellent physical properties and could be cleaved under mild
conditions to give octaacid 12. However, the final rigid-rod octa-
Leu 2 was again difficult to purify, presumably due to the
ionophoric properties of its dimer 22 (see below), and extensive
exposure of the product to PTLC and RP-HPLC accounts for the
low yield of the final step.
Although the oligomeric nature of 2 limited the applicability
of NMR spectroscopy, 2D 1H,1H-COSY NMR experiments (D2O/
CD3OD) allowed us to assign the C(R)-H resonances at 4.32-
4.52 ppm. Their downfield shift with respect to random coil values
(4.17 ( 0.1 ppm)8 is indicative for, in this case, self-assembly
by intermolecular â-sheet formation, because intramolecular
â-sheet formation along the rigid-rod scaffold is sterically unlikely.
The negative Cotton effect (CE) at 216 nm in the concentration-
(7) For selected examples of self-assembled ionophores, see: (a) Davis, J.
T.; Tirumala, S. K.; Marlow, A. L. J. Am. Chem. Soc. 1997, 119, 5271. (b)
Gottarelli, G.; Masiero, S.; Spada, G. P. J. Chem. Soc., Chem. Commun. 1995,
2555. (c) Schepartz, A.; McDevitt, J. P. J. Am. Chem. Soc. 1989, 111, 5976.
(8) Wishart, D. S.; Sykes, B. D.; Richards, F. M. Biochemistry 1992, 31,
1647.
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Communications to the Editor
J. Am. Chem. Soc., Vol. 121, No. 17, 1999 4295
Figure 3. Change in fluorescent intensity of EYPC-SUV entrapped
HPTS as a function of time after the addition of 2 (solid; curve a: 1 µM,
curve b: 500 nM, curve c: 100 nM), 3 (dotted; curve d: 30 µM), and
melittin (dashed; curve e: 30 nM) followed by 20 µL of 0.5 M NaOH
(pHout ) 7.4, pHin ) 6.4; 10 mM Na2HPO4/NaH2PO4, 100 mM NaCl).
Intravesicular pH was monitored ratiometrically [It ) Ia (λem ) 510 nm,
λex ) 460 nm)/Ib (λem ) 510 nm, λex ) 405 nm)] and normalized {[(It -
I0)/(I∞ - I0)] × 100%}. I∞ was determined by adding 50 µL of 1.2%
Triton X-100.
concentrations were sufficient to mediate rapid intravesicular pH
changes (Figure 3). Ion transport by 2 was >75-times more and
∼3-times less efficient than that by 3 and the pore forming
melittin, respectively. The linear dependence of transport rates
with respect to the concentration of 2 is consistent with the
incorporation of preformed dimer 22 as indicated by the spectro-
scopic data.
The ionophoric properties of 2, implied by ESI-MS as well as
transport experiments, were further corroborated by picrate
extraction.7 Comparison of the relative concentrations of picrate
and oligophenylene 2 after extraction of solid sodium picrate into
CDCl3 containing 3.8 mM 2 revealed a Na+/2 ratio of 1.2 ( 0.1.
This indicates that 2 to 3 cations are bound within the â-barrel
of dimer 22, which thus may represent an intriguing self-assembled
“cation-wire”13 capable of spanning the hydrophobic core of
EYPC bilayers.
Figure 2. (A) CD spectra of octa-Leu 2 (solid) and tetra-Leu 3 (dashed).
(B) ESI-MS of 2 (MeOH/0.1% HCOOH).
independent circular dichroism (CD) spectra of 2 further supported
â-sheet formation and thus self-assembly in, e.g., water as well
as egg yolk phosphatidylcholine (EYPC) bilayers.9 Bisignate CEs
centered around the 1La/1Lb absorptions at 312 nm1a implied
exciton coupling, i.e., stereoselective organization of the rigid-
rod chromophore (Figure 2A).10
In light of mounting evidence for the identity of gas-phase
complexes seen in ESI-MS with those in solution,11 the four major
peaks for 2 strongly supported the presence of ionophoric dimers
(22) and the absence of trimeric or higher oligomers in solution
(2n, Figure 2B). The increasingly poor resolution of the isotopic
patterns with multiple charges, shown for the doubly charged peak
around m/z 2123.4, is in full agreement with this conclusion. Tetra-
Leu 3 was only measurable as monomer with negative detection
mode.
Consistent with ESI-MS results, the absence of toroidal
suprastructures with internal pores of diameters >5 Å (2n) was
further implied by the incapacity of octa-Leu 2 to mediate release
of CF (a self-quenching, watersoluble fluorophore) across the lipid
bilayer of uniformly sized EYPC-SUVs (SUV ) small unilamellar
vesicle).12 However, when added to EYPC-SUVs having en-
trapped pH sensitive fluorophore 8-hydroxypyrene-1,3,6-trisul-
fonic acid (HPTS) and a transmembrane pH gradient, nanomolar
In summary, the above results imply that, in sharp contrast to
tetra-Leu 3, rigid-rod octa-Leu 2 forms stable, mainly (or even
exclusively) dimeric rigid-rod â-barrels with ionophoric properties
(22), capable of mediating efficient ion transport across lipid
bilayers. Studies toward larger toroidal rigid-rod supramolecules
similar to 2n are ongoing.
Acknowledgment. We are grateful to B. Ghebremariam for providing
51b and M. M. Tedesco for experimental assistance. 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. Mass spectrometry was performed
by Dr. N. Leffers at Georgetown University Lombardi Cancer Center’s
Macromolecular Analysis Shared Resource, supported in part by U.S.
PHS Grant P30-CA-51008.
(9) Yang, J. T.; Wu, C. C.; Martinez, H. M. Methods Enzymol. 1986, 130,
208.
(10) Nakanishi, K.; Berova, N. In Circular Dichroism-Principles and
Applications; Nakanishi, K., Berova, N., Woody, R. W., Eds.; VCH Publish-
ers: New York, 1994; p 361.
(11) Fitzgerald, M. C.; Siuzdak, G. Chem. Biol. 1996, 3, 707.
(12) Dye leakage and ion transport experiments were performed as
previously described.1a
JA983893S
(13) (a) Doyle, D. A.; Cabral, J. M.; Pfuetzner, R. A.; Kuo, A.; Gulbis, J.
M.; Cohen, S. L.; Chait, B. T.; MacKinnon, R. Science 1998, 280, 69. (b)
Hess, P.; Tsien, R. W. Nature 1984, 309, 453.