COMMUNICATIONS
vesicles was removed by two passes through a gel filtration unit (Sephadex
G-25) with a phosphate buffer (pH 7.7, 0.1m KCl) as eluent. For the
fluorescence measurements (Digital Spectrofluorophotometer R-510,
Shimadzu), 200 mL of this solution was diluted with the same phosphate
buffer to 1 mL. Thereupon, 2 ± 10 mL of
a solution of 1, 2a ± d, or
gramicidin D in methanol are added and stirred for 5 min. (which
corresponds in the vesicle solution to 2 Â 10À3 ± 1 Â 10À2 gLÀ1Ðapprox-
imately 4 ± 20 mm 1 and 2a ± d, respectively, and 1 ± 5 mm gramicidin D).
Thereafter, by addition of HCl the pH value outside the vesicle was
lowered to 6.7and the decrease of the fluorescence was measured. Light
scattering experiments show that the DPPC vesicles are not dissolved by
concentrations up to 30 mol-% of 1 or 2a ± d, which is higher than the
concentrations used for the fluorescence and the conductivity measure-
ments. Additionally at room temperature, the critical micelle concentration
of 2c was determined, from the concentration dependence of the surface
tension, to be 1.4 Â 10À2 gLÀ1
.
C) Single channel events of the channel-forming compounds in planar lipid
membranes: The planar lipid membranes are prepared using the mono-
layer-folding technique.[18] For that purpose, in each of the compartments of
the cell, saline solution (0.7mL, sterile filtered 1 m KCl, LiCl, or CsCl, T
296 K) was added. On top of the buffer, a lipid solution (10 mL, soybean
lipid extract with 20% phosphatidyl choline/cholesterol (9/1, w/w; Avanti
Polar Lipids Inc., Alabaster, USA), 4 mgmLÀ1 in n-pentane) was added.
After waiting 10 min, when the pentane had evaporated, a planar lipid
membrane with a diameter of about 100 mm spontaneously formed across
the hole in the teflon septum by raising and lowering the liquid level in the
cell.
Figure 7. Symmetrical potential dependance of the single channel current
of 2a; linear regression leads to
Experimental conditions: membrane: soybean lipid extract with 20%
phosphatidyl choline/cholesterol (9/1, w/w) and 2.5mm 2a in the buffer;
buffer: 1m KCl; 296 K; filter frequency: 100 Hz; sample rate: 500 Hz.
a
conductance of G 26 Æ 1.5 pS.
To both compartments of the cell, the channel-forming compounds are
added as 0.5mm solutions in ethanol. The final concentrations were 2.5 ±
5mm. About 2 ± 3 min after addition of the compounds, the first single
channel events were observed.
In summary, based on the motif of 1, new compounds have
been prepared and identified as ion channels. They have
several advantages compared to other artificial channel-
forming compounds: They most probably span the mem-
brane, their synthesis is fairly simple, and their structure
provides the possibility to vary the head group, the number of
the ethylene glycol units, or the ratio of the hydrophilic to
hydrophobic part of the chain. The conducting pores are
shown to consist of several molecules of the respective ion
channel-forming compound; they require no rigid elements
for ion conductivity.
In all experiments, the sign of the potential difference refers to the side of
the membrane which is connected through the Ag/AgCl electrode to the
voltage supply. This side is called the cis side, the other the trans side. A
positive current corresponds to a cation transfer from the cis to the trans
side and is registered in the corresponding figures as an upward signal.
Received: October 25, 1999
Revised: March 28, 2000 [Z14178]
[1] B. Hille, Ionic Channels of Excitable Membranes, 2nd ed., Sinauer,
Sunderland, 1992.
Experimental Section
A) Synthesis of 2 and 3: The purity (>99%) and structure of all new
compounds have been determined by gas chromatography, 1H and
13C NMR spectroscopy, mass spectrometry, and elemental analysis or high
resolution mass spectrometry.
[2] B. Eisenberg, Acc. Chem. Res. 1998, 31, 117± 123.
[3] D. G. Nicholls, Proteins, Transmitters and Synapses, Blackwell, Ox-
ford, 1994.
[4] a) Membrane Protein Structure (Ed.: S. H. White), Oxford University
Press, New York, 1994; b) A. Kreusch, P. J. Pfaffinger, C. F. Stevens, S.
Choe, Nature 1998, 392, 945 ± 948; c) D. A. Doyle, J. M. Cabral, R. A.
Pfuetzner, A. L. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, R.
MacKinnon, Science 1998, 280, 69 ± 7 7 .
3: The appropriate ethylene glycol (1 equiv) was slowly added with stirring
and reflux under argon to a suspension of sodium in water-free THF (5 mL)
until the sodium was totally dissolved. To this solution at 296 K,
benzylbromide (1 equiv) was added and subsequently refluxed for 5 h.
Water (10 mL) was then added, the aqueous phase extracted with ethyl
acetate (3 Â 5 mL), the combined organic phases dried over MgSO4, and
the solvent removed by rotary evaporation. Flash chromatography with
ethyl acetate provided the product as colorless oil.
[5] a) U. Koert, Chem. Unserer Zeit 1997, 31, 20 ± 26; b) G. W. Gokel, O.
Murillo, Acc. Chem. Res. 1996, 29, 425 ± 432.
[6] a) K. S. Akerfeldt, J. D. Lear, Z. R. Wassermann, L. A. Chung, W. F.
DeGrado, Acc. Chem. Res. 1993, 26, 191 ± 197; b) J. D. Lear, Z. R.
Wassermann, W. F. DeGrado, Science 1988, 240, 1177 ± 1181; c) N.
Voyer, M. Robitaille, J. Am. Chem. Soc. 1995, 117, 6599 ± 6600.
[7] a) A. Nakano, Q. Xie, J. V. Mallen, L. Echegoyen, G. W. Gokel, J. Am.
Chem. Soc. 1990, 112, 1287± 1289; b) Y. Kobuke, K. Ueda, M. Sokabe,
J. Am. Chem. Soc. 1992, 114, 7618 ± 7622; c) M. J. Pregel, L. Jullen,
J.-M. Lehn, Angew. Chem. 1992, 104, 1695 ± 1639; Angew. Chem. Int.
Ed. Engl. 1992, 31, 1637± 1639; d) K. Kaye, T. Fyles, J. Am. Chem. Soc.
1993, 115, 12315 ± 12321; e) Y. Tanaka, Y. Kobuke, M. Sokabe, Angew.
Chem. 1995, 107, 717 ± 719; Angew. Chem. Int. Ed. Engl. 1995, 34,
693 ± 695; f) G. Deng, M. Merritt, K. Yamashita, V. Janout, A.
Sadownik, S. L. Regen, J. Am. Chem. Soc. 1996, 118, 3307± 3308;
g) H. Wagner, K. Harms, U. Koert, S. Meder, G. Boheim, Angew.
Chem. 1996, 108, 2836 ± 2839; Angew. Chem. Int. Ed. Engl. 1996, 35,
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2: To 4a ± d (1.25 equiv) in water-free dichloromethane (10 mL) under
argon, a slight excess of thionyl chloride was added and the solution
refluxed until gas evolution stopped. The solvent and excessive thionyl-
chloride were removed under vacuum. Thereupon the acid chloride was
cautiously added, with ice-water cooling, to a solution of 3a or 3b (1 equiv)
in water-free dichloromethane (10 mL) and triethylamine (1 mL) and was
then stirred for 24 h at 296 K. Water (10 mL) was added to this solution and
the mixture neutralized with dilute HCl. The aqueous phase was extracted
with ethyl acetate (3 Â 15 mL). The combined organic phases were dried
over MgSO4, the solvent removed under vacuum, and the product purified
by flash chromatography.
B) Fluorescence emission of pyranine-containing vesicles: The vesicles
were prepared from DPPC (1 mg) and phosphate buffer (pH 7.7, 293 K)
containing 0.1m KCl and 5 Â 10À4 m pyranine.[11] Pyranine outside the
Angew. Chem. Int. Ed. 2000, 39, No. 14
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