Fluorobenzyl Terminal Residues as Probes of Function in Tris(macrocycle) Channels: Evidence from NMR and Planar Bilayer Conductance Studies Clare L. Murray, Eric S. Meadows, Oscar Murillo, and George W. Gokel* Bioorganic Chemistry Program and Department of Molecular Biology & Pharmacology Washington UniVersity School of Medicine 660 South Euclid AVenue, Campus Box 8103 St. Louis, Missouri 63110 ReceiVed April 28, 1997 Most cation transport in ViVo is thought to be mediated by proteins having multiple transmembrane helices that organize into pores. This process has been studied a great deal, but its inherent complexity has frustrated the elucidation of mechanistic details. 1,2 The fundamental biological importance of this area and the elusive nature of functional details have brought forth several synthetic organic chemical models for ion transporters; this work has recently been reviewed. 3 In previous work, we have described a successful cation- conducting channel that functions in phospholipid bilayers. 4 We have inferred information about both the position of the headgroups in the bilayer and the active conformation of the channel-former from these studies. Information about the position of the molecule comes largely from fluorescence studies. It was found that the central macroring could be reduced in size from diaza-18-crown-6 to diaza-15-crown-5 without altering cation flux. We concluded from this and other data that the central macroring was not required to be parallel to the distal macrocycles. We wished to inquire similarly into the role of the distal macrorings which appeared to interact directly with Na + as judged from an application of the Hammett principle. 4c We report here the results of that investigation. Two tris(macrocycle)s were prepared for the present study: 4-F-C 6 H 4 CH 2 <N15N>C 12 <N18N>C 12 <N15N>CH 2 C 6 H 4 - 4-F, 1, and 4-F-C 6 H 4 CH 2 <N18N>C 12 <N18N>C 12 <N18N> CH 2 C 6 H 4 -4-F, 2. 4-Fluorobenzyl bromide was treated with 1.1 equiv of 4,10-diaza-15-crown-5 to afford the monosubstituted crown, F-C 6 H 4 CH 2 <N15N>H, in 47% yield. The central subunit was prepared by treating 4,13-diaza-18-crown-6 with excess 12-bromododecanoyl chloride to afford Br(CH 2 ) 11 - CO<N18N>CO(CH 2 ) 11 Br (89%) which was, in turn, reduced using B 2 H 6 ‚THF to Br(CH 2 ) 12 <N18N>(CH 2 ) 12 Br (43%). The crown dibromide was then treated with 2 equiv of the appropri- ate mono(fluorobenzyl)crown, FC 6 H 4 CH 2 <N15N>H, to yield (24%) channel 1 having two 15-membered distal macrocycles or FC 6 H 4 CH 2 <N18N>H to produce (28%) 2. 5 The activity of channel compounds 1 and 2 in phosphati- dylglycerol/phosphatidylcholine bilayer vesicles was confirmed by using the dynamic 23 Na-NMR technique devised originally by Riddell and Hayer 6 and later elaborated for gramicidin. 7 The rates determined for Na + -transport relative to gramicidin (k rel ) 100) for 1 and 2 were, respectively, 16 and 26. The 23 Na-NMR method is an equilibrium experiment and does not involve a concentration gradient or electromotive pressure (see below). The results reflect overall transport efficacy of the system; single channels are not observed. It was inferred from experimental data for relatives of 2 4c that Na + passes through the distal 18-membered macrorings. There is currently no direct evidence that Na + also passes through the smaller macrorings of 1, but the hole diameter of 15-crown-5 is sufficient to permit it. An alternative to the 23 Na-NMR method for evaluating Na + transport is the planar lipid bilayer conductance (pbc) method. 8 The latter method measures current as a function of time in which the opening and closing of individual cation channels are observed directly rather than the overall transport phenom- enon as determined by dynamic NMR. In principle, the pbc and NMR methods should produce similar information. A significant difference between the activities of 1 and 2 was observed by using the pbc method, but the calculated conduc- tance of each channel was essentially identical: 13 pS. The latter point is illustrated by the superimposable “I-V” curves shown in Figure 1. A difference between the two channels is apparent in the traces shown as Figure 2. The top panel (A) shows data for the 15-membered ring compound. In the lower panel, data for 18-membered ring structure 2 are shown. In each case, a 2 pmol sample of compound was studied at +100 mV applied potential in the presence of 500 mM NaCl. Each trace shows approximately 2 min of activity (of a total of several hours in each case). In the top pair of traces, four open/close transitions are apparent. The identity of the peak heights shows that each is a single channel event. The width of the peaks shows the duration (dwell) of the opening. The longest opening (top right- most peak) has a duration of approximately 5 s. No multiple channel openings are apparent. In marked contrast, multiple channel openings are abundant in the lower trace. We have analyzed a much more extensive data set than shown here by calculating open-close histograms for 1 and for 2 (Figure 3). The ordinate in the plot shows the number of transitions observed for each compound. For 1, which has 15-membered * G. W. Gokel, Tel. 314/362-9297; FAX 314/362-9298 or 7058. E-mail: ggokel@pharmdec.wustl.edu. (1) (a) Stein, W. D. Channels, Carriers, and Pumps: An Introduction to Membrane Transport; Academic Press: San Diego, CA, 1990. (b) Hille, B. Ionic Channels of Excitable Membranes, 2nd ed.; Sinauer Associates, Inc.: Sunderland, MA, 1992. (c) Nicholls, D. G. Proteins, Transmitters, and Synapses; Blackwell Science: Oxford, 1994. (2) (a) Henderson, R.; Baldwin, J. M.; Ceska, T. A.; Zemlin, F.; Beckmann, E.; Downing, K. H.; J. Mol. Biol. 1990, 213, 899-929. (b) Deisenhofer, J.; Epp, O.; Miki, K.; Huber, R.; Michel, H. Nature 1985, 318, 618-624. (c) Baldwin, J. M. EMBO J. 1993, 12, 1693-1703. (3) (a) Gokel, G. W.; Murillo, O. Acc. Chem. Res. 1996, 29, 425-432. (b) Voyer, N. Top. Curr. Chem. 1996, 184,1-37. (4) (a) Nakano, A.; Xie, Q.; Mallen, J.; Echegoyen, L.; Gokel, G. W. J. Am. Chem. Soc. 1990, 112, 1287. (b) Murillo, O.; Watanabe, S.; Nakano, A.; Gokel, G. W. J. Am. Chem. Soc. 1995, 117, 7665-7679. (c) Murillo, O.; Suzuki, I.; Abel, E.; Gokel, G. W. J. Am. Chem. Soc. 1996, 118, 7628- 7629. (d) Murillo, O.; Abel, E.; Maguire, G. E. M.; Gokel, G. W. J. Chem. Soc., Chem. Commun. 1996, 2147-2148. (e) Abel, E.; Meadows, E. S.; Suzuki, I.; Jin, T.; Gokel, G. W. J. Chem. Soc., Chem. Commun. 1997, 1145. (f) Murillo, O.; Suzuki, I.; Abel, E.; Murray, C. L.; Meadows, E. S.; Jin, T.; Gokel, G. W. J. Am. Chem. Soc. 1997, 119, 5540. (g) Abel, E.; Maguire, G. E. M.; Meadows, E. S.; Murillo, O.; Jin, T.; Gokel, G. W. J. Am. Chem. Soc. In press. (5) All new compounds were fully characterized by IR, NMR, and either combustion analysis ((0.4%) or HRMS (1 part in 10000). (6) Riddell, F.; Hayer, M. Biochim. Biophys. Acta 1985, 817, 313-317. (7) Buster, D.; Hinton, J.; Millett, F.; Shungu, D. Biophysical J. 1988, 53, 145-152. (8) The planar bilayer chamber was prepared with 0.5 M NaCl, 0.001 M sodium phosphate (pH ) 7.0) solution on each side of the membrane. The membrane was formed by the painting method using a solution (30 mg/mL) of L-R-lecithin in decane. The channels were added (2 μL) as 1.0 μM solutions in trifluoroethanol and stirred for 5-10 min. After a 5 min equilibration period, a holding voltage was applied, and the channel responses were recorded using a Warner PC-505 patch clamp amplifier, a DigiData A/D converter and the acquisition software Axoscope. 7887 J. Am. Chem. Soc. 1997, 119, 7887-7888 S0002-7863(97)01325-5 CCC: $14.00 © 1997 American Chemical Society