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Synthesis of a 19-Residue Peptide with Alamethicin-Like Activity (Solid-Phase Peptide Synthesis/Lipid Bilayer Membranes/Voltage-Dependent Conductances) BALTHASAR F

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Synthesis of a 19-Residue Peptide with Alamethicin-Like Activity (Solid-Phase Peptide Synthesis/Lipid Bilayer Membranes/Voltage-Dependent Conductances) BALTHASAR F Proc. Nati. Acad. Sci. USA Vol. 74, No. 1, pp. 115-119, January 1977 Biochemistry Synthesis of a 19-residue peptide with alamethicin-like activity (solid-phase peptide synthesis/lipid bilayer membranes/voltage-dependent conductances) BALTHASAR F. GISIN*, SHIGERU KOBAYASHI*, AND JAMES E. HALO * Department of Biochemistry, The Rockefeller University, New York, N.Y. 10021; and t Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710 Communicated by R. B. Merrifield, October 18,1976 ABSTRACT This paper describes the chemical synthesis ALA-o, on the other hand, the same side group is engaged in of a compound with voltage-gating characteristics similar to an amide bond with phenylalaninol and the imino function of those observed in nerve membranes. For alamethicin (ALA), a Pro2 is blocked by an Ac-Aib residue. Gln'9 again is identical natural antibiotic that induces such properties in lipid bilayer membranes, there are two proposed structures, one a cyclic and in both sequences and provides the a-carboxyl group that makes the other an open chain peptide. The open chain sequence ALA a monobasic acid. (ALA-o) proposed by Martin and Williams [(1976) Biochem. J. In view of the significance ALA has gained as an agent that 153, 181-1901 was synthesized by stepwise solid-phase con- imparts nerve-like electrical properties upon artificial mem- densation of four fragments prepared by solid-phase synthesis. branes, its detailed molecular mechanism of action is of con- The product, purified to homogeneity, was not identical with siderable interest. However, the same mechanism would almost the main component of natural ALA. Nevertheless, in lipid bi- layer membranes the exponential dependence of conductance certainly not be compatible with both of the proposed primary on voltage and the dependence of conductance on a high power structures. Chemical synthesis seemed to be the appropriate of the peptide concentration were qualitatively similar for approach to decide which structure, if either, exhibits electrical ALA-o and for natural ALA. Like ALA, ALA-o showed the activity. characteristics of a channel-former, although the single-channel The available evidence for and against ALA-c and ALA-o conductances were less well defined for the synthetic com- indicated the latter more probably ALA pound. This work establishes that a cyclic structure is not a represented for two necessary condition for a peptide to induce voltage-dependent reasons. First, in partial hydrolysates of ALA, a peptide com- conductances in membranes and that ALA-o possesses all the prising -Glu(y-Pro)-, the cycle-forming link, was not found structural elements required for such an activity. (19). Second, the presence of blocking groups on the Glul' y-carboxyl (phenylalaninol) and the Pro2 imino group (Ac-Aib) Alamethicin (ALA), a peptide with antibiotic activity against was demonstrated (21, 22, 17), thus precluding a direct covalent certain bacteria, is found in the culture broth of Trichoderma link between these two residues. On these grounds, the synthesis viride (2, 3). Because of its amphiphilic character, ALA shows of ALA-o rather than ALA-c was undertaken first. This effort pronounced surface activity and adsorbs strongly to biological and the evaluation of the product is the subject of the current and artificial membranes. As a consequence, it causes lysis of preliminary contribution (a detailed report for publication human erythrocytes and damages the outer membrane of elsewhere is in preparation). Ehrlich ascites tumor cells (4). Moreover, ALA induces aggre- gation and fusion of lecithin vesicles (5). Synthesis The peptide also affects the electrical properties of planar The synthesis of ALA-o was performed by the solid-phase artificial lipid membranes in a unique and specific way: minute method (23). A fragment condensation approach rather than concentrations of ALA induce voltage-dependent conductances a stepwise synthesis was chosen for the following reasons. First, that mimic the action potential phenomena observed in nerve due to the sterically hindered amino group of Aib, some of the membranes. These effects were observed by Mueller and Rudin coupling reactions could have failed to go to completion. Had (6) and have been studied also by other groups (7-9). It is now that occurred at each of the eight Aib residues in a stepwise generally believed that several ALA molecules aggregate in the approach, one would have been confronted in the end with the membrane to form ion-conducting channels (8, 10-12). difficult task of separating the target peptide from a large Despite a number of physicochemical (13, 14), conforma- number of deletion peptides similar in size and other properties. tional (15-18), and chemical (17-22) studies, the primary Fragments, on the other hand, could be purified individually structure of ALA remains unknown. Currently, there are two before their stepwise assembly on the resin. In this way, ho- proposed sequences: a cyclic (ALA-c) and an open chain mogeneity of the sequences covered by each fragment could (ALA-o) peptide structure (Fig. 1). The former, proposed by be assured and the purification of the crude final product would Payne et al. (19), has been confirmed by Ovchinnikov et al. be greatly simplified. Second, the uncertainty of the exact (20), while the latter, proposed by Martin and Williams (21, 22), structures of the N-terminal and COOH-terminal portions of found the support of the Jung group (17). The two proposals the peptide demanded a strategy that would allow the prepa- agree on a sequence of 15 consecutive residues (3-17 according ration of additional sequences, including ALA-c, without re- to ALA-o numbering). In ALA-c the y-carboxyl group of Glu'8 quiring resynthesis of the whole molecule. And third, analogues is in amide linkage with Pro2 to form a cyclic structure. In designed to furnish information on the mode of action of ALA could be prepared efficiently by combining the already avail- Abbreviations: (cf. ref. 1) ALA, alamethicin; ALA-o and ALA-c, open able fragments with new fragments and cyclic ALA, respectively; Aib, a-aminoisobutyric acid; Boc-, t- containing, e.g., single butoxycarbonyl; DCC, dicyclohexylcarbodiimide; Ac, acetyl; HOBT, amino acid substitutions. N-hydroxybenzotriazole; Bzl, benzyl; PE, phosphatidylethanolamine; Based on these considerations, four protected fragments of GMO, glycerol monooleate; Phol, L-phenylalaninol (phenylalanine ALA-o were synthesized (Figs. 1 and 2): the tetrapeptide 1-4, with -CH20H instead of -COOH); Res (resin), CH2-polystyrene-co- Ac-Aib-Pro-Aib-Ala-O-Res (A), the tetrapeptide 5-8, Boc- 1%-divinylbenzene. Aib-Ala-Gln-Aib-O-Res (B), the nonapeptide 9-17, Boc-Val- 115 Downloaded by guest on September 24, 2021 116 Biochemistry: Gisin et al. Proc. Natl. Acad. Sc. USA 74 (1977) (A) PRo-AIB-ALA-AIB-ALA-GLN-AIB-VAL-AIB-GLY-LEu-AIB-PRO-VAL-AIB-AIB-GLU-GLN-OH (2) (3) (4) (17) (18) (19) PHOL (B) AC-AIB-PRO-AIB-ALA-AIB-ALA-GLN-AIB-VAL-AIB-GLY-LEU-AIB-PRO-VAL-AIB-AIB-GLU-GLN-OH 1 2 3 4 5 8 9 17 18 19 (C) i j I E I FIG. 1. Proposed sequences for alamethicin. Aib is a-aminoisobutyric acid; Phol, L-phenylalaninol. (A) the cyclic structure (ALA-c) by Payne et al. (19) and (B) the open chain structure (ALA-o) by Martin and Williams (21, 22). (C) Fragments used in the synthesis of ALA-o. Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-O-Res (C), and the dipep- gel permeation chromatography (Ac-A-B-C-OH, ALA-o) using tide Boc-Glu(Phol)-Gln-O-Res (D). These fragments were a Sephadex G-25 column (1.6 X 90 cm) in ethanol/water (1:1, prepared starting with chloromethylated (23, 24) polysty- vol/vol) in connection with a differential refractometer. The rene-co-1%-divinylbenzene resin (Res) to which the Boc-pro- peaks superimposed with the trace obtained from quantitative tected COOH-terminal amino acid was esterified with the radioactivity analysis of the fractions. Homogeneity of each cesium salt procedure (25). Trifluoroacetic acid (50%) in fragment and ALA-o was established by thin-layer chroma- methylene chloride was used for deprotection and diisopro- tography on silica gel G plates. One single, ninhydrin-negative pylethylamine (5%) in the same solvent was used for neutral- iodine/tolidine-positive spot was observed in each case. ization. Coupling was with 1.5 equivalents each of Boc-amino Condensation of the fragments (Fig. 3) was effected by acid and dicyclohexylcarbodiimide (DCC) (26) in methylene preactivating the carboxyl component with DCC and HOBT chloride for four hours followed by another 16-hr period of (1:1.5:3.0, eq/eq/eq) in methylene chloride/N,N-dimethyl- coupling with fresh reagents. This protocol was normally suf- formamide at 0-0 for 1 hr and 1 hr at room temperature prior ficient to give coupling yields greater than 99% as determined to addition to the resin-bound amine component in dimethyl- with picrate (27). However, either when the carboxyl compo- formamide. The reaction was allowed to proceed at room nent was Boc-Aib or when the amine component ended in an temperature and was monitored by measuring the radioactivity Aib residue, longer reaction times were necessary. In these cases, of the solution phase. The molar equivalents used, coupling coupling was repeated until the picrate test indicated a yield greater than 98%. In order to suppress nitrile formation the glutamine residue of fragment B was coupled in the presence AIB PRO AIB ALA of N-hydroxybenzotriazole (HOBT) (28). Model experiments using infrared spectroscopy showed indeed that under the BOC-! O-RES conditions used this side reaction did not occur. The low chemical reactivity of Aib is also reflected in the low BOC +-OH H + O-RES color value it yields when run on an amino acid analyzer under standard conditions (ref, 18, 19; B.
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