SynopsisBoc-L-Leu-Aib-Pro-Val-AibAibGlu(OBz1)-Gln-Phl (Boc = t-butyloxycarbonyl, Aib = a-aminoisobutyric acid, Bzl = benzyl, Phl = phenylalaninol), C59H,Nlo0,,, the protected C-terminal nonapeptide with the sequence 12-20 of alamethicin, crystallize: in the orthorhombic space group P2,2121 with a = 15.666, b = 16.192, c = 26.876 A, and 2 1 4. The molecular conformation is right-handed helical with three a45 + 1 hydrogen bonds) and three p-turns (4 -1 hydrogen bonds). All but two of the hydrogen bonds are significantly longer than the usual value and show bifurcation to some extent. The a/3io-helical nonapeptide molecules are arranged head-to-tail along the a direction. The resulting linear antiparallel chains are linked by a weak intermolecular hydrogen bridge, thus forming a two-dimensional layer structure in the ab plane. The conformation of this nonapeptide is almost identical with that of the corresponding C-terminal part found by x-ray crystallography of the eicosapeptide alamethicin.

SynopsisThe x-ray structure of Boc-~-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-AlaOMe(1) represents the first a-helix determined by direct methods. This undecapeptide is a model of the N-terminus of alamethicin, and it exhibits voltage-dependent pores in bilayer membranes a t a higher voltage and concentration than alamethicin. The molecule crystallizes in the monoclinic space group P2, with a = 10.602(1), b = 23.884(3), c = 13.622(1) A, p = 95.61(6)", and 2 = 2. It adopts a right-handed a-helical conformation in the solid state with intramolecular 5 -1 hydrogen bonds. An additional intramolecular hydrogen bond is bifurcated, forming a stronger 4 -1 interaction (i.e., a p-turn 111) and a weaker 5 -1 interaction, thus prolonging the a-helical part up to 9 residues.The a-helix radius of 2.1 A, the height per res,Pue (distance N, . . . N,+$ of 1.53 A, the resulting length of the a-helical part of 13.8 A (9 residues) resp. 15.3 A (10 residues), the van der Waals radius (4.7 A), and the minimal diameter of pores formed by aggregation of 3-10 a-helices were calculated omitting the Glu(OBz1) side chain. In the crystal, the a-helices are linked head to tail via two hydrogen bridges forming continuous chains. Adjacent helices are oriented in antiparallel with their helix axes and have only van der Waals contacts.

The voltage-dependent membrane pore-forming polypeptide antibiotic alamethicin F 30 (Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-Phl, 1) was synthesiied by segment condensations. It was obtained free of racemate (<0.8% o-components) via the segments 2-11 (4a) and 12-20 (3a) using the dicyclohexylcarbodiimide/l-hydroxybenzotriazole and the mixed anhydride methods. The intermediates were unequivocally characterized by chromatographic and physical methods including I3C NMR, circular dichroism, and chiral phase gas chromatography. The segments 2 -6 (Sa) and 12 -20 (3) were also proven by X-ray structure determinations. -The final product 1 (150 mg) was obtained without HPLC separations in very high purity and in crystalline form. According to all analytical data and the comparison by spectroscopy and chromatographic analyses (HPLC, DC) the product 1 is identical with the main component F 30 of the microheterogeneous antibiotic metabolite isolated from Trichoderma tliride. The antibiotic activities of synthetic alamethicin (I) and the stimulating effect on calcium/calmodulin-dependent guanylate cyclase of Paramecium are identical with those of natural alamethicin. -The uniform synthetic alamethicin (1) shows perfectly homogeneous conductance states of single pores on measurements in lipid bilayer membranes which had not been reported so far for any synthetic or purified natural preparation. Totalsynthese des a-helikalen Eikosapeptid-Anlibiotikums Alamethicin *)Das spannungsabhangige Mernbranporen-bildende Polypeptidantibiotikum Alamethicin F 30 (Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-Phl, 1) wurde durch Segmentkondensationen synthetisiert. Es wurde uber die Segmente 2-11 (4a) und 12 -20 (3a) racematfrei (<0.8% D-Anteile) mit der Dicyclohexylcarbodiimid/l-Hydroxybenzotriazol-und der Mischanhydrid-Methode erhalten. Die Zwischenstufen wurden eindeutig charakterisiert durch chromatographische und physikalische Methoden einschlieBlich I3C-NMR, Circulardichroismus und Gaschromatographie an chiraler Phase. Die Segmente 2 -6 (Sa) und 12 -20 (3) wurden auch durch Rontgenstrukturbestimmung untersucht. -Das Endprodukt 1 (1 50 mg) wurde ohne HPLC-Trennung in sehr hoher Reinheit und kristallin erhalten. Nach allcn analytischen Daten und bei spektroskopischem und chromatographischem (HPLC, DC) Vergleich ist das Produkt 1 identisch mit der Hauptkomponente F 30 des mikroheterogenen, aus Trichoderma ciride isolierten antibiotischen Metaboliten. Die antibiotischen Aktivitaten von synthetischem * ) Results of this synthesis including I3C NMR and single pore measurements of synthetic alamethicin were reported on the following symposia:Alamethicin (1) und die stimulierende Wirkung auf die Calcium/Calmodulin-abhangige Guanylatcyclase von Paramecium sind identisch niit denen von natiirlichem Alamethicin. -Das einheitliche synthetische Alamethicin (1) zeigt bei Messungen an Lipiddoppelschicht-Membranen perfekt homogene Leitwerte von Einzelporen, wie sie bisher weder v...

Z 724 IE] German version: Angew. Chem. 96 (1984) 439 CAS-Registry number: 90343-29-6. [l] a) J. Ferraris, D. 0. Cowan, V. Walatka, Jr., J. H. Perlstein, J. Am. Chem. 121 131 141 151 [61 Soc. 95 (1973) 948; b) L.

Suzukacillin A is a potential-dependent membrane pore-forming polypeptide antibiotic exhibiting similar properties as alamethicin in biophysical1.2) and biological3-6) experiments. The essential a-helical part of these polypeptides with its high dipole moment is responsible for potential-dependent pore formation in black lipid membranes as described by the flip-flop gating model7). On the other hand the polarity and size of the C-and N-termini determine specific characteristics such as medium pore size and stability of pore statesa.9). Thus the exact knowledge of structure/activity relations contributes considerably to a better understanding of the ionconduction properties of these linear amphiphilic polypeptides.A preliminary sequence based solely on GC-MS studies of partial fragments was published 1976 by our groups'). As alamethicin'O.'') and trichotoxin A40'2-'4) the eicosapeptide suzukacillin A belongs to the longest peptide antibiotics known and shows a characteristic natural microheterogeneity in its amino acid composition. Because of several ambiguities in the former instrumental sequencing') it was necessary to isolate and 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1985