Peptaibolin: synthesis, 3D-structure, and membrane modifying properties of the natural antibiotic and selected analogues Marco Crisma, a Alessandra Barazza, a Fernando Formaggio, a Bernard Kaptein, b Quirinus B. Broxterman, b Johan Kamphuis c and Claudio Toniolo a,p a Department of Organic Chemistry, University of Padova, Biopolymer Research Centre, C.N.R., Via Marzolo 1, 35131 Padova, Italy b DSM Research, Organic Chemistry and Biotechnology Section, P.O. Box 18, 6160 MD Geleen, The Netherlands c DSM Food Specialties, Nutritional Ingredients, P.O. Box 1, 2600 MA Delft, The Netherlands Received 13 November 2000; revised 20 December 2000; accepted 18 January 2001 Abstract ÐWe synthesized by solution methods and fully characterized the naturally occurring, tetrapeptide antibiotic peptaibolin and selected analogues with replacements at the N- and C-terminal groups and the C a -tetrasubstituted a-amino acids. The preferred conformation of all of the peptides was assessed in solution by using FT-IR absorption and 1 H NMR techniques. Results of the X-ray diffraction analyses of peptaibolin itself and three analogues are also presented. Permeability measurements of such multiple turn forming, very short peptides indicate that peptaibolin is devoid of membrane activity because a lipoyl N-terminal blocking group is an essential requisite. q 2001 Elsevier Science Ltd. All rights reserved. 1. Introduction Peptaibols 1 are a unique class of membrane active peptides biosynthesized by fungi. 2 These antibiotics are character- ized by a high proportion of Aib (a-aminoisobutyric acid or C a,a -dimethylglycine), a strong a/3 10 -helix 3 supporting C a -tetrasubstituted a-amino acid, 4±7 and a C-terminal 1,2- amino alcohol. Some peptaibols typically contain Iva (isovaline or C a -methyl, C a -ethylglycine), another repre- sentative of the Aib family. An additional common feature is represented by an N a -terminal acyl group (more speci®c- ally, an acetyl group in the longest members of the class, while a fatty acyl group of eight or ten carbon atoms in the shortest members). Sequences of these peptides range from 19 amino acids (e.g. in the extensively investigated alamethicin) to as low as six amino acids (in the lipopeptai- bol trichodecenin). Peptaibols are known to induce leakage of the cytoplasmic material and eventually to lead to cell death. It has been demonstrated that the long-sequence peptaibols form voltage-dependent channels, 2 whereas the short-sequence (lipo)peptaibols tend to ¯oat on the lipid bilayer (carpet-like mechanism). 8 In the course of a screening for fungal peptides Hu Èlsmann et al. 9 have recently reported on the isolation and primary structure of peptaibolin, an unusual representative of the peptaibol class in that it contains only four amino acids in combination with the small acetyl blocking group at the N-terminus. The sequence of peptaibolin is as follows: where Ac is acetyl and Phol is the 1,2-amino alcohol phenylalaninol. This compound exhibits antimicrobial activity, albeit moderate, against Gram-positive bacteria and yeasts. Tetrahedron 57 (2001) 2813±2825 Pergamon TETRAHEDRON 0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S0040-4020(01)00124-7 Keywords: conformation; membranes; peptides; X-ray crystallography. p Corresponding author. Tel.: 139-049-827-5247; fax: 139-049-827- 5239; e-mail: biop02@chor.unipd.it