JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2004; 39: 949–960 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.674 Extraribosomal cyclic tetradepsipeptides beauverolides: profiling and modeling the fragmentation pathways Alexandr Jegorov, 1 Bela Paizs, 2† Marek Kuzma, 3 Martin Zabka, 4 Zdenek Landa, 4 Miroslav Sulc, 3 Mark P. Barrow 5 and Vladimir Havlicek 3* 1 IVAX-Pharmaceuticals a.s., Branisovska 31, 370 05 Ceske Budejovice, Czech Republic 2 Department of Molecular Biophysics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany 3 Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, CZ-142 20 Prague 4, Czech Republic 4 University of Southern Bohemia, Faculty of Agriculture, Studentska 13, CZ-370 05 Ceske Budejovice, Czech Republic 5 Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK Received 24 February 2004; Accepted 12 May 2004 Profiling of cyclic tetradepsipeptides beauverolides was tested as a chemotaxonomic tool for fungal strain identification/discrimination. Two new tetradepsipeptides, beauverolides Q and R, were characterized by tandem mass spectrometry. Specific elimination of 113 atomic mass units from both protonated and sodiated molecules of beauverolides is ubiquitous for all 12 most dominant congeners evaluated in this profiling study. Reconstruction of the total ion chromatogram, according to this neutral fragment release, was used for data filtering and selectivity enhancement. Selective ring opening and fragment ion formation of beauverolide I are discussed in detail utilizing high-level theoretical modeling of the fragmentation pathways. Copyright  2004 John Wiley & Sons, Ltd. KEYWORDS: cyclodepsipeptide; beauverolide; Beauveria; Paecilomyces; peptide fragmentation pathways; spore; fungus INTRODUCTION Cyclic peptides or depsipeptides belong to fungal sec- ondary metabolites, the biosynthesis of which proceeds non-ribosomally and are catalyzed by complex multi- functional enzymes, termed non-ribosomal peptide syn- thetases (NRPS). In addition to the fungal sources, non- ribosomal peptides are produced also by bacteria, algae and some marine microorganisms, but have not been found in mammals. It is believed, based on the enormous size of NRPSs, that production of these secondary metabolites is not accidental, but provides a phylogenetically devel- oped advantage to the producer. As an example, many pathogenic bacteria employ NRPSs in the biosynthesis of siderophore-dependent virulence factors. 1 The repeating series of functional domains are highly conserved across various peptide synthetases. 2 However, the complexity of peptide synthetases, variation in the substrate specifici- ties of individual enzymes and metabolic differences of individual strains leading to different precursor availabil- ity may result in different secondary metabolite patterns L Correspondence to: Vladimir Havlicek, Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, CZ-142 20 Prague 4, Czech Republic. E-mail: vlhavlic@biomed.cas.cz † To whom correspondence regarding the fragmentation pathways should be addressed. Contract/grant sponsor: Czech Science Foundation; Contract/grant numbers: 203/02/1417; 203/04/0799. Contract/grant sponsor: Czech Ministry of Education; Contract/grant number: MSM 122200002/2. Contract/grant sponsor: Institutional Research Concept; Contract/grant number: AV0Z5020903. within the same species. For example, it was reported that various cyclosporin-producing fungi have apparently dif- ferent spectra of analogues produced than the originally described Tolypocladium inflatum. 3 Examples include the cyclosporins [MeLeu 1 ]CsA, [Leu 4 ]CsA, [Ile 4 ]CsA, [Leu 5 ]CsA and [Leu 9 ]CsA produced by Tolypocladium terricola, 4–6 [Ala 2 , Val 11 ]CsA produced by Mycelium sterilae MS2929, 7 [Thr 2 , Leu 5 , Leu 10 ]CsA produced by Stachybotrys chartarum, 8 [Thr 2 , Leu 5 , Ala 10 ]CsA produced by Acremonium luzulae (Fuckel) W Gams 9 and [Thr 2 , Ile 5 ]CsA produced by the Lep- tostroma anamorph of Hypoderma eucalytii Cooke and Harkn. 9 Cyclosporin analogues were also produced by the fungus Cylindrotrichum oligospermum (Corda), but by the different multienzyme system of peptolide, SDZ 214–103 D [Thr 2 , Leu 5 , D-hydroxyisovaleric acid 8 , Leu 10 ]CsA synthetase (note: the amino acid(s) and its (their) position in superscript indi- cate the substitution with respect to cyclosporin A (CsA)). 10 Hence the pattern of these secondary metabolites, prepared by cultivation under identical conditions potentially offers a chemotaxonomic tool or diagnostic marker for identification of various species or isolates. In this work, the group of cyclic tetradepsipeptides known as beauverolides (Fig. 1) were selected as potential markers of Beauveria and Paecilomyces species. 11,12 Various biological tests of these lipophilic neutral cyclotetradepsipep- tides revealed that beauverolides are not mycotoxins since they do not exhibit bactericidal, fungicidal, insecticidal or any other direct toxic effects. 13,14 On the contrary, recent studies have indicated that beauverolides could potentially serve as new drugs for the treatment of atherosclerosis. 15 – 18 Copyright  2004 John Wiley & Sons, Ltd.