Structural characterization of cyclosporin A, C and microbial bio-transformed cyclosporin A analog AM6 using HPLC-ESI-ion trap-mass spectrometry

Cyclosporin A (CyA), a cyclic undecapeptide produced by a number of fungi, contains 11 unusual amino acids, and has been one of the most commonly prescribed immunosuppressive drugs. To date, there are over sixty different analogs reported as congeners and analogs resulting from precursor-directed biosynthesis, human CYP-mediated metabolites, or microbial bio-transformed analogs. However, there is still a need for more structurally diverse CyA analogs in order to discover new biological potentials and/or improve the physicochemical properties of the existing cyclosporins. As a result of the complexity of the resulting mass spectrometric (MS) data caused by its unusual amino acid composition and its cyclic nature, structural characterization of these cyclic peptides based on fragmentation patterns using multiple tandem MS analyses is challenging task. Here, we describe, an efficient HPLC-ESI-ion trap MSn (up to MS8) was developed for the identification of CyA and CyC, a (Thr(2))CyA congener in which L-aminobutyric acid (Abu) is replaced by L-threonine (Thr). In addition, we examined the fragmentation patterns of a CyA analog obtained from the cultivation of a recombinant Streptomyces venezuelae strain fed with CyA, assigning this analog as (gamma-hydroxy-MeLeu(6))CyA (otherwise, known as an human CYP metabolite AM6). This is the first report on both the MSn-aided identification of CyC and the structural characterization of a CyA analog by employing HPLC-ESI-ion trap MSn analysis. (C) 2014 Elsevier B.V. All rights reserved.