Identification of isocyclosporins by collision-induced dissociation of doubly protonated species

Abstract

Nonribosomal cyclopeptide cyclosporin A (CsA), produced by fungus Tolypocladium inflatum, is an extremely important immunosuppressive drug used in organ transplantations and for therapy of autoimmune diseases. Here we report for the first time production of CsA, along with related cyclosporins B and C, by Tolypocladium inflatum strains of marine origin (White Sea). Cyclosporins A-C contain an unusual amino acid, (4R)-4-((E)-2-butenyl)-4,N-dimethyl-l-threonine (MeBmt), and are prone to isomerization to non-active isocyclosporin by N→O acyl shift of valine connected to MeBmt in acidic conditions. CsA and isoCsA are not distinguishable in MS analysis of [M+H]⁺ ions due to rapid [CsA + H]⁺→[isoCsA + H]⁺ conversion. We found that the N→O acyl shift is completely suppressed in cyclosporine [M+2H]²⁺ ions, and their collision-induced dissociation (CID) can be used for rapid and unambiguous analysis of cyclosporins and isocylosporins. Fragmentation patterns of [CsA+2H]²⁺ and [isoCsA+2H]²⁺ ions were analyzed and explained. The developed approach could be useful for MS analysis of other peptides containing β-hydroxy-α-amino acids.

Keywords: Collision-induced dissociation; Cyclosporin; Doubly charged ions; Identification; Isocyclosporin.