Solid-phase synthesis of amanitin derivatives and preliminary evaluation of cellular uptake and toxicity

The study of transcriptional arrest is of great importance and can provide insight into the cellular response to various toxins, most notably chemotherapeutics. Therefore, specific inhibitors of RNA Polymerase II (RNAP II) could prove to be extremely useful. Given that alpha-amanitin is one of the most potent and selective inhibitors of RNAP II, we prepared two amanitin derivatives on solid phase as a proof of principle towards the development of a One-Bead-One-Compound (OBOC) amanitin chemical library. The amatoxin family comprises several related toxic peptides that are characterized by a defined rigid bicyclic structure based on a head-to-tail cyclized octapeptide, with a transannular tryptathionine crosslink. The latter is prepared via the Savige-Fontana tryptathionylation of the oxidized tryptophan derivative 3a-hydroxypyrrolo[2,3-b] indoline in neat TFA. We synthesized a new fluorescently labelled S-deoxo-[Asn(1)-TEG-DEAC, Ile(3)]-amaninamide (Ile(3)Ama) and studied its ability to be taken up by CHO cells and to stain the nucleus, the site of transcriptionally active DNA. Towards the solid phase synthesis of an amanitin-inspired library, we synthesized a linker that is stable both in TFA, used to promote the tryptathionine crosslink, and base used for Fmoc-deprotection. The linker allowed the full synthesis of an amanitin derivative on solid phase, including tryptathionylation and macrolactamization.