Alpha-amanitin is an exceedingly toxic, naturally occurring, bicyclic octapeptide that inhibits RNA polymerase and results in cellular and organismal death. Here we report the straightforward synthesis of an amanitin analogue that exhibited near-native toxicity. A pendant alkyne was readily installed to enable copper-catalyzed alkyne-azide cycloaddition (CuAAC) to azido-rhodamine and two azide-bearing versions of the RGD peptide. The fluorescent toxin analogue entered cells and provoked morphological changes consistent with cell death. The latter two conjugates are as toxic as the parent alkyne precursor, which demonstrates that conjugation does not diminish toxicity. In addition, we showed that toxicity depends on a single diastereomer of the unnatural amino acid, dihydroxyisoleucine (DHIle), at position 3. The convenient synthesis of a heptapeptide precursor now provides access to bioactive amanitin analogues that may be readily conjugated to biomolecules of interest.
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 α‐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‐[Asn1‐TEG‐DEAC, Ile3]‐amaninamide (Ile3‐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.
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