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UBC Theses and Dissertations

Methodologies for synthesis of transcription inhibiting peptides, amatoxins, and their bioconjugates for targeted cancer therapy Pryyma, Alla


This thesis describes my contribution to the development of synthetic methods directed at accessing bicyclic toxic peptides, amatoxins, as well as amatoxin-based bioconjugates for targeted treatment of cancer. Alpha-amanitin, the principal toxin of the amatoxin family and a potent transcription inhibitor, is of significant interest for applications in cancer therapy in a form of conjugates targeting cancer-specific receptors. Despite being known for over 70 years, the complex structure of α-amanitin has challenged the development of synthetic methods. In Chapter 2, C-3a-halopyrroloindolines are successfully employed as precursors to a tryptathionine crosslink, a key structural feature shared by amatoxins. Notably, fluoropyrroloindoline (FPI) was utilized to access previously inaccessible, oxidation-sensitive 6'-hydroxy-tryptahtionine found in α amanitin. Furthermore, the distinct reactivity of FPIs under H-bonding conditions was explored whereby hexafluoroisopropanol was used to ionize the C-F bond for nucleophilic attack yielding C-2-thiol-substituted tryptophans and C-3a-functionalized pyrroloindolines. Chapter 3 describes the synthesis and evaluation of (RSO)-5'-hydroxy-6'-deoxy-amanitin, a novel, bioactive analog of α-amanitin for use as a payload in targeted therapies. This synthesis employs naturally abundant 5-OH-tryptophan to incorporate an indole conjugation handle in three steps, in contrast to the synthetically challenging 6-OH-tryptophan. In vitro cell viability and RNA polymerase II transcription inhibition assays complete the biological evaluation of (RSO)-5'-hydroxy-6'-deoxy-amanitin and sulfur-modified analogs thereof. Chapter 4 describes two diastereoselective sulfoxidation methods providing access to amatoxins with the biologically active (R)-sulfoxide, thereby solving the last-standing challenge in the synthesis of amatoxins. The late-stage sulfoxidation approach utilizes Ti(IV)/L-DET-based system and is applied to the bicyclic precursor as the last step of the synthesis yielding the (R)-sulfoxide in large excess ((R:S) 19:1). Additionally, facile and cost-effective early-stage sulfoxidation employing meta-chloroperoxybenzoic acid in dimethylformamide is described. In Chapters 5 and 6, two synthetic amanitin analogs, conjugated at different sites, are used to assemble peptide drug conjugates targeted to cancer-specific receptors. The development of a modular synthetic platform for the construction of cleavable and non-cleavable amanitin-peptide conjugates that can be adapted to various targeting moieties is reported. In vitro cell proliferation studies on antigen-positive cell line revealed target-specific toxicity of conjugates with up to 1000-fold bioactivity enhancement.

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