When the chemistry is right – towards bioorthogonal prodrug activation

Abstract

An estimated 10 million people are diagnosed with cancer every year, one of the world’s leadingcauses of death. It comes as no surprise that the demand for new and better chemotherapeuticagents with minimal side effects is at an all-time high. The concept of targeted (pro-)drug deliveryappears to be a suitable strategy to satisfy this request. Despite major breakthroughs in the past,prodrug activation comes with a number of limitations, as it is usually achieved by disintegrationof chemical linkers, that are labile under cell-specific conditions (e.g. certain pH, presence ofcertain enzymes, ...). Drug release may also appear in healthy tissue and as a result, currenttargeted prodrug delivery systems often suffer from a lack of selectivity, leading to undesired offtargettoxicity. Chemically triggered controlled drug release seems to be the solution to thisdilemma and might potentially boost the overall performance of these otherwise highly promisingdrug delivery systems.This thesis focuses on utilizing bioorthogonal chemistry, in particular the inverse electron-demandDiels-Alder (IEDDA) reaction between tetrazines (Tz) and trans-cyclooctenes (TCO), to tackle thistask. Fundamental development of the required reaction partners will be discussed, mechanisticinsights explored and finally, proof-of-concept studies in live cells will be presented.The application of bioorthogonal chemistry in living systems requires exceptionally high reactionrates, as concentrations of reaction partners typically range between micro- and sub-nanomolarlevels. Tweaking the kinetics of these reactions requires a well-considered design of bothtetrazines and TCO. The presented work contributes to the development of novel transcyclooctenederivatives by showcasing the incorporation of structural motifs, that dramaticallyimpact reaction rates. Specifically, installation of cis-fused ring systems (cyclic carbamates, cyclicurea) to the TCO backbone is exploited. As a result, higher ring strains arise and cause enhancedreactivities. The chemical nature of the newly incorporated moieties contributes to a higher polaritycompared to previously developed compounds, and thus provides more suitable TCO forapplications in biological environments.Impacts on reaction kinetics of different factors are discussed based on theoretical calculationsas well as empirical observations. As an example, the role of the surrounding media and theformation of hydrophilic TCO “patches” in the reaction of TCO-functionalized craft copolymerswith tetrazines of different hydrophilicity is investigated.In addition to the “classic” tetrazine ligation, the versatility of the emerging bioorthogonal Tz/TCOclick-to-release reaction is employed in a “turn-off “and “turn-on” fashion by bond-cleavage in acellular environment. For that matter, new cleavable TCO derivatives are introduced and theirapplication is demonstrated in live cells in proof-of-concept studies. Specifically, intracellularbioorthogonal disassembly of a fluorescent molecular probe is achieved by introducing acleavable C2-symmetric TCO, that enables fast and complete cleavage upon reaction withtetrazines.Furthermore, different strategies for bioorthogonal intracellular drug release are presented, interalia by applying “iTCO”, which impresses with unprecedented click-to-release efficiencies due toa newly revealed release mechanism. Bioorthogonal prodrug activation of an antimitotic reagentwith the help of iTCO is demonstrated in live cells, leading to a more than 100-fold increase intoxicity in a controlled manner.In summary, the presented work lays the foundation towards intracellular bioorthogonal prodrugactivation, helps to understand underlying mechanistic backgrounds and showcases, based onlive cell experiments, that, when the (bioorthogonal) chemistry is right, it can help to improvetargeted drug release and therapy.