Two sides of click chemistry: Synthesis of linkable tomaymycin derivatives as in situ activated drugs and a trimethyl lock/ tetrazine-based click-to-release system

In drug development various challenges have to be overcome, including ensuring sufficient half-life, cellular permeability, or avoiding rapid development of resistance. Prodrug approaches, i.e., forming or releasing the active drug at the target site, are being pursued as vital strategies to address these challenges. Such strategies either utilize biochemical processes or bioorthogonal reactions, which provide a wider array of chemical modifications. Bioorthogonal reactions refer to any chemical reaction that can occur in living systems without interfering with native biochemical processes. Initially, bioorthogonal reactions were used to link different chemical entities. Contemporary studies describe the so-called "click-to-release" approach, which allows selective cleavage of two chemical entities triggered by a click reaction. This study explored the in situ activation of two linkable derivatives of the naturally occurring DNA binder tomaymycin. A set of four different tomaymycin-based heterodimers were synthetized utilizing different bioorthogonal reactions as the key step. Using a set of complementary biological evaluation methods, i.e., a plasmid and a oligonucleotide based electrophoretic mobility shift assay, a HPLC-MS assay, a fluorescence based thermal DNA denaturation assay, and a DNase I foot printing assay, this study could prove target binding, i.e., DNA-binding as well as DNA cross-linking for two of the novel tomaymycin based heterodimers. In addition this study reports a novel chemically triggered, bioorthogonal click-to-release system. Herein the trimethyl lock (TML) system is combined with the bioorthogonal inverse electron demand Diels-Alder (IEDDA) reaction of a vinyl ether and a tetrazine, thus expanding the scope of the TML system. Kinetic studies were carried out showing that the reaction rate can be controlled and adjusted depending on the choice of diene (here selected tetrazines). Thereby a 42-fold increase in reaction rate could be achieved.