Investigating the Intricacies of Aryl Diazonium Ion Chemistry in Biological Environments for Improved Bioconjugation and Targeted Delivery

Abstract

The field of bioconjugation is a rapidly growing area of research. Aryl diazonium ions (ADIs) are one of the earliest reported bioconjugation agents, forming covalent azo-adducts with electron rich aromatic amino acid residues such as tyrosine and histidine. In addition to azo-adduct formation, certain ADIs have also been reported to generate radical species that not only conjugate to proteins, but also demonstrate the ability to fragment DNA. The many fates of ADIs make them attractive for a many biological applications, however ADIs are not extensively used due to their instability and harsh generation conditions. Herein describes strategies to expand the utility of ADIs as tools for bioconjugation, including approaches for evaluating reactivity and selectivity of ADIs, as well as developing tools to aid in ADI generation and delivery to various biological systems through triazabutadiene probes. Prior work in the Jewett lab focused on expanding the triazabutadiene toolbox by adding functional handles and protecting groups to the triazabutadiene scaffold. This work expands on those previous findings by outlining how the triazabutadiene structure was further modified and adapted for biological delivery, as well as strategies for evaluating the reactivity and selectivity of ADIs in biology. The triazabutadiene scaffold and various mechanisms of protection and deprotection were evaluated, as well as how the protecting group was varied in attempt to couple ADI release to specific events (i.e., enzymatic cleavage). Also included is work on developing tools and technology to utilize triazabutadienes as a biological analysis tool. Furthermore, this work reports how challenges of ADI generation were addressed through the triazabutadiene scaffold, and how this scaffold was further functionalized through a reduction-sensitive protecting group to achieve direct, intracellular delivery, as well as the biological consequences and effects of intracellular ADIs. Additionally, intracellular triazabutadiene probes were further developed with functional handles for more in-depth analyses of intracellular biology. Finally, this work is concluded with a holistic study on ADI reactivity that delves into the development of experiments to understand ADI selectivity small molecule and biomolecule level. These studies will also include factors that influence the ability of an ADI to form an aryl radical and subsequently degrade DNA. This holistic study develops a rulebook of reactivity and selectivity for ADI probes, so that they may be fully utilized in biological systems.