Development of an Efficient 18F labeling technology based on tetrazine trans-cyclooctene ligation

Abstract: Proposed is the development of readily available tools for radiochemistry based on tetrazine ligation— a bioorthogonal reaction with rapid kinetics that has been studied for over a decade as a uniquely powerful tool for radiochemical labeling of biomolecules. Limitations in accessing tetrazine and trans-cyclooctene (TCO) labeling

precursors with appropriate kinetic and physicochemical properties have prevented the broader application of this technology. This proposal builds on several technological innovations in reaction chemistry that have been developed in the groups of PIs, including a methodology for introducing 18F into aromatic molecules via

photocatalysis via C-H activation or aromatic substitution, a new methodology for the practical and scalable synthesis of trans-cyclooctenes with favorable kinetic and physiochemical properties, and new methodologies for the preparation of functionalized tetrazines. Through this collaborative technology proposal, our groups will

leverage these methodologies for the development of new tools for the efficient radiolabeling of proteins in site- selective fashion and the application of these tools in vitro. In Aim 1, we will develop improved methods for the synthesis of 18F-labeled trans-cyclooctenes with balanced reactivity, hydrophilicity and stability. Late-stage

photoisomerization will also be used to obtain highly reactive 18F-TCOs, and we will develop methodology for the site-selective attachment to the C-terminus of cancer-targeting proteins. In Aim 2, a rapid and mild method for the preparation of 18F-labeled tetrazine reporters will be developed using photoredox radiofluorination

reactions for PET probe construction. In Aim 3, we will validate the newly developed 18F-TCOs and 18F-tetrazines using the hydrophilic FAPI ligand, the hydrophobic SR142948A, and proteins. The brain permeability of the 18F- TCO/tetrazine reporters will also be evaluated in normal rats. Because this application focuses solely on the

development of 18F-labeling technologies, the studies in Aim 3 are primarily intended to provide important feedback to Aims 1-2, which will result in the design of 18F-TCOs and 18F-tetrazines of high utility to the broader radiolabeling community (in other words, comprehensive evaluation/characterization of specific probes will not

be performed in this application). In summary, we are developing a general labeling toolbox to generate 18F labeled PET agents. We aim to develop labeling precursors that can be readily commercialized, so that the developed technologies will be widely used as efficient and simple labeling methods to modify biologically active

small molecules/peptides/proteins/drugs, which could have a significant impact on medical imaging, drug discovery and development.