A Bone-Seeking trans-Cyclooctene for Pretargeting and Bioorthogonal Chemistry: A Proof of Concept Study Using 99mTc- and 177Lu-Labeled Tetrazines

A high yield synthesis of a novel, small molecule, bisphosphonate-modified trans-cyclooctene (TCO-BP, 2) that binds to regions of active bone metabolism and captures functionalized tetrazines in vivo, via the bioorthogonal inverse electron demand Diels-Alder (IEDDA) cycloaddition, was developed. A ^99mTc-labeled derivative of 2 demonstrated selective localization to shoulder and knee joints in a biodistribution study in normal mice. Compound 2 reacted rapidly with a ¹⁷⁷Lu-labeled tetrazine in vitro, and pretargeting experiments in mice, using 2 and the ¹⁷⁷Lu-labeled tetrazine, yielded high activity concentrations in shoulder and knee joints, with minimal uptake in other tissues. Pretargeting experiments with 2 and a novel ^99mTc-labeled tetrazine also produced high activity concentrations in the knees and shoulders. Critically, both radiolabeled tetrazines showed negligible uptake in the skeleton and joints when administered in the absence of 2. Compound 2 can be utilized to target functionalized tetrazines to bone and represents a convenient reagent to test novel tetrazines for use with in vivo bioorthogonal pretargeting strategies.