A Bone-Seeking <i>trans</i>-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 <i>trans</i>-cyclooctene (TCO-BP, <b>2</b>) 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 <b>2</b> demonstrated selective localization to shoulder and knee joints in a biodistribution study in normal mice. Compound <b>2</b> reacted rapidly with a ¹⁷⁷Lu-labeled tetrazine in vitro, and pretargeting experiments in mice, using <b>2</b> and the ¹⁷⁷Lu-labeled tetrazine, yielded high activity concentrations in shoulder and knee joints, with minimal uptake in other tissues. Pretargeting experiments with <b>2</b> 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 <b>2</b>. Compound <b>2</b> 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