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

¹²⁵I‑Tetrazines and Inverse-Electron-Demand Diels–Alder Chemistry: A Convenient Radioiodination Strategy for Biomolecule Labeling, Screening, and Biodistribution Studies

A convenient method to prepare radioiodinated tetrazines was developed, such that a bioorthogonal inverse electron demand Diels–Alder reaction can be used to label biomolecules with iodine-125 for in vitro screening and in vivo biodistribution studies. The tetrazine was prepared by employing a high-yielding oxidative halo destannylation reaction that concomitantly oxidized the dihydrotetrazine precursor. The product reacts quickly and efficiently with <i>trans</i>-cyclooctene derivatives. Utility was demonstrated through antibody and hormone labeling experiments and by evaluating products using standard analytical methods, in vitro assays, and quantitative biodistribution studies where the latter was performed in direct comparison to Bolton-Hunter and direct iodination methods. The approach described provides a convenient and advantageous alternative to conventional protein iodination methods that can expedite preclinical development and evaluation of biotherapeutics

A ^99mTc-Labelled Tetrazine for Bioorthogonal Chemistry. Synthesis and Biodistribution Studies with Small Molecule <i>trans</i>-Cyclooctene Derivatives

<div><p>A convenient strategy to radiolabel a hydrazinonicotonic acid (HYNIC)-derived tetrazine with ^99mTc was developed, and its utility for creating probes to image bone metabolism and bacterial infection using both active and pretargeting strategies was demonstrated. The ^99mTc-labelled HYNIC-tetrazine was synthesized in 75% yield and exhibited high stability <i>in vitro</i> and <i>in vivo</i>. A <i>trans</i>-cyclooctene (TCO)-labelled bisphosphonate (TCO-BP) that binds to regions of active calcium metabolism was used to evaluate the utility of the labelled tetrazine for bioorthogonal chemistry. The pretargeting approach, with ^99mTc-HYNIC-tetrazine administered to mice one hour after TCO-BP, showed significant uptake of radioactivity in regions of active bone metabolism (knees and shoulders) at 6 hours post-injection. For comparison, TCO-BP was reacted with ^99mTc-HYNIC-tetrazine before injection and this active targeting also showed high specific uptake in the knees and shoulders, whereas control ^99mTc-HYNIC-tetrazine alone did not. A TCO-vancomycin derivative was similarly employed for targeting <i>Staphylococcus aureus</i> infection <i>in vitro</i> and <i>in vivo</i>. Pretargeting and active targeting strategies showed 2.5- and 3-fold uptake, respectively, at the sites of a calf-muscle infection in a murine model, compared to the contralateral control muscle. These results demonstrate the utility of the ^99mTc-HYNIC-tetrazine for preparing new technetium radiopharmaceuticals, including those based on small molecule targeting constructs containing TCO, using either active or pretargeting strategies.</p></div

Synthesis scheme for ^99mTc-HYNIC-tetrazine-TCO-vancomycin (7) from TCO-vancomycin (6) and 4.

<p>Synthesis scheme for ^99mTc-HYNIC-tetrazine-TCO-vancomycin (7) from TCO-vancomycin (6) and 4.</p

Biodistribution data for 4.

<p>Data are presented as the mean (± SEM) percent injected dose per gram (%ID/g) for selected tissues and fluids from CD1 mice at 0.5, 1, 2 and 6 h post injection (n = 3 per time point). Approximately 0.88 MBq were administered per mouse. Full biodistribution data can be found in the supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167425#pone.0167425.s004" target="_blank">S4 File</a>).</p

Radiolabelling scheme for the HYNIC-tetrazine ligand 3 and HPLC chromatogram of the isolated product 4.

<p>^99mTc labelling of the HYNIC-tetrazine <b>3</b> (A). A γ-HPLC chromatogram of the purified final product <b>4</b> (B).</p

Synthesis scheme for the preparation of 3.

<p>A protected form of HYNIC (<b>1</b>) was coupled to a commercially available tetrazine to form <b>2</b>. The Boc group was removed prior to labelling by treatment with TFA in DCM to produce <b>3</b>. Tz* = (4-(1,2,4,5-tetrazin-3-yl)phenyl) methanamine.</p

Biodistribution data comparing active targeting to pretargeting.

<p>Active targeting with ^99mTc-HYNIC-tetrazine-TCO-BP (<b>5</b>) (black bars) is compared to pretargeting of TCO-BP administered 1 h prior to <b>4</b> (gray bars). Data are expressed as the mean (± SEM) %ID/g for selected tissues and fluids from Balb/c mice (n = 3 per time point). Tabulated biodistribution data can be found in the supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167425#pone.0167425.s004" target="_blank">S4 File</a>).</p

Biodistribution data for active targeting of <i>S</i>. <i>aureus</i> infection using ^99mTc-HYNIC-tetrazine-TCO-vancomycin (7).

<p>Compounds <b>4</b> and <b>6</b> were combined prior to i.v. injection of Balb/c mice (n = 3 per time point). Select fluids and tissues were collected at 1 (gray bars) and 6 h (black bars) post injection, including the infected calf muscle (right), and the non-infected calf muscle (left). Data are expressed as the mean percent injected dose per gram (%ID/g) ± SEM. Tabulated biodistribution data can be found in the supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167425#pone.0167425.s004" target="_blank">S4 File</a>).</p

Synthesis scheme for the actively targeted derivative, ^99mTc-HYNIC-tetrazine-TCO-BP (5).

<p>The TCO derivative of the bisphosphonate, alendronate (TCO-BP) was mixed with 4 prior to administration to mice.</p