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

PLoS One. 2016 Dec 9;11(12):e0167425. doi: 10.1371/journal.pone.0167425. eCollection 2016.


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 in vitro and in vivo. A trans-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 Staphylococcus aureus infection in vitro and in vivo. 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.

MeSH terms

  • Animals
  • Bone and Bones / diagnostic imaging*
  • Cyclooctanes / chemistry
  • Cyclooctanes / pharmacokinetics*
  • Diphosphonates / chemistry
  • Diphosphonates / pharmacokinetics*
  • Female
  • Heterocyclic Compounds, 1-Ring / chemistry
  • Heterocyclic Compounds, 1-Ring / pharmacokinetics*
  • Hydrazines / chemistry
  • Hydrazines / pharmacokinetics*
  • Mice
  • Nicotinic Acids / chemistry
  • Nicotinic Acids / pharmacokinetics*
  • Radionuclide Imaging / methods
  • Staphylococcal Infections / diagnostic imaging*
  • Staphylococcus aureus / isolation & purification
  • Technetium / chemistry
  • Technetium / pharmacokinetics*
  • Tissue Distribution
  • Vancomycin / chemistry
  • Vancomycin / pharmacokinetics*


  • 6-hydrazinopyridine-3-carboxylic acid
  • Cyclooctanes
  • Diphosphonates
  • Heterocyclic Compounds, 1-Ring
  • Hydrazines
  • Nicotinic Acids
  • Vancomycin
  • Technetium

Grants and funding

Funding was provided by the following government and cancer research agencies within Canada through standard, peer-reviewed research grants: 1)The Natural Sciences and Engineering Research Council of Canada (NSERC) [www.nserc-crsng.gc.ca] research grants: #227514 held by JFV and #227511 held by PJB. 2)The Canadian Cancer Society (CCS) [www.cancer.ca] grant #703857 held by JFV. 3)The Canadian Institutes for Health Research (CIHR) [www.cihr-irsc.gc.ca] grant #493840-16 held by JFV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.