Tetrazine Glycoconjugate for Pretargeted Positron Emission Tomography Imaging of trans-Cyclooctene-Functionalized Molecular Spherical Nucleic Acids

Pretargeted concept in positron emission tomography (PET) together with bioorthogonal chemistry is an elegant solution to study processes with slow pharmacokinetics by utilizing radiotracers labeled with short-lived radionuclides. Namely, radiotracers based on tetrazine ligation with trans-cyclooctene (TCO) via the inverse electron demand Diels-Alder (IEDDA) reaction have become a state-of-the-art for the pretargeted PET imaging. For radiolabeling of tetrazine scaffolds, indirect radiofluorination methods are often preferred, as tetrazines are vulnerable to harsh conditions typically necessary for the direct radiofluorination. F-18-Fluoroglycosylation is an indirect radiofluorination method, which allows the introduction of a widely accessible glucose analog 2-[F-18]fluoro-2-deoxy-d-glucose ([F-18]FDG) to aminooxy-functionalized precursors via oxime formation. Here, we report the biological evaluation of [F-18]FDG-Tz as a tracer for pretargeted PET imaging of TCO-functionalized molecular spherical nucleic acids (MSNA) against human epidermal growth factor receptor 2 (HER2) mRNA. The oxime ether formation between [F-18]FDG and tetrazine oxyamine resulted in [F-18]FDG-Tz with high radiochemical purity (>99%) and moderate yields (6.5 +/- 3.6%, n = 5). Biological evaluation of [F-18]FDG-Tz in healthy mice indicated favorable pharmacokinetics with quick blood clearance, urinary excretion as the main elimination route, and the absence of GLUT1 transportation. The successful pretargeted experiments with TCO-functionalized MSNA revealed higher tumor uptake compared to preclicked MSNA in HER2-expressing human breast cancer xenograft-bearing mice.

Automated summary

Pretargeted concept in positron emission tomography (PET) utilizing radiotracers labeled with short-lived radionuclides is an elegant solution to study processes with slow pharmacokinetics. Radiotracers based on tetrazine ligation with trans-cyclooctene (TCO) have become a state-of-the-art for pretargeted PET imaging. F-18-Fluoroglycosylation is an indirect radiofluorination method preferred for labeling tetrazine scaffolds. This study reports the biological evaluation of [F-18]FDG-Tz as a tracer for pretargeted PET imaging of TCO-functionalized molecular spherical nucleic acids (MSNA) against human epidermal growth factor receptor 2 (HER2) mRNA. Evaluation in healthy mice showed favorable pharmacokinetics, quick blood clearance, urinary excretion, and absence of GLUT1 transportation. Successful pretargeted experiments with TCO-functionalized MSNA demonstrated higher tumor uptake compared to preclicked MSNA.