Strain-promoted catalyst-free click chemistry for the rapid construction of 64Cu-labeled PET imaging probes

Objectives The presence of catalytic amounts of non-radiolabeled copper ions, often required by the azide-alkyne cycloaddition (click chemistry), interferes with 64Cu, making click chemistry unfavorable for the development of 64Cu-labeled PET probes. Herein, we reported a fast, efficient, and catalyst-free method for generating 64Cu-labeled PET probes based on the aza-dibenzocyclooctyne (DBCO) ligation.

Methods Macrocyclic chelating agent 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine (DiAmSar)-conjugated DBCO was synthesized and radiolabeled with 64Cu in ammonium acetate buffer. To test the efficacy of aza-DBCO ligation, 64Cu-labeled DBCO was mixed with an azide-terminated RGD peptide. The coupling conditions were systematically explored. The resulting conjugate was subjected to small animal PET and direct tissue sampling biodistribution studies in a U87MG-xenografted mouse tumor model.

Results The 64Cu-labeled DBCO was obtained with 90% labeling yield and the identity was confirmed by HPLC co-injection. The conjugation between 64Cu-labeled DBCO and azide-containing RGD peptide was completed (≥ 98%) within 5 minutes of mixing at low micromolar concentrations. The resulting product 64Cu-Sar-DBCO-cRGD can be used for in vivo studies without further purification. For microPET studies, the 64Cu-Sar-DBCO-cRGD peptide demonstrated prominent U87MG tumor uptake with the highest value of 4.25±0.65%ID/g at 2 h pi. The blocking experiment was achieved by co-injection of 64Cu-Sar-DBCO-cRGD with RGDyK (10 mg/kg). The biodistribution results were consistent with the quantitative analysis of microPET imaging.

Conclusions A reliable catalyst-free click chemistry approach was developed for 64Cu-labeling of biomolecules. This method is general and can be applied to other 64Cu-labeled probes for PET applications.

Research Support This research was supported by American Cancer Society Pilot Project Fund (IRG-58-007-51) and USC Department of Radiology