Fluorous solid-phase extraction (F-SPE) platform for the facile preparating heterodimeric radiotracer

Objectives: Molecular imaging is a powerful tool for visualizing biochemical processes involved in normal physiology and/or disease both in vitro and in vivo. The development of novel imaging probes that have high affinity through multivalency or target two receptors on a single cell evokes increasing interest because such probes would be very desirable in diseases where both receptors are over-expressed but the density of each is relatively low. However, current preparations of such probes require complex organic chemistry, conjugation and radiolabeling strategies, which hinder widespread and routine utilization of promising probes for preclinical and/or clinical studies. Herein, we develop a new strategy to simplify the preparation of radiotracers that eliminates chromatographic purification via fluorous solid-phase extraction (F-SPE) by using the fluorous-tagged Staudinger ligation.

Methods: A fluorous-tagged acetyl thioester compound 1 (C8F18-SL-Ac) bearing C8F18 tag on the phosphine was synthesized for Staudinger ligation, in which the phosphine moiety was protected with borane-protection group to avoid oxidation. The Staudinger ligation between the compound 1 and azide-Cy5 were performed under different conditions, including various borane-deprotecting agents, reaction solvents, temperature and time, and the optimal Staudinger ligation will be used to prepare the heterodimeric radiotracers. The resulting Staudinger ligation products were purified by F-SPE (flourous-tagged solid phase extraction). Before and after F-SPE treatment, the purities of products were compared. The integrin α_vβ₃ targeted cyclo(RGDyK) was attached to the C8F18 tag containing Staudinger ligation moiety via TCO/Tz ligation, and the resulting compound 2 (C8F19-SL-RGD) has been used to prepared the integrin α_vβ₃ targeted radiotracer.

Results: The product after reactions of the model compound with azide-Cy5 was obtained and its identity was confirmed by MALDI-TOF. In the optimization studies, the borane-deprotection using DABCO showed higher ligation yields compared to that using TFA, probably due to that the TFA deprotection induced significant oxidation on phosphine (core of Staudinger ligation moiety). Compared to acetonitrile and THF, DMF was more favorable for this fluorous-tagged Staudinger ligation, and it offered 42%, 73% and 90% yield in DMF at 80 °C for 0.5h, 1h, and 2h, respectively, while at lower temperatures of 25 °C and 50 °C, the yields were less than 20% after reacting for 2h. Over 90% excess compound 1 could be quickly removed by F-SPE. Compound 2 (C8F19-SL-RGD) was successfully prepared via TCO/Tz ligation in high yield, and it has been successfully used for the preparation of Cu-64 labeled RGD via Staudinger ligation, while the ligation conditions are currently under optimization.

Conclusion: After thoroughly conditions optimization, over 90% ligation yields were achieved in Staudinger ligation between the compound 1 and azide-Cy5. By using F-SPE, over 90% excess compound 1 was removed rapid, consequently eliminating the chromatographic purification. Despite the conditions still need to be optimized, Cu-64 labeled RGD tracer has been successfully prepared using the proposed F-SPE platform, demonstrating its feasibility on the facile preparation of heterodimeric radiotracer. Research Support: This research was supported by NIH R21-EB017317.