Radiochemical, Computational, and Spectroscopic Evaluation of High-Denticity Desferrioxamine Derivatives DFO2 and DFO2p toward an Ideal Zirconium-89 Chelate Platform

Abstract

Desferrioxamine (DFO) has long been considered the gold standard chelator for incorporating [89Zr]Zr4+ in radiopharmaceuticals for positron emission tomography (PET) imaging. To improve the stability of DFO with zirconium-89 and to expand its coordination sphere to enable binding of large therapeutic radiometals, we have synthesized the highest denticity DFO derivatives to date: dodecadentate DFO2 and DFO2p. In this study, we describe the synthesis and characterization of a novel DFO-based chelator, DFO2p, which is comprised of two DFO strands connected by an p-NO2-phenyl linker and therefore contains double the chelating moieties of DFO (potential coordination number up to 12 vs 6). The chelator DFO2p offers an optimized synthesis comprised of only a single reaction step and improves water solubility relative to DFO2, but the shorter linker reduces molecular flexibility. Both DFO2 and DFO2p, each with 6 potential hydroxamate ligands, are able to reach a more energetically favorable 8-coordinate environment for Zr(IV) than DFO. The zirconium(IV) coordination environment of these complexes were evaluated by a combination of density functional theory (DFT) calculations and synchrotron spectroscopy (extended X-ray absorption fine structure), which suggest the inner-coordination sphere of zirconium(IV) to be comprised of the outermost four hydroxamate ligands. These results also confirm a single Zr(IV) in each chelator, and the hydroxide ligands which complete the coordination sphere of Zr(IV)-DFO are absent from Zr(IV)-DFO2 and Zr(IV)-DFO2p. Radiochemical stability studies with zirconium-89 revealed the order of real-world stability to be DFO2 > DFO2p ≫ DFO. The zirconium-89 complexes of these new high-denticity chelators were found to be far more stable than DFO, and the decreased molecular flexibility of DFO2p, relative to DFO2, could explain its decreased stability, relative to DFO2.

DFO-Km: A Modular Chelator as a New Chemical Tool for the Construction of Zirconium-89-Based Radiopharmaceuticals

“…Previous zirconium-89 chelation and radiochemical stability data reported from the chelators DFO-Em, DFO2, and DFO2p have shown that increasing the coordination number from what DFO offers (max CN = 6) to saturate the coordination sphere of [ 89 Zr]­Zr 4+ improved the stability of the complexes. The glutamic acid linker used in DFO-Em also greatly improved water solubility relative to DFO .…”

“…To assess whether Zr-(DFO-Km) can attain this preferred cis–cis–trans–trans octadentate Zr­(IV) coordination geometry, we performed DFT calculation using Materials Studio (DMOL3) to determine the energy of 16 possible geometric isomers (Λ-enantiomers) of Zr-(DFO-Km) (Figures and ) using similar methods as previously published. ,,, All calculations contained identical molecular formulas and the same number of atoms, and were calculated with water as solvent (COSMO). As with our DFT study of Zr-(DFO-Em), the large size and flexibility of Zr-(DFO-Km) makes it difficult to settle on a single most stable confirmation of each isomer.…”

“…32,53,54 In our previous work, we reported the synthesis and evaluation of a glutamic acid-based octadentate chelator we named DFO-Em. 15,19,30,31 We attempted synthesis of the bifunctional version of DFO-Em via N-hydroxysuccinimide (NHS) ester activation of the glutamic acid side chain to enable conjugation to antibodies and other vectors. However, this method was not successful due to intramolecular cyclization resulting from reaction of hydroxamate groups with the activated ester.…”

“…In a DFT study on Zr-DFO2 and Zr-DFO2p, we calculated the exchange energy for a single HA bound to Zr(IV) of ∼40−50 kJ/mol, suggesting a substantial energy barrier at ambient temperature to the process of decomplexation/recomplexation and rearrangement of the coordination sphere. 31 As such, regardless of whether isomer (A) is really the most stable and abundant geometric isomer of Zr-(DFO-Km), interconversion between isomers could be very slow without heating. Note that each of the 16 Λ-isomers have a Δ-enantiomer that makes up a total of 32 possible isomers; however, for this study only the Λ-enantiomers were calculated and modeled (Figures 2 and 3) for practical reasons due to the long calculation and optimization times for each of these large structures.…”

“…Other hydroxamic acid-based chelators such as DFO2, DFO2p, DFO-Em, HOPO, oxoDFO*/DFO*, and others have been developed with at least 4 hydroxamic acid groups (Figure ). ,− ,− ,− Many of these chelators have been shown to form more stable complexes with Zr­(IV) than DFO, likely owing to saturation of the coordination sphere around Zr­(IV) with 4 HAs in an octadentate fashion. These higher denticity DFO derivatives typically also have poor water solubility, which makes their chemistry and purification challenging and typically results in low conjugation efficiencies.…”