Chemists have reported the solid-phase synthesis of a new chelator, DFO*sp, that binds two of the most widely used positron-emitting radionuclides in nuclear medicine, gallium-68 and zirconium-89, and have shown that its radioactive complexes behave favourably in mice. The work, published in EJNMMI Radiopharmacy and Chemistry, compares the new compound with the established chelators desferrioxamine-B (DFO), DFO* and oxoDFO* in a head-to-head evaluation of radiolabelling efficiency, stability, protein binding and whole-body distribution.
Gallium-68 and zirconium-89 are beta-plus emitters used for diagnostic imaging by positron emission tomography (PET). Their physical half-lives dictate which biological molecules they can accompany: gallium-68, with a half-life of 68 minutes, suits small molecules and peptides with fast pharmacokinetics, while zirconium-89, with a half-life of 78.4 hours, is matched to proteins such as antibodies that circulate in the blood for days. Zirconium-89 immunoPET has become an important tool for visualising and quantifying cancer biomarkers in vivo.
A prerequisite for medical use of these radiometals is a chelator that holds the metal ion securely both in the vial and in the body. DOTA and DFO can bind both metals, but each has drawbacks. DOTA forms highly stable complexes yet requires elevated temperatures unsuitable for sensitive biomolecules such as antibodies, and quantitative zirconium-89 complexation with DOTA demands a rather low pH of 4.5 using a zirconium chloride reagent that is not yet commercially available. DFO, by contrast, binds both metals at room temperature and neutral pH, and gallium-68-labelled DFO has shown utility in infection imaging. However, when DFO is attached to proteins for zirconium-89 immunoPET, the resulting complex has proven unstable in vivo, releasing osteophilic zirconium ions that accumulate unspecifically in bone.
To address that weakness, researchers developed DFO*, an extended version of DFO that satisfies the preferred octadentate coordination sphere of zirconium-89. Zirconium-89-labelled DFO* is more stable in vivo than the DFO complex and is currently considered a potential new standard, under investigation in several clinical trials for immunoPET applications. A water-soluble variant, oxoDFO*, containing oxygen atoms within the linker moieties connecting its four hydroxamate units, has also been reported, and both DFO* and oxoDFO* have been shown to chelate gallium-68 as well as zirconium-89. Before the new study, however, biological data on the gallium-68 complexes of DFO* and oxoDFO*, the zirconium-89 complex of oxoDFO*, and comparisons among them and with labelled DFO had not been published.
The team, led by Robin A. J. Maradan and Barbora Neuzilova with senior author Thomas L. Mindt, now reports DFO*sp, a close analog of DFO* prepared by automated, microwave-assisted solid-phase synthesis using standard Fmoc chemistry on a low-loading Rink amide resin. The approach yielded the chelator in 20 percent yield after preparative HPLC purification, with purity above 95 percent, comparable to the roughly 25 to 30 percent achieved for DFO* by fragment condensation. The authors note that solid-phase synthesis circumvents solubility problems with starting materials, intermediates and products and offers a straightforward route to scaling up production. The same strategy was attempted for oxoDFO* but did not succeed: an oxygen-containing aldehyde intermediate underwent spontaneous intramolecular cyclisation, forming a side product isolated in 40 percent yield, and the overall yield of the oxo-monomer remained moderate at 11 percent versus 34 percent for the carbon analog. The researchers conclude that the originally described benzyl protecting group is better suited for oxoDFO* synthesis.
Radiolabelling proceeded efficiently for all four chelators. With gallium-68 chloride in sodium acetate buffer, the complexes formed in 15 minutes at 95 degrees Celsius with radiochemical purity and yield both above 95 percent. With zirconium-89 oxalate in HEPES buffer, complexes of DFO, DFO* and DFO*sp formed quantitatively at room temperature within one hour, while the oxoDFO* complex required two hours of incubation, consistent with previous observations. All complexes showed radiochemical purity and yield above 95 percent, with specific activities of 2.5 to 3.6 megabecquerels per microgram, except the zirconium-89 DFO complex at 0.5 to 0.8 megabecquerels per microgram.
In vitro testing revealed favourable physicochemical properties across the panel. All complexes were hydrophilic, with logP values between minus 3.3 and minus 2.4, the oxoDFO* complexes showing the lowest values in each radiometal series. Stability in phosphate-buffered saline and human serum exceeded 95 percent for most complexes over two hours for gallium-68 labels and 48 hours for zirconium-89 labels, although the DFO*sp complexes showed slightly lower stability that the authors judged not significant given experimental standard deviations. Protein binding was generally low, between 2.3 and 13.0 percent, but unexpectedly high in human serum for gallium-68-labelled DFO at 26.7 percent and gallium-68-labelled DFO* at 30.2 percent. The authors suggest this interspecies difference may explain the long blood half-life observed in first clinical trials of gallium-68 DFO for infection imaging, since binding to murine serum proteins was low.
In vivo experiments in female BALB/c mice confirmed favourable behaviour. Blood stability measured five minutes after intravenous injection exceeded 90 percent for all complexes except gallium-68-labelled DFO*sp, and the authors note that differences in the proportion of intact radiolabelled siderophore among the gallium-68 complexes indicated that favourable in vitro stability did not necessarily translate into comparable in vivo stability. Ex vivo biodistribution showed rapid renal elimination of radioactivity with minimal retention in blood and other organs and tissues at early time points, half an hour for the gallium-68 complexes and one hour for the zirconium-89 ones. The highest accumulation occurred in the kidneys, decreasing over time, and at the latest time points background signal was very low everywhere except the kidneys. Only the DFO* complexes showed retention of radioactivity in the intestines over time. Crucially, no or negligible uptake of radioactivity in bone was observed for any of the zirconium-89-labelled chelators, including the DFO complex known to release the radiometal when conjugated to antibodies with long circulation times.
PET/CT imaging in the mice corroborated the biodistribution data. All complexes cleared rapidly from the bloodstream and were excreted primarily via the kidneys into the bladder, with some accumulation in the intestines and gall bladder indicating partial hepatobiliary excretion. This effect was more pronounced for the less hydrophilic all-carbon analogs than for the oxoDFO* complexes, a finding the authors say indicates potential advantages of using oxoDFO* instead of DFO* for some nuclear medicine applications. The imaging produced high-quality PET/CT images with low background in organs other than those involved in renal excretion, particularly at later time points.
The authors conclude that the excellent in vitro and in vivo characteristics of the gallium-68 and zirconium-89 complexes of the hydroxamate-based chelators make the class promising candidates for radiopharmaceutical development, including immunoPET, infection imaging and other nuclear medicine applications. The new DFO*sp complexes and the previously not preclinically studied oxoDFO* complexes performed comparably to the established DFO and DFO* references. As with any preclinical mouse study, the findings require further validation before translation, but the work provides both a practical synthetic route to a family of chelators and the first direct biological comparison among them under identical conditions.
Subject of Research: Solid-phase synthesis and preclinical evaluation of DFO-analog chelators for gallium-68 and zirconium-89 PET imaging
Article Title: Gallium-68 and zirconium-89 complexes of DFO-analogs for molecular imaging applications using PET/CT
Article References: Maradan, R. A. J., Neuzilova, B., Kronberger, J., Feiner, I., Guarrochena, X., Kılçar, A. Y., Ralis, J., Petrik, M., & Mindt, T. L. (2026). Gallium-68 and zirconium-89 complexes of DFO-analogs for molecular imaging applications using PET/CT. EJNMMI Radiopharmacy and Chemistry, 11(1), Article 80. https://doi.org/10.1186/s41181-026-00509-0
Image Credits: AI Generated
DOI: 10.1186/s41181-026-00509-0
Keywords: gallium-68, zirconium-89, PET imaging, chelators, desferrioxamine, DFO*, radiopharmaceuticals, solid-phase synthesis, immunoPET, biodistribution, hydroxamate, nuclear medicine
News Source: Bethany Barker. (October 27, 2026). New DFO chelator family shows promise for gallium-68 and zirconium-89 PET imaging. Scienmag.
