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Aspergillus fumigatus ( A. fumigatus ) is a human pathogen causing severe invasive fungal infections, lacking sensitive and selective diagnostic tools. A. fumigatus secretes the siderophore desferri-triacetylfusarinine C (TAFC) to acquire iron from the human host. TAFC can be labelled with gallium-68 to perform positron emission tomography (PET/CT) scans. Here, we aimed to chemically modify TAFC with fluorescent dyes to combine PET/CT with optical imaging for hybrid imaging applications. Starting from ferric diacetylfusarinine C ([Fe]DAFC), different fluorescent dyes were conjugated (Cy5, SulfoCy5, SulfoCy7, IRDye 800CW, ATTO700) and labelled with gallium-68 for in vitro and in vivo characterisation. Uptake assays, growth assays and live-cell imaging as well as biodistribution, PET/CT and ex vivo optical imaging in an infection model was performed. Novel fluorophore conjugates were recognized by the fungal TAFC transporter MirB and could be utilized as iron source. Fluorescence microscopy showed partial accumulation into hyphae. µPET/CT scans of an invasive pulmonary aspergillosis (IPA) rat model revealed diverse biodistribution patterns for each fluorophore. [ 68 Ga]Ga-DAFC-Cy5/SufloCy7 and -IRDye 800CW lead to a visualization of the infected region of the lung. Optical imaging of ex vivo lungs corresponded to PET images with high contrast of infection versus non-infected areas. Although fluorophores had a decisive influence on targeting and pharmacokinetics, these siderophores have potential as a hybrid imaging compounds combining PET/CT with optical imaging applications., Competing Interests: The authors declare no conflict of interest.

Fusarinine C (FSC) has recently been shown to be a promising and novel chelator for 89 Zr. Here, FSC has been further derivatized to optimize the complexation properties of FSC-based chelators for 89 Zr-labeling by introducing additional carboxylic groups. These were expected to improve the stability of 89 Zr-complexes by saturating the 8-coordination sphere of [ 89 Zr] Zr 4+ , and also to introduce functionalities suitable for conjugation to targeting vectors such as monoclonal antibodies. For proof of concept, succinic acid derivatization at the amine groups of FSC was carried out, resulting in FSC(succ)₂ and FSC(succ)₃. FSC(succ)₂ was further derivatized to FSC(succ)₂ AA by reacting with acetic anhydride (AA). The Zr 4+ complexation properties of these chelators were studied by reacting with ZrCl₄. Partition coefficient, protein binding, serum stability, acid dissociation, and transchelation studies of 89 Zr-complexes were carried out in vitro and the results were compared with those for 89 Zr-desferrioxamine B ([ 89 Zr]Zr-DFO) and 89 Zr-triacetylfusarinine C ([ 89 Zr]Zr-TAFC). The in vivo properties of [ 89 Zr]Zr-FSC(succ)₃ were further compared with [ 89 Zr]Zr-TAFC in BALB/c mice using micro-positron emission tomography/computer tomography (microPET/CT) imaging. Fusarinine C (succ)₂AA and FSC(succ)₃ were synthesized with satisfactory yields. Complexation with ZrCl₄ was achieved using a simple strategy resulting in high-purity Zr-FSC(succ)₂AA and Zr-FSC(succ)₃ with 1:1 stoichiometry. Distribution coefficients of 89 Zr-complexes revealed increased hydrophilic character compared to [ 89 Zr]Zr-TAFC. All radioligands showed high stability in phosphate buffered saline (PBS) and human serum and low protein-bound activity over a period of seven days. Acid dissociation and transchelation studies exhibited a range of in vitro stabilities following the order: [ 89 Zr]Zr-FSC(succ)₃ > [ 89 Zr]Zr-TAFC > [ 89 Zr]Zr-FSC(succ)₂AA >> [ 89 Zr]Zr-DFO. Biodistribution studies of [ 89 Zr]Zr-FSC(succ)₃ revealed a slower excretion pattern compared to [ 89 Zr]Zr-TAFC. In conclusion, [ 89 Zr]Zr-FSC(succ)₃ showed the best stability and inertness. The promising results obtained with [ 89 Zr]Zr-FSC(succ)₂AA highlight the potential of FSC(succ)₂ as a monovalent chelator for conjugation to targeted biomolecules, in particular, monoclonal antibodies.