**1. Introduction**

Various imaging modalities have evolved as valuable tools for molecular imaging of human diseases. When used as a stand-alone technique, every method shows benefits and limitations [1] so combining different modalities, namely dual-modality- (DMI) or hybrid imaging (HI), is a field with increasing attraction. Optical imaging (OI), for example, is characterized by excellent sensitivity to acquire morphological information but poor tissue penetration limits its applicability for non-invasive imaging purposes. In contrast, single photon emission- (SPECT) and positron emission tomography (PET) show excellent tissue penetration and allow to obtain functional information with high sensitivity. However, due to the lack of morphological information unsurprisingly computed tomography (CT) or magnetic resonance (MR) are used to add morphological details. Although hybrid imaging systems like PET–CT/MRI and SPECT–CT are well established in clinical routine for high resolution imaging their value for real-time intra-operative guided surgery is limited. Despite recent advancements regarding Cerenkov luminescence imaging (CLI) [2] and the development of directional gamma probes (DGP) [3] for radio-guided surgery, optical imaging, particularly near-infrared fluorescence (NIRF) enables the delineation of tumor margins with high accuracy, thus bearing an enormous potential for image-guided surgery [4–7]. Developing targeted imaging probes to combine the complementary nature of PET and NIRF has gained increasing interest and various studies have shown promising results [8–13]. However, most of these studies rely on the use of small peptides as targeting structures being characterized by fast blood clearance and short biological half-life, thus exhibiting fast pharmacokinetics. In contrast, monoclonal antibodies (mAbs) are characterized by their prolonged circulation time exhibiting slow pharmacokinetics but their high affinity and excellent selectivity towards molecular targets bear great potential as imaging agents. The inverse electron-demand Diels-Alder (IEDDA) [14] reaction between 1,2,4,5-tetrazine (Tz) and trans-cyclooct-2-en (TCO) has been established successfully as a highly promising pretargeting strategy, where the TCO-modified mAb is administered prior to the injection of the Tz-bearing radioactive payload to form the radioimmunoconjugate in vivo with exquisite selectivity and tremendously rapid reaction kinetics [15]. With this the applicability of mAbs as targeting vectors has reached a new dimension. Zeglis and co-workers went one step further and have recently shown the suitability of mAbs as targeting vectors for PET/OI hybrid-imaging either using direct labelling [16] or following the IEDDA approach [17]. However, in both studies the fluorescent dye was directly attached to the mAb. In contrast, we herein report on the design of dual-modality imaging agents for pretargeting applications by chelator scaffolding i.e., the fluorescent residue was conjugated to the chelator. Therefore, we utilized the macrocyclic chelator fusarinine C (FSC), which has been already proven to be a suitable scaffold for the development of targeted hybrid imaging agents [18] and allows straight forward radiolabelling with Gallium-68 for PET applications without further modifications, providing advantages over, e.g., F-18 labelling. FSC provides three amine functionalities at the chelating backbone for site specific modification and despite the optical signalling residue we conjugated one or two Tz-motifs for potentially improved IEDDA-based pretargeting as presented in Scheme 1. We used Cyanine based fluorescent dyes as they provide a choice of wavelengths both for microscopy (Cy5) and near-infrared for OI (Cy7, IRDye800CW). A Polyethleneglycol-5 (PEG5)-Tetrazine was chosen for conjugation to FSC, which has provided suitable properties in a previous approach [19].

**Scheme 1.** Route of synthesis for fusarinine C (FSC)-based clickable dual-modality imaging agents (**a**: MetOH/Ac2O; **b**: PEG5-Tz-NHS, DMF/DIPEA; **c**: fluorophore, DMF/DIPEA and *O*-(7- Azabenzotriazol-1-yl)-*N*,*N*,*N*′,*N*′-tetramethyluronium-hexafluorphosphate (HATU); **d**: EDTA). **Scheme 1.** Route of synthesis for fusarinine C (FSC)-based clickable dual-modality imaging agents (**a**: MetOH/Ac2O; **b**: PEG<sup>5</sup> -Tz-NHS, DMF/DIPEA; **c**: fluorophore, DMF/DIPEA and *O*-(7-Azabenzotriazol-1-yl)-*N*,*N*,*N*0 ,*N*0 -tetramethyluronium-hexafluorphosphate (HATU); **d**: EDTA).

#### **2. Results 2. Results**

#### *2.1. Synthesis 2.1. Synthesis*

The FSC-based Tz-bearing hybrid imaging agents were accessible by a straightforward three– four-step synthesis starting from the macrocyclic chelator [Fe]FSC in a similar approach as described in [18,19]. The monomeric conjugates were prepared by initial coupling of the Tetrazine-PEG5-NHS ester to the mono acetyl protected form of FSC, [Fe]MAFC, the dimeric conjugates by starting from [Fe]FSC. Using equal molar amounts the predominant products were [Fe]FSC-(Tetrazine-PEG5)2 or [Fe]MAFC-Tetrazine-PEG5, which were isolated by high-performance liquid chromatography (HPLC), whereby for [Fe]FSC yields were lower due to formation of [Fe]FSC-(Tetrazine-PEG5). The resulting conjugates were straight forward coupled with the respective Dyes by *O*-(7- Azabenzotriazol-1-yl)-*N*,*N*,*N′*,*N*′-tetramethyluronium-hexafluorphosphate (HATU) activation and finally iron was removed. The conjugates could be obtained in acceptable yield i.e., 30–60% for monomeric and 40–60% for dimeric FSC-based Tz hybrid imaging agents in sufficient purity (>90%) determined by analytical reversed phase (RP)-HPLC using UV absorption at λ = 220 nm. The FSC-based Tz-bearing hybrid imaging agents were accessible by a straightforward three–four-step synthesis starting from the macrocyclic chelator [Fe]FSC in a similar approach as described in [18,19]. The monomeric conjugates were prepared by initial coupling of the Tetrazine-PEG5-NHS ester to the mono acetyl protected form of FSC, [Fe]MAFC, the dimeric conjugates by starting from [Fe]FSC. Using equal molar amounts the predominant products were [Fe]FSC-(Tetrazine-PEG5)<sup>2</sup> or [Fe]MAFC-Tetrazine-PEG5, which were isolated by high-performance liquid chromatography (HPLC), whereby for [Fe]FSC yields were lower due to formation of [Fe]FSC-(Tetrazine-PEG5). The resulting conjugates were straight forward coupled with the respective Dyes by *O*-(7-Azabenzotriazol-1-yl)-*N*,*N*,*N*0 ,*N*0 -tetramethyluronium-hexafluorphosphate (HATU) activation and finally iron was removed. The conjugates could be obtained in acceptable yield i.e., 30–60% for monomeric and 40–60% for dimeric FSC-based Tz hybrid imaging agents in sufficient purity (>90%) determined by analytical reversed phase (RP)-HPLC using UV absorption at λ = 220 nm.

#### *2.2. Radiolabeling 2.2. Radiolabeling*

Radiolabeling with the 68Ge/68Ga-generator derived radiometal gallium-68 was conducted at ambient temperature within five minutes and radiochemical yields greater than 98% determined by radio-RP-HPLC and radio-ITLC (instant thin layer chromatography) could be achieved—as exemplarily shown in Figure 1. Radiolabeling with the <sup>68</sup>Ge/ <sup>68</sup>Ga-generator derived radiometal gallium-68 was conducted at ambient temperature within five minutes and radiochemical yields greater than 98% determined by radio-RP-HPLC and radio-ITLC (instant thin layer chromatography) could be achieved—as exemplarily shown in Figure 1.
