3.2.2. Conjugation of PEGylated tetrazine (PEG5-Tz)

Either [Fe]FSC (8.0 mg, 10.3 µmol) or [Fe]MAFC (11.0 mg, 13.4 µmol) was dissolved in 500 µL dry DMF and after pH adjustment with DIPEA (pH 9) the mixture was stirred for 30 min at RT. Tetrazine-PEG5-NHS (1 equivalent) was dissolved in 200 µL anhydrous DMF and was slowly added dropwise to the solution over a period of 15 min. After 2 h reaction time at ambient temperature the organic solvent was concentrated in vacuo and purified by preparative RP-HPLC using gradient B to give [Fe]FSC-(PEG5-Tz)<sup>2</sup> (t<sup>R</sup> = 32.9 min) and [Fe]MAFC-PEG5-Tz (t<sup>R</sup> = 26.9 min) as red-brown colored solid after lyophilization. Analytical data:


3.2.3. Synthesis of Monomeric FSC-based Tz Hybrid Imaging Agents

For conjugation of the fluorescent dyes to the monomeric FSC-based Tz-ligand, 2.3 mg of [Fe]MAFC-PEG5-Tz (1.75 µmol) were each dissolved in 500 µL dry DMF, pH was adjusted with DIPEA (pH 9) and 1.1 equivalent of the corresponding dye dissolved in 500 µL DMF was added. SulfoCyanine5-NHS ester (1.50 mg, 1.93 µmol) was added directly while the carboxylic acid of SulfoCyanine7 (1.41 mg, 1.93 µmol), as well as IRDye800CW (2.1 mg, 1.93 µmol) were pre-activated with 1.5 equivalent of *O*-(7-Azabenzotriazol-1-yl)-*N*,*N*,*N*0 ,*N*0 -tetramethyluronium-hexafluorphosphate (HATU, 1.1 mg, 2.89 µmol) for 10 min at ambient temperature. The reaction mixtures were maintained for complete conjugation at RT for 6 h followed by evaporation of DMF in vacuo. The crude bioconjugates were re-dissolved in 1 mL 50% ACN/H2O (*v*/*v*). Half of the solution (500 µL) was purified by preparative RP-HPLC (gradient B) to obtain SulfoCyanine5-[Fe]MAFC-Tz (t<sup>R</sup> = 32.0 min) as dark blue and SulfoCyanine7-[Fe]MAFC-Tz (t<sup>R</sup> = 34.9 min) as well as IRDye800CW-[Fe]MAFC-Tz (t<sup>R</sup> = 28.8 min) as dark green colored solids after lyophilization. Analytical data:


The remaining half of the reaction mixture (500 µL) was used for demetallation. Therefore, 1 mL of disodium EDTA (Na2EDTA, 200 mM) was added and the mixture was stirred for 4 h at RT to completely remove the iron from the conjugates followed by preparative RP-HPLC purification to give intensively green to blue colored solids after freeze drying. Analytical data:

