and G.F. of the most promising chelating agents developed so far. Here, we report on the comparison of five different chelators with high potential for stable complexation of 89Zr and determined two of themDFO* and 3,4,3-(LI-1,2-HOPO)to be highly interesting for the preparation of 89Zr-based radiolabeled agents and routine clinical application. Abstract In this work, five different chelating agents, namely DFO, CTH-36, DFO*, 3,4,3-(LI-1,2-HOPO) and DOTA-GA, were compared with regard to the relative kinetic inertness of their corresponding 89Zr complexes to evaluate their potential for in vivo application and stable 89Zr complexation. The chelators were identically functionalized with tetrazines, enabling a fully comparable, efficient, chemoselective and biorthogonal conjugation chemistry for the modification of any complementarily derivatized biomolecules of interest. A small model peptide of clinical relevance (TCO-c(RGDfK)) was derivatized via iEDDA click reaction with the developed chelating agents (TCO = trans-cyclooctene and iEDDA = inverse electron demand Diels-Alder). The bioconjugates were labeled with 89Zr4+, and their radiochemical properties (labeling conditions and efficiency), log= 52.84. This is the result of a nearly optimal complex geometry that is close to the lowest energy structure of Zr(MeAHA)4, being formed by Zr4+ and bidentate Protopanaxdiol MeAHA (= 51.56, which can be explained by the complete saturation of the coordination sphere of the Zr4+ ion. These theoretical considerations are supported by experimental studies demonstrating that the chelators CTH36 and DFO* form complexes of significantly increased stability compared to DFO Protopanaxdiol in in silico complex challenges and/or in vivo imaging studies [7,8,9,10]. Therefore, these two chelating agents are of high interest with regard to further comparative investigation and also potential clinical application. Other chelating agents that showed a significantly higher stability of the formed 89Zr complexes were 3,4,3-(LI-1,2-HOPO) [11,12] and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid) [13] (Figure 1B), which are thus additional promising candidates for further comparative investigation of complex stability and clinical applicability. The aim of the current study was therefore to directly compare the mentioned four chelating agents, as well as the Protopanaxdiol commonly used DFO with regard to the relative kinetic inertness of the 89Zr complexes formed under identical conditions for direct comparability of the obtained results, and to be able to identify the most useful chelating agent Protopanaxdiol for stable 89Zr complexation. For this, analogs of these chelating agents were to be developed enabling an efficient introduction into biomolecules by a chemoselective and biorthogonal conjugation reaction to facilitate a high-yield derivatization of even sensitive biomolecules such as antibodies. For this purpose, a necessary functional group for bioconjugation had to be introduced in a position of the molecular structure of the chelators not interfering with 89Zr complex formation. This requires a backbone functionalization of the respective chelators, leaving the hydroxamate or carboxylate functional groups needed for 89Zr complexation uncompromised. Furthermore, the same biorthogonal and chemoselective conjugation reaction should find application in all cases, thus excluding the possibility that the bioconjugation chemistry itself influences 89Zr complex formation or kinetic inertness. A popular and customizable click chemistry reaction is the inverse electron demand Diels-Alder (iEDDA) conjugation reaction between tetrazines and TCOs (TCO = trans-cyclooctene), which has already found widespread application in radiochemistry [14,15,16]. For this reason, we decided (i) to synthesize backbone tetrazine-modified analogs Rabbit Polyclonal to RPL30 of DFO, CTH-36, DFO*, 3,4,3-(LI-1,2-HOPO) and DOTA, leaving the coordination sphere of the respective agents unaltered to achieve a high Protopanaxdiol kinetic inertness of the resulting 89Zr complexes, and (ii) to introduce them into.