Thiosemicarbazone-Based Responsive MRI Contrast Agents Activated Through a Change in Cu^II Binding Site on Albumin

The development of copper-responsive magnetic resonance imaging (MRI) probes remains limited by the scarcity of robust strategies capable of operating under physiological conditions and within complex biological environments. Here we report Cu^II-responsive MRI contrast agents (CAs) which are able to extract Cu^II from human serum albumin (HSA) and subsequently form ternary adducts. The CAs combine a Gd^III-DO3A-monoamide complex linked to α-pyridyl thiosemicarbazone (pyrTSC) ligands, introduced here as innovative Cu^II binding units. The TSC units display strong Cu^II affinity and form ternary TSC-Cu^II-HSA adducts at a site distinct from the canonical ATCUN motif. Using UV-visible and EPR spectroscopies, we show that the Cu^II extraction and ternary adduct formation remain possible even in the presence of the Gd-binding moiety. The formation of ternary complexes enhances the rotational correlation time (τ_R), leading to measurable increases in relaxivity for the various complexes. This work introduces a new strategy in which Cu^II-triggered protein binding is exploited to activate responsive MRI CAs, thereby broadening their chemical space.