Accessing Self-Illuminated, Luminescent Lanthanide Probes by Enzymatic Radiophosphorylation

Here, we describe the design and synthesis of Tb³⁺ and Eu³⁺ complexes appended to a kinase-substrate peptide, enabling the incorporation of ³²P, a potent Cerenkov emitter, by enzymatic phosphorylation to form a metallacyclized peptide structure with a dual turn-on effect. The construct was optimized to accommodate one inner-sphere donor, identifying 8-coordinate, tricazamacrocycles as ideal to produce a selective turn-on response by displacement of an inner-sphere water molecule by phosphate. The optimization of the peptide sequence allowed for the maximization of PKCα kinase-induced luminescence enhancement. The resulting peptide gave a selective turn-on response of 15% upon displacement of inner-sphere waters. Sequence elongation or rigidification results in disruption of the O-coordination of phosphoserine, as evidenced by NMR spectroscopy and supported by Molecular dynamics (MD) simulations. The enzymatic incorporation of ³²P to the lead peptide-chelate structure was successfully demonstrated with a 95% radiochemical yield and radiochemical purity. Subsequent optical imaging experiments demonstrate the highest probe sensitivity reported to date for a lanthanide probe employing conventional, optical imaging tools, with 0.2 nmol Tb³⁺ complex producing an optical signal above the limit of detection in the presence of 10 μCi ³²P. This work validates enzymatic radiophosphorylation as a suitable strategy to synthesize self-illuminated lanthanide complexes.