# Accessing Self-Illuminated, Luminescent Lanthanide Probes by Enzymatic Radiophosphorylation

## TL;DR

Scientists created a new type of glowing probe using lanthanide metals and radioactive phosphorus, which can be activated by enzymes for better imaging.

## Contribution

The novel use of enzymatic radiophosphorylation to create self-illuminated lanthanide probes with a dual turn-on effect.

## Key findings

- A 15% selective turn-on response was achieved by displacing inner-sphere water molecules with phosphate.
- Enzymatic incorporation of 32P into the peptide–chelate structure reached 95% radiochemical yield and purity.
- Optical imaging showed the highest sensitivity for lanthanide probes using standard tools, detecting 0.2 nmol Tb3+ with 10 μCi 32P.

## Abstract

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.