Evaluation of single amino acid chelate derivatives and regioselective radiolabelling of a cyclic peptide for the urokinase plasminogen activator receptor.

Introduction: The aim of this work was to investigate the relative radiolabelling kinetics and affinity of a series of ligands for the [(99m)Tc(CO)(3)](+) core, both in the absence and in the presence of competing donors. This information was used to select a suitable ligand for radiolabelling complex peptide-based targeting vectors in high yield under mild conditions.

Methods: A series of alpha-N-Fmoc-protected lysine derivatives bearing two heterocyclic donor groups at the epsilon-amine (1a, 2-pyridyl; '1b, quinolyl; '1c, 6-methoxy-2-pyridyl; 1d, 2-thiazolyl; 1e, N-methylimidazolyl; '1f, 3-pyridyl) were synthesized and labelled with (99m)Tc. A resin-capture purification strategy for the separation of residual ligand from the radiolabelled product was also developed. The binding affinities of targeted peptides 4, 5a and 5b for uPAR were determined using flow cytometry.

Results: Variable temperature radiolabelling reactions using 1a-'1f and [(99m)Tc(CO)(3)](+) revealed optimal kinetics and good selectivity for compounds '1a and 1d; in the case of '1a, 1d, and 1e, the labelling can be conducted at ambient temperature. The utility of this class of ligands was further demonstrated by the radiolabelling of a cyclic peptide that is known to target the serine protease receptor uPAR; essentially quantitative incorporation of (99m)Tc occurred exclusively at the SAAC site, despite the presence of a His residue, and without disruption of the disulfide bond.

Conclusion: A series of single amino acid chelate (SAAC) ligands have been evaluated for their ability to incorporate (99m)Tc into peptides. The lead agent to emerge from this work is the thiazole SAAC derivative 1d which has demonstrated the ability to regioselectively label the widest range of peptides.