Efficient preparation of ^99mTc(III) ‘4+1’ mixed-ligand complexes for peptide labeling with high specific activity

An improved labeling procedure for peptides attached to organometallic ^99mTc(III) ‘4+1’ mixed-ligand complexes in which the radiometal is coordinated by a tripodal tetradentate chelator 2,2',2''-nitrilotriethanethiol (NS₃) and a monodentate isocyanide ligand is presented. The labeling procedure was evaluated by the synthesis of [^99mTc(NS₃)(L2-RGD)]. The containing radiopharmaceutically interesting RGD-peptide cyclo[Arg-Gly-Asp-D-Tyr-Lys] was modified with 4-isocyanobutanoic acid (L2) as linker conjugated to N⁶-Lys to get the monodentate ligand L2-RGD. The structural identity of the ^99mTc-conjugate was confirmed by comparison to a Re reference compound. The Tc- and Re-conjugates had matching retention times under identical HPLC conditions. The ^99mTc-labeling was performed in a novel one-step procedure using the eluate of a ⁹⁹Mo/^99mTc generator, NS₃, the isocyanide modified peptide, SnCl₂, Na₂EDTA, mannitol and ascorbic acid in the reaction mixture. Using optimized reagents it is possible to label 50 nmol peptide with ^99mTc within 60 min at room temperature with a radiochemical yield higher than 95% and a specific activity of ~20 GBq/mmol.