A novel and simplified route to the synthesis of N3S chelators for 99mTc labeling

As one example of a N3S chelator, MAG3 has been used successfully for labeling peptides, proteins, DNAs and other carriers with 99mTc. We now report on a simplified route to the synthesis of N3S chelators. As a test of the approach, we have synthesized the succinimidyl ester of S-acetylmercaptoacetyl-(L)-glutamyl(gamma-O-t-Bu)glycylglycolic acid (MAGluG2) (thus MAG3 with a t-butyl protected carboxyl group on the backbone via an ethylene linker) and the succinimidyl ester of S-acetylmercaptoacetyl-phenylalanyl-glycylglycolic acid (MAPheG2) (thus MAG3 with a benzyl group on the backbone). The first chelator was selected to provide a free carboxyl group in the backbone after conjugation to peptides and after t-butyl deprotection whereas the second chelator was selected for its expected lipophilicity. The Fmoc protected NHS ester of the corresponding glutamic acid and phenylalanine were purchased and each was reacted with diglycine followed by Fmoc deprotection to provide the tripeptide. This was reacted with SATA and the NHS ester added via DCC to provide the final NHS ester of MAGluG2 or MAPheG2. After purification, both NHS-derivatives were conjugated to HNE2 (a 7 kDa neutrophil elastase inhibitor) as a test polypeptide. In the MAGluG2 case, t-butyl deprotection was performed after peptide conjugation. Both of the conjugated HNE2 peptides were radiolabeled with 99mTc by transchelation from tartrate as is routine for the labeling of MAG3-conjugated carriers. Labeling efficiencies and stability of the chelated 99mTc towards cysteine transchelation were identical for HNE2 labeled via MAGluG2, MAPheG2 and MAG3. A 3 hr biodistribution of 99mTc radiolabels in normal mice showed significant differences between the three labeled HNE2, especially in major organs (liver and kidneys). We conclude that this synthesis route provides a simplified path to the synthesis of N3S chelators which in principle may be used to incorporate any natural or unnatural amino acid.