A novel and simplified route to the synthesis of N3S chelators for Tc-99m labeling

As one example of a N3S chelator. MAG(3) has been used successfully for lab eling peptides, proteins, DNAs and other carriers with Tc-99m. We now repor t on a simplified route to the synthesis of N3S chelators. As a test of the approach, we have synthesized the succinimidyl ester of S-acetylmercaptoac etyl-(L)-glutamyl(-gamma -O-t-Bu)glycylglycolic acid (MAGluG(2)) (thus MAG( 3) with a t-butyl protected carboxyl group on the backbone via an ethylene linker) and the succinimidyl ester of S-acetylmercaptoacetyl-phenylalanyl-g lycylglycolic acid (MAPheG(2)) (thus MAG(3) with a benzyl group on the back bone). The first chelator was selected to provide a free carboxyl group in the backbone after conjugation to peptides and after t-butyl deprotection w hereas the second chelator was selected for its expected lipophilicity. The Fmoc protected NHS ester of the corresponding glutamic acid and phenylalan ine were purchased and each was reacted with diglycine followed by Fmoc dep rotection to provide the tripeptide. This was reacted with SATA and the NHS ester added via DCC to provide the final NHS ester of MAGluG(2) or MAPheG( 2). After purification, both NHS-derivatives were conjugated to HNE2 (a 7 k Da neutrophil elastase inhibitor) as a test polypeptide. In the MAGluG2 cas e, t-butyl deprotection was performed after peptide conjugation. Both of th e conjugated HNE2 peptides were radiolabeled with Tc-99m by transchelation from tartrate as is routine for the labeling of MAG(3)-conjugated carriers. Labeling efficiencies and stability of the chelated Tc-99m towards cystein e transchelation were identical for HNE2 labeled via MAGluG(2), MAPheG(2) a nd MAG(3). A 3 hr biodistribution of Tc-99m radiolabels in normal mice show ed significant differences between the three labeled HNE2, especially in ma jor organs (liver and kidneys). We conclude that this synthesis route provi des a simplified path to the synthesis of N3S chelators which in principle may be used to incorporate any natural or unnatural amino acid. (C) 2001 El sevier Science Inc. All rights reserved.