The labelling of proteins with 99m Tc represents a technology often applied for tumor imaging. Among those ligands which are used for the preconjugation route BAT-type N 2 S 2 ligands, HYNIC and His 6 tags are the most prominent examples which provide stable complexation sites in proteins. There are various arguments supporting this approach saying that preformed chelator-tagged protein can be hold on stock and that 99m Tc complexation is fast and efficient. Complexation of ligands preceding conjugation on the other hand using e.g. 4,5-bis(ethylcarbonylmercaptoaceta-mide)pentanoic acid 1 is associated with laborious manipulations which, however, is compensated by the very small complex/chelator load. This feature may be termed as a non-chelator(carrier) added preparation. High chelators/protein ratio may have negative influence on the binding characteristics. Here we describe a novel, activated heterobifunctional complex ligand which proved stable against hydrolysis during 99m Tc complexation.
Synthesis of OC-2: 5 mmol S-benzoyl mercaptoactetyldiglycine-N-hydroxysuccinimide ester 2 and 5 mmol p-aminobenzoic acid were dissolved in 35 mL CH 3 CN and the solution was refluxed 5 hr forming a white precipitate. 100 mL CH 3 CN was added and refluxed for additional 30 min.The reaction mixture was filtered warm, and the product was washed with CH 3 CN to obtain N-(S-benzoylthioacetyl)glycylglycyl-p-aminobenzoic acid as a white solid (73%); m.p.: 266-268Β°C (dec). 2.5 mmol of this compound and 5 mmol 2,3,5,6-tetrafluorophenol were dissolved in 20 mL DMF and the solution was cooled in an ice-water bath. 5 mmol N,N'-dicyclohexylcarbodiimide dissolved in 5 mL DMF was added dropwise over 30 min. The reaction mixture was stirred at 0Β°C for 2 hr and then at room temperature overnight. After removal of insoluble N,N'-dicyclohexylurea by filtration the solvent was evaporated in vacuo. The residue was recrystallized twice from iPrOH yielding OC-2 (34%); m.p.:243-245Β°C (dec). Complexation: 100 Β΅l 2 mM OC-2 in DMF was mixed with 100 Β΅l 99m TcO 4 -eluate (300 MBq) and 2 Β΅l 50 mM SnCl 2 in 0.2 M K-gluconate solution. This solution was heated for 20 min at 80Β°C and separated by RP18 HPLC. As shown above the RCY of complexation is temperature dependent showing a maximum between 70-80Β°C almost independent of reaction time. Conjugation: The HPLC eluate was evaporated to dryness and reacted at pH 9-9.5 with 1 mg of an anti-EGF receptor antibody (60Β΅M). 46% RCY was obtained at 30Β°C after 15 min. Conclusion: OC-2 is a novel activated heterobifunctional chelator which is stable against hydrolysis during 99m Tc complexation. Conjugation of 99m Tc-OC-2 with a monoclonal antibody yielded a no carrier added labelling product.