Publikationsrepositorium - Helmholtz-Zentrum Dresden-Rossendorf

Introduction:

The aim of this work is the development of bifunctional chelating agents (BFCAs) for mild and selective labelling of proteins with copper radioisotopes. In this perspective, the triazacyclononane derivative with two pyridyl pendant arms 1
(DMPTACN) seems to be useful. DMPTACN 1 and its bombesin conjugates can readily form very stable radiocopper(II) complexes under physiological conditions.1 The remaining secondary amine group of ligand 1 may be utilized to introduce the isothiocyanate group allowing the conjugation to appropriate biomolecules, e.g. proteins/antibodies by thiourea-bridging.
Results and Discussion:
Compound 1 was synthesized by a eleven-step sequence according to the previously published procedure.1 Treating the secondary amino group of 1 with 2-bromoacetamide reagents gave the appropriate N-Boc protected predecessors 2 (Figure 1). Subsequent cleavage of the Boc protection group with trifluoroacetic acid led smoothly to the 4-aminophenyl and 4-aminobenzyl derivatives of DMPTACN 3. In the last step, the isothiocyanates 4, synthesized by the reaction of
2 with thiophosgen proved to be high yielding. First labelling experiments of DMPTACN isothiocyanates 4 with [⁶⁴Cu]CuCl2 were performed in 0.1M MES/NaOH buffer at a pH = 6.2 and 251C, resulting in a radiochemical purity of higher than 99% within few minutes. The reactivity of the ⁶⁴Cu-labeled isothiocyanates with primary amino groups was tested using glycine as model compound. Applying a ten-fold excess of glycine, the reaction was completed after 4 h at room temperature. To prove the potential to label proteins, the monoclonal antibody C225 was investigated. In a first approach, ligands 4 were radiolabelled with ⁶⁴Cu, and subsequently conjugated to C225. For this pre-labelling strategy, a specific activity of 2.5 GBq/mmol was obtained. The post-labelling approach is more practical. The achievable ligand-toantibody ratios were 5:1, analyzed by Maldi-Tof mass spectrometry. Rapid labelling of the DMPTACN-containing antibody with [⁶⁴Cu]CuCl₂ was achieved to give a specific activity of 304 GBq/mmol.
Conclusion:
DMPTACN isothiocyanates are attractive bifunctional agents for the radiocopper(II)-labelling of proteins featuring very high chemical stabiliy.
Reference
[1] G. Gasser, L. Tjioe, B. Graham, M. J. Belousoff, S. Juran, M. Walther, J.-U. Ku¨ nstler, R. Bergmann, H. Stephan, L. Spiccia, Bioconjugate Chem. 2008, 9, 719–730.