Transglutaminase-mediated conjugation and nitride-technetium-99m labelling of a bis(thiosemicarbazone) bifunctional chelator

An assessment study involving the use of the transglutaminase (TGase) conjugation method and the nitride-technetium-99m labelling on a bis(thiosemicarbazone) (BTS) bifunctional chelating agent is presented. The previously described chelator diacetyl-2-(N-4-methyl-3-thiosemicarbazone)-3-(N-4-amino-3-thiosemicarbazone), H(2)ATSM/A, has been functionalized with 6-aminohexanoic acid (epsilon-Ahx) to generate the bifunctional chelating agent diacetyl-2-(N-4-methyl-3-thiosemicarbazone)-3-[N-4-(amino)-(6-aminohexanoic acid)-3-thiosemicarbazone], H(2)ATSM/A-epsilon-Ahx (1), suitable for conjugation to glutamine (Gln) residues of bioactive molecules via TGase. The feasibility of the TGase reaction in the synthesis of a bioconjugate derivative was investigated using Substance P (SP) as model peptide. Compounds 1 and H(2)ATSM/A-epsilon-Ahx-SP (2) were labelled with nitride-technetium-99m, obtaining the complexes [Tc-99m] [Tc(N)(ATSM/A-epsilon-Ahx)] ((99m)Tc1)and [Tc-99m][Tc(N)(ATSM/ A-epsilon-Ahx-SP)] ((99m)Tc2). The chemical identity of (99m)Tc1 and (99m)Tc2 was confirmed by radio/UV-RP-HPLC combined with ESI-MS analysis on the respective carrier-added products (99g/99m)Tc1 and (99g/99m)Tc2. The stability of the radiolabelled complexes after incubation in various environments was investigated. All the results were compared with those obtained for the corresponding Cu-64-analogues, (64)Cu1 and (64)Cu2. The TGase reaction allows the conjugation of 1 with the peptide, but it is not highly efficient due to instability of the chelator in the required conditions. The SP-conjugated complexes are unstable in mouse and human sera. However, indeed the BTS system can be exploited as nitride-technetium-99m chelator for highly efficient technetium labelling, thus making compound 1 worthy of further investigations for new targeted technetium and copper radio-pharmaceuticals encompassing Single Photon Emission Computed Tomography and Positron Emission Tomography imaging.