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

• Published in: Journal of inorganic biochemistry 2018-06, Vol.183, p.18-31
• Main Authors: Salvarese, Nicola, Spolaore, Barbara, Marangoni, Selena, Pasin, Anna, Galenda, Alessandro, Tamburini, Sergio, Cicoria, Gianfranco, Refosco, Fiorenzo, Bolzati, Cristina
• Format: Article
• Language: English
• Subjects: Bis(thiosemicarbazone); Copper; Substance P; Technetium; Transglutaminase
• ISSN: 0162-0134; 1873-3344
• DOI: 10.1016/j.jinorgbio.2018.02.023

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-(N4-methyl-3-thiosemicarbazone)-3-(N4-amino-3-thiosemicarbazone), H2ATSM/A, has been functionalized with 6-aminohexanoic acid (ε-Ahx) to generate the bifunctional chelating agent diacetyl-2-(N4-methyl-3-thiosemicarbazone)-3-[N4-(amino)-(6-aminohexanoic acid)-3-thiosemicarbazone], H2ATSM/A-ε-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 H2ATSM/A-ε-Ahx-SP (2) were labelled with nitride-technetium-99m, obtaining the complexes [99mTc][Tc(N)(ATSM/A-ε-Ahx)] (99mTc1) and [99mTc][Tc(N)(ATSM/A-ε-Ahx-SP)] (99mTc2). The chemical identity of 99mTc1 and 99mTc2 was confirmed by radio/UV-RP-HPLC combined with ESI-MS analysis on the respective carrier-added products 99g/99mTc1 and 99g/99mTc2. The stability of the radiolabelled complexes after incubation in various environments was investigated. All the results were compared with those obtained for the corresponding 64Cu-analogues, 64Cu1 and 64Cu2. 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 radiopharmaceuticals encompassing Single Photon Emission Computed Tomography and Positron Emission Tomography imaging. We assessed the transglutaminase-mediated conjugation and the labelling with nitride-technetium-99m on a bis(thiosemicarbazone) chelator. Diacetyl-2-(N4-methyl-3-thiosemicarbazone)-3-(N4-amino-3-thiosemicarbazone) was modified with 6-aminohexanoic acid and conjugated with Substance P via transglutaminase. The products were labelled with nitride technetium(V)-99g/99m and the complexes were characterized, compared with the 64Cu-analogues and their in vitro stability was tested. [Display omitted] •A Bis(ThioSemicarbazone)-based BiFunctional Chelator (BTS-BFC) is prepared.•The