The Influence of the Combination of Carboxylate and Phosphinate Pendant Arms in 1,4,7-Triazacyclononane-Based Chelators on Their 68Ga Labelling Properties

Máté G, Šimeček J, Pniok M, Kertész I, Notni J, Wester HJ, Galuska L, Hermann P

21.07.2015 [Original Artikel]

In order to compare the coordination properties of 1,4,7-triazacyclononane (tacn) derivatives bearing varying numbers of phosphinic/carboxylic acid pendant groups towards 68Ga, 1,4,7-triazacyclononane-7-acetic-1,4-bis(methylenephosphinic) acid (NOPA) and 1,4,7- triazacyclononane-4,7-diacetic-1-[methylene(2-carboxyethyl)phosphinic] acid (NO2AP) were synthesized using Mannich reactions with trivalent or pentavalent forms of H-phosphinic acids as phosphorus components. Stepwise protonation constants logK1–312.06, 3.90 and 1.95, and stability constants with GaIII and CuII, logKGaL 24.01 and logKCuL 16.66, were potentiometrically determined for NOPA. Both ligands were labelled with 68Ga and compared with NOTA (tacn-N,N′,N″-triacetic acid) and NOPO, a TRAP-type [tacn-N,N′,N″- tris(methylenephosphinic acid)] chelator. At pH 3, NOPO and NOPA showed higher labelling efficiency (binding with lower ligand excess) at both room temperature and 95 °C, compared to NO2AP and NOTA. Labelling efficiency at pH = 0–3 correlated with a number of phosphinic acid pendants: NOPO >> NOPA > NO2AP >> NOTA; however, it was more apparent at 95 °C than at room temperature. By contrast, NOTA was found to be labelled more efficiently at pH > 4 compared to the ligands with phosphinic acids. Overall, replacement of a single phosphinate donor with a carboxylate does not challenge 68Ga labelling of TRAP-type chelators. However, the presence of carboxylates facilitates labelling at neutral or weakly acidic pH.

A shortcut to high-affinity Ga-68 and Cu-64 radiopharmaceuticals: one-pot click chemistry trimerisation on the TRAP platform

Baranyai Z, Reich D, Vágner A, Weineisen M, Tóth I, Wester HJ, Notni J

05.05.2015 [Original Artikel]

Due to its 3 carbonic acid groups being available for bioconjugation, the TRAP chelator (1,4,7-triazacyclononane-1,4,7-tris(methylene(2-carboxyethylphosphinic acid))) is chosen for the synthesis of trimeric bioconjugates for radiolabelling. We optimized a protocol for bio-orthogonal TRAP conjugation via Cu(I)-catalyzed Huisgen-cycloaddition of terminal azides and alkynes (CuAAC), including a detailed investigation of kinetic properties of Cu(II)–TRAP complexes. TRAP building blocks for CuAAC, TRAP(alkyne)3 and TRAP(azide)3 were obtained by amide coupling of propargylamine/3-azidopropyl-1-amine, respectively. For Cu(II) complexes of neat and triply amide-functionalized TRAP, the equilibrium properties as well as pseudo-first-order Cu(II)-transchelation, using 10 to 30 eq. of NOTA and EDTA, were studied by UV-spectrophotometry. Dissociation of any Cu(II)–TRAP species was found to be independent on the nature or excess of a competing chelator, confirming a proton-driven two-step mechanism. The respective thermodynamic stability constants (log KML: 19.1 and 17.6) and dissociation rates (k: 38 × 10−6 and 7 × 10−6 s−1, 298 K, pH 4) show that the Cu(II) complex of the TRAP-conjugate possesses lower thermodynamic stability but higher kinetic inertness. At pH 2–3, its demetallation with NOTA was complete within several hours/days at room temperature, respectively, enabling facile Cu(II) removal after click coupling by direct addition of NOTA trihydrochloride to the CuAAC reaction mixture. Notwithstanding this, an extrapolated dissociation half life of >100 h at 37 °C and pH 7 confirms the suitability of TRAP-bioconjugates for application in Cu-64 PET (cf. t1/2(Cu-64) = 12.7 h). To showcase advantages of the method, TRAP(DUPA-Pep)3, a trimer of the PSMA inhibitor DUPA-Pep, was synthesized using 1 eq. TRAP(alkyne)3, 3.3 eq. DUPA-Pep-azide, 10 eq. Na ascorbate, and 1.2 eq. Cu(II)-acetate. Its PSMA affinity (IC50), determined by the competition assay on LNCaP cells, was 18-times higher than that of the corresponding DOTAGA monomer (IC50: 2 ± 0.1 vs. 36 ± 4 nM), resulting in markedly improved contrast in Ga-68-PET imaging. In conclusion, the kinetic inertness profile of Cu(II)–TRAP conjugates allows for simple Cu(II) removal after click functionalisation by means of transchelation, but also confirms their suitability for Cu-64-PET as demonstrated previously (Dalton Trans., 2012, 41, 13803).

MA-NOTMP: A Triazacyclononane Trimethylphosphinate Based Bifunctional Chelator for Gallium Radiolabelling of Biomolecules

Poty S, Désogère P, Šimeček J, Bernhard C, Goncalves V, Goze C, Boschetti F, Notni J, Wester HJ, Denat F

01.05.2015 [Original Artikel]

In the past few years, gallium-68 has demonstrated significant potential as a radioisotope for positron emission tomography (PET), and the optimization of chelators for gallium coordination is a major goal in the development of radiopharmaceuticals. Methylaminotriazacyclononane trimethylphosphinate (MA-NOTMP), a new C-functionalized triazacyclononane derivative with phosphinate pendant arms, presents excellent coordination properties for 68Ga (low ligand concentration, labelling at low pH even at room temperature). A “ready-to-be-grafted” bifunctional chelating agent (p-NCS-Bz-MA-NOTMP) was prepared to allow 68Ga labelling of sensitive biological vectors. Conjugation to a bombesin(7–14)derivative was performed, and preliminary in vitro experiments demonstrated the potential of MA-NOTMP in the development of radiopharmaceuticals. This new chelator is therefore of major interest for labelling sensitive biomolecules, and further in vivo experiments will soon be performed.