How do HYNIC-conjugated peptides bind technetium? Insights from LC-MS and stability studies

Abstract

Hydrazinonicotinamide (HYNIC) is an established bifunctional complexing agent for technetium-99m ((99m)Tc) but the structure of the technetium coordination sphere remains uncertain. To gain further insight into this, we have prepared conjugates of HYNIC and hydrazinobenzoic acid (HYBA) with a model peptide, and radiolabelled them with (99m)Tc using three well-established co-ligand systems: EDDA, tricine and tricine-nicotinic acid. The labelled peptides were studied by LC-MS and by subjecting them to serum stability and protein binding assays. For each co-ligand system, HYNIC conjugates formed fewer and more stable labelled species than the corresponding HYBA conjugates. LC-MS analysis showed that all conjugates contained one hydrazine moiety bound to Tc, that binding of Tc to HYNIC-peptide and co-ligand occurs with displacement of 5H(+) indicating a Tc formal oxidation state of +5, and that the Tc has no oxo- or halide ligands. LC-MS also shows that complexes formed with the HYNIC conjugate contain fewer coordinating co-ligand molecules than the HYBA conjugate indicating that HYNIC is able to more effectively satisfy the coordination requirement of technetium, perhaps by binding in chelating mode.

Fig. 1

Some possible structures of ^99mTc–HYNIC–peptide complexes showing monodentate and bidentate binding of HYNIC to technetium.

Fig. 2

Structures of nanogastrin peptide, HYNIC and HYBA and of co-ligands used for technetium labelling.

Fig. 3

RP-HPLC radiochromatograms of ^99mTc–HYNIC-nanogastrin using tricine as co-ligand, labelled at room temperature for 10 min (top) and 3 h (bottom).

Fig. 4

Sample HPLC profiles of conjugate radiolabelling at 95 °C for 30 min, using tricine as co-ligand, comparing steep (method 1) and gentle (method 2) gradients. From top, ^99mTc–tricine–HYNIC-nanogastrin, (i) method 1 and (ii) method 2; ^99mTc–tricine–HYBA-nanogastrin, (iii) method 1 and (iv) method 2.

Fig. 5

Solution stability of labeled peptides. Top: on dilution in phosphate buffer; bottom: on SEPPAK purification to remove excess co-ligand and stannous chloride.

Fig. 6

Stability of labeled peptides on incubation in human serum. Top, % intact radiopeptide by radioHPLC after precipitation of proteins with MeCN; bottom: % protein binding of radioactivity by size exclusion chromatography.

Fig. 7

Schematic structures of crystallographically characterised model complexes (ref. and 12).

Fig. 8

Example schematic structures of possible peptide complexes that are both analogous to crystallographically characterized model complexes and are consistent with ES-MS data; left: Tc–HYNIC-nanogastrin–tricine; right: Tc–HYNIC-nanogastrin–tricine–(nicotinic acid)₂.