Technetium-binding in labelled HYNIC-peptide conjugates: role of coordinating amino acids.

Electrospray mass spectrometry (ESMS) of certain peptides labelled with (99m)Tc via hydrazinonicotinamide (HYNIC) with tricine as co-ligand shows one Tc-bound tricine, whereas typically two are observed. We speculated that this was due to coordination of a neighbouring histidine (His) or glutamate (Glu). To investigate this possibility, several short peptides incorporating lysine (HYNIC), with and without His and Glu at different positions in the sequence, were radiolabelled with (99m)Tc, using tricine, ethylenediaminediacetic acid (EDDA) and nicotinic acid as co-ligands.

99mTc-HYNIC-Gastrin Peptides: Assisted Coordination of 99mTc by Amino Acid Side Chains Results in Improved Performance Both In Vitro and In Vivo.

Unlabelled: The aim of this study was to determine the effects of assisted coordination by amino acids such as histidine and glutamic acid on the function of (99m)Tc-labeled gastrin peptide-hydrazinonicotinamide (HYNIC) conjugates and their ability to target cholecystokinin-R in small-animal models.

Methods: Three peptide-HYNIC conjugates containing the -AYGWMDF-NH2 C-terminal sequence and combinations of histidine, glutamic acid, and glycine were synthesized, radiolabeled with (99m)Tc/(99)Tc using either tricine or ethylenediaminediacetic acid as a coligand, and analyzed by the high-performance liquid chromatography and liquid chromatography-mass spectrometric techniques. Stability, receptor binding, and internalization and in vivo targeting in AR42J-bearing mice were assessed.

A technetium intermediate specifically promotes deprotection of trifluoroacetyl HYNIC during radiolabelling under mild conditions.

The trifluoroacetyl group protects the nucleophilic HYNIC from unwanted side reactions yet allows radiolabelling with technetium-99m under mild conditions, due to efficient and specific promotion of deprotection by a technetium complex intermediate.

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

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.

Trifluoroacetyl-HYNIC peptides: synthesis and 99mTc radiolabeling.

Fmoc-lys(HYNIC-Boc)-OH, a precursor for solid-phase synthesis of 99mTc-labeled peptides, was synthesized efficiently without HPLC purification. HPLC-ESMS showed that deprotection and decoupling of peptide from the resin with trifluoroacetic acid gave initially HYNIC-peptide, which was trifluoroacetylated upon prolonged incubation. The trifluoroacetyl-HYNIC group was hydrolyzed during 99mTc labeling, rendering deprotection unnecessary.