Labeling of proteins with metallic radionuclides for use in radiopharmaceuticals involves covalently attaching a bifunctional chelator. In principle, use of smaller peptides allows this chelator to be incorporated during solid-phase peptide synthesis (SPPS) with total site specificity. To realize the advantages of this approach, a lysine-hynic conjugate Fmoc-N-epsilon-(Hynic-Boc)-Lys was synthesized for incorporating the well-known technetium-99m-binding hydrazinonicotinamide ligand into peptides during SPPS. It was used to synthesize a technetium-99m-labeled salmon calcitonin with the hynic-linked amino acid in place of lysine-18. A trifluoroacetate group protected the hynic during alkaline oxidation to the cyclic disulfide and was readily removed by mild acid treatment. The peptide was efficiently labeled (91-98% radiochemical yield) with Tc-99m in the presence of tricine and SnCl(2) with high specific activity (>100 MBq/microg). The product showed good serum stability and specific affinity for human calcitonin receptors. Fmoc-N-epsilon-(Hynic-Boc)-Lys is a highly versatile technetium-binding amino acid for incorporation into peptides during SPPS. This allows total flexibility and control in the site of attachment and is suitable for a combinatorial approach to peptide radiopharmaceuticals.

The development of the rhenium-188 generator has provided a convenient and economic source of radionuclide for targeted radionuclide therapy of cancer. To exploit this more widely, mild, convenient and efficient methods for stable incorporation of the radionuclide into biomolecules are required. The aim of this work is to exploit the tumour targeting therapeutic radiopharmaceutical [ReO(dmsa) 2 ] Ϫ (H 2 dmsa = meso-dimercaptosuccinic acid), which is easily synthesised in radioactive form and highly stable in vivo, for this purpose. A new efficient synthesis of [ReO(dmsa) 2 ] Ϫ , as a single isomer (syn-endo), from perrhenate using acetylhydrazine as reductant, is reported. Dehydration of the two dicarboxylic acid groups of [ReO(dmsa) 2 ] Ϫ with dicyclohexylcarbodiimide gives the yellow cyclic dianhydride [ReO(dmsaa) 2 ] Ϫ (H 2 dmsaa = meso-dimercaptosuccinic anhydride) without disrupting the ReOS 4 core. As its tetraethylammonium salt, [ReO(dmsaa) 2 ] Ϫ was characterised crystallographically as a distorted square pyramidal complex with an axial oxo-group and a syn-endo orientation of the two anhydride rings with respect to this oxo-group. [ReO(dmsaa) 2 ] Ϫ reacts with primary and secondary amines including lysine residues of peptides, to form amide-linked conjugates, which were identified by ES-MS. The syntheses are fast, efficient and employ mild conditions, making them readily adaptable for a kit-based approach to rhenium and technetium radiopharmaceuticals.

Direct labeling of salmon calcitonin (sCT) is possible in one step using water-soluble phosphines (sulfonated triphenylphosphines) as the reducing agent both for disulfide and for pertechnetate. Phosphines were the most efficient reducing agent for disulfide bonds among those examined. The phosphines both reduced the pertechnetate to Tc(III), and contributed to the technetium coordination sphere in the labeled product. In contrast, the phosphines did not reduce rhenium below oxidation state V, nor did they participate in the rhenium coordination sphere in the labeled peptide. Instead, the expected oxorhenium(V) moiety was incorporated. Both Tc and Re labeling processes gave rise to dimers with two peptides linked by the metal center, as well as simple monomeric species. Positive mode electrospray mass spectrometry not only revealed the presence of phosphine bound to technetium and oxygen bound to rhenium in the metallopeptides but also revealed the oxidation states of the metals. Electrospray mass spectrometry is proving to be an exceptionally valuable technique for characterizing radiopharmaceuticals. Although the one-step direct labeling method described gives mixed products and poor receptor affinity when applied to the small peptide sCT, it might be readily adapted to monoclonal antibodies.