## 3 Chemical Approaches to Bioactive Molecule Labelling with Technetium-99m
### The [\({}^{99\text{m}}\)Tc(N)PNP]\({}^{2+}\) Metal Fragment  
The nitride technetium mixed ligand complexes [\({}^{99\text{m}}\)Tc][Tc(N)(PNP)Cl\({}_{2}\)] provide a further example of the metal fragment approach, where the Tc(N) group is coordinated to a chelating diphosphate ligand of the PNP type and the pentacoordinated geometry is saturated by two chloride atoms. These atoms can be easy replaced by bidentate ligands (Y-Z) having in their molecular structure electron-rich coordinating atoms (Y, Z) highly reactive towards the electrophilic [\({}^{99\text{m}}\)Tc(N)(PNP)]\({}^{2+}\) metallic block to afford the asymmetrical complexes [\({}^{99\text{m}}\)Tc(N)(PNP)(Y-Z)]\({}^{0/+}\)[50; 51]. From this class of asymmetrical complexes, a novel class of myocardial tracers exhibiting superior qualities was obtained, among which \({}^{99\text{m}}\)Tc-N-DBODC and \({}^{99\text{m}}\)Tc-N-MPO are the most representative (Figure 8) [52; 53].  
The first compound is prepared through a two-vial kit formulation following a two-step preparation procedure. To the first vial, the generator eluted \({}^{99\text{m}}\)Tc-pertchentate is added in the presence of SnCl\({}_{2}\) and succini dihydrazide (SDH), required to form the Tc\(\equiv\)N group. The second vial contains the sodium salt of DBODC (bis-N-ethoxyethyl-dithiocarbamato), the bis(dimethoxypropylphosphinoethyl)-ethoxytylamine [PNP5; (CH\({}_{3}\)OC\({}_{3}\)H\({}_{6}\))\({}_{2}\)P(CH\({}_{2}\))\({}_{2}\)-N(C\({}_{2}\)H\({}_{4}\)-OCH\({}_{2}\)CH\({}_{3}\))-(CH\({}_{2}\))\({}_{2}\)P(C\({}_{3}\)H\({}_{6}\)OCH\({}_{3}\))\({}_{2}\)], and \(\gamma\)-cyclodextrintrin, used as a solubilizing agent for the diphosphine ligand. After the reconstitution of the contents of the second lyophilized vial with saline, a part of the resulting solution is added to the first vial and heated at 100 \({}^{\circ}\)C for 15 min to obtain the final \({}^{99\text{m}}\)Tc-N-DBODC compound with yield \(>\)95%. A similar procedure is applied to the preparation of the cationic nitride complex Tc-N-MPO ([\({}^{99\text{m}}\)Tc-N(mpo)(PNP5)]\({}^{+}\): mpo = 2-mercaptoethanoloxide). \({}^{99\text{m}}\)Tc-N-MPO showed favourable biodistribution properties and myocardial uptake with rapid liver clearance in Sprague-Dawley rats [52; 53].  
The [\({}^{99\text{m}}\)Tc(N)PNP]\({}^{2+}\) metal fragment has been efficiently employed for connecting a bioactive molecule with technetium-99m, providing that the biomolecule includes the appropriate set of coordinating atoms. In particular, it was found that the amino acid cysteine shows excellent coordinating properties toward the electrophilic [\({}^{99\text{m}}\)Tc(N)PNP]\({}^{2+}\) metal fragment, either through the (NH\({}_{2}\), S-) pair or the (O-, S-) pair alternatively, giving rise to highly specific and quantitative reactions. Therefore, a selected bioactive molecule, such as a peptide or a small protein, can be conveniently combined with a cysteine residue through the terminal carboxylic group to form the corresponding COO-functionalized \(N\),\(S\)-cysteine ligand or through the terminal amino group to give an N- functionalized O,\(S\)-cysteine ligand. The resulting bioactive cysteine derivative is then reacted with [\({}^{99\text{m}}\)Tc(N)PNP]\({}^{2+}\) to produce the mixed [\({}^{99\text{m}}\)Tc(N)PNP-cysteine- bioactive]\({}^{0/+}\) complex. This approach has been used for the preparation  
Figure 8: Chemical structure of two not-commercial imaging agents for the myocardial perfusion, \({}^{99\text{m}}\)Tc-N-DBODC and \({}^{99\text{m}}\)Tc-N-MPO.  
of receptor-specific complexes for imaging benzodiazepepine receptors and receptor-specific tracers for 5 HT1A receptors [54; 55].