# Labeling Peptides with Radioiodine: An Overview of Traditional and Emerging Techniques
## 4 Indirect Radioiodination Methods: The Labelling of the Prosthetic Groups
### Boronic Acids  
[PERSON] et al. [59] explored the impact of water on the efficiency of copper-mediated radioiododeboronation, a method used to directly radiolabel small molecules and peptides with radioiodine. The study seeks to optimize reaction conditions for various peptides, especially considering the solubility of peptides in water-containing solvents. The central goal was to improve the design of boronic peptide precursors and radiolabeling protocols for reproducible direct radiolabeling of diverse peptides.  
The study uses copper-mediated nucleophilic reactions with a boronic precursor, a promising method for labelling an aromatic ring with radioiodine. This method occurs in open air at room temperature without complex materials and can be adapted for direct radiolabeling of peptide mimetics, solving issues of classical direct radiolabeling such as byproduct formation. Small molecules were firstly used as models before synthesizing \({}^{125}\)I-labelled octreatate derivatives ([\({}^{125}\)I] \(m/p\)-IBTA).  
Key findings and observations were the following. Methanol (MeOH) and ethanol (EtOH) are optimal solvents, yielding high radiochemical conversions (RCCs \(>\) 95%). Acetonitrile (MeCN), \(N\), \(N\)-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) resulted in significantly lower RCCs. Adding MeOH to aprotic polar solvents improved RCCs, but adding H\({}_{2}\)O did not. Increasing water content in MeOH solvent decreases RCCs. This decrease may be due to poor solubility of boronic precursors or target compounds. RCCs can be improved by increasing the amount of the copper catalyst Cu(py)\({}_{4}\)(OTf)\({}_{2}\) and extending the labelling reaction time. In H\({}_{2}\)O/MeOH solvents, substrates with electron-donating groups have higher RCCs than those with electron-withdrawing groups. This aligns with previous research on Chan-Evans-Lam (CEL) coupling reactions [60; 61; 62], where electron-rich arylboronic acids lead to faster catalyst turnover.  
In the proposed mechanism, the reaction involves the formation of complexes, with the presence of MeOH promoting complex formation required for transmetalation. Water can interfere with this process, reducing the formation of the necessary complex and lowering RCCs.  
Direct radiolabeling of peptides was then performed using copper-mediated radioiodo-deboronation. The synthesis of [\({}^{125}\)I] \(m/p\)-IBTA required a larger amount of copper catalyst to exceed 97% RCC (Figure 15).  
The study concludes that high RCCs can be achieved using alcohol solvents, but water content should be minimized. Adjusting the amount of copper catalyst and reaction time can improve RCCs in water-containing solvents. The amount of copper catalyst used was lower than the levels of concern defined in the draft guidance ICH Q3D [63]. Inductively  
Figure 14: Radioiodination of proteins with arylodium salts.  
Figure 15: Standard example of copper-mediated radioiododeboronation.  
coupled plasma mass spectrometry (ICP-MS) analysis showed that Cu(py)\({}_{4}\)(OTf)\({}_{2}\) can be effectively removed, with the labelled material containing substantially less copper residue than any level of concern.