# Labeling Peptides with Radioiodine: An Overview of Traditional and Emerging Techniques
## 2 Direct Radioiodination Methods  
### Electrophilic Isolation  
Direct radioiodination involves the direct incorporation of radioiodine into the peptide structure, usually through electrophilic substitution. This methodology is most feasible when the peptide contains amino acids that are susceptible to iodination, such as tyrosine and histidine [28]. The most common method for direct radioiodination of peptides involves targeting tyrosine residues [29]. Tyrosine contains a phenol group that is highly reactive towards electrophilic attack by iodine (Figure 1). The reaction typically proceeds via an electrophilic aromatic substitution mechanism. The radioiodine, usually in the form of iodide (I\({}^{-}\)), is first oxidized to a more electrophilic species, such as I\({}^{+}\), using an oxidizing agent. This electrophilic iodine then attacks the ortho positions on the tyrosine's phenolic ring, leading to the formation of a carbon-iodine bond. Commonly used oxidizing agents are Chloramine-T and Iodogen. Chloramine-T can be quite harsh and might lead to unwanted side reactions, such as oxidation of cysteine residues or hydrolysis of peptide bonds [30]. This oxidizing agent is typically used in aqueous solutions, and the reaction is quenched with sodium metabisulfite. Iodogen, on the other hand, is a milder oxidizing agent and is often preferred for peptide iodination. Another advantage of using Iodogen is that this is an insoluble reagent that can be easily removed by filtration after the reaction [31], helping in reducing the exposure of the peptide to harsh conditions. The reaction is normally carried out in a heterogeneous system. The reaction of both oxidizing agents is generally performed in a buffered solution at a slightly basic pH (around 7.0-7.5) to facilitate electrophilic substitution and is typically carried out at room temperature for a short time (5-10 min) to minimize any potential degradation of the peptide [16; 17]. Direct iodination of tyrosine can result in mono- or, less commonly, di-iodination--particularly in carrier-added conditions where excess iodine is present--potentially affecting the peptide's biological activity and pharmacokinetics. Additionally, oxidants such as chloramine-T and Iodogen generate reactive chlorine species that may also lead to the formation of mixed halogenated products, such as monochloro-monoido derivatives. Control over the degree and nature of halogenation is typically achieved by adjusting the stoichiometry of the reagents and optimizing reaction conditions. Histidine is another amino acid that can be directly radioiodinated, but it is less reactive than tyrosine. The iodination of histidine occurs at the imidazole ring via an electrophilic substitution mechanism similar to that of \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Method** & **Key Features** & **Use Case** & **Key References** \\ \hline Electrophilic iodination & \begin{tabular}{c} Mild conditions, wide \\ substrate scope \\ \end{tabular} & \begin{tabular}{c} Small molecules, tyrosine/ \\ histidine-containing peptides \\ \end{tabular} & [16; 17] \\ \hline Isotope/halogen exchange & Simple, no precursor synthesis & \begin{tabular}{c} Molecules with existing \\ halogen groups \\ \end{tabular} & [18; 19] \\ \hline Nucleophilic substitution & Requires good leaving groups & Prosthetic groups & [13; 20; 21] \\ \hline Iododestannylation & One-step, highly efficient & \begin{tabular}{c} Aryl-containing molecules, \\ pre-functionalized systems \\ \end{tabular} & [22; 23; 24] \\ \hline Iododesilylation & \begin{tabular}{c} Lower radiochemical yields \\ than Iododestannylation \\ \end{tabular} & \
\begin{tabular}{c} Aryl-containing molecules, \\ pre-functionalized systems \\ \end{tabular} & [2; 14] \\ \hline Diazotisation & High reactivity of intermediate &
\begin{tabular}{c} Aryl-containing molecules, \\ pre-functionalized systems \\ \end{tabular} & [25; 26; 27] \\ \hline \hline \end{tabular}
\end{table}