# Labeling Peptides with Radioiodine: An Overview of Traditional and Emerging Techniques
## 3 Indirect Radioiodination Methods: The Conjugation of the Prosthetic Groups
### Activated Esters and Maleimide Derivatives  
In indirect radioiodination, activated esters serve as prosthetic groups to facilitate the introduction of radioiodine into macromolecules. This approach is particularly useful when direct labelling is not feasible due to harsh reaction conditions or when modifications to the molecule, such as adding tyrosine, are not possible. The use of prosthetic groups allows reactions to proceed under milder conditions, preventing macromolecules from being affected by oxidizing agents, and can lead to a radioisotope-labelled macromolecule that is stable to deiodination in vivo [13]. Activated esters like NHS (\(N\)-ester of hydroxysuccinimide), Tfp, pentafluorophenyl ester, and their water-soluble SO\({}_{3}\)H-substituted derivatives are commonly employed (Figure 4) [28; 37; 38; 39]. Conjugation is typically achieved by reacting the peptide with a labelled prosthetic agent in a polar solvent like DMF or acetonitrile, in the presence of a tertiary amine. For peptides soluble in water, the reaction can occur in aqueous buffer solutions with a pH of 8-9 or in aqueous-organic reaction media with acetonitrile. Unlike peptides, protein radioiodination is ideally performed in aqueous buffer solutions at a pH close to the physiological range, with conjugation efficiency depending on pH, protein concentration, and temperature. Figure 4: Indirect radioiodination using activated esters. (**a**) Conjugation of activated esters with NH\({}_{2}\) groups of macromolecules. (**b**) Isolation using the Bolton–Hunter method. (* means that the iodine could either be at 3 or 4 position). The formation of peptide/amide bonds between the carboxyl fragment of the prosthetic group and the amino group of the macromolecule is a widely used approach. While Tfp esters are more stable than NHS esters at high pH, NHS esters are more commonly used. Higher protein concentrations can yield better conjugation reaction results but may decrease the molar activity of the target product. An example is the preparation of a radioiodinated bovine serum albumin (BSA) conjugate using \(N\)-Tetrachlorophthaloly (TCP) as a bifunctional linker (a chemical moiety that contains two reactive ends), yielding the \({}^{125}\)I-containing conjugate with 58-75% yield and 99.8% radiochemical purity after purification. Biodistribution studies in mice showed significantly less absorption of the conjugate in the thyroid gland compared to [125]I-iodinated BSA [38]. One of the first and most widely used prosthetic groups is the Bolton-Hunter reagent (\(N\)-succcinimidyl-3-(4-hydroxy-5-[\"I]iodophenyl)propionate), which can be conjugated to the \(N\)-terminus peptide sequence or side chain amino groups, such as lysine [40; 41; 42]. The activated aromatic fragment of the Bolton-Hunter reagent is radioiodinated with the necessary isotope of iodine and then conjugated with a macromolecule to obtain the target compound (Figure 4). The most common prosthetic agents used for radioiodination include \(N\)-succcinimidyl-4-[\"I]iodobenzoate (PIB), \(N\)-succcinimidyl-3-[\"I]iodobenzoate (SIB), and \(N\)-succcinimidyl-5-[\"I]iodo-3-pyridinecarboxylate (SIPC) (Figure 5) [20; 21; 43]. Maleimide derivatives are also used as prosthetic groups for -SH fragments modification in indirect radioiodination, with conjugation reactions typically performed under physiological conditions and generally giving relatively high radiochemical yields (RCYs). An example of using a maleimide derivative is in the synthesis of a tumor-targeting radioiodine derivative of the F3 peptide, called ([125]I)IBMF3), which can be used as a radioligand for tumor imaging by SPECT synthesized with an overall RCY of 73% [43]. Other maleimide derivatives, such as \(N\)-(2-aminoethyl) maleimide (IBM) or 1-(3-[\"I]iodophenyl) maleimide (IPM), have been proposed as prosthetic groups for the radioactive iodination of macromolecules (Figure 5) [2; 44].