# Labeling Peptides with Radioiodine: An Overview of Traditional and Emerging Techniques
## 3 Indirect Radioiodination Methods: The Conjugation of the Prosthetic Groups
### Cycloadditions  
[PERSON] et al. [49] explored the use of Strain-Promoted Sydnone-Alkyne Cycloaddition (SPSAC) for radioiodination, offering a novel approach to radiopharmaceutical development. The research focuses on sydonnes, which are easily synthesized and chemically stable, as a means to introduce radiohalogens into biomolecules. As already mentioned, direct electrophilic substitution, has limitations, including the requirement for accessible tyrosine residues and potential instability due to dehalogenation. To overcome these limitations, the use of prosthetic groups such as the proposed sydonnes has become common. The researchers compared various bioorthogonal systems and focused on SPSAC due to its high reactivity and suitability for low reactant concentrations, which is ideal for radiotracer development. They designed and synthesized a series of iodinated sydonnes with different substituents at the N\({}_{3}\) and C\({}_{4}\) positions to modulate reactivity (Figure 7). The synthesis of bifunctional precursors involved creating \(N\)-arylsydrones with a carboxylic acid function. Boronic acid precursors and iodinated references were obtained through amide bond formation. Figure 6: Labeling/maleimide conjugation with \({}^{125}\)I using an organotin precursor. Kinetic studies were performed using model peptides with dibenzoazacyclooctyne (DIBAC) or bicyclononyne (BCN) clickable handles to evaluate the impact of N\({}_{3}\) and C\({}_{4}\) substituents on syndromes reactivity. The study found that \(N\)-alkyl syndromes showed the slowest reaction kinetics, while \(N\)-arylchlorosydrones exhibited higher reaction rates with the BCN peptide. The high reaction rate of cycloaddition with BCN proved nearly 11 times higher with the chlorosydrone compared to azide. Radiochemistry experiments involved obtaining radioiodinated syndromes and benzyl azide via the iododeboronation reaction. The radioiodinated prosthetic groups were then evaluated in cycloaddition reactions with model peptides, showing similar trends to the non-radioactive evaluations. The \(N\)-arylchlorosydrone reported in Figure 7 was identified as the most reactive, achieving high radiochemical yields with both DIBAC and BCN peptides. The study concludes that SPSAC is a promising approach for radiopharmaceutical decision, offering a modular system to tune reactivity and functionality for optimal bioconjugation. The optimized system, particularly with BCN, outperformed previously reported SPAAC reactions. The correlation between non-radioactive and radioactive kinetics assays suggests that non-radioactive kinetics assay is a robust predictor of radioactive conditions. The methods developed are also of interest for labeling with astatine-211, a potential alpha emitter for cancer therapy, because astatine exhibits similar reactivity to iodine. [PERSON] et al. developed a convenient method to prepare radioiodinated tetrazines so that a biorthogonal reverse electron demand Diels-Alder reaction could be used to label biomolecules with iodine-125 [50]. Tetrazine was prepared by employing a high-throughput oxidative destannylation reaction that simultaneously oxidized the precursor dihydroretrazine, as reported in Figure 8. Later, their utility was demonstrated through antibody and hormone labeling experiments. Figure 7: Radioiodination through the Strain-Promoted Sydrone-Alkyne Cycloaddition (SPSAC) approach. N\({}_{3}\) and C\({}_{4}\) positions are highlighted. Similarly, [PERSON] and coworkers present a rapid and highly efficient method for radioactive iodine labeling of biomolecules conjugated with trans-cyclootene group using the reverse electron-demand Diels-Alder reaction [51]. The tetrazine structure radioiodination reaction (Figure 9) was conducted always using a stannylated precursor to obtain an \({}^{125}\)I-labeled product with high radiochemical yield (65 \(\pm\) 8%) and radiochemical purity (>99%).