# Labeling Peptides with Radioiodine: An Overview of Traditional and Emerging Techniques
## 6 Modern Examples of Biological Applications of Radioiodinated Peptides
The conjugation of the synthesized [**\"**]SIB to 50 \(\upmu\)g of VIP116 was carried out by incubating the labelling mixture at room temperature for 20 min. The conjugation yield obtained was \(46.2\pm 8.0\%\) relative to the starting [\({}^{125}\)]SIB activity. Following the conjugation reaction, the radioiodinated peptide, [**\"**]SIB-VIP116, was purified. Reversed-phase high-performance liquid chromatography HPLC (RP-HPLC) was utilized for this purification step. Subsequently, the purified [**\"**]SIB-VIP116 underwent solid-phase extraction (Emporc(tm) C18) to remove residual acetonitrile from the formulation. The purified product was stored in an elution mixture comprising ethanol and PBS in a 60:40 ratio at 4 \({}^{\circ}\)C. The synthesized[\"]SIB-VIP116 exhibited high stability when stored at 4 \({}^{\circ}\)C in its elution mixture, remaining 97.7% intact at day 7. In vitro studies in human serum at 37 \({}^{\circ}\)C also confirmed high stability, with 98.2% intact at 1 h and only decreasing slightly to 96.0% intact after 72 h of incubation. The final labeled peptide was measured to have a lipophilicity (logD) of 1.45 \(\pm\) 0.05 (\(n\) = 4), suggesting a generally lipophilic character. It is speculated that the SIB conjugation may have contributed to this lipophilicity. Prostate-specific membrane antigen (PSMA) imaging probes are crucial for prostate cancer diagnosis and therapy [76; 77]. Two boronic precursors were synthesized by [PERSON] et al. [78] for the radiosynthesis of the \({}^{125}\)I-labeled PSMA probe, namely [\({}^{125}\)I]mIB-PS: a carboxylic acid-unprotected precursor and a carboxylic acid-protected precursor. The protected precursor was synthesized from a fBu-protected peptidomimetic urea precursor and 3-boronobenzoic acid, followed by purification (yield: 60.2%). The unprotected precursor was obtained by treating the protected one with trifluoroacetic acid (yield: 73.0%). The copper-mediated radioiodination was performed using these precursors (Figure 16). Method I (unprotected precursor): the molecule reacted with Cu(py)\({}_{4}\)(OTf)\({}_{2}\) and [\({}^{125}\)I]NaI in methanol at room temperature for 10 min, followed by HPLC purification. The radiochemical yield for [\({}^{125}\)I]mIB-PS was 77.1% \(\pm\) 6.0%. Method II (protected precursor): the molecule reacted with the copper catalyst and [\({}^{125}\)I]NaI at room temperature for 10 min. After solvent removal, TFA was added to perform continuous deprotection of fBu groups at room temperature for 20 min, without intermediate purification. The product was then purified by HPLC to yield [\({}^{125}\)I]mIB-PS. This method resulted in a significantly higher RCY of 93.8% \(\pm\) 2.6%. Method III (protected precursor, no deprotection): using the protected molecule under the same conditions as Method II but omitting the TFA deprotection step yielded the protected product, [\({}^{125}\)I]mIB-PS(tBu), with an even higher RCY of 98.0 \(\pm\) 1.6%. The study confirmed that the copper-mediated radioiodination successfully produced [\({}^{125}\)I]mIB-PS under mild conditions (room temperature, 10 min reaction time). Using the carboxylic acid-protected boronic precursor in Method II provided a higher RCY for the final product compared to the unprotected precursor (Method I). The continuous deprotection step in Method II also streamlined the synthesis process. Figure 16: Radiosynthesis of [\({}^{125}\)I]mIB-PS via boronic precursors using the three methodologies described (Method I–III) by [PERSON] et al. [78]. (**A**) Method I; (**B**) Method II; (**C**) Method III. The synthesis of the radioiodinated SARS-CoV-2 receptor binding domain (RBD) probe involved labeling the RBD protein with the positron-emitting medical nuclide, \({}^{124}\)I [79]. The radioiodination was achieved using an \(N\)-bromosuccinimide(NBS)-mediated method. Successful synthesis yielded \({}^{124}\)I-RBD with a radiochemical yield of 83.9% \(\pm\) 4.6%. Radio-TLC analysis demonstrated a radiochemical purity of over 99% for the purified \({}^{124}\)I-RBD.