# Labeling Peptides with Radioiodine: An Overview of Traditional and Emerging Techniques
## 6 Modern Examples of Biological Applications of Radioiodinated Peptides
Stability studies showed that \({}^{124}\)I-RBD maintained a radiochemical purity of over 99% after incubation in saline or 5% human serum albumin at room temperature for 120 h (5 days), indicating high in vitro stability. Quality control parameters, including appearance, volume, pH, radiochemical purity, ethanol content, endotoxins, sterility, and specific activity, were within the specified limits. This radioiodination technique produced a stable \({}^{124}\)I-labeled RBD probe suitable for further biological evaluation. Cyclic RGD peptides (cRGDs) are recognized ligands for integrins [80]. The radioiodination technique employed for the synthesis of the radiolabeled bicyclic RGD peptide derivatives in the study of [PERSON] et al. [81] involved site-specific radioiodination using [\({}^{125}\)I]SIB. This approach was selected based on the design of the peptide precursors, which lacked other reactive functional groups except for specific \(\varepsilon\)-amino groups at the \(N\)-terminal Lys in bcRGDpal and bcRGDiba, and the amino group of the terminal \(\beta\)-alanine in bcRGDazide, thus directing the radioiodination to these sites. For the synthesis of the single-unit probes, specifically [\({}^{125}\)I]bcRGDpal and [\({}^{125}\)I]bcRGDiba, the corresponding peptide precursor (200 \(\upmu\)g) was dissolved in a mixture of borate buffer (0.1 M, pH 8.5) and acetonitrile. This solution was then combined with [\({}^{125}\)I]SIB (10-20 MBq) dissolved in acetonitrile. The reaction mixture was incubated at 40 \({}^{\circ}\)C for 1 h. Following the reaction, the desired radiolabeled peptides were purified using reversed-phase HPLC. This purification step successfully separated the radiolabeled products from any unreacted precursor, ensuring they were obtained in a non-carrier-added form. The RCY from [\({}^{125}\)I]SIB was 59% for [\({}^{125}\)I]bcRGDpal and 70% for [\({}^{125}\)I]bcRGDiba. Both probes demonstrated radiochemical purities exceeding 99% after HPLC purification. The synthesis of the dimeric probe, [\({}^{125}\)I]bcRGDdimer, involved a multi-step process. First, the radiolabeled precursor [\({}^{125}\)I]bcRGDazide was prepared using the same procedure as for [\({}^{125}\)I]bcRGDpal and [\({}^{125}\)I]bcRGDiba, utilizing [\({}^{125}\)I]SIB. The RCY for [\({}^{125}\)I]bcRGDazide from [\({}^{125}\)I]SIB was 40%, with an RCP \(>\) 99%. Subsequently, [\({}^{125}\)I]bcRGDdimer was synthesized via a click chemistry reaction. The reaction mixture contained equimolar amounts of [\({}^{125}\)I]bcRGDazide and the non-radiolabeled bcRGDalkyne precursor, along with CuSO\({}_{4}\).5H\({}_{2}\)O (50 eq.) and ascorbic acid (100 eq. ), dissolved in dry DMF. This mixture was stirred for 1 h at room temperature. Similar to the single-unit probes, the dimeric product was purified by RP-HPLC. The RCY for [\({}^{125}\)I]bcRGDdimer from [\({}^{125}\)I]bcRGDazide was 61%. The overall yield for [\({}^{125}\)I]bcRGDdimer from [\({}^{125}\)I]SIB was lower (24%). Despite the lower overall yield, the RCP of [\({}^{125}\)I]bcRGDdimer also exceeded 99% after purification.