# Labeling Peptides with Radioiodine: An Overview of Traditional and Emerging Techniques
## 1 Introduction  
The synthesis of radioiodinated peptides is a critical area in radiopharmaceutical chemistry, playing a fundamental role in the development of novel agents for molecular imaging and targeted therapy. Peptides exhibit remarkable specificity and affinity for biological targets, making them highly valuable in the diagnosis and treatment of various diseases, including cancer, cardiovascular disorders, and neurological conditions [1]. When combined with the unique nuclear properties of radioiodine isotopes, these radiolabeled biomolecules serve as powerful tools for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging, as well as radiotherapy [2, 3, 4]. Iodine-based radioisotopes (\({}^{123}\)I, \({}^{124}\)I, \({}^{131}\)I) provide versatile options for both diagnostic and therapeutic applications due to their diverse half-lives and emission properties [5, 6, 7, 8, 9]. \({}^{123}\)I is commonly used for SPECT imaging owing to its favorable gamma emission and short half-life (13.2 h), whereas \({}^{124}\)I enables PET imaging with a relatively long half-life (4.2 days), beneficial for tracking slow biological processes, despite its higher positron energy and reduced resolution compared to \({}^{18}\)F, \({}^{131}\)I, with its beta and gamma emissions, serves as a dual-purpose isotope for imaging and radiotherapy but is less ideal for high-resolution imaging. In contrast, \({}^{18}\)F is prized for its optimal PET imaging characteristics--short half-life (109.8 min), low positron energy, and high resolution--though its short half-life can limit applications requiring extended imaging times. \({}^{64}\)Cu, offering both \(\beta^{+}\) and \(\beta^{-}\) decay, supports PET imaging and radiotherapy, and its intermediate half-life (12.7 h)balances logistical flexibility with imaging resolution. Compared to \({}^{18}\)F and \({}^{64}\)Cu, iodine isotopes are advantageous in settings requiring longer imaging windows, theranostic use, or easy labeling chemistry, but may be less suited for applications prioritizing high spatial resolution or short biological half-lives [10]. Among the earliest and most widespread applications of radioiodinated peptides is their use in radioimmunoassays, particularly with iodine-125 (\({}^{125}\)I). This technique has been instrumental in the sensitive and specific quantification of peptide hormones and other biologically active peptides in complex biological matrices, and continues to serve as a benchmark in immunoassay development [11; 12]. While radiometal-labeled peptides (e.g., \({}^{68}\)Ga-, \({}^{64}\)Cu-, \({}^{177}\)Lu-labeled) have gained prominence in nuclear medicine due to their excellent stability, favorable pharmacokinetics, and suitability for theranostic applications, radioiodinated peptides continue to offer unique advantages in specific contexts. Iodine isotopes allow for direct labeling without the need for bifunctional chelators, simplifying synthesis and avoiding potential alterations in peptide conformation or receptor affinity. Additionally, radioiodination enables high-specific-activity labeling, which is advantageous for receptor-saturable targets. However, a key limitation of iodine-labeled peptides is their susceptibility to in vivo deiodination, particularly with tyrosine-based labeling, leading to thyroid uptake and background signal [13]. In contrast, radiometal complexes, when properly chelated (e.g., via DOTA or NOTA), tend to exhibit greater in vivo stability. Despite these advantages, the synthesis of radioiodinated peptides presents significant challenges. The primary concern is maintaining the structural integrity and biological activity of the peptide while ensuring efficient incorporation of the radioiodine. Peptide radioiodination methods can be broadly categorized into direct and indirect approaches, each offering distinct benefits and limitations.