4.1. Direct Labeling

The radioactive isotopes of iodine (¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I) can be directly integrated into a Nb molecule by electrophilic substitution at tyrosine and histidine residues. The first step consists of creating the electrophile *I⁺ using oxidizing agents like chloramine T (Iodogen^®®) and N-halosuccinimides. Next, the electrophile attacks the aromatic ring of the amino acid tyrosine, since it contains the electron-donating hydroxyl group, forming and stabilizing the σ-complex. This attachment of iodine to tyrosine is highly suitable because the labeling takes place under mild conditions [71]. A similar approach was performed by Pruszynski et al. [74], using 5F7GGC Nb radioiodinated with ¹²⁵I via Iodogen^®® in a radiochemical yield (RCY) of 83.6 ± 5.0%. However, in the same study, better tumor-targeting properties were obtained by radioiodination of the same Nb using [¹³¹I]IB-Mal-D-GEEEK as a prosthetic group. Therefore, some disadvantages have to be taken into consideration on this direct strategy. This radioiodination method is only possible when the protein contains accessible tyrosine or histidine residues. Moreover, an increasing in vivo toxicity can occur due to the accumulation of iodine in the thyroid and stomach.

Similar to iodination, the positron emitter ¹⁸F can also be directly incorporated into a Nb through a tyrosine residue. In theory, this could be achieved via electrophilic or nucleophilic substitution but, in practice, the nucleophilic fluorination approach is the most routinely used. As harsh conditions are required for nucleophilic substitution, this approach is not suitable for the radiolabeling of proteins [71].

Small antibody fragments can also be radiolabeled via an indirect approach, using a prosthetic group or a bifunctional chelator complex. Both strategies will be discussed in the next subsections.