Table 2.

Comprehensive overview of prosthetic groups used for radiolabeling thiol-containing biomolecules: applications, strengths, and limitations.

S. No.	Prosthetic group/ radiolabeling conditions	Applications	Strengths	Limitations	Ref.
1	Maleimide Reaction conditions: pH: 7.0 to 7.5 Media: 10% DMF in aqueous buffer Temperature: 25 °C Time: 10 to 90 min Additive: 5-10 eq TCEP	Used for the 18F, 68Ga, 90Y, 177Lu, or 89Zr-based radiolabeling of peptides, proteins, and antibodies.	Exhibiting fast reaction kinetics, high specificity to the cysteine moiety, and the ability to react under physiological conditions, it can be attached to various 18F precursors and radiometal chelators.	For 18F radiolabeling The synthesis is multistep and complex; however in vivo stability supports imaging study. For 68Ga radiolabeling The synthesis is straightforward and facile, with in vivo stability supporting imaging studies. However, it cannot be employed for the imaging of macromolecules due to its short half-life. For 89Zr, 90Y, and 177Lu radiolabeling The synthesis is easy and simple; however, it exhibits poor in vivo stability after 48 h.	21-50
2	Vinyl sulfone Reaction conditions: pH: 8.0 to 8.5 Media: 10% DMF in aqueous buffer Temperature: 25 °C Time: 5 to 30 min Additive: 5-10 eq TECP	Used for the 18F, 64Cu, and 111In-based radiolabeling of peptides, proteins and antibodies.	The prosthetic groups demonstrate a simple and easy synthesis, offering moderate to high radiochemical yield. They react with thiol under physiological conditions and can be attached to various 18F precursors and radiometal chelators, ensuring high in vivo and in vitro stability of both the prosthetic groups and the final radiolabeled product.	Exhibiting slower reaction kinetics compared to the maleimide-thiol reaction, it is not specific to cysteine only and can react with lysine under certain conditions.	54-63
3	Phenyl vinyl sulfone Reaction conditions: pH: 8.5 Media: 50% Methanol in borate buffer Temperature: 30-35 °C Time: 30-40 min Additive: 2 mM TECP	Used for the 18F-based radiolabeling of peptides.	The prosthetic groups demonstrate a simple and easy synthesis, offering moderate to high radiochemical yield. They react with thiol under physiological conditions and can be attached to various 18F precursors.	Exhibiting slower reaction kinetics compared to the maleimide-thiol reaction, it is not specific to cysteine only and can react with lysine under certain conditions.	64-65
4	Perfluoroarylation Reaction conditions: pH: 7.0 to 7.5 Media: DMF Temperature: 25°C Time: 45 min Additive: 2 mM TCEP	Used only for the 18F-based radiolabeling of peptides.	Featuring a simple and easy synthesis, it exhibits high in vivo stability, and allows for simultaneous radiolabeling and dimerization of peptides.	The reaction has slow kinetics and overall low radiochemical yield. It is only suitable for the radiolabeling of peptides.	69
5	Aryl sulfone Reaction conditions: pH: 8.5 Media: 10-50% DMSO in borate buffer Temperature: 30-35 °C Time: 30-120 min Additive: 10 eq TECP	Used for the 18F, 125I, and 89Zr- based radiolabeling of peptides, proteins, and antibodies.	The prosthetic groups undergo a simple and easy synthesis, offering moderate to high yields. They react with thiols under physiological conditions and can be attached various 18F precursors and radiometal chelators, ensuring high in vivo and in vitro stability for both the prosthetic groups and the final radiolabeled product.	Exhibiting slower reaction kinetics compared to the maleimide-thiol reaction, it is not specific to cysteine only and can react with lysine under certain conditions.	73-76
6	2-Cyanobenzothiazoles Reaction conditions: pH: 7.0 to 7.5 Media: 10% DMF in aqueous buffer Temperature: 37°C Time: 30 min Additive: 2 mM TCEP	Used for the 18F, 125I, and 68Ga- based radiolabeling of peptides, and proteins.	The prosthetic groups undergo a simple and easy synthesis, yielding radiochemical results ranging from moderate to high. They react with N-terminal cysteine under physiological conditions, can be attached to various 18F precursors and radiometal chelators, and demonstrate high in vivo and in vitro stability for both the prosthetic groups and the final radiolabeled product. Notably, they are highly specific towards N-terminal cysteine.	Special modification is required to incorporate N-terminal cysteine into the peptide and protein to be radiolabeled. Bulky conjugation increases the lipophilicity of the radiolabeled products, impacting their binding efficiency and pharmacokinetics.	81-84
7	Thiol-ene Reaction conditions: pH: > 7.0 Media: MeOH Temperature: 25°C Time: 60 min Special conditions: Dimethylolpropionic acid and UV irradiation at 366 nm	Used for the 99mTc- based radiolabeling of small molecules.	The prosthetic group is highly stable under harsh reaction conditions, allowing for both click-then-chelate and chelate-then-click strategies, resulting in moderate to high radiochemical yield.	Exhibiting slow reaction kinetics, there is currently no available radiolabeling data for macromolecules, and data on in vitro and in vivo stability are not yet available.	90
8	Strained release reaction Reaction conditions: pH: 8.0 Media: 10%DMF in aqueous buffer Temperature: 60°C Time: 30 to 60 min Special conditions: 0.2 M potassium carbonate	Used for the radioiodine-based radiolabeling of small molecules.	A high radiochemical yield is achieved for peptide radiolabeling, along with high in vivo and in vitro stability.	The synthesis involves a multi-step complex process and has only been attempted for radioiodine-based radiolabeling of peptides. The reaction kinetics is slow, necessitating high temperatures to complete the reaction with thiol. However, no data are available for protein and antibody radiolabeling.	93