From molecules to medicine: thiol selective bioconjugation in synthesis of diagnostic and therapeutic radiopharmaceuticals

Radiolabeling of biomolecules and cells with radiolabeled prosthetic groups has significant implications for nuclear medicine, imaging, and radiotherapy. Achieving site-specific and controlled incorporation of radiolabeled prostheses under mild reaction conditions is crucial for minimizing the impact on the bioactivity of the radiolabeled compounds. The targeting of natural and abundant amino acids during radiolabeling of biomolecules often results in nonspecific and uncontrolled modifications. Cysteine is distinguished by its low natural abundance and unique nucleophilicity. It is therefore an optimal target for site-selective and site-specific radiolabeling of biomolecules under controlled parameters. This review extensively discusses thiol-specific radiolabeled prosthetic groups and provides a critical analysis and comprehensive study of the synthesis of these groups, their in vitro and in vivo stability profiles, reaction kinetics, stability of resulting adducts, and overall impact on the targeting ability of radiolabeled biomolecules. The insights presented here aim to facilitate the development of highly efficient radiopharmaceuticals, initially in preclinical settings and ultimately in clinical applications.


Introduction
Advancements in modification of biomolecules such as proteins and antibodies have garnered considerable attention as a pivotal technology that is useful in deciphering in vivo and in vitro biochemical mechanisms and pioneering the development of innovative diagnostic and/or therapeutic radiopharmaceuticals [1][2][3].However, the synthesis of a variety of bioconjugates those are both structurally and functionally diverse using two or more small molecules and/or bio-macromolecules continues to be a challenge persisting in the fields of chemistry and biochemistry [4].Pivotal factors such as the specificity and site-selectivity of prosthetic groups and stability of conjugates need to be stringently controlled to safeguard the in vivo functionalities and inherent properties of biomolecules marked for modification.Hence, genetic engineering methodologies have been used adeptly to introduce specific chemical moieties at precise locations within the peptide or protein sequences.These strategically placed moieties act as anchoring points for subsequent radiolabeling via highly efficient and quantitative bioorthogonal chemical reaction [5].Alternatively, the direct modification of native antibodies and proteins has proven to be practical because it delves into the intrinsic reactivity profiles of various side-chain functionalities.Initial efforts to synthesize protein-or antibody-based radiopharmaceuticals involved the modification of relatively abundant, solventaccessible, and highly reactive residues such as lysine [6].Therefore, lysine-specific prosthetic groups such as N-hydroxysuccinimide esters (NHS esters) or other groups that show excess availability were used for this purpose [7,8].However, these approaches led to unpredictable modifications at multiple sites, resulting in heterogeneity and the potential blocking of biologically active sites [9].Currently, the availability of various prosthetic groups facilitates the radiolabeling of biomolecules with heightened specificity and efficiency [10].This advancement enables radiolabeling not only through lysine residues but also via other natural amino acids such as tyrosine, cysteine, and N-terminal cysteine [11].
Among the spectrum of available amino acids, cysteine holds a distinct advantage as the most widely accepted amino acid for the precise and site-selective labeling of folded proteins and antibodies with a variety of prosthetic groups (Figure 1).Its prominence stems from its lower abundance compared with that of lysine and its unique nucleophilicity compared with its affinity to other reactive sites (Figure 2) [12].Moreover, the clinical acceptance of both developed and underdeveloped emerging therapeutic antibodydrug conjugates (ADCs) validates the superiority of cysteine-based modifications over other alternatives.This preference is rooted in the fact that monoclonal antibodies offer approximately eight cysteine and forty lysine residues suitable for prosthetic group conjugation.Consequently, it is anticipated that cysteine-based modifications yield a minimal level of heterogeneity compared to their lysine counterparts, thereby emphasizing their superior suitability for such applications [13].For example, several FDA-approved second-generation ADCs, including brentuximab vedotin, polatuzumab vedotin enfortumab vedotin, trastuzumab deruxtecan, sacituzumab govitecan, belantamab mafodotin, and tisotumab vedotin were predominantly modified via cysteine chemistry (Figure 3), demonstrating the effectiveness of this approach [14][15][16].Nevertheless, challenges persist in the form of conjugate instability, suboptimal reaction kinetics, and non-specific labeling reactions, which remain as primary concerns.These challenges have prompted innovation such as the design of novel and highly efficient prosthetic groups for the radiolabeling of cysteine-containing biomolecules.Although thiol-specific prosthetic groups have in general been reviewed extensively with regard to protein modifications [17][18][19], a comprehensive overview of the most recent advancements in thiol-specific prosthetic groups for radiolabeling micro-and macro-biomolecules is lacking.This review aims to fill the gap by discussing various facets, including the synthesis of radiolabeled thiol-specific prosthetic groups, their stability profiles, bioconjugation kinetics, and stability of the resulting adducts in relation to their chemical structures.Moreover, this review provides a critical analysis and comparative study aimed at aiding the scientific community in identifying the most effective methodologies for radiolabeling biomolecules.The insights presented here are intended to facilitate the development of superior radiopharmaceuticals, initially in preclinical settings and ultimately in clinical applications.

Maleimide based radiolabeling
Michael acceptors, such as maleimide, represent an important class of prosthetic groups that react with cysteine to form thiosuccinimide bonds.Moore and Ward pioneered the initial exploration of the maleimide-thiol-based reaction and elucidated its potential for cross-linking thiol-containing proteins [20].Subsequently, maleimide-based prosthetic groups have been further developed for sulfhydryl group blocking, fluorescent mapping of cysteine residues in biomolecules, and the synthesis of therapeutic or diagnostic radiopharmaceuticals.Despite concerns regarding thiosuccinimide bond degradation, maleimide-thiol-based prostheses have gained significant traction and are currently well-established.Numerous studies have demonstrated their application in nuclear medicine.Currently, maleimide-based prosthetic groups are preferred by radiochemists for synthesizing radiopharmaceuticals using thiolcontaining biomolecules.Several factors drive this preference.Maleimide-based reactions demonstrate high selectivity towards less-abundant thiol groups, yield minimal side products, and allow reactions to occur in aqueous media under mild conditions, and exhibit high reaction rates in the absence of heat and catalysts.The subsequent sections present a comprehensive examination of maleimide-based prosthetic groups.]DDPFB) compounds, respectively (Figure 4).Although both prosthetic groups were radiolabeled with high yields, only [ 18 F]DDPFB was investigated further.
[ 18 F]DDPFB successfully radiolabeled rabbit IgG-derived Fab with >50% radioactivity attached to the antibody fragment.However, this study did not assess the in vitro stability or perform in vivo testing using the radiolabeled antibody.

[ 18 F]FBABM, [ 18 F]FBAM, and [ 18 F]FBOM
During the synthesis of [ 18 F]DDPFB, utilizing m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) following 18 F radiolabeling was considered inefficient because of the poor stability of the NHS ester.An alternative approach involved the synthesis of 18 F labeled maleimide-based prosthetic group through the reaction of benzaldehyde with aminooxy group.The selection of the aminooxy functional group over the amine group occurs because of its high reactivity and selectivity towards the carbonyl group.Moreover, oxime ethers exhibit exceptional stability, which is crucial for efficient radiolabeling.In this study, 4-[ 18   F] FBABM) (Scheme 1) at a moderate radiochemical yield (>35%). 18 F labeled maleimide was further used for radiolabeling the linear peptide Glutathione (GSH) and thiol containing antisense oligodeoxynucleotides (ODNs).Radiolabeled GSH and ODNs were obtained at moderate yields of 70% and 5%, respectively.However, the radiolabeled compounds were not further analyzed in biological studies [22].Wuest et al. used a similar strategy to synthesize N-[6-(4-[ 18 F]fluoro-benzylidene) aminooxyhexyl]maleimide ([ 18 F]FBAM) with 29% total radiochemical yield.This prosthetic group was used to radiolabel various small peptides such as GSH and human low-density lipoprotein (LDL). 18F-labeled LDL (17 ± 10% radiochemical yield) demonstrated high in vitro and in vivo stabilities [23].To enhance the hydrophilicity of [ 18 F]FBAM, some researchers tried to synthesize 4-[ 18  F]FBOM).However, the radiochemical yield of [ 18 F]FBOM and its radiolabeling efficiency for LDL protein were suboptimal compared with those of [ 18 F]FBAM [24].

[ 18 F]FPyME
To synthesize radio-fluoropyridinyl moieties, nucleophilic heteroaromatic substitution with [ 18 F]fluoride has emerged as a more precise strategy for synthesizing tracers with high specific radioactivity.Based on this, Bruin et al. detailed the synthesis of [ 18 F]fluoropyridine-based maleimide for the highly specific radiolabeling of biomolecules [25].They synthesized [(2-nitro-pyridin-3-yloxy)alkyl] carbamic acid tert-butyl esters for radiofluorination and concurrently investigated the influence of alkyl chain length on the radiolabeling reaction.The nitro moiety acts as an efficient leaving group without requiring additional electron-withdrawing groups to activate the heteroaromatic ring.Precursor [(2-nitro-pyridin-3-yloxy)alkyl]carbamic acid tert-butyl ester (alkyl: ethyl, propyl, butyl, pentyl or hexyl) was heated in the presence of dried K[ 18 F]F-K 222 complex at 145 °C for 30 min to obtain the corresponding 18 F-labeled compound.Notably, the precursor with a propyl chain exhibited a high radiochemical yield (50-75%) and was chosen for subsequent investigation.Incorporation of the maleimide group into the radiofluorinated compound was achieved through the reaction of maleic anhydride or N-methoxycarbonylmaleimide in o-xylene under reflux conditions.However, N-methoxycarbonylmaleimide provided a superior radiochemical yield (46-77%) for the synthesis of 4).[ 18 F]FPyME was also synthesized using ([3-(3-tert-butoxycarbonylaminopropoxy)-pyridin-2-yl]trimethylammonium trifluoromethane sulfonate) as an alternative to the nitro-based leaving group, resulting in a high radiochemical yield under relatively mild reaction conditions.The efficacy of [ 18 F]FPyME was assessed using (N-Ac)-KAAAAC as the model peptide.Radiolabeling was achieved with high yields (60-70%, non-decay-corrected); however, the radiolabeled compounds were not subjected to further biological studies.
[ 18 F]FDG-HMO [ 18 F]Fluoro-2-deoxy-D-glucose ([ 18 F]FDG) is a cornerstone radiopharmaceutical for clinical PET imaging because of its exceptional efficiency and ease of automated and streamlined radiosynthesis process, which enables its widespread availability in cancer hospitals.Despite this prevalence, the incorporation of [ 18 F]FDG as a building block or prosthetic group in radiopharmaceutical synthesis remains relatively limited.In 2008, Wuest et al. proposed that the abundant accessibility of [ 18 F]FDG in hospitals could be harnessed to synthesize maleimide-based prosthetic groups.Notably, [ 18 F]FDG exists as a mixture of α/β anomers in aqueous solutions with an accompanying transformation into an acyclic aldehyde form through a ring opening mechanism.
They further aimed to synthesize [ 18 F]FDG-maleimide prosthetic groups through a chemoselective aminooxy-aldehyde coupling reaction in anticipation of the utility of the aldehyde form.Treatment of aminooxy maleimide hydrochloride with [ 18 F]FDG at 100 °C for 15 min produced an isomeric mixture of [ 18 F]FDG-MHO (Scheme 2) with high radiochemical yield (45-69%) and purity (>95%), which was purified using HPLC.The efficiency of [ 18 F]FDG-MHO was demonstrated by its successful conjugation with the model peptide glutathione (GSH) and protein annexin A5.Notably, incubation of [ 18 F]FDG-MHO with annexin A5 yielded the corresponding 18 F-labeled protein with high radiochemical yield (43-58%) and purity.Its subsequent application in PET imaging of human colorectal adenocarcinoma (HT-29) animal model revealed excellent in vivo stability of the [ 18 F]FDG-MHO-anxA5 complex.Although a significant radioactivity signal was observed in the kidneys and liver, tumor uptake remained relatively limited in these experimental settings [32].
[ 18 F]FDG-HMO exists in the form of isomers.
Successful immuno PET imaging relies on the selection of radioisotopes with half-lives that are compatible with those of the biomolecules.For instance, antibodies (approximately 140 kDa) have a biological half-life ranging from several days to weeks; yet an optimal tumor-to-background ratio typically manifests 2-6 days after administration [38].In this context, positron-emitting radioisotopes such as zirconium-89 ( 89 Zr, T 1/2 = 3.3 days) represent a better solution compared with 18 F or 68 Ga [39]. 89Zr has been integrated into biomolecules using lysine [40] and cysteine [41].Monoclonal antibodies offer approximately 8 cysteine and 40 lysine sites for prosthetic group conjugation.Predictably, cysteinebased modifications aim to reduce heterogeneity compared with lysine-based modifications.However, cysteine-based modifications may decrease the plasma half-lives of the diagnostic or therapeutic drugs.Tinianow et al. evaluated the impact of thiol-or amino-based radiolabeling on the bioactivity of a radiopharmaceutical by conducting a comparative study using the desferrioxamine B (DFO) chelator (Figure 6A) containing thiol-based prosthetic groups such as DFO-Chx-Mal (Figure 6B) [42] and DFO-Iac (Figure 6C) along with frequently reported lysinebased prosthetic groups such as DFO-N-Suc-TFP (Figure 6D) and DFO-Bz-SCN (Figure 6E).All four  PET imaging and biodistribution data were acquired for all four 89 Zr-trastuzumab derivatives using the BT474M1 xenograft model (Figure 6F).Despite slight differences in tumor, kidney, bone, and muscle uptake, all prosthetic groups exhibited comparable biodistribution and imaging outcomes.However, using site-selective DFO-Chx-Mal offers distinct advantages.Notably, site-sensitive radiolabeling of genetically modified antibodies avoids potential alterations in antigen binding sites and ensures homogeneous and nearly identical products with minimal batch-to-batch variability [43].  8Zr-trastuzumab derivatives using BT474M1 xenograft models (96 h post intravenous injection).Adapted with permission from [43], copyright 2010, Elsevier Inc.In pursuit of a chelating agent with efficiency that surpasses that of DFO, Orvig et al. devised the synthesis of 2,2'-((((2-aminoethyl)azanediyl)bis (ethane-2,1-diyl))bis(((8-hydroxyquinolin-2-yl)methyl )azanediyl))diacetic acid (H 4 neunox).A maleimidecontaining chelator (H 4 neunox-Mal) was tailored for 89 Zr radiolabeling of biomolecules.The synthesis involved the reaction of N-Succinimidyl 6-maleimidohexanoate with H 4 neunox under basic conditions to produce H 4 neunox-Mal (Figure 7A).Subsequently, H 4 neunox-trastuzumab was synthesized using H 4 neunox-Mal.For comparison, DFO-trastuzumab was synthesized using the corresponding maleimide precursor.
Its biodistribution profile and tumor-to-blood (24.61 ± 14.45) and tumor-to-muscle ratios (34.96 ± 8.60) were better than that of [ 18 F]AlF-NODA-Z HER3:8698 ,confirming its superiority over IEDDA based conjugation [47].For example, [ 18 F]AlF-NOTA-MAL was synthesized at 15% radiochemical yield and used for the radiolabeling of the Cys-annexin V protein (Figure 8C).The resulting [ 18 F]-AlF-NOTA-MAL-Cys-annexin V demonstrated robust in vitro and in vivo stability.This radiotracer has been effectively used to visualize apoptotic animal models using a PET imaging system [48].9B) and substantial tumor-to-background and tumor-to-liver ratios; however, renal uptake remained notably high [49].Despite yielding critical outcomes, [ 18 F]FPenM has some limitations such as low radiochemical yield and complex fabrication procedures.
To address these limitations, Chen et al.

F labeled thiol groups for maleimide containing molecules
A multistep radiolabeling process is used to counteract the poor stability of the maleimide group under the basic conditions necessary for nucleophilic radiofluorination.This process is cumbersome and diminishes the overall radiochemical yield.In 2019, Jacobson et al. proposed an alternative approach using 18 F radiolabeled thiol-containing precursor.Two prosthetic groups, namely, [ 18  Based on these results, Schirrmacher et al. proposed 18 F labeling of maleimide-containing biomolecules such as rat serum protein.To achieve this, a maleimide group was incorporated into the protein using 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxy-succinimide ester sodium salt (sulfo-SMCC).The silicon fluoride acceptor reagent [ 18 F]SiFA-SH was synthesized using an isotopic exchange reaction, which was subsequently used for radiolabeling the protein with the installed maleimide group.Nevertheless, the radiolabeled compound was not subjected to further studies [52].This radiolabeling method involves the incorporation of a maleimide group using a lysine functional group.
Although this approach appears suitable for radiolabeling peptides, it may pose challenges when applied to macromolecules because of the potential for nonspecific radiolabeling of molecules similar to that encountered with other lysine-based radiolabeling approaches.
Overall, maleimide-based prosthetic groups remain the preferred option for cysteine-based radiolabeling of biomolecules, owing to their distinct properties that are outlined in Table 1.Maleimide-thiol-based modifications are recognized for their rapid kinetics and achieving quantitative and highly specific reactions under physiological conditions.However, maleimide-thiol adducts are notably unstable under physiological conditions owing to thiol exchange and retro-Michael addition reactions (Figure 11).Pharmacologically, the instability of thiosuccinimide adducts and premature cleavage of therapeutic or diagnostic radionuclides lead to reduced drug cytotoxicity, heightened off-target toxicity, or a decline in imaging quality.Moreover, the hydrolysis of thiosuccinimide and subsequent ring-opening pose additional challenges.High in vitro and in vivo stability were observed alongside a high radiochemical yield.
The process involves multistep synthesis, resulting in a final product that is a mixture of isomers.32 9 DOTA-malimide Utilized for 90 Y and 177 Lu-based radiolabeling of GSH peptide and 12 mer L-RNA.
High radiochemical yield was achieved with a straightforward synthesis.
Poor in vivo stability was observed after 48 hours.
High radiochemical yield was achieved with a straightforward synthesis.
Poor in vivo stability.37
High radiochemical yield was achieved with a straightforward synthesis.
Poor in vivo stability was observed after 48 hours.
High radiochemical yield was achieved with a straightforward synthesis.
Compared to DFO, the process resulted in low radiochemical yield and poor in vivo stability.The radiolabeled compound exhibited superior pharmacokinetics.
The multistep synthesis entails a complex procedure and yields poor radiochemical results.

Vinyl sulfone-based radiolabeling
Vinyl sulfone consists of a vinyl moiety that is covalently bonded to an electrophilic sulfone group.They act as building blocks in various organic syntheses and as crucial structural components in various biologically important molecules and drugs.Essentially, diaryl vinyl sulfone represents a structural derivative of the chalcone-like structure, when the α,β-unsaturated carbonyl moiety is replaced by vinyl sulfone [54].The vinyl sulfone functional group is important for amine-or thiol-based conjugation with macro-biomolecules such as proteins.Vinyl sulfone can specifically conjugate with free thiol groups and avoid nonspecific reactions with other nucleophilic functional groups such as amines or histidine under mildly acidic conditions [55].Vinylsulfone-based radiolabeled prosthetic groups exhibit higher stability than that of their maleimide-based counterparts, which are prone to hydrolysis.The resulting adduct was stable both in vitro and in

Radiometal chelator-based vinyl sulfone
The potential of a vinyl sulfone-based bifunctional chelating agent (BCA) for the synthesis of radiopharmaceuticals was explored.Initially, a maleimide-based DOTA chelator was used to radiolabel the monoclonal antibody.However, pH-dependent cleavage of the resulting bioconjugate was observed over time, leading to a high blood clearance rate, which ultimately hindered effective tumor uptake [56,57].In a modified approach, a vinyl sulfone-based prosthetic group was synthesized using a DOTA chelator.This novel prosthetic group was used for radiolabeling the chimeric anti-CEA antibody cT84.66 with indium-111 ( 111 In) (half-life, 2 days) (Figure 12A).Despite concerns regarding the reaction of vinyl sulfone with amino groups, nonspecific radiolabeling was effectively controlled by manipulating the pH of the reaction mixture.The same prosthetic groups react with amino groups at pH = 9 in the absence of a reducing agent.However, the reaction towards the sulfhydryl group was dominant at neutral pH in the presence of a reducing agent.Comparative biodistribution using human colon tumor xenograft models revealed superior tumor-to-blood (T/B) and tumor-to-liver (T/L) ratios for SH-specific conjugates compared with that of the NH-based conjugates.This difference was attributed to the slow blood clearance and comparatively higher tumor uptake observed for SH-specific conjugates [58].

F-DEG-VS and F-PEG n -VS
The use of a PEG linker has been reported for the synthesis of 18 F-labeled vinyl sulfone-based prosthetic groups, specifically that of (2-(2-(2-[ 18 F]fluoroethoxy)ethoxy)ethylsulfonyl)ethane ( 18 F-DEG-VS), for the radiolabeling of thiol-containing biomolecules.The application of 18 F-DEG-VS in the radiolabeling of c(RGDyC), c(RGDyK) 2 , and thiolated neurotensin (NT) peptides was investigated [60].This study revealed that 18 F-DEG-VS efficiently reacted with the free thiol of c(RGDyC) within 30 min at neutral pH, resulting in a high radiochemical yield.However, the reaction with the free amino group of c(RGDyK) provided moderate radiochemical yield under basic conditions after 24 h of incubation. 18F-labeled NT peptide ( 18 F-DEG-VS-NT) was obtained at a substantial radiochemical yield.Both radiolabeled and non-modified NT peptides showed similar binding affinities towards NTR1-positive HT-29 cells.PET imaging data were acquired using the HT-29 xenograft model and the targeting efficiency 18 F-DEG-VS-NT (Figure 13A) was compared with that of maleimide-based 18 F labeled NT peptide ( 18 F-FBEM-NT) (Figure 13B).Compared with that of the maleimide-based precursor, 18 F-DEG-VS-NT exhibited superior tumor targeting, lower accumulation in abdominal tissues, and higher tumor-to-background contrast (Figure 13C).
Further analysis of the potential of this prosthetic group showed that the addition of a PEG linker could significantly enhance its biomedical application.Accordingly, three compounds, namely, 18 F-PEG 1 -VS, 18 F-PEG 2 -VS, and 18 F-PEG 3 -VS, were synthesized using their corresponding nosylate precursors, for a moderate radiochemical yield (19-26%). 18F-PEG 1 -VS was optimized for thiol-or amine-based radiolabeling of biomolecules.The prosthetic group provided moderate to high radiochemical yields of 13-97% and 7-58%, respectively, within 30 min at pH = 8.5 and room temperature. 18F-PEG 1 -VS was used to radiolabel homocysteine-SH, GSH, glucose-SH, and coenzyme-SH using thiol-based linkages.Similarly, triphenyl phosphate-NH 2 (TTP2 and TTP6), cRGDyK, and mouse serum protein were radiolabeled using amine-based linkages.Homocysteine-SH was radiolabeled with >95% radiochemical yield and effectively targeted the H1299 tumor model in vivo (Figure 14A).Similarly, the 18 F-radiolabeled TTP-NH 2 , cRGDyK peptide, and mouse serum proteins were obtained at quantitative radiolabeling yields and were used for PET imaging of the cardiac area, U87MG tumors, and blood pool imaging (Figure 14B), respectively.Notably, all three prosthetic groups were used to radiolabel red blood cells in vivo, demonstrating their potential applications in gastrointestinal bleeding scintigraphy (GIBS) (Figure 14C).All three prosthetic groups were intravenously injected into mice.Blood pool activity revealed that 18 F-PEG 1 -VS and 18 F-PGE 2 -VS showed similar pharmacokinetics and high uptake in the heart with minimal background.However, 18 F-PEG 3 -VS showed high cardiac uptake but poor imaging contrast.Additionally, 18 F-PEG 1 -VS efficiently radiolabeled red blood cells in vitro with a high radiochemical yield (75%) under mildly basic conditions (Figure 14D).The radiolabeled red blood cells ( 18 F-PEG 1 -VS-RBCs) were efficiently used for PET imaging of extravascular blood in the abdominal region and the cardiovascular system.The radiotracer exhibited prolonged residence within the cardiac blood pool, with its uptake remaining unaffected by variations in blood glucose levels [61].
In another study, red blood cells were radiolabeled with the same prosthetic group for detailed in vivo analysis.Notably, 18 F-PEG 1 -VS crossed the blood-brain barrier and accumulated in the cerebellum.The uptake of 18 F-PEG 1 -VS is presumed to be related to the presence of free thiol groups in brain tissues.This suggests that brain imaging can be performed using this precursor [62].Using a similar approach, Wu et al. synthesized a series of PSMAtargeting tracers using 18 F-(PEG) n -VS prosthetic groups (Figure 15A).The incorporation of a PEG n linker (where n=1, 2, 3, 6, and 12) into the main structure was aimed at enhancing hydrophilicity.Consequently, improved tumor-to-background contrast was achieved.Binding affinity, pharmacokinetics, and PET imaging data were acquired using PSMA-positive LNCaP animal models, and the results were benchmarked against the clinically approved 68 Ga-PSMA-11 precursor (Figure 15B).Notably, 18 F-(PEG) 2 -VS-PSMA and 18 F-(PEG) 3 -VS-PSMA exhibited higher binding affinities for PSMA-positive C4-2B cells than with their counterparts.PET imaging and organ distribution data in LNCaP tumors revealed elevated tumor uptake for (PEG) 2 and (PEG) 3 , which registered at 8.8 ± 3.3 and 12 ± 2.2 %ID/g at 0.5 h, respectively.Moreover, this heightened uptake persisted for up to 3 h.Notably, the tumor-to-muscle ratio of 18 F-(PEG) 1 -VS-PSMA surpassed those of (PEG) 2 and (PEG) 3 owing to the shortness of the PEG chain and rapid washout from the muscles.The radiolabeled precursor demonstrated high specificity, and blocking caused a significant reduction in both tumor and kidney uptake.Notably, the tumor uptake of 18 F-(PEG) 2 -VS-PSMA and 18 F-(PEG) 3 -VS-PSMA was nearly double that of the clinically approved 68 Ga-PSMA-11 (Figure 15C-D), whereas comparable uptake was maintained in the muscles and kidneys [63].[ 18 F]FVSB 18 F-PEG n -VS and its derivatives are recognized for their radiolabeling efficiencies.However, despite notable advancements, the synthesis of these prosthetic groups traditionally involves azeotropic drying and HPLC purification steps before the thiol conjugation reaction.This multistep synthesis process and the potential instability of the precursors often result in low radiochemical yields.To address these challenges, Murphy et al. introduced a novel prosthetic group, namely, [ 18 F]fluoro-4-(vinylsulfonyl)benzene ([ 18 F]FVSB), which was designed for radiolabeling of thiol-containing biomolecules [64].The synthesis of [ 18 F]FVSB used a highly stable uranium-based intermediate precursor, which eliminates the need for additional steps such as azeotropic drying or HPLC purification.[ 18 F]FVSB was synthesized within 30 min at 130 °C with high radiochemical yield (46 ± 4%, decay corrected) and purity (85%).The radiofluorinated precursor was directly used for the thiol-based radiolabeling of biologically active peptides.[ 18 F]FVSB exhibited excellent radiolabeling efficiency under physiological conditions when used to radiolabel clinically important peptide analogs, including the linear RGD peptide (RCY = 84 ± 8%), cyclic (RGDfC) peptide (RCY = 87 ± 2%), PSMA analogue (RCY = 80 ± 3%), MG11 analogue (RCY = 83 ± 10%), neuromedin B analogue (RCY = 93 ± 1%), and bombesin analogue (RCY = 55 ± 11%) (Figure 16).However, this study did not include in vitro or in vivo investigations.
Recently, Mei et al. investigated the potential of [ 18 F]FVSB to aid the synthesis of 18 F labeled amino acid analogs intended for PET/CT imaging in inflammation and tumor-bearing animal models.In this study, various amino acids, and derivatives such as L-cysteine, L-homocysteine, D-cysteine, and the small peptide glutathione were radiolabeled to produce the corresponding 18 F labeled precursors.A series of initial tests including stability assessment, cancer cell uptake testing, specificity evaluation, biodistribution analysis, and PET/CT imaging were performed.The results revealed that the radiolabeled precursors corresponding to L-cysteine and L-homocysteine, denoted as [ 18 F]a and [ 18 F]b, respectively, were shortlisted as suitable candidates for PET imaging of tumors and inflammatory lesions (Figure 17A).Both precursors exhibited high uptake in the tumor (T) and inflammatory regions (I) of mice bearing NCI-H1975 tumors and inflammation in the shoulder.However, the uptake values were lower than those observed with [ 18 F]FDG [65] (Figure 17B).
Despite obtaining encouraging radiolabeling results with vinyl sulfone analogs, concerns persist regarding their reaction with amino groups, leading to nonspecific radiolabeling.Additionally, slow reaction kinetics further emphasizes the need for research to explore better and more efficient thiol-specific radiolabeling prosthetic groups.

Perfluoroarylation based radiolabeling
Pentelute et al. were the first to report an aromatic nucleophilic substitution involving sulfhydryl groups in small proteins, peptides, and hexafluorobenzene (HFB) molecules.Subsequently, HBF was used to exclusively staple various peptides, each featuring a free thiol group, via 1,4-disubstitution [66].Although some genetic modifications are necessary to apply this strategy to proteins, this methodology has been extensively used in peptide synthesis, particularly in the context of macrocyclization [67].The yield of this reaction was lower than that of other thiol-based substitution reactions owing to the poor solubility in water and slow reactivity [68].Chen  18 F-tetrafluorobenzene-RGD ( 18 F-TFB-RGD, Figure 18A) at high radiochemical yield (40 ± 2%).The cell binding affinity of 18 F-TFB-RGD in U87MG α v β 3 expressing cells (IC 50 = 81 nM) was comparable to that of dimeric c(RGDfK) peptide (IC 50 = 62 nM) (Figure 18B).PET using 18 F-TFB-RGD in the U87MG xenograft model showed high tumor uptake (4 %ID/g, 1 h postinjection) and a high T/M ratio (9.8) (Figure 18C).These results are comparable to those of the dimeric c(RGDfK) peptide radiolabeled with N-succinimidyl 4-[ 18 F]-fluorobenzoate.The tumor specificity of 18 F-TFB-RGD was confirmed through blocking studies.Moreover, no defluorination or 18 F uptake was observed in the bone for up to 2 h postinjection.These findings suggest that [ 18 F]HFB is a highly specific and stable prosthetic group that enables the radiolabeling of clinically significant peptides and generation of corresponding cyclic or dimeric products [69].Further studies are anticipated to explore the full potential of radiolabeled HBF prosthetic groups, particularly in the context of radiolabeling proteins and antibodies.

Aryl sulfone based radiolabeling
The limitations associated with the addition of maleimides have prompted researchers to design rapid and highly efficient thiol-specific reactions.
Initially, functionalized sulfone reagents were investigated for thiol blocking, and their applicability was subsequently extended to drug conjugation and radiolabeling of biomolecules [70].For instance, Zhang et al. studied a series of thiol-blocking reagents and found that 2-(methanesulfonyl)benzothiazole (MSBT) is a novel thiol-specific blocking agent with high reactivity and selectivity [71].Nevertheless, the chemistry of methylsulfonyl-functionalized heteroaromatic compounds and their reactivity towards thiols remain unexplored.In a separate investigation, Toda et al. explored a new class of thiol-reactive heteroaromatic methylsulfones for thiol-based conjugation of proteins and peptides.Within this category, phenyloxadiazole methylsulfone (POS) was identified as the most efficient prosthetic group as it demonstrated a thiol-specific addition reaction within a few minutes in aqueous media at pH = 7.0 and room temperature [72].Subsequently, this compound has been used in radiopharmaceutical development.

F-FPOS and 125 I-MSTP
The radiolabeling of phenyloxadiazole methylsulfone (POS) with 18 F was initially performed to explore the potential applications of POS in radiopharmaceutical development.A nosylated phenyloxadiazole methylsulfone derivate was synthesized using commercially available methyl 4hydroxybenzoate.This nosylate derivative was radiolabeled with 18 F using the [ 18 F]KF-K 2.2.2 complex to obtain the corresponding [ 18 F]fluoro phenyloxadiazole methylsulfone ( 18 F-FPOS) at a moderate radiochemical yield (27 ± 6%) and high radiochemical purity.Then, the efficacy of 18 F-FPOS was assessed by radiolabeling clinically important biomolecules such as bombesin (BBN) and the affibody Z HER2:2395 (Figure 19A). 18F-FPOS-BBN and 18 F-FPOS-Z HER2:2395 were obtained at substantial radiochemical yields of 33% and 40%, respectively, within a short period of 15 min under physiological conditions. 18F-FPOS-Z HER2:2395 was isolated using a size-exclusion cartridge, and its tumor-targeting properties were studied using HER2+ SCOV3 xenograft models.PET/CT revealed significant accumulation of the radiotracer in the tumor within the first 2 h postinjection, providing a high tumor-to-background contrast (Figure 19B).Remarkably, there was an absence of a typical bone uptake, underscoring the exceptional in vivo stability of the compound [73].

Radiometal chelator-based POS
Following the promising outcomes of 18 F-FPOSbased radiolabeling of biomolecules, scientists have extended this technique to radiolabel biologically important molecules using radiometals.To facilitate this, radiometal-chelator-based POS prosthetic groups were synthesized, and commercially available compounds such as 5-phenyl-1,3,4-oxadiazole-2-thiol, and its derivatives, including 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol, were explored.Among them, 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol was selected and modified by incorporating a terminal amine-containing polyethylene glycol (PEG) group.The PEG group in addition to enhancing water solubility also provided additional space between the biomolecule and radioisotope chelator, which improved radiolabeling efficiency.The amine group in the modified compound was used to attach the bifunctional chelators DFO and DTPA (Figure 20A-B) for 89 Zr and 177 Lu radiolabeling, respectively [75].Both chelators were conjugated with a trastuzumab antibody via POS and thiol reactions.The final products, namely, 8 9 Zr-DFO-POS-trastuzumab and 177 Lu-DTPA-POS-trastuzumab, were obtained in high radiochemical yields and purities.In vitro stability studies revealed that both radiotracers exhibited superior stability for more than 7 days compared with that observed for the maleimide-based conjugate (Figure 20C).The huA33 antibody was also radiolabeled using either 89 Zr-DFO-Mal or 89 Zr-DFO-POS before being intravenously injected into an A33 antigen-expressing SW1222 human colorectal cancer xenograft model for a comparative study.The PET images were acquired for several hours, demonstrating that the POS-based conjugate exhibited enhanced in vivo stability compared with that of its competitor (Figure 20D).
In 2020, Pieve et al. used the POS-thiol reaction for the 18 F radiolabeling of antibodies using the [ 18 F]AlF complex [76].They conjugated POS with NOTA and NODAGA chelators, resulting in the synthesis of NOTA-POS and NODAGA-POS prosthetic groups.Then, NOTA-POS was incorporated into the Z EGFR:03115 -Cys antibody, which was subsequently radiolabeled using the [ 18 F]AlF complex for a low radiochemical yield (11.0-12.7%)and high purity (>98%).The radiochemical yield of [ 18 F]AlF-NODAGA-POS-Z EGFR:03115 was similarly low (4.3-8.1%).Moreover, the nature of the chelator significantly influenced the pharmacokinetics of both radiotracers, elevating the uptake in the liver, kidneys, and tumor.However, the biodistribution profiles of POS and the maleimide-based conjugates were almost identical up to 1 h postinjection.Despite the slow reaction kinetics, it is anticipated that POS-based radiolabeling strategies hold great potential in clinical settings.

2-cyanobenzothiazoles based radiolabeling
In Rao et al. reported for the first time a thiol-based condensation reaction between 2-cyanobenzothiazole (CBT) and an N-terminal cysteine residue to form luciferin linkage.This reaction occurred in PBS solvent under mild pH conditions and room temperature [77,78].The reaction exhibited moderate kinetics with a second-order reaction rate of approximately 9.2 M -1 s -1 , which surpassed that of several well-defined click reactions reported previously [79,80].This reaction demonstrated site selectivity and specificity, which was further confirmed by labeling the N-terminal cysteine-containing proteins expressed on cell surfaces.As the reaction occurs exclusively at the N-terminal cysteine residue, its true strength lies in site selectivity but requires prior incorporation of the N-terminal cysteine in peptides and proteins.Chin et al. used this reaction to perform nuclear imaging and reported the 18 F radiolabeling of N-terminal cysteinecontaining proteins and peptides.The tosylate precursor was synthesized using commercially available 6-methoxy-CBT.However, the 18 F radiolabeling attempts using [ 18 F]KF-K 2.2.2 , tetrabutylammonium bicarbonate or Cs 2 CO 3 were unsuccessful because of the hydrolysis of the cyano group on CBT.Nevertheless, successful radiolabeling was achieved using the 18-Crown-6/ K 2 CO 3 complex at 90 °C for a moderate yield (20%) of 18 F-CBT at high radiochemical purity.Furthermore, 18 F-CBT was used to radiolabel the dimeric c(RGD) peptide with a high radiochemical yield (92% HPLC-based, 80% decay corrected) and purity (>98%) under physiological conditions.The PET imaging data from U87MG tumor-bearing mice showed high tumor targeting efficiency and high tumor-to-background ratio (Figure 21A).Elevated radioactivity signals were observed in the liver and kidneys owing to the increased lipophilicity of the radiolabeled compound resulting from the addition of the 18 F-CBT moiety.The efficacy of the prosthetic group was also determined using Renilla luciferase (RLuc8) protein after installing an N-terminal cysteine residue, which provided a moderate radiochemical yield (12%).PET images were acquired in normal mice, and the biodistribution profile of the radiolabeled protein was similar to that of 124 I-labeled RLuc8 (Figure 21A) [81].
The 18 F-CBT prosthetic group was synthesized using an aliphatic nucleophilic substitution reaction.Subsequently, new nucleophilic radiofluorination reactions were explored using heteroaromatic substituents, particularly those with a pyridine structure. 18F-CBT was more efficiently synthesized using nucleophilic heteroaromatic substitution than with aliphatic nucleophilic substitution.A new prosthetic group, [ 18 F]FPyPEGCBT (Figure 21B) was synthesized using a trimethylammonium triflate precursor [82].The optimized radiolabeling conditions resulted in a high radiochemical yield (>45%) and purity (>98%).This prosthetic group was used to radiolabel the N-terminal cysteine-bearing c(RGD) peptide.Another study by Mushtaq et al. reported a 125 I-labeled CBT for the radiolabeling of biomolecules [83].The direct radioiodination of CBT provided 125 I-labeled CBT at high radiochemical yield (92%) and purity (>99%) within 10 min when a chloramine-T oxidizing agent was used (Figure 21C).The prosthetic group was tested on an N-terminal cysteine-containing c(RGD) peptide; however, the radiolabeled peptide was not subjected to further in vivo studies.Using the same theory, Seimbille et al. synthesized DOTA and NODAGA chelator-based CBT prosthetic groups (Figure 21D) for the efficient radiolabeling of biomolecules using 68 Ga [84].Both chelators were tested for the radiolabeling of small peptides.Overall, the CBT-based radiolabeling of N-terminal cysteine-containing biomolecules represents an efficient, site-selective, and site-specific strategy with a high radiolabeling yield.However, the increase in the lipophilicity of molecules led to abnormal uptake in abdominal areas.Moreover, incorporating N-terminal cysteine into bioactive molecules before the radiolabeling reaction poses additional challenge in clinical settings.

Thiol-ene click reaction based radiolabeling
In the early 1900s, efficient and site-selective reactions were reported between free thiol and carbon-to-carbon double bonds or "enes".These thiol-ene reactions exhibit the key attributes of a reliable click reaction.The photo induced thiol-ene reaction, characterized by ease of implementation, rapid kinetics, high yield, stable product formation, and photo initiation control, demonstrates potential in general synthetic chemistry and radiopharmacy [85].Initially used in the synthesis of macrocyclic compounds [86], sugars [87], peptides [88], dendrimers, and polymers [89], thiol-ene reactions have recently been used for the synthesis of 99m Tc based radiotracers.To achieve this, 2,2′-dipicolylamine (DPA) based BFCs were synthesized to link the central amine of DPA with the terminal alkene via a propyl allyl ether or allyl linker.The 99m Tc radiolabeled tracers were synthesized using either a click-then-chelate or a chelate-then-click strategy (Figure 22B,C).Benzyl mercaptan was used as a model thiol compound for photo-initiated thiol-ene conjugation reactions [90].

Thiol specific strained-release reaction
The strained bonds have long been recognized for their unique applications in organic syntheses.These molecules store significant potential energy and are suitable for versatile applications in biology, materials science, chemistry, and bioorthogonal chemistry [91].Notably, these molecules exhibit remarkable chemoselectivity for thiols and excellent stability under harsh reaction conditions [92].A new type of hetero-bifunctional agent was developed by leveraging these scaffolds for the radioiodination of thiol-containing peptides.Specifically  23).The radiolabeling process was catalyzed by Cu2O and yielded the desired product within 1 h at room temperature.Strain-release reactions with thiols were performed in phosphate buffer (pH = 8.0), and K 2 CO 3 was used to ensure an alkaline pH, which facilitates the deprotonation of cysteine thiol in the peptide.Under optimized conditions, various peptides, including N-acetylcysteine, L-cysteine, L-cysteine methyl ester hydrochloride, CAQK, and c(RGDyC) were radiolabeled with a high radiochemical yield (>95%) and purity (>99%).The 125 I-labeled c(RGDyC) was subjected to further in vivo and in vitro studies.The radioiodinated compound demonstrated high in vitro and in vivo stability and elevated uptake in the tumors of U87MG tumor-bearing mice [93].Despite these favorable results, the prosthetic group was not tested for the radiolabeling of macromolecules such as proteins and antibodies.

Conclusion and future perspectives
Thus, this review presents our findings of what we perceive to be the most important and highly innovative thiol-based radiolabeling strategies.This comprehensive review primarily focuses on thiol-based prosthetic groups by examining their direct applications, analyzing radiolabeling outcomes, and considering their potential impact on the pharmacokinetics and binding affinity of radiolabeled biomolecules (summarized in Table 2).We acknowledge that the scientific data elaborated here primarily stems from preclinical studies.Additionally, some of the studies discussed here that feature thiol-based prosthetic groups do not use them directly but rather as intermediates to support or supplement other click reactions such as in the IEDDA or aldehyde and 1,2-diamine conjugation reactions; this aspect has not been explicitly addressed in this review.Similarly, we have intentionally omitted an in-depth discussion of reactions that use catalysts for the direct modification of thiol groups with radioisotopes such as 18 F or 11 C.These deliberate omissions highlight both the strengths and limitations of our study aim, warranting further studies with nuanced consideration to comprehensively understand the broad landscape of thiol-based radiolabeling strategies.   Ga, 90 Y, 177 Lu, or 89 Zr-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 18 F precursors and radiometal chelators.

For 18 F radiolabeling
The synthesis is multistep and complex; however in vivo stability supports imaging study.
For 68 Ga 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 89 Zr, 90  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 18 F 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.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.

S.
No. 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 18 F precursors and radiometal chelators, ensuring high in vivo and in vitro stability for both the prosthetic groups and the final radiolabeled product.

Prosthetic
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.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 18 F 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.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
The intricate development of targeted nuclear imaging and radiotherapeutic agents necessitates a meticulous approach involving the precise incorporation of radioisotopes into biomolecules of interest with chemo-and site-specificity.The prosthetic group approach, particularly the modification of cysteine (Cys) residues, is the prevalently used strategy for radiolabeling thiol-containing biomolecules.Although significant progress has been achieved in radiolabeling using various prosthetic groups based on maleimide, vinylsulfone, aryl sulfone, CBT, and thiol-ene conjugation reactions, critical hurdles remain.These limitations include in vivo stability issues, reactivity towards non-thiol functional groups, sluggish reaction kinetics, and the need for excess substrate to achieve a sufficient radiolabeling yield.These challenges hinder the use of these prosthetic groups in preclinical and clinical settings.
Further developments leading to the use of thiol-based reactions in clinical nuclear medicine depend on overcoming the current limitations and fostering innovative applications.Efforts should be directed towards refining prosthetic groups, exploring new synthesis routes, and assessing their compatibility with other bioconjugation methods.The inherent potential of thiol-based reactions, especially those with short-lived radioisotopes such as 18 F, 11 C, and 68 Ga, needs to be thoroughly investigated and compared through detailed studies.
As this field of application evolves, enhanced efficacy can be attained through the combination of thiol-based prosthetic groups and other click reactions.The continuous expansion of thiol-based reactions into clinical trials is not only desirable but also crucial for establishing their true impact.Ease of synthesis, site-specific bioconjugation, high stability, automation, and probe purification have emerged as key areas in which thiol-based reactions can revolutionize clinical nuclear medicine and offer novel and impactful solutions.

Figure 1 .
Figure 1.Representative thiol-specific prosthetic groups for the site-selective radiolabeling of cysteine containing biomolecules.

Figure 2 .
Figure 2. Lysine-or thiol-based radiolabeling of biomolecules.(A) Schematic diagram illustrating a monoclonal antibody with surface-exposed lysine residues and four disulfide bonds.(B) Non-specific and uncontrolled radiolabeling, as demonstrated by lysine-based methods, results in a heterogeneous mixture of products, potentially impacting the overall performance of radiopharmaceuticals. (C) Thiol-based radiolabeling of antibodies, following partial or complete reduction of disulfide bonds.Partial reduction, followed by radiolabeling, produces a heterogeneous mixture with a minimal degree of heterogeneity compared to lysine methods.Complete reduction, followed by a thorough radiolabeling reaction, yields a homogeneous product.
prosthetic groups were used to incorporate DFO chelator into trastuzumab under the given reaction conditions.Notably, DFO-Chx-Mal demonstrated superior conjugation under mild conditions than did DFO-Iac.Moreover, DFO-Chx-Mal exhibited greater radiochemical yield and purity than that of DFO-Iac.The in vitro stability of DFO-Chx-Mal matched or surpassed that of DFO-N-Suc-TFP, DFO-Bz-SCN, and DFO-Iac for up to 5 days in mouse serum under physiological conditions; although a gradual loss of antibody-bound89 Zr (1.8% per day) was observed.

Figure 7 .
Figure 7. Illustration depicting the synthesis process of H4neunox-Mal and its in vivo studies.(A) The synthesis of H4neunox-Mal involves the use of N-Succinimidyl 6-maleimidohexanoate and H4neunox under basic conditions.(B) In vivo PET imaging data of [ 89 Zr]Zr-DFO-trastuzumab and [ 89 Zr]Zr-neunox-trastuzumab, conducted in a SKOV-3 cancer xenograft model on one and four days post-intravenous injection.Adapted with permission from [44], copyright 2022, Elsevier Inc.
F]fluoropropyl-thiol and [ 18 F]fluoro-PEG4-thiol were synthesized and used for radiolabeling maleimide-containing biomolecules under physiological conditions.[ 18 F]fluoropropyl benzothioate and [ 18 F]Fluoro-PEG4 were obtained in a moderate radiochemical yield of 37-47% and 28-35%, respectively.Benzothioate facilitates efficient protection and rapid deprotection using sodium methoxide, followed by a conjugation reaction in the same vessel.The radiolabeling efficiency of [ 18 F]fluoropropyl-thiol and [ 18 F]fluoro-PEG4-thiol was assessed using a maleimide-containing molecules such as Evans Blue, a PSMA analog, and the [c(RGDfK)]2 peptide (Figure 10A-B).The 18 F-labeled compounds showed high in vitro stability in PBS and mouse serum.Moreover, 18 F-labled peptides were subjected to further in vivo studies.PET imaging data from the U87MG tumor model revealed the rapid clearance of both [ 18 F]fluoro-PEG4-S-[c(RGDfK)]2 and [ 18 F]fluoro-propyl-S-[c(RGDfK)]2 from background tissues and high accumulation in the tumor.However, the PEG4-based tracer showed superior pharmacokinetics while exhibiting minimal accumulation in the intestine over time owing to its enhanced hydrophilic nature (Figure 10C-D) [51].

Figure 11 .
Figure 11.Proposed mechanism elucidating the in vivo instability of thiosuccinimide adducts under physiological conditions.Two mechanisms have been posited to account for the cleavage of the resulting product: (I) retro-Michael β-elimination reaction, and (II) oxidative cleavage of the product [53].

Figure 13 .
Figure 13. 18F-labeling of thiolated neurotensin (NT) peptide and in vivo imaging study.(A) Chemical structure of 18 F-DEG-VS-NT peptide.(B) Chemical structure of 18 F-FBEM-NT peptide.(C) PET imaging data for 18 F-DEG-VS-NT peptide (under normal or blocking conditions) and 18 F-FBEM-NT peptide utilizing HT-29 xenograft model.Adapted with permission from [60], copyright 2014, The Society of Nuclear Medicine and Molecular Imaging, Inc.

Figure 14 .
Figure 14. 18F-labeling of thiol-containing biomolecules utilizing 18 F-PEG1-VS for in vivo imaging study.(A) 18 F-labeling of homocysteine and PET imaging utilizing H1299 tumor model.(B) 18 F-labeling of albumin protein and PET imaging of blood pool.(C) In vivo radiolabeling of red blood cell utilizing 18 F-PEG1-VS and PET imaging of cardiac area (D) In vitro radiolabeling of red blood cell utilizing 18 F-PEG1-VS and PET imaging of cardiac area.Adapted with permission from [61], copyright 2020, American Chemical Society.

Figure 17 .
Figure 17.Chemical structures of clinically important 18 F-labeled amino acids and in vivo imaging data.(A) Synthesis of 18 F-labeled L-cysteine and L-homocysteine molecules, denoted as [ 18 F]a and [ 18 F]b, respectively (B) PET imaging of [ 18 F]a and [ 18 F]b compared with [ 18 F]FDG in NCI-H1975 tumor-bearing mice with inflammation introduced in the shoulder.Tumor and inflammation regions are denoted as T and I, respectively.Adapted with permission from [65], copyright 2022, Elsevier Inc.

Figure 20 .
Figure 20.POS-based radiolabeling of biomolecules and in vitro and in vivo testing of resulting conjugates.(A) Synthesis of DFO-POS prosthetic group.(B) Synthesis of DTPA-POS prosthetic groups.(C) In vitro stability test of 89 Zr-DFO-POS-trastuzumab compared with 89 Zr-DFO-mal-trastuzumab under identical conditions.(D) PET images of A33 antigen-expressing SW1222 colorectal cancer xenograft bearing nude mice following the injection of89 Zr-DFO-POS-huA33 and89 Zr-DFO-mal-huA33 and images were acquired 120 h post injection.Adapted with permission from[75], copyright 2018, American Chemical Society.

Figure 21 .
Figure 21.CBT-based radiolabeling of biomolecules and in vivo study.(A)18 F-CBT based radiolabeling of dimeric c(RGD) peptide and PET images were acquired using U87MG tumor bearing (left side) 0.5 h post injection.18F-CBT was also used for the radiolabeling of Renilla lucifierase (RLuc8) protein and PET imaging were acquired using normal mouse (right side).T: tumors; K: kidneys.Adapted with permission from[81], copyright 2012, American Chemical Society.(B) Chemical structure of [18 F]FPyPEGCBT.(C) Chemical structure of 125 I-labeled CBT.(D) Chemical structure of DOTA and NODA based CBT prosthetic groups.
only for the18 F-based radiolabeling of peptides.

Table 1 .
Comprehensive overview of maleimide-based prosthetic groups for radiolabeling: applications, strengths, and limitations.

Table 2 .
Comprehensive overview of prosthetic groups used for radiolabeling thiol-containing biomolecules: applications, strengths, and limitations.

Y, and 177 Lu radiolabeling
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.