The Development and Application of Tritium-Labeled Compounds in Biomedical Research

With low background radiation, tritiate compounds exclusively emit intense beta particles without structural changes. This makes them a useful tool in the drug discovery arsenal. Thanks to the recent rapid progress in tritium chemistry, the preparation and analysis of tritium-labeled compounds are now much easier, simpler, and cheaper. Pharmacokinetics, autoradiography, and protein binding studies have been much more efficient with the employment of tritium-labeled compounds. This review provides a comprehensive overview of tritium-labeled compounds regarding their properties, synthesis strategies, and applications.


Introduction
Radionuclide-labeled compounds have been widely used in a vast number of studies, especially in the life sciences [1][2][3][4].Radionuclides are attractive in drug development as they exclusively emit intense signals without structural changes to the drugs.The high specific activity of molecules labeled with tritium is easy to visualize, track, and quantify, which makes them powerful tools for pharmaceutical studies.In addition to the drug metabolism and pharmacokinetics studies, tritium-labeled compounds are widely employed in other research areas such as receptor binding and autoradiography [5][6][7][8][9][10].
The International System of Units quantifies the radioactivity of tritium-labeled compounds in terms of the Becquerel (Bq), whereas the Curie (Ci) is the traditional unit of measure [11].Another important parameter in radionuclide chemistry is specific activity (SA), which is expressed in terms of radioactivity per molar unit.It is the level of SA that determines the application of radionuclides.The SA of tritium is much higher than that of carbon-14 [12], which enables tritium-labeled compounds to be employed in the studies of autoradiography, protein binding, and permeability assays.
The main method to detect the emission of tritium is liquid scintillation counting (LSC).The beta particle emitted by tritium can excite the fluor, which then emits light when relaxing to the ground state [13].The light detected by the photomultiplier tube is directly related to the amount of radioactivity.A higher sensitivity can be obtained by solid scintillation.Techniques that utilize solid scintillation after automating fraction collection provide better approaches to online radioactivity detection.A higher radioactivity can even be detected by accelerator mass spectrometry techniques, which have a sensitivity of about 1000-fold that of LSC [14].This technique, however, is limited by the high price of the equipment and the complexity of sample preparation.
One of the important reasons that labeling with tritium is preferred over other radionuclides is its faster, simpler, and cheaper synthesis approaches.On one hand, many classic approaches to labeling, such as tritiodehalogenation, tritiated methylation, and hydride reduction, have experienced a great improvement [15][16][17].On the other hand, ortho-exchange chemistry has brought revolutionary developments to tritium chemistry [18,19].Catalyzed Tritium-NMR is a useful tool for the detection and analysis of tritiated compounds [22,23].As tritium has almost the same chemical shifts as protium, tritium-NMR is widely used to assign the unambiguous site of the tritium label.In addition, diverse 2D-NMR techniques have been employed to analyze complex tritium-NMR spectra [24,25].
Different chromatographic behaviors have been observed between tritiated compounds and their unlabeled counterparts [26].The shorter and stronger bonds of tritium and carbon result in a change in lipophilicity, which may explain the difference in chromatographic separation [27].For compounds with high-tritiated labeling, isotopic fractionation is much clearer than that of single-labeled compounds, which are often neglected [28].It is worth noting that tritiated compounds usually elute ahead of unlabeled compounds through reversed-phase high-performance liquid chromatography.

Storage of Tritiated Compounds
In addition to chemical degradation, the beta particle emitted by the tritium label continuously accelerates the decomposition of tritiated compounds, of which there are three types: internal, external, and secondary decomposition [29].The first is caused by isotopic decay and accounts for about 5% of tritium decomposition per year [30].The second is caused by the direct interaction with the high-energy beta particle.The emission energy of beta particles from tritium is about two to four orders of magnitude greater than the average energy of an organic bond.Hence, these bonds are quite vulnerable, especially in the solid or high-concentration liquid state.In addition to the drug molecules, the emitted beta particle can interact with other nearby solvent or air molecules, which are then activated [29].These activated molecules can then cause secondary decomposition of drug molecules.This approach accounts for the primary aim of these decomposition processes.
The vulnerability of the tritium-labeled compounds emphasizes the importance of short and proper storage.A practical method to reduce external decomposition is to disperse tritiated compounds into an appropriate solvent [10].It is important that the chosen solvent is highly pure, deoxygenated, and chemically compatible with the compounds.To address the problem of secondary decomposition, solvents are required to absorb and stabilize the energy of beta particles.Benzene and toluene are able to stabilize excited states through their π-orbitals [31] and are therefore often used to dissolve less polar radiochemical compounds.Alcohols such as methanol or ethanol acting as radical scavengers are widely used as solvents or as cosolvents of more polar compounds.On the other hand, solvents like chloroform, dichloromethane, ether, or water are poor choices due to their inclination toward forming radicals [32].As the decomposition of radioactive compounds is temperature-dependent, it is recommended to store radioactive compounds at temperatures as low as −80 • C, which is usually adequate; however, storage in liquid nitrogen (−140 • C) is optimal [33,34].

Biological Properties of the Tritium Isotope
Potential isotope effects also raise concern in the switching metabolic process of labeled compounds [35].There are two crucial factors in the kinetic isotope effect: the status of bond hybridization and the type of enzyme involved [36,37].Many aromatic hydroxylation processes are not likely to show a notable kinetic isotope effect [38].The rate-determining step of aromatic hydroxylation reactions is an epoxidation reaction through cytochrome p450 oxidation, which does not involve a direct C-H bond breaking [35].On the other hand, an isotope effect on an aromatic heterocyclic ring was reported to shift the enzymatic oxidation [38].The scale of isotope effects increases with the number of tritium atoms integrated into the compound.Pharmacological or even safety issues may arise.It is worthwhile to minimize isotope effects and avoid metabolic switching to establish the correct and accurate behavior of a target compound.
The primary concern about the biological properties of the tritium isotope is the risk of its biological instability.With modern labeling methodology and analytic techniques, loss of the tritium label resulting from chemical and radiochemical processes is no longer a key issue.Tritium loss is mainly due to the introduction of tritium atoms into metabolically vulnerable positions.Furthermore, tritium atom degradation by oxidation or cleavage may even undergo an incorporation with endogenous biomolecules [39][40][41], which would, however, raise the overall radiation in studies involving human beings.In the same molecules, labels at different positions lead to very diverse stabilities [7].For instance, tritium in the ortho-position of a phenoxy moiety suffers from metabolic instability, while that in the meta position remains quite stable.It is, therefore, of great value to take into detailed consideration the possible metabolic pathways.
Many metabolically vulnerable positions deserve thoughtful consideration to place a tritium label.N-and O-tritiomethylated compounds are usually uninstable in the metabolic environment, which makes them unsuitable for in vivo studies [42].The hydroxylation of aromatic systems is mainly through CYP-450-mediated metabolic pathways [43].The meta or para position of an aromatic system, which often lies on the periphery of the molecule, is not advised when introducing a tritium label.This is worth considering when the tritium labels are incorporated through tritiodehalogenation.The primary hydroxyl group often suffers from hydroxylation by dehydrogenate, so the tritium label adjacent to it is not in an optimal position, while secondary and tertiary hydroxyl tolerates the dehydrogenase pretty well.

Tritium-Halogen Exchange
Great developments in tritium chemistry have been made in the last 30 years [44][45][46], and many improved classical approaches to tritium labeling are also used, such as tritiodehalogenation.With heterogeneous (usually Pd/C) catalysts, tritium can easily take the place of halide in the aryl iodides, aryl bromides, and aryl chlorides.
The main drawback of this approach is the tolerance of reducible groups (other aryl halides, unsaturated bonds, and ArNO 2 ), which has been improved in recent decades [47].By controlling the reaction conditions, tritiodeiodinate can be selectively achieved in the presence of chlorine and even bromine, while tritiodebromination in the presence of chlorine is also possible.A major advantage of this synthetic approach is that the precursor (aryl halides) can be readily synthesized using one-step halogenations.
ways [43].The meta or para position of an aromatic system, which often lies on the periphery of the molecule, is not advised when introducing a tritium label.This is worth considering when the tritium labels are incorporated through tritiodehalogenation.The primary hydroxyl group often suffers from hydroxylation by dehydrogenate, so the tritium label adjacent to it is not in an optimal position, while secondary and tertiary hydroxyl tolerates the dehydrogenase pretty well.

Tritium-Halogen Exchange
Great developments in tritium chemistry have been made in the last 30 years [44][45][46], and many improved classical approaches to tritium labeling are also used, such as tritiodehalogenation.With heterogeneous (usually Pd/C) catalysts, tritium can easily take the place of halide in the aryl iodides, aryl bromides, and aryl chlorides.
The main drawback of this approach is the tolerance of reducible groups (other aryl halides, unsaturated bonds, and ArNO2), which has been improved in recent decades [47].By controlling the reaction conditions, tritiodeiodinate can be selectively achieved in the presence of chlorine and even bromine, while tritiodebromination in the presence of chlorine is also possible.A major advantage of this synthetic approach is that the precursor (aryl halides) can be readily synthesized using one-step halogenations.

Tritide Reductions
The tritide reduction approach introduces tritium labels through tritium gas or tritium hydrides.Unlike tritium-halogen exchange methods, tritide reductions require a transformation of some precursor chemical.One of the major advantages of catalytic tritiation is that the region and chiral selectivity of tritium labeling can be achieved with a proper catalyst.However, due to the requirement of precursor preparation, this method is infrequently used.
Carbon-carbon multiple bonds can be readily reduced with the catalyst in a suitable solvent in the presence of tritium gas, while carbon heteroatom multiple bonds, because of their polar nature, are more favorable for reduction with complex tritides rather than catalytic tritiation.The efficiency of catalytic tritiation is determined by the substrate structure, the activity of the heterogeneous metal catalyst, the support material, and the property of the solvent.Generally, Pd or Rh dispersed on alumina or carbon in protic polar solvents has the greatest ability of reduction, while Ir or Ru dispersed on calcium in aprotic apolar solvents exhibits a much lower reducibility [50,51].The main drawback of catalytic tritiation is that it can be spread over original carbon-carbon multiple bonds, which

Tritide Reductions
The tritide reduction approach introduces tritium labels through tritium gas or tritium hydrides.Unlike tritium-halogen exchange methods, tritide reductions require a transformation of some precursor chemical.One of the major advantages of catalytic tritiation is that the region and chiral selectivity of tritium labeling can be achieved with a proper catalyst.However, due to the requirement of precursor preparation, this method is infrequently used.
Carbon-carbon multiple bonds can be readily reduced with the catalyst in a suitable solvent in the presence of tritium gas, while carbon heteroatom multiple bonds, because of their polar nature, are more favorable for reduction with complex tritides rather than catalytic tritiation.The efficiency of catalytic tritiation is determined by the substrate structure, the activity of the heterogeneous metal catalyst, the support material, and the property of the solvent.Generally, Pd or Rh dispersed on alumina or carbon in protic polar solvents has the greatest ability of reduction, while Ir or Ru dispersed on calcium in aprotic apolar solvents exhibits a much lower reducibility [50,51].The main drawback of catalytic tritiation is that it can be spread over original carbon-carbon multiple bonds, which is called isotopic scrambling.According to a generally accepted theory, isotope scrambling occurs during catalytic reductions of carbon-carbon multiple bonds through the processes of isomerizations and migrations involving reversible hydrometallations [52,53].Although tritium-halogen exchange may occur during catalytic tritiation, the selective reduction of unsaturated bonds in the presence of halogens can be accomplished through appropriate selection of the catalyst.Using a Wilkinson catalyst, Egan successfully reduced a double bond without tritiodehalogenation [54] (Scheme 2).
bling occurs during catalytic reductions of carbon-carbon multiple bonds through the processes of isomerizations and migrations involving reversible hydrometallations [52,53].Although tritium-halogen exchange may occur during catalytic tritiation, the selective reduction of unsaturated bonds in the presence of halogens can be accomplished through appropriate selection of the catalyst.Using a Wilkinson catalyst, Egan successfully reduced a double bond without tritiodehalogenation [54] (Scheme 2).Compared with the tritium gas, it is much easier, using other tritium sources such as LiBT4, NaBT4, and LiAlT4, to introduce tritium in a chemoselective and regioselective manner.Isotope scrambling is also difficult to find in the products of tritide reductions.In addition, higher degrees of stereocontrol can often be achieved by using bulky reagents, chiral tritides, or common achiral tritides in the presence of appropriate chiral catalysts [55].

Methylation
A well-established method for the labeling of drug molecules is the use of several small tritiated building blocks.These building blocks, such as tritiated water, tritiated diimide, tritiated methyl iodide, and tritiated formaldehyde, are of unique utility and commercially available.Especially tritiated methyl iodide and tritiated methyl triflate are the most common building blocks and enable fast and easy reactions with frequently high yields [56,57].However, N-and O-tritiomethylated compounds are often metabolically unstable, making them unsuitable for many in vivo investigations [58].In contrast, C-tritiomethylated compounds show much higher metabolic stability, which makes them feasible for in vivo studies as an alternative to tritium labeling.MacMillan et al. [59] reported the radio-methylation of aryl and alkyl bromide catalyzed by metal photoredox and achieved the rapid synthesis of a series of PET radioligands under mild conditions with a wide application range of substrates applied (Scheme 3).This is also the first example of tritium labeling by an alkyl-alkyl cross-coupling strategy to date.

Metal-Catalyzed Hydrogen Isotope Exchange
Although tritium-labeled compounds can be prepared through chemical synthesis, they may also be prepared directly by exchanging hydrogen atoms with tritium without any change in the chemical structure of the substrates.This method is undoubtedly one of the most dramatic improvements in tritium chemistry [19].In the presence of an appropriate metal catalyst (usually an Ir or Rh complex), tritium labeling can be achieved with the treatment of tritium gas or tritiated water [60].Chemoselectivity and regioselectivity can be directed by a wide range of functional groups, most of which tolerate the tritium exchange condition.Compared with the aforementioned method, metal-catalyzed Scheme 2. Selective reduction of a double bond without tritiodehalogenation.
Compared with the tritium gas, it is much easier, using other tritium sources such as LiBT 4 , NaBT 4 , and LiAlT 4 , to introduce tritium in a chemoselective and regioselective manner.Isotope scrambling is also difficult to find in the products of tritide reductions.In addition, higher degrees of stereocontrol can often be achieved by using bulky reagents, chiral tritides, or common achiral tritides in the presence of appropriate chiral catalysts [55].

Methylation
A well-established method for the labeling of drug molecules is the use of several small tritiated building blocks.These building blocks, such as tritiated water, tritiated diimide, tritiated methyl iodide, and tritiated formaldehyde, are of unique utility and commercially available.Especially tritiated methyl iodide and tritiated methyl triflate are the most common building blocks and enable fast and easy reactions with frequently high yields [56,57].However, N-and O-tritiomethylated compounds are often metabolically unstable, making them unsuitable for many in vivo investigations [58].In contrast, Ctritiomethylated compounds show much higher metabolic stability, which makes them feasible for in vivo studies as an alternative to tritium labeling.MacMillan et al. [59] reported the radio-methylation of aryl and alkyl bromide catalyzed by metal photoredox and achieved the rapid synthesis of a series of PET radioligands under mild conditions with a wide application range of substrates applied (Scheme 3).This is also the first example of tritium labeling by an alkyl-alkyl cross-coupling strategy to date.[52,53].Although tritium-halogen exchange may occur during catalytic tritiation, the selective reduction of unsaturated bonds in the presence of halogens can be accomplished through appropriate selection of the catalyst.Using a Wilkinson catalyst, Egan successfully reduced a double bond without tritiodehalogenation [54] (Scheme 2).Compared with the tritium gas, it is much easier, using other tritium sources such as LiBT4, NaBT4, and LiAlT4, to introduce tritium in a chemoselective and regioselective manner.Isotope scrambling is also difficult to find in the products of tritide reductions.In addition, higher degrees of stereocontrol can often be achieved by using bulky reagents, chiral tritides, or common achiral tritides in the presence of appropriate chiral catalysts [55].

Methylation
A well-established method for the labeling of drug molecules is the use of several small tritiated building blocks.These building blocks, such as tritiated water, tritiated diimide, tritiated methyl iodide, and tritiated formaldehyde, are of unique utility and commercially available.Especially tritiated methyl iodide and tritiated methyl triflate are the most common building blocks and enable fast and easy reactions with frequently high yields [56,57].However, N-and O-tritiomethylated compounds are often metabolically unstable, making them unsuitable for many in vivo investigations [58].In contrast, C-tritiomethylated compounds show much higher metabolic stability, which makes them feasible for in vivo studies as an alternative to tritium labeling.MacMillan et al. [59] reported the radio-methylation of aryl and alkyl bromide catalyzed by metal photoredox and achieved the rapid synthesis of a series of PET radioligands under mild conditions with a wide application range of substrates applied (Scheme 3).This is also the first example of tritium labeling by an alkyl-alkyl cross-coupling strategy to date.

Metal-Catalyzed Hydrogen Isotope Exchange
Although tritium-labeled compounds can be prepared through chemical synthesis, they may also be prepared directly by exchanging hydrogen atoms with tritium without any change in the chemical structure of the substrates.This method is undoubtedly one of the most dramatic improvements in tritium chemistry [19].In the presence of an appropriate metal catalyst (usually an Ir or Rh complex), tritium labeling can be achieved with the treatment of tritium gas or tritiated water [60].Chemoselectivity and regioselectivity can be directed by a wide range of functional groups, most of which tolerate the tritium exchange condition.Compared with the aforementioned method, metal-catalyzed Scheme 3. Tritiation of aliphatic pharmaceuticals and radiotracers.

Metal-Catalyzed Hydrogen Isotope Exchange
Although tritium-labeled compounds can be prepared through chemical synthesis, they may also be prepared directly by exchanging hydrogen atoms with tritium without any change in the chemical structure of the substrates.This method is undoubtedly one of the most dramatic improvements in tritium chemistry [19].In the presence of an appropriate metal catalyst (usually an Ir or Rh complex), tritium labeling can be achieved with the treatment of tritium gas or tritiated water [60].Chemoselectivity and regioselectivity can be directed by a wide range of functional groups, most of which tolerate the tritium exchange condition.Compared with the aforementioned method, metal-catalyzed hydrogen-isotope exchange (HIE) labeling has a great advantage in terms of speed and confidence.Regarding these merits, hydrogen isotope exchange has been the method of choice [61].
The exchange labeling reactions usually utilize group 8, 9, and 10 metals in tritiated water or mildly acidic solutions.Platinum, palladium, rhodium, ruthenium nickel, and iridium are the most investigated metals for catalytic tritium-hydrogen isotope exchange [62,63].
The area of isotopic exchange over homogeneous iridium catalysts has undergone significant expansion since the inception of the technique in the early 1990s [64,65].These iridium-containing catalysts have the ability to insert into the C-H bond of arenes regioselectivity by directing groups such as ketones, esters, and amides [61].Along with new iridium-containing catalysts, the activity and selectivity of the iridium complex in the area of tritium labeling have been under detailed investigations, and the overall mechanism of the hydrogen isotope exchange process has been proposed [66][67][68][69].Pieters et al.
[70] reported a general method for the multiple site hydrogen isotope labelling of complex molecules using the commercially available and air-stable iridium precatalyst [Ir(COD)(OMe)] 2 .The multiple site isotope incorporation explained by the in situ formation of a catalytically active system including both monometallic Ir complexes and Ir nanoclusters, and can form complementary catalytic active substances in situ, monometallic iridium complexes, and iridium nanoparticles, which enabled the labeling of a wide range of pharmacologically relevant substructures, including pyridine, pyrazine, indole, carbazole, aniline, oxygen/thiazole, thiophene, and electron-rich phenyl (Scheme 4a).Additionally, many drug molecules on the market, such as Perebron, Celecoxib, and Nilutamide, were labeled with isotopes through hydrogen exchange labeling with iridium-containing catalysts [66].Recently, HIE reactions using water-soluble Kerr catalysts at different pH solutions have been reported for the first time [71].The tritium-hydrogen exchange of imidazole, benzoic acid, pyridine n-oxide, tetrazole, and other molecules was realized successfully.
The area of isotopic exchange over homogeneous iridium catalysts has undergone significant expansion since the inception of the technique in the early 1990s [64,65].These iridium-containing catalysts have the ability to insert into the C-H bond of arenes regioselectivity by directing groups such as ketones, esters, and amides [61].Along with new iridium-containing catalysts, the activity and selectivity of the iridium complex in the area of tritium labeling have been under detailed investigations, and the overall mechanism of the hydrogen isotope exchange process has been proposed [66][67][68][69].Pieters et al.
[70] reported a general method for the multiple site hydrogen isotope labelling of complex molecules using the commercially available and air-stable iridium precatalyst [Ir(COD)(OMe)]2.The multiple site isotope incorporation explained by the in situ formation of a catalytically active system including both monometallic Ir complexes and Ir nanoclusters, and can form complementary catalytic active substances in situ, monometallic iridium complexes, and iridium nanoparticles, which enabled the labeling of a wide range of pharmacologically relevant substructures, including pyridine, pyrazine, indole, carbazole, aniline, oxygen/thiazole, thiophene, and electron-rich phenyl (Scheme 4a).Additionally, many drug molecules on the market, such as Perebron, Celecoxib, and Nilutamide, were labeled with isotopes through hydrogen exchange labeling with iridiumcontaining catalysts [66].Recently, HIE reactions using water-soluble Kerr catalysts at different pH solutions have been reported for the first time [71].The tritium-hydrogen exchange of imidazole, benzoic acid, pyridine n-oxide, tetrazole, and other molecules was realized successfully.Palladium was found to be generally the most active metal.The C-H tritiation of complex pharmaceuticals with T 2 gas involving electrophilic C-H palladation offers a novel scope for this tritiation [72].This practical conversion shows a new substrate range and greater functional group tolerance compared to other tritiation methods, and it has been applied to direct tritiated complex drugs and unprotected dipeptides.Hydrogen hydrolysis catalyzed by palladium is an effective method of molecular tritiation.Ritters et al. [73] reported a well-defined molecular palladium catalyst for homogeneous hydrolysis, with thianthracene as the leaving group for selective drug introduction through late C-H functionalization (Scheme 4b).Unlike the coordination capacity of the traditional leaving groupassociated palladium (II) catalyst, an unprecedented dihydrogen catalysis was achieved.This unique reactivity does not require an inert atmosphere or dry conditions, making it practical and stable for having a direct impact on drug discovery and development.Except for single metal catalysis, platinum and palladium catalysts can also be utilized together to achieve synergy efficiency in labeling [74,75].
With the increase in structural complexity of bioactive molecules, it is of great significance to exploit more practical C-H tritiation methods so that valuable tritium radioactive ligands can continue to be used in the investigation of bioactivity.Professor Chirik and collaborators reported an iron-catalyzed method for direct tritium labeling [18].The regioselectivity of the iron catalyst is orthogonal to that of iridium-containing catalysts (Scheme 4c).This catalyst is tolerated by a range of pharmaceutically relevant functional groups and operates efficiently in polar aprotic solvents at low pressures of tritium gas.
The efficient and specific introduction of isotopes at specific sites is one of the difficulties of HIE reactions.The usual tritiation conditions often lead to the introduction of multiple isotopes with low regional selectivity.The use of nanoparticles has appeared recently as an attractive solution to overcome this shortcoming.Many types of organometallic nanoparticles (NPs) have been developed to achieve high chemical and regionally selective isotope labeling by adjusting the catalytic activity of nanoparticle size and concentration and the surface ligand properties of metal nanoclusters, or different metals [76,77].Pieters et al. performed a comprehensive review in this regard [78], so we do not detail it here.

Photoredox-Catalyzed Hydrogen Isotope Exchange
In addition to the methods mentioned above, there are several strategies that have been developed to achieve a high selectivity of reactions.In recent years, visible light-mediated photoredox catalytic reactions have rapidly developed in organic synthesis.Macmillan first reported in 2017 that photoredox and thiol hydrogen transfer (HAT) synergistic catalytic strategies enable efficient aliphatic hydrocarbon bond isotope exchange (Scheme 5a) [79].It is worth noting that this strategy can only use tritium water as tritium sources, which faces drawbacks such as radioactive contamination due to the danger and instability of tritium water.Based on this, the MSD team proposed the use of metal catalyst Rh(PPh 3 ) 3 Cl to activate tritium gas, and the generated metal hydride in situ was used to complete the hydrogen transfer catalysis, which realized the efficient isotope exchange of aliphatic hydrocarbon bonds and obtained tritium substitution drug molecules with high specific activity (Scheme 5b) [80].
making it practical and stable for having a direct impact on drug discovery and development.Except for single metal catalysis, platinum and palladium catalysts can also be utilized together to achieve synergy efficiency in labeling [74,75].
With the increase in structural complexity of bioactive molecules, it is of great significance to exploit more practical C-H tritiation methods so that valuable tritium radioactive ligands can continue to be used in the investigation of bioactivity.Professor Chirik and collaborators reported an iron-catalyzed method for direct tritium labeling [18].The regioselectivity of the iron catalyst is orthogonal to that of iridium-containing catalysts (Scheme 4c).This catalyst is tolerated by a range of pharmaceutically relevant functional groups and operates efficiently in polar aprotic solvents at low pressures of tritium gas.
The efficient and specific introduction of isotopes at specific sites is one of the difficulties of HIE reactions.The usual tritiation conditions often lead to the introduction of multiple isotopes with low regional selectivity.The use of nanoparticles has appeared recently as an attractive solution to overcome this shortcoming.Many types of organometallic nanoparticles (NPs) have been developed to achieve high chemical and regionally selective isotope labeling by adjusting the catalytic activity of nanoparticle size and concentration and the surface ligand properties of metal nanoclusters, or different metals [76,77].Pieters et al. performed a comprehensive review in this regard [78], so we do not detail it here.

Photoredox-Catalyzed Hydrogen Isotope Exchange
In addition to the methods mentioned above, there are several strategies that have been developed to achieve a high selectivity of reactions.In recent years, visible lightmediated photoredox catalytic reactions have rapidly developed in organic synthesis.Macmillan first reported in 2017 that photoredox and thiol hydrogen transfer (HAT) synergistic catalytic strategies enable efficient aliphatic hydrocarbon bond isotope exchange (Scheme 5a) [79].It is worth noting that this strategy can only use tritium water as tritium sources, which faces drawbacks such as radioactive contamination due to the danger and instability of tritium water.Based on this, the MSD team proposed the use of metal catalyst Rh(PPh3)3Cl to activate tritium gas, and the generated metal hydride in situ was used to complete the hydrogen transfer catalysis, which realized the efficient isotope exchange of aliphatic hydrocarbon bonds and obtained tritium substitution drug molecules with high specific activity (Scheme 5b) [80].

Applications of the Tritium-Labeled Compounds 4.1. Cell Cytotoxicity Estimation
Evaluation of the anti-proliferative activity of drugs is one of the important methods for early drug discovery.The tritiated thymidine incorporation ([ 3 H]-TdR) assay is used as an indicator of cellular proliferation and is particularly attractive for screening antimitotic agents for cancer [81,82].Tritiated thymidine is a radiolabeled DNA precursor incorporated into newly synthesized DNA during the S-phase of the cell cycle [83][84][85].The response value of the radioactive signal is related to the rate of proliferation, which makes it a successful agent to screen and optimize potential new drugs.

Human ADME Studies
The profile of a new drug's absorption, distribution, metabolism, and excretion (ADME) properties is required for the registration, which is generally obtained in late phase I or in phase II clinical trials [86].Preliminary metabolism data can be provided by in vitro studies.In vivo data are usually acquired by using a radiolabeled ADME study.Compared to non-radioactive techniques, such as HPLC-MS, radiolabeled compounds provide an easier and more accurate method due to their good tracer ability [87].
It is often the case that only when the carbon-14 option fails will the tritium-labeled compounds be used in ADME studies [88].Many ADME scientists often carry the stereotype that tritium is a problematic isotope in terms of chemical and biological stability.However, based on the experiences and scientific data, the same ADME information can be achieved with tritium-labeled compounds as with carbon-14-labeled compounds.Along with the rapid advance in synthetic and analytic tritium chemistry and the convenient commercial reagents and equipment, the use of tritium has made major progress [2].
Metabolite profiling based on radioactivity monitoring of the LC eluent will provide a number of metabolites excreted via each route as well as the quantity (% of dose) or concentration (e.g., µmolEq/L).The amount of radioactivity in the analyzed sample tends to be measured using the radiological aerosol monitor (RAM) for high levels and LSC for low levels.By analyzing urine, bile, and feces, we could assess whether the drug clearance is governed by metabolism or excretion of the unchanged parent compound.Based on an agonist of the α7 nicotinic acetylcholinergic receptor, G. D. Fate et al. synthesized two tritium-labeled compounds (Scheme 6) [89].Because of the intrinsic properties of tritium, all biological samples must undergo lyophilization to determine the content of urinary tritiated water.At the same time, using [ 3 H]-1 confirmed the species-dependent metabolism for 3 H loss from [ 3 H]-1 in rats but not dogs.As the majority of rat metabolites resulted from furan-pyridine biotransformation, the loss of Ta was closely related to compound 3. porated into newly synthesized DNA during the S-phase of the cell cycle [83][84][85].The response value of the radioactive signal is related to the rate of proliferation, which makes it a successful agent to screen and optimize potential new drugs.

Human ADME Studies
The profile of a new drug's absorption, distribution, metabolism, and excretion (ADME) properties is required for the registration, which is generally obtained in late phase I or in phase II clinical trials [86].Preliminary metabolism data can be provided by in vitro studies.In vivo data are usually acquired by using a radiolabeled ADME study.Compared to non-radioactive techniques, such as HPLC-MS, radiolabeled compounds provide an easier and more accurate method due to their good tracer ability [87].
It is often the case that only when the carbon-14 option fails will the tritium-labeled compounds be used in ADME studies [88].Many ADME scientists often carry the stereotype that tritium is a problematic isotope in terms of chemical and biological stability.However, based on the experiences and scientific data, the same ADME information can be achieved with tritium-labeled compounds as with carbon-14-labeled compounds.Along with the rapid advance in synthetic and analytic tritium chemistry and the convenient commercial reagents and equipment, the use of tritium has made major progress [2].
Metabolite profiling based on radioactivity monitoring of the LC eluent will provide a number of metabolites excreted via each route as well as the quantity (% of dose) or concentration (e.g., µmolEq/L).The amount of radioactivity in the analyzed sample tends to be measured using the radiological aerosol monitor (RAM) for high levels and LSC for low levels.By analyzing urine, bile, and feces, we could assess whether the drug clearance is governed by metabolism or excretion of the unchanged parent compound.Based on an agonist of the α7 nicotinic acetylcholinergic receptor, G. D. Fate et al. synthesized two tritium-labeled compounds (Scheme 6) [89].Because of the intrinsic properties of tritium, all biological samples must undergo lyophilization to determine the content of urinary tritiated water.At the same time, using [ 3 H]-1 confirmed the species-dependent metabolism for 3 H loss from [ 3 H]-1 in rats but not dogs.As the majority of rat metabolites resulted from furan-pyridine biotransformation, the loss of Ta was closely related to compound 3. Scheme 6. Tritium-labeled compounds in different sections (Ta and Tb) with metabolic structures that were detected analytically.
The late introduction property of tritium makes it highly favored in the field of labeling complex molecules.Based on this, molecules with complex structures, such as the macrolide antibiotic [90,91], oligonucleotides [92], and polypeptides [10], have been labeled to enable pharmacokinetic, distribution, and formulation studies.
Apart from drug metabolism and pharmacokinetics studies, tritium-labeled compounds are broadly used in many other fields, such as receptor-binding studies and autoradiography.

Autoradiography Distribution Studies
The effects of most drugs depend on their concentrations in target tissues, while concentrations of drugs and metabolites in plasma do not directly determine those in the tissues.To this end, autoradiography distribution studies are used to provide useful information regarding tissue distribution and pharmacokinetics in drug-related material [93,94].By using tritium-labeled drugs, the autoradiography technique can nondestructively obtain drug distributional information, which can be crucial to the studies of pharmacology, pharmacokinetics, efficacy, and toxicity [93,95].It is worth noticing that this technique can also be used to evaluate the uptake of radioactive material in tumors [96].
The classical radionuclide used in autoradiography distribution studies is iodine-125 [97].However, as shown in Figure 1A, its distribution of iodine is often influenced by up-taking the free iodine to the thyroid, salivary glands, gastric mucosa, mammary glands, and ovarian follicles, resulting in an ambiguous image [8].Fortunately, this is no issue in tritium-labeled compounds.The autoradiograms obtained from tritium have a high resolution in the images, resulting from the short path length of the beta particle emitted by tritium.

Autoradiography Distribution Studies
The effects of most drugs depend on their concentrations in target tissues, while concentrations of drugs and metabolites in plasma do not directly determine those in the tissues.To this end, autoradiography distribution studies are used to provide useful information regarding tissue distribution and pharmacokinetics in drug-related material [93,94].By using tritium-labeled drugs, the autoradiography technique can nondestructively obtain drug distributional information, which be crucial to the studies of pharmacology, pharmacokinetics, efficacy, and toxicity [93,95].It is worth noticing that this technique can also be used to evaluate the uptake of radioactive material in tumors [96].
The classical radionuclide used in autoradiography distribution studies is iodine-125 [97].However, as shown in Figure 1A, its distribution of iodine is often influenced by uptaking the free iodine to the thyroid, salivary glands, gastric mucosa, mammary glands, and ovarian follicles, resulting in an ambiguous image [8].Fortunately, this is no issue in tritium-labeled compounds.The autoradiograms obtained from tritium have a high resolution in the images, resulting from the short path length of the beta particle emitted by tritium.Recently, Kristian et al. [98] reported that probes based on the high-affinity inhibitor AVLX-144 of PSD-95 were labeled with tritium as well as fluorescent tags (Figure 2a).Tracer binding showed saturable, translocatable, and heterogeneous distributions in rat brain sections and proved effective in quantitative autoradiography and cellular imaging studies (Figure 2b).Recently, Kristian et al. [98] reported that probes based on the high-affinity inhibitor AVLX-144 of PSD-95 were labeled with tritium as well as fluorescent tags (Figure 2a).Tracer binding showed saturable, translocatable, and heterogeneous distributions in rat brain sections and proved effective in quantitative autoradiography and cellular imaging studies (Figure 2b).Tritium-sucrose could be used to validate the dosing strategy in lung distribution studies of an isolated perfused mouse lung.After introducing the dose, the lung perfusion was immediately halted, and the trachea and major bronchi were isolated.All five lobes (superior, middle, inferior, post-caudal, and left) were separated using a surgical blade and separately weighed.The sample was transferred for radioactivity liquid scintillation analysis.The mass of tritium-sucrose distributed into each lobe was calculated and corrected for the lobar mass [99].

Tagging for Protein Binding
The high specific activity of tritium makes it a valuable tool in biological applications for detection of a variety of small molecules with their receptors.The easy detectability of the tritium also enabled the investigations of enzyme kinetics at the nanomolar level.The photoaffinity probe usually consists of three parts: a ligand with targeting properties, a Tritium-sucrose could be used to validate the dosing strategy in lung distribution studies of an isolated perfused mouse lung.After introducing the dose, the lung perfusion was immediately halted, and the trachea and major bronchi were isolated.All five lobes (superior, middle, inferior, post-caudal, and left) were separated using a surgical blade and separately weighed.The sample was transferred for radioactivity liquid scintillation analysis.The mass of tritium-sucrose distributed into each lobe was calculated and corrected for the lobar mass [99].

Tagging for Protein Binding
The high specific activity of tritium makes it a valuable tool in biological applications for detection of a variety of small molecules with their receptors.The easy detectability of the tritium also enabled the investigations of enzyme kinetics at the nanomolar level.The photoaffinity probe usually consists of three parts: a ligand with targeting properties, a photoreactive group, and a report label [100].Compared with other identification tags, such as fluorescent dyes and affinity tags, a tritium label can minimize interference with the structure and activity of the due to its small volume, thereby preserving the affinity of the ligand to the target receptor and improving the accuracy of receptor recognition [12].In addition, the high specific activity and sensitivity of tritium can effectively reduce the background interference of non-target ligands, thus enabling in vivo activity evaluation in living cells.
A tritiated chemical probe origin from a DcpS inhibitor PF-0665247 was tested against a 9000 human protein microarray to assess its binding selectivity [101].Then, its excellent selectivity was confirmed by assessing against whole cell extracts.The tritiated probe was prepared through the tritium-halogen exchange method (Scheme 7a).Two tritium atoms were incorporated to ensure adequate specific radioactivity.High-resolution array images were received after exposure on a tritium-sensitive phosphor screen and processing through software.
studies of an isolated perfused mouse lung.After introducing the dose, the lung perfusion was immediately halted, and the trachea and major bronchi were isolated.All five lobes (superior, middle, inferior, post-caudal, and left) were separated using a surgical blade and separately weighed.The sample was transferred for radioactivity liquid scintillation analysis.The mass of tritium-sucrose distributed into each lobe was calculated and corrected for the lobar mass [99].

Tagging for Protein Binding
The high specific activity of tritium makes it a valuable tool in biological applications for detection of a variety of small molecules with their receptors.The easy detectability of the tritium also enabled the investigations of enzyme kinetics at the nanomolar level.The photoaffinity probe usually consists of three parts: a ligand with targeting properties, a photoreactive group, and a report label [100].Compared with other identification tags, such as fluorescent dyes and affinity tags, a tritium label can minimize interference with the structure and activity of the ligand due to its small volume, thereby preserving the affinity of the ligand to the target receptor and improving the accuracy of receptor recognition [12].In addition, the high specific activity and sensitivity of tritium can effectively reduce the background interference of non-target ligands, thus enabling in vivo activity evaluation in living cells.
A tritiated chemical probe origin from a DcpS inhibitor PF-0665247 was tested against a 9000 human protein microarray to assess its binding selectivity [101].Then, its excellent selectivity was confirmed by assessing against whole cell extracts.The tritiated probe was prepared through the tritium-halogen exchange method (Scheme 7a).Two tritium atoms were incorporated to ensure adequate specific radioactivity.High-resolution array images were received after exposure on a tritium-sensitive phosphor screen and processing through software.To investigate the high-affinity γ-hydroxybutyrate GHB binding sites in the central nervous system, tritium-labeled 3-hydroxycyclopent-1-enecarboxylic acid (tritium-HOCPCA) was prepared using in situ generated lithium trimethoxyborotritide (Scheme 7b) [102].The tritium incorporation approach produced an almost theoretical radioactive yield with high radiochemical purity.Based on its brain permeability, the probe is useful for understanding the molecular mechanism of GHB at its high-affinity binding site and elucidating the pharmacology of the GHB system.
The very high but still reversible binding of probes to their receptors may be broken during the process of detection.A photoaffinity approach is commonly used to address this [103].Photoaffinity probes replacing hydrogen atoms with tritium atoms without altering their structure will preserve the binding pattern of the drug to the maximum extent.The most extensively investigated photolabile groups are benzophenone, diazirine, and azide, among which the azide group is most widely used.By incorporating the tritium label through tritiodehalogenation, an azido-and tritium-labeled photoaffinity probe based on MX-126374 was successively synthesized (Scheme 8) [104].Through the cell-based screening assay, a series of agents that selectively induce apoptosis in a novel pathway were found.Tail-interacting protein 47 (TIP47), a 47 kDa protein that is commonly considered a chaperone or cargo protein, is revealed as the primary target of MX-126374.The identification of the probe's target discloses a novel and potential therapeutic selective apoptosis target.
based on MX-126374 was successively synthesized (Scheme 8) [104].Through the cellbased screening assay, a series of agents that selectively induce apoptosis in a novel pathway were found.Tail-interacting protein 47 (TIP47), a 47 kDa protein that is commonly considered a chaperone or cargo protein, is revealed as the primary target of MX-126374.The identification of the probe's target discloses a novel and potential therapeutic selective apoptosis target.

Conclusions
Tritium labeling is an attractive field with tremendous potential for both pharmacy and chemical biology applications.With the advances in understanding of their properties, the great value of tritium-labeled compounds is emphasized in drug discovery and development.High-quality labeled compounds can be prepared rapidly and easily as a result of the rapid progress in analytical and synthesis approaches.Due to the merit of high specific activity and sensitivity, tritium-labeled compounds have been widely used in diverse fields such as human ADME studies, quantitative whole-body autoradiography, and activity-based protein profiling.We believe that their potential has not been completely exploited.

Scheme 2 .
Scheme 2. Selective reduction of a double bond without tritiodehalogenation.

Scheme 2 .
Scheme 2. Selective reduction of a double bond without tritiodehalogenation.

Scheme 4 .
Scheme 4. Hydrogen/tritium exchange using an iridium-containing catalyst and a palladium-containing catalyst.(a) Multiple site hydrogen isotope labelling of complex molecules using iridium precatalyst [Ir(COD)(OMe)]2; (b) Chemo-and site-selective C-H tritiation via arylthianthrenium salt by homogeneous palladium catalysis; (c) Homogeneous transition Fe- catalysed hydrogen/tritium exchange using tritium gas.Palladium was found to be generally the most active metal.The C-H tritiation of complex pharmaceuticals with T2 gas involving electrophilic C-H palladation offers a novel scope for this tritiation [72].This practical conversion shows a new substrate range and Scheme 4. Hydrogen/tritium exchange using an iridium-containing catalyst and a palladiumcontaining catalyst.(a) Multiple site hydrogen isotope labelling of complex molecules using iridium precatalyst [Ir(COD)(OMe)] 2 ; (b) Chemo-and site-selective C-H tritiation via arylthianthrenium salt by homogeneous palladium catalysis; (c) Homogeneous transition Fe-catalysed hydrogen/tritium exchange using tritium gas.

Scheme 5 .
Scheme 5. (a) Tritiation of HIE through photoredox and HAT catalysis; (b) HIE with tritium gas through the merger of photoredox and hydrogenation catalysts.

Table 1 .
Physical properties of tritium versus those of carbon-14.