Modern Developments in Bifunctional Chelator Design for Gallium Radiopharmaceuticals

The positron-emitting radionuclide gallium-68 has become increasingly utilised in both preclinical and clinical settings with positron emission tomography (PET). The synthesis of radiochemically pure gallium-68 radiopharmaceuticals relies on careful consideration of the coordination chemistry. The short half-life of 68 min necessitates rapid quantitative radiolabelling (≤10 min). Desirable radiolabelling conditions include near-neutral pH, ambient temperatures, and low chelator concentrations to achieve the desired apparent molar activity. This review presents a broad overview of the requirements of an efficient bifunctional chelator in relation to the aqueous coordination chemistry of gallium. Developments in bifunctional chelator design and application are then presented and grouped according to eight categories of bifunctional chelator: the macrocyclic chelators DOTA and TACN; the acyclic HBED, pyridinecarboxylates, siderophores, tris(hydroxypyridinones), and DTPA; and the mesocyclic diazepines.


Introduction
Non-invasive molecular nuclear imaging has become a valuable tool to assist clinicians diagnosing and treating certain diseases. Single photon emission computed tomography (SPECT) is the most routinely used nuclear imaging technique. SPECT utilises radionuclides that emit γ radiation which penetrates the body for external detection [1]. The wide availability of technetium-99m ( 99m Tc) (t 1/2 = 6.01 h) from 99 Mo/ 99m Tc generators and the ideal single 140 keV γ emission has made 99m Tc coordination complexes for SPECT the most common radiopharmaceuticals used in nuclear medicine [2]. Positron emission tomography (PET) utilises positron emitting radionuclides. As the radionuclide decays, the positrons (β + ) annihilate with their antiparticles, electrons, up to a few millimetres away from the site of emission, causing two coincident 511 keV γ photons, approximately 180 • apart. These photons are converted to a three-dimensional image by a circular ring of detectors [3].
PET offers higher sensitivity and resolution than SPECT, which means that the images generated are often of a higher quality at lower radiotracer concentration (10 −10 M for PET cf. 10 −6 M for SPECT) [4,5]. Both techniques are often combined with anatomical imaging techniques such as CT to correlate functional data with anatomical information. The availability of PET has historically been limited because of the need for cyclotron-produced radionuclides, such as fluorine-18 ( 18 F) (t 1/2 = 109.8 min), for use in imaging agents [6]. The growth in the use of PET has been stimulated by the introduction of 18 F-fluorodeoxyglucose ( 18 F-FDG), which is a glucose analogue that allows for the imaging of sites where there is a large cellular energy requirement (e.g., in primary tumour and metastatic cells) [7]. There are several other clinically approved radiopharmaceuticals containing 18 F, ranging in function from brain imaging to prostate and breast cancers and associated metastases [8].
The high cost of running and maintaining cyclotron facilities, as well as the synthetically challenging formation of covalent C-F bonds for labelling biologically active targeting molecules, has encouraged the use of metallic radionuclides. The formation of coordination complexes using positron emitting radionuclides offers the opportunity for the simple preparation and straightforward incorporation of biologically active molecules to the ligand structure. There are several positron emitting radiometals with suitable half-lives that match the biological residence times of the targeting molecule. The success of the 99 Mo/ 99m Tc generator for SPECT imaging spurred interest in the development of generator-produced radiometals suitable for PET, such as gallium-68 ( 68 Ga), which has since become a practical and successful alternative to cyclotron-produced radionuclides for PET imaging [9]. Compared to cyclotrons, generators do not require special premises with large radiation shielding equipment, specialist personnel for the maintenance of cyclotron equipment, nor a large consumption of energy.
To apply metallic radionuclides to specific biological applications, it is most often necessary to use chelators that form complexes with high thermodynamic stability and kinetic inertness to avoid transmetallation to competing proteins and hydrolysis of the radiometal. To influence the biodistribution and pharmacokinetics and improve selectivity to disease states in vivo, it is important that the chelators, termed 'bifunctional chelators' (BFC), feature a reactive functional group (primary amine, carboxylic acid, isothiocyanate, etc.) that can be tethered to a targeting vector biomolecule, such as a peptide or antibody.

Aqueous Gallium Chemistry and Gallium Radioisotopes
Gallium is a semi-metallic group 13 element. The chemistry of gallium in physiological systems is dominated by Ga 3+ . Lower oxidation states of gallium are known but are generally unstable in water [10][11][12]. Ga 3+ is readily hydrolysed at pH > 3 forming insoluble Ga(OH) 3 . Radiolabelling at pH values between 3 and 7 is often difficult due to the low solubility of the Ga(OH) 3 precipitate (K sp = 7.28 × 10 −36 ) [13,14]. The precipitate is amphoteric, forming [Ga(OH) 4 ] − and re-dissolving at pH > 7, as demonstrated in the following equilibrium equations: [Ga(OH 2 ) 6  Stabilising buffers are used to avoid precipitate formation, with citrate and particularly acetate common choices [7]. With its high charge and small octahedral ionic radius (0.62 Å), Ga 3+ is classified as a hard Lewis acid [15,16]. Therefore, Ga 3+ will generally coordinate preferentially to hard Lewis bases, such as those containing nitrogen and oxygen donor atoms. However, sulphur, selenium, and tellurium-containing coordinating systems are known [17][18][19]. Ga 3+ is generally found to form 4-and 6-coordinate complexes [20][21][22]. The solution and coordination chemistries of Ga 3+ are somewhat similar to Al 3+ and In 3+ , and very similar to high-spin Fe 3+ [7]. Ga 3+ is expected to follow many of the same chemical pathways as Fe 3+ in the body, having similar electronegativities (Ga 3+ = 1.81 Pauling units; Fe 3+ = 1.83 Pauling units), 4th ionisation potentials (Ga 3+ = 64 eV; Fe 3+ = 55 eV), and electron affinities (3rd ionisation potential Ga 3+ = 30.71 eV; Fe 3+ = 30.65 eV) [23]. Indeed, the biochemical chelation and protein binding similarities between Ga 3+ and Fe 3+ are likely causative of physiological Ga 3+ activity and uptake. Tissue distribution studies have shown that the majority of administered Ga 3+ ions bind to iron-transporting proteins, such as transferrin, lactoferrin, and ferritin, with some localising in osteoblasts [23,24]. However, differences in reduction potential (unlike Fe 3+ , Ga 3+ will not be reduced to Ga 2+ under physiological conditions) mean that Ga 3+ does not compete with Fe 2+ containing molecules, such as heme, in the body [23].
Currently, 30 different isotopes of gallium are known, of which 3 are medically relevant: 66 Ga, 67 Ga and 68 Ga (Table 1) [25]. 66 Ga and 67 Ga are cyclotron produced, and 67 Ga has been used for SPECT imaging. 66 Ga and 68 Ga are β + -emitters with half-lives of 9.5 h and 67.7 min, respectively. 68 Ga is by far the most extensively studied for medical purposes, mostly due to the widespread availability of the 68 Ge/ 68 Ga generator, which can be stored on-site in hospitals [26]. In a typical 68 Ge/ 68 Ga generator, 68 Ge 4+ is immobilised on a column filled with metal oxide/hydroxide matrices where it spontaneously decays to 68 [7]. The long half-life of the 68 Ge parent isotope (t 1/2 = 270.95 d) means that a 68 Ge/ 68 Ga generator has a working life of up to one year depending on the initial activity (cf.~14 d for the 99 Mo/ 99m Tc generator) [9]. Additionally, 68 Ga can be produced in a cyclotron in large amounts [27]. This second supply source could potentially be important to mitigate supply issues, as well as allowing for a centralised local site for radiopharmaceutical production. 68 Ga decays to the stable isotope 68 Zn via β + emission and electron capture (EC). The 67.7-min half-life allows patients to be scanned in clinics quickly, minimising wait times and radiation exposure to both patients and personnel. 68 Ga also allows for repetitive examinations due to relatively low radiation exposure. 68 Ga has a maximum β + energy of 1880 keV, an average β + energy of 890 keV, and an annihilation radiation of 511 keV [26]. Although the higher β + energy means a slightly lower resolution than 18 F, this provides an adequate level of radioactivity for high quality PET images.
Clinical radiosynthesis of the three radiopharmaceuticals takes 5-20 min at pH 3-5 with heating, followed by purification to remove by-products and unreacted 68 Ga [29]. These conditions add process complexity, limit molar activity (the measured radioactivity per mole of radiopharmaceutical), and the heat and low pH may damage vector biomolecules. The ideal synthesis should be a one-step procedure, matching the simplicity of the long-established kit-based 99m Tc radiolabelling protocols [29,[32][33][34]. This would enable fast, simple, and reproducible formulations of the radiopharmaceutical in keeping with good manufacturing practice (GMP) standards [35,36]. The design of BFCs that can quantitatively coordinate metallic radioisotopes such as 68 Ga, with the resulting complexes exhibiting high thermodynamic stability and kinetic inertness in vitro and in vivo, is now considered a mature field [37].
Clinical radiosynthesis of the three radiopharmaceuticals takes 5-20 min at pH 3-5 with heating, followed by purification to remove by-products and unreacted 68 Ga [29]. These conditions add process complexity, limit molar activity (the measured radioactivity per mole of radiopharmaceutical), and the heat and low pH may damage vector biomolecules. The ideal synthesis should be a one-step procedure, matching the simplicity of the long-established kit-based 99m Tc radiolabelling protocols [29,[32][33][34]. This would enable fast, simple, and reproducible formulations of the radiopharmaceutical in keeping with good manufacturing practice (GMP) standards [35,36]. The design of BFCs that can quantitatively coordinate metallic radioisotopes such as 68 Ga, with the resulting complexes exhibiting high thermodynamic stability and kinetic inertness in vitro and in vivo, is now considered a mature field [37].

DOTA and Other Tetraazamacrocyclic-Based Bifunctional Chelator Development
The coordination sphere of the Ga 3+ -DOTA complex is a N 4 O 2 distorted octahedron ( Figure 3), and it is well-understood that the large size of the macrocycle is not well-suited for Ga 3+ complexation (logK 1 = 26.05) [45] compared with more flexible acyclic chelators or smaller macrocycles [25]. As previously mentioned, DOTA and its associated bifunctional derivatives are often used as a motivating factor for researchers to develop better chelating systems for 68 Ga radiolabelling. However, the fact remains that two of the four 68 Ga radiopharmaceuticals currently approved by the FDA are based on the DOTA macrocycle. Consequently, effort has been made towards developing DOTA-based BFCs for radiopharmaceutical applications, as well as novel cyclen-based and other tetraazamacrocyclic non-bifunctional 68 Ga chelators. The following sections will discuss the recent developments (2012-2022) of bifunctional chelators for radiolabelling with Ga radioisotopes and are divided by chelator type (beginning with developments in HBED and DOTA bifunctional chelators). A discussion of the acyclic families of chelators (DTPA, siderophores, pyridinecarboxylates, and hydroxypyridinones) is followed by the hybrid acyclic/macrocyclic diazepines, and finally, macrocyclic TACN. A discussion of the ligand design and the coordination chemistry with Ga 3+ , where applicable, is provided as well as progress that has been made to deliver the radiation to specific sites in vivo by tethering the complexes to targeting vectors, such as peptides, antibody fragments, and receptor-specific molecules. In this context, we hope to obtain a comprehensive understanding of the library of bifunctional chelating systems available for Ga radioisotopes, with a view to designing novel systems for specific applications.

DOTA and Other Tetraazamacrocyclic-Based Bifunctional Chelator Development
The coordination sphere of the Ga 3+ -DOTA complex is a N4O2 distorted octahedron (Figure 3), and it is well-understood that the large size of the macrocycle is not well-suited for Ga 3+ complexation (logK1 = 26.05) [45] compared with more flexible acyclic chelators or smaller macrocycles [25]. As previously mentioned, DOTA and its associated bifunctional derivatives are often used as a motivating factor for researchers to develop better chelating systems for 68 Ga radiolabelling. However, the fact remains that two of the four 68 Ga radiopharmaceuticals currently approved by the FDA are based on the DOTA macrocycle. Consequently, effort has been made towards developing DOTA-based BFCs for radiopharmaceutical applications, as well as novel cyclen-based and other tetraazamacrocyclic non-bifunctional 68 Ga chelators.  [46,47]. Ellipsoids are drawn at the 50% probability level. Hydrogen atoms and solvent molecules are omitted for clarity.
Edem and co-workers prepared a poly(ethylene glycol)-tetrazine DOTA conjugate (DOTA-PEG11-Tz, Figure 4) that was investigated as a pre-targeting probe for both the 68 Ga radiolabelling of bone (via conjugation to a trans-cyclooctene (TCO)-derived bonetargeting bisphosphonate, alendronate), as well as the TCO-conjugated antibody, CC49  [46,47]. Ellipsoids are drawn at the 50% probability level. Hydrogen atoms and solvent molecules are omitted for clarity.
Edem and co-workers prepared a poly(ethylene glycol)-tetrazine DOTA conjugate (DOTA-PEG 11 -Tz, Figure 4) that was investigated as a pre-targeting probe for both the 68 Ga radiolabelling of bone (via conjugation to a trans-cyclooctene (TCO)-derived bonetargeting bisphosphonate, alendronate), as well as the TCO-conjugated antibody, CC49 (which targets the tumour associated glycoprotein 72 antigen on colorectal cancer cells) [48]. The advantage of pre-targeting is that you can heat the complex without damaging the antibody. Additionally, pre-targeting provides an opportunity to use 68 Ga, with its short half-life, with biomolecules that have long biological half-lives, such as antibodies [49][50][51]. The radiotracers showed target-specific uptake of [ 68 Ga][Ga(DOTA-PEG11-Tz)] in the bone (3.7% injected dose/gram (%ID/g) in the knee) in mice pre-treated with TCO-alendronate, as well as tumour-specific uptake (5.8% ID/g) in mice containing LS174 xenografts that were pre-treated with TCO-CC49.
(which targets the tumour associated glycoprotein 72 antigen on colorectal cancer cells) [48]. The advantage of pre-targeting is that you can heat the complex without damaging the antibody. Additionally, pre-targeting provides an opportunity to use 68 Ga, with its short half-life, with biomolecules that have long biological half-lives, such as antibodies [49][50][51]. The radiotracers showed target-specific uptake of [ 68 Ga][Ga(DOTA-PEG11-Tz)] in the bone (3.7% injected dose/gram (%ID/g) in the knee) in mice pre-treated with TCOalendronate, as well as tumour-specific uptake (5.8% ID/g) in mice containing LS174 xenografts that were pre-treated with TCO-CC49. Pathuri and co-workers reported DOTA-hippurate and DOTA-glycine conjugates ( Figure 4) with the aim of developing alternative renographic agents to 99m Tc-DTPA (used in SPECT) for PET [3]. Both 68 Ga complexes demonstrated high radiochemical purity (RCP > 98%) at pH 4-5 within 10 min and cleared from circulation primarily through the kidneys (with <0.2% ID/g remaining in the blood at 1 h post-injection (p.i.)). DOTA-hippurate and DOTA-glycine both showed kidney-to-blood %ID/g ratios of ~3:1, which were deemed insufficient for further investigation.

HBED-Based Bifunctional Chelator Development
The acyclic chelator, HBED, based on an ethylenediaminetetraacetic acid (EDTA)-type framework with two pendant phenol arms, has several structural characteristics (N and O donor atoms, potential hexadentate coordination environment) that make it an ideal chelator for Ga 3+ . The original chelator synthesis was described in the 1960s [75]. The resulting Ga 3+ complex is highly thermodynamically stable (logβ 1 = 38.51) [76]. An X-ray crystal structure of HBED with Ga 3+ revealed an N 2 O 4 octahedral coordination sphere with the N 2 O 3 pentadentate chelator and an apical water molecule ( Figure 5). The development and characterisation of novel bifunctional variants of the dicarboxylate analogue, HBED-CC, has been growing over the past decade since Eder and co-workers' reported the conjugation of the PSMA-targeting motif, Glu-urea-Lys, to HBED-CC for the imaging of prostate cancer ([ 68 Ga]Ga-HBED-CC-PSMA) in 2012 [31,77].

HBED-Based Bifunctional Chelator Development
The acyclic chelator, HBED, based on an ethylenediaminetetraacetic acid (EDTA)type framework with two pendant phenol arms, has several structural characteristics (N and O donor atoms, potential hexadentate coordination environment) that make it an ideal chelator for Ga 3+ . The original chelator synthesis was described in the 1960s [75]. The resulting Ga 3+ complex is highly thermodynamically stable (logβ1 = 38.51) [76]. An X-ray crystal structure of HBED with Ga 3+ revealed an N2O4 octahedral coordination sphere with the N2O3 pentadentate chelator and an apical water molecule ( Figure 5). The development and characterisation of novel bifunctional variants of the dicarboxylate analogue, HBED-CC, has been growing over the past decade since Eder and co-workers' reported the conjugation of the PSMA-targeting motif, Glu-urea-Lys, to HBED-CC for the imaging of prostate cancer ([ 68 Ga]Ga-HBED-CC-PSMA) in 2012 [31,77]. In an effort to improve on the characteristics of Eder's HBED-CC-PSMA platform, Zha and co-workers introduced an O-(carboxymethyl)-L-tyrosine linker group into the structure (termed HBED-PSMA-093, Figure 6), which demonstrated increased cell internalisation (12.5% ID/10 6 cells at 1 h) compared to HBED-CC-PSMA (7.4% ID/10 6 cells at 1 h), whilst also demonstrating comparable fast clearance from non-target organs [78]. The development of a robust kit-based synthesis followed, and direct comparison with [ 68 Ga]Ga-PSMA-617 (a DOTA analogue) in PET/CT of patients with prostate cancers In an effort to improve on the characteristics of Eder's HBED-CC-PSMA platform, Zha and co-workers introduced an O-(carboxymethyl)-L-tyrosine linker group into the structure (termed HBED-PSMA-093, Figure 6), which demonstrated increased cell internalisation (12.5% ID/10 6 cells at 1 h) compared to HBED-CC-PSMA (7.4% ID/10 6 cells at 1 h), whilst also demonstrating comparable fast clearance from non-target organs [78]. The development of a robust kit-based synthesis followed, and direct comparison with [ 68 Ga]Ga-PSMA-617 (a DOTA analogue) in PET/CT of patients with prostate cancers showed higher tumour uptake, less blood pooling, and reduced bladder accumulation [33,79]. Targeting the HBED-CC chelator for bone imaging was achieved by tethering a bisphosphonate (HBED-CC-BP, Figure 6) [80]. The 68  showed higher tumour uptake, less blood pooling, and reduced bladder accumulation [33,79]. Targeting the HBED-CC chelator for bone imaging was achieved by tethering a bisphosphonate (HBED-CC-BP, Figure 6) [80]. The 68 Ga complex (termed [ 68 Ga]Ga-P15-041) demonstrated excellent in vivo and in vitro stability, and compared favourably to the more widely used tracer, [ 18 F]NaF. A kit-based formulation of the complex showed high RCP and radiochemical conversion (RCC) (> 90%) within 10 min and required no further purification [81]. Satpati and co-workers reported the conjugation of HBED-CC via amide bond formation to RGD (HBED-CC-cRGD, Figure 6) and asparagine-glycine-arginine (NGR)- Satpati and co-workers reported the conjugation of HBED-CC via amide bond formation to RGD (HBED-CC-cRGD, Figure 6) and asparagine-glycine-arginine (NGR)-containing peptides (HBED-CC-cNGR, Figure 6) for 68 Ga radiolabelling and PET imaging of tumour vasculature markers, integrin α v β 3 and CD13/aminopeptidase N, respectively. They showed that whilst uptake was similar in HT-1080 human xenografts, the radiolabelled HBED-CC-c(RGD) conjugate displayed higher uptake in B16F10 tumours and higher specificity to α v β 3 -positive cells than the NGR conjugate [82].
The conjugation of HBED-CC to monoclonal antibodies (mAbs) and antibody fragments for antibody-based PET imaging (immuno-PET) has also been explored. Fay and co-workers used photoactivatable aryl azides (HBED-CC-PEG 3 -ArN 3 , Figure 6) for the fast (~10 min) conjugation of the HBED-CC chelator to a model anti-c-MET antibody, onartuzumab, via the formation of an azepin group [83]. Klika and co-workers have recently reported a thiol-reactive HBED-CC derivative containing a phenyloxadiazolyl methylsulfone (PODS) group (HBED-CC-PODS, Figure 6) to mitigate the instability of the resulting succinimidyl linkage of the traditional maleimide coupling procedure (the reaction between a thiol of cysteine-containing biomolecules and a maleimide-containing chelator) [84,85].
Makarem and co-workers have published the syntheses of two novel azide-containing derivatives, namely the symmetric diazido HBED-NN ( Figure 6) and asymmetric monoazidomonocarboxylate HBED-NC ( Figure 6), for labelling via the Cu + -catalysed azide-alkyne cycloaddition (CuAAC) reaction [87,88]. HBED-NC, in particular, shows significant promise through the potential construction of heterodimeric architectures (i.e., for use in multimodal imaging procedures such as PET/CT/fluorescence imaging, studies of which were recently reported) [89]. The synthesis of HBED-CC and derivatives has been adapted to solid-phase techniques, which could potentially ease the multistep synthetic protecting group strategies currently employed for the synthesis of HBED-CC derivatives [90].
The approval of [ 68 Ga]Ga-HBED-CC-PSMA in December 2020 by the FDA for the PET imaging of prostate cancer has catalysed the developed of novel BFCs based on the HBED scaffold, with renewed interest in the once-overlooked chelating platform [25]. The successful clinical translation of other HBED bioconjugates has yet to be seen. However, it seems likely that the favourable 68 Ga radiolabelling kinetics of HBED will lead to further radiopharmaceuticals with novel biological targets in future.

DTPA-Based Bifunctional Chelator Development
Another acyclic chelator that has been investigated as a 68 Ga 3+ chelator for radiopharmaceutical development is 2-(bis{2-[bis(carboxymethyl)amino]ethyl}-amino)acetic acid (DTPA) [25,91]. The X-ray crystal structure of the Ga 3+ complex was reported by Wallin and co-workers and revealed a N 2 O 4 coordination sphere comprising the 5-coordinate DTPA chelator and an apical water molecule (Figure 7) [92]. This is somewhat surprising given the eight potential donor atoms and is potentially due to the small ionic radius of the Ga 3+ ion or the conditions of crystallisation. The analogous Fe 3+ complex, [Fe(DTPA)] 2− , is 7-coordinate [93,94].
The synthetic development of DTPA BFCs has involved derivatisation of the carboxylic acids or the incorporation of a functional group to the diethylenetriamine backbone. Bis-amide derivatives (Figure 8) synthesised from DTPA bis-anhydride were shown to form complexes with Ga 3+ , In 3+ , and Lu 3+ [95]. DTPA bis-anhydride reacts with aminecontaining compounds, including biomolecules containing surface lysine residues, to generate the resulting DTPA bis-amide. However, radiolabelling studies with 68 Ga were not performed. DTPA bis-anhydride was also used to label β-neurotoxins of Micrurus fulvius with 67 Ga to track the biodistribution of the venoms via reaction with the proteins' surface lysine residues [96]. The synthetic development of DTPA BFCs has involved derivatisation of the carboxylic acids or the incorporation of a functional group to the diethylenetriamine backbone. Bis-amide derivatives (Figure 8) synthesised from DTPA bis-anhydride were shown to form complexes with Ga 3+ , In 3+ , and Lu 3+ [95]. DTPA bis-anhydride reacts with amine-containing compounds, including biomolecules containing surface lysine residues, to generate the resulting DTPA bis-amide. However, radiolabelling studies with 68 Ga were not performed. DTPA bis-anhydride was also used to label β-neurotoxins of Micrurus fulvius with 67 Ga to track the biodistribution of the venoms via reaction with the proteins' surface lysine residues [96].   The synthetic development of DTPA BFCs has involved derivatisation ylic acids or the incorporation of a functional group to the diethylenetriam Bis-amide derivatives (Figure 8) synthesised from DTPA bis-anhydride w form complexes with Ga 3+ , In 3+ , and Lu 3+ [95]. DTPA bis-anhydride reacts wi taining compounds, including biomolecules containing surface lysine resid ate the resulting DTPA bis-amide. However, radiolabelling studies with performed. DTPA bis-anhydride was also used to label β-neurotoxins of M with 67 Ga to track the biodistribution of the venoms via reaction with the pro lysine residues [96].   Figure 4) [53], DOTAGA-(11.1 ± 0.2, n = 3, Figure 4) [53], and DFO-(67-88%, n = 2) analogues [99], DTPA-PEG 3 -ArN 3 showed the lowest RCC values of the antibody conjugates (3.9 ± 1.0%, n = 4), with the precise reasons currently unknown.
Another way to generate DTPA BFCs is via functionalisation of the alkyl backbone. This has the advantage of not interfering with the available donor atoms, allowing for a potential non-coordinating carboxylic acid to be derivatised further. An isothiocyanate (NCS) analogue of DTPA (DTPA-Bn-NCS, Figure 8) was derivatised from the diethylenetriamine backbone. The NCS group reacts with amines under mild conditions to form a thiourea bond.
Jain and co-workers compared the relative stability of various 68 Ga-labelled bis-RGD peptide conjugates (utilising the DTPA-Bn-NCS, NOTA-Bn-NCS, and DOTA-Bn-NCS BFCs) and found that the DTPA analogue had by far the worst metabolic stability, as well as requiring high temperature radiolabelling conditions to achieve high RCY (along with the DOTA analogue) [100]. The attachment of DTPA-Bn-NCS to a short-chain fatty acid, undecanoic acid (DTPA undecanoic acid, Figure 8), was developed for imaging cardiac metabolic events [101]. However, the biodistribution of the 68 Ga complex revealed lower myocardial uptake (1.3 ± 0.5% ID/g) compared to the TACN derivatives, NOTA undecanoic acid (3.8 ± 0.6% ID/g), and NODAGA undecanoic acid (3.8 ± 0.6% ID/g).
In recent years, DTPA and derivatives have also been used to decorate iron oxide nanoparticles for 68 Ga radiolabelling studies, as well as for the molecular imaging of glomeruli using the targeting agent, tilmanocept (using a DTPA-mannosyl-dextran adduct) [102,103]. Taken together, these results seem to suggest that DTPA is not an optimal chelator platform for 68 Ga, as it often shows relatively poor RCC results compared to other acyclic as well as macrocyclic chelators. With the success of HBED and promising results of other acyclic chelators (vide infra), DTPA has been supplanted.

Siderophore-Based Bifunctional Chelator Development
A promising class of acyclic chelators for 68 Ga are the siderophores. Siderophores are most well-known as Fe 3+ sequestration agents used by fungi, bacteria, and plants [104,105]. Due to the chemical similarities between Fe 3+ and Ga 3+ (vide supra), siderophores have been identified as potential 68 Ga 3+ chelating agents. Desferrioxamine-B (DFO) is a bacterial siderophore that was originally characterised in 1958 as a metabolite of Streptomyces pilosus [106]. DFO is a linear trihydroxamic acid that chelates Fe 3+ to form ferrioxamine B ([Fe(DFO)] + or FOB) (Figure 9). DFO was one of the first reported bifunctional siderophores to be radiolabelled with radioisotopes of Ga in high RCY [107]. The DFO-human serum albumin (HSA) conjugate was radiolabelled with 67 Ga at pH 7-8, achieving a RCY of 99.8 ± 0.3%. Despite the fact that DFO conjugates have been seen to leach radioisotopes of Ga in vivo [108][109][110][111], several bifunctional chelating agents have been synthesised over the past decade based on the DFO scaffold, as well as other siderophore-based ligands that are structurally distinct from DFO [112]. Gourni and co-workers reported succinic acid (DFO-Nsucc, Figure 10) and isothiocyanate (DFO-pNCS-Bn-NCS, Figure 10) derivatives of DFO and subsequent conjugation to PSMA for 68 Ga preclinical imaging of prostate cancer in mice bearing subcutaneous LNCaP tumours [114]. Compared to HBED-CC-PSMA, however, both conjugates showed decreased tumour uptake. Ioppolo and co-workers produced a library of alkyl-substituted DFO carbamates (-Me, -Et, -nPr, -iPr, -nBu, -iBu, -nhexyl, -boc, -Bn, (CH2)6NHboc and (CH2)6NH2, Figure 10) to tune the uptake of the 67 Ga-labelled complexes in bacteria (Staphylococcus aureus) and sites of bacterial infection [115].  [113]. Ellipsoids are drawn at the 50% probability level. Hydrogen atoms, counterions, and solvent molecules are omitted for clarity.
The versatility of the DFO-squarate platform (DFOSq, Figure 10), where primary amine-containing compounds can be tethered together via the squaric acid group, was demonstrated with 68 Ga radiolabelling and in vivo studies of octreotate, octreotide, and PSMA conjugates [119,120]. Similar derivatives of the free primary amine, including isothiocyanates and maleimides (DFO-maleimide, Figure 10), have been reported for the 68 Ga PET imaging of tumour-induced angiogenesis and 66 Ga radiolabelling of EGFR, respectively [121,122].
Bauman and co-workers and in a subsequent publication, Kaeppeli and co-workers, reported on DFO-Exendin 4 conjugates (using the bifunctional chelator, DFO-pNCS-Bn-NCS, Figure 10) for the radiolabelling of insulinomas and compared the radiolabelling as well as in vitro and in vivo stability to NODAGA and DTPA analogues [123,124]. Both studies demonstrated comparable tumour uptake with DFO outperforming NODAGA in terms of target-to-kidney ratio. DFO-pNCS-Bn-NCS was also used to radiolabel a shortchain variable fragment (scFv) targeting the human epidermal growth factor 2 (HER2) receptor [125]. The radiolabelled DFO-scFv conjugate showed high accumulation in HER2positive xenograft-bearing mice and was used to monitor changes in HER2 expression following anti-HER2 therapy.
Siderophores not based on the DFO scaffold have also seen developments over the last decade ( Figure 11). Pandey and co-workers reported the conjugation of the desferrichrome chelator to the fluoroquinolone, ciprofloxacin, for 68 Ga monitoring of a potential therapeutic for bacterial infection [105]. The catecholamide, enterobactin, is produced by the Enterobacteriaceae family of bacteria and has been shown to have one of the highest pFe values (35.5) [126] for all Fe 3+ complexes (where the higher the pFe value, the more stable the complex), where pFe is defined as:  Figure 11). The 68 Ga complexes of TREN-CAM, 2,2-Glu-CAM, 3,3-Glu-CAM and TREN-bisGlyGly-CAM performed similarly to other acyclic ligands, including DFO, in terms of in vitro (human serum) and in vivo stability and in vivo renal clearance [108]. Petrik and co-workers reported the 68 Ga radiolabelling of the siderophores, triacetylfusarinine C (TAFC, Figure 11) and ferrioxamine E (FOXE, Figure 11) for PET imaging of invasive pulmonary aspergillosis caused by the bacterium Aspergillus fumigatus [127]. TAFC was radiolabelled at room temperature for 15 min whereas FOXE required elevated temperature (80 • C) for 20 min for quantitative (>95% RCC) radiolabelling. Both hexadentate complexes were investigated in an in vitro model of A. fumigatus and showed rapid uptake in irondeficient cultures. Follow-up studies were performed in vivo, showing vastly different organ uptake depending on the siderophore used, including DFO and fusarinine C (FSC, Figure 11) [128,129]. FSC is the deacetylated form of TAFC containing three primary amines that are separated from the hexadentate coordination sphere. Knetsch and co-workers followed by Zhai and co-workers demonstrated favourable 68 Ga radiolabelling of FSC (>90% RCY after 5 min at room temperature) and conjugation to three RGD moieties through amide bond formation.
Given the similarity in coordination chemistry between Fe 3+ and Ga 3+ , siderophores are a natural choice of chelator, and subsequent bifunctional chelator, for 68 Ga radiopharmaceutical development. The resulting complexes are thermodynamically stable (e.g., logK 1 ([Ga(DFO)] = 28.65) [130] but suffer from a lack of kinetic inertness, resulting in radiation leaching in vivo [108]. With the development of higher-denticity DFO ligands on the rise [116][117][118]131,132], these systems are seemingly better suited to other metallic radioisotopes, such as 89 Zr 4+ . Exploring non-DFO siderophores may lead to better candidates for 68 Ga radiolabelling, with the underexplored catechol-and catecholamide-based ligands a promising alternative.
Siderophores not based on the DFO scaffold have also seen developments over the last decade (Figure 11). Pandey and co-workers reported the conjugation of the desferrichrome chelator to the fluoroquinolone, ciprofloxacin, for 68 Ga monitoring of a potential therapeutic for bacterial infection [105]. The catecholamide, enterobactin, is produced by the Enterobacteriaceae family of bacteria and has been shown to have one of the highest pFe values (35.5) [126] for all Fe 3+ complexes (where the higher the pFe value, the more stable the complex), where pFe is defined as:

Pyridinecarboxylate-Based Bifunctional Chelator Development
In 2010, Boros and Orvig reported the synthesis, 67/68 Ga radiolabelling, bioconjugation, stability, and biodistribution of an acyclic pyridinecarboxylate ("pa") ligand, H 2 dedpa, based on earlier synthetic work by Platas-Iglesias and co-workers [133,134]. The X-ray crystal structure of the monocationic complex shows an N 2 O 4 , pseudo-octahedral coordination environment ( Figure 12). The resulting 68 Ga 3+ complex (log K ML = 28.11 (8)) demonstrated quantitative radiolabelling at ligand concentrations as low as 10 −7 M and has inspired the design and synthesis of several derivatives (Figure 13). Follow-up studies demonstrated the versatility of the bifunctional platform via conjugation to RGD peptides, synthesis of lipophilic analogues for myocardial imaging, as well as translation to copper-64 ( 64 Cu) radiolabelling and subsequent in vitro and in vivo studies [135][136][137].     Bailey and co-workers reported a triazole dedpa derivative, H 2 azapa (Figure 13), that quantitatively radiolabelled 67 Ga and 64 Cu, as well as the large radiometal ions indium-111 ( 111 In) and lutetium-177 ( 177 Lu) [138]. Bis(propylamine) derivatives were also prepared (H 2 dedpa-propyl pyr -NH 2 and H 2 dedpa-propyl pyr -NH 2 -(N,N'-propyl-2-NI, Figure 13) that show promise for bifunctionality via the reactive primary amine [139]. Bifunctional bi-modal fluorescent/nuclear imaging H 2 dedpa-fluorescein conjugates have also been reported, but showed that the bulky fluorescein moieties prevented effective 67 Ga radiolabelling [140]. However, a recent report has shown that increasing the distance between the chelating unit (in this case, DOTA) and the fluorophore improves radiolabelling capabilities [89]. The analogous dedpa study has yet to be reported.
Saito and co-workers developed bifunctional H 2 dedpa derivatives bearing pyridylbenzofuran groups (dedpa-(PBF) 2 , Figure 13) for targeting islet amyloid deposition in pancreas islets, a key marker for Type 2 diabetes mellitus [143]. The authors were able to radiolabel the BFC with 67/68 Ga but noted that improvement in non-target organ clearance was needed. Recently, Wang and co-workers reported 8-hydroxyquinoline ("hox") analogues of both H 2 dedpa and H 2 CHXdedpa and investigated the preferential heart uptake of the Ga 3+ complexes compared to the 'pa' family analogues [144,145]. Additionally, the inherent fluorescence of the 'hox' group shows promise for potential multi-modal PET/SPECT-fluorescence imaging in future.
Given the early promising results of the pyridinecarboxylate chelators, it is perhaps somewhat surprising that there have not been more pre-clinical and clinical studies reported. Moreover, the excellent kinetic inertness of the rigidified Ga 3+ complexes make dedpa and its derivatives excellent choices for radiopharmaceutical development. Consequently, further exploration of their potential is warranted.

Hydroxypyridinone-Based Bifunctional Chelator Development
Hydroxypyridinones are a class of aromatic heterocycles that have been extensively studied for iron overload disease. They contain hydroxyl and ketone functional groups as donor atoms resulting in bidentate chelators [44,146,147]. Depending on the substitution pattern of the aromatic ring, three isomeric forms can exist: 1,2-hydroxypyridinones, 2,3-hydroxypyridinones, and 3,4-hydroxypyridinones. Binding to metal ions occurs with deprotonation of the acidic hydroxyl. The relative stability of the three Ga 3+ complexes is known to follow the order 3,4-hydroxypyridinone > 2,3-hydroxypyridinone > 1,2hydroxypyridinone, which reflects the charge density on the O atoms caused by the relative delocalisation of aromaticity around the ring [91,[148][149][150]. 3,4-Hydroxypyridinones, such as deferiprone, form isostructural octahedral complexes with Fe 3+ and Ga 3+ (Figure 15). Saito and co-workers developed bifunctional H2dedpa derivatives bearing pyridylbenzofuran groups (dedpa-(PBF)2, Figure 13) for targeting islet amyloid deposition in pancreas islets, a key marker for Type 2 diabetes mellitus [143]. The authors were able to radiolabel the BFC with 67/68 Ga but noted that improvement in non-target organ clearance was needed. Recently, Wang and co-workers reported 8-hydroxyquinoline ("hox") analogues of both H2dedpa and H2CHXdedpa and investigated the preferential heart uptake of the Ga 3+ complexes compared to the 'pa' family analogues [144,145]. Additionally, the inherent fluorescence of the 'hox' group shows promise for potential multi-modal PET/SPECT-fluorescence imaging in future.
Given the early promising results of the pyridinecarboxylate chelators, it is perhaps somewhat surprising that there have not been more pre-clinical and clinical studies reported. Moreover, the excellent kinetic inertness of the rigidified Ga 3+ complexes make dedpa and its derivatives excellent choices for radiopharmaceutical development. Consequently, further exploration of their potential is warranted.

Hydroxypyridinone-Based Bifunctional Chelator Development
Hydroxypyridinones are a class of aromatic heterocycles that have been extensively studied for iron overload disease. They contain hydroxyl and ketone functional groups as donor atoms resulting in bidentate chelators [44,146,147]. Depending on the substitution pattern of the aromatic ring, three isomeric forms can exist: 1,2-hydroxypyridinones, 2,3hydroxypyridinones, and 3,4-hydroxypyridinones. Binding to metal ions occurs with deprotonation of the acidic hydroxyl. The relative stability of the three Ga 3+ complexes is known to follow the order 3,4-hydroxypyridinone > 2,3-hydroxypyridinone > 1,2-hydroxypyridinone, which reflects the charge density on the O atoms caused by the relative delocalisation of aromaticity around the ring [91,[148][149][150]. 3,4-Hydroxypyridinones, such as deferiprone, form isostructural octahedral complexes with Fe 3+ and Ga 3+ (Figure 15).  Hexadentate tris(hydroxypyridinones) (THPs) have been of interest for Ga 3+ complexation due to the 1:1 complexation stoichiometry compared to the 3:1 that would eventuate from tris(3,4-hydroxypyridinones). The first report of THP was in 2011 by Berry and Blower, where a radiolabelling study showed that THP could achieve higher RCY values than HBED, DOTA, and NOTA at pH 6.5 after 5 min at room temperature [146]. THPs have been synthesised from either tripodal polyamines or tripodal carboxylic acids, and dendritic constructs have also been reported [152].
Over the past decade, several bifunctional THP derivatives have been reported (Figure 16) with the accompanying 68  Hexadentate tris(hydroxypyridinones) (THPs) have been of interest for Ga 3+ complexation due to the 1:1 complexation stoichiometry compared to the 3:1 that would eventuate from tris(3,4-hydroxypyridinones). The first report of THP was in 2011 by Berry and Blower, where a radiolabelling study showed that THP could achieve higher RCY values than HBED, DOTA, and NOTA at pH 6.5 after 5 min at room temperature [146]. THPs have been synthesised from either tripodal polyamines or tripodal carboxylic acids, and dendritic constructs have also been reported [152].
Imberti and co-workers explored dendritic variants of THP and synthesised three phenyl-isothiocyanate constructs conjugated to the RGD peptide (HP9-RGD3, HP3-RGD and HP3-RGD3) [152]. The HP9-RGD3 bioconjugate, containing three hexadentate metal binding sites, was shown to radiolabel at three times the specific activity value as the other two (180-240 MBq/nmol). However, it was demonstrated to have large uptake in non-target organs that compared unfavourably to the HP3-RGD3 construct. Although each of the chelators could quantitatively radiolabel 68 Ga to 97% RCY, studies indicated that the distance between the RGD units was not large enough to sufficiently bind more than one integrin receptor.
Efforts have also been made towards synthesising THP bioconjugates for the PET imaging of prostate cancer. Nawaz and co-workers reported the conjugation of THP-maleimide ( Figure 16) to a C-terminal cysteine residue of the scFv of the monoclonal antibody J591 that specifically binds to an external epitope of PSMA [156]. Radiolabelling of the THP-scFv conjugate proceeded at room temperature and neutral pH in a one-step synthesis, and the resulting radiotracer showed high affinity for PSMA in vitro and demonstrated uptake in a xenograft model (DU145-PSMA) of prostate cancer in mice. Blower and co-workers had arguably more success with their THP-PSMA conjugates, which can be radiolabelled in a kit-based, one-step synthesis at room temperature and near-neutral pH [29,[157][158][159]. Phase I clinical trials have been reported and, in 2020, a 118-patient study was reported demonstrating the effectiveness of [ 68 Ga][Ga(THP-PSMA)] in influencing clinical management of prostate cancer before therapy by identifying metastases in bone [159].
Despite  Figure 16) [147,155,160]. Preliminary radiolabelling studies have been reported, and work is ongoing to form radiolabelled bioconjugates with these promising systems.
The favourable radiolabelling properties of the THP chelators (near neutral pH and room temperature) are ideal for large biomolecules, such as proteins and antibodies [37]. Work is ongoing to translate THP and various bioconjugates to simple, rapid kit-based systems for clinical use, with the most promising to date being [ 68 Ga][Ga(THP-PSMA)].

Diazepine-Based Bifunctional Chelator Development
The family of chelators based on the 6-amino-6-methyl-perhydro-1,4-diazepine (diazepine) scaffold has attracted much attention over the last two decades for 68 Ga radiopharmaceutical development. This has been primarily spurred on by studies investigating the coordination chemistry of the parent and derivatised ligands between 2004 and 2009 (including transition metals and Gd 3+ for MRI contrast agents), which highlighted the similarities between the resultant complexes and those of TACN, a constitutional isomer of diazepine [161][162][163][164][165]. The ligands form hexadentate Ga 3+ complexes, generally in a facial (fac) arrangement of donor atoms with the N 3 plane remaining consistent among the various complexes ( Figure 17).  Several groups have developed bifunctional variants of the diazepine chelators with various applications over the past decade. Wu and co-workers reported the synthesis of bisphosphonate AAZTA chelators (PhenA, PhenA-BPAMD, and PhenA-HBP, Figure 18) for the 68 Ga PET imaging of bone [171]. The introduction of the bifunctional phenylcarboxylate pendant arm was enabled through tosylation of a methanol-diazepine derivative, originally reported in 2009 by Gugliotta and co-workers [172].
A pentanoic acid version of AAZTA, AAZTA 5 ( Figure 18) [173], was synthesised and Several groups have developed bifunctional variants of the diazepine chelators with various applications over the past decade. Wu and co-workers reported the synthesis of bisphosphonate AAZTA chelators (PhenA, PhenA-BPAMD, and PhenA-HBP, Figure 18) for the 68 Ga PET imaging of bone [171]. The introduction of the bifunctional phenylcarboxylate pendant arm was enabled through tosylation of a methanol-diazepine derivative, originally reported in 2009 by Gugliotta and co-workers [172].
AAZTA-curcumin conjugates ( Figure 18) were reported by Orteca and co-workers for potential 68 Ga PET imaging of colorectal cancer. The tetra-tert-butyl ester protected AAZTA was pre-activated with the coupling agent, HBTU, and reacted directly with curcumin to form the BFC [179]. Compared to the TACN derivative, NODAGA-curcumin, the AAZTAcurcumin conjugate showed greater stability in human blood over 2 h (with comparable stability in plasma and serum).
Yadav and co-workers were interested in comparing the biodistribution and gastroenteropancreatic neuroendocrine tumour (GEP-NET) uptake of [ 68  . This is promising, as DATA chelators can be more easily synthesised in a kit-based synthesis than DOTA-based systems [181,182].
The excellent radiolabelling conditions (very low ligand concentrations, neutral pH, and room temperature) make the diazepine family of chelators, particularly DATA m , a promising candidate for 68 Ga radiopharmaceutical development. This facilitates preparation of instant kit-type preparations, which are crucial for successful clinical translation.

TACN-Based Bifunctional Chelator Development
Interest in TACN-based chelators has been spurred on by the particularly attractive coordination properties of triazamacrocyclic chelators with tricarboxylic acid (NOTA), triphosphonic acid (NOTP) and triphosphinic acid (TRAP) pendant groups for Ga 3+ . They are known to be highly rigid (due to the preformed geometry offered by the macrocycle), kinetically inert, and thermodynamically stable due in part to the macrocyclic effect (stability constants logK 1 > 26), which encompasses entropic gain from a pre-organised structure around the metal ions (Table 3) [52,183,184].  [188] 26.05 [188] In the case of [Ga(NOTA)], the X-ray crystal structure of the complex indicates that the distorted octahedral coordination environment contains three deprotonated carboxyl groups [189]. [Ga(TRAP)] exhibits similar coordination environments to [Ga(NOTA)], with deprotonated phosphinic acids contributing the O 3 donor atoms. The radiolabelling properties of these chelators with 68 Ga 3+ have also been investigated. The triphosphinic acid, TRAP, was shown to radiolabel with 68 Ga 3+ to >95% RCC at a reduced ligand concentration (3 µM) compared to DOTA (500 µM) and NOTA (100 µM) at 25 • C and pH 0.5-5 [38,184]. Phosphonate-containing ligands such as NOTP have been shown to chelate 68 Ga 3+ at room temperature (25 • C) and neutral pH (6.5) at higher RCYs than DOTA, NOTA, and TRAP chelators at chelator concentrations of 0.5 µM (Figure 19) [38]. The X-ray crystal structure of [Ga(NOTP)] shows a hexadentate N 3 O 3 coordination environment that is closer to ideal octahedral than [Ga(NOTA)] [190]. The simplest method of forming bifunctional variants of NOTA is through derivatisation of one carboxylic acid pendant arm ( Figure 20). Coupling to an amine is achieved either through agents, such as HBTU or HATU or activated (sulfo)esters [191,192]. Less commonly used methods include the use of click compounds or thiol coupling [53,193,194]. However, these methods have a marked disadvantage, which is that the resulting amide is a worse coordinating group for Ga 3+ than the carboxylic acid due to the lower thermodynamic stability of the resulting complexes [195,196]. Adapted from Ref. [38] with permission from the Royal Society of Chemistry [38].
The simplest method of forming bifunctional variants of NOTA is through derivatisation of one carboxylic acid pendant arm ( Figure 20). Coupling to an amine is achieved either through agents, such as HBTU or HATU or activated (sulfo)esters [191,192]. Less commonly used methods include the use of click compounds or thiol coupling [53,193,194]. However, these methods have a marked disadvantage, which is that the resulting amide is a worse coordinating group for Ga 3+ than the carboxylic acid due to the lower thermodynamic stability of the resulting complexes [195,196].
The simplest method of forming bifunctional variants of NOTA is through derivatisation of one carboxylic acid pendant arm ( Figure 20). Coupling to an amine is achieved either through agents, such as HBTU or HATU or activated (sulfo)esters [191,192]. Less commonly used methods include the use of click compounds or thiol coupling [53,193,194]. However, these methods have a marked disadvantage, which is that the resulting amide is a worse coordinating group for Ga 3+ than the carboxylic acid due to the lower thermodynamic stability of the resulting complexes [195,196].  Several well-known NOTA-based BFCs have been developed where an additional reactive functional group has been provided for conjugation reactions but does not interfere with the coordination environment. NODAGA (Figure 20), a glutaric acid analogue of NOTA, which was first reported in 2002 and has seen extensive conjugation and radiolabelling optimisation [55,56,62,124,[197][198][199][200][201][202]. The succinic acid derivative, NODASA ( Figure 20), was reported in 1998, but has seen less application than NODAGA, presumably due to their structural similarities and comparable radiolabelling efficiencies. The X-ray crystal structure of [Ga(NODASA)] shows a hexadentate N 3 O 3 coordination environment that is typical of Ga 3+ complexes of TACN and derivatives ( Figure 21). Several well-known NOTA-based BFCs have been developed where an additional reactive functional group has been provided for conjugation reactions but does not interfere with the coordination environment. NODAGA (Figure 20), a glutaric acid analogue of NOTA, which was first reported in 2002 and has seen extensive conjugation and radiolabelling optimisation [55,56,62,124,[197][198][199][200][201][202]. The succinic acid derivative, NODASA ( Figure 20), was reported in 1998, but has seen less application than NODAGA, presumably due to their structural similarities and comparable radiolabelling efficiencies. The Xray crystal structure of [Ga(NODASA)] shows a hexadentate N3O3 coordination environment that is typical of Ga 3+ complexes of TACN and derivatives ( Figure 21).  [203]. Ellipsoids are drawn at the 50% probability level. Hydrogen atoms and solvent molecules are omitted for clarity. NOTA-p-Bn-NCS ( Figure 20) is one of the most widely-studied BFCs for 68 Ga, with a plethora of studies reporting its conjugation to targeting vector biomolecules, such as small molecules, peptides, and antibodies [100,[204][205][206][207][208][209]. Massa and co-workers reported a triglycine derivative of NOTA-p-Bn-NCS, GGGYK-NOTA, for use in enzyme-mediated site-specific labelling of camelid single-domain antibody fragments with 68 Ga [210]. Bonetargeting bisphosphonate-NOTA conjugates have been reported by Holub and co-work- Figure 21. ORTEP representation of [Ga(NODASA)] (CSD-NUHLOR) [203]. Ellipsoids are drawn at the 50% probability level. Hydrogen atoms and solvent molecules are omitted for clarity. NOTA-p-Bn-NCS ( Figure 20) is one of the most widely-studied BFCs for 68 Ga, with a plethora of studies reporting its conjugation to targeting vector biomolecules, such as small molecules, peptides, and antibodies [100,[204][205][206][207][208][209]. Massa and co-workers reported a triglycine derivative of NOTA-p-Bn-NCS, GGGYK-NOTA, for use in enzyme-mediated site-specific labelling of camelid single-domain antibody fragments with 68 Ga [210]. Bonetargeting bisphosphonate-NOTA conjugates have been reported by Holub and co-workers and Passah and co-workers, demonstrating improved bone uptake compared to commercially available bone imaging agents [211,212]. Recently, the imidazole-based BFC, NODIA-Me (Figure 20), was reported and tethered to a PSMA targeting vector [213][214][215][216]. The resulting 68 Ga complex and conjugate was kinetically inert to transmetallation in vivo, which underlined its potential use as a radiopharmaceutical agent.
NOPO was evaluated as an αvβ3 integrin-targeting BFC via conjugation to the cyclic pentapeptide, c(RGDfK) [219]. The Ga 3+ complex showed high affinity to αvβ3 integrin (IC 50 = 1.02 nM) and showed a tumour-to-blood ratio of 19.6 ± 6.8 at 60 min p.i. in the same M21 mice model. A monomeric TRAP-azide construct has also been radiolabelled and tethered to an αvβ8 integrin targeting cyclic peptide [225].
The presence of phosphonate pendant groups increases the thermodynamic stability and rigidity of the Ga 3+ TACN chelates. in competition experiments. Unlike the phosphinate-containing chelators, however, minimal bifunctional TACN chelators have been reported containing phosphonates [38]. This may be due to perceived difficulties with designing and synthesising BFCs. To date, the only BFCs that have been derivatives of NO2AP and NOA2P ( Figure 20) were achieved by Gai and co-workers, who reported synthesis of bisphosphonate and monophosphonatecontaining TACN BFCs (p-R-PhPr-NE2A1P and p-R-PhPr-NE2P1A, where R = NO 2 or NCS, Figure 20) [183,[226][227][228]

Conclusions
Proper chelator design and an understanding of Ga 3+ aqueous coordination chemistry are essential for the successful development of 68 Ga radiopharmaceuticals. The key components of this development process include the use of well-designed chelators, capable of forming thermodynamically stable and kinetically inert 68 Ga 3+ complexes using mild conditions (near-neutral pH, room temperature and low concentrations of ligand) in a short timeframe (<10 min), which are tethered to vector biomolecules, such as peptides and antibodies.
The most promising 68 Ga radiotracers are those that can be streamlined to pre-clinical and clinical applications using robust, reliable, and cost-efficient radiopharmaceutical kits. These include derivatives of THP, DATA, and TRAP, which have been applied to clinically relevant targeting vectors, such as RGD, octreotate, NaI 3 -octreotide, and PSMAtargeting motifs. Newer generation BFCs, such as PIDAZTA, non-DFO siderophores, and TACN phosphonates, fulfil the requirements for effective nat/67/68 Ga 3+ complexation (N and O donor atoms, six-coordinate complexes, mild radiolabelling conditions), yet remain underexplored in terms of bifunctionality. Further developments in this area will continue to enable the effective and simple incorporation of 68 Ga into new radiopharmaceuticals.
Finally, the choice of chelator has been shown to play a role in determining the biodistribution of small molecule and peptide-based radiopharmaceuticals. Therefore, assessing multiple BFCs to tune optimal biodistribution should be considered when utilising new targeting vectors.