Combining Nuclear Medicine With Other Modalities: Future Prospect for Multimodality Imaging.

This meeting report summarizes a consultants meeting that was held at International Atomic Energy Agency Headquarters, Vienna, in July 2022 to provide an update on the development of multimodality imaging by combining nuclear medicine imaging agents with other nonradioactive molecular probes and/or biomedical imaging techniques.

Radiolabelled 177 Lu-Bispidine-Trastuzumab for Targeting Human Epidermal Growth Factor Receptor 2 Positive Cancers.

Radioimmunotherapy (RIT) is a promising alternative to conventional treatment options. Here, we present experimental work on the synthesis, radiochemistry, and in vivo performance of a lanthanoid-selective nonadentate bispidine ligand suitable for 177 Lu3+ ion complexation. The ligand (bisp,1) was derivatised with a photoactivatable aryl azide (ArN3 ) group as a bioconjugation handle for light-induced labelling of proteins. Quantitative radiosynthesis of [177 Lu]Lu-1+ was accomplished in 10 minutes at 40 °C. Subsequent incubation of [177 Lu]Lu-1+ with trastuzumab, followed by irradiation with light at 365 nm for 15 min, at room temperature and pH 8.0-8.3, gave the radiolabelled mAb, [177 Lu]Lu-1-azepin-trastuzumab ([177 Lu]Lu-1-mAb) in a decay-corrected radiochemical yield of 14 %, and radiochemical purity (RCP)>90 %. Stability studies and cellular binding assays in vitro using the SK-OV-3 human ovarian cancer cells confirmed that [177 Lu]Lu-1-mAb remained biological active and displayed specific binding to HER2/neu. Experiments in immunocompromised female athymic nude mice bearing subcutaneous xenograft models of SK-OV-3 tumours revealed significantly higher tumour uptake in the normal group compared with the control block group (29.8±11.4 %ID g-1 vs. 14.8±6.1 %ID g-1 , respectively; P-value=0.037). The data indicate that bispidine-based ligand systems are suitable starting points for constructing novel, high-denticity chelators for specific complexation of larger radiotheranostic metal ion nuclides.

Cooperative Capture Synthesis of Functionalized Heterorotaxanes─Chemical Scope, Kinetics, and Mechanistic Studies.

The self-assembly of molecularly interlocked molecules offers new opportunities for creating bioactive molecules for applications in medicine. Cooperative capture synthesis of heterorotaxanes in water is an attractive methodology for developing multifunctional supramolecular imaging agents or drugs, but derivatizing the rotaxane scaffold with biologically active vectors like peptides and proteins, or reporter probers like radioactive metal ion complexes and fluorophores, requires the installation of reactive functional groups. Here, we explored the chemical scope of ß-cyclodextrin (ß-CD) derivatization on the cucurbit[6]uril (CB[6])-mediated cooperative capture synthesis of hetero[4]rotaxanes with the objective of identifying which reactive groups can be used for further functionalization without compromising the efficiency of rotaxane synthesis. Nine ß-CD derivatives featuring an electrophilic leaving group (tosylate), aliphatic amines, a carboxylic acid, aliphatic azides, anilines, and aryl isothiocyanate were evaluated in the synthesis of hetero[4]rotaxanes. Experimental measurements on the kinetics of rotaxane synthesis were combined with detailed computational studies using the density functional theory to elucidate the mechanistic pathways and rate determining step in the cooperative capture process. Computational studies on the structure and bonding also revealed why intermolecular interactions between the ß-CD and CB[6] macrocycles improve the rate and efficiency of rotaxane formation through cooperative capture. Understanding the mechanistic details and synthetic scope will facilitate broader access to functionalized hetero[4]rotaxanes for applications in biomedicine and beyond.

Radiometal Complexes as Pharmacokinetic Modifiers: A Potent 68Ga-Labeled Gastrin-Releasing Peptide Receptor Antagonist Based on the Macrocyclic Metal Chelator NODIA-Me.

The gastrin-releasing peptide receptor (GRPr) is overexpressed in various cancer types including prostate and breast carcinomas, making it an attractive target for molecular imaging and therapy. In this work, we designed a novel GRPr antagonistic probe comprising metal chelator NODIA-Me. This 1,4,7-triazacyclononane-based chelator forms positively charged metal complexes due to its neutral methylimidazole arms. Because a positive charge at the N-terminus of GRPr conjugates is responsible for high receptor affinity as exemplified by the current gold standard DOTA-RM2, we investigated if a positively charged radiometal complex can be used as a pharmacokinetic modifier to also produce high-affinity GRPr conjugates. In this respect, the bioconjugate NODIA-Me-Ahx-JMV594 was prepared by a combination of solid-phase peptide synthesis and solution-based reactions in a 94% yield. Radiolabeling provided the 68Ga-labeled conjugate in radiochemical yields of >95% and radiochemical purities of >98% with mean molar activities of Am ∼17 MBq nmol-1. The competitive GRPr affinity of the metal-free and 69/71Ga-labeled conjugate was determined to be IC50 = 0.41 ± 0.06 and 1.45 ± 0.06 nM, respectively. The metal-free GRPr antagonist DOTA-RM2 and its corresponding 69/71Ga complex had IC50 values of 1.42 ± 0.07 and 0.98 ± 0.19 nM, respectively. Small-animal PET imaging of mice bearing GRPr(+) PC-3 tumors revealed high radioactivity accumulation in the tumors and in the pancreas as an organ with high levels of GRPr expression. These findings were corroborated by the corresponding ex vivo biodistribution data, in which the tumors and the pancreas exhibited the highest radioactivity accumulation. By coinjection of an excess of NODIA-Me-Ahx-JMV594, uptake in the tumors and GRPr(+) organs was significantly reduced, confirming specific receptor-mediated uptake. The estrogen receptor-positive tumor of a female breast cancer patient was clearly visualized by PET imaging using 68Ga-labeled NODIA-Me-Ahx-JMV594. To summarize, the positive charge at the N-terminus of the conjugate induced by the Ga(NODIA-Me) complex resulted in high GRPr affinity comparable to that of the potent antagonist DOTA-RM2. The conjugate NODIA-Me-Ahx-JMV594 is a promising probe for imaging of GRPr tumors that warrants further evaluation in larger patient cohorts as well as in combination with other radiometals.

HNODThia: A Promising Chelator for the Development of 64Cu Radiopharmaceuticals.

Herein, we present the synthesis and coordination chemistry of copper(II) and zinc(II) complexes of two novel heterocyclic triazacyclononane (tacn)-based chelators (HNODThia and NODThia-AcNHEt). The chelator HNODThia was further derivatized to obtain a novel PSMA-based bioconjugate (NODThia-PSMA) and a bifunctional photoactivatable azamacrocyclic analogue, NODThia-PEG3-ArN3, for the development of copper-64 radiopharmaceuticals. 64Cu radiolabeling experiments were performed on the different metal-binding chelates, whereby quantitative radiochemical conversion (RCC) was obtained in less than 10 min at room temperature. The in vitro stability of NODThia-PSMA in human plasma was assessed by ligand-challenge and copper-exchange experiments. Next, we investigated the viability of the photoactivatable analog (NODThia-PEG3-ArN3) for the light-induced photoradiosynthesis of radiolabeled proteins. One-pot photoconjugation reactions to human serum albumin (HSA) as a model protein and the clinically relevant monoclonal antibody formulation MetMAb were performed. [64Cu]Cu-7-azepin-HSA and [64Cu]Cu-7-azepin-onartuzumab were prepared in less than 15 min by irradiation at 395 nm, with radiochemical purities (RCP) of >95% and radiochemical yields (RCYs) of 42.7 ± 5.3 and 49.6%, respectively. Together, the results obtained here open the way for the development of highly stable 64Cu-radiopharmaceuticals by using aza-heterocyclic tacn-based chelators, and the method can easily be extended to the development of 67Cu radiopharmaceuticals for future applications in molecularly targeted radio(immuno)therapy.

Sortase-Mediated Site-Specific Conjugation and 89Zr-Radiolabeling of Designed Ankyrin Repeat Proteins for PET.

Designed ankyrin repeat proteins (DARPins) are genetically engineered proteins that exhibit high specificity and affinity toward specific targets. Here, the G3-DARPin, which binds the HER2/neu receptor, was site-specifically modified with enzymatic methods and 89Zr-radiolabeled for applications in positron emission tomography (PET). Sortase A transpeptidation was used to install a desferrioxamine B (DFO) chelate bearing a reactive triglycine group to the C-terminal sortase tag of the G3-DARPin, and 89Zr-radiolabeling produced a novel 89ZrDFO-G3-DARPin radiotracer that can detect HER2/neu-positive tumors. The triglycine probe, DFO-Gly3 (1), was synthesized in 29% overall yield. After sortase A transpeptidation and purification from the nonfunctionalized protein component, the DFO-G3-DARPin product was radiolabeled to give 89ZrDFO-G3-DARPin. Binding specificity was assessed in HER2/neu-expressing BT-474 and SK-OV-3 cellular assays. The pharmacokinetics, tumor uptake, and specificity of 89ZrDFO-G3-DARPin were measured in vivo by PET imaging and confirmed by final time point (24 h) biodistribution experiments in female athymic nude mice bearing BT-474 xenografts. Sortase A transpeptidation afforded the site-specific and stoichiometrically precise functionalization of DFO-G3-DARPin with one chelate per protein. The modified DFO-G3-DARPin was purified from the nonfunctionalized DARPin by using Ni-NTA affinity chromatography. 89ZrDFO-G3-DARPin was obtained with a radiochemical purity of >95% measured by radio-size-exclusion chromatography. BT-474 tumor uptake at 24 h postadministration reached 4.41 ± 0.67 %ID/g (n = 3) with an approximate ∼70% reduction in tumor-associated activity in the blocking group (1.26 ± 0.29 %ID/g; 24 h postadministration, n = 5, P-value of <0.001). Overall, the site-specific, enzyme-mediated functionalization and characterization of 89ZrDFO-G3-DARPin in HER2/neu positive BT-474 xenografts demonstrate that DARPins are an attractive platform for generating a new class of protein-based radiotracers for PET. The specific uptake and retention of 89ZrDFO-G3-DARPin in tumors and clearance from most background tissues produced PET images with high tumor-to-background contrast.

Supramolecular Rotaxane-Based Multi-Modal Probes for Cancer Biomarker Imaging.

Mechanically interlocked molecules present opportunities to construct therapeutic drugs and diagnostic imaging agents but harnessing supramolecular chemistry to make biologically active probes in water is a challenge. Here, we describe a rotaxane-based approach to synthesise radiolabelled proteins and peptides for molecular imaging of cancer biomarkers in vivo. Host-guest chemistry using ß-cyclodextrin- and cucurbit[6]uril-catalysed cooperative capture synthesis produced gallium-68 or zirconium-89 radiolabelled metallo[4]rotaxanes. Photochemical conjugation to trastuzumab led to a viable positron emission tomography (PET) radiotracer. The rotaxane architecture can be tuned to accommodate different radiometal ion complexes, other protein- or peptide-based drugs, and fluorophores for optical detection. This technology provides a platform to explore how mechanical bonding can improve drug delivery, enhance tumour specificity, control radiotracer pharmacokinetics, and reduce dosimetry.

The Influence of a Polyethylene Glycol Linker on the Metabolism and Pharmacokinetics of a 89Zr-Radiolabeled Antibody.

Most experimental work in the space of bioconjugation chemistry focuses on using new methods to construct covalent bonds between a cargo molecule and a protein of interest such as a monoclonal antibody (mAb). Bond formation is important for generating new diagnostic tools, yet when these compounds advance to preclinical in vitro and in vivo studies, and later for translation to the clinic, understanding the fate of potential metabolites that arise from chemical or enzymatic degradation of the construct is important to obtain a full picture of the pharmacokinetic performance of a new compound. In the context of designing new bioconjugate methods for labeling antibodies with the positron-emitting radionuclide 89Zr, we previously developed a photochemical process for making 89Zr-mAbs. Experimental studies on [89Zr]ZrDFO-PEG3-azepin-mAb constructs revealed that incorporation of the tris-polyethylene glycol (PEG3) linker improved the aqueous phase solubility and radiochemical conversion. However, the use of a PEG3 linker also has an impact on the whole-body residence time of the construct, leading to a more rapid excretion of the 89Zr activity when compared with radiotracers that lack the PEG3 chain. In this work, we investigated the metabolic fate of eight possible metabolites that arise from the logical disconnection of [89Zr]ZrDFO-PEG3-azepin-mAb at bonds which are susceptible to chemical or enzymatic cleavage. Synthesis combined with 89Zr-radiolabeling, small-animal positron emission tomography imaging at multiple time points from 0 to 20 h, and measurements of the effective half-life for whole-body excretion are reported. The conclusions are that the use of a PEG3 linker is non-innocent in terms of its impact on enhancing the metabolism of [89Zr]ZrDFO-PEG3-azepin-mAbs. In most cases, degradation can produce metabolites that are rapidly eliminated from the body, thereby enhancing image contrast by reducing nonspecific accumulation and retention of 89Zr in background organs such as the liver, spleen, kidney, and bone.

Tuning Tetrazole Photochemistry for Protein Ligation and Molecular Imaging.

Photochemistry provides a wide range of alternative reagents that hold potential for use in bimolecular functionalisation of proteins. Here, we report the synthesis and characterisation of metal ion binding chelates derivatised with disubstituted tetrazoles for the photoradiochemical labelling of monoclonal antibodies (mAbs). The photophysical properties of tetrazoles featuring extended aromatic systems and auxochromic substituents to tune excitation toward longer wavelengths (365 and 395 nm) were studied. Two photoactivatable chelates based on desferrioxamine B (DFO) and the aza-macrocycle NODAGA were functionalised with a tetrazole and developed for protein labelling with 89 Zr, 64 Cu and 68 Ga radionuclides. DFO-tetrazole (1) was assessed by direct conjugation to formulated trastuzumab and subsequent radiolabelling with 89 Zr. Radiochemical studies and cellular-based binding assays demonstrated that the radiotracer remained stable in vitro retained high immunoreactivity. Positron emission tomography (PET) imaging and biodistribution studies were used to measure the tumour specific uptake and pharmacokinetic profile in mice bearing SK-OV-3 xenografts. Experiments demonstrate that tetrazole-based photochemistry is a viable approach for the light-induced synthesis of PET radiotracers.

Microfluidic Preparation of 89Zr-Radiolabelled Proteins by Flow Photochemistry.

89Zr-radiolabelled proteins functionalised with desferrioxamine B are a cornerstone of diagnostic positron emission tomography. In the clinical setting, 89Zr-labelled proteins are produced manually. Here, we explore the potential of using a microfluidic photochemical flow reactor to prepare 89Zr-radiolabelled proteins. The light-induced functionalisation and 89Zr-radiolabelling of human serum albumin ([89Zr]ZrDFO-PEG3-Et-azepin-HSA) was achieved by flow photochemistry with a decay-corrected radiochemical yield (RCY) of 31.2 ± 1.3% (n = 3) and radiochemical purity >90%. In comparison, a manual batch photoreactor synthesis produced the same radiotracer in a decay-corrected RCY of 59.6 ± 3.6% (n = 3) with an equivalent RCP > 90%. The results indicate that photoradiolabelling in flow is a feasible platform for the automated production of protein-based 89Zr-radiotracers, but further refinement of the apparatus and optimisation of the method are required before the flow process is competitive with manual reactions.

Light-Activated Protein Conjugation and 89 Zr-Radiolabelling with Water-Soluble Desferrioxamine Derivatives.

Protein-conjugates are vital tools in biomedical research, drug discovery and imaging science. For example, functionalised monoclonal antibodies (mAbs) coupled to the desferrioxamine B (DFO) chelate and radiolabelled with 89 Zr4+ ions are used as radiopharmaceuticals for diagnostic positron emission tomography (PET). In this context, protein functionalisation requires the formation of a covalent bond that must be achieved without compromising the biological properties of the mAb. Photochemistry offers new synthetic routes toward protein conjugates like 89 Zr-mAbs but to harness the potential of light-induced conjugation reactions new photoactivatable reagents are required. Herein, we introduce two photoactivatable DFO-derivatives functionalised with an aryl azide (ArN3 ) for use in light-activated conjugation and radiosynthesis of 89 Zr-mAbs. Incorporation of a tris-polyethylene glycol (PEG)3 linker between DFO and the ArN3 group furnished water-soluble chelates that were used in the one-pot, photoradiosynthesis of different 89 Zr-radiolabelled protein conjugates with radiochemical yields up to 72.9±1.9 %. Notably, the DFO-PEG3 chelates can be readily synthesised in accordance with Good Laboratory Practice (GLP), which will facilitate clinical trials with photoradiolabelled 89 Zr-mAbs.

Photochemical Reactions in the Synthesis of Protein-Drug Conjugates.

The ability to modify biologically active molecules such as antibodies with drug molecules, fluorophores or radionuclides is crucial in drug discovery and target identification. Classic chemistry used for protein functionalisation relies almost exclusively on thermochemically mediated reactions. Our recent experiments have begun to explore the use of photochemistry to effect rapid and efficient protein functionalisation. This article introduces some of the principles and objectives of using photochemically activated reagents for protein ligation. The concept of simultaneous photoradiosynthesis of radiolabelled antibodies for use in molecular imaging is introduced as a working example. Notably, the goal of producing functionalised proteins in the absence of pre-association (non-covalent ligand-protein binding) introduces requirements that are distinct from the more regular use of photoactive groups in photoaffinity labelling. With this in mind, the chemistry of thirteen different classes of photoactivatable reagents that react through the formation of intermediate carbenes, electrophiles, dienes, or radicals, is assessed.

Predicting the Thermodynamic Stability of Zirconium Radiotracers.

The thermodynamic stability of a metal-ligand complex, as measured by the formation constant (log ß), is one of the most important parameters that determines metal ion selectivity and potential applications in, for example, radiopharmaceutical science. The stable coordination chemistry of radioactive 89Zr4+ in an aqueous environment is of paramount importance when developing positron-emitting radiotracers based on proteins (usually antibodies) for use with positron emission tomography. Desferrioxamine B (DFO) remains the chelate of choice for clinical applications of 89Zr-labeled proteins, but the coordination of DFO to Zr4+ ions is suboptimal. Many alternative ligands have been reported, but the challenges in measuring very high log ß values with metal ions such as Zr4+ that tend to hydrolyze mean that accurate thermodynamic data are scarce. In this work, density functional theory (DFT) calculations were used to predict the reaction energetics for metal ion complexation. Computed values of pseudoformation constants (log ß') are correlated with experimental data and showed an excellent linear relationship (R2 = 0.97). The model was then used to estimate the absolute and relative formation constants of 23 different Zr4+ complexes using a total of 17 different ligands, including many of the alternative bifunctional chelates that have been reported recently for use in 89Zr4+ radiochemistry. In addition, detailed computational studies were performed on the geometric isomerism and hydration state of Zr-desferrioxamine. Collectively, the results offer new insights into Zr4+ coordination chemistry that will help guide the synthesis of future ligands. The computational model developed here is straightforward and reproducible and can be readily applied in the design of other metal coordination compounds.

Revisiting the Radiosynthesis of [18F]FPEB and Preliminary PET Imaging in a Mouse Model of Alzheimer's Disease.

[18F]FPEB is a positron emission tomography (PET) radiopharmaceutical used for imaging the abundance and distribution of mGluR5 in the central nervous system (CNS). Efficient radiolabeling of the aromatic ring of [18F]FPEB has been an ongoing challenge. Herein, five metal-free precursors for the radiofluorination of [18F]FPEB were compared, namely, a chloro-, nitro-, sulfonium salt, and two spirocyclic iodonium ylide (SCIDY) precursors bearing a cyclopentyl (SPI5) and a new adamantyl (SPIAd) auxiliary. The chloro- and nitro-precursors resulted in a low radiochemical yield (<10% RCY), whereas both SCIDY precursors and the sulfonium salt precursor produced [18F]FPEB in the highest RCYs of 25% and 36%, respectively. Preliminary PET/CT imaging studies with [18F]FPEB were conducted in a transgenic model of Alzheimer's Disease (AD) using B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J (APP/PS1) mice, and data were compared with age-matched wild-type (WT) B6C3F1/J control mice. In APP/PS1 mice, whole brain distribution at 5 min post-injection showed a slightly higher uptake (SUV = 4.8 ± 0.4) than in age-matched controls (SUV = 4.0 ± 0.2). Further studies to explore mGluR5 as an early biomarker for AD are underway.

Expanding the Chemistry Palette for Radiotracer Synthesis.

The synthesis, characterisation and application of radiolabelled compounds for use in diagnostic and therapeutic medicine requires a diverse skill set. This article highlights a selection of our ongoing projects that aim to provide new synthetic methods and radiochemical tools for building molecular imaging agents with various radionuclides.

Photoradiosynthesis of 68Ga-Labeled HBED-CC-Azepin-MetMAb for Immuno-PET of c-MET Receptors.

In an alternative approach for radiotracer design, a photoactivatable HBED-CC-PEG3-ArN3 chelate was synthesized and photoconjugated to the anti-c-MET antibody MetMAb (onartuzumab). Photoconjugation gave the functionalized protein HBED-CC-azepin-MetMAb with a photochemical conversion of 18.5 ± 0.5% ( n = 2) which was then radiolabeled with 68Ga3+ ions. The purified and formulated [68Ga]GaHBED-CC-azepin-MetMAb radiotracer was evaluated in vitro and in vivo. Standard stability tests and cellular binding assays confirmed that the radiotracer remained radiochemically pure and immunoreactive after photochemical conjugation. [68Ga]GaHBED-CC-azepin-MetMAb showed specific uptake in c-MET-positive MKN-45 (high-expression) and PC-3 (low/moderate expression) tumors with tumor-associated activities at 6 h post-administration of 10.33 ± 1.27 ( n = 5) and 3.88 ± 1.27 ( n = 3) %ID/g, respectively. In competitive blocking experiments, MKN-45 tumor uptake was reduced by approximately 55% ( P-value <0.001 compared with nonblocked experiments) confirming specific radiotracer binding to c-MET in vivo. Radiochemical, cellular, and in vivo experiments confirmed that the photoradiochemical approach is a viable tool to synthesize new radiotracers for immuno-PET.

Synthesis and Photochemical Studies on Gallium and Indium Complexes of DTPA-PEG3-ArN3 for Radiolabeling Antibodies.

Photochemistry is a rich source of inspiration for developing alternative methods to functionalize proteins with drug molecules, fluorophores, and radioactive probes. Here, we report the synthesis and photochemical reactivity of a modified diethylenediamine pentaacetic acid chelate that was derivatized with a light-responsive aryl azide group (DTPA-PEG3-ArN3, compound 1). The corresponding nonradioactive and radioactive nat/68Ga3+ and nat/111In3+ complexes of DTPA-PEG3-ArN3 were synthesized and their physical and photochemical properties were studied to evaluate the potential of employing this ligand system in the photochemical synthesis of radiolabeled antibodies. Photodegradation kinetics revealed that irradiation with ultraviolet light (365 nm) induced rapid photoactivation of compound 1 and the metal complexes nat/68Ga-1- and nat/111In-1-. Light-induced reactions were complete in <100 s, with measured first-order rate constants of 0.078 ± 0.045 s-1, 0.093 ± 0.009 s-1, and 0.117 ± 0.054 s-1 (n = 2, per species) for compound 1, natGa-1-, and natIn-1-, respectively. Photochemically induced bioconjugation reactions between DTPA-PEG3-ArN3 and the monoclonal antibody trastuzumab, as well as pre- and postconjugation 68Ga- and 111In-radiolabeling experiments, were performed using either a one-pot or two-step strategy. Both approaches yielded radiolabeled trastuzumab ([68Ga]GaDTPA-azepin-trastuzumab) with average radiochemical conversions of 3.9 ± 1.0% (n = 4, one-pot), and 10.0 ± 1.0% (n = 3, two-step). One-pot radiolabeling reactions with [111In]InCl3 produced the corresponding [111In]InDTPA-azepin-trastuzumab radiotracer in a similar radiochemical conversion of 5.4 ± 0.8% (n = 3). Radiochemical conversions for the desired bimolecular coupling between the chelate and the protein were comparatively low. This observation is likely caused by the high photoinduced reactivity of the compounds and subsequent competition with background reactions. Nevertheless, access to DTPA-PEG3-ArN3 increases the scope of photoradiochemical methods to include metal ions like In3+ that form complexes with higher coordination numbers.

endo-Hydroxamic Acid Monomers for the Assembly of a Suite of Non-native Dimeric Macrocyclic Siderophores Using Metal-Templated Synthesis.

An expedited synthesis of endo-hydroxamic acid aminocarboxylic acid (endo-HXA) compounds has been developed. These monomeric ligands are relevant to the synthesis of metal-macrocycle complexes using metal-templated synthesis (MTS), and the downstream production of apomacrocycles. Macrocycles can display useful drug properties and be used as ligands for radiometals in medical imaging applications, which supports methodological advances in accessing this class of molecule. Six endo-HXA ligands were prepared that contained methylene groups, ether atoms, or thioether atoms in different regions of the monomer (1-6). MTS using a 1:2 Fe(III)/ligand ratio furnished six dimeric hydroxamic acid macrocycles complexed with Fe(III) (1a-6a). The corresponding apomacrocycles (1b-6b) were produced upon treatment with diethylenetriaminepentaacetic acid (DTPA). Constitutional isomers of the apomacrocycles that contained one ether oxygen atom in the diamine-containing (2b) or dicarboxylic acid-containing (3b) region were well resolved by reverse-phase high-performance liquid chromatography (RP-HPLC). Density functional theory calculations were used to compute the structures and solvated molecular properties of 1b-6b and showed that the orientation of the amide bonds relative to the pseudo-C2 axis was close to parallel in 1b, 2b, and 4b-6b but tended toward perpendicular in 3b. This conformational constraint in 3b reduced the polarity compared with 2b, consistent with the experimental trend in polarity observed using RP-HPLC. The improved synthesis of endo-HXA ligands allows expanded structural diversity in MTS-derived macrocycles and the ability to modulate macrocycle properties.

Photochemical Conjugation and One-Pot Radiolabelling of Antibodies for Immuno-PET.

Monoclonal antibodies (mAbs), immunoglobulin fragments, and other proteins are important scaffolds in the development of radiopharmaceuticals for diagnostic immuno-positron emission tomography (immuno-PET) and targeted radioimmunotherapy (RIT). Conventional methods for radiolabelling proteins with metal ions such as 68 Ga, 64 Cu, 89 Zr, and 90 Y require multi-step procedures involving pre-purification, functionalisation with a chelate, and subsequent radiolabelling. Standard coupling chemistries are time-consuming, difficult to automate, and involve synthesis, isolation, and storage of an intermediate, new molecular entity (the conjugated mAb) whose biochemical properties can differ from those of the parent protein. To circumvent these issues, we developed a photoradiochemical approach that uses fast, chemoselective, light-induced protein modification under mild conditions with novel metal-ion-binding chelates derivatised with aryl azide (ArN3 ) groups. Experiments show that one-pot photochemical conjugation and radiolabelling of formulated mAbs can be achieved in <20 min.

Chemical Kinetics of Radiolabelling Reactions.

The application of chemical kinetics is one of the most powerful and versatile tools for investigating reaction mechanisms in complex mixtures. Kinetic studies are commonplace in traditional synthetic chemistry but are seldom used in radiopharmaceutical sciences. When deriving standard reaction rate laws, the focus is normally placed on calculating the chemical concentration of different species over time. In radiopharmaceutical synthesis, the desired product is one of the radioactive components of the mixture. Reaction conditions are optimised to obtain the radioactive product in the highest activity yield. When short-lived radionuclides are used, radioactive decay during the reaction window means that the maximum activity yield does not necessarily coincide with the chemical or decay-corrected radiochemical yields. To account for this difference in the kinetic models, it is shown how standard integrated rate laws can be modified to incorporate the contribution from radioactive decay. An example is then presented to show how radiochemical kinetics can be used to model complex systems, like [18 F]FDG radiosynthesis, that involve parallel or competing reactions at the different chemical scales of the radionuclide and substrate. Increased knowledge of reaction rates, and a more wide-spread application of radiochemical kinetics, can facilitate the development of new radiolabelling reactions. Accurate identification of maximum activity yields using kinetic models also has the potential to improve the optimisation and radiochemical efficiency of all current and future radiopharmaceutical syntheses.