Enzyme‐Mediated Site‐Specific Bioconjugation of Metal Complexes to Proteins: Sortase‐Mediated Coupling of Copper‐64 to a Single‐Chain Antibody

The enzyme‐mediated site‐specific bioconjugation of a radioactive metal complex to a single‐chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single‐chain variable fragment. The antibody fragment scFv_anti‐LIBS targets ligand‐induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron‐emitting isotope ⁶⁴Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site‐specific enzyme‐mediated conjugation and should be broadly applicable to other metal complexes and proteins.     

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 ⁸⁹Zr, ⁶⁴Cu and ⁶⁸Ga radionuclides. DFO-tetrazole ( 1 ) was assessed by direct conjugation to formulated trastuzumab and subsequent radiolabelling with ⁸⁹Zr. 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.     

A homogeneous high-DAR antibody–drug conjugate platform combining THIOMAB antibodies and XTEN polypeptides

The antibody–drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above ∼4 typically destroys the biophysical properties and in vivo efficacy for ADCs. Herein, we describe the development of a novel bioconjugation platform combining cysteine-engineered (THIOMAB) antibodies and recombinant XTEN polypeptides for the unprecedented generation of homogeneous, stable “TXCs” with DAR of up to 18. Across three different bioactive payloads, we demonstrated improved AMD to tumors and Staphylococcus aureus bacteria for high-DAR TXCs relative to conventional low-DAR ADCs.

Efficiency of targeted cell delivery of small molecules was enhanced in cells and animals via a novel well-defined bioconjugation platform combining site-specific antibody conjugation and XTEN polypeptides to enable high payload loading.     

A novel tyrosine hyperoxidation enables selective peptide cleavage

A novel tyrosine hyperoxidation enabling selective peptide cleavage is reported. The scission of the N-terminal amide bond of tyrosine was achieved with Dess–Martin periodinane under mild conditions, generating a C-terminal peptide fragment bearing the unprecedented hyperoxidized tyrosine motif, 4,5,6,7-tetraoxo-1H-indole-2-carboxamide, along with an intact N-terminal peptide fragment. This reaction proceeds with high site-selectivity for tyrosine and exhibits broad substrate scope for various peptides, including those containing post-translational modifications. More importantly, this oxidative cleavage was successfully applied to enable sequencing of three naturally occurring cyclic peptides, including one depsipeptide and one lipopeptide. The linearized peptides generated from the cleavage reaction significantly simplify cyclic peptide sequencing by MS/MS, thus providing a robust tool to facilitate rapid sequence determination of diverse cyclic peptides containing tyrosine. Furthermore, the highly electrophilic nature of the hyperoxidized tyrosine unit disclosed in this work renders it an important electrophilic target for the selective bioconjugation or synthetic manipulation of peptides containing this unit.

A Tyr-selective peptide cleavage was reported using Dess–Martin periodinane. The cleavage generates an unprecedented hyperoxidized tyrosine motif in the C-terminal fragment and showed excellent site-specificity and broad scope for various peptides.     

[89Zr]-Pertuzumab PET Imaging Reveals Paclitaxel Treatment Efficacy Is Positively Correlated with HER2 Expression in Human Breast Cancer Xenograft Mouse Models

Paclitaxel (PTX) treatment efficacy varies in breast cancer, yet the underlying mechanism for variable response remains unclear. This study evaluates whether human epidermal growth factor receptor 2 (HER2) expression level utilizing advanced molecular positron emission tomography (PET) imaging is correlated with PTX treatment efficacy in preclinical mouse models of HER2+ breast cancer. HER2 positive (BT474, MDA-MB-361), or HER2 negative (MDA-MB-231) breast cancer cells were subcutaneously injected into athymic nude mice and PTX (15 mg/kg) was administrated. In vivo HER2 expression was quantified through [⁸⁹Zr]-pertuzumab PET/CT imaging. PTX treatment response was quantified by [¹⁸F]-fluorodeoxyglucose ([¹⁸F]-FDG) PET/CT imaging. Spearman’s correlation, Kendall’s tau, Kolmogorov–Smirnov test, and ANOVA were used for statistical analysis. [⁸⁹Zr]-pertuzumab mean standard uptake values (SUV_mean) of BT474 tumors were 4.9 ± 1.5, MDA-MB-361 tumors were 1.4 ± 0.2, and MDA-MB-231 (HER2−) tumors were 1.1 ± 0.4. [¹⁸F]-FDG SUV_mean changes were negatively correlated with [⁸⁹Zr]-pertuzumab SUV_mean (r = −0.5887, p = 0.0030). The baseline [¹⁸F]-FDG SUV_mean was negatively correlated with initial [⁸⁹Zr]-pertuzumab SUV_mean (r = −0.6852, p = 0.0002). This study shows PTX treatment efficacy is positively correlated with HER2 expression level in human breast cancer mouse models. Molecular imaging provides a non-invasive approach to quantify biological interactions, which will help in identifying chemotherapy responders and potentially enhance clinical decision-making.     

Chemotopological study of positron emitter radionuclides used in PET diagnostic imaging: physical, physico-chemical, dosimetric, quantum and nuclear properties

We carried out a chemotopological study of two groups of positron emitter radionuclides of current and potential use in positron emission tomography (PET) diagnostic imaging. The aim was to look for potential β⁺ radionuclides not yet in use for PET imaging, taking into account the similarities of these radionuclides with radionuclides already used in PET imaging. The similarity was studied through physical, physico-chemical, dosimetric, quantum and nuclear properties of the radionuclides. We applied cluster analysis (two similarity functions and three grouping methodologies) and generated six dendrograms. One dendrogram was selected in each group, which was used to build up a basis for a topology. From the calculation of topological properties of several subsets of interest, we propose β⁺ radionuclides ⁴⁷V, ⁴⁸V, ⁶³Zn, ⁷⁰As, ⁹⁰Nb, ¹⁰⁶Ag, ¹¹⁵Sb, ¹¹⁶Sb, ¹²⁰Sb, ¹³⁰Cs, ¹³⁴La and ¹⁴⁰Pr, as potential new radionuclides of use in PET imaging.     

中枢多巴胺系统正电子发射计算机断层扫描显像剂的研究进展

中枢多巴胺系统与多种神经行为障碍的病理生理学有关。一直以来,多巴胺系统正电子发射计算机断层扫描(PET)成像在研究活体大脑中多巴胺生物化学过程上有着重要价值。PET成像的基础是¹¹C、¹⁸F等发射正电子的放射性核素标记的显像剂,这些显像剂通过与多巴胺神经系统不同的靶点特异性结合从而反映多巴胺合成、囊泡储存、突触释放和受体结合以及再摄取过程,推动神经病学、精神病学、药物滥用和成瘾以及药物开发的研究进展。本文综述了以氨基酸脱羧酶、多巴胺转运体、多巴胺受体以及囊泡单胺转运体为靶点的¹¹C、¹⁸F标记的PET显像剂的研究进展。     

New Synthesis of 2-Deoxy-2-fluoro-D-hexoses by Fluorination in Water

Interest in fluorinated sugars labelled with {¹⁸F} positron-emitting radionuclides as tracers for the measurement of glucose utilization in man by positron emission tomography¹ (PET) and in animals by autoradiography² has resulted in the development of numerous syntheses^3-11 of 2-deoxy-2-fluoro-D-glucose (3a). Some of these syntheses are not practical because the carbohydrate substrates for the fluorination reactions are not readily available.     

Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography

Positron emission tomography (PET) is a powerful and rapidly developing area of molecular imaging that is used to study and visualize human physiology by the detection of positron-emitting radiopharmaceuticals. Information about metabolism, receptor/enzyme function, and biochemical mechanisms in living tissue can be obtained directly from PET experiments. Unlike magnetic resonance imaging (MRI) or computerized tomography (CT), which mainly provide detailed anatomical images, PET can measure chemical changes that occur before macroscopic anatomical signs of a disease are observed. PET is emerging as a revolutionary method for measuring body function and tailoring disease treatment in living subjects. The development of synthetic strategies for the synthesis of new positron-emitting molecules is, however, not trivial. This Review highlights key aspects of the synthesis of PET radiotracers with the short-lived positron-emitting radionuclides (11)C, (18)F, (15)O, and (13)N, with emphasis on the most recent strategies.     

中枢多巴胺系统正电子发射计算机断层扫描显像剂的研究进展

中枢多巴胺系统与多种神经行为障碍的病理生理学有关。一直以来,多巴胺系统正电子发射计算机断层扫描(PET)成像在研究活体大脑中多巴胺生物化学过程上有着重要价值。PET成像的基础是¹¹C、¹⁸F等发射正电子的放射性核素标记的显像剂,这些显像剂通过与多巴胺神经系统不同的靶点特异性结合从而反映多巴胺合成、囊泡储存、突触释放和受体结合以及再摄取过程,推动神经病学、精神病学、药物滥用和成瘾以及药物开发的研究进展。本文综述了以氨基酸脱羧酶、多巴胺转运体、多巴胺受体以及囊泡单胺转运体为靶点的¹¹C、¹⁸F标记的PET显像剂的研究进展。     

Chelate-free metal ion binding and heat-induced radiolabeling of iron oxide nanoparticles

A novel reaction for chelate-free, heat-induced metal ion binding and radiolabeling of ultra-small paramagnetic iron oxide nanoparticles (USPIOs) has been established. Radiochemical and non-radioactive labeling studies demonstrated that the reaction has a wide chemical scope and is applicable to p-, d- and f-block metal ions with varying ionic sizes and formal oxidation states from 2+ to 4+. Radiolabeling studies found that ⁸⁹Zr–Feraheme (⁸⁹Zr–FH or ⁸⁹Zr–ferumoxytol) can be isolated in 93 ± 3% radiochemical yield (RCY) and >98% radiochemical purity using size-exclusion chromatography. ⁸⁹Zr–FH was found to be thermodynamically and kinetically stable in vitro using a series of ligand challenge and plasma stability tests, and in vivo using PET/CT imaging and biodistribution studies in mice. Remarkably, ICP-MS and radiochemistry experiments showed that the same reaction conditions used to produce ⁸⁹Zr–FH can be employed with different radionuclides to yield ⁶⁴Cu–FH (66 ± 6% RCY) and ¹¹¹In–FH (91 ± 2% RCY). Electron magnetic resonance studies support a mechanism of binding involving metal ion association with the surface of the magnetite crystal core. Collectively, these data suggest that chelate-free labeling methods can be employed to facilitate clinical translation of a new class of multimodality PET/MRI radiotracers derived from metal-based nanoparticles. Further, this discovery is likely to have broader implications in drug delivery, metal separation science, ecotoxicology of nanoparticles and beyond.     

Low power threshold photochemical upconversion using a zirconium(iv) LMCT photosensitizer

The current investigation demonstrates highly efficient photochemical upconversion (UC) where a long-lived Zr(iv) ligand-to-metal charge transfer (LMCT) complex serves as a triplet photosensitizer in concert with well-established 9,10-diphenylanthracene (DPA) along with newly conceived DPA–carbazole based acceptors/annihilators in THF solutions. The initial dynamic triplet–triplet energy transfer (TTET) processes (ΔG ∼ −0.19 eV) featured very large Stern–Volmer quenching constants (K_SV) approaching or achieving 10⁵ M⁻¹ with bimolecular rate constants between 2 and 3 × 10⁸ M⁻¹ s⁻¹ as ascertained using static and transient spectroscopic techniques. Both the TTET and subsequent triplet–triplet annihilation (TTA) processes were verified and throughly investigated using transient absorption spectroscopy. The Stern–Volmer metrics support 95% quenching of the Zr(iv) photosensitizer using modest concentrations (0.25 mM) of the various acceptor/annihilators, where no aggregation took place between any of the chromophores in THF. Each of the upconverting formulations operated with continuous-wave linear incident power dependence (λ_ex = 514.5 nm) down to ultralow excitation power densities under optimized experimental conditions. Impressive record-setting η_UC values ranging from 31.7% to 42.7% were achieved under excitation conditions (13 mW cm⁻²) below that of solar flux integrated across the Zr(iv) photosensitizer''s absorption band (26.7 mW cm⁻²). This study illustrates the importance of supporting the continued development and discovery of molecular-based triplet photosensitizers based on earth-abundant metals.

The LMCT photosensitizer Zr(^MesPDP^Ph)₂ paired with DPA-based acceptors enabled low power threshold photochemical upconversion with record-setting quantum efficiencies.     

Biomedical radioisotope generator systems

Although the⁹⁹Mo^99mTc radionuclide generator system has evolved as the workhorse of nuclear medicine over the past three decades, recent developments in radionuclide generator technology have provided positron emitters for PET studies, radionuclides for therapy, and ultra short-lived radionuclides for repeated clinical applications. Attention is being paid to development ofin vivo generator systems and potential use of tandem generator systems. Separation of radionuclides in a generator system exploits differences in chemical properties of the parent-daughter pair; information is presented for these generator types based on the three chemical categories of chromatography, extraction, and volatilization and sublimation methods. This review focuses on chromatography-based systems.     

Metal-chelating benzothiazole multifunctional compounds for the modulation and 64Cu PET imaging of Aβ aggregation

While Alzheimer''s Disease (AD) is the most common neurodegenerative disease, there is still a dearth of efficient therapeutic and diagnostic agents for this disorder. Reported herein are a series of new multifunctional compounds (MFCs) with appreciable affinity for amyloid aggregates that can be potentially used for both the modulation of Aβ aggregation and its toxicity, as well as positron emission tomography (PET) imaging of Aβ aggregates. Firstly, among the six compounds tested HYR-16 is shown to be capable to reroute the toxic Cu-mediated Aβ oligomerization into the formation of less toxic amyloid fibrils. In addition, HYR-16 can also alleviate the formation of reactive oxygen species (ROS) caused by Cu²⁺ ions through Fenton-like reactions. Secondly, these MFCs can be easily converted to PET imaging agents by pre-chelation with the ⁶⁴Cu radioisotope, and the Cu complexes of HYR-4 and HYR-17 exhibit good fluorescent staining and radiolabeling of amyloid plaques both in vitro and ex vivo. Importantly, the ⁶⁴Cu-labeled HYR-17 is shown to have a significant brain uptake of up to 0.99 ± 0.04 %ID per g. Overall, by evaluating the various properties of these MFCs valuable structure–activity relationships were obtained that should aid the design of improved therapeutic and diagnostic agents for AD.

A series of multifunctional compounds and their ⁶⁴Cu complexes exhibit good affinity for Aβ aggregates and can also control Aβ toxicity.     

Heat‐Induced Radiolabeling of Nanoparticles for Monocyte Tracking by PET

Heat‐induced radiolabeling (HIR) yielded ⁸⁹Zr‐Feraheme (FH) nanoparticles (NPs) that were used to determine NP pharmacokinetics (PK) by positron emission tomography (PET). Standard uptake values indicated a fast hepatic uptake that corresponded to blood clearance, and a second, slow uptake process by lymph nodes and spleen. By cytometry, NPs were internalized by circulating monocytes and monocytes in vitro. Using an IV injection of HIR ⁸⁹Zr‐FH (rather than in vitro cell labeling), PET/PK provided a view of monocyte trafficking, a key component of the immune response.     

An experimental approach probing the conformational transitions and energy landscape of antibodies: a glimmer of hope for reviving lost therapeutic candidates using ionic liquid

Understanding protein folding in different environmental conditions is fundamentally important for predicting protein structures and developing innovative antibody formulations. While the thermodynamics and kinetics of folding and unfolding have been extensively studied by computational methods, experimental methods for determining antibody conformational transition pathways are lacking. Motivated to fill this gap, we prepared a series of unique formulations containing a high concentration of a chimeric immunoglobin G4 (IgG4) antibody with different excipients in the presence and absence of the ionic liquid (IL) choline dihydrogen phosphate. We determined the effects of different excipients and IL on protein thermal and structural stability by performing variable temperature circular dichroism and bio-layer interferometry analyses. To further rationalise the observations of conformational changes with temperature, we carried out molecular dynamics simulations on a single antibody binding fragment from IgG4 in the different formulations, at low and high temperatures. We developed a methodology to study the conformational transitions and associated thermodynamics of biomolecules, and we showed IL-induced conformational transitions. We showed that the increased propensity for conformational change was driven by preferential binding of the dihydrogen phosphate anion to the antibody fragment. Finally, we found that a formulation containing IL with sugar, amino acids and surfactant is a promising candidate for stabilising proteins against conformational destabilisation and aggregation. We hope that ultimately, we can help in the quest to understand the molecular basis of the stability of antibodies and protein misfolding phenomena and offer new candidate formulations with the potential to revive lost therapeutic candidates.

Probing the energy landscape and thermodynamics of biomolecules for drug design.     

Umpolung strategies for the functionalization of peptides and proteins

Umpolung strategies, defined as synthetic approaches which reverse commonly accepted reactivity patterns, are broadly recognized as enabling tools for small molecule synthesis and catalysis. However, methods which exploit this logic for peptide and protein functionalizations are comparatively rare, with the overwhelming majority of existing bioconjugation approaches relying on the well-established reactivity profiles of a handful of amino acids. This perspective serves to highlight a small but growing body of recent work that masterfully capitalizes on the concept of polarity reversal for the selective modification of proteinogenic functionalities. Current applications of umpolung chemistry in organic synthesis and chemical biology as well as the vast potential for further innovations in peptide and protein modification will be discussed.

This perspective highlights the growing body of literature that leverages polarity reversal (umpolung reactivity) for the selective modification of proteinogenic functionalities and identifies opportunities for further innovation.     

A Semi Rigid Novel Hydroxamate AMPED-Based Ligand for 89Zr PET Imaging

In this work, we designed, developed, characterized, and investigated a new chelator and its bifunctional derivative for ⁸⁹Zr labeling and PET-imaging. In a preliminary study, we synthesized two hexadentate chelators named AAZTHAS and AAZTHAG, based on the seven-membered heterocycle AMPED (6-amino-6-methylperhydro-1,4-diazepine) with the aim to increase the rigidity of the ⁸⁹Zr complex by using N-methyl-N-(hydroxy)succinamide or N-methyl-N-(hydroxy)glutaramide pendant arms attached to the cyclic structure. N-methylhydroxamate groups are the donor groups chosen to efficiently coordinate ⁸⁹Zr. After in vitro stability tests, we selected the chelator with longer arms, AAZTHAG, as the best complexing agent for ⁸⁹Zr presenting a stability of 86.4 ± 5.5% in human serum (HS) for at least 72 h. Small animal PET/CT static scans acquired at different time points (up to 24 h) and ex vivo organ distribution studies were then carried out in healthy nude mice (n = 3) to investigate the stability and biodistribution in vivo of this new ⁸⁹Zr-based complex. High stability in vivo, with low accumulation of free ⁸⁹Zr in bones and kidneys, was measured. Furthermore, an activated ester functionalized version of AAZTHAG was synthesized to allow the conjugation with biomolecules such as antibodies. The bifunctional chelator was then conjugated to the human anti-HER2 monoclonal antibody Trastuzumab (Tz) as a proof of principle test of conjugation to biologically active molecules. The final ⁸⁹Zr labeled compound was characterized via radio-HPLC and SDS-PAGE followed by autoradiography, and its stability in different solutions was assessed for at least 4 days.     

15N-Azides as practical and effective tags for developing long-lived hyperpolarized agents

Azide moieties, unique linear species containing three nitrogen atoms, represent an attractive class of molecular tag for hyperpolarized magnetic resonance imaging (HP-MRI). Here we demonstrate (¹⁵N)₃-azide-containing molecules exhibit long-lasting hyperpolarization lifetimes up to 9.8 min at 1 T with remarkably high polarization levels up to 11.6% in water, thus establishing (¹⁵N)₃-azide as a powerful spin storage for hyperpolarization. A single (¹⁵N)-labeled azide has also been examined as an effective alternative tag with long-lived hyperpolarization. A variety of biologically important molecules are studied in this work, including choline, glucose, amino acid, and drug derivatives, demonstrating great potential of ¹⁵N-labeled azides as universal hyperpolarized tags for nuclear magnetic resonance imaging applications.

This work demonstrates that ¹⁵N-labeled azides are practical and effective tags for developing long-lived hyperpolarized MRI agents and can offer hyperpolarization lifetimes up to 9.8 min at 1 T and high polarization levels up to 11.6% in water.     

Flash chemistry enables high productivity metalation-substitution of 5-alkyltetrazoles

Tetrazoles play a prominent role in medicinal chemistry due to their role as carboxylate bioisosteres but have largely been overlooked as C–H functionalisation substrates. We herein report the development of a high-yielding and general procedure for the heterobenzylic C–H functionalisation of 5-alkyltetrazoles in up to 97% yield under batch conditions using a metalation/electrophilic trapping strategy. Through the use of thermal imaging to identify potentially unsafe exotherms, a continuous flow procedure using a flash chemistry strategy has also been developed, allowing products to be accessed in up to 95% yield. This enabled an extremely high productivity rate of 141 g h⁻¹ to be achieved on an entry-level flow system.

We report a α-metalation-substitution of readily deprotected 5-alkyltetrazoles under batch and continuous flow conditions. In flow, thermal imaging enabled identification of an unsafe exotherm and optimisation of a productivity rate of 141 g h⁻¹.