23 research outputs found
Heptadentate chelates for 89Zr-radiolabelling of monoclonal antibodies
Herein, we report the synthesis of three new bifunctional heptadentate metal ion binding chelates derived from desferrioxamine B (DFO) linked to a tripeptide unit that comprises of a glutamic acid and two glycine residues. The three DFO derivatives were also functionalised with a photoactivatable aryl azide unit for light-triggered labelling of proteins. The chelates were obtained in 3 synthetic steps in good overall yields by using solid phase peptide synthesis (SPPS). Density Functional Theory (DFT) calculations were used to estimate thermodynamic formation constants (logâÎČ) of the corresponding Zr4+ complexes. Quantitative zirconium-89 radiolabelling (>95%) was obtained in <5 min at room temperature, and the stability of the radioconjugates toward different competitors (human serum, EDTA and Fe3+) was assessed in vitro. One-pot 89Zr-photoradiosynthesis produced [89Zr]Zr-2-onartuzumab directly from the formulated, clinical-grade sample MetMAbâą, without pre-purifying the monoclonal antibody (mAb) component, with an isolated decay-corrected radiochemical yield of 36.4 ± 2.4%. PET imaging and biodistribution studies were performed in female athymic nude mice bearing subcutaneous xenografts derived from the MKN-45 human gastric cancer cell line to assess the pharmacokinetic profile and tumour binding of [89Zr]Zr-2-onartuzumab. Specific tumour uptake of [89Zr]Zr-2-onartuzumab was confirmed by using competitive inhibition (blocking) studies and bone uptake was significantly reduced compared to the parent DFO analogue
Light-induced synthesis of protein conjugates and its application in photoradiosynthesis of 89Zr-radiolabeled monoclonal antibodies
Efficient methods to functionalize proteins are essential for the development of many diagnostic and therapeutic compounds, such as fluorescent probes for immunohistochemistry, zirconium-89 radiolabeled mAbs (89Zr-mAbs) for positron emission tomography and antibody-drug conjugates (ADCs). This protocol describes a step-by-step procedure for the light-induced functionalization of proteins with compounds bearing the photochemically active aryl azide group. As an illustration of the potential utility of our approach, this protocol focuses on the synthesis of 89Zr-mAbs using photoactivatable derivatives of the metal ion binding chelate desferrioxamine B (DFO). The light-induced synthesis of 89Zr-mAbs is a unique, one-pot process involving simultaneous radiolabeling and protein conjugation. The photoradiochemical synthesis of purified 89Zr-mAbs, starting from unmodified proteins, [89Zr][Zr(C2O4)4]4â (89Zr-oxalate), and a photoactivatable DFO derivative, can be performed in <90 min. The method can be easily adapted to prepare other radiolabeled proteins, ADCs or fluorescently tagged proteins by using drug molecules or fluorophores functionalized with photoactive moieties
Photoactivatable bis(thiosemicarbazone) derivatives for copper-64 radiotracer synthesis
In recent years, copper-64 and copper-67 have been considered as a useful theranostic pair in nuclear medicine, due to their favourable and complementary decay properties. As and are chemically identical, development of both existing and new bifunctional chelators for imaging agents can be readily adapted for the -radionuclide. In this study, we explored the use of photoactivatable copper chelators based on the asymmetric bis(thiosemicarbazone) scaffold, HATSM/en, for the photoradiolabelling of protein. Photoactivatable CuATSM-derivatives were prepared by both direct synthesis and transmetallation from the corresponding Zn complex. Then, irradiation with UV light in the presence of a protein of interest in a pH buffered aqueous solution afforded the -labelled protein conjugates in decay-corrected radiochemical yield of 86.9 ± 1.0% via the transmetallation method and 35.3 ± 1.7% from the direct radiolabelling method. This study successfully demonstrates the viability of photochemically induced conjugation methods for the development of copper-based radiotracers for potential applications in diagnostic positron emission tomography (PET) imaging and targeted radionuclide therapy
Photoactivatable Fluorescent Tags for Dual-Modality Positron Emission Tomography Optical Imaging
Fluorescent protein conjugates are vital tools in a wide range of scientific disciplines from basic biochemical research to applications in clinical pathology and intraoperative surgery. We report the synthesis and characterization of photoactivatable fluorophores (PhotoTags) based on the functionalization of coumarin, fluorescein, BODIPY, rhodamine B, and cyanine dyes with a photochemically active aryl azide group. Photochemical labeling experiments using human serum albumin produced fluorescent proteins in high yields under irradiation with ultraviolet light for <15 min. We also synthesized DFO-RhodB-PEG3-ArN3âa photoactivatable compound that can be radiolabeled with 89Zr for applications in optical imaging and positron emission tomography. One-pot 89Zr-radiolabeling and light-induced protein conjugation produced [89Zr]ZrDFO-RhodB-PEG3-azepin-trastuzumab. Proof-of-concept studies in vitro and in vivo confirmed that [89Zr]ZrDFO-RhodB-PEG3-azepin-trastuzumab is a potential dual-modality agent for detecting human epidermal growth factor receptor 2 (HER2/neu) expression. Overall, the PhotoTag technology represents a rapid, synthetically versatile, and user-friendly approach for generating novel protein conjugates
Charting the Chemical and Mechanistic Scope of Light-Triggered Protein Ligation
The creation of discrete, covalent bonds between a protein and a functional molecule like a drug, fluorophore, or radiolabeled complex is essential for making state-of-the-art tools that find applications in basic science and clinical medicine. Photochemistry offers a unique set of reactive groups that hold potential for the synthesis of protein conjugates. Previous studies have demonstrated that photoactivatable desferrioxamine B (DFO) derivatives featuring a para-substituted aryl azide () can be used to produce viable zirconium-89-radiolabeled monoclonal antibodies () for applications in noninvasive diagnostic positron emission tomography (PET) imaging of cancers. Here, we report on the synthesis, -radiochemistry, and light-triggered photoradiosynthesis of -labeled human serum albumin (HSA) using a series of 14 different photoactivatable DFO derivatives. The photoactive groups explore a range of substituted, and isomeric reagents, as well as derivatives of benzophenone, a para-substituted trifluoromethyl phenyl diazirine, and a tetrazole species. For the compounds studied, efficient photochemical activation occurs inside the UVA-to-visible region of the electromagnetic spectrum (âŒ365â450 nm) and the photochemical reactions with HSA in water were complete within 15 min under ambient conditions. Under standardized experimental conditions, photoradiosynthesis with compounds 1â14 produced the corresponding conjugates with decay-corrected isolated radiochemical yields between 18.1 ± 1.8% and 62.3 ± 3.6%. Extensive density functional theory (DFT) calculations were used to explore the reaction mechanisms and chemoselectivity of the light-induced bimolecular conjugation of compounds 1â14 to protein. The photoactivatable DFO-derivatives operate by at least five distinct mechanisms, each producing a different type of bioconjugate bond. Overall, the experimental and computational work presented here confirms that photochemistry is a viable option for making diverse, functionalized protein conjugates
Microfluidic preparation of89 zr-radiolabelled proteins by flow photochemistry
89 Zr-radiolabelled proteins functionalised with desferrioxamine B are a cornerstone of diagnostic positron emission tomography. In the clinical setting,89 Zr-labelled proteins are produced manually. Here, we explore the potential of using a microfluidic photochemical flow reactor to prepare89 Zr-radiolabelled proteins. The light-induced functionalisation and89 Zr-radiolabelling of human serum albumin ([89 Zr]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-based89 Zr-radiotracers, but further refinement of the apparatus and optimisation of the method are required before the flow process is competitive with manual reactions
Synthesis and coordination of triazacyclononanes and bifunctional derivatives : toward applications in medical imaging
Le 1,4,7-triazacyclononane (tacn) fait partie de la famille des polyazamacrocycles et a la facultĂ©, lorsquâil est N-fonctionnalisĂ© par des groupements coordinants adĂ©quats, de former des complexes de coordination stables thermodynamiquement et inertes cinĂ©tiquement avec les mĂ©taux de transition. Câest pourquoi les dĂ©rivĂ©s du tacn trouvent de nombreuses applications dans de nombreux domaines, par exemple celui de lâimagerie mĂ©dicale. Notre Ă©quipe a dĂ©veloppĂ© des ligands N-fonctionnalisĂ©s par des groupements picolinate et mĂ©thylpyridine pour des applications en imagerie TEP et IRM.Le premier travail de cette thĂšse porte sur lâĂ©laboration de nouvelles sondes bimodales TEP-imagerie optique et IRM-imagerie optique Ă motifs issus de tacn-picolinate dĂ©crits au laboratoire.Une seconde partie est axĂ©e sur la synthĂšse de nouveaux chĂ©lates du Cu(II) Ă base de tacn portant des bras mĂ©thylthiazole pour une potentielle application en imagerie TEP. Les complexes de Cu(II) correspondant ont Ă©tĂ© Ă©tudiĂ©s par diffĂ©rentes mĂ©thodes analytiques et spectroscopiques afin de juger leurs stabilitĂ©s thermodynamiques et leurs inerties cinĂ©tiques.Les chĂ©latants rĂ©pondant aux critĂšres imposĂ©s par le milieu biologique ont Ă©tĂ© sĂ©lectionnĂ©s pour une application en imagerie TEP dans le cadre du cancer de la prostate. Des analogues bifonctionnels ont ainsi Ă©tĂ© Ă©laborĂ©s afin de les incorporer dans des radiopharmaceutiques radiomarquĂ©s au cuivre-64.The 1,4,7-triazacyclononane (tacn) is part of the family of polyazamacrocycles, and has the faculty, when appropriately N-functionalized with coordination pendants, to form thermodynamically stable and kinetically inert coordination complexes with transition metals. Thanks to their properties tacn derivatives can be found in numerous applications such as medical imaging. Our group has developed some ligands N-functionalized with picolinate and picolyl pendants for an application in PET imaging and MRI.The first part of this thesis is focused on the development of a new class of bimodal PET-optical imaging and MRI-optical imaging probes based on tacn-picolinate units described in the laboratory.The second part deals with the synthesis of new Cu(II) chelators based on tacn-methylthiazolyl units for a potential application in PET imaging. The corresponding Cu(II) complexes were studied by different analytical and spectroscopic methods in order to evaluate their thermodynamic stabilities and kinetic inertness.The chelators that fullfiled all the criterias imposed by the biological media were selected for an applicationPET imaging of prostate cancer. Some bifunctional analogues were elaborated so as to incorporate them in radiopharmaceuticals radiolabeled with copper-64
SynthÚse et coordination de triazacyclononanes et de dérivés bifonctionnels : vers des applications en imageries médicales
The 1,4,7-triazacyclononane (tacn) is part of the family of polyazamacrocycles, and has the faculty, when appropriately N-functionalized with coordination pendants, to form thermodynamically stable and kinetically inert coordination complexes with transition metals. Thanks to their properties tacn derivatives can be found in numerous applications such as medical imaging. Our group has developed some ligands N-functionalized with picolinate and picolyl pendants for an application in PET imaging and MRI.The first part of this thesis is focused on the development of a new class of bimodal PET-optical imaging and MRI-optical imaging probes based on tacn-picolinate units described in the laboratory.The second part deals with the synthesis of new Cu(II) chelators based on tacn-methylthiazolyl units for a potential application in PET imaging. The corresponding Cu(II) complexes were studied by different analytical and spectroscopic methods in order to evaluate their thermodynamic stabilities and kinetic inertness.The chelators that fullfiled all the criterias imposed by the biological media were selected for an applicationPET imaging of prostate cancer. Some bifunctional analogues were elaborated so as to incorporate them in radiopharmaceuticals radiolabeled with copper-64.Le 1,4,7-triazacyclononane (tacn) fait partie de la famille des polyazamacrocycles et a la facultĂ©, lorsquâil est N-fonctionnalisĂ© par des groupements coordinants adĂ©quats, de former des complexes de coordination stables thermodynamiquement et inertes cinĂ©tiquement avec les mĂ©taux de transition. Câest pourquoi les dĂ©rivĂ©s du tacn trouvent de nombreuses applications dans de nombreux domaines, par exemple celui de lâimagerie mĂ©dicale. Notre Ă©quipe a dĂ©veloppĂ© des ligands N-fonctionnalisĂ©s par des groupements picolinate et mĂ©thylpyridine pour des applications en imagerie TEP et IRM.Le premier travail de cette thĂšse porte sur lâĂ©laboration de nouvelles sondes bimodales TEP-imagerie optique et IRM-imagerie optique Ă motifs issus de tacn-picolinate dĂ©crits au laboratoire.Une seconde partie est axĂ©e sur la synthĂšse de nouveaux chĂ©lates du Cu(II) Ă base de tacn portant des bras mĂ©thylthiazole pour une potentielle application en imagerie TEP. Les complexes de Cu(II) correspondant ont Ă©tĂ© Ă©tudiĂ©s par diffĂ©rentes mĂ©thodes analytiques et spectroscopiques afin de juger leurs stabilitĂ©s thermodynamiques et leurs inerties cinĂ©tiques.Les chĂ©latants rĂ©pondant aux critĂšres imposĂ©s par le milieu biologique ont Ă©tĂ© sĂ©lectionnĂ©s pour une application en imagerie TEP dans le cadre du cancer de la prostate. Des analogues bifonctionnels ont ainsi Ă©tĂ© Ă©laborĂ©s afin de les incorporer dans des radiopharmaceutiques radiomarquĂ©s au cuivre-64
Light-activated protein-conjugation and 89Zr-radiolabelling with water-soluble desferrioxamine derivatives
Proteinâconjugates 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 89Zr are used as radiopharmaceuticals for diagnostic positron emission tomography (PET). In this context, protein functionalisation requires the formation of a covalent bond which must be achieved without compromising the biological properties of the mAb. Photochemistry offers new synthetic routes toward proteinâconjugates like 89ZrâmAbs but to harness the potential of lightâinduced conjugation reactions new photoactivatable reagents are required. Here, we introduce two photoactivatable DFOâderivatives functionalised with an aryl azide (ArN3) for use in lightâactivated conjugation and radiosynthesis of 89ZrâmAbs. Incorporation of a trisâpolyethylene glycol (PEG)3 linker between DFO and the ArN3 group furnished waterâsoluble chelates that were used in the oneâpot, photoradiosynthesis of different 89Zrâradiolabelled proteinâconjugates with radiochemical yields up to 72.9±1.9%. Notably, the DFOâPEG3 chelates can be readily synthesised in accordance with Good Laboratory Practice (GLP), which will facilitate clinical trials with photoradiolabelled 89ZrâmAbs
Photochemical Reactions in the Synthesis of ProteinâDrug 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 preâassociation (nonâcovalent ligandâprotein 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