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The tetrazine/trans-cyclooctene ligation stands out from the bioorthogonal toolbox due to its exceptional reaction kinetics, enabling multiple molecular technologies in vitro and in living systems. Highly reactive 2-pyridyl-substituted tetrazines have become state of the art for time-critical processes and selective reactions at very low concentrations. It is widely accepted that the enhanced reactivity of these chemical tools is attributed to the electron-withdrawing effect of the heteroaryl substituent. In contrast, we show that the observed reaction rates are way too high to be explained on this basis. Computational investigation of this phenomenon revealed that distortion of the tetrazine caused by intramolecular repulsive N-N interaction plays a key role in accelerating the cycloaddition step. We show that the limited stability of tetrazines in biological media strongly correlates with the electron-withdrawing effect of the substituent, while intramolecular repulsion increases the reactivity without reducing the stability. These fundamental insights reveal thus far overlooked mechanistic aspects that govern the reactivity/stability trade-off for tetrazines in physiologically relevant environments, thereby providing a new strategy that may facilitate the rational design of these bioorthogonal tools.
Assuntos
Compostos Heterocíclicos , Reação de Cicloadição , Elétrons , Compostos Heterocíclicos/química , CinéticaRESUMO
Bioorthogonal chemistry is bridging the divide between static chemical connectivity and the dynamic physiologic regulation of molecular state, enabling in situ transformations that drive multiple technologies. In spite of maturing mechanistic understanding and new bioorthogonal bond-cleavage reactions, the broader goal of molecular ON/OFF control has been limited by the inability of existing systems to achieve both fast (i.e., seconds to minutes, not hours) and complete (i.e., >99%) cleavage. To attain the stringent performance characteristics needed for high fidelity molecular inactivation, we have designed and synthesized a new C2-symmetric trans-cyclooctene linker (C2TCO) that exhibits excellent biological stability and can be rapidly and completely cleaved with functionalized alkyl-, aryl-, and H-tetrazines, irrespective of click orientation. By incorporation of C2TCO into fluorescent molecular probes, we demonstrate highly efficient extracellular and intracellular bioorthogonal disassembly via omnidirectional tetrazine-triggered cleavage.
Assuntos
Ciclo-Octanos/química , Sondas Moleculares/química , Anticorpos/química , Anticorpos/metabolismo , Carbono/química , Química Click , Corantes Fluorescentes/química , IsomerismoRESUMO
Radiotheranostics are designed by labeling targeting (bio)molecules with radionuclides for diagnostic or therapeutic application. Because the pharmacokinetics of therapeutic compounds play a pivotal role, chemically closely related imaging agents are used to evaluate the overall feasibility of the therapeutic approach. "Theranostic relatives" that utilize different elements are frequently used in clinical practice. However, variations in pharmacokinetics, biodistribution, and target affinity due to different chemical properties of the radioisotopes remain as hurdles to the design of optimized clinical tools. Herein, the design and synthesis of structurally identical compounds, either for diagnostic (18 F and a stable metal isotope) or therapeutic application (radiometal and stable 19 F), are reported. Such "molecular twins" have been prepared by applying a modular strategy based on click chemistry that enables efficient radiolabeling of compounds containing a metal complex and a tetrazine moiety. This additional bioorthogonal functionality can be used for subsequent radiolabeling of (bio)molecules or pretargeting approaches, which is demonstrated in vitro.
Assuntos
Química Click/métodos , Marcação por Isótopo/métodos , Compostos Radiofarmacêuticos , Nanomedicina Teranóstica/métodos , Humanos , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Distribuição TecidualRESUMO
In recent years, radiofluorinated alkyl azides have been reported for click radiolabeling and pretargeted PET imaging, but only little is known about the biodistribution and metabolism of these compounds. In this work, we present a significantly improved procedure for the synthesis of [18F]fluoroethyl azide and reinvestigated this radiolabeled probe in detail showing poor stability and very restricted suitability for in vivo application. Therefore, modified low-molecular-weight [18F]fluoroalkyl azides were developed. Propargyl-tagged endomorphin-1 (as model compound) was successfully radiolabeled in high yield and short reaction time making these probes useful and efficient bioorthogonal tools for rapid radiolabeling. Biodistribution, pharmacokinetics and in vivo stability were studied by preclinical PET/MR scanning and metabolite analysis. The results of this study revealed only limited applicability of [18F]fluoroalkyl azides for in vivo application.
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The crystallization of terbium 5,5'-azobis[1H-tetrazol-1-ide] (ZT) in the presence of trace amounts (ca. 50â Bq, ca. 1.6â pmol) of americium results in 1)â the accumulation of the americium tracer in the crystalline solid and 2)â a material that adopts a different crystal structure to that formed in the absence of americium. Americium-doped [Tb(Am)(H2 O)7 ZT]2 ZTâ 10 H2 O is isostructural to light lanthanide (Ce-Gd) 5,5'-azobis[1H-tetrazol-1-ide] compounds, rather than to the heavy lanthanide (Tb-Lu) 5,5'-azobis[1H-tetrazol-1-ide] (e.g., [Tb(H2 O)8 ]2 ZT3 â 6 H2 O) derivatives. Traces of Am seem to force the Tb compound into a structure normally preferred by the lighter lanthanides, despite a 108 -fold Tb excess. The americium-doped material was studied by single-crystal X-ray diffraction, vibrational spectroscopy, radiochemical neutron activation analysis, and scanning electron microcopy. In addition, the inclusion properties of terbium 5,5'-azobis[1H-tetrazol-1-ide] towards americium were quantified, and a model for the crystallization process is proposed.
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The ability to observe cells in live organisms is essential for understanding their function in complex in vivo milieus. A major challenge today has been the limited ability to perform higher multiplexing beyond four to six colors to define cell subtypes in vivo. Here, a click chemistry-based strategy is presented for higher multiplexed in vivo imaging in mouse models. The method uses a scission-accelerated fluorophore exchange (SAFE), which exploits a highly efficient bioorthogonal mechanism to completely remove fluorescent signal from antibody-labeled cells in vivo. It is shown that the SAFE-intravital microscopy imaging method allows 1) in vivo staining of specific cell types in dorsal and cranial window chambers of mice, 2) complete un-staining in minutes, 3) in vivo click chemistries at lower (µm) and thus non-toxic concentrations, and 4) the ability to perform in vivo cyclic imaging. The potential utility of the method is demonstrated by 12 color imaging of immune cells in live mice.
Assuntos
Química Click , Corantes Fluorescentes , Animais , Anticorpos , Química Click/métodos , Corantes Fluorescentes/química , Microscopia Intravital , Camundongos , Coloração e RotulagemRESUMO
Cells in complex organisms undergo frequent functional changes, but few methods allow comprehensive longitudinal profiling of living cells. Here we introduce scission-accelerated fluorophore exchange (SAFE), a method for multiplexed temporospatial imaging of living cells with immunofluorescence. SAFE uses a rapid bioorthogonal click chemistry to remove immunofluorescent signals from the surface of labeled cells, cycling the nanomolar-concentration reagents in seconds and enabling multiple rounds of staining of the same samples. It is non-toxic and functional in both dispersed cells and intact living tissues. We demonstrate multiparameter (n ≥ 14), non-disruptive imaging of murine peripheral blood mononuclear and bone marrow cells to profile cellular differentiation. We also show longitudinal multiplexed imaging of bone marrow progenitor cells as they develop into neutrophils over 6 days and real-time multiplexed cycling of living mouse hepatic tissues. We anticipate that SAFE will find broad utility for investigating physiologic dynamics in living systems.
Assuntos
Corantes Fluorescentes , Leucócitos Mononucleares , Camundongos , Animais , Corantes Fluorescentes/química , Coloração e Rotulagem , Imagem Óptica/métodos , ImunofluorescênciaRESUMO
The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of pretargeted in vivo chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody-drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile in vivo chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the in vivo ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the in vivo fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted trans-cyclooctene (TCO)-modified antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their in vivo performance. In particular, high rate constants (>50â¯000 M-1 s-1) for the reaction with TCO and low calculated logD 7.4 values (below -3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected 18F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for in vivo application and will thereby assist the clinical translation of bioorthogonal pretargeting.
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In the past decade, several developments have expanded the chemical toolbox for astatination and the preparation of 211At-labeled radiopharmaceuticals. However, there is still a need for advanced methods for the synthesis of astatinated (bio)molecules to address challenges such as limited inâ vivo stability. Herein, we report the development of multifunctional 211At-labeled reagents that can be prepared by applying a modular and versatile click approach for rapid assembly. The introduction of tetrazines as bioorthogonal tags enables rapid radiolabeling and radio-crosslinking, which is demonstrated by steric shielding of 211At to significantly increase label stability in human blood plasma.
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Química Click/métodos , Compostos Radiofarmacêuticos/química , Astato/química , Meia-Vida , Compostos Heterocíclicos com 1 Anel/química , Humanos , Marcação por Isótopo , Compostos Radiofarmacêuticos/sangue , Compostos Radiofarmacêuticos/síntese químicaRESUMO
Invited for this month's cover is the group of Dr. Hannes Mikula at Vienna University of Technology (TU Wien), Austria. The cover picture shows immobilized astatine-labeled reagents that have been sterically shielded with polyethylene chains by rapid bioorthogonal ligation leading to increased stability of the radiolabel in biological media. These multifunctional and bioorthogonal 211 At reagents can be efficiently prepared by rapid click assembly and simultaneous astatination. Read the full text of the article at 10.1002/cplu.201900114.