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Triple Orthogonal Labeling of Glycans Applying Photoclick Chemistry
摘要： Bioorthogonal labeling of multiple biomolecules is of current interest in chemical biology. For the visualization of carbohydrates, metabolic glycoengineering (MGE) has been shown to be an appropriate approach. Here, we report that the nitrile imine–alkene cycloaddition (photoclick reaction) is a suitable ligation reaction in MGE. Using a mannosamine derivative with an acrylamide reporter group, that is efficiently metabolized by cells and that quickly reacts in the photoclick reaction, we achieved the labeling of sialic acids on the surface of living cells. Screening of several alkenes unraveled that a previously reported carbamate-linked methylcyclopropene reporter, that is well suited for the inverse-electron-demand Diels-Alder (DAinv) reaction, has a surprisingly low reactivity in the photoclick reaction. This enabled us to achieve for the first time a triple labeling of glycans by the combination of DAinv, photoclick, and copper-free click chemistry.
关键词： carbohydrates,tetrazines,bioorthogonal reactions,click chemistry,metabolic engineering
更新于2025-09-23 15:21:21
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Preclinical Evaluation of a Novel <sup>18</sup> F-Labeled dTCO-Amide Derivative for Bioorthogonal Pretargeted Positron Emission Tomography Imaging
摘要： Pretargeted positron emission tomography (PET) imaging based on the bioorthogonal inverse-electron-demand Diels?Alder reaction between tetrazines (Tz) and trans-cyclooctenes (TCO) has emerged as a promising tool for solid tumor imaging, allowing the use of short-lived radionuclides in immune-PET applications. With this strategy, it became possible to achieve desirable target-to-background ratios and at the same time to decrease the radiation burden to nontargeted tissues because of the fast clearance of small PET probes. Here, we show the synthesis of novel 18F-labeled dTCO-amide probes for pretargeted immuno-PET imaging. The PET probes were evaluated regarding their stability, reactivity toward tetrazine, and pharmacokinetic pro?le. [18F]MICA-213 showed an extremely fast kinetic rate (10,553 M?1 s?1 in 50:50 MeOH/water), good stability in saline and plasma up to 4 h at 37 °C with no isomerization observed, and the biodistribution in healthy mice revealed a mixed hepatobiliary and renal clearance with no de?uorination and low background in other tissues. [18F]MICA-213 was further used for in vivo pretargeted immune-PET imaging carried out in nude mice bearing LS174T colorectal tumors that were previously treated with a tetrazine-modi?ed anti-TAG-72 monoclonal antibody (CC49). Pretargeted μPET imaging results showed clear visualization of the tumor tissue with a signi?cantly higher uptake when compared to the control.
关键词： immuno-PET,tetrazines,bioorthogonal chemistry,Pretargeted PET imaging,trans-cyclooctenes
更新于2025-09-23 15:21:01
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Microscope laser assisted photooxidative activation of bioorthogonal ClickOx probes
摘要： A photoactivatable fluorogenic tetrazine-rhodaphenothiazine probe was synthesized and studied in light-assisted, bioorthogonal labeling schemes. Experimental results revealed that the bioorthogonally conjugated probe efficiently sensitizes 1O2 generation upon illumination with green or orange light and undergoes self-oxidation leading to an intensely fluorescent sulfoxide product. An added value of the present probe is that it is also suitable for STED super-resolution microscopy using a 660 nm depletion laser.
关键词： tetrazine-rhodaphenothiazine,fluorogenic,STED,super-resolution microscopy,photoactivatable,bioorthogonal,labeling
更新于2025-09-23 15:19:57
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Small-Molecule Fluorescent Probes for Live-Cell Super-Resolution Microscopy
摘要： Super-resolution fluorescence microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to super-resolution microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this perspective, we discuss the role of small-molecule fluorescent probes for live-cell super-resolution microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell super-resolution microscopy are given.
关键词： Bioorthogonal chemistry,Super-resolution microscopy,Live-cell imaging,Fluorogenic probes,Protein labeling
更新于2025-09-23 15:19:57
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Systematic investigation of bioorthogonal cellular DNA metabolic labeling in a photo-controlled manner
摘要： Two 5-ethynyl-2′-deoxyuridine (EdU) derivatives photocaged on N-3 or 3′ position were synthesized by simple and effective methods, through direct UV irradiation on cells treated with them, cellular DNA metabolic labeling was successfully controlled. The systematic investigation of optical-cleavage position and type in living systems contributes to form a more integrated repertoire of bioorthogonal cleavage reaction.
关键词： DNA metabolic labeling,Cell imaging,Photocage group,Modified nucleoside,Bioorthogonal chemistry
更新于2025-09-19 17:13:59
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Dual-Modal Imaging-Guided Precise Tracking of Bioorthogonally Labeled Mesenchymal Stem Cells in Mouse Brain Stroke
摘要： Non-invasive and precise stem cell tracking after transplantation in living subject is very important to monitor both stem cell destinations and their in vivo fate, which was closely related to their therapeutic efficacy. Herein, we developed bicyclo[6.1.0]nonyne (BCN)-conjugated glycol chitosan nanoparticles (BCN-NPs) as a delivery system of dual-modal stem cell imaging probes. Near-infrared fluorescent (NIRF) dye, Cy5.5, was chemically conjugated to the BCN-NPs and then oleic acid-coated superparamagnetic iron oxide nanoparticles (OA-Fe3O4 NPs) were encapsulated into BCN-NPs, in resulting Cy5.5-labeled and OA-Fe3O4 NP-encapsulated BCN-NPs (BCN-dual-NPs). For bioorthogonal labeling of human adipose-derived mesenchymal stem cells (hMSCs), firstly, hMSCs were treated with tetra-acetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) for generating azide (-N3) groups onto their surface via metabolic glycoengineering. Second, azide groups on the cell surface were successfully chemically labeled with BCN-dual-NPs via bioorthogonal click chemistry in vitro. This bioorthogonal labeling of hMSCs could greatly increase the cell labeling efficiency, safety, and imaging sensitivity, compared to only nanoparticle-derived labeling technology. The dual-modal imaging-guided precise tracking of bioorthogonally labeled hMSCs was tested in the photothrombotic stroke mouse model via intraparenchymal injection. Finally, BCN-dual-NPs-labeled hMSCs could be effectively tracked of their migration from implanted site to brain stroke lesion using NIRF/T2-weighted magnetic resonance (MR) dual-modal imaging for 14 days. Our observation would provide a potential application of bioorthogonally labeled stem cell imaging in regenerative medicine by providing safety and high labeling efficiency in vitro and in vivo.
关键词： metabolic engineering,dual-modal imaging,bioorthogonal click chemistry,stem cell tracking,imaging probe,brain stroke
更新于2025-09-16 10:30:52
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Nitrone-Modified Gold Nanoparticles: Synthesis, Characterization, and Their Potential as <sup>18</sup> F-Labeled Positron Emission Tomography Probes via I-SPANC
摘要： A novel bioorthogonal gold nanoparticle (AuNP) template displaying interfacial nitrone functional groups for bioorthogonal interfacial strain-promoted alkyne?nitrone cycloaddition reactions has been synthesized. These nitrone?AuNPs were characterized in detail using 1H nuclear magnetic resonance spectroscopy, transmission electron microscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy, and a nanoparticle raw formula was calculated. The ability to control the conjugation of molecules of interest at the molecular level onto the nitrone?AuNP template allowed us to create a novel methodology for the synthesis of AuNP-based radiolabeled probes.
关键词： bioorthogonal chemistry,radiolabeled probes,PET imaging,nitrone,gold nanoparticles
更新于2025-09-12 10:27:22
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Clickable PNA Probes for Imaging Human Telomeres and Poly(A) RNAs
摘要： The ability to bind strongly to complementary nucleic acid sequences, invade complex nucleic acid structures, and resist degradation by cellular enzymes has made peptide nucleic acid (PNA) oligomers as very useful hybridization probes in molecular diagnosis. For such applications, the PNA oligomers have to be labeled with appropriate reporters as they lack intrinsic labels that can be used in biophysical assays. Although solid-phase synthesis is commonly used to attach reporters onto PNA, development of milder and modular labeling methods will provide access to PNA oligomers labeled with a wider range of biophysical tags. Here, we describe the establishment of a postsynthetic modification strategy based on bioorthogonal chemical reactions in functionalizing PNA oligomers in solution with a variety of tags. A toolbox composed of alkyne- and azide-modified monomers were site-specifically incorporated into PNA oligomers and postsynthetically click-functionalized with various tags, ranging from sugar, amino acid, biotin, to fluorophores, by using copper(I)-catalyzed azide?alkyne cycloaddition, strain-promoted azide?alkyne cycloaddition, and Staudinger ligation reactions. As a proof of utility of this method, fluorescent PNA hybridization probes were developed and used in imaging human telomeres in chromosomes and poly(A) RNAs in cells. Taken together, this simple approach of generating a wide range of functional PNA oligomers will expand the use of PNA in molecular diagnosis.
关键词： peptide nucleic acid,bioorthogonal chemical reactions,molecular diagnosis,poly(A) RNAs,human telomeres,PNA,click chemistry
更新于2025-09-09 09:28:46
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Recent Developments and Applications of Photoconjugation Chemistry
摘要： Understanding of biological systems has always been the general interest in chemical biology, and chemical modifications of biomolecules are often required to elucidate their functions and properties. There has thus been a rapid development of covalent chemistries for the modification of macromolecules. Among these strategies, photochemistry provides the advantage of using biocompatible light as an energy source to trigger bioconjugation reactions; this circumvents the use of toxic reagents (e.g. metal catalysts and chelating ligands). Light-induced chemistry can achieve precise spatial and temporal conjugation of biomolecules in their native environment.
关键词： Chemical biology,Photoconjugation,Bioorthogonal ligation,Small molecule probe
更新于2025-09-09 09:28:46