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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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Off-colony screening of biosynthetic libraries by rapid laser-enabled mass spectrometry
摘要： Leveraging advances in DNA synthesis and molecular cloning techniques, synthetic biology increasingly makes use of large construct libraries to explore large design spaces. For biosynthetic pathway engineering the ability to screen these libraries for a variety of metabolites of interest is essential. If the metabolite of interest or the metabolic phenotype is not easily measurable, screening soon becomes a major bottleneck involving time-consuming culturing, sample preparation, and extraction. To address this, we demonstrate the use of automated Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) – a form of ambient laser desorption ionisation mass spectrometry – to perform rapid mass spectrometry analysis direct from agar plate yeast colonies without sample preparation or extraction. We use LA-REIMS to assess production levels of violacein and betulinic acid directly from yeast colonies at a rate of 6 colonies per minute. We then demonstrate the throughput enabled by LA-REIMS by screening over 450 yeast colonies in under 4 hours, while simultaneously generating recoverable glycerol stocks of each colony in real-time. This showcases LA-REIMS as a pre-screening tool to complement downstream quantification methods such as LCMS. Through pre-screening several hundred colonies with LA-REIMS, we successfully isolate and verify a strain with a 2.5-fold improvement in betulinic acid production. Finally, we show that LA-REIMS can detect 20 out of a panel of 27 diverse biological molecules, demonstrating the broad applicability of LA-REIMS to metabolite detection. The rapid and automated nature of LA-REIMS makes this a valuable new technology to complement existing screening technologies currently employed in academic and industrial workflows.
关键词： synthetic biology,metabolic engineering,REIMS,ambient mass spectrometry,high throughput screening
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