While bioorthogonal reactions are routinely employed in living cells and organisms, their application within individual organelles remains limited. In this review, we highlight diverse examples of bioorthogonal reactions used to investigate the roles of biomolecules and biological processes as well as advanced imaging techniques within cellular organelles. These innovations hold great promise for therapeutic interventions in personalized medicine and precision therapies. We also address existing challenges related to the selectivity and trafficking of subcellular dynamics. Organelle-targeted bioorthogonal reactions have the potential to significantly advance our understanding of cellular organization and function, provide new pathways for basic research and clinical applications, and shape the direction of cell biology and medical research.

• Keywords
• Bioconjugations, Bioorthogonal reactions, Cellular organelles, Click chemistry, Targeting,
• MeSH
• Cell Biology MeSH
• Organelles * chemistry   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

The development of abiotic chemical reactions that can be performed in an organelle-specific manner can provide new opportunities in drug delivery and cell and chemical biology. However, due to the complexity of the cellular environment, this remains a significant challenge. Here, we introduce structurally redesigned bioorthogonal tetrazine reagents that spontaneously accumulate in mitochondria of live mammalian cells. The attributes leading to their efficient accumulation in the organelle were optimized to include the right combination of lipophilicity and positive delocalized charge. The best performing mitochondriotropic tetrazines enable subcellular chemical release of TCO-caged compounds as we show using fluorogenic substrates and mitochondrial uncoupler niclosamide. Our work demonstrates that a shrewd redesign of common bioorthogonal reagents can lead to their transformation into organelle-specific probes, opening the possibility to activate prodrugs and manipulate biological processes at the subcellular level by using purely chemical tools.

• Publication type
• Journal Article MeSH

Fluorescent probes that light-up upon reaction with complementary bioorthogonal reagents are superior tools for no-wash fluorogenic bioimaging applications. In this work, a thorough study is presented on a set of seventeen structurally diverse coumarin-tetrazine probes that produce fluorescent dyes with exceptional turn-on ratios when reacted with trans-cyclooctene (TCO) and bicyclononyne (BCN) dienophiles. In general, formation of the fully aromatic pyridazine-containing dyes resulting from the reaction with BCN was found superior in terms of fluorogenicity. However, evaluation of the probes in cellular imaging experiments revealed that other factors, such as reaction kinetics and good cell permeability, prevail over the fluorescence turn-on properties. The best compound identified in this study showed excellent performance in live cell-labeling experiments and enabled no-wash fluorogenic imaging on a timescale of seconds.

• Keywords
• click chemistry, coumarins, fluorescence, live cell bioimaging, tetrazines,
• MeSH
• Fluorescent Dyes analysis   MeSH
• Heterocyclic Compounds analysis   MeSH
• Microscopy, Confocal MeSH
• Coumarins analysis   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Optical Imaging * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Heterocyclic Compounds MeSH
• Coumarins MeSH

Inverse-electron-demand Diels-Alder (iEDDA) cycloaddition between 1,2,4,5-tetrazines and strained dienophiles belongs among the most popular bioconjugation reactions. In addition to its fast kinetics, this cycloaddition can be tailored to produce fluorescent products from non-fluorescent starting materials. Here we show that even the reaction intermediates formed in iEDDA cycloaddition can lead to the formation of new types of fluorophores. The influence of various substituents on their photophysical properties and the generality of the approach with use of various trans-cyclooctene derivatives were studied. Model bioimaging experiments demonstrate the application potential of fluorogenic iEDDA cycloaddition.

• Keywords
• bioorthogonal chemistry, click chemistry, cycloaddition, heterocycles, tetrazines,
• MeSH
• Cycloaddition Reaction MeSH
• Cyclooctanes chemistry   MeSH
• Fluorescent Dyes chemical synthesis   chemistry   MeSH
• Microscopy, Fluorescence methods   MeSH
• HeLa Cells MeSH
• Heterocyclic Compounds, 2-Ring chemical synthesis   chemistry   MeSH
• Heterocyclic Compounds, 1-Ring chemistry   MeSH
• Microscopy, Confocal methods   MeSH
• Humans MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cyclooctanes MeSH
• Fluorescent Dyes MeSH
• Heterocyclic Compounds, 2-Ring MeSH
• Heterocyclic Compounds, 1-Ring MeSH