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.

• Klíčová slova
• Bioconjugations, Bioorthogonal reactions, Cellular organelles, Click chemistry, Targeting,
• MeSH
• biologie buňky MeSH
• organely * chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

A series of 2'-deoxyribonucleoside triphosphates (dNTPs) bearing 2- or 4-linked trans-cyclooctene (TCO) or bicyclononyne (BCN) tethered through a shorter propargylcarbamate or longer triethyleneglycol-based spacer were designed and synthesized. They were found to be good substrates for KOD XL DNA polymerase for primer extension enzymatic synthesis of modified oligonucleotides. We systematically tested and compared the reactivity of TCO- and BCN-modified nucleotides and DNA with several fluorophore-containing tetrazines in inverse electron-demand Diels-Alder (IEDDA) click reactions to show that the longer linker is crucial for efficient labeling. The modified dNTPs were transported into live cells using the synthetic transporter SNTT1, incubated for 1 h, and then treated with tetrazine conjugates. The PEG3-linked 4TCO and BCN nucleotides showed efficient incorporation into genomic DNA and good reactivity in the IEDDA click reaction with tetrazines to allow staining of DNA and imaging of DNA synthesis in live cells within time periods as short as 15 min. The BCN-linked nucleotide in combination with TAMRA-linked (TAMRA = carboxytetramethylrhodamine) tetrazine was also efficiently used for staining of DNA for flow cytometry. This methodology is a new approach for in cellulo metabolic labeling and imaging of DNA synthesis which is shorter, operationally simple, and overcomes several problems of previously used methods.

• Publikační typ
• časopisecké články 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.

• Klíčová slova
• click chemistry, coumarins, fluorescence, live cell bioimaging, tetrazines,
• MeSH
• fluorescenční barviva analýza   MeSH
• heterocyklické sloučeniny analýza   MeSH
• konfokální mikroskopie MeSH
• kumariny analýza   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• optické zobrazování * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fluorescenční barviva MeSH
• heterocyklické sloučeniny MeSH
• kumariny MeSH

Fluorogenic bioorthogonal reactions enable visualization of biomolecules with excellent signal-to-noise ratio. A bicyclononyne-tetrazine ligation that produces fluorescent pyridazine products has been developed. In stark contrast to previous approaches, the formation of the dye is an inherent result of the chemical reaction and no additional fluorophores are needed in the reagents. The crucial structural elements that determine dye formation are electron-donating groups present in the starting tetrazine unit. The newly formed pyridazine fluorophores show interesting photophysical properties the fluorescence intensity increase in the reaction can reach an excellent 900-fold. Model imaging experiments demonstrate the application potential of this new fluorogenic bioorthogonal reaction.

• Klíčová slova
• Diels-Alder reactions, cyclic alkynes, cycloaddition reactions, fluorogenic probes, tetrazines,
• MeSH
• cykloadiční reakce MeSH
• fluorescenční barviva chemie   MeSH
• heterocyklické sloučeniny monocyklické chemie   MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• můstkové bicyklické sloučeniny chemie   MeSH
• nádorové buněčné linie MeSH
• pyridaziny chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fluorescenční barviva MeSH
• heterocyklické sloučeniny monocyklické MeSH
• můstkové bicyklické sloučeniny MeSH
• pyridaziny 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.

• Klíčová slova
• bioorthogonal chemistry, click chemistry, cycloaddition, heterocycles, tetrazines,
• MeSH
• cykloadiční reakce MeSH
• cyklooktany chemie   MeSH
• fluorescenční barviva chemická syntéza   chemie   MeSH
• fluorescenční mikroskopie metody   MeSH
• HeLa buňky MeSH
• heterocyklické sloučeniny bicyklické chemická syntéza   chemie   MeSH
• heterocyklické sloučeniny monocyklické chemie   MeSH
• konfokální mikroskopie metody   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklooktany MeSH
• fluorescenční barviva MeSH
• heterocyklické sloučeniny bicyklické MeSH
• heterocyklické sloučeniny monocyklické MeSH