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Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labelling in live cells

Fluorogenic bioorthogonal reactions are promising tools for tracking small molecules or biomolecules in living organisms. Two-photon excitation, by shifting absorption towards the red, significantly increases the signal-to-noise ratio and decreases photodamage, while allowing imaging about 10 times...

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Autores principales: Auvray, Marie, Naud-Martin, Delphine, Fontaine, Gaëlle, Bolze, Frédéric, Clavier, Gilles, Mahuteau-Betzer, Florence
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10395273/
https://www.ncbi.nlm.nih.gov/pubmed/37538830
http://dx.doi.org/10.1039/d3sc01754k
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author Auvray, Marie
Naud-Martin, Delphine
Fontaine, Gaëlle
Bolze, Frédéric
Clavier, Gilles
Mahuteau-Betzer, Florence
author_facet Auvray, Marie
Naud-Martin, Delphine
Fontaine, Gaëlle
Bolze, Frédéric
Clavier, Gilles
Mahuteau-Betzer, Florence
author_sort Auvray, Marie
collection PubMed
description Fluorogenic bioorthogonal reactions are promising tools for tracking small molecules or biomolecules in living organisms. Two-photon excitation, by shifting absorption towards the red, significantly increases the signal-to-noise ratio and decreases photodamage, while allowing imaging about 10 times deeper than with a confocal microscope. However, efficient two-photon excitable fluorogenic probes are currently lacking. We report here the design and synthesis of fluorogenic probes based on a two-photon excitable fluorophore and a tetrazine quenching moiety. These probes react with bicyclo[6.1.0]no-4-yn-9ylmethanol (BCN) with a good to impressive kinetic rate constant (up to 1.1 × 10(3) M(−1) s(−1)) and emit in the red window with moderate to high turn-on ratios. TDDFT allowed the rationalization of both the kinetic and fluorogenic performance of the different probes. The best candidate displays a 13.8-fold turn-on measured by quantifying fluorescence intensities in live cells under one-photon excitation, whereas a value of 3 is sufficient for high contrast live-cell imaging. In addition, live-cell imaging under two-photon excitation confirmed that there was no need for washing to monitor the reaction between BCN and this probe since an 8.0-fold turn-on was measured under two-photon excitation. Finally, the high two-photon brightness of the clicked adduct (>300 GM) allows the use of a weak laser power compatible with in vivo imaging.
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spelling pubmed-103952732023-08-03 Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labelling in live cells Auvray, Marie Naud-Martin, Delphine Fontaine, Gaëlle Bolze, Frédéric Clavier, Gilles Mahuteau-Betzer, Florence Chem Sci Chemistry Fluorogenic bioorthogonal reactions are promising tools for tracking small molecules or biomolecules in living organisms. Two-photon excitation, by shifting absorption towards the red, significantly increases the signal-to-noise ratio and decreases photodamage, while allowing imaging about 10 times deeper than with a confocal microscope. However, efficient two-photon excitable fluorogenic probes are currently lacking. We report here the design and synthesis of fluorogenic probes based on a two-photon excitable fluorophore and a tetrazine quenching moiety. These probes react with bicyclo[6.1.0]no-4-yn-9ylmethanol (BCN) with a good to impressive kinetic rate constant (up to 1.1 × 10(3) M(−1) s(−1)) and emit in the red window with moderate to high turn-on ratios. TDDFT allowed the rationalization of both the kinetic and fluorogenic performance of the different probes. The best candidate displays a 13.8-fold turn-on measured by quantifying fluorescence intensities in live cells under one-photon excitation, whereas a value of 3 is sufficient for high contrast live-cell imaging. In addition, live-cell imaging under two-photon excitation confirmed that there was no need for washing to monitor the reaction between BCN and this probe since an 8.0-fold turn-on was measured under two-photon excitation. Finally, the high two-photon brightness of the clicked adduct (>300 GM) allows the use of a weak laser power compatible with in vivo imaging. The Royal Society of Chemistry 2023-07-04 /pmc/articles/PMC10395273/ /pubmed/37538830 http://dx.doi.org/10.1039/d3sc01754k Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by/3.0/
spellingShingle Chemistry
Auvray, Marie
Naud-Martin, Delphine
Fontaine, Gaëlle
Bolze, Frédéric
Clavier, Gilles
Mahuteau-Betzer, Florence
Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labelling in live cells
title Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labelling in live cells
title_full Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labelling in live cells
title_fullStr Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labelling in live cells
title_full_unstemmed Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labelling in live cells
title_short Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labelling in live cells
title_sort ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labelling in live cells
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10395273/
https://www.ncbi.nlm.nih.gov/pubmed/37538830
http://dx.doi.org/10.1039/d3sc01754k
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