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

06 March 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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 photodamages, while allowing to image about 10 times deeper than with a confocal. 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. Among our series of 6 new probes, our best candidate possesses a turn-on ratio of 22 and an impressive kinetic rate constant of 1.1x103M-1s-1, about 10 times higher than that reported for the fluorogenic iEDDA reaction with bicyclo[6.1.0]no-4-yn-9yl)methanol (BCN). Live-cell imaging under two-photon excitation confirmed that there was no need for washing to monitor the reaction between BCN and this probe with high-contrast images. The high two-photon brightness of the clicked adduct (362 GM) allows the use of a weak laser power compatible with in vivo imaging.

Keywords

Bioorthogonal reaction
Fluorescence
Two-photon excitation
Live-cell imaging
Tetrazine

Supplementary materials

Title
Description
Actions
Title
Ultrabright two-photon excitable fluorogenic probes for fast and wash-free biorthogonal labeling in live cells
Description
Experimental procedures and supplementary data
Actions

Comments

Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.