A Systematic Study of Coumarin–Tetrazine Light‐Up Probes for Bioorthogonal Fluorescence Imaging

Abstract

Fluorescent probest hat light-up upon reaction with complementary bioorthogonal reagents are superior tools for no-wash fluorogenic bioimaging applications.I n this work, at horough study is presented on as et of seventeen structurally diverse coumarin-tetrazine probes that produce fluorescent dyes with exceptional turn-onr atios when reacted with trans-cyclooctene (TCO) and bicyclononyne (BCN) dienophiles. In general,f ormation of the fully aromatic pyridazine-containing dyes resulting fromt he reaction with BCN was found superiori nt erms of fluorogenicity.H owever, evaluation of the probesi nc ellular imaging experiments revealed that other factors, such as reaction kinetics and good cell permeability,p revail over the fluorescence turn-on properties. The best compound identified in this study showed excellent performance in live cell-labeling experimentsa nd enabledn o-wash fluorogenic imaging on at imescale of seconds.

Fluorescence Quenching Effects of Tetrazines and Their Diels–Alder Products: Mechanistic Insight Toward Fluorogenic Efficiency

“…[5] Beyond the remarkable kinetics,anattractive feature of iedDAreactions involving tetrazines is the possibility of obtaining fluorescent products from non-emissive precursors,t hat is,afluorogenic reaction, given the right choice of reaction partners. [9][10][11][12][13][14][15][16][17][18][19][20][21][22] This feature can be particularly useful for in situ fluorescence labeling of cellular species to enable their visualization and tracking by various microscopy techniques.T he fluorogenic character stems from the strong fluorescence quenching effect that tetrazines can exert on tethered fluorophores,w hich leads to net fluorescence enhancement once the tetrazine core is broken.…”

Fluorescence Quenching Effects of Tetrazines and Their Diels–Alder Products: Mechanistic Insight Toward Fluorogenic Efficiency

“…With remarkably fast reaction rates, innocuous byproduct formation, and no catalyst requirement, these transformations offer an excellent alternative to azide‐alkyne cycloadditions and other “click” reactions, especially for bioconjugation reactions performed in cellulo [5] . Beyond the remarkable kinetics, an attractive feature of iedDA reactions involving tetrazines is the possibility of obtaining fluorescent products from non‐emissive precursors, that is, a fluorogenic reaction, given the right choice of reaction partners [9–22] . This feature can be particularly useful for in situ fluorescence labeling of cellular species to enable their visualization and tracking by various microscopy techniques.…”

“…[5] Beyond the remarkable kinetics,a na ttractive feature of iedDAr eactions involving tetrazines is the possibility of obtaining fluorescent products from non-emissive precursors,t hat is,afluorogenic reaction, given the right choice of reaction partners. [9][10][11][12][13][14][15][16][17][18][19][20][21][22] This feature can be particularly useful for in situ fluorescence labeling of cellular species to enable their visualization and tracking by various microscopy techniques.T he fluorogenic character stems from the strong fluorescence quenching effect that tetrazines can exert on tethered fluorophores,w hich leads to net fluorescence enhancement once the tetrazine core is broken.…”

Bioorthogonal red and far-red fluorogenic probes for wash-free live-cell and super-resolution microscopy

“…While we used BCN as dienophile for in vitro characterization due to its formation of a single product, we tested both, BCN and trans-cyclooctene (TCO) as dienophiles in live cells. TCO derivatives have been reported to possess superior kinetics over BCN [37][38][39] and it is further known that the choice of the DAinv-dienophile can influence the fluorescence turn-on of tetrazine dyes 26,40,41 .…”