Photo‐Triggered, Fast, and Fluorogenic Thiophene‐Based Cycloalkynes for the Bioorthogonal Fluorescent Labeling of 1,3‐Dipole‐Tagged Molecules in No‐Wash Conditions

ABSTRACT Bioorthogonal reactions have revolutionized our way of performing chemistry in a highly complex biological environment. In particular, strain‐promoted 1,3‐dipolar cycloadditions, employing cyclooctyne probes in conjunction with azido‐reporters (strain‐promoted alkyne‐azide cycloaddition, SPAAC), have permitted the labeling and visualization of bio‐macromolecules in vitro, in living cells, as well as in animals. However, SPAAC's slow kinetics (< 1 M −1 s −1 ), in combination with the necessity to eliminate the excess of the used fluorescent probes, have hampered its widespread application, especially for the real‐time imaging of low concentration targets. Here we describe two novel thiophene‐based cycloalkynes that not only exhibit very high kinetics toward a variety of 1,3‐dipoles (up to 1528 M −1 s −1 ), but are also efficiently turned‐on (up to 150‐fold increase in brightness) upon reaction with their target. We demonstrated their fast and fluorogenic capabilities by monitoring the labeling overtime of a glycoprotein in physiological and no‐wash conditions, using as little as 5 µM of the probes and reaching full labeling in less than 15 min. These fluorogenic cycloalkynes significantly expand our chemical biology toolbox, and we anticipate them to open new avenues for the fast and real‐time imaging of biomolecules in complex environments.