First author, Brismar Pinto-Pacheco (Bucella Group) and NYU Chemistry colleagues have published a study in Angewandte Chemie International Edition entitled, "Fluorescence Quenching Effects of Tetrazines and Their Diel-Alder Products: Mechanistic Insight Toward Fluorogenic Efficiency." The study uncovers the influence of the dienophile on the fluorescence enhancement in reactions of tetrazine-decorated fluorophores, and reveals options for maximizing contrast in widely used biolabeling applications. Co-authors include Will Carbery (an alumnus currently a postdoc at City College's Center for Discovery and Innovation), Sameer Khan (CAS 2017), Daniel Turner and Professor Daniela Buccella.
Abstract: Inverse electron demand Diels–Alder (iedDA) reactions between s ‐tetrazines and strained dienophiles have numerous applications in chemical biology and materials science. Tetrazines most often quench the emission of pendant fluorophores. This effect, however, is diminished upon iedDA, leading to the formation of emissive products with important applications in fluorescence labelling of biomolecules. Herein we investigate the effect of the dienophile on the fluorescence enhancement obtained upon reaction with a tetrazine‐quenched fluorophore and study the possible mechanisms of fluorescence quenching by both the tetrazine and its reaction products through a combination of linear and ultrabroadband two‐dimensional electronic spectroscopy (2D ES) experiments. The dihydropyridazine product obtained from reaction of the tetrazine with a strained cyclooctene shows a residual fluorescence quenching effect, greater than that exerted by the pyridazine arising from reaction of the tetrazine with the analogous alkyne. Ultrabroadband 2D ES measurements reveal that resonance energy transfer is the mechanism responsible for the fluorescence quenching effect of tetrazines, whereas a mechanism involving more intimate electronic coupling, most likely photoinduced electron transfer, is responsible for the quenching effect of the dihydropyridazine. These studies uncover parameters that can be tuned to maximize fluorogenic efficiency in bioconjugation reactions and they reveal that strained alkenes, though optimal for fast kinetics, are not the best reaction partners for achieving maximum contrast ratio in fluorescence labeling.
This research was supported by the National Science Foundation and the Alfred P. Sloan Foundation.