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Fluorescence Quenching Effects of Tetrazines and Their Diels–Alder Products: Mechanistic Insight Toward Fluorogenic Efficiency
Author(s) -
PintoPacheco Brismar,
Carbery William P.,
Khan Sameer,
Turner Daniel B.,
Buccella Daniela
Publication year - 2020
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.202008757
Subject(s) - tetrazine , photochemistry , quenching (fluorescence) , chemistry , fluorescence , fluorophore , photoinduced electron transfer , electron transfer , förster resonance energy transfer , bioconjugation , combinatorial chemistry , organic chemistry , quantum mechanics , physics
Abstract Inverse electron demand Diels–Alder reactions between s ‐tetrazines and strained dienophiles have numerous applications in fluorescent labeling 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. The dihydropyridazine obtained from reaction with a strained cyclooctene shows a residual fluorescence quenching effect, greater than that exerted by the pyridazine arising from reaction with the analogous alkyne. Linear and ultrabroadband two‐dimensional electronic spectroscopy experiments reveal that resonance energy transfer is the mechanism responsible for the fluorescence quenching effect of tetrazines, whereas a mechanism involving more intimate electronic coupling, 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 reveal that strained alkynes are better reaction partners for achieving maximum contrast ratio.