TY - JOUR
T1 - Fluorescence Quenching Effects of Tetrazines and Their Diels–Alder Products
T2 - Mechanistic Insight Toward Fluorogenic Efficiency
AU - Pinto-Pacheco, Brismar
AU - Carbery, William P.
AU - Khan, Sameer
AU - Turner, Daniel B.
AU - Buccella, Daniela
N1 - Publisher Copyright:
© 2020 Wiley-VCH GmbH
PY - 2020/12/1
Y1 - 2020/12/1
N2 - 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.
AB - 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.
KW - 2D electronic spectroscopy
KW - FRET
KW - photoinduced electron transfer
KW - quenching mechanisms
KW - tetrazine
UR - http://www.scopus.com/inward/record.url?scp=85091684937&partnerID=8YFLogxK
U2 - 10.1002/anie.202008757
DO - 10.1002/anie.202008757
M3 - Article
C2 - 33245600
AN - SCOPUS:85091684937
SN - 1433-7851
VL - 59
SP - 22140
EP - 22149
JO - Angewandte Chemie - International Edition
JF - Angewandte Chemie - International Edition
IS - 49
ER -