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

Abstract

Inverse electron demand Diels-Alder reactions between s-tetrazines and strained dienophiles have numerous applications in fluorescent labeling of biomolecules.H erein, we investigate the effect of the dienophile on the fluorescence enhancement obtained upon reaction with at etrazinequenched fluorophore and study the possible mechanisms of fluorescence quenching by both the tetrazine and its reaction products.T he dihydropyridazine obtained from reaction with astrained cyclooctene shows aresidual fluorescence quenching effect, greater than that exerted by the pyridazine arising from reaction with the analogous alkyne.L inear and ultrabroadband two-dimensional electronic spectroscopye xperiments reveal that resonance energy transfer is the mechanism responsible for the fluorescence quenching effect of tetrazines, whereas am echanism involving more intimate electronic coupling,l ikely photoinduced electron transfer,i sr esponsible for the quenchinge ffect of the dihydropyridazine.T hese 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.

Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy

“…This was in good accordance with the different emission intensities obtained after reaction of HD555 with BCN and TCO* in vitro ( Figure S15 ) and recent work by others that reported a strong dependency of fluorescence enhancement in DA inv on the used dienophile. 42 , 43 Due to the higher obtainable fluorescence brightness, we employed BCN as a DA inv dienophile for cellular labeling in all following experiments.…”

Versatile naphthalimide tetrazines for fluorogenic bioorthogonal labelling

“…Fluorescence is restored to the fluorophore as pyridazines do not quench fluorescence through energy transfer and hence the tetrazine-BCN ligation is an excellent strategy for fluorogenic labelling. 14 Many tetrazine-containing fluorogenic probes have been synthesized with emission wavelengths spanning the visible and infrared spectrum, commonly employing coumarin, 15 fluorescein, 16 rhodamine, 17 cyanine, 18 BODIPY 12 and other commercial and novel scaffolds. [19][20][21][22][23][24][25] All of these have been utilized in confocal microscopy, and some for super resolution imaging.…”

“…Fluorescence is restored to the fluorophore as pyridazines do not quench fluorescence through energy transfer and hence the tetrazine-BCN ligation is an excellent strategy for fluorogenic labelling. 14 …”

Intracellular fluorogenic supramolecular assemblies for self-reporting bioorthogonal prodrug activation

“…In the bioorthogonal reaction pair of tetrazine (Tz) and trans-cyclooctene (TCO), the Tz moiety usually functions as a fluorescence quencher, which reacts with TCO and turns on the emission. [38][39][40] [41][42][43] the Tz-conjugated assembly precursor, e.g., BQA-K(Tz)GGGFF (1), could self-assemble selectively in vitro in ROS over-produced cancerous cells without fluorescence emission. Upon treatment with the TCO moiety, the conversion from Tz to Tz-TCO adducts eliminated photo-induced electron transfer (PET), 44 which turned on fluorescence from those Tzbearing assemblies.…”