Abstract
Fluorogenic bioorthogonal probes are ideal for fluorescent imaging in live cell conditions. By taking advantage of the dual functionality of tetrazine (Tz), as a bioorthogonal reaction unit as well as a fluorescence quencher, a fluorophore-Tz conjugate (FLTz) has been utilized for fluorescent live cell imaging via inverse electron-demand Diels-Alder (iEDDA) type bioorthogonal reactions. However, most FLTz strategies rely on a donor-acceptor-type energy transfer mechanism, which limits red-shifting of probes' emission wavelength without deterioration of the fluorescent turn-on/off ratio. To address this constraint, herein we present a monochromophoric design strategy for making a series of FLTzs spanning a broad range of emission colors. For the systematic comparison of design strategies with minimized structural differences, we selected indolizine-based emission-tunable Seoul-Fluor (SF) as a model fluorophore system. As a result, by inducing strong electronic coupling between Tz and π-conjugation systems of an indolizine core, we efficiently quench the fluorescence of SF-tetrazine conjugates (SFTzs) and achieved more than 1000-fold enhancement in fluorescence after iEDDA reaction with trans-cyclooctene (TCO). Importantly, we were able to develop a series of colorful SFTzs with a similar turn-on/off ratio regardless of their emission wavelength. The applicability as bioorthogonal probes was demonstrated with fluorescence bioimaging of innate microtubule and mitochondria using docetaxel-TCO and triphenylphosphonium-TCO in live cells without washing steps. We believe this study could provide new insight for the reliable and generally applicable molecular design strategy to develop bioorthogonal fluorogenic probes having an excellent turn-on ratio, regardless of their emission wavelength.
Highlights
Fluorescent imaging techniques have revolutionized the way to understand biological systems at the nanoscopic,[1] microscopic,[2] and macroscopic levels.[3]
In the case of through-bond energy transfer (TBET)-based SFTz01, we proposed that the Tz moiety at the R1 position is geometrically tilted and electronically decoupled from the π-system of indolizine, which was strongly supported by marginal changes in the absorption spectrum of SFTz01 around 400 nm after the inverse electron-demand Diels−Alder (iEDDA) reaction
The monochromophoric design strategy enables the development of fluorogenic probe SF−tetrazine conjugates (SFTzs), having an extraordinary turn-on/off ratio covering the full visible color range, for the live cell imaging via rapid and catalyst-free bioorthogonal reaction
Summary
Between the fluorophore (donor) and Tz (acceptor) for simple conjugation,11a and the fluorescence of FLTz is assumed to be quenched by transferring the fluorophore’s excited energy to the Tz quencher via a long-range dipole−dipole interaction, known as Förster resonance energy transfer (FRET) (Figure 1b, left).[13]. Introduced electron-donating substituents guided by the Hammett constant24 [methoxy (σp = −0.27), amino (−0.66), and diethylamino group (−0.72)] at the R1 position of SFPy the corresponding iEDDA reaction product of SFTz with TCO having a 1,4-dihydropyridazine moiety to induce a bathochromic shift of the emission wavelength of SFTz−TCO adducts. The slight reduction in turn-on/off ratio of SFTz08 might originate from the innate low quantum yield of its fluorogenic form This comparison experiment demonstrated the unique advantage of monochromophoric design strategy, which guarantees excellent fluorescence off/on ratio, independent from the emission wavelength of fluorophores. Highly efficient and selective fluorescent staining of intracellular TOI protein and intracellular organelles was possible without multiple washing steps using SFTzs in live cell conditions
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