Monochromophoric Design Strategy for Tetrazine-Based Colorful Bioorthogonal Probes with a Single Fluorescent Core Skeleton

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: mechanism, Which limits red-shifting of probes emission wavelength without deterioration of the fluorescent turnon/off ratio. To address this constraint, herein we present a monochromophoric design strategy for making a series of FL(Tz)s 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 pi-conjugation systems of an indolizine core, we efficiently quench the fluorescence of SF tetrazine conjugates (SFT(Tz)s) and achieved more than 1000-fold enhancement in fluorescence after iEDDA reaction with trans-cydooctene (TCO). Importantly, we were able to develop a series of colorful SF(Tz)s 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.