Ultrafluorogenic Monochromophore-Type BODIPY-Tetrazine Series for Dual-Color Bioorthogonal Imaging with a Single Probe

Various fluorogenic probes utilizing tetrazine (Tz) as a fluorescence quencher and bioorthogonal reaction partner have been extensively studied over the past few decades. Herein, we synthesized a series of boron-dipyrromethene (BODIPY)-Tz probes using monochromophoric design strategy for bioorthogonal cellular imaging. The BODIPY-Tz probes exhibited excellent bicyclo6.1.0]nonyne (BCN)-selective fluorogenicity with three- to four-digit-fold enhancements in fluorescence over a wide range of emission wavelengths, including the far-red region. Furthermore, we demonstrated the applicability of BODIPY-Tz probes in bioorthogonal fluorescence imaging of cellular organelles without washing steps. We also elucidated the aromatized pyridazine moiety as the origin of BCN-selective fluorogenic behavior. Additionally, we discovered that the fluorescence of the trans-cyclooctene (TCO) adducts was quenched in aqueous media via photoinduced electron transfer (PeT) process. Interestingly, we observed a distinctive recovery of the initially quenched fluorescence of BODIPY-Tz-TCO upon exposure to hydrophobic media, accompanied by a significant bathochromic shift of its emission wavelength relative to that exhibited by the corresponding BODIPY-Tz-BCN. Leveraging this finding, for the first time, we achieved dual-color bioorthogonal cellular imaging with a single BODIPY-Tz probe.