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

Abstract 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 bicyclo[6.1.0]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.