Fluorophores can be used as optical “paints” to visualize otherwise invisible structures or processes and hence have attracted scientists’ attention over the past 70 years. Fluorophores that absorb and emit light within the near-infrared (NIR) window (650 nm ~ 900 nm) are desirable for monitoring biological events since NIR light has deeper tissue penetration and mitigates issues associated with autofluorescence of natively occurring fluorophores. Herein we report the design, synthesis, and evaluation of three novel far-red to near-infrared fluorophore platforms: termed Rachael Fluor620 (RF620), SiOH2R, and Nebraska Red (NR). These new scaffolds were obtained by replacing the bridging oxygen atom of rhodamine with boronate, silanediol, or phosphinate functionalities. The resulting fluorophores showed remarkable brightness (ε × Φ) in the far-red to NIR region. Indeed, the NR derivative termed NR666 was found to be 1.3-fold brighter than its parent scaffold tetramethylrhodamine (TMR). Leveraging the outstanding photophysical properties of these new fluorophore templates coupled with the new chemistry afforded by the introduced bridging functional groups, we were also able to apply these fluorophore scaffolds in chemical biology applications. Specifically, RF620 and SiOH2R have been evaluated as ratiometric probes for hydrogen peroxide (H2O2) in living cells. In situ formation of TMR upon oxidation of these probes results in dramatic shifts in emission wavelength of 66 nm and 111 nm, respectively. Given the selective localization of these probes to the mitochondria, these new platforms allow for the sensitive detection of endogenous H2O2 production. In addition, we show that the phosphinate group of the NR series of dyes can be leveraged to tune cell permeability, enabling the generation of reagents for cytosolic or membrane protein labeling. The intrinsic reactivity of the phosphinate functionality is also leveraged in order to generate a self-reporting small molecule delivery vehicle that responds to endogenous production of hypochlorous acid. Taken together, this work provides evidence for the ability to generate new chemical biology tools by leveraging the introduction of novel chemical functionalities in the xanthene scaffold.

Development of novel far-red to near-infrared fluorophores for chemical biology

253rd National Meeting (2017)

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