Oxime as a general photocage for the design of visible light photo-activatable fluorophores

Abstract

Photoactivatable fluorophores have been widely used for tracking molecular and cellular dynamics with subdiffraction resolution. In this work, we have prepared a series of photoactivatable probes using the oxime moiety as a new class of photolabile caging group in which the photoactivation process is mediated by a highly efficient photodeoximation reaction. Incorporation of the oxime caging group into fluorophores results in loss of fluorescence. Upon light irradiation in the presence of air, the oxime-caged fluorophores are oxidized to their carbonyl derivatives, restoring strong fluorophore fluorescence. To demonstrate the utility of these oxime-caged fluorophores, we have created probes that target different organelles for live-cell confocal imaging. We also carried out photoactivated localization microscopy (PALM) imaging under physiological conditions using low-power light activation in the absence of cytotoxic additives. Our studies show that oximes represent a new class of visible-light photocages that can be widely used for cellular imaging, sensing, and photo-controlled molecular release.

Fig. 1. Design of oxime-caged fluorogenic dyes. Oxime substitution at the carbonyl group of fluorophores results in very weak fluorescence via an ICT off mechanism. Upon irradiation with light, the oxime group can be oxidized to its carbonyl derivatives, thus restoring the strong fluorescence of fluorophores.

Fig. 2. Photoactivation of oxime-caged fluorophores. (A) Overlay of ¹H NMR spectra (6.8–11.5 ppm) of ACD-Oxm taken at the indicated light irradiation times (430 nm, 62.2 μW cm⁻²). (B) Normalized absorbance spectra of ACD, ACD-Oxm, and ACD-Oxm after photoactivation. (C) Absorbance changes in ACD-Oxm after light irradiation for different times (50 μM in DMSO, 430 nm, 62.2 μW cm⁻²). (D) Fluorescence spectra of ACD-Oxm (50 μM in DMSO) after light irradiation for different times (430 nm, 62.2 μW cm⁻²). (E–I) Fluorescence spectra of Cou-Oxm, API-Oxm, BAD-Oxm, NP-Oxm, and NR-Oxm (50 μM in DMSO) after light irradiation for different times (Cou-Oxm, BAD-Oxm, NP-Oxm and NR-Oxm by 430 nm, 62.2 μW cm⁻², API-Oxm by 405 nm, 70.0 μW cm⁻²).

Fig. 3. Mechanism of oxime-caged fluorophore activation. (A) Control experiments for studying the reaction mechanism. (B) Near-IR phosphorescence spectra of singlet oxygen generated by excitation of ACD-Oxm and the reference (Ru(bpy)₃²⁺) in oxygen-saturated methanol (50 μM) with 405 nm laser pulses at 25 °C. (C) Fluorescence change in ACD-Oxm (50 μM in DMSO) in the presence (1.1 equivalent TEMPO) and absence of TEMPO under light irradiation (430 nm, 62.2 μW cm⁻²). (D) Proposed photoactivation mechanism of oxime-caged fluorophores.

Fig. 4. Live-cell confocal imaging using oxime-caged fluorophores. (A) Synthesis of organelle-targeting BAD-Oxm fluorogenic fluorophores. (B) Photoactivation of BAD-Oxm (5 μM) in A431 cells using 405 nm laser activation. Confocal images were captured before and after 405 nm light activation of BAD-Oxm. Scale bar = 5 μm. (C) Photoactivation of organelle-targeting BAD-Oxm probes (2 μM) in A431 cells. Confocal images were captured from cells incubated with BAD-Oxm-TPP, BAD-Oxm-MOR, and BAD-Oxm-Ts along with corresponding commercial markers for mitochondria, lysosomes, and endoplasmic reticulum, respectively. Commercial MitoView™ 633, LysoView™ 633, and ERTracker™ Red (BODIPY™ TR Glibenclamide) were used as markers for mitochondria, lysosomes, and endoplasmic reticulum, respectively. Scale bar = 5 μm.

Fig. 5. Super-resolution imaging of HaloTag proteins using oxime-caged fluorophores. (A) Scheme for labeling the protein of interest (POI) with BAD-Oxm-Halo ligand for fluorescence imaging. (B) Fluorescence change of BAD-Oxm-Halo in the presence of 430 nm light (62.2 μW cm⁻²). (C) Intracellular fluorescence change in BAD-Oxm-Halo (5 μM) during photoactivation at 405 nm in CHO–K1 cells. (D) Widefield image and corresponding PALM images of H2B labeled with H2B-HaloTag in CHO–K1 cells. (E) Histogram plot of the localization accuracy of PALM images in (D). Scale bar = 1 μm.