Efficient Far-Red/Near-IR Absorbing BODIPY Photocages by Blocking Unproductive Conical Intersections.

Photocages are light-sensitive chemical protecting groups that give investigators control over activation of biomolecules using targeted light irradiation. A compelling application of far-red/near-IR absorbing photocages is their potential for deep tissue activation of biomolecules and phototherapeutics. Toward this goal, we recently reported BODIPY photocages that absorb near-IR light. However, these photocages have reduced photorelease efficiencies compared to shorter-wavelength absorbing photocages, which has hindered their application. Because photochemistry is a zero-sum competition of rates, improvement of the quantum yield of a photoreaction can be achieved either by making the desired photoreaction more efficient or by hobbling competitive decay channels. This latter strategy of inhibiting unproductive decay channels was pursued to improve the release efficiency of long-wavelength absorbing BODIPY photocages by synthesizing structures that block access to unproductive singlet internal conversion conical intersections, which have recently been located for simple BODIPY structures from excited state dynamic simulations. This strategy led to the synthesis of new conformationally restrained boron-methylated BODIPY photocages that absorb light strongly around 700 nm. In the best case, a photocage was identified with an extinction coefficient of 124000 M-1 cm-1, a quantum yield of photorelease of 3.8%, and an overall quantum efficiency of 4650 M-1 cm-1 at 680 nm. This derivative has a quantum efficiency that is 50-fold higher than the best known BODIPY photocages absorbing >600 nm, validating the effectiveness of a strategy for designing efficient photoreactions by thwarting competitive excited state decay channels. Furthermore, 1,7-diaryl substitutions were found to improve the quantum yields of photorelease by excited state participation and blocking ion pair recombination by internal nucleophilic trapping. No cellular toxicity (trypan blue exclusion) was observed at 20 µM, and photoactivation was demonstrated in HeLa cells using red light.

Direct Photorelease of Alcohols from Boron-Alkylated BODIPY Photocages.

BODIPY photocages allow the release of substrates using visible light irradiation. They have the drawback of requiring reasonably good leaving groups for photorelease. Photorelease of alcohols is often accomplished by attachment with carbonate linkages, which upon photorelease liberate CO2 and generate the alcohol. Here, we show that boron-alkylated BODIPY photocages are capable of directly photoreleasing both aliphatic alcohols and phenols upon irradiation via photocleavage of ether linkages. Direct photorelease of a hydroxycoumarin dye was demonstrated in living HeLa cells.

Coumarin-based Fluorescent Probes for Selectively Targeting and Imaging the Endoplasmic Reticulum in Mammalian Cells.

Developing improved fluorescent probes for imaging the endoplasmic reticulum (ER) is necessary for structure-activity studies of this dynamic organelle. Two coumarin-based compounds with sulfonamide side groups were synthesized and characterized as ER-targeting probes. Their selectivity to target the ER in HeLa and GM07373 mammalian cells was shown with co-localization experiments using commercially available probes that localize in the ER, mitochondria, or lysozymes. The hydrophobicity of the coumarin-based probes was comparable to known probes that partition into the ER membrane. Their cytotoxicity in mammalian cells was low with IC50 values that range from 205 to 252 µm. The fluorescent quantum yields of the coumarin-based probes when excited with 400 nm light were 0.60, and they have a much narrower emission spectrum (from 435 to 525 nm in methanol) than that of the only commercially available ER probe that is exited with 400 nm light (ER-Tracker™ Blue-White DPX). Thus, the coumarin-based probes are more useful for multicolor imaging with yellow and red emitting fluorophores. In addition to the above benefits, ER labeling was achieved with the coumarin-based probes in both live cells and fixed cells, revealing their versatility for a wide range of cellular imaging applications.

A Photoactivatable BODIPY Probe for Localization-Based Super-Resolution Cellular Imaging.

The synthesis and application of a photoactivatable boron-alkylated BODIPY probe for localization-based super-resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously unknown boron-photodealkylation reaction with a single low-power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization-based microscopy of biological systems. The probe can be easily linked to nucleophile-containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super-resolution imaging of microtubules is demonstrated. This is the first example of single-molecule localization-based super-resolution microscopy using a visible-light-activated BODIPY compound as a fluorescent probe.