Members of a family of Ru(II)-appended pyrenylethynylene dyads were synthesized, characterized according to their photophysical and photobiological properties, and evaluated for their collective potential as photosensitizers for metal-organic photodynamic therapy. The dyads in this series possess lowest-lying (3)IL-based excited states with lifetimes that can be tuned from 22 to 270 μs in fluid solution and from 44 to 3440 μs in glass at 77 K. To our knowledge, these excited-state lifetimes are the longest reported for Ru(II)-based dyads containing only one organic chromophore and lacking terminal diimine groups. These excited states proved to be extremely sensitive to trace amounts of oxygen, owing to their long lifetimes and very low radiative rates. Herein, we demonstrate that (3)IL states of this nature are potent photodynamic agents, exhibiting the largest photocytotoxicity indices reported to date with nanomolar light cytotoxicities at very short drug-to-light intervals. Importantly, these new agents are robust enough to maintain submicromolar PDT in pigmented metastatic melanoma cells, where the presence of melanin in combination with low oxygen tension is known to compromise PDT. This activity underscores the potential of metal-organic PDT as an alternate treatment strategy for challenging environments such as malignant melanoma.

Here we show the design, preparation, and characterization of a dormant singlet oxygen ((1)O2) photosensitizer that is activated upon its reaction with reactive oxygen species (ROS), including (1)O2 itself, in what constitutes an autocatalytic process. The compound is based on a two segment photosensitizer-trap molecule where the photosensitizer segment consists of a Br-substituted boron-dipyrromethene (BODIPY) dye. The trap segment consists of the chromanol ring of α-tocopherol, the most potent naturally occurring lipid soluble antioxidant. Time-resolved absorption, fluorescence, and (1)O2 phosphorescence studies together with fluorescence and (1)O2 phosphorescence emission quantum yields collected on Br2B-PMHC and related bromo and iodo-substituted BODIPY dyes show that the trap segment provides a total of three layers of intramolecular suppression of (1)O2 production. Oxidation of the trap segment with ROS restores the sensitizing properties of the photosensitizer segment resulting in ∼40-fold enhancement in (1)O2 production. The juxtaposed antioxidant (chromanol) and prooxidant (Br-BODIPY) antagonistic chemical activities of the two-segment compound enable the autocatalytic, and in general ROS-mediated, activation of (1)O2 sensitization providing a chemical cue for the spatiotemporal control of (1)O2.The usefulness of this approach to selectively photoactivate the production of singlet oxygen in ROS stressed vs regular cells was successfully tested via the photodynamic inactivation of a ROS stressed Gram negative Escherichia coli strain.

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Several mononuclear Ru(II) dyads possessing 1,10-phenanthroline-appended pyrenylethynylene ligands were synthesized, characterized, and evaluated for their potential in photobiological applications such as photodynamic therapy (PDT). These complexes interact with DNA via intercalation and photocleave DNA in vitro at submicromolar concentrations when irradiated with visible light (λ irr g 400 nm). Such properties are remarkably sensitive to the position of the ethynylpyrenyl substituent on the 1,10-phenanthroline ring, with 3-substitution showing the strongest binding under all conditions and causing the most deleterious DNA damage. Both dyads photocleave DNA under hypoxic conditions, and this photoactivity translates well to cytotoxicity and photocytotoxicity models using human leukemia cells, where the 5-and 3-substituted dyads show photocytotoxicity at 5-10 μM and 10-20 μM, respectively, with minimal, or essentially no, dark toxicity at these concentrations. This lack of dark cytotoxicity at concentrations where significant photoactivity is observed emphasizes that agents with strong intercalating units, previously thought to be too toxic for phototherapeutic applications, should not be excluded from the arsenal of potential photochemotherapeutic agents under investigation.

Protein and DNA alkylation by endogenously produced electrophiles is associated with the pathogenesis of neurodegenerative diseases, to epigenetic alterations and to cell signaling and redox regulation. With the goal of visualizing, in real-time, the spatiotemporal response of the cell milieu to electrophiles, we have designed a fluorogenic BODIPY-acrolein probe, AcroB, that undergoes a >350-fold fluorescence intensity enhancement concomitant with protein adduct formation. AcroB enables a direct quantification of single post-translational modifications occurring on cellular proteins via recording fluorescence bursts in live-cell imaging studies. In combination with super-resolution imaging, protein alkylation events may be registered and individually counted, yielding a map of protein-electrophile reactions within the cell lipid milieu. Alkylation is predominantly observed within mitochondria, a source, yet not a sink, of AcroB adducts, illustrating that a mitochondrial constitutive excretion mechanism ensures rapid disposal of compromised proteins. Sorting within the Golgi apparatus and trafficking along microtubules and through the cell endo- and exocytic pathways can be next visualized via live-cell imaging. Our results offer a direct visualization of cellular response to a noncanonical acrolein warhead. We envision AcroB will enable new approaches for diagnosis of pathologies associated with defective cellular trafficking. AcroB may help elucidate key aspects of mitochondria electrophile adduct excretion and cell endocytic and exocytic pathways. Conceptually, AcroB provides a new paradigm on fluorescence microscopy studies where chemical perturbation is achieved and simultaneously reported by the probe.

Reactive oxygen species (ROS) and their associated byproducts have been traditionally associated with a range of pathologies. It is now believed, however, that at basal levels these molecules also have a beneficial cellular function in the form of cell signaling and redox regulation. Critical to elucidating their physiological role is the opportunity to visualize and quantify the production of ROS with spatiotemporal accuracy. Armed with a newly developed, extremely sensitive fluorogenic α-tocopherol analogue (HBPMHC), we report herein the observation of steady concentrations of lipid peroxyl radicals produced in live cell imaging conditions. Imaging studies with HBPMHC indicate that the rate of production of lipid peroxyl radicals in HeLa cells under basal conditions is 33 nM/h within the cell. Our work further provides indisputable evidence on the antioxidant role of Vitamin E, as lipid peroxidation was suppressed in HeLa cells both under basal conditions and in the presence of Haber-Weiss chemistry, generated by the presence of cumyl hydroperoxide and Cu in solution, when supplemented with the α-tocopherol surrogate, PMHC (2,2,5,7,8-pentamethyl-6-hydroxy-chromanol, an α-tocopherol analogue lacking the phytyl tail). HBPMHC has the sensitivity needed to detect trace changes in oxidative status within the lipid membrane, underscoring the opportunity to illuminate the physiological relevance of lipid peroxyl radical production during cell homeostasis and disease.

The photostability of reporter fluorophores in single-molecule fluorescence imaging is of paramount importance, as it dictates the amount of relevant information that may be acquired before photobleaching occurs. Quenchers of triplet excited states are thus required to minimize blinking and sensitization of singlet oxygen. Through a combination of single-molecule studies and ensemble mechanistic studies including laser flash photolysis and time-resolved fluorescence, we demonstrate herein that Ni(2+) provides a much desired physical route (chemically inert) to quench the triplet excited state of Cy3, the most ubiquitous green emissive dye utilized in single-molecule studies.

We used a case study approach to examine the nutritional effect of a policy to increase fruit and vegetable consumption in the Students Today Achieving Results for Tomorrow after-school program. The snack menu was changed in 44 after-school programs serving 8000 low-income and ethnically diverse elementary-school students. A comparison of previous and current snack menus identified a significant increase in fruit servings (83%) and no change in vegetable servings. We discuss the unintended consequences resulting from the menu changes.