Taming of Singlet Oxygen: Towards Artificial Oxygen Carriers Based on 1,4-Dialkylnaphthalenes.

Naphthalene endoperoxides are known as convenient sources of singlet oxygen (O2 , 1 Δg ), which is the major product of endoperoxide cycloreversion reaction. However, their potential as carriers of ground-state molecular oxygen (O2 , 3 Σg ) similar to artificial oxygen carriers remains largely unexplored. This is due to the extreme reactivity and cytotoxic effects of the released singlet oxygen. We now report that a compound with a bimodular design, which incorporates an endoperoxide and an efficient physical quencher of singlet oxygen, 1,4-diazabicyclo[2.2.2]octane (DABCO), produces exclusively ground-state molecular oxygen. This result, coupled with the fact that oxygen release rates from endoperoxides are highly amenable to fine-tuning in a very broad range, and open to targeting by ligand attachment, raises the potential of these compounds far above any comparable chemical, or even biochemical sources. In cell culture experiments, we showed that the addition of the endoperoxide-quencher conjugate can enhance and sustain cell proliferation.

Targeted Singlet Oxygen Delivery: A Bioorthogonal Metabolic Shunt Linking Hypoxia to Fast Singlet Oxygen Release.

Singlet oxygen can be generated by thermal cycloreversion of aromatic endoperoxides. However, for any practical potential of chemically generated singlet oxygen within a therapeutic context, the time and place of the release of this cytotoxic species must be tightly regulated. We now show that using a bimodular design with a hypoxia responsive unit and fluoride-triggered endoperoxide unit, a bioorthogonal metabolic shunt can be established, where an enzymatically generated submicromolar fluoride signal plays a crucial role. Thus, cellular nitroreductase is repurposed in a bioorthogonal enzymatic activity, where it releases fluoride ions upon the reduction of a targeted compound. The fluoride ions released in the initial reaction remove the silyl stopper, yielding a highly accelerated release of singlet oxygen. The result is a remarkable difference in cytotoxicity between hypoxic and normoxic conditions as evidenced by microscopy, viability assays and the use of control compounds.

Molecular logic gates: the past, present and future.

The field of molecular logic gates originated 25 years ago, when A. P. de Silva published a seminal article in Nature. Stimulated by this ground breaking research, scientists were inspired to join the race to simulate the workings of the fundamental components of integrated circuits using molecules. The rules of this game of mimicry were flexible, and have evolved and morphed over the years. This tutorial review takes a look back on and provides an overview of the birth and growth of the field of molecular logics. Spinning-off from chemosensor research, molecular logic gates quickly proved themselves to be more than intellectual exercises and are now poised for many potential practical applications. The ultimate goal of this vein of research became clearer only recently - to "boldly go where no silicon-based logic gate has gone before" and seek out a new deeper understanding of life inside tissues and cells.

Energy Harvesting in a Bodipy-Functionalized Rotaxane.

A rotaxane composed of two separate Bodipy-functionalized units can be synthesized with a high yield. The resulting structure shows a very efficient through-space energy transfer (FRET), acting as an energy funnel. Thus, maximum solar output in the visible region can be collected and converted into red light, which can be transformed efficiently with a fine-tuned photovoltaic device. The versatility of the synthetic pathway demonstrates the potential utility of rotaxane-based energy harvesting supramolecules assemblies.

Fluorescent chemosensors: the past, present and future.

Fluorescent chemosensors for ions and neutral analytes have been widely applied in many diverse fields such as biology, physiology, pharmacology, and environmental sciences. The field of fluorescent chemosensors has been in existence for about 150 years. In this time, a large range of fluorescent chemosensors have been established for the detection of biologically and/or environmentally important species. Despite the progress made in this field, several problems and challenges still exist. This tutorial review introduces the history and provides a general overview of the development in the research of fluorescent sensors, often referred to as chemosensors. This will be achieved by highlighting some pioneering and representative works from about 40 groups in the world that have made substantial contributions to this field. The basic principles involved in the design of chemosensors for specific analytes, problems and challenges in the field as well as possible future research directions are covered. The application of chemosensors in various established and emerging biotechnologies, is very bright.

A Bifunctional Photosensitizer for Enhanced Fractional Photodynamic Therapy: Singlet Oxygen Generation in the Presence and Absence of Light.

The photosensitized generation of singlet oxygen within tumor tissues during photodynamic therapy (PDT) is self-limiting, as the already low oxygen concentrations within tumors is further diminished during the process. In certain applications, to minimize photoinduced hypoxia the light is introduced intermittently (fractional PDT) to allow time for the replenishment of cellular oxygen. This condition extends the time required for effective therapy. Herein, we demonstrated that a photosensitizer with an additional 2-pyridone module for trapping singlet oxygen would be useful in fractional PDT. Thus, in the light cycle, the endoperoxide of 2-pyridone is generated along with singlet oxygen. In the dark cycle, the endoperoxide undergoes thermal cycloreversion to produce singlet oxygen, regenerating the 2-pyridone module. As a result, the photodynamic process can continue in the dark as well as in the light cycles. Cell-culture studies validated this working principle in vitro.

Remote-Controlled Release of Singlet Oxygen by the Plasmonic Heating of Endoperoxide-Modified Gold Nanorods: Towards a Paradigm Change in Photodynamic Therapy.

The photodynamic therapy of cancer is contingent upon the sustained generation of singlet oxygen in the tumor region. However, tumors of the most metastatic cancer types develop a region of severe hypoxia, which puts them beyond the reach of most therapeutic protocols. More troublesome, photodynamic action generates acute hypoxia as the process itself diminishes cellular oxygen reserves, which makes it a self-limiting method. Herein, we describe a new concept that could eventually lead to a change in the 100 year old paradigm of photodynamic therapy and potentially offer solutions to some of the lingering problems. When gold nanorods with tethered endoperoxides are irradiated at 808 nm, the endoperoxides undergo thermal cycloreversion, resulting in the generation of singlet oxygen. We demonstrate that the amount of singlet oxygen produced in this way is sufficient for triggering apoptosis in cell cultures.

Intracellular modulation of excited-state dynamics in a chromophore dyad: differential enhancement of photocytotoxicity targeting cancer cells.

The photosensitized generation of reactive oxygen species, and particularly of singlet oxygen [O2 (a(1) Δg )], is the essence of photodynamic action exploited in photodynamic therapy. The ability to switch singlet oxygen generation on/off would be highly valuable, especially when it is linked to a cancer-related cellular parameter. Building on recent findings related to intersystem crossing efficiency, we designed a dimeric BODIPY dye with reduced symmetry, which is ineffective as a photosensitizer unless it is activated by a reaction with intracellular glutathione (GSH). The reaction alters the properties of both the ground and excited states, consequently enabling the efficient generation of singlet oxygen. Remarkably, the designed photosensitizer can discriminate between different concentrations of GSH in normal and cancer cells and thus remains inefficient as a photosensitizer inside a normal cell while being transformed into a lethal singlet oxygen source in cancer cells. This is the first demonstration of such a difference in the intracellular activity of a photosensitizer.

Near-IR absorbing BODIPY derivatives as glutathione-activated photosensitizers for selective photodynamic action.

Enhanced spatiotemporal selectivity in photonic sensitization of dissolved molecular oxygen is an important target for improving the potential and the practical applications of photodynamic therapy. Considering the high intracellular glutathione concentrations within cancer cells, a series of BODIPY-based sensitizers that can generate cytotoxic singlet oxygen only after glutathione-mediated cleavage of the electron-sink module were designed and synthesized. Cell culture studies not only validate our design, but also suggest an additional role for the relatively hydrophobic quencher module in the internalization of the photosensitizer.

Near-IR-triggered, remote-controlled release of metal ions: a novel strategy for caged ions.

A ligand incorporating a dithioethenyl moiety is cleaved into fragments which have a lower metal-ion affinity upon irradiation with low-energy red/near-IR light. The cleavage is a result of singlet oxygen generation which occurs on excitation of the photosensitizer modules. The method has many tunable factors that could make it a satisfactory caging strategy for metal ions.

Self-reporting intracellular delivery agent for singlet oxygen.

1,4-Dimethylphenazine endoperoxide releases singlet oxygen with a half-life of 89 hours at 37 °C. The thermal cycloreversion reaction is accompanied by a strong increase in the emission intensity with a peak at 490 nm, due to the formation of the phenazine core. The endoperoxide is effective against cancer cells in culture medium and tumor spheroids, with singlet oxygen-mediated cytotoxicity.

Cascading of molecular logic gates for advanced functions: a self-reporting, activatable photosensitizer.

Logical progress: Independent molecular logic gates have been designed and characterized. Then, the individual molecular logic gates were coerced to work together within a micelle. Information relay between the two logic gates was achieved through the intermediacy of singlet oxygen. Working together, these concatenated logic gates result in a self-reporting and activatable photosensitizer. GSH=glutathione.

Upconverting Nanoparticle-Based Photoactive Probes for Highly Efficient Labeling and Isolation of Target Proteins.

Photoaffinity labeling (PAL) has blossomed into a powerful and versatile tool for capture and identification of biomolecular targets. However, low labeling efficiency for specific targets such as lectins, the tedious process for protein purification, inevitable cellular photodamage, and less tissue penetration of UV light are significant challenges. Herein, we reported a near-infrared (NIR) light-driven photoaffinity labeling approach using upconverting nanoparticle (UCNP)-based photoactive probes, which were constructed by assembling photoactive groups and ligands onto NaYF4:Yb,Tm nanoparticles. The novel probes were easily prepared and functionalized, and the labeled proteins can be isolated and purified through simple centrifugation and washing. The advantages of this approach were demonstrated by labeling and isolation of peanut agglutinin (PNA), asialoglycoprotein receptor (ASGPR), and human carbonic anhydrase II (hCAII) from mixed proteins or cell lysates with good selectivity and efficiency, especially for PNA and ASGPR, two lectins that showed low binding affinity to their ligands. More importantly, successful labeling of PNA through pork tissues and ASGPR in mice strongly proved the good tissue penetrating capacity of NIR light and the application potential of UCNP-based photoactive probes for protein labeling in vivo. Biosafety of this approach was experimentally validated by enzyme, cell, and animal work, and we demonstrated that NIR light caused minimal photodamage to enzyme activity compared to UV light, and the UCNP-based photoactive probe presents good biosafety both in vitro and in vivo. We believe that this novel PAL approach will provide a promising tool for study of ligand-protein interactions and identification of biomolecular targets.

Heavy atom free singlet oxygen generation: doubly substituted configurations dominate S1 states of bis-BODIPYs.

S(0), S(1), and T(1) states of various orthogonal 8,8' and 8,2'-bis-boradiaza-s-indacene (BODIPY) dyes, recently (Angew. Chem., Int. Ed.2011, 50, 11937) proposed as heavy atom free photosensitizers for O(2)((1)Δ(g)) generation, were studied by multireference quantum chemical approaches. S(0)→S(1) excitation characteristics of certain bis-BODIPYs are shown to be drastically different than the parent BODIPY chromophore. Whereas a simple HOMO→LUMO-type single substitution perfectly accounts for the BODIPY core, S(1) states of certain orthogonal bis-BODIPYs are described as linear combinations of doubly substituted (DS) configurations which overall yield four electrons in four singly occupied orbitals. Computed DS character of S(1), strongly correlated with facile (1)O(2) production, was presumed to occur via S(1)→T(1) intersystem crossing (ISC) of the sensitizer. Further confirmation of this relation was provided by newly synthesized BODIPY derivatives and comparison of spectroscopic properties of their dimers and monomers. Near-IR absorption, desired for potential photodynamic therapy applications, was not pursuable for bis-chromophores by the standard strategy of π-extension, as DS singlet states are destabilized. Decreased exchange coupling in π-extended cases appears to be responsible for this destabilization. Comparisons with iodine incorporated bis-BODIPYs suggest that the dynamics of (1)O(2) generation via DS S(1) states are qualitatively different from that via ISC originating from heavy atom spin-orbit coupling. Although red-shifting the absorption wavelength to enter the therapeutic window does not seem attainable for orthogonal bis-BODIPYs with DS S(1) states, modifications in the chromophore cores are shown to be promising in fine-tuning the excitation characteristics.

Degradation of amyloid peptide aggregates by targeted singlet oxygen delivery from a benzothiazole functionalized naphthalene endoperoxide.

Aggregate structures formed by amyloid-ß (Aß) are correlated with the progression of pathogenesis in Alzheimer's disease. Previous works have shown that photodynamic photosensitizers were effective in oxidatively degrading amyloid-ß aggregates and thus decreasing their cytotoxicity under various conditions. In this work, we designed and synthesized a benzothiazole-naphthalene conjugate, with high level of structural analogy to Thioflavin T which is known to have high affinities for the amyloid peptide aggregates. The endoperoxide form (BZTN-O2) of this compound, which releases singlet oxygen with a half-life of 77 minutes at 37 °C, successfully inhibited and/or reversed amyloid aggregation. The endoperoxide is capable of singlet oxygen release without any need for light, and its charge-neutral form could allow blood-brain barrier (BBB) permeability. The therapeutic potential of such endoperoxide compounds with amyloid binding affinity is exciting.

Prostate-specific membrane antigen (PSMA) targeted singlet oxygen delivery via endoperoxide tethered ligands.

Singlet oxygen is the primary agent responsible for the therapeutic effects of photodynamic therapy (PDT). In this work, we demonstrate that singlet oxygen release due to thermal endoperoxide cycloreversion can be targeted towards specific features of selected cancer cells, and this targeted singlet oxygen delivery can serve as an effective therapeutic tool. Thus, cytotoxic singlet oxygen can be delivered regioselectively into prostate specific membrane antigen (PSMA) overexpressing lymph node carcinoma (LNCaP) cells. However, unlike typical photodynamic processes, there is no need for light or oxygen. The potential of the approach is exciting, considering the limitations on the availability of light and oxygen in deep-seated tumors.

From virtual to physical: integration of chemical logic gates.

Integration by parts: advanced information processing at the molecular level requires integrated logic gates, which has to date been possible only virtually. Now, two independently working AND molecular logic gates are brought together by "click" chemistry to form integrated logic gates which respond exactly as predicted from such an integration scheme.

Naphthalene Endoperoxide Heterodimer Designed for Sustained Singlet Oxygen Release.

Naphthalene endoperoxides undergo thermal cycloreversion reactions to produce singlet oxygen and their parent naphthalene compounds. The rate of the reaction is dependent on the structural features, such as steric and electronic modulators. We believe that achieving a sustained release rate of singlet oxygen is important in potential biological applications. This can be achieved by tethering of two endoperoxides with different singlet oxygen release rates in a single molecular construct. Here, we report the synthesis of such a dimeric endoperoxide. Our data shows that with the biexponential reaction kinetics of singlet oxygen generation from a heterodimeric endoperoxide, it is possible to hold singlet oxygen release rates within a selected range for a longer period of time.

Anticoagulant activity of singlet oxygen released from a water soluble endoperoxide by thermal cycloreversion.

Singlet oxygen generated by photosensitization has limited potential in vivo due to light attenuation in tissues. However, controlled chemical generation of this reactive oxygen species is likely to open new therapeutic spaces to explore. The fact that its activity is limited by the rate of cycloreversion reaction and the diffusion distance of the excited state molecular oxygen species, is a clear advantage, considering the serious side effects of off-target anticoagulants. In this work, we present novel 1,4-naphthalene endoperoxides as potential anti-coagulant agents due to thermal release of singlet oxygen.

Silyl-naphthalene endoperoxides as switchable sources of singlet oxygen for bactericidal activity.

Singlet oxygen is a short half-life cytotoxic agent which can be generated by chemical and photochemical methods. In order to make use of its antibacterial action at a selected location, it is desirable to have singlet oxygen in a relatively stable, "caged" structure, in the form of an endoperoxide. Here, the trimethylsilyl (TMS) group supplies the steric bulk, inhibiting the cycloreversion reaction to produce very little singlet oxygen under ambient conditions. However, when fluoride ions are added as tetrabutylammonium fluoride, very rapid removal of the TMS group takes place, followed by the unhindered cycloreversion, releasing singlet oxygen much faster. The bactericidal action on surfaces was demonstrated using E. coli, and imaged under fluorescence microscopy. Considering the issues related to emergence of antibiotic resistant bacterial strains, "on demand singlet oxygen" appears to be an exciting alternative.