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.

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.

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.

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.

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.

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.

Proof-of-principle for two-stage photodynamic therapy: hypoxia triggered release of singlet oxygen.

We propose to overcome oxygen deficiency and light attenuation problems in photodynamic therapy (PDT), by separating photoexcitation and singlet oxygen delivery of the PDT process into two distinct operations to be carried out sequentially, at different locations. We now demonstrate the viability of this approach, using 2-pyridone derivative which yields a relatively stable endoperoxide. The initial storage endoperoxide obtained is transformed enzymatically into a more labile compound when placed in hypoxic cell cultures, and releases singlet oxygen significantly faster. The potential of this approach in advancing PDT beyond its current limits is exciting.

Mechanochemical generation of singlet oxygen.

Controlled generation of singlet oxygen is very important due to its involvement in scheduled cellular maintenance processes and therapeutic potential. As a consequence, precise manipulation of singlet oxygen release rates under mild conditions, is crucial. In this work, a cross-linked polyacrylate, and a polydimethylsiloxane elastomer incorporating anthracene-endoperoxide modules with chain extensions at the 9,10-positions were synthesized. We now report that on mechanical agitation in cryogenic ball mill, fluorescence emission due to anthracene units in the PMA (polymethacrylate) polymer is enhanced, with a concomitant generation of singlet oxygen as proved by detection with a selective probe. The PDMS (polydimethylsiloxane) elastomer with the anthracene endoperoxide mechanophore, is also similarly sensitive to mechanical force.

"Off-on" switching of intracellular singlet oxygen release under biocompatible conditions.

Precise spatiotemporal control of singlet oxygen generation is of immense importance considering its involvement in photodynamic therapy. In this work, we present a rational design for an endoperoxide which is highly stable at ambient temperatures yet, can rapidly be converted into a highly labile endoperoxide, thus releasing the "stored" singlet oxygen on demand. The "off-on" chemical switching from the stable to the labile form is accomplished by the reaction with fluoride ions. The potential utility of controlled singlet oxygen release was demonstrated in cell cultures.

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.

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.

Molecular demultiplexer as a terminator automaton.

Molecular logic gates are expected to play an important role on the way to information processing therapeutic agents, especially considering the wide variety of physical and chemical responses that they can elicit in response to the inputs applied. Here, we show that a 1:2 demultiplexer based on a Zn2+-terpyridine-Bodipy conjugate with a quenched fluorescent emission, is efficient in photosensitized singlet oxygen generation as inferred from trap compound experiments and cell culture data. However, once the singlet oxygen generated by photosensitization triggers apoptotic response, the Zn2+ complex then interacts with the exposed phosphatidylserine lipids in the external leaflet of the membrane bilayer, autonomously switching off singlet oxygen generation, and simultaneously switching on a bright emission response. This is the confirmatory signal of the cancer cell death by the action of molecular automaton and the confinement of unintended damage by excessive singlet oxygen production.

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.

Singlet Oxygen Generation with Chemical Excitation of an Erythrosine-Luminol Conjugate.

Chemical generation of singlet oxygen under biologically relevant conditions is very important, considering the role played by singlet oxygen in cancer therapeutics. We now demonstrate that a luminol derivative can be chemically excited and transfer the excitation energy to the covalently attached photosensitizer derived from erythrosin. A photosensitizer module, when excited in this manner, can generate singlet oxygen in solution. As hydrogen peroxide is present in a relatively high concentration in cancer cells, singlet oxygen generation through chemical excitation may evolve into an important therapeutic approach.

Amplified Chemiluminescence Signal for Sensing Fluoride Ions.

Bringing together the concepts of self-immolative linkers and chemiluminogen dioxetane modules, a chemiluminescence-based sensor for fluoride with signal amplification is presented. Signal amplification is obtained by triggering two chemiluminescence events for each reacting fluoride ion that in turn releases two fluoride ions for each ion. As expected, the chemiluminescence signal starts to rise following an induction period. In addition to the analytical potential, this chemical system is also of interest as a demonstration of positive feedback loop character.

Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase.

Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.

Orthogonal Bodipy Trimers as Photosensitizers for Photodynamic Action.

Orthogonally linked BODIPY units show exceptional intersystem crossing efficiencies. We now report orthogonal BODIPY trimers with strong absorption in the visible region and high singlet oxygen generation capability. The X-ray diffraction structure confirms that the two peripheral BODIPY units are at a perpendicular angle to the core structure.

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.