Gonzalo Cosa

Photophysical Properties of Fluorescent DNA-dyes Bound to Single- and Double-stranded DNA in Aqueous Buffered Solution¶

Abstract The absorption and fluorescence spectra, fluorescence quantum yields, lifetimes and time-resolved fluorescence spectra are reported for nine different fluorescent DNA-dyes. The work was initiated in search of a quantitative method to detect the ratio of single-to-double stranded DNA (ssDNA/dsDNA) in solution based on the photophysics of dye–DNA complexes; the result is a comprehensive study providing a vast amount of information for users of DNA stains. The dyes examined were the bisbenzimide or indole-derived stains (Hoechst 33342, Hoechst 33258 and 4′,6-diamidino-2-phenylindole), phenanthridinium stains (ethidium bromide and propidium iodide) and cyanine dyes (PicoGreen, YOYO-1 iodide, SYBR Green I and SYBR Gold). All were evaluated under the same experimental conditions in terms of ionic strength, pH and dye–DNA ratio. Among the photophysical properties evaluated only fluorescence lifetimes for the cyanine stilbene dyes allowed a convenient differentiation between ssDNA and dsDNA. The bisbenzimide dyes showed multiexponential decays when bound to either form of DNA, making lifetime-based analysis cumbersome with inherent errors. These dyes also presented biexponential decay when free in aqueous buffered solutions at different pH. A mechanism for their deactivation is proposed based on two different conformers decaying with different kinetics. The phenanthridinium dyes showed monoexponential decays with ssDNA and dsDNA, but there was no discrimination between them. High dye–DNA ratios (e.g. 1:1) resulted in multiexponential decays for cyanine dyes, resulting from energy transfer or self-quenching deactivation. Shifts in both absorption and fluorescence maxima for both ssDNA and dsDNA DNA–cyanine dye complexes were small. Broadening of dye–ssDNA absorption and fluorescence bands for the cyanine dyes relative to dye–dsDNA bands was detected and attributed to higher degrees of rotational freedom in the former.

Modular construction of DNA nanotubes of tunable geometry and single- or double-stranded character

DNA nanotubes can template the growth of nanowires, orient transmembrane proteins for nuclear magnetic resonance determination, and can potentially act as stiff interconnects, tracks for molecular motors and nanoscale drug carriers. Current methods for the construction of DNA nanotubes result in symmetrical and cylindrical assemblies that are entirely double-stranded. Here, we report a modular approach to DNA nanotube synthesis that provides access to geometrically well-defined triangular and square-shaped DNA nanotubes. We also construct the first nanotube assemblies that can exist in double- and single-stranded forms with significantly different stiffness. This approach allows for parameters such as geometry, stiffness, and single- or double-stranded character to be fine-tuned, and could enable the creation of designer nanotubes for a range of applications, including the growth of nanowires of controlled shape, the loading and release of cargo, and the real-time modulation of stiffness and persistence length within DNA interconnects.

Bodipy Dyes with Tunable Redox Potentials and Functional Groups for Further Tethering: Preparation, Electrochemical, and Spectroscopic Characterization

The preparation, spectroscopic, and electrochemical characterization of a family of 16 new bodipy dyes with tunable redox potentials and versatile functional groups is reported. Electron-withdrawing or -donating groups (Et, H, Cl, or CN) at positions C2 and C6 enabled tuning the redox potentials within a ca. 0.7 eV window without significantly affecting either the HOMO-LUMO gap or the absorption and emission spectra. Hydroxymethyl or formyl groups at the meso (C8) position in turn provided a handle for covalent tethering to receptors and biomolecules of interest, which dispenses with the more commonly used meso-aryl moiety as a means to tag molecules. The dyes can thus be coupled to both electrophiles and nucleophiles. Importantly, it is shown that meso-formyl bodipy dyes are nonemissive and have significantly lower molar extinction coefficients compared to their meso-hydroxymethyl and meso-acetoxymethyl counterparts (which in turn are bright, with emission quantum yields in the range of 0.7-1). The nonemissive meso-formyl bodipy dyes thus provide unique opportunities as fluorogenic probes of nucleophilic attack and as fluorescent labeling agents where uncoupled fluorophores will not contribute to the fluorescence background. Overall, the new bodipy dyes reported here are promising candidates for the preparation of fluorescent sensors relying on photoinduced electron transfer and may find use in a number of fluorescent-labeling protocols.

Photo-induced Metal-Catalyst-Free Aromatic Finkelstein Reaction

The facile iodination of aromatic compounds under mild conditions is a great challenge for both organic and medicinal chemistry. Particularly, the synthesis of functionalized aryl iodides by light has long been considered impossible due to their photo-lability, which actually makes aryl iodides popular starting materials in many photo-substitution reactions. Herein, a photo-induced halogen exchange in aryl or vinyl halides has been discovered for the first time. A broad scope of aryl iodides can be prepared in high yields at room temperature under exceptionally mild conditions without any metal or photo-redox catalysts. The presence of a catalytic amount of elemental iodine could promote the reaction significantly.

Photodegradation and photosensitization in pharmaceutical products: Assessing drug phototoxicity*

Toxic reactants are a common result of the interaction of sunlight with pharmaceutical agents transported in the blood system or applied topically. Over the past decade there has been a considerable amount of research toward understanding both the unimolecular deactivation pathway of photoexcited pharmaceutical products and their photosensitizing capability in the presence of biological substrates. This work summarizes recent developments in the study of the photodegradation mechanism of ketoprofen, fenofibric acid, and tiaprofenic acid. An analysis of excited-state electronic energy levels, the type of intermediates formed following excitation, and transient intermediate lifetimes is presented. The analysis involves both parent drugs and their major photoproducts. Phototoxicity, usually the result of adverse photochemical reactions following direct photoexcitation of the drugs, is shown to be strongly related to the photoexcitation of photoproducts when high radiation dose conditions prevail. The photoproducts are the species directly involved in photosensitizing reactions.

Influence of solvent polarity and base concentration on the photochemistry of ketoprofen: independent singlet and triplet pathways

The photochemistry of ketoprofen in aqueous solutions is strongly influenced by its acid–base chemistry. While the acid form of ketoprofen behaves as a typical benzophenone chromophore, the ketoprofen carboxylate undergoes efficient photodecarboxylation involving a carbanion intermediate. In the present work we have conducted studies in organic solvent–water mixtures in attempts to establish the nature of the carbanion precursor. We find a dual photochemical behavior of ketoprofen depending on the protic state of the ground state absorbing species. The detection of both the ketoprofen triplet and the carbanion during the photolysis of ketoprofen in these mixtures, and the experimentally determined independence of both pathways indicates that the carbanion originates from the singlet state in the system under study.

How Drug Photodegradation Studies Led to the Promise of New Therapies and Some Fundamental Carbanion Reaction Dynamics along the Way

The photodegradation of nonsteroidal anti-inflammatory drugs (NSAIDs), a class of medications that includes aspirin and ibuprofen, has generated considerable interest since the 1990s, largely because of the phototoxic and photoallergic effects that frequently accompany their therapeutic use. Among NSAIDs, ketoprofen, which contains a benzophenone chromophore, has been extensively studied, reflecting both its notorious adverse effects and the fascination that photochemists have with benzophenone. The photochemistry of ketoprofen involves the intermediacy of an easily detectable carbanion with a remarkable lifetime of 200 ns in water; its life expectancy can in fact be extended to minutes under carefully controlled anhydrous conditions. Over the past decade, we have used some key properties of the ketoprofen carbanion to conduct mechanistic studies on carbanions under various conditions. In particular, its ease of photogeneration provides the temporal control required for kinetic studies, which, combined with its long lifetime and readily detectable visible absorption, have enabled extensive laser flash photolysis work. These studies have led to an intimate understanding of the reaction dynamics for carbanions in solution, including the determination of absolute rate constants for protonation, S(N)2, and elimination reactions. Together they provide excellent exemplars of reactivity patterns that today are part of all introductory curricula in organic chemistry and illustrate the fundamentals of nucleophilic substitution paradigms. More recently, we have begun to exploit the photochemistry of ketoprofenate and have developed the ketoprofenate photocage, a valuable tool for the photocontrolled cleavage of protecting groups and concomitant drug release. The photorelease has been illustrated with ibuprofen, among many other molecules. These photocages have been further improved with the use of the xanthone chromophore; the goal is the release of antiviral agents taking advantage of the improved UVA absorption of xanthone (xanthonate photocages). In this Account, we survey our work of the past few years on the photochemistry of ketoprofen and related chromophores. Beginning with studies on the phototoxicity of ketoprofen, we have made the journey to new prodrug candidates, unraveling mechanistic elements of aroyl-substituted benzyl carbanions along the way.

Reactive Oxygen Species Mediated Activation of a Dormant Singlet Oxygen Photosensitizer: From Autocatalytic Singlet Oxygen Amplification to Chemicontrolled Photodynamic Therapy

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.

Phenol-Based Lipophilic Fluorescent Antioxidant Indicators: A Rational Approach

The reactivity, electrochemistry, and photophysics of the novel antioxidant indicator B-TOH, a BODIPY-alpha-tocopherol adduct, were investigated. We also studied a newly prepared BODIPY-3,5-di-tert-butyl-4-hydroxybenzoic acid adduct (B-BHB) and compared the results for both sets of probes. Our results highlight the potential of B-TOH as a fluorescent antioxidant indicator and help illustrate the considerations to be taken into account in preparing a receptor-reporter-type fluorescent antioxidant indicator. Based on the experimental values of the redox potentials for the reporter BODIPY and from the redox potentials estimated for the phenol receptor segment, the off-to-on emission enhancement recently reported for B-TOH upon peroxyl radical scavenging can be unequivocally assigned to the deactivation of an intramolecular photoinduced electron transfer (PeT) which operates in the reduced form of B-TOH. Theoretical calculations performed at the B3LYP/6-31G(d) level on HOMO energy levels relative to vacuum further support the deactivation of a PeT mechanism upon peroxyl radical scavenging by B-TOH. Fluorescence lifetimes and fluorescence quantum yields measured in a range of solvent polarities, from hexane to acetonitrile, for B-TOH, B-BHB, and their BODIPY precursors PM605 or PMOH, are consistent with an intramolecular nonradiative decay pathway operative in B-TOH. This pathway is not operative in B-BHB where PeT is deemed highly endergonic based on electrochemical studies. A subsequent analysis on the antioxidant properties of both fluorophore-phenol adducts studied herein indicates that B-TOH antioxidant activity is on par with that of alpha-tocopherol, the most potent naturally occurring lipid soluble antioxidant, whereas B-BHB is a poor antioxidant. Oxygen uptake studies upon peroxyl radical initiated styrene autoxidation and laser flash photolysis studies on the rate of H-atom abstraction by cumyloxyl radicals reveal similar reactivity patterns for B-TOH and 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMHC), an alpha-tocopherol analogue lacking the phytil tail. Analogous reactivity studies on B-BHB underscore its poor antioxidant activity. In general, this work provides substantial amount of information useful in designing off/on lipid soluble fluorescent antioxidant indicators based on phenol moieties.

Fluorogenic α-Tocopherol Analogue for Monitoring the Antioxidant Status within the Inner Mitochondrial Membrane of Live Cells

We report here the preparation of a lipophilic fluorogenic antioxidant (Mito-Bodipy-TOH) that targets the inner mitochondrial lipid membrane (IMM) and is sensitive to the presence of lipid peroxyl radicals, effective chain carriers in the lipid chain autoxidation. Mito-Bodipy-TOH enables monitoring of the antioxidant status, i.e., the antioxidant load and ability to prevent lipid chain autoxidation, within the inner mitochondrial membrane of live cells. The new probe consists of 3 segments: a receptor, a reporter, and a mitochondria-targeting element, constructed, respectively, from an α-tocopherol-like chromanol moiety, a BODIPY fluorophore, and a triphenylphosphonium cation (TPP). The chromanol moiety ensures reactivity akin to that of α-tocopherol, the most potent naturally occurring lipid soluble antioxidant, while the BODIPY fluorophore and TPP ensure partitioning within the inner mitochondrial membrane. Mechanistic studies conducted either in homogeneous solution or in liposomes and in the presence of free radical initiators show that the antioxidant activity of Mito-Bodipy-TOH is on par with that of α-tocopherol. Studies conducted on live fibroblast cells further show the antioxidant depletion in the presence of methyl viologen (paraquat), a known agent of oxidative stress and source of superoxide radical anion (and indirectly, a causative of lipid peroxidation) within the mitochondria matrix. We recorded a ca. 8-fold emission enhancement with Mito-Bodipy-TOH in cells stressed with methyl viologen, whereas no enhancement was observed in control studies with untreated cells. Our findings underscore the potential of the new fluorogenic antioxidant Mito-Bodipy-TOH to study the chemical link between antioxidant load, lipid peroxidation and mitochondrial physiology.