Nitroxide amide-BODIPY probe behavior in fibroblasts analyzed by advanced fluorescence microscopy.

A novel synthesized nitroxide amide-BODIPY prefluorescent probe was used to study cellular redox balance that modulates nitroxide/hydroxylamine ratio in cultured human fibroblasts. FLIM quantitatively differentiated between nitroxide states of the cytoplasm-localized probe imaged by TIRF, monitoring nitroxide depletion by hydrogen peroxide; eluding incorrect interpretation if only fluorescence intensity is considered.

Reaction mechanism in crystalline solids: kinetics and conformational dynamics of the Norrish type II biradicals from α-adamantyl-p-methoxyacetophenone.

In an effort to determine the details of the solid-state reaction mechanism and diastereoselectivity in the Norrish type II and Yang cyclization of crystalline α-adamantyl-p-methoxyacetophenone, we determined its solid-state quantum yields and transient kinetics using nanocrystalline suspensions. The transient spectroscopy measurements were complemented with solid-state NMR spectroscopy spin-lattice relaxation experiments using isotopically labeled samples and with the analysis of variable-temperature anisotropic displacement parameters from single-crystal X-ray diffraction to determine the rate of interconversion of biradical conformers by rotation of the globular adamantyl group. Our experimental findings include a solid-state quantum yield for reaction that is 3 times greater than that in solution, a Norrish type II hydrogen-transfer reaction that is about 8 times faster in crystals than in solution, and a biradical decay that occurs on the same time scale as conformational exchange, which helps to explain the diastereoselectivity observed in the solid state.

Design of xanthone propionate photolabile protecting group releasing acyclovir for the treatment of ocular herpes simplex virus.

We have attached the antiviral drug acyclovir (ACV) to a xanthone photolabile protecting group (or photocage) through the O6 position of acyclovir, a procedure designed for the treatment of ocular herpes simplex virus infections. Acyclovir is photoreleased from the photocage, under physiological conditions, with a quantum yield (Φ(ACV release)) of 0.1-0.3 and an uncaging cross section (Φ·Îµ) of 450-1350 M cm(-1). We demonstrate that this photorelease method outcompetes alternative reaction pathways, such as protonation. Furthermore, complete release of the drug is theoretically possible given a sufficient dose of light . Surprisingly the acyclovir photocage, also showed some antiviral activity towards HSV-1.

Accurate O-H bond dissociation energy differences of hydroxylamines determined by EPR spectroscopy: computational insight into stereoelectronic effects on BDEs and EPR spectral parameters.

Differences in O-H bond dissociation enthalpies (ΔBDEs) between the hydroxylamine of (15)N-labeled TEMPONE and 10 N,N-di-tert-alkyl hydroxylamines were determined by EPR. These ΔBDEs, together with the g and a(N) values of the derived nitroxide radicals, are discussed in relation to various geometric, intramolecular dipole/dipole, and steric effects and in relation to the results from DFT calculations. We find that dipole/dipole interactions are the dominant factors in dictating a(N) values and O-H BDEs in all of these structurally similar nitroxides and hydroxylamines, respectively. The importance of including the Boltzmann distribution of conformations for each nitroxide in the a(N) calculations is emphasized.

Photophysics and photochemistry of aflatoxins B1 and B2.

Aflatoxins are mycotoxins produced by fungi of the genus Aspergillus, which is widely spread in the tropics and subtropics. To date, aflatoxin phototoxicity has been recognized, but the mechanism responsible for this phototoxicity has not been fully characterized. In the present paper, nanosecond laser flash photolysis studies allowed us to elucidate the photochemical processes undergone by two mycotoxins, namely aflatoxin B(1) and B(2), upon UV irradiation. In brief, photolysis (308 nm) of the aflatoxins leads to intersystem crossing, giving rise to their triplet excited state. The triplet state can readily be quenched by indole and hydroquinone, and also by molecular oxygen yielding singlet oxygen (singlet oxygen quantum yields of 0.51 and 0.59 were found for aflatoxin B(1) and B(2), respectively). In addition, our data indicate the ability of the two aflatoxins to photoionize upon 248 nm excitation. The photoionization quantum yield for aflatoxin B(1) and B(2) have been estimated to be 0.11 and 0.29, respectively.

Opportunistic use of tetrachloroaurate photolysis in the generation of reductive species for the production of gold nanostructures.

The photolysis of gold salts is rarely viewed as the initiation for gold nanoparticle (AuNP) formation. Yet, photolysis of AuCl(4)(-) generates chlorine atoms whose rich hydrogen transfer chemistry can readily generate strongly reducing radicals. Interesting precursors include hydrogen peroxide, 2-propanol, 1,4-cyclohexadiene and tetrahydrofuran; all of them yield strongly reducing radicals. Further, this group of substrates has been selected because of the innocuous and volatile nature of reagents and products, thus allowing a remarkably clean synthesis of gold nanostructures. In the case of H(2)O(2) the by-products are water and oxygen. The methodology reported here opens the door to particles that can be modified in situ or post-synthesis with custom surface covering without concern for chemical debris from the nanostructure synthesis.

Surface plasmons control the dynamics of excited triplet states in the presence of gold nanoparticles.

Aqueous gold nanoparticles (AuNPs) cause a large increase in the yield of methylene blue triplets ((3)MB*) obtained upon 650 nm laser excitation as a result of surface plasmon field interactions that can be described as transmitter-receiver antenna effects. Two distinct (3)MB* populations are observed; a fast decaying one (tau(T) approximately 25 ns) is believed to be due to molecules on the AuNP surface at the time of excitation and is described as static quenching. A longer lived (3)MB* population has lifetimes in the tens of microseconds but is subject to an anomalously high rate constant for a AuNP quenching of 6.4 x 10(13) M(-1) s(-1). This ultrafast quenching is attributed to a nonrandom distribution caused by the AuNP plasmon field that preferentially excites MB molecules located in the proximity of the AuNP where they are subject to antenna type interactions with the nanoparticle and are spatially predisposed for efficient quenching.

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.

Photobehavior of merocyanine 540 bound to human serum albumin.

The photobehavior of merocyanine 540 (MC) was studied in homogeneous media (ethanol, buffer and glycerol), and in microheterogenous systems (Triton X-100 micelles and in the presence of human serum albumin) using stationary and time-resolved techniques. Merocyanine 540 in aqueous solution mostly forms aggregates, which in the presence of Triton X-100 or HSA are disaggregated. The extent of binding to HSA and its characteristics were estimated from dye absorption and fluorescence changes following protein addition; the Trp-214 fluorescence quenching was also employed to assess the extent of dye association, and physical separation was employed to evaluate the dye's apparent association constant. These results showed that dye adsorption on HSA takes place at both main protein-binding sites (I and II). This adsorption leads to dye monomerization, changing its photobehavior remarkably, with a noticeable increase in fluorescence and triplet lifetimes. These slower decays can be ascribed to a reduction of the dye photoisomerization rate. In addition, the adsorption of the dye partially protects it from the oxygen present in solution, thus reducing the apparent dye triplet-quenching rate constant. However, singlet oxygen and MC triplet quantum yields remain very low in all the systems tested. Finally, we found that the photoconsumption of merocyanine bound to HSA takes place predominantly by a type I mechanism, being more than seven times more efficient than that taking place in ethanol.

Photophysics and photochemistry of dyes bound to human serum albumin are determined by the dye localization.

The photophysics and photochemistry of rose bengal (RB) and methylene blue (MB) bound to human serum albumin (HSA) have been investigated under a variety of experimental conditions. Distribution of the dyes between the external solvent and the protein has been estimated by physical separation and fluorescence measurements. The main localization of protein-bound dye molecules was estimated by the intrinsic fluorescence quenching, displacement of fluorescent probes bound to specific protein sites, and by docking modelling. All the data indicate that, at low occupation numbers, RB binds strongly to the HSA site I, while MB localizes predominantly in the protein binding site II. This different localization explains the observed differences in the dyes' photochemical behaviour. In particular, the environment provided by site I is less polar and considerably less accessible to oxygen. The localization of RB in site I also leads to an efficient quenching of the intrinsic protein fluorescence (ascribed to the nearby Trp residue) and the generation of intra-protein singlet oxygen, whose behaviour is different to that observed in the external solvent or when it is generated by bound MB.

On-off QD switch that memorizes past recovery from quenching by diazonium salts.

The understanding of the interaction of CdSe/ZnS semiconductor quantum dots (QD) with their chemical environment is fundamental, yet far from being fully understood. p-Methylphenyldiazonium tetrafluoroborate has been used to get some insight into the effect of diazonium salts on the spectroscopy of QD. Our study reveals that the surface of CdSe/ZnS quantum dots can be modified by diazonium salts (although not functionalized), showing and on-off fluorescence behaviour that memorizes past quenching recoveries. Facile modification of the surface confers protection against quenching by new molecules of diazonium salt and other known quenchers such as 4-amino-TEMPO. The reaction mechanism has been explored in detail by using different spectroscopic techniques. At the first time after addition of diazonium salt over QD the fluorescent is turned off with Stern-Volmer behaviour; the fluorescence recovers following irradiation. Subsequent additions of diazonium salts do not cause the same degree of quenching. We have noted that the third addition (following two cycles of addition and irradiation) is unable to quench the fluorescence. Monitoring the process using NMR techniques reveals the formation of p-difluoroborane toluene as a result of the irradiation of diazonium-treated QD; the treatment leads to the fluorination of the QD surface.

Photoinduced formation and characterization of electron-hole pairs in azaxanthylium-derivatized short single-walled carbon nanotubes.

2-Azaxanthone, a nitrogenated derivative of the well-studied organic chromophore xanthone, has been covalently bound through 2-(ethylthio)ethylamido linkers to the carboxylic acid groups of short, soluble single-walled carbon nanotubes (CNTs) of 450 nm average length, and the resulting azaxanthylium-functionalized CNTs (AZX-CNT, 8.5 wt % AZX content) characterized by solution (1)H NMR, Raman and IR spectroscopy and thermogravimetric analysis. Comparison of the quenching of the triplet excited state of AZX (steady-state and time-resolved) and of the transient optical spectra of CNTs and AZX-CNT shows that the covalent linkage boosts the interaction between the azaxanthylium moiety and the short CNT units. The triplet excited state of the azaxanthylium derivative is quenched by CNT with and without covalent bonding, but when it is covalently bonded, the singular transient spectrum is compatible with the photogeneration of electron holes through electron transfer from CNT to excited azaxanthylium units.

7-mercapto-4-methylcoumarin as a reporter of thiol binding to the CdSe quantum dot surface.

Interaction of 7-mercapto-4-methylcoumarin with the surface of core-shell CdSe-ZnS quantum dots leads to characteristic absorption and emission bands that are excellent reporters for thiol interactions at the nanoparticle surface.

Raising the ceiling of diastereoselectivity in hydrogen transfer on acyclic radicals.

We report here that the monodentate complexation of Me2AlCl to an ester group significantly enhances the selectivity of hydrogen transfer on acyclic radicals flanked by both an ester functionality and a stereogenic center, leading to C-2,C-3-anti products with high diastereoselectivity. In certain cases improved ratios were obtained using bulkier aluminum Lewis acid such as MAD (methylaluminum-di(di-2,6-tert-butyl-4-methylphenoxide). Electron spin resonance studies on these acyclic radicals indicate that Lewis acid complexation leads to conformational changes in the radicals. The stereochemistry of the preferred enolate radicals complexed with Lewis acids and their impact on the acyclic transition states involved are suggested.

Cucurbituril complexes cross the cell membrane.

Cucurbiturils (CBs) of the appropriate size (CB[7] and CB[8]) form strong guest-host complexes in phosphate buffer solution (PBS) with acridine orange (AO) and pyronine Y (PYY) with 1 : 1 and 2 : 1 stoichiometries for CB[7] and CB[8] complexes, respectively. Binding constants in the range 0.87-1.60 x 10(6) M(-1) and 5.2-6.3 x 10(13) M(-2) were determined by titration with fluorescence spectroscopy for 1 : 1 and 2 : 1 complexes, respectively. These binding constants in PBS and the eight-fold excess of CBs minimize the presence of free dye in solution and also stabilize the host-guest complex in the culture medium. Images showing that the CB complexes can cross the cell membrane of 3T3 cells have been acquired using fluorescence microscopy. Given the current importance of supramolecular CB complexes and the search for new drug delivery systems, the present findings open avenues for the use of CBs as nanocapsules to transport drugs into the cells.

Quantitative determination of singlet oxygen generated by excited state aromatic amino acids, proteins, and immunoglobulins.

Singlet oxygen quantum yields generated by excited state aromatic amino acids (tryptophan, tyrosine, phenylalanine), N-acetylated amino acids (N-acetyl-tryptophan, N-acetyl-tyrosine, N-acetyl-phenylalanine), and from selected proteins and immunoglobulins have been quantified by time-resolved phosphorescence measurements. A small, but significant, quantum yield found for proteins and immunoglobulins demonstrates that molecular oxygen can diffuse through the polypeptide matrix and can be sensitized by residues buried within the folds of protein structure.

Hydrogen-transfer reactions from phenols to TEMPO prefluorescent probes in micellar systems.

A nitroxide prefluorescent probe has been used to evaluate local reactivity of antioxidants in micellar systems. An apparent rate constant that directly reflects the relevance of antioxidant hydrophobicity on the reaction toward nitroxide radicals has been defined. Dramatic increases in this parameter for quercetin are shown on moving from methanol to micellar media: 90 and 230 fold enhancements for SDS and Triton X100 micelles, respectively. This is a clear consequence of the favorable partition of reactants in the micelles.

Photophysical properties of the prefluorescent nitroxide probes QT and C343T.

The photophysical properties of the nitroxide prefluorescent probes 4-(3-hydroxy-2-methyl-4-quinolinoyloxy)-2,2,6,6-tetramethyl-piperidin-4-yl) ester free radical (QT) and 2,3,4,6,7,8-hexahydro-quinolizino [1,10,9-gh] coumarin-3-carboxylic acid (1-oxyl-2,2,6,6-tetramethyl-piperidin-4-yl) ester free radical (C343T) were evaluated as a function of pH and solvent properties. The absorbance of QT showed high pH sensitivity. The pKa values for the different ionization forms involved in the acid-base equilibrium of the quinoline chromophore were determined in the ground and excited states. The fluorescence lifetimes of QT, and N-hydroxylamine (QTH) and quinoline methyl ester (QMe) derivatives, showed that the intramolecular quenching efficiency by the nitroxide moiety is independent of the quinoline ionization form. The fluorescence and absorbance of C343T were highly sensitive to solvent polarity in agreement with a charged transfer excited state of the chromophore. However, we noted a decrease in the intramolecular fluorescence quenching efficiency by the nitroxide moiety when changing the polarity of the solvent from hexane to water. This behavior has been attributed to a suppression of an energy transfer mechanism in the nitroxide quenching process in very polar solvents. The results obtained in micelles allow us to propose QT and C343T as sensors for pH and micropolarity, respectively, in addition to their role as monitors for free radicals or hydrogen transfer from phenols.

Reactivity of adrenaline toward alkoxyl radicals and carbonyl triplet states.

The reactivities of adrenaline toward hydrogen abstraction by the tert-butoxyl radical and the excited triplet state of benzophenone have been examined in solution using laser flash photolysis techniques. The rate constants obtained in acetonitrile-rich solvents are 13 and 1.5 x 10(8) M(-1) s(-1) for benzophenone triplet and the tert-butoxyl radical, respectively. Adrenaline is a better hydrogen donor than phenol, but not as efficient as alpha-tocopherol.

Transient enol isomers of dibenzoylmethane and avobenzone as efficient hydrogen donors toward a nitroxide pre-fluorescent probe.

Dibenzoylmethane and avobenzone photochemistry involves the formation of transient enol isomers (Z and E). Conjugation of the OH group with the carbonyl group in these transient isomers reduces the OH bond energy. A fast reduction of the pre-fluorescent probe (C343T) was observed after addition to photolysed DBM samples in nonpolar solvents. This can be attributed to a hydrogen transfer reaction from the transient E accumulated. While no quenching for Z by TEMPO was detected in the laser flash photolysis timescale. Theoretical calculations of spin densities distribution of the radical formed from Z and E showed a more delocalized distribution that would indicate a low reactivity towards oxygen. Our results suggest that DBM and avobenzone can effectively behave as photoantioxidants or photoactivated antioxidants under conditions where its enol isomers can be accumulated.